pensée wu mb chb - imperial college london · pensée wu mb chb . thesis submitted for the degree...
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Muscular dystrophy cell therapy - an in utero approach
using human fetal mesenchymal stem cells
Pensée Wu MB ChB
Thesis submitted for the degree of Doctor of Medicine (Research)
Imperial College London
Institute of Reproductive and Developmental Biology Imperial College London, Hammersmith Campus
London W12 0NN, United Kingdom
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ABSTRACT
Duchenne muscular dystrophy (DMD) is the most prevalent genetic neuromuscular
disorder and affects 1 in 3,500 live male births. Lack of the protein dystrophin in
muscle fibres causes permanent muscle damage, is lethal and despite various potential
therapeutic strategies aimed at restoring dystophin expression, has no cure. As DMD
affects all skeletal muscles as well as the heart, a systemic treatment would be
necessary and in utero stem cell transplantation is a promising way of achieving this.
The identification of human fetal mesenchymal stem cells (hfMSC) in early gestation
fetal blood offers the prospect of allogeneic or autologous cell therapy, while
intrauterine administration would capitalise on ontological opportunities unique to the
developing fetus.
The aim of the study was to improve hfMSC engraftment and contribution to skeletal
muscle fibres following intrauterine transplantation (IUT) in a mouse model of DMD.
My project demonstrated that hfMSCs are easily isolated and expandable with the
ability to undergo myogenesis in vitro. HfMSCs differentiated into mature myotubes
following exposure to galectin-1 conditioned medium, while galectin-1 transduced
hfMSCs showed significantly higher expression of myogenic markers compared to
non-transduced hfMSCs. Co-culture experiments provided an in vitro model to
explore the underlying mechanism for muscle differentiation of hfMSCs following
IUT. HfMSCs were able to form chimeric myotubes by fusing with myoblasts
isolated from E15 mouse embryos, evidence that they should be able to fuse with
developing muscle fibres in vivo. Engraftment and differentiation into muscle fibres
of hfMSCs injected intra-peritoneally into E15 mouse embryos in vivo was enhanced
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by using immunodeficient dystrophic host mice, postnatal muscle injury and
additional neonatal hfMSC transplantation following IUT.
In conclusion, my thesis supports the use of hfMSC as an attractive source for cell
therapy and provides the background for further studies to optimise their engraftment
and differentiation to underpin future clinical applications.
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ACKNOWLEDGEMENTS
I would like to thank both my supervisors, without whom this thesis would not have
been completed. Professor Nicholas Fisk and Dr. Jennifer Morgan advised and guided
me throughout this project. When confronted with the unfortunate circumstance that
they were both to leave Imperial College during the latter half of my study, they went
to considerable effort to reduce the resultant impact. I am grateful for their support in
the modification of my plan of work to allow submission as an MD instead of a PhD
thesis. I am indebted to Dr. Nick Dibb, who kindly became my principal supervisor in
the latter stages of my project despite muscle work being a new area to him. I
acknowledge Wellbeing of Women and Institute of Obstetrics and Gynaecology Trust
for funding this work, and appreciate Professor Fisk’s generous offer of financial
support for my third year as a PhD student in case it had been needed.
It was a challenge after eighteen months of the project to change strategy and
reorganise a series of demanding experiments planned for three years into a
condensed two-year scheme of work. I am grateful to Mr. Sailesh Kumar and
Professor Fisk for their collection of fetal blood samples, to Dr. Morgan and Carl
Adkin for help with transplantation in postnatal mice, Dr. Pascale Guillot, Dr. Hitoshi
Kurata and Dr. Hassan Abdulrazzak for their assistance in intrauterine transplantation
work, and Professor Diana Lawrence-Watt for providing the galectin-1 plasmid.
I would like to thank the other members of the Experimental Fetal Medicine
Group, Dafni Moschidou, Paula Galea, Faisal Allaf, Margarida Santos, Sofia
Kanellopoulou, Oyebode Abass and Hanan Sultan, as well as the Muscle Biology
Group, Louisa Boldrin and Sofia Muses whose guidance and comradeship made it
possible for me to carry on. I am also thankful for people on the fifth floor of IRDB,
Ferda, Ellen, Sharon, Nicola, Elcie and Anil for being so helpful and friendly. The
advice from Dr. Keelin O’Donoghue and Dr. Jerry Chan at the beginning of my work
was highly valued, as was the consistent support from Mrs. Jan Preece throughout.
I am appreciative of the staff and patients of Queen Charlotte’s and Chelsea
Hospital, and the Hammersmith Hospital for taking part in this study. I thank the
Eastern Deanery for supporting the writing of this thesis during my clinical training.
Finally, I would like to thank my family for their constant encouragement and support,
and my fiancé, David, for always being there for me.
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TABLE OF CONTENTS
ABSTRACT. ............................................................................................................. 2
ACKNOWLEDGEMENTS ....................................................................................... 4
TABLE OF CONTENTS ........................................................................................... 5
LIST OF FIGURES ................................................................................................. 13
LIST OF TABLES .................................................................................................. 17
ABBREVIATIONS ................................................................................................. 18
Units / symbols ................................................................................................ 21
CHAPTER 1 INTRODUCTION ............................................................................ 22
1.1. Stem cells ......................................................................................................... 23
1.1.1. Self-renewal ........................................................................................... 23
1.1.2. Plasticity ................................................................................................ 24
1.1.3. Niche ..................................................................................................... 25
1.1.4. Cancer stem cells ................................................................................... 26
1.1.5. Types of stem cells ................................................................................. 27
1.1.5.1. Totipotent embryonic stem cells .................................................. 27
1.1.5.2. Multipotent somatic stem cells ..................................................... 28
1.1.5.2.1. Adult mesenchymal stem cells .......................................... 29
1.1.5.2.1.1. Characterisation ...................................................... 29
1.1.5.2.1.2. Immunology ........................................................... 30
1.1.5.2.1.3. Challenges .............................................................. 32
1.1.5.2.2. Fetal mesenchymal stem cells ........................................... 33
1.1.5.2.2.1. Extraembryonic fetal tissues ................................... 35
1.1.5.2.2.1.1. Amniotic fluid .............................................. 35
1.1.5.2.2.1.2. Placenta and amnion .................................... 36
1.1.5.2.2.1.3. Umbilical cord ............................................. 37
1.1.5.3. Unipotent satellite cells ................................................................ 42
1.1.6. Applications ........................................................................................... 42
1.1.6.1. Tissue repair ................................................................................ 43
1.1.6.1.1. Autologous versus allogeneic cell therapy ......................... 44
1.1.6.1.2. Ex vivo gene therapy ......................................................... 45
6
1.1.6.2. Paracrine effects .......................................................................... 47
1.1.6.3. Tumour targeting ......................................................................... 47
1.1.6.4. Microchimerism .......................................................................... 47
1.2. Duchenne muscular dystrophy .......................................................................... 48
1.2.1. Clinical features ..................................................................................... 49
1.2.2. Molecular pathology .............................................................................. 50
1.2.3. Animal models ....................................................................................... 51
1.2.3.1. Mdx ............................................................................................. 51
1.2.3.1.1. Mdx/nude .......................................................................... 52
1.2.3.1.2. Mdx/scid ........................................................................... 52
1.2.3.2. Utrophin and dystrophin knockout ............................................... 52
1.2.3.3. Golden retriever muscular dystrophic dogs .................................. 53
1.2.4. Treatment modalities .............................................................................. 53
1.2.4.1. Supportive measures .................................................................... 55
1.2.4.2. Corticosteroids............................................................................. 56
1.2.4.3. Gene therapy ............................................................................... 56
1.2.4.3.1. Gene-replacement strategies using adeno-associated virus
vector ............................................................................................... 56
1.2.4.3.1.1. Challenges .............................................................. 58
1.2.4.3.2. Modification of dystrophin messenger ribonucleic acid
(mRNA) splicing using antisense oligonucleotides ........................... 58
1.2.4.3.2.1. Challenges .............................................................. 59
1.2.4.3.3. Read-through stop-codon strategies ................................... 60
1.2.4.4. Cell therapy ................................................................................. 61
1.2.4.4.1. Myoblasts ......................................................................... 61
1.2.4.4.2. Side population ................................................................. 62
1.2.4.4.3. Skeletal muscle-derived stem cells .................................... 63
1.2.4.4.4. Pericytes ........................................................................... 63
1.2.4.4.5. Mesoangioblasts ................................................................ 64
1.2.4.4.6. CD133+ cells ..................................................................... 64
1.2.4.4.7. Other cell types ................................................................. 65
1.2.4.5. Modification of compensatory mechanisms ................................. 66
1.2.4.5.1. Utrophin upregulation ....................................................... 66
1.2.4.5.2. Insulin-like growth factor-1 ............................................... 66
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1.2.4.5.3. Other candidates................................................................ 66
1.3. Intrauterine transplantation ............................................................................... 67
1.3.1. Animal models ....................................................................................... 68
1.3.1.1. Wild type ..................................................................................... 68
1.3.1.2. Mdx ............................................................................................. 69
1.3.1.3. Osteopetrosis ............................................................................... 70
1.3.2. Humans .................................................................................................. 70
1.3.3. Strategies to overcome low engraftment ................................................. 71
1.3.3.1. Optimisation of cell source .......................................................... 71
1.3.3.2. Immunocompromised host ........................................................... 73
1.3.3.3. Serial transplantation ................................................................... 74
1.3.3.4. Postnatal muscle injury ................................................................ 74
1.3.3.5. Models with clinical phenotype ................................................... 75
1.4. Aims of this study ............................................................................................. 76
1.5. Hypotheses ....................................................................................................... 77
CHAPTER 2 MATERIALS AND METHODS ...................................................... 78
2.1. Ethics..... ........................................................................................................... 79
2.2. Human fetal mesenchymal stem cells ................................................................ 79
2.2.1. Harvest of first trimester fetal blood ....................................................... 79
2.2.2. Harvest of first trimester fetal bone marrow and liver ............................. 79
2.2.3. Isolation and cell culture ........................................................................ 80
2.2.4. Cryopreservation and thawing ................................................................ 80
2.3. Cell culture of other cell types........................................................................... 81
2.3.1. 293T cell line ......................................................................................... 81
2.3.2. E15 myoblasts ........................................................................................ 81
2.3.3. Myoblast cell lines ................................................................................. 82
2.3.3.1. C2C12 myoblasts ......................................................................... 82
2.3.3.2. H2K 2B4 murine myoblasts ......................................................... 82
2.3.3.3. Human myoblasts ........................................................................ 83
2.3.3.4. L6 rat skeletal myoblasts ............................................................. 84
2.4. Cell characterisation ......................................................................................... 84
2.4.1. Crystal violet .......................................................................................... 84
2.4.2. Growth kinetics ...................................................................................... 84
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2.4.2.1. Nucleated cell counts ................................................................... 84
2.4.2.2. Doubling time and growth rate ..................................................... 84
2.4.3. Differentiation ........................................................................................ 85
2.4.3.1. Osteogenic differentiation ............................................................ 86
2.4.3.2. Adipogenic differentiation ........................................................... 86
2.4.3.3. Chondrogenic differentiation ....................................................... 86
2.4.3.3.1. Wax embedding and microtome processing ....................... 87
2.4.3.3.2. Alcian blue staining .......................................................... 87
2.5. Immunophenotyping ......................................................................................... 87
2.5.1. Flow cytometric analysis ........................................................................ 87
2.5.2. Immunostaining ..................................................................................... 88
2.5.2.1. Controls ....................................................................................... 88
2.5.2.2. Immunocytochemistry ................................................................. 89
2.5.2.3. Immunohistochemistry ................................................................ 90
2.5.2.3.1. Analysis of muscle sections ............................................... 91
2.6. Muscle differentiation ....................................................................................... 92
2.6.1. Fibronectin coating ................................................................................. 92
2.6.2. 5-azacytidine .......................................................................................... 92
2.6.3. Myoblast-conditioned medium ............................................................... 93
2.6.4. Galectin-1 conditioned medium .............................................................. 93
2.6.4.1. Synthesis of medium.................................................................... 93
2.6.4.2. Verification of GALM by Western blot analysis .......................... 95
2.6.4.3. GALM induced myogenesis ........................................................ 96
2.6.5. Notch ICD induction .............................................................................. 96
2.6.5.1. Verification of Notch ICD plasmid .............................................. 96
2.6.5.2. Myogenic differentiation ............................................................. 96
2.7. Generation of lentiviral vectors ......................................................................... 98
2.7.1. Lentiviral titres ....................................................................................... 98
2.7.2. Lentiviral transduction ........................................................................... 98
2.8. Amplification of plasmids ................................................................................. 99
2.8.1. Transformation of competent cells.......................................................... 99
2.8.2. Plasmid maxiprep ................................................................................... 99
2.9. Polymerase chain reaction (PCR) .....................................................................100
2.9.1. RNA extraction and reverse transcription PCR (RT-PCR) .................... 100
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2.9.1.1. Cells .......................................................................................... 100
2.9.1.2. Tissues....................................................................................... 101
2.9.2. DNA extraction and PCR analysis ........................................................ 102
2.10. Co-cultures ....................................................................................................103
2.10.1. Human myoblasts and murine myoblasts ............................................ 103
2.10.2. HfMSCs and rat L6 ............................................................................ 104
2.10.3. HfMSCs and E15 myoblasts ............................................................... 104
2.11. Animal work ..................................................................................................104
2.11.1. Mdx .................................................................................................... 104
2.11.2. Mdx/nude ........................................................................................... 105
2.11.3. Timed mating ..................................................................................... 105
2.11.4. Muscle injury protocols ...................................................................... 105
2.11.4.1. Notexin injection ..................................................................... 106
2.11.4.2. Irradiation ................................................................................ 106
2.11.4.3. Cryodamage ............................................................................ 107
2.11.5. HfMSC transplantation ....................................................................... 107
2.11.5.1. Intramuscular injections ........................................................... 107
2.11.5.2. Neonatal transplantation .......................................................... 108
2.11.5.3. Intrauterine transplantation ...................................................... 108
2.12. Tissue analysis ...............................................................................................108
2.12.1. Optical bioluminescence imaging (BLI) ...................................... 108
2.12.2. Cryofixation and cryostat processing .......................................... 109
2.12.3. Haematoxylin and eosin staining ................................................ 109
2.13. Statistical analysis ..........................................................................................109
CHAPTER 3 CELL CHARACTERISATION ......................................................111
3.1. Introduction .....................................................................................................112
3.2. Experimental design and results .......................................................................115
3.2.1. Cell isolation ........................................................................................ 115
3.2.2. Growth kinetics .................................................................................... 115
3.2.3. Immunophenotype................................................................................ 121
3.2.4. Differentiation ...................................................................................... 121
3.2.4.1. Osteogenic differentiation .......................................................... 121
3.2.4.2. Adipogenic differentiation ......................................................... 127
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3.2.4.3. Chondrogenic differentiation ..................................................... 127
3.3. Discussion .......................................................................................................131
3.3.1. Conclusion ........................................................................................... 132
CHAPTER 4 IN VITRO MYOGENENIC DIFFERENTIATION .........................133
4.1. Introduction .....................................................................................................134
4.2. Experimental design and results .......................................................................136
4.2.1. 5-azacytidine and myoblast conditioned medium .................................. 136
4.2.2. Notch intracellular domain induction .................................................... 136
4.2.3. Verification of GALM .......................................................................... 138
4.2.4. Optimisation of GALM concentration for myogenesis .......................... 140
4.2.5. Effect of GALM on myogenesis ........................................................... 142
4.2.6. Different forms of galectin-1 for myogenic differentiation.................... 147
4.2.7. Effect of galectin-1 inhibitors on GALM-induced myogenesis ............. 149
4.2.8. Galectin-1 expression in human fetal mesenchymal stem cells ............. 149
4.2.9. Galectin-1 gene transduced human fetal mesenchymal stem cells ......... 151
4.2.10. In vivo myogenesis of galectin-1 gene transduced human fetal
mesenchymal stem cells ................................................................................. 151
4.3. Discussion .......................................................................................................157
4.3.1. Myoblast conditioned medium and notch intracellular domain ............. 157
4.3.2. Galectin-1 conditioned medium ............................................................ 157
4.3.3. Recombinant human galectin-1 ............................................................ 158
4.3.4. Human fetal mesenchymal stem cells express galectin-1 ...................... 160
4.3.5. Galectin-1 gene transduced human fetal mesenchymal stem cells and their
in vivo differentiation ..................................................................................... 161
4.3.6. Conclusion ........................................................................................... 162
CHAPTER 5 CO-CULTURE SYSTEMS .............................................................164
5.1. Introduction .....................................................................................................165
5.2. Experimental design and results .......................................................................168
5.2.1. Human myoblasts fused with murine myoblasts ................................... 168
5.2.2. Human fetal mesenchymal stem cells fused with rat myoblasts............. 172
5.2.3. E15 myoblasts ...................................................................................... 174
5.2.3.1. E15 myoblasts are positive for desmin, MyoD and myogenin .... 174
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5.2.3.2. E15 myoblasts form myotubes under permissive conditions ....... 177
5.2.3.3. HfMSCs undergo myogenic conversion with or without fusion to
E15 myoblasts ........................................................................................ 177
5.3. Discussion .......................................................................................................187
5.3.1. Fusion of murine myoblasts with human myobalsts or hfMSCs ............ 187
5.3.2. E15 myoblasts ...................................................................................... 187
5.3.3. Staining ................................................................................................ 188
5.3.4. Conclusion ........................................................................................... 188
CHAPTER 6 CELL TRANSPLANTATION ........................................................191
6.1. Introduction .....................................................................................................192
6.2. Experimental design and results .......................................................................195
6.2.1. Adult transplantation ............................................................................ 195
6.2.2. Neonatal transplantation ....................................................................... 197
6.2.3. Intrauterine transplantation ................................................................... 207
6.2.3.1. Pilot experiments ....................................................................... 210
6.2.3.1.1. Intrauterine transplantation and postnatal muscle injury in
mdx/nude mice ............................................................................... 210
6.2.3.1.2. Double transplantation in mdx mice................................. 214
6.2.3.2. Optimisation .............................................................................. 214
6.3. Discussion .......................................................................................................227
6.3.1. Adult and neonatal transplantation........................................................ 227
6.3.2. Intrauterine transplantation ................................................................... 228
6.3.3. Mdx animal model ............................................................................... 230
6.3.4. Antibody .............................................................................................. 231
6.3.5. Conclusion ........................................................................................... 231
CHAPTER 7 GENERAL DISCUSSION ..............................................................233
7.1. Implications .....................................................................................................234
7.2. Limitations ......................................................................................................239
7.3. Future research directions ................................................................................243
7.4. Conclusion .......................................................................................................247
REFERENCES .......................................................................................................249
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APPENDICES.. ......................................................................................................291
Appendix 1 – Patient information sheets and consent forms ....................................292
Appendix 2 – Plasmids used to generate lentiviral vectors ......................................299
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LIST OF FIGURES
Figure 1-01. Symmetric and asymmetric cell division .............................................. 24
Figure 1-02. Multiple actions of mesenchymal stromal cells and perhaps other adult
stem-progenitor cells in repairing tissues ............................................. 43
Figure 1-03. Ex vivo gene therapy strategy .............................................................. 46
Figure 1-04. Clinical and pathological features of Duchenne muscular dystrophy .... 49
Figure 1-05. Schematic representation of dystrophin-associated protein complex .... 51
Figure 1-06. Molecular landmarks of myogenic differentiation ................................ 54
Figure 1-07. Diagram summarising the cell sources that have been studied for
Duchenne muscular dystrophy cell therapy.......................................... 61
Figure 2-01. Calculation of growth kinetics ............................................................. 85
Figure 2-02. Map of pcDNA3/rat galectin-1 plasmid ............................................... 94
Figure 2-03. Summary of NICD induction protocol ................................................. 97
Figure 3-01. Morphology of hfMSCs stained with crystal violet .............................116
Figure 3-02. Growth rate of hfMSCs.......................................................................119
Figure 3-03. Doubling time and cumulative population doublings of hfMSCs. ........120
Figure 3-04. HfMSCs are positive for mesenchymal markers. .................................122
Figure 3-05. HfMSCs express intracellular matrix proteins. ....................................123
Figure 3-06. HfMSCs are positive for CD90 and Pax7. ...........................................124
Figure 3-07. HfMSCs are HLA-DR-negative, non-haematopoietic and do not express
B cell marker. .....................................................................................125
Figure 3-08. Immunophenotypes of hfMSCs by FACS analysis. .............................126
Figure 3-09. Osteogenic differentiation of hfMSCs shown by von Kossa staining. ..128
Figure 3-10. Adipogenic differentiation of hfMSCs stained with oil red O. .............129
Figure 3-11. Chondrogenic differentiation of hfMSCs stained with Alcian blue ......130
Figure 4-01. Verification of NICD plasmid .............................................................137
Figure 4-02. Verification of Gal-1 conditioned medium ..........................................139
Figure 4-03. Desmin expression in hfMSCs following exposure to different
conditioned media ..............................................................................141
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Figure 4-04. MF20 expression in hfMSCs following exposure to different conditioned
media .................................................................................................143
Figure 4-05. Desmin expression in hfMSCs following treatment with 1% GALM in
SFM ...................................................................................................145
Figure 4-06. MF20 expression in hfMSCs following treatment with 1% GALM in
SFM ...................................................................................................146
Figure 4-07. Effect of H-GALM and recombinant Gal-1 on hfMSCs or C2C12
cells ...................................................................................................148
Figure 4-08. Effects of Gal-1 inhibitors on Gal-1-induced hfMSC myogenesis .......150
Figure 4-09. Immunolabelling for Gal-1 .................................................................152
Figure 4-10. Lentiviral transduction of hfMSCs with Gal-1 gene ............................153
Figure 4-11. Immunolabelling for myogenic markers in transduced hfMSCs ..........154
Figure 4-12. Transverse section of TA transplanted intramuscularly with hfMSC-
Gal .....................................................................................................156
Figure 5-01. Human adult myoblasts fused with mouse myoblasts in co-culture .....170
Figure 5-02. Human fetal myoblasts fused with mouse myoblasts in co-culture ......171
Figure 5-03. HfMSCs fused with L6 rat myoblasts in co-culture .............................173
Figure 5-04. Morphology of E15 myoblasts ............................................................175
Figure 5-05. E15 myoblasts express desmin, MyoD and myogenin, but not Pax7 ...176
Figure 5-06. E15 myoblasts form myotubes ...........................................................178
Figure 5-07. HfMSCs can fuse with E15 mouse myoblasts in co-culture,
example 1 ...........................................................................................180
Figure 5-08. HfMSCs can fuse with E15 mouse myoblasts in co-culture,
example 2 ...........................................................................................181
Figure 5-09. HfMSCs do not always fuse with E15 mouse myoblasts in
co-culture ...........................................................................................182
Figure 5-10. HfMSCs differentiate in the presence of E15 mouse myoblasts in co-
culture, example 1 ..............................................................................184
Figure 5-11. HfMSCs differentiate in the presence of E15 mouse myoblasts in co-
culture, example 2 ..............................................................................185
Figure 5-12. HfMSCs differentiate in the presence of E15 mouse myoblasts in co-
culture, example 3 ..............................................................................186
15
Figure 6-01. Positive and negative controls for antibodies used................................198
Figure 6-02. Intramuscular injection of hfMSCs engrafted to a greater extent in mdx
nu/nu TA muscles damaged by notexin, irradiation or cryoinjury than in
non-injured muscles ...........................................................................199
Figure 6-03. Experimental design of neonatal hfMSC transplantation in mdx/nude
mice followed by subsequent TA muscle injury .................................200
Figure 6-04. H&E staining of non-injured and non-hfMSC-injected mdx nu/+ muscles
showing variations in fibre size and peripheral nucleation ..................201
Figure 6-05. Neonatally administered hfMSCs engrafted in injured mdx nu/nu and
nu/+ TA muscles ................................................................................203
Figure 6-06. Neonatally administered hfMSCs engrafted in mdx nu/nu and nu/+ TA
muscles, diaphragm and heart in mice that underwent subsequent TA
muscle injury .............................................. …………………………204
Figure 6-07. PCR to detect human cells in mouse organs from neonatally transplanted
mice ...................................................................................................208
Figure 6-08. Sensitivity of PCR in amplifying human DNA sequence in the mouse
tissue ..................................................................................................209
Figure 6-09. Intrauterine transplantation model .......................................................209
Figure 6-10. Experimental design of intrauterine transplantation and postnatal muscle
injury .................................................................................................211
Figure 6-11. HfMSCs transplanted in utero underwent myogenesis in notexin-
damaged TA muscles .........................................................................212
Figure 6-12. HfMSCs transplanted in utero engrafted and underwent myogenesis in
notexin-damaged TA muscles ............................................................213
Figure 6-13. Experimental design and bioluminescence imaging results of double
transplantation ....................................................................................215
Figure 6-14. HfMSCs transplanted in utero and neonatally engrafted underwent
myogenesis in TA muscles .................................................................217
Figure 6-15. Doubly transplanted hfMSCs engrafted and underwent myogenesis in
TA muscles ........................................................................................218
Figure 6-16. Gross dissection of mdx nu/+ mouse 7 days following intrauterine
transplantation procedure ...................................................................225
Figure 6-17. Gross dissection of mdx nu/+ mouse 2 days following intrauterine
transplantation procedure ...................................................................226
16
Figure 6-18. Post-mortem examination of mice following intrauterine injection......226
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LIST OF TABLES
Table 1-01. Immunophenotype of the different types of fetal stem cells from first or
second trimester and extra-embryonic tissues ...................................... 39
Table 1-02. Summary of human in utero stem cell transplantations in
immunodeficiencies ........................................................................... 71
Table 2-01. Culture of other cell types ..................................................................... 81
Table 2-02. Primary antibody used for immunostaining and Western blotting .......... 90
Table 2-03. Secondary antibody used for immunostaining and Western blotting. .... 91
Table 2-04. Primers used for RT-PCR ....................................................................101
Table 2-05. Primers used for PCR ...........................................................................103
Table 3-01. HfMSC samples. .................................................................................117
Table 4-01. HfMSC samples examined ...................................................................144
Table 5-01. Co-culture conditions of human myoblasts with mouse myoblasts .......169
Table 5-02. Co-culture conditions of hfMSCs with rat L6 myoblasts ......................173
Table 5-03. Co-culture conditions of hfMSCs with E15 mouse myoblasts ..............179
Table 6-01. HfMSC transplantation to adult mdx/nude with TA muscle injury ........196
Table 6-02. Neonatal hfMSC transplantation followed by TA injury in adult life ....200
Table 6-03. Intrauterine transplantation experiment ................................................219
Table 6-04. Summary of strategies for the improvement of live birth rate ...............221
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ABBREVIATIONS
2OMe 2-O-methyl phosphorothioate
3-D Three-dimensional
AAV Adeno-associated virus
AF Amniotic fluid
AO Antisense oligonucleotides
APC Antigen presenting cell
ATCC American type culture collection
AT-SVF Adipose tissue stromal vascular fraction
BCA Bicinchoninic acid
BLI Bioluminescence imaging
BM Bone marrow
BMD Becker muscular dystrophy
BSA Bovine serum albumin
cDNA Complementary DNA
CFU-F Colony-forming unit-fibroblasts
CMV Cytomegalovirus
CO2 Carbon dioxide
COSM COS-1 conditioned medium
CRL Crown-rump length
CSC Cancer stem cell
CVS Chorionic villus sampling
D10 DMEM supplemented with 10% FBS
DAPC Dystrophin-associated protein complex
DAPI 4’,6 diamidino-2-phenylindole
DEPC Diethylpyrocarbonate
DKO Double knockout
DMD Duchenne muscular dystrophy
DMEM Dulbecco’s modified eagle’s medium
DMSO Dimethylsulphoxide
DNA Deoxyribonucleic acid
EB Embryoid bodies
19
EDB Extensor digitorum brevis
EDTA Ethylenediaminetetraacetic acid
EF1 Cellular polypeptide chain elongation factor 1 alpha
EGF Epidermal growth factor
ESC Embryonic stem cell
FACS Fluorescence-activated cell sorter
FB Fetal blood
FBS Fetal bovine serum
FGF Fibroblast growth factor
FITC Fluorescein isothiocyanate
FL Fetal liver
F-Luc Firefly luciferase
Gal-1 Galectin-1
GALM Galectin-1 conditioned medium
GFP Green fluorescent protein
GRMD Golden retriever muscular dystrophic
GVHD Graft versus host disease
H&E Haematoxylin and eosin
HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulphonic acid
HfMSC Human fetal mesenchymal stem cell
H-GALM Human galectin-1 conditioned medium
HLA Human leukocyte antigen
HS Horse serum
HSC Haematopoietic stem cell
HUCPV Human umbilical cord perivascular cells
ICM Inner cell mass
IFN-γ Interferon-gamma
IGF Insulin-like growth factor
IgG Immunoglobulin G
ISCT International society for cellular therapy
ITSS Insulin-transferrin-sodium selenite medium supplement
IUT Intrauterine or in utero transplantation
LB Lysogeny broth
MACS Magnetic cell sorting
20
MAPCs Multipotent adult progenitor cells
MF20 Sarcomeric myosin antibody
MHC Major histocompatibility complex
MOI Multiplicity of infection
MPC Muscle precursor cell
MRF Myogenic regulatory factors
MRI Magnetic resonance imaging
mRNA Messenger ribonucleic acid
MSC Mesenchymal stem cell
Neg Negative
NICD Notch intracellular domain
NK Natural killer
NVS Named veterinary surgeon
OI Osteogenesis imperfecta
oim Osteogenesis imperfecta murine
PBS Phosphate buffered saline
PBS-T 0.05% Tween 20 in PBS
PCR Polymerase chain reaction
PDGF Platelet-derived growth factor
PE Phycoerythrin
PFA Paraformaldehyde
PMO Phosphorodiamidate morpholino oligomers
Pos Positive
R-Luc Renilla luciferase
RNA Ribonucleic Acid
RPMI Roswell Park Memorial institute 1640 medium
RT-PCR Reverse transcriptase polymerase chain reaction
Sca-1 Stem cell antigen-1
Scid Severe combined immunodeficiency
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SEM Standard error of the mean
SFM Serum free medium
SP Side population
TA Tibialis anterior
21
TBE Tris-borate-EDTA
TGF Transforming growth factor
TnT Troponin T
TPX Permanox
ts Trans-splicing
TU Transforming units
UCB Umbilical cord blood
UV Ultraviolet
VCAM-1 Vascular cell adhesion molecule-1
Units / symbols
°C degrees Celsius
×g gravities
bp base pairs
cm2 centimetres squared
d days
g grams
G gauge
h hours
kb kilo-base pairs
kDa kilodaltons
μg micrograms
μl microlitres
μm micrometres
min minute(s)
ml millilitres
mm millimetres
mM milllimolars
rpm revolutions per minute
sec seconds
w weeks
22
CHAPTER 1
INTRODUCTION
23
Many diseases can now be diagnosed in utero as a result of the combination of
advances in high resolution ultrasound (Neilson, 2000) and serum screening (Weisz et
al., 2007), and of progress in invasive (Alfirevic et al., 2003) and molecular diagnostic
(Bianchi et al., 2002; Dennis Lo, 2006; Larrabee et al., 2004; Ng et al., 2004;
Oudejans et al., 2003) methods of prenatal diagnosis. Regrettably, often the only
option offered is termination of pregnancy, with the notable exceptions of fetal blood
(FB) transfusion for RhD alloimmunisation (Bang et al., 1982) and fetal laser surgery
for twin-to-twin transfusion (De Lia et al., 1995; Ville et al., 1995). Therefore, it is
pertinent to develop novel fetal therapeutic strategies such as intrauterine stem cell
therapy which may offer the possibility of cure for a number of devastating congenital
diseases that have been diagnosed prenatally, e.g. muscular dystrophy, osteogenesis
imperfecta (OI) and haemoglobinopathies.
1.1. Stem cells
Stem cells are cells that can self-renew and also have the ability to produce daughter
cells that have different, more restricted properties. They are maintained by their local
microenvironment, the “stem cell niche” and have characteristics of self-renewal and
plasticity.
1.1.1. Self-renewal
Self-renewal is thought to be achieved via symmetrical and asymmetrical cell division,
which produces a copy of the stem cell and a second cell for differentiation. Evidence
for asymmetric cell division comes from studies of the immunolocalisation of proteins,
such as Numb, a component of the Notch pathway, that are differentially distributed
between two daughter cells (Betschinger and Knoblich, 2004; Yu et al., 2006).
24
Asymmetrical cell divisions have been reported in muscle satellite cells (Chapter
1.1.5.3), where template deoxyribonucleic acid (DNA) and Numb segregate
selectively to one daughter cell (Kuang et al., 2007; Shinin et al., 2006). Stem cells
are relatively quiescent; this helps to prevent over-proliferation, which may otherwise
lead to DNA damage and oncogenic events. However, in order to generate large
numbers of differentiated cells over extended periods of time, stem cell populations
must also expand via symmetric cell division (Figure 1-01).
Figure 1-01. Symmetric and asymmetric cell division (modified from Martinez-
Agosto et al., 2007).
1.1.2. Plasticity
Totipotent cells can form the entire organism and were originally thought only to exist
in animal zygote and plant meristem cells. Pluripotent embryonic stem cells (ESC)
can differentiate to all cell lineages in the body, and recently have been shown to
generate extraembryonic trophoblasts in vitro making them totipotent (Schenke-
25
Layland et al., 2007). Somatic stem cells are more restricted in their potency. For
example, multipotent haematopoietic stem cells (HSC) can give rise to various cell
types belonging to only the lymphoid and myeloid lineages. In the case of unipotent
cells, such as germline stem cells, a single lineage is formed: an egg or a sperm.
Recent literature suggests that by perturbing the epigenetic state of a cell, the potency
can be altered. Fibroblasts and more recently, lineage committed somatic cells from
liver and stomach can be reprogrammed into a pluripotent state (iPS, induced
pluripotent stem cells) if proteins associated with ESC stemness, such as Oct-4, Sox2
and Klf4, are over-expressed in these cells (Aoi et al., 2008; Maherali et al., 2007;
Okita et al., 2007; Wernig et al., 2007).
1.1.3. Niche
The niche hypothesis suggests that stem cells reside within fixed compartments called
niches, which are conducive to the maintenance of stem cell identity (Schofield, 1978).
In Drosophila germline stem cells, signals from the niche regulate stem cell self-
renewal, survival and maintenance (Henderson et al., 1994; Kiger et al., 2001; Kimble
and White, 1981; Tulina and Matunis, 2001; Xie and Spradling, 2000), while the
interaction between stem cells and support cells can polarise the stem cells towards
asymmetric divisions (Deng and Lin, 1997; Yamashita et al., 2003). Furthermore, the
signals are reinforced through adhesion between stem cells and stromal cells and/or
the extracellular matrix to anchor stem cells within their niche (Song and Xie, 2002;
Song et al., 2002). Therefore, stemness is preserved through preventing precocious
differentiation or loss of quiescence and potency. For example, in bone marrow (BM),
mesenchymal stem cells (MSCs) support HSCs via secreting cytokines and cell
contact-mediated signals for differentiation or self-renewal (Scadden, 2007). However,
it remains unclear whether cells of the embryonic inner cell mass (ICM) require a
26
niche, or if the interaction between these stem cells is sufficient for its maintenance in
vivo (Biswas and Hutchins, 2007). ESCs usually require a feeder layer of fibroblasts
for maintenance in vitro. When this feeder layer is removed but fibroblast growth
factor (FGF) added to the growth medium, ESCs can produce fibroblast-like cells that
secrete insulin-like growth factor (IGF)-II and transforming growth factor (TGF)-ß,
which then allow other ESCs to self-renew (Bendall et al., 2007).
1.1.4. Cancer stem cells
Due to their self-renewal and plasticity, stem cells may be at risk of malignant
transformation. Tumours are composed of heterogeneous populations of cells with
varying differentiation, just as in normal tissues. In fact, histological staging of
tumours is based on the differentiation stages of carcinoma cells. Therefore, tumours
are akin to “abnormal organs” sustained by a diseased “cancer stem cell” (CSC)
population which self-renew and undergo aberrant differentiation (Clarke and Fuller,
2006; Reya et al., 2001). Furthermore, cancer results from the accumulation of
multiple genetic mutations in a single cell, often over a long period (Fearon and
Vogelstein, 1990). The relative quiescence of stem cells makes them a probable
candidate for transforming mutations. Initially developed in human myeloid
leukaemia, the concept of CSC is used to describe a tumour-initiating cell subset that
can form a heterogeneous progeny, similar to the phenotypes from its tissue of origin
(Bonnet and Dick, 1997). The genes involved in controlling asymmetric division in
the niche may mutate and result in uncontrolled symmetrical cell division (Trosko et
al., 2000).
There are two hypotheses to explain the origin of cancer, namely, the
dedifferentiation theory and the stem cell theory. Because cancer cells have many
phenotypic traits similar to undifferentiated embryonic cells, it is still unclear whether
27
cancer cells are derived from differentiated cells that have dedifferentiated, or from
adult stem cells in tissue. The Oct-4 gene, a marker of pluripotent stem cells, is
expressed in ESCs and in tumour cells but not in cells from differentiated tissues. Oct-
4 expression in some tumours has been described as being re-expressed or restored by
the transformation process (Gidekel et al., 2003; Monk and Holding, 2001), which
favoured the dedifferentiation theory of carcinogenesis. However, a more recent study
reported by Tai et al. found that expression of Oct-4 gene was detected in adult stem
cells and immortalised non-tumourigenic cells, as well as tumour cells and cell lines,
but not in differentiated cells (Tai et al., 2005). These results provided evidence to
support the stem cell theory of carcinogenesis.
1.1.5. Types of stem cells
1.1.5.1. Totipotent embryonic stem cells
ESCs are derived from the ICM of pre-implantation embryos. Their totipotency
allows them to differentiate into cells from all three germ layers both in vitro and in
vivo. ESC lines from blastocysts of mice and humans were generated and
characterised in 1981 and 1998, respectively (Evans and Kaufman, 1981; Martin,
1981; Thomson et al., 1998). Injection of mouse or human ESCs into mice produced
teratomas, which contain cell types from all three germ layers. While injection of
mouse ESCs into a blastocyst gave rise to chimeric offspring with all their tissues
carrying cells differentiated from ESCs, the same could not be tested in humans for
ethical reasons (Smith, 2001).
ESC research has been hampered by both safety concerns, and ethical
reservations due to requisite destruction of the blastocyst during harvesting.
Transplantation of ESCs into animals is almost invariably followed by the
28
development of embryonal teratomas, precluding cell transplantation. Furthermore,
directed differentiation of ESCs down a targeted cell lineage presents a further
challenge. They are commonly differentiated by formation of embryoid bodies (EB),
cell aggregates in suspension culture, which mimic early embryo development in vivo
but without proper axial organisation or elaboration of a body plan. Using this
technique, cells have differentiated down neural (Zhang et al., 2001b), cardiac
(Maltsev et al., 1994), pancreatic (Schroeder et al., 2006), haematopoietic (Wiles and
Keller, 1991) and skeletal muscle lineages (Rohwedel et al., 1994). However,
heterogeneous populations are obtained where undifferentiated ESCs co-exist with
small numbers of differentiated cells, as individual cells may receive different signals
depending on their locations in an EB. Hence, the following strategies are currently
being investigated: (1) serum-free culture medium with specific growth factors
(Schulz et al., 2004), (2) co-culture with other cells (Kawasaki et al., 2000), (3)
adherent culture (Gerrard et al., 2005), and (4) three-dimensional (3-D) culture to
recapitulate cell-to-cell interaction (Chen et al., 2003).
1.1.5.2. Multipotent somatic stem cells
Somatic stem cells can be found in adult, neonatal and fetal tissues or organs.
Throughout the 1950s and 1960s, transplantation of HSCs, isolated from the BM, was
shown to reconstitute the depleted BM following irradiation. This culminated in 1963
when Mathé demonstrated the long-term survival of a leukaemia patient treated with
HSCs (Mathe et al., 1963). Clinically, BM transplantation is now routine practice. In
the 1970s, Friedenstein and colleagues observed another cell type in BM, initially
termed colony-forming unit-fibroblasts (CFU-F) due to their adherence to the plastic
surfaces of cell culture plates (Friedenstein et al., 1974). Their ability to differentiate
into multiple mesoderm-derived tissue cell types at a clonal level (Dennis et al., 1999;
29
Pittenger et al., 1999) is in line with Caplan’s notion of MSCs (Caplan, 1994).
However, some investigators have opted for the term marrow stromal cells instead of
mesenchymal stem cells, since there is still debate as to whether these are true stem
cells, taking into account the wide variation of cell harvesting techniques in the field.
To clarify the confusion in nomenclature, a recent position paper by International
Society for Cellular Therapy (ISCT) has encouraged the general use of the term
“multipotent mesenchymal stromal cells”, while reserving “mesenchymal stem cells”
for those that demonstrate definite stem cell properties, i.e. abilities for long-term self-
renewal and differentiation into specific, multiple cell types in vivo, though the
acronym MSC can be used for both populations (Horwitz et al., 2005).
1.1.5.2.1. Adult mesenchymal stem cells
1.1.5.2.1.1. Characterisation
Multipotent MSCs reside mainly in the BM (Pittenger et al., 1999) and adipose tissues
(Zuk et al., 2002) in adult life, but have also been found in other sites such as
synovium (De Bari et al., 2001) and peripheral blood (Eghbali-Fatourechi et al., 2005).
Despite their rarity, constituting 0.0001-0.01% of mononuclear cells in adult BM,
MSCs are readily isolated and expandable in vitro (Pittenger et al., 1999). MSCs are
identified by a combination of physical, functional and phenotypic properties.
They have a spindle-shaped morphology and adhere selectively to a plastic
surface. During expansion they retain their growth and differentiation characteristics
into bone, fat or cartilage. The minimal criteria for MSCs state that they should
express CD105 (SH2 or endoglin, a TGF-ß receptor III also present on endothelial
cells, syncytiotrophoblasts, and macrophages), CD73 (SH3 and SH4) and CD90 (Thy-
1, thymocyte differentiation antigen-1, an early BM progenitor cell marker); and
30
should be negative for CD14 (lipopolysaccharide receptor), CD34 (haematopoietic
marker), CD45 (leucocyte antigen), CD11b (monocyte markers), CD79α or CD19 (B
cell markers) and human leucocyte antigen (HLA)-DR (major histocompatibility
complex class II) (Dominici et al., 2006). Although SH-2, SH-3 and SH-4 antibodies
were raised in mice immunised with MSCs, they were later found to be non-exclusive
for MSCs (Barry et al., 1999).
Other MSC markers that have been used in studies include stroma associated
markers CD29 (ß1-integrin) and CD44 (HCAM1), extracellular matrix proteins
vimentin, laminin and fibronectin, and the activated leucocyte cell adhesion molecule,
CD166 (Bruder et al., 1997; Bruder et al., 1998). The monoclonal antibody against
Stro-1 identifies non-haematopoietic stromal cell precursors in BM cell populations
(Simmons and Torok-Storb, 1991), but the antigen is also found on haematopoietic
cells at low levels. Recently, SSEA-4 has been identified as a new MSC marker
despite previously thought to be restricted to ESCs, erythrocytes and some neural
ganglion cells (Kannagi et al., 1983). SSEA-4-expressing BM cells appeared to
represent a MSC population devoid of haematopoietic cells (Gang et al., 2007). Apart
from tri-lineage differentiation into bone, fat or cartilage, dependent on their origin
some MSC populations have demonstrated abilities to differentiate down
neuroectodermal, epithelial and myogenic lineages (Arnhold et al., 2006; Dezawa et
al., 2005; Zhao et al., 2002).
1.1.5.2.1.2. Immunology
Adult MSCs express intermediate levels of HLA class I but lack epitopic HLA class II.
The normally intracellular HLA class II can be expressed on the cell surface
following interferon-γ (IFN-γ) stimulation (Le Blanc et al., 2003). MSCs are known
for their non-immunogenic properties and are even immunosuppressive against
31
allogeneic immune responses in vitro, which is maintained after tri-lineage
differentiation in vitro (Le Blanc et al., 2003). Human MSCs engrafted in adult albino
rats for up to seventy-two days (Azizi et al., 1998) and persisted to adulthood after
administration in utero to fetal sheep (Liechty et al., 2000). Allogeneic mismatched
human fetal liver (FL)-derived MSCs in utero transplanted into an immunocompetent
fetus with OI in third trimester of gestation engrafted in recipient bones (Le Blanc et
al., 2005), while mouse MSCs were tolerated in immunocompetent rats (Saito et al.,
2002). These data suggest that MSCs can be tolerated when transplanted across major
histocompatibility complex (MHC) barriers. However, recent publications have
refuted this concept.
Human MSCs have been shown to express many natural killer (NK) cell receptor
ligands which are recognised by activated NK cells (Spaggiari et al., 2006). Though
ex vivo-expanded MSCs suppress the function of many immune cells, such as T cells,
B cells, NK cells and antigen-presenting cells (APC), depending on their stimuli, they
can also activate immune responses. For example, under syngeneic conditions murine
MSCs have been found to act as conditional APCs and can induce antigen-specific
immunity (Stagg et al., 2006). Murine MSCs also elicited both cellular and humoral
immune response under allogeneic conditions (Badillo et al., 2007). Moreover, human
MSCs can act as APCs under low IFN-γ conditions in vitro (Chan et al., 2006b;
Romieu-Mourez et al., 2007).
In a case report of graft versus host disease (GVHD), Le Blanc et al. showed that
repeated administration of haploidentical human MSCs following allogeneic HSC
transplantation completely reversed GVHD (Le Blanc et al., 2004). Nevertheless, this
finding has not been reproduced in mice in which it was found that co-transplantation
32
of allogeneic MSCs with allogeneic BM cells actually increased graft rejection
(Sudres et al., 2006).
1.1.5.2.1.3. Challenges
Compared to ESCs, adult stem cells are more accessible, but have a more limited
proliferative capacity and restricted plasticity. After successive in vitro passages, adult
MSCs lose their multipotency and proliferation rate, which is a potential problem for
cell therapy as a large stem cell dose is required (Neuhuber et al., 2008). Following
prolonged culture, cells can undergo crisis and stop proliferating along with the
occurrence of cell death. The few surviving cells acquire genetic mutations, are
immortalised, and may go through malignant transformation. This process has been
shown in mouse embryonic fibroblasts (Rubin, 2001), murine MSCs (Tolar et al.,
2007) and in stressed human MSCs (Rubio et al., 2005). On the other hand, human
BM-MSCs did not undergo transformation and did not express telomerase activity or
human telomerase reverse transcriptase transcripts in long term cultures (Bernardo et
al., 2007).
Current literature suggests that adult MSCs are either multipotent, or are
composed of a mixed population of committed progenitor cells, each with varying
differentiation capacity (Kemp et al., 2005). Individual colonies derived from MSC
precursors differ in their plasticity with only one third of the original population being
able to undergo tri-lineage differentiation (Pittenger et al., 1999). This is supported by
another study which showed only 30% of in vitro expanded clones can differentiate
down both osteogenic, adipogenic as well as chondrogenic pathways (Muraglia et al.,
2000).
33
1.1.5.2.2. Fetal mesenchymal stem cells
With increasing age, MSCs are found at decreasing frequencies in fetal blood.
Campagnoli et al. described 8.2 CFU-F per 106 mononuclear cells in first trimester
blood compared with 1.3 and 0.35 CFU-F per 106 cells in second trimester and term
cord blood, respectively (Campagnoli et al., 2001). In BM from a second trimester
fetus, 1 MSC is found per 400 nucleated cells (In't Anker et al., 2003a), while 1 in
10,000 cells is a MSC from newborn BM, compared with 1 in 250,000 from adult
marrow and 1 in 2 × 106 in BM from an 80-year-old individual (Caplan, 1994). Their
presence in fetal circulation before initiation of BM haemopoiesis, their ability to
support haemopoiesis in co-culture, their decline in frequency as haemopoiesis is
established, along with comparable populations of MSCs being found in first trimester
BM and liver is consistent with the hypothesis that fetal MSCs are migrating to future
definitive sites of haemopoiesis, where they adhere to facilitate HSC engraftment and
initiation of haemopoiesis (De la Fuente et al., 2002). MSCs have also been isolated
from second trimester lung, spleen (In't Anker et al., 2003a), pancreas (Huang and
Tang, 2003), kidney (Almeida-Porada et al., 2002) and cartilage (Cui et al., 2006)
(Table 1-01).
Considerable debate has focused on the contrasting merits of embryonic versus
adult stem cells, whereas fetal stem cells represent an intermediate cell type in this
controversy. Unfortunately, there is a lack of comparative studies between varying
fetal cell sources and gestation, as most research has focused on comparing fetal stem
cells with adult stem cells, particularly BM-MSCs. Only one study compared MSCs
from first and third trimester placenta and showed little difference in lineage
differentiation, though the early gestation amnion epithelial cells expressed more
MSC markers than those from late gestation (Portmann-Lanz et al., 2006).
34
Fetal stem cells can be found at various stages of human development with a
declining gradient of potency as gestation advances, and teleologically lie between
ESCs and adult stem cells (Guillot et al., 2006). Like adult MSCs, human fetal
mesenchymal stem cells (hfMSC) express low levels of HLA I, but unlike them, they
lack intracellular HLA II, taking a longer time to express this on stimulation
(Gotherstrom et al., 2004). HfMSCs self-renew faster in culture than adult MSCs and
senesce later whilst retaining a stable phenotype. Therefore they are more readily
expandable to therapeutic scale for either ex vivo gene or cell therapy, fetal tissue
engineering or IUT. They have greater differentiation capacity, a higher level of
telomerase activity and express the purportedly embryonic-specific pluripotency
markers such as Oct-4, Nanog, SSEA-3, Rex-1, Tra-1-81 and Tra-1-61 at protein level
(Campagnoli et al., 2001; Guillot et al., 2007). They are less lineage-committed and
less immunogenic than adult MSCs, (Gotherstrom et al., 2003; Gotherstrom et al.,
2005; Lee et al., 2006a; Mirmalek-Sani et al., 2006) and express a shared α2, α4, and
α5β1 integrin profile with first trimester HSC, implicating them in homing and
engraftment, which is consistent with their significantly greater binding to their
respective extracellular matrix ligands than adult MSCs (De la Fuente et al., 2003).
Unlike ESCs, they do not form teratomas in vivo (Aguilar et al., 2007). In addition to
the standard tri-lineage differentiation of adult MSCs, there have been reports
demonstrating oligodendritic (Kennea et al., 2003), haemopoietic (MacKenzie et al.,
2001), hepatic (S. Kanellopoulou, personal communication, 2007) and myogenic
differentiation (Chan et al., 2006a). HfMSC has been shown to have a higher potential
for osteogenic differentiation than adult MSC, which is evidenced by their greater
ability to generate extracellular calcium crystals and by their higher basal osteogenic
gene expression in vitro (Guillot et al., 2008c). Comparable to adult MSCs, hfMSCs
35
can be efficiently transduced with integrating vectors without effects on long-term
self-renewal or multipotentiality (Chan et al., 2005).
The collection of stem cells from fetuses in ongoing pregnancies for autologous
use should obviate the ethical concerns that have hampered ESC research. However,
ethical concerns remain because fetal stem cells are more usually collected after first
or second trimester termination of pregnancy, subject to informed consent,
institutional ethical approval, and compliance with national guidelines covering fetal
tissue research as is necessary for further development of this approach.
1.1.5.2.2.1. Extraembryonic fetal tissues
1.1.5.2.2.1.1. Amniotic fluid
Amniotic fluid (AF) contains cells from fetal urogenital, respiratory and digestive
systems as well as fetal skin. It has been regarded with much hope as a minimally-
invasive cell source in utero, since isolation of cells from autologous AF would
involve few, if any, ethical concerns. Various stem cells types such as HSC and MSC
have been isolated from AF but their actual origin has yet to be determined due to the
heterogeneous nature of cells in AF. A subpopulation of Oct-4-positive cells has been
identified in second trimester AF, together with cells that express markers for
neuronal stem cells. This contributes to the idea that pluripotent cells are present in
AF (Prusa et al., 2003). Recently, De Coppi et al. isolated cells from second trimester
AF using c-kit, a tyrosine kinase receptor for the stem cell factor, as a selection
marker. These AF-derived stem cells have similar characteristics to hfMSCs, can be
clonally expanded and differentiated into cell types representative of all three
embryonic germ layers (adipogenic, osteogenic, myogenic, endothelial, neuronal and
hepatic lineages) in vitro and form bone over scaffold structures in vivo (De Coppi et
36
al., 2007). The stem cell properties of MSCs in second trimester AF have been
confirmed, whereby they express a number of mesenchymal cell surface markers
including CD90 and CD105, and differentiate into osteoblasts and adipocytes
following in vitro expansion, and they appear similar to MSCs from other fetal
sources (In't Anker et al., 2003b; Kaviani et al., 2003; Tsai et al., 2004).
1.1.5.2.2.1.2. Placenta and amnion
Chorionic villus sampling (CVS) is used clinically to isolate placental cells with
sufficiently minor pregnancy disruption to allow diagnostic use; hence placenta could
be an accessible source of autologous stem cells. Rhodes et al. have shown that HSCs
are not only present in murine placenta, but also that the placenta is an HSC
generation site (Rhodes et al., 2008). These CD34+ cells lack expression of lineage
markers such as CD38 and HLA-DR. They have a slow doubling time, differentiate
down the myeloid and lymphoid lineages in vitro, and can repopulate the entire
haemopoietic system when transplanted into xenogeneic recipients (Taylor et al.,
2002). MSCs have been successfully isolated from second and third trimester
placental samples, and represent <1% of cells present in the placenta (Alviano et al.,
2007; Zhang et al., 2004b). Limited research also suggests that MSCs are present in
first trimester placenta (Portmann-Lanz et al., 2006). Like hfMSCs and AF-MSCs,
placental MSCs express typical markers such as CD166, CD105, CD73, and CD90,
while they are negative for CD14, CD34 and CD45. Placental MSCs can differentiate
into cells of the osteogenic, adipogenic, chondrogenic, myogenic, endothelial and
neurogenic lineages, as confirmed by several groups (Alviano et al., 2007; Portmann-
Lanz et al., 2006; Yen et al., 2005; Zhang et al., 2004b). Additionally, placenta-
derived MSCs express primitive markers such as FZD-9, SSEA-4, Oct-4, Nanog-3
and nestin when grown in medium used for ESC expansion (Battula et al., 2007).
37
Several multipotent cells from the amnion have also been described, such as
amniotic epithelial cells (Miki et al., 2005) and amniotic mesenchymal cells
(Tamagawa et al., 2007). MSCs from the amnion membrane have recently been
suggested to have some characteristics of cardiomyoblasts, and can differentiate into
cells similar to cardiomyocytes and successfully integrate into cardiac tissues (Zhao et
al., 2005).
1.1.5.2.2.1.3. Umbilical cord
Umbilical cord blood (UCB) is a rich source of HSCs and progenitor cells that can be
isolated non-invasively (Ueno et al., 1981). Some HSCs remain in the umbilical cord
at the time of delivery where they can be collected for allogeneic or occasionally
autologous use. Cord blood stem cells were first successfully employed in a BM
transplant in 1988 (Broxmeyer et al., 1989) and are now used in clinical practice as an
alternative to BM and peripheral blood mobilised HSCs. They are used to treat a wide
range of immunodeficiencies, certain carcinomas besides haematological, genetic and
metabolic disorders. The frequency of MSCs in UCB is far lower than that found in
first trimester FB or in the BM (Campagnoli et al., 2001). While having similar
properties to first trimester fetal MSCs, cord blood MSCs are difficult to isolate and
are present in very low concentrations in term cord blood compared to UCB from
preterm infants (Erices et al., 2000; Javed et al., 2008). Furthermore, Kogler et al.
demonstrated that only 35% of term UCB samples contain differentiating MSCs
(Kogler et al., 2006). This reality has considerable implications for private
commercial blood banks offering directed autologous storage of UCB (Fisk and Atun,
2008).
Wharton’s Jelly is the proteoglycan-rich connective tissue that protects umbilical
vessels. Umbilical cord matrix stem cells have been isolated from this extracellular
38
matrix (Karahuseyinoglu et al., 2007; Weiss et al., 2006). Promisingly, MSCs have
been obtained from Wharton’s jelly at much higher efficiencies than term UCB
(Secco et al., 2008).
Perhaps the richest postnatal source of mesenchymal progenitors is human
umbilical cord perivascular cells (HUCPV). These cells have a doubling time of 20 h
at passage 2, and produce over 1010 cells in one month in culture. They express SH2,
SH3, Thy-1 and CD44, but are negative for CD34 and class I and II major
histocompatibility antigens, which could be an advantage for allogeneic applications
(Sarugaser et al., 2005).
Table 1-01 summarises the immunophenotype of various fetal stem cells. All
fetal stem cells are CD45- and CD34- (except for second trimester fetal lung and
spleen) and BM cells from first and second trimester exhibit similar characteristics.
The majority of fetal stem cells are positive for SH2, CD29, CD44 and CD90,
however they are HLA-DR-negative. Many fetal cells express pluripotency markers
SSEA-4 and Oct-4, whereas other markers such as CD113 and CD133 are
inconsistently expressed across different cell sources.
Table 1-01. Immunophenotype of the different types of fetal stem cells from first or second trimester and extra-embryonic tissues. +
positive, -/+ weakly positive or low expression, - negative (extended and updated from Guillot et al., 2006).
1st Trimester 2nd Trimester Unknown Postnatal
Antigen
CD No.
FB
FL
BM
Amnion
BM
Lung / Spleen
Pancreas
Kidney
Cartilage
AF
Muscle
UCB
Wharton’s Jelly
HUCPV
Placenta
Amnion
LCA CD45 - - - - - - - - - - - - - - - -
T10 CD38 - - - -
LPS-R CD14 - - - - - - - - - - -
Macrosialin CD68 - - -
gp 105-120 CD34 - - - - - + - - - - - - - - - -
PECAM CD31 - - - - - - - - -
LFA-3 CD58 -/+ +
ICAM-3 CD50 - - - -
ß1-integrin CD29 + + + + -/+ + + + + + + +
HCAM-1 CD44 + + + + + + + + + + + + + + +
CD51
T9 CD71 -/+ - +
Bp50 CD40 -
VCAM-1 CD106 + + + - - - - -
40
1st Trimester 2nd Trimester Unknown Postnatal
Antigen
CD No.
FB
FL
BM
Amnion
BM
Lung / Spleen
Pancreas
Kidney
Cartilage
AF
Muscle
UCB
Wharton’s Jelly
HUCPV
Placenta
Amnion
B7.1 CD80 - - - - - -
CD166 + + + + + + +
B7.2 CD86 - -
SH2 CD105 + + + + + + + + + + + + +
SH3 CD73 + + + + + + + + + + +
SH4 CD73 + + + + + + + +
HLA-DR - - - - - - - - - - -
VLA-4 CD49d - - - -
VLA-5 CD49e + + + + + + -
IL-2R CD25 - - - -
IL-3R CD123 -/+ -/+ -/+ -/+ -
IL-7R CD127 - - -
TNF-R1,2 CD120a,b - - -
LFA-1 CD11a - - -
Aminopeptidase N CD13 + + + + + +
Neurothelin CD147 + +
SCFR, c-kit CD117 - - - -/+ - - + -/+
41
1st Trimester 2nd Trimester Unknown Postnatal
Antigen
CD No.
FB
FL
BM
Amnion
BM
Lung / Spleen
Pancreas
Kidney
Cartilage
AF
Muscle
UCB
Wharton’s Jelly
HUCPV
Placenta
Amnion
AC 133 CD133 - - - - -/+ - -/+
Vimentin + + + + +
Laminin + + +
Fibronectin + + +
vWF - - - - +
ICAM-1 CD54 + + + + + +
Thy1 CD90 + + + + + + + + + + + + -/+
Glycophorin A CD235a -
CD146 + +
Nestin + + + + + + +
CK18 + + + + + +
Stro-1 -
SSEA-4 + + + + + + - + +
Oct-4 + + + + + + - +
Nanog + + + +
1.1.5.3. Unipotent satellite cells
Satellite cells are thought to be the primary skeletal muscle stem cell responsible for
the maintenance and regeneration of adult skeletal muscles (Zammit et al., 2006) and
are defined by their anatomical position of being enclosed between sarcolemma and
basal lamina surrounding each myofibre (Mauro, 1961). Upon activation, the
normally quiescent satellite cells divide to produce muscle precursor cells (MPC)
(Schultz et al., 1978). These satellite cell-derived myoblasts proliferate, differentiate
and fuse into multinucleated myotubes and myofibres (Bischoff, 1975, 1986;
Rosenblatt et al., 1995). Due to asymmetric division, a few satellite cells are sufficient
to regenerate over 100 myofibres as well as maintain their own satellite cell
compartment. It is still debatable whether satellite cells are true stem cells, although
they are able to self-renew (Collins et al., 2005) and have mesenchymal plasticity
(Shefer and Yablonka-Reuveni, 2007; Sinanan et al., 2006).
Quiescence seems to be the key to effective satellite cell stem cell function, as
efficient muscle regeneration was shown following graft of quiescent satellite cells on
their parent fibre without in vitro culture (Montarras et al., 2005). This is further
evidenced in mdx/nude mice (Chapter 1.2.3.1.1) intramuscularly transplanted with
murine myoblasts, where all donor-derived muscle is produced from a minority of
donor cells that divide slowly in vitro but proliferated rapidly in vivo after grafting
(Beauchamp et al., 1999).
1.1.6. Applications
The different stem cell types have been investigated with a view towards cell therapy,
each type with its own profile of benefits and disadvantages. The particular strengths
of MSCs lie in its ease of isolation and expansion and its suitability for transplantation
43
treatment of musculoskeletal diseases such as Duchenne muscular dystrophy (DMD,
Chapter 1.2), especially with BM-derived MSCs. The following paragraphs will
concentrate on the applications of MSCs (Figure 1-02).
Figure 1-02. Multiple actions of mesenchymal stromal cells and perhaps other
adult stem-progenitor cells in repairing tissues (Prockop, 2007).
1.1.6.1. Tissue repair
The homing ability of MSCs has been demonstrated in non-injured non-human
primates, where infused allogeneic MSCs localised to gastrointestinal tissues and
distributed throughout other organs, though engraftment levels were low (Devine et
al., 2003). MSCs have shown therapeutic effects in animal models of lung injury
(Ortiz et al., 2007; Ortiz et al., 2003), renal failure (Kunter et al., 2006), diabetes (Lee
et al., 2006b), myocardial infarction (Minguell and Erices, 2006) and various
neurological disorders (Phinney and Isakova, 2005). Furthermore, improvement in
cardiac function was observed after infusion of human MSCs into immunodeficient
mice with acute myocardial infarction, though no engraftment was evident three
weeks after administration (Iso et al., 2007). In humans, autologous intracoronary
44
injection of MSCs into ischaemic hearts improved cardiac functions (Chen et al.,
2004). MSCs have also been used in patients previously successfully treated with
allotransplant for Hurler’s syndrome (mucopolysaccharidosis type IH) and
metachromatic leucodystrophy (Koc et al., 2002). Moreover, the administration of
allogeneic whole BM (Horwitz et al., 1999) or BM-MSCs (Horwitz et al., 2002) in
children with type III OI led to improvements in growth velocity, bone mineral
density and ambulation despite <1% donor cell engraftment (Horwitz et al., 2001). As
MSCs are known to produce a variety of cytokines and adhesion molecules, it has
been suggest that secreted soluble factors which modulate the tissue
microenvironment may be more significant than transdifferentiation in effecting tissue
repair (Prockop, 2007). The long-term fate of infused MSCs is currently unknown,
though it is thought that they may home to inflamed and damaged tissue to furnish a
protective effect, after which they are difficult to detect. Whether this is due to an
expected physiological decrease in MSC numbers from apoptosis or MSCs
undergoing rejection remains to be elucidated.
With regards to fetal MSCs, cell lines derived from human fetal BM migrated to
the lesion site in a mouse intracerebral haemorrhage stroke model, underwent neural
differentiation and induced functional improvements up to seven weeks post-
transplantation (Nagai et al., 2007). AF-MSCs transplanted into ischaemic brain
differentiated into neural lineages, particularly astrocytes (Cipriani et al., 2007) and
human amniotic stem cell clones pre-differentiated down the neural lineage can
engraft in the mouse brain (De Coppi et al., 2007).
1.1.6.1.1. Autologous versus allogeneic cell therapy
Theoretically, cells can be retrieved from a patient for ex vivo expansion with or
without genetic manipulation, and then placed back into the same individual for tissue
45
repair. While the harvest of fetal BM or brain is unlikely ever to be feasible in
continuing pregnancies, the harvest of FL has been reported in fetal sheep, albeit with
significant morbidity and mortality (Schoeberlein et al., 2004). This autologous use
negates the need for immunosuppression. The adult MSC population is not an ideal
candidate in view of their poor proliferation, limited plasticity, low abundance and
difficult accessibility. Fetal cells appear more attractive than adult cells on several
fronts for autologous cell therapy, while allogeneic fetal cells provide an easily
accessible and disease-free source for cell therapy. The ethical reservations from use
of abortal tissue would seize to be an issue if alternative sources of fetal MSCs, such
as third trimester placenta, were used.
1.1.6.1.2. Ex vivo gene therapy
Integrating vectors have been widely used in gene therapy because they ensure stable
and sustained gene expression through cell divisions in the absence of selection.
However, random integration of the therapeutic gene can give rise to undesired effects
of transgene silencing, host gene disruption and insertional mutagenesis. Stem cells
have considerable utility as targets for gene therapy because they can self-renew, thus
precluding the need for repeated administration of the gene vector (Figure 1-03).
While this approach has already been validated in postnatal clinical trials with HSCs
(Aiuti et al., 2002; Gaspar et al., 2004; Hacein-Bey-Abina et al., 2002), the occurrence
of leukaemia in four of ten children treated in the French trial
(http://www.esgct.org/upload/4th_CaseofLeukemial.pdf(Hacein-Bey-Abina et al.,
2003) and in one of ten children from the London trial
(http://www.esgct.org/upload/ESGCT%20Commentary-UK-SCID-Leukaemia-18DE
C2007%20(GD-FINAL).pdf) due to insertion of the transgene near a proto-oncogene
has raised serious concerns about the risk of insertional oncogenesis. Several lines of
46
investigation are currently being pursued to reduce this risk, such as the use of
insertional site screening prior to re-infusion, the use of safer (Zhang et al., 2007a)
and/or site specific vectors and the use of regulable vectors which could be switched
off should an adverse event arise (Kohn et al., 2003). HfMSCs are readily transduced
with integrating vectors without affecting their stem cell properties of self-renewal
and multilineage differentiation (Chan et al., 2005). Transduced hfMSCs can be
clonally expanded and work is now underway to select clones where integration
occurred at “safe” regions of the human genome, in order to minimise the risk of any
oncogenic event. The use of hfMSCs as vehicles for gene delivery would be
applicable to a number of diseases such as OI, the muscular dystrophies and various
enzyme deficiency syndromes such as the mucopolysaccharidoses and lysosomal
storage diseases.
Figure 1-03. Ex vivo gene therapy strategy.
47
1.1.6.2. Paracrine effects
MSCs may exert their beneficial effects through paracrine mechanisms (Togel et al.,
2005). Convincing evidence can be found from studies which examined injection of
MSC-conditioned medium to sites of myocardial infarcts. Conditioned medium
reduced the number of apoptotic cells and the infarct size in addition to improving left
ventricular function (Gnecchi et al., 2005; Gnecchi et al., 2006). This mode of action
is unsurprising given that MSCs have been thought to provide for the HSC niche via
soluble signals (Weimar et al., 1998).
1.1.6.3. Tumour targeting
A potential undesirable effect of MSCs is the promotion of tumour development.
MSCs may provide the niche for cancer stem cells or suppress immune functions.
Several studies have described a direct effect of stromal fibroblasts in cancer initiation
and progression. On the other hand, the selective migration of MSCs to tumour sites
can be utilised for delivery of therapeutic agents using MSCs transduced with specific
genes (Studeny et al., 2004). Although there have been some reports of benefits in
murine and rat models (Nakamura et al., 2004; Sonabend et al., 2008; Stoff-Khalili et
al., 2007), more work is required to refine the use of this strategy.
1.1.6.4. Microchimerism
Fetal stem cells trafficking into maternal blood during pregnancy engraft in maternal
tissues, where they persist for decades. While these cells were initially implicated in
maternal autoimmune disease (Johnson et al., 2001), more thorough studies with
controls suggest that microchimeric fetal cells are found widely in controls without
autoimmune disease, and in a wide range of organs. Furthermore, recent studies have
suggested that fetal microchimerism is both ubiquitous and may play a role in
48
response to tissue injury (Khosrotehrani et al., 2004; O'Donoghue et al., 2008).
Interestingly, microchimerism is not always associated with disease and may also
occur in healthy women since fetomaternal trafficking occurs in all pregnancies
(Bianchi et al., 1996). In addition, though the fetus and its cells are semi-allogeneic
and should be recognised by the maternal immune system, no reaction or rejection is
apparent (Liegeois et al., 1977).
The cell type responsible for microchimerism is currently unknown, although
hfMSCs are likely candidates. Firstly, expression of adhesion molecules and in vitro
adherent properties imply hfMSCs readily implant in tissues (Campagnoli et al.,
2001). Next, their persistence in maternal tissues after many years may be due to the
non-immunogenic and immunomodulatory nature of hfMSCs (Gotherstrom et al.,
2004). Thirdly, fetal cells found in murine maternal brain were negative for CD45
expression, suggesting these cells were non-haematopoietic (Tan et al., 2005).
Additionally in animal models, infused allogeneic MSCs migrate to and engraft in
host tissues (Devine et al., 2001). Finally, male cells present in normal and diseased
female tissues exhibited MSC phenotypes (O'Donoghue et al., 2004; O'Donoghue et
al., 2008).
1.2. Duchenne muscular dystrophy
Skeletal muscle is the largest tissue in the human body, responsible for posture,
movement and breathing. It consists of myofibre bundles and the contractile muscle
units, each containing hundreds of syncytial postmitotic myonuclei.
49
1.2.1. Clinical features
DMD belongs to a group of human X-linked recessive muscular dystrophies that
include Becker muscular dystrophy (BMD) and Emery Dreifuss muscular dystrophy.
It is the most prevalent genetic neuromuscular disorder affecting 1 in 3,500 live male
births. Affected individuals have progressive myopathy, presenting with proximal
muscle weakness of the lower limbs, pseudohypertrophy of calf muscles, waddling
gait and raised serum creatinine kinase levels (Figure 1-04). They are wheelchair
bound by the age of 12 and often die between ages 14-25 due to respiratory failure
from intercostal muscle weakness and thoracic deformity such as scoliosis. About a
third of patients die from cardiac failure (Hoffman and Schwartz, 1991). DMD causes
permanent muscle damage and despite having discovered the molecular defect
responsible for DMD over twenty years ago (Hoffman et al., 1987; Koenig et al.,
1987), there is still no curative treatment.
Figure 1-04. Clinical and pathological features of Duchenne muscular dystrophy.
Affected individuals exhibit muscle wasting deformity (Emery, 1993) (A), while on
histological examination, muscles are replaced by fat or connective tissues (B) and on
immunostaining, the lack of dystrophin (C, right panel) is evident compared to normal
muscle (C, left panel).
50
1.2.2. Molecular pathology
The disease is caused by the absence of or reduction in dystrophin (Hoffman et al.,
1987; Koenig et al., 1987). The dystrophin gene is the largest known mammalian gene
consisting of 2.5 million bp with 79 exons thus taking up over 1% of the X-
chromosome i.e. 0.1% of the entire genome (Lansman and Franco, 1991; Roberts et
al., 1993). Most mutations (deletions, insertions, frame-shift arrangements or point
mutations) result in premature termination of transcription, and the rest are
duplications. Dystrophin is a 427 kDa sub-sarcolemmal protein which forms a
structural link between the intracellular actin cytoskeleton and the extracellular matrix
via several other proteins that makes up the dystrophin-associated protein complex
(DAPC) (Hoffman and Dressman, 2001) (Figure 1-05). The lack of dystrophin in
DAPC, which connects the cytoskeleton of myofibre to the basal lamina, causes
instability in the muscle structure due to sarcolemma fragility and muscle necrosis
(Ohlendieck and Campbell, 1991; Ohlendieck et al., 1993). Initially the muscle is able
to regenerate via satellite cells but this ability is lost with time, and muscle is
progressively replaced by fibrotic adipose tissue and loss of function eventually
occurs (Petrof, 1998). Therefore, in cell therapy of DMD, ideally the donor cells
should replenish the satellite cell compartment as well as correct the dystrophin defect.
51
Figure 1-05. Schematic representation of dystrophin-associated protein complex.
Dystrophin links the actin cytoskeleton via the protein complex to the extracellular
matrix and maintain stability of the DAPC and therefore the sarcolemma of the
muscle fibre. This protects the muscle fibre from contraction-related stresses (Davies
and Nowak, 2006).
1.2.3. Animal models
1.2.3.1. Mdx
The mdx mouse is a genetic homologue of DMD, which is widely used as its animal
model despite the minimal phenotype. It was derived from a spontaneously occurring
mutant with lack of dystrophin (though <1% dystrophin is expressed from revertant
fibres, discussed in Chapter 1.2.4.3.2) on the background of a C57BL/10 colony
(Bulfield et al., 1984). Mdx muscle undergoes repeated cycles of degeneration and
regeneration, beginning postnatally at three weeks and continuing throughout life
(Collins and Morgan, 2003). However, the mdx mouse has little impairment of muscle
strength and mechanical function, thus severe cardiomyopathy, muscle damage and
associated fibrosis only occur near the end of its almost normal lifespan. Analysis of
52
mdx muscle pathology demonstrates the sparing of some muscles (e.g. masseter)
coexisting with severe pathology in others (e.g. gastrocnemius and diaphragm), while
in between this spectrum of severity lies tibialis anterior (TA) muscle which is more
susceptible to contraction-induced injury than the wild type (Dellorusso et al., 2001).
1.2.3.1.1. Mdx/nude
This is an athymic immunodeficient model which cannot produce mature T
lymphocytes (Flanagan, 1966; Pantelouris, 1968; Wortis et al., 1971). These mdx
mice generated on a nu/nu background are maintained by breeding mdx nu/nu males
with mdx nu/+ females (Chapter 2.11.2). Half of the offsprings are nude and
immunocompromised, while the other half are hairy and immunocompetent (Partridge
et al., 1989). The fibrotic changes seen in human DMD muscles are recapitulated in
the mdx diaphragm and limb muscles near the end of its lifespan. However, there is a
lower level of fibrosis in mdx/nude diaphragm and heart muscles compared to mdx
tissues, possibly due to their reduced levels of immune cells to cause inflammatory
responses (Morrison et al., 2000; Morrison et al., 2005).
1.2.3.1.2. Mdx/scid
Mdx/scid is another immunodeficient model lacking both T and B cells (Bosma et al.,
1983), generated by crossing mdx with severe combined immunodeficiency (scid)
mice over ten generations. Compared to mdx mice, their rate of fibrosis is similar in
the diaphragm though lower in the TA muscles (Farini et al., 2007).
1.2.3.2. Utrophin and dystrophin knockout
Utrophin and dystrophin are structurally similar and the pathology in mdx mice is
abolished when utrophin expression is upregulated (Tinsley et al., 1998; Tinsley et al.,
1996). Though utrophin-deficient mice display no severe abnormalities in muscle and
53
non-muscle tissues except for subtle defects in their skeletal neuromuscular junctions
(Grady et al., 1997a), utrophin and dystrophin double-knockout (DKO) mice have
severe muscle pathology compared to mdx phenotype, showing skeletal deformities,
growth restriction and premature death (Deconinck et al., 1997; Grady et al., 1997b).
1.2.3.3. Golden retriever muscular dystrophic dogs
Spontaneous mutations of the dystrophin gene resulting in X-linked muscular
dystrophy were found in golden retriever muscular dystrophic (GRMD) dogs (Cooper
et al., 1988; Kornegay et al., 1988) which also exhibit abnormal utrophin expressions
(Wilson et al., 1994). Pathology occurs in utero with the development of lingual
muscle lesions. From birth, the progressive myopathy in limb, cardiac and respiratory
muscles leads to severe muscle fibrosis and joint contractures by six months of age,
often culminating in premature death by respiratory or cardiac failure comparable to
DMD patients (Nguyen et al., 2002; Valentine et al., 1988).
1.2.4. Treatment modalities
Apart from using dystrophin as a marker for successful cell therapy to repair
dystrophic muscle, other markers are used to determine degrees of de novo muscle
generation. Pax7 is a marker for quiescent satellite cells and is also expressed in the
early stages of myogenesis. Desmin, an intermediate filament protein, is one of the
earliest protein markers for muscle tissue in embryogenesis (Kaufman and Foster,
1988) as it is detected in the somites of myoblasts. MyoD (Choi et al., 1990; Davis et
al., 1987; Sassoon et al., 1989) and myogenin (Hasty et al., 1993; Jin et al., 2000;
Nabeshima et al., 1993) are myogenic regulatory factors (MRF) that mark myoblast
commitment down the muscle lineage. Myosin is a marker used to determine terminal
muscle differentiation, as myofibres consist of sarcomeres, an organisation of myosin
54
and actin filaments (Figure 1-06). Troponin T (TnT) is a component of the troponin
complex which binds to tropomyosin in the thin filament of striated muscle and is
integral to muscle contraction.
Figure 1-06. Molecular landmarks of myogenic differentiation. During
myogenesis, desmin and Pax7 are expressed in the early stages. Activation towards
myogenesis is marked by MyoD, while myogenin later marks the commitment to
differentiation. Myosin is expressed in fully differentiated myofibres (Zammit et al.,
2006).
Most research so far has concentrated on local intramuscular administration of
candidate treatment. Promisingly, by repeated local administration of MPCs,
otherwise known as myoblasts, dystrophin expression was achieved in 26-30% of
DMD muscle fibres injected (Skuk et al., 2006; Skuk et al., 2007). However, systemic
delivery would be preferable both in terms of patient acceptance and the systemic
manifestations of DMD, involving a wide range of disparate tissues including heart,
diaphragm and skeletal muscle. In contrast to intra-arterially injected mesoangioblasts,
MPCs have not been delivered systemically. The drawback of cell trapping in organs
55
such as lung, spleen and liver can be circumvented by using intra-arterial
administration instead of intravenous, as donor cells can access skeletal muscles
through the muscle capillary network (Galvez et al., 2006; Sampaolesi et al., 2006).
However, cell embolism to lungs, heart, brain, kidneys and liver remains a potential
problem. For organs such as heart and diaphragm, cells can be delivered on synthetic
“patch” scaffolds seeded with cells (Boldrin et al., 2007; Hill et al., 2006a, b). In large
animal models such as pigs and rhesus monkeys, intra-arterial delivery of plasmid
encoding ß-galactosidase using large volumes of plasmid DNA solution under high
hydrostatic pressure has resulted in high levels of reporter gene expression but
variable expression within different muscle groups possibly due to varying
intramuscular pressure (Danialou et al., 2005; Zhang et al., 2001a), while intra-arterial
delivery of dystrophin gene into mdx mice led to a modest 5% transfection rate
(Zhang et al., 2004a). This route has also been used to administer adeno-associated
virus (AAV) vectors, which induced exon skipping (Chapter 1.2.4.3.1 and 1.2.4.3.2),
and mesoangioblasts (Chapter 1.2.4.4.5) in mdx mice (Goyenvalle et al., 2004) and
GRMD dogs (Sampaolesi et al., 2006), respectively. Intravenous reporter gene
delivery gave a high rate of transfection and has been well tolerated in mdx mice, rat,
dog and monkey (Hagstrom et al., 2004). In terms of therapeutic gene transfer,
intravenous administration of antisense oligonucleotides (Alter et al., 2006) and AAV
vectors carrying microdystrophin (Gregorevic et al., 2006) have been successfully
used in mdx and DKO mice.
1.2.4.1. Supportive measures
Current treatment is mainly palliative, such as exercise to minimise contractures, and
treatment to alleviate cardiac and respiratory diseases, such as non-invasive positive
pressure ventilation (Wagner et al., 2007).
56
1.2.4.2. Corticosteroids
Corticosteroids have a catabolic effect on non-exercised muscle and act to preserve
existing muscle fibres and reduce inflammation. Although their exact mechanism of
action is unknown, steroids may reduce the inflammation seen in DMD which
contributes to myofibre necrosis (Tidball and Wehling-Henricks, 2005). Randomised
controlled clinical trials show that glucocorticoids improve muscle strength and
function in children treated from six months to two years of age (Griggs et al., 1991;
Mendell et al., 1989). Due to known systemic risks of long term steroid use
(Cushing’s syndrome, glaucoma, hypertension, diabetes, pancreatitis, gastritis and
osteoporosis) but unknown, yet possible benefits to respiratory and cardiac muscles,
the 2006 Cochrane review of corticosteroid use in DMD could not evaluate long term
benefits from currently published studies (Manzur et al., 2008).
1.2.4.3. Gene therapy
Successful gene therapy for DMD has been hampered by the large size of the
dystrophin gene (14 kb complementary DNA (cDNA)) and the systemic nature of the
disease, manifesting in all muscle groups, including heart, in addition to the brain.
1.2.4.3.1. Gene-replacement strategies using adeno-associated virus vector
AAV, a small, non-enveloped single-stranded DNA virus, is a non-pathogenic human
parvovirus that requires co-infection with a helper virus, such as adenovirus or
herpesvirus, to undergo a productive infection in cultured cells. They have become the
mainstay in the choice of vector due to their efficiency in transducing skeletal and
cardiac muscles via intravenous administration. Furthermore, AAV merely causes a
mild immune response on infection. However, these small vectors can only carry a
57
small genome, which is particularly problematic in the case of a large transgene such
as dystrophin.
Despite having large deletions in the dystrophin gene, BMD patients exhibit a
mild phenotype, as their in-frame deletion gene defects remove non-essential portions
of the dystrophin gene (England et al., 1990). This has inspired the engineering of
minigenes (~6 kb) which replicate some of these mutations, and also smaller
microdystrophins (3.6-4.2 kb) with larger, non-contiguous deletions. Having been
shown to be effective in the mdx model (Banks et al., 2007; Denti et al., 2006;
Townsend et al., 2007), there is now a need for evaluation in large animal models. A
recent paper suggests at least one version of microdystrophin is not effective in
GRMD dogs (Sampaolesi et al., 2006).
The small capacity of AAV vectors can be overcome by a trans-splicing (ts)
approach which splits the gene between two vectors. In the tsAAV vectors, the split
sequences are engineered with splicing signals. Upon co-infection, the AAV genomes
undergo head-to-tail recombination, forming the complete transgene, interrupted by
the intron which is removed by the engineered splicing signals, thus resulting in a
functional gene larger than that can be delivered by a single AAV vector. This
strategy was used successfully to deliver a minidystrophin to all muscles in the mdx
mouse (Ghosh et al., 2007).
In terms of gene targeting to muscles and reducing non-specific tissue toxicity or
immune response, skeletal muscle-specific promoters have been used to drive the
therapeutic gene. Promising results were recently reported by using a modified
creatine kinase promoter to drive high levels of dystrophin gene expression
selectively in the skeletal and cardiac muscles (Salva et al., 2007).
58
1.2.4.3.1.1. Challenges
So far, high titres of viral vectors have been used in the preclinical studies. Therefore
in clinical scenarios, high output vector production will need to be optimised. Also,
further efforts will need to be made in improving the transduction efficiency and
lowering the AAV doses. Recently developed serotypes such as AAV6, AAV8 and
AAV9 have an improved efficiency of gene delivery to muscle (Gregorevic et al.,
2006; Inagaki et al., 2006; Wang et al., 2005b). Another challenge lies in the rare
integration of AAV vectors into chromosomes, thus even if satellite cells were
transduced, the viral genome will eventually be lost following multiple cell divisions.
The ongoing trial in Ohio, USA which began in March 2006 should be
informative. This is a Phase I/IIa study in which a modified AAV vector is used to
deliver microdystrophin to the biceps of boys with DMD
(http://www.mda.org/research/view_ctrial.aspx?id=212). Though a low dose was
deemed to be safe in the initial 6 patients, there are some uncertainties regarding
administering a higher dosage.
1.2.4.3.2. Modification of dystrophin messenger ribonucleic acid (mRNA) splicing
using antisense oligonucleotides
Antisense oligonucleotides (AO, oligomers) are used to knock down target gene
expression. In DMD, they have been used to redirect splicing and induce exon
skipping. Most gene defects in DMD involve out-of-frame deletions (~65%) or
duplications (~10%). Hence AO deletion to restore the open reading frame would
result in internally deleted but semi-functional dystrophins similar to the mild BMD
phenotype. Different modifications of antisense chemistry have been investigated,
including 2-O-methyl phosphorothioate (2OMe) and phosphorodiamidate morpholino
oligomers (PMO). The systemic administration via intraperitoneal or intravenous
59
routes of morpholinos in mdx mice gave rise to appreciable induction of exon
skipping with dystrophin expression and functional improvements throughout the
body musculature (Alter et al., 2006; Fletcher et al., 2007; Fletcher et al., 2006).
Internally truncated genes are also expressed in “revertant” fibres which are
individual dystrophin-positive fibres found in 50% of DMD patients (Fanin et al.,
1995), mdx mice (Hoffman et al., 1990) and GRMD dogs (Schatzberg et al., 1998).
Revertant fibres arise via an alternative-splicing mechanism occurring in dystrophin
pre-mRNAs, skipping of frame-shifting exons to remove protein-truncating mutations
and restoration of the open reading frame (Yokota et al., 2006).
1.2.4.3.2.1. Challenges
The treatment will need to be patient-tailored, as different gene deletions will require
different AO. However, it has been shown that by skipping seven DMD exons, the
reading frame will be restored in 70% of all patients, due to a recognised pattern of
deletions. Next, this treatment is limited by how long AO persist in the tissue, in
addition to the half-life of the skipped mRNA and the resulting protein. Therefore,
patients will require lifelong treatment. Current data in mdx mice suggest that 2OMe
AO maintained dystrophin expression for a minimum of eight weeks (Lu et al., 2003),
compared with fourteen weeks using PMO AO (Muntoni and Wells, 2007). Finally,
the long term side effects of AO remains unknown, hence there is caution regarding
systemic use.
A recent clinical trial from the Netherlands administered a single dose of 2OMe
AO into the TA muscle of four patients with DMD. In all cases, exon 51 skipping and
partial dystrophin restoration (3-12% of healthy control muscle) were achieved
without adverse events by 28 days (Van Deutekom et al., 2007). The ongoing U.K.
trial focuses on assessing the safety and efficacy of the intramuscular administration
60
of a PMO AO directed against exon 51 in the extensor digitorum brevis (EDB) of
boys with DMD. This dose escalation study will compare EBD biopsies one month
after administration and use contralateral EDB as controls
(http://clinicaltrial.gov/ct/show/NCT00159250;jsessionid=ED5806BC32E7EFF11F42
859C2F990C9A?order=12).
1.2.4.3.3. Read-through stop-codon strategies
The rationale of read-through strategies stems from the observation that some
antibiotics such as aminoglycosides can suppress stop codons. Since a proportion of
mutations in the dystrophin gene are nonsense mutations, administration of
aminoglycosides could allow read-through past the premature stop codon, and the
production of a functional protein. Despite promising data in mdx mice, with a
dystrophin expression rate up to 20% (Barton-Davis et al., 1999), its use in humans
has proved inconclusive (Politano et al., 2003; Wagner et al., 2001). Furthermore,
concerns for gentamicin-induced toxicity (nephrotoxicity and ototoxicity) have led to
the development of an alternative agent, PTC124, an orally administered small
molecule which allows ribosomes to bypass the nonsense mutations in mRNAs.
Recent data indicated that PTC124 administration to mdx mice restored the
production of dystrophin to levels of 20-25% of control mouse muscles along with
some functional improvement (Welch et al., 2007). The main concern is a theoretical
possibility regarding the loss of fidelity which may lead to global mistranslation, since
any other mRNA could be misread. However, genes typically contain multiple stop
codons at the 3’ end, thus reducing this risk. A phase IIa study of PTC124 and
nonsense mutations in DMD patients has been completed in the U.S.A. and is
awaiting analysis (http://clinicaltrials.gov/ct/show/NCT00264888?order=3).
61
1.2.4.4. Cell therapy
Cell therapy utilises allogeneic donor or autologous cells with or without ex vivo
genetic manipulation (Figure 1-07).
Figure 1-07. Diagram summarising the cell sources that have been studied for
Duchenne muscular dystrophy cell therapy (modified from Boldrin and Morgan,
2007).
1.2.4.4.1. Myoblasts
In 1989, myoblast transplantation was shown to restore dystrophin expression in
mdx/nude mice (Partridge et al., 1989). It is worth noting that these early studies used
cells obtained by enzymatically-disaggregating donor muscle, which would have
given a population including all the cells present within skeletal muscle: satellite cells,
cells from the muscle vessels, cells resident in the interstitial space or enrolled from
the circulation, non-myogenic cells e.g. fibroblasts, and cells that are known today for
their myogenicity and stem potential, like muscle-derived stem cells.
After failed human trials, three main problems were identified: (1) at least 75%
of the transplanted MPCs die in the first three days after transplantation (Beauchamp
et al., 1999; Fan et al., 1996; Guerette et al., 1997; Huard et al., 1994); (2) poor
62
migration as they do not migrate more than 200 μm away from the site of in jection
and cannot cross the endothelial barrier (Skuk et al., 1999); and (3) without adequate
immunosuppression, myoblast rejection occurs within two weeks (Guerette et al.,
1994). Moreover, ciclosporin, which was used for immunosuppression in several
clinical trials, induced apoptosis of the myoblasts at the time of their differentiation.
Migration has been improved by using matrix metalloproteinase (El Fahime et al.,
2000) and by modulating MyoD expression (Smythe and Grounds, 2001).
Immunosuppression with FK506 (Tacrolimus or Prograf®; Astellas Pharma,
Deerfield, IL, USA) allowed Kinoshita et al. to obtain good transplantation results in
both mice and monkeys (Kinoshita et al., 1996; Kinoshita et al., 1994). However,
concerns for side effects of immunosuppression have led to other strategies such as
inducing specific immunological tolerance toward donor myoblasts or transplantation
of genetically modified autologous myoblasts. Approaches include using lentiviral
vectors containing the microdystrophin gene (Quenneville et al., 2007), AAV vectors
encoding dystrophin gene (Floyd et al., 1998; Goncalves et al., 2006) and exon
skipping using lentivirus coding for the appropriate short hairpin ribonucleic acid
(RNA) (Peault et al., 2007).
Satellite cells are situated beneath the basal lamina that surrounds each myofibre
and give rise to its progeny, MPCs or myoblasts. In mdx/nude hosts, freshly prepared,
non-cultured and non-expanded satellite cells can regenerate skeletal muscles and
reconstitute the host pool of satellite cells (Collins et al., 2005; Montarras et al., 2005).
1.2.4.4.2. Side population
Side population (SP) cells can be isolated from the interstitial muscle space and these
cells, distinguished by their efficient efflux of the vital DNA dye Hoescht 33342, can
be purified by fluorescence-activated cell sorter (FACS) (Gussoni et al., 1999). The
63
majority of mouse muscle SP cells (>90%) are positive for stem cell antigen-1 (Sca-1)
and are negative for the haematopoietic SP markers CD45, CD43 and c-kit, and share
markers with mesoangioblasts (Montanaro et al., 2004). After the initial
demonstration that intravenously injected SP cells partially restored dystrophin
expression in mdx mice (Gussoni et al., 1999), another study demonstrated that SP
cells transduced with a microdystrophin transgene can be delivered intra-arterially,
with or without lethal irradiation of mice (Bachrach et al., 2006). SP cells are distinct
from satellite cells as they are present in Pax7-/- mice which have few satellite cells.
Human fetal muscle SP cells have also been isolated and their roles are currently
under study (Pavlath and Gussoni, 2005).
1.2.4.4.3. Skeletal muscle-derived stem cells
In the interstitial muscle space in mice, there is a cell population heterogeneous for
CD34 and Sca-1 expression that shows plasticity towards both mesenchymal and
haematopoietic lineages (Torrente et al., 2001). They have been shown to contribute
towards mdx muscle regeneration following local or systemic administration and
appear to be more efficient at regenerating skeletal muscle than other adherent
skeletal muscle-derived cells. However, these have not yet been isolated in humans
and there was no physiological improvement in the dystrophic condition of mdx mice
following treatment with these cells derived from normal mice (Mueller et al., 2002).
Therefore, further research is needed before they can be used as a candidate cell
source for the treatment of DMD.
1.2.4.4.4. Pericytes
Pericytes are parietal cells localised under the basal lamina of human skeletal muscle
microvasculature and have been characterised by molecular markers
64
(CD146+/CD45−/CD34−/CD144−/CD56−) as a distinct cell population from satellite
cells and from endothelial cells (Gussoni et al., 1999). These perivascular cells
regulate microvessel contractility and are not myogenic in culture. However, they
become myogenic upon in vitro myogenic induction and contribute greatly towards
skeletal muscle regeneration and to satellite cells after intra-arterial delivery into scid
mice (Dellavalle et al., 2007). Despite being a promising candidate for treatment, it
remains to be seen whether pericytes can fully reconstitute the satellite cell
compartment (Morgan and Muntoni, 2007).
1.2.4.4.5. Mesoangioblasts
Mesoangioblasts are distinct from pericytes in that they are vessel-associated cells
isolated from the embryonic dorsal aorta in avian and mammalian species, express
endothelial markers and exhibit mesodermal phenotypes. Delivered through the intra-
arterial route, they have been shown to improve functionality or histology in
dystrophic muscle of α-sarcoglycan-null mice (a model for limb girdle muscular
dystrophy) (Galvez et al., 2006; Sampaolesi et al., 2003) and GRMD dogs
(Sampaolesi et al., 2006). Nevertheless, some have argued that the isolation from
muscle blood vessels cannot exclude the presence of other muscle stem cells.
Moreover, the immunosuppressive drugs given to dystrophic dogs are confounding
factors as anti-inflammatory and immunosuppressive drugs can also reduce the
severity of muscle dystrophy (Brunelli et al., 2007; Davies and Grounds, 2006;
Radley et al., 2007).
1.2.4.4.6. CD133+ cells
Circulating cells expressing CD133 have been isolated from peripheral blood, and
following intra-arterial injection in mdx/scid mice, they were able to contribute to
65
muscle regeneration (Torrente et al., 2004). It was shown that human dystrophin
expression was significantly increased when CD133+ cells were injected intra-
arterially 24 h after muscle exercise (Gavina et al., 2006). The acute inflammation
produced by muscle exercise increased the expression of vascular cell adhesion
molecule-1 (VCAM-1) on murine endothelium and mediated the efficient recruitment
of injected cells (Galvez et al., 2006). Therefore, another approach to improve cell
therapy for muscular dystrophy may be through the manipulation of VCAM-1
expression and its ligands, in order to modify the homing of injected cells.
1.2.4.4.7. Other cell types
Synovial membrane derived-MSCs have been shown to restore dystrophin expression
in mdx mice and contribute to both myofibres and satellite cells in nude mouse
muscles undergoing regeneration following cardiotoxin treatment (De Bari et al.,
2003). Satellite cells on isolated fibres can regenerate skeletal muscles and
reconstitute the host pool of satellite cells (Collins et al., 2005; Montarras et al., 2005).
Circulating myogenic precursor cells exist in the BM (Ferrari et al., 1998). Despite
being able to contribute to muscle regeneration, these MPCs exhibit low level
engraftment and their satellite cell derivatives are non-functional. A recent report
suggests that ESCs can restore dystrophin in 11-16% of total mdx myofibres
combined with functional improvements without forming teratomas over a 4-month
follow-up period (Darabi et al., 2008). However, those ESCs had to be genetically-
modified to express Pax3, then selected for the PDGF-αR+Flk-1- fraction prior to
transplantation in order to avoid teratoma formation.
66
1.2.4.5. Modification of compensatory mechanisms
1.2.4.5.1. Utrophin upregulation
There is a high degree of sequence similarity between dystrophin and utrophin at both
DNA and protein levels (Blake et al., 1996; Blake et al., 2002). Utrophin expression is
high during embryonic development but declines in adult skeletal muscle and is
limited to the neuromuscular junctions and myotendinous junctions (Love et al.,
1989). When utrophin is over-expressed in transgenic mdx mice, it localises to the
sarcolemma of muscle cells and restores the components of the DAPC, which
prevents dystrophic development and in turn leads to functional improvement of
skeletal muscles (Tinsley et al., 1998). More recently, the delivery of a small peptide
region of the heregulin ectodomain to mdx mice increased utrophin expression and
ameliorated the dystrophic phenotype (Krag et al., 2004).
1.2.4.5.2. Insulin-like growth factor-1
IGF-1, a signalling peptide involved in myogenesis, has been used with the aim to
increase muscle mass. Moderate over-expression of IGF-1 in mdx muscles yielded
increased muscle mass and strength with decreased necrosis and fibrosis (Barton-
Davis et al., 1998; Musaro et al., 2001). Another strategy for inducing muscle
hypertrophy is to use a myostatin antibody (Patel et al., 2005), which resulted in a
significant increase in muscle mass, size and strength after weekly injections in mdx
mice for three months (Bogdanovich et al., 2002).
1.2.4.5.3. Other candidates
Increased expression in the major laminin-binding integrin of mature muscle, integrin
α, is seen in both DMD patients and mdx mice (Hodges et al., 1997) and the
expression of rat α7 integrin in DKO mice led to phenotypic improvements (Burkin et
67
al., 2005; Burkin et al., 2001). Angiotensin II type 1 receptor blockade by losartan has
been shown to induce muscle regeneration and reduce fibrosis via antagonism of
TGF-ß, which in turn ameliorated the mdx phenotype (Cohn et al., 2007). Recently, a
strategy of reducing mitochondrial-dependent muscle necrosis by inhibiting
cyclophilin was highlighted by Millay et al. The inhibitor, Debio-025, is a synthetic
ciclosporin analogue and reduced muscle fibrosis significantly in mdx mice (Millay et
al., 2008).
1.3. Intrauterine transplantation
Intrauterine treatment is an attractive approach towards a variety of prenatally
diagnosed diseases, including those in the musculoskeletal, haematological, metabolic
and immunological systems. As DMD can be diagnosed prenatally (Beggs et al., 1990;
Sancho et al., 1993), a possible strategy for treatment is in utero transplantation (IUT).
This allows systemic delivery and may be more attractive than local postnatal therapy
due to the wide range of disparate muscle groups and organs involved.
There are many potential advantages for treating the fetus whilst in utero. There
is a possibility of reconstitution of a missing or defective cell type, or correction of
genetic defects, and it obviates the need for myeloablation or chemotherapeutics and
their associated morbidity and mortality. Moreover, it can be used to induce prenatal
tolerance to specific antigens because of tolerance to foreign antigens in the first
trimester fetus (Gotherstrom et al., 2003), thus facilitating postnatal cellular or organ
transplantations from the same donor.
There are several advantages to a fetal to fetal stem cell transplantation approach.
As well as allowing pre-emptive treatment before the occurrence of irreversible end-
organ damage in the fetus, there is an exponential expansion of cellular compartments
68
and natural migration of stem cells (Le Blanc and Ringden, 2005). The stoichiometric
benefit of the fetus being only about 30 g in size at 13 weeks is such that a much
larger proportion of cells can be delivered prenatally compared to postnatally.
HfMSCs may be ontologically advantageous within the fetal environment compared
with adult MSCs and either allogeneic hfMSCs without the gene defect or genetically-
corrected autologous hfMSCs can be used. However, in terms of gene therapy, there
are still many hurdles before it can be used in the clinical setting, including risks from
unintentional transduction of the germ line, insertional oncogenesis and transplacental
transduction of maternal cells. Compared with adult and neonatal transplantation, IUT
has the advantage of avoiding sequestration of stem cells in the fetal lungs, as most of
the fetal circulation bypasses the lungs.
1.3.1. Animal models
1.3.1.1. Wild type
Initial experiments have been performed using transplacental injection of HSCs to
cure anaemic mice (Fleischman and Mintz, 1979). Forty-three percent of recipient
mice were engrafted for more than six months. Furthermore, intrauterine HSC therapy
has been used to treat inherited immune deficiencies with some success (Diukman and
Golbus, 1992; Slavin et al., 1992; Thilaganthan et al., 1993; Touraine et al., 1989).
However, in immunocompetent and non-anaemic mice, there was restricted
engraftment of HSCs after IUT due to competition between host and donor cells for
the limited cell compartments (Carrier et al., 1995; Hajdu et al., 1996; Kim et al.,
1998). After allogeneic IUT with FL-derived HSCs into normal fetal lambs, levels of
mixed chimerism as high as 30% were achieved after a single intrauterine dose, while
using other HSC sources, levels of 5-15% can be achieved routinely (Flake et al.,
69
1986; Zanjani et al., 1992; Zanjani et al., 1993). Promising results with increased
levels (2-10 fold compared to no co-transplantation) of haematopoietic chimerism two
months after stromal co-transplantation in wild type sheep have been reported in a
fetal ovine model (Almeida-Porada et al., 2002). Similarly, human MSCs engraft into
multiple organ compartments after IUT into fetal sheep and also showed evidence of
site-specific differentiation (Mackenzie and Flake, 2001; Schoeberlein et al., 2005).
Past experience in my laboratory showed that when first trimester human FB-MSCs
were transplanted in utero into immunocompetent MF1 wild type fetal mice, MSCs
can be detected postnatally by staining for human-specific markers. Upon
transplantation into injured muscle in immunodeficient mice, MSCs participated in
regeneration of muscle by forming spectrin-positive muscle fibres, albeit at low levels
(<1% fibres per muscle section) (Chan et al., 2007).
1.3.1.2. Mdx
Previous work in my group on IUT in mdx achieved a low level of engraftment (0.5-
1.0%), although the level was significantly higher in muscle than in non-muscle
tissues (7.05 ± 1.80 versus 1.48 ± 0.67 per 1,000 murine cells) (Chan et al., 2007).
The level of chimerism was comparable to that obtained using murine allogeneic
MSCs similarly given in utero (Mackenzie et al., 2002). This is likely to be
insufficient for functional improvements in DMD, based on the evidence that in mdx
mice, a 20-30% expression of human minidystrophin transgene reduces their
myopathy (Wells et al., 1995). On the other hand, phenotypic recovery towards wild
type has been seen following IUT in an OI mouse model (Chapter 1.3.3.5) despite
relatively low engraftment levels (~5%) in the target organ (Guillot et al., 2008a).
In terms of underlying mechanisms for engraftment and in vivo myogenesis
following IUT, transplanted stem cells undergoing muscle differentiation should be
70
able to fuse with each other or with growing or regenerating myofibres since myoblast
fusion is fundamental both to the production of skeletal muscle during development
and to its repair later in life (Horsley and Pavlath, 2004). Because of difficulties in
distinguishing cell fusion in vivo, in vitro techniques have been utilised in the
literature (discussed in Chapter 5).
1.3.1.3. Osteopetrosis
Autosomal recessive osteopetrosis is a genetic disease where mineralised cartilage
and bone cannot be degraded thus blocking BM formation. In its mouse model, IUT
of unselected adult murine BM cells led to improvements in 35% of mice transplanted,
by rendering them either clinically asymptomatic or having a normal phenotype in
terms of growth, ability to breed, and the presence of donor cells during the normal
lifespan (Frattini et al., 2005).
1.3.2. Humans
The first attempt to treat human disease using an in utero procedure was performed in
1967 by Davies using fetal BM for Rh disease (Davies, 1967), whereas the first
successful human fetal transplantation was reported by Touraine in 1989, using
human FL cells for transplantation into a fetus with bare lymphocyte syndrome
(Touraine et al., 1989). Since then, results from different centres performing fetal
HSC transplantation in humans have been less encouraging, except in scid patients
(Flake et al., 1996; Lanfranchi et al., 1998; Muench et al., 2001; Porta et al., 2000;
Touraine et al., 2004; Wengler et al., 1996; Westgren et al., 2002) who can instead be
transplanted more safely postnatally (Table 1-02). MSCs have already been used with
allogeneic BM transplantation in children with OI, where improvements in height and
71
reduction in fracture frequency have been reported (Horwitz, 2001; Horwitz et al.,
2002; Horwitz et al., 1999; Horwitz et al., 2001).
Table 1-02. Summary of human in utero stem cell transplantations in
immunodeficiencies (Tiblad and Westgren, 2008).
1.3.3. Strategies to overcome low engraftment
1.3.3.1. Optimisation of cell source
Due to the variety of sources of first trimester hfMSCs (blood, liver and BM), there
may be inherent differences in their engraftment and myogenic potential. Other
strategies that can be exploited therapeutically include transplanting hfMSCs pre-
differentiated down the myogenic lineage to increase myogenic engraftment rates.
Local transplantation of pre-differentiated rat MSCs using 5-azacytidine, a
hypomethylation-inducing agent, improved function and histology of damaged
72
cardiac muscles compared to non-stimulated or naïve cells (Tomita et al., 1999).
Following IUT of pre-differentiated hfMSCs down the oligodendritic pathway, the
rate of oligodendrocyte differentiation in vivo was increased compared to
undifferentiated hfMSCs (Kennea et al., 2003). In addition, hfMSCs pre-stimulated
with galectin-1 (Gal-1) underwent myogenesis efficiently in vitro and gave rise to
significantly more muscle fibres of donor origin than non-stimulated hfMSCs in vivo
(Chan et al., 2006a).
Gal-1, a 14-15 kDa lectin, belongs to a phylogenetically conserved family of
lectins responsible for β-galactoside binding (Barondes et al., 1994a; Barondes et al.,
1994b; Camby et al., 2006). It is secreted by myoblasts and myotubes in culture
(Harrison and Wilson, 1992) and has various biological functions, e.g. mitogenesis,
inhibition of cell proliferation, apoptosis induction and migration regulation (Smetana
et al., 1997). A number of lines of evidence suggest that Gal-1 has a myogenic effect.
Firstly, Gal-1 increases desmin expression in human fetal and murine dermal
fibroblasts (Goldring et al., 2002a; Goldring et al., 2000). It also increases the fusion
index (total number of nuclei in myotubes divided by total number of nuclei in culture)
of myotubes formed by C2C12, a murine myoblast cell line (Blau et al., 1985; Yaffe
and Saxel, 1977), and primary mouse myoblasts (Goldring et al., 2002b). Secondly,
myoblasts derived from Gal-1 null mouse showed a reduced ability to fuse in vitro,
whereas in vivo Gal-1 null mutant has delayed muscle regeneration following injury,
delay in neonatal muscle fibre development and reduced muscle fibre diameter
(Georgiadis et al., 2007; Watt et al., 2004). In particular, Gal-1 receptors, such as
laminin, fibronectin and actin, are present in hfMSCs (Elola et al., 2005). Finally,
previous work in my group has demonstrated that Gal-1 induced a high level of
myogenic conversion in hfMSCs (66.1 ± 5.7% of total cells became desmin-positive)
73
with formation of myotubes expressing both desmin and sarcomeric myosin (MF20)
via activation of MRF. This is the most efficient in vitro myogenic conversion of a
naïve untransduced stem cell population reported so far. In addition, Gal-1-treated
hfMSCs formed four times more human muscle fibres than non-treated hfMSCs after
intramuscular injection in a muscle injury model in an immunodeficient adult mouse
(Chan et al., 2006a).
1.3.3.2. Immunocompromised host
Although the immunological naiveté of the early gestation fetus should allow use of
allogeneic stem cells for IUT, the limited data that exist suggest this may not
necessarily be the case (Flake et al., 1996; Wengler et al., 1996; Westgren et al.,
2002). Monocytes and macrophages, belonging to the innate immune system, seem to
be the first cell type to appear in high numbers in the fetal circulation (Forestier et al.,
1991). NK cells appear early and in high numbers. Their activity was seen from the
ninth week of gestation in FL (Uksila et al., 1983) and by thirteen weeks, about 30%
of lymphocytes in the fetal circulation are NK cells (Thilaganathan et al., 1993). In
terms of cell-mediated immunity, at 8-9 weeks of gestation, T-cell progenitors from
FL are present in fetal thymus, followed by expression of early T-cell surface antigens
CD2 and CD7 in 20-60% of cells in the thymus (Stites et al., 1974; Toivanen et al.,
1981). Lindton et al. found that mixed lymphocyte cultures of FL cells could
recognise and react to allogeneic stimulation from 9-12 weeks of gestation. There was
an increased responsiveness with increased gestational age. Depletion of HLA class II
in the stimulator population resulted in a decrease in allogeneic response, suggesting
that HLA compatibility might play a role in successful graft after IUT (Lindton et al.,
2000).
74
Even though hfMSCs have immunomodulatory properties and do not express
HLA II, (Gotherstrom et al., 2004; Gotherstrom et al., 2003) new evidence has
emerged suggesting that adult MSCs do induce cellular and humoral immune
responses in vivo in contrast to their in vitro behaviour. Together with the fact that in
utero donor cells can induce an immune response (Peranteau et al., 2007), using an
immunocompromised model such as the mdx/nude mouse may enhance the human
xenograft.
1.3.3.3. Serial transplantation
A more novel approach is to transplant cells in the neonatal period as well as in utero
in order to promote further engraftment. In addition to being able to administer more
cells over two injections, the first dose of hfMSCs may induce tolerance during fetal
life and thus avoid the second dose being rejected neonatally. It has been shown by
Milner and co-workers that after an initial in utero transplantation of 106 HSCs in
wild type mice, booster injections of 5 × 106 cells at postnatal days 2, 4, and 7 resulted
in a significantly elevated multilineage engraftment with granulocyte predominance
(Milner et al., 1999). This finding is further supported by more recent publications on
HSCs (Ashizuka et al., 2006; Hayashi et al., 2002; Peranteau et al., 2002).
1.3.3.4. Postnatal muscle injury
Due to the fact that mdx regenerate so well, irradiation was used to mimic the DMD
phenotype in which there is little regeneration (Wakeford et al., 1991). Irradiation of
the muscle inhibits host muscle satellite cell regeneration and permits donor derived
MPC and satellite cell proliferation (Beauchamp et al., 1999; Collins et al., 2005;
Morgan et al., 2002; Wakeford et al., 1991). Although both cryodamage and notexin
(snake venom) cause muscle necrosis and regeneration, cryodamage destroys mature
75
muscle fibres as well as satellite cells (Irintchev et al., 1997a; Pye and Watt, 2001),
whereas notexin damages mature muscles only (Harris and Johnson, 1978).
1.3.3.5. Models with clinical phenotype
The low level of engraftment seen post-IUT of hfMSCs in mdx (Chan et al., 2007;
Mackenzie et al., 2002) may be due to lack of overt pathology at the time of transplant
(Tanabe et al., 1986). In non-injured models, MSCs engraft at low levels (Devereux et
al., 2001; Devine et al., 2003; Schoeberlein et al., 2005), though they can be recruited
from remote sites to injury areas where the engraftment is higher compared with
normal tissues (Liechty et al., 2000). Endogenous MSCs have been found at higher
levels in pathological human muscles (Ramirez et al., 2006). Moreover, BM cells are
recruited and home to dystrophic muscles in mdx mice but not to normal muscles in
normal mice (Bittner et al., 1999). Following irradiation injury in immunodeficient
mice systemically transplanted with human adult BM-derived MSCs, there is
increased recruitment and homing of donor cells to irradiated tissues (Francois et al.,
2006). The mechanisms for systemic recruitment of stem cells in response to tissue
damage are currently under investigation (Abbott et al., 2004; Lapidot and Petit, 2002;
Neuss et al., 2004).
Using a murine model of OI (oim), the brittle bone disease which presents with
intrauterine bone fractures, osteopenia and bone fragility, the Fisk group recently
reported that IUT of hfMSCs ameliorated the disease phenotype, producing a
clinically-relevant two thirds reduction in fracture incidence, along with increased
bone strength, length and thickness (Guillot et al., 2008a). The marked improvement
in skeletal phenotype from hfMSC transplantation was associated with engraftment
levels in bone of only around 5%. This is in keeping with 7.4% engraftment following
hfMSC transplantation in a human fetus with OI, resulting in long-term chimerism in
76
bone and BM, and a lack of alloreactivity to donor MSCs (Horwitz et al., 1999; Le
Blanc et al., 2005).
In contrast, DMD has a milder clinical course in utero as the pathology worsens
only after a high level of ambulation (Bradley et al., 1972; Gardner-Medwin, 1983;
Vassilopoulos and Emery, 1977). In addition, mdx mice have a near-normal lifespan
with little muscle weakness unless they undergo eccentric exercise (Bulfield et al.,
1984). Indeed, using a model with clinical phenotype, such as utrophin and dystrophin
DKO mouse (Deconinck et al., 1997) or GRMD canine model (Cooper et al., 1988),
may also improve engraftment levels and/or muscle function.
1.4. Aims of this study
Stem cell research is a dynamic area of research with a promising outlook for fetal
therapy. HfMSCs can be easily isolated, expanded and differentiated to multiple
lineages. The intermediately primitive properties of hfMSCs, along with the
receptiveness of the first trimester fetus, make them an ideal candidate for in utero
therapeutic approach.
Given data suggesting hfMSCs can undergo myogenic differentiation using Gal-
1 conditioned medium and that hfMSCs engraft poorly following IUT in mdx model,
my original aim was to enhance hfMSC engraftment in mdx muscles by using Gal-1
conditioned medium-treated hfMSCs for IUT in an immunodeficient mdx model with
postnatal muscle injury, which is serially transplanted with hfMSCs during neonatal
and adult life. This approach towards a phase 1 clinical trial had to be altered in my
project since pre-differentiation of hfMSCs down the myogenic lineage using Gal-1
conditioned medium was not as efficient as described previously. Therefore, this
study was modified such that the initial part centred on the collection and
77
characterisation of hfMSC samples. Next, the method of in vitro myogenesis of
hfMSC was optimised. To prepare for in vivo work, my focus was on elucidating the
possible underlying mechanism for in vivo myogenesis of hfMSC following IUT,
such as fusion of hfMSCs with myoblasts. Finally, I explored strategies to improve
survival of donor cells following IUT.
1.5. Hypotheses
1. HfMSC engraftment leading to de novo skeletal muscle formation following
transplantation in a DMD murine model can be improved by:
Use of in vitro hfMSCs pre-differentiated towards the myogenic lineage via
galectin-1 conditioned medium or hfMSCs expressing galectin-1 transgene
Use of an immunodeficient model to prevent immunological rejection of
grafted cells
Serial cell transplantations
Muscle injury paradigms
2. HfMSCs are able to fuse in vitro with murine fetal myoblasts derived from mice
at the gestational period when IUT is normally performed.
78
CHAPTER 2
MATERIALS AND METHODS
79
2.1. Ethics
Fetal blood and fetal and adult tissue collection for research purposes was approved
by Research Ethics Committee of Hammersmith and Queen Charlotte’s Hospitals
(2001/6234, 2002/6482 and 06/Q0406/33). All hfMSC samples were collected
following clinically-indicated termination of pregnancy after each woman had given
written informed consent (see Appendix 1 for information sheets and forms). Animal
experiments were conducted in accordance with Home Office and institutional
guidelines (Project Licences PPL 70/6228 and 70/6256) in accordance with the
Animals (Scientific Procedures) Act of 1986.
2.2. Human fetal mesenchymal stem cells
2.2.1. Harvest of first trimester fetal blood
First trimester FB samples (200-800 μl) were obtained by ultrasound-guided cardiac
aspiration between 9-11 weeks gestation (n=6) prior to clinically-indicated surgical
termination of pregnancy. Heparinised (by flushing with heparin sodium 1,000
units/ml from CP Pharmaceuticals), disposable siliconised 20G 15-cm needles
(COOK (UK) Ltd.) attached to 1-ml syringes were used for cardiocentesis. Fetal
gestational age was determined by crown-rump length (CRL) measurement
referenced to standard biometry chart (Robinson and Fleming, 1975).
2.2.2. Harvest of first trimester fetal bone marrow and liver
Fetal tissues were collected after clinically-indicated surgical termination of
pregnancy during first trimester. Fetal BM (n=10) were flushed from marrows of fetal
long bones or spines while FL (n=4) was diced. Tissues were then washed with
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phosphate-buffered saline (PBS), and filtered through a 40-μm cell strainer (BD
Biosciences) to yield cell suspensions.
2.2.3. Isolation and cell culture
Harvested cell suspensions were centrifuged at 1,500 rpm (400 ×g) for 5 min and the
cell pellet was resuspended in high glucose Dulbecco’s modified Eagle’s medium
(DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; BioSera), 2 mM
L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin (Invitrogen) (D10
medium) and plated in 10-cm tissue culture dishes (Corning®). Cells were incubated
at 37°C with 5% carbon dioxide (CO2). After 3 days, they were washed in PBS twice
to remove non-adherent cells. The selected adherent colonies were passaged at 60-
70% confluence with a subcultivation ratio of 1:5.
2.2.4. Cryopreservation and thawing
Cells were stored in liquid nitrogen at 1 × 106 per ml in freezing medium (containing
30% FBS, 10% dimethylsulphoxide (DMSO; Sigma) and 60% DMEM). After
transfer of 1 × 106 cells per cryopreservation vial (Nunc™), vials were placed into a
controlled freezing box (VWR) at -70°C overnight and then into liquid nitrogen for
long-term storage. For thawing, cells were thawed rapidly in a water bath at 37°C and
then added drop wise in 10 ml of warm D10 to allow gentle mixing, which was then
seeded in 10-cm dishes. After ensuring cell adherence, the medium is changed the
following day.
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2.3. Cell culture of other cell types
All cells used were maintained, cryopreserved and thawed under the same conditions
as hfMSCs unless otherwise specified (Table 2-01).
Table 2-01. Culture of other cell types.
Cell Cell type Source Culture condition
COS-1 Monkey kidney
fibroblasts
ATCC, CRL-1650 D10 medium
HeLa Human cervical
carcinoma
Gift from F. Allaf,
Imperial College
D10 medium
PC3 Human prostate
carcinoma
Gift from C. Bevan,
Imperial College
Roswell Park Memorial
institute (RPMI) 1640 medium
with the same supplement as
D10 medium
SAOS-2 Human
osteosarcoma
ATCC, HTB-85 D10 medium
2.3.1. 293T cell line
293T human embryonic kidney cells were obtained from American Type Culture
Collection (ATCC, CRL-11268) and used to generate lentiviruses in my work. This
immortalised cell line was plated on 0.1% bovine collagen (StemCells Technologies)
coated T75 flasks (BD Biosciences) and subcultured to a ratio of 1 in 10 at 100%
confluence.
2.3.2. E15 myoblasts
For co-culture experiments, primary myoblasts were harvested from skeletal muscles
of fetal mdx/nude mice at 15 days gestation by enzymatic disaggregation with 0.1%
trypsin and collagenase (Roche) (Montarras et al., 2005). Dissected muscles were
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incubated in 5 ml enzyme at 30°C for 20 min, triturated and neutralised with an equal
volume of 20% FBS in DMEM to obtain a cell suspension, which was repeated up to
five times to digest all tissues. Cell suspension was filtered through a 40-μm cell
strainer (BD Biosciences), centrifuged at 1,500 rpm (400 ×g) for 15 min, and the cell
pellet resuspended in growth medium for immediate use in co-culture experiments.
The average yield was 1.20 ± 0.44 × 105 per embryo (n=33).
2.3.3. Myoblast cell lines
Except for C2C12 myoblasts, the following cells were kind gifts from the Dubowitz
Neuromuscular Centre, Imperial College London.
2.3.3.1. C2C12 myoblasts
C2C12 murine myoblast cells (ATCC, CRL-1772) (Blau et al., 1985; Yaffe and Saxel,
1977) were used as the positive control for immunocytochemical staining as they
readily differentiate into myotubes under low serum conditions. They were
maintained at low densities of 500/cm2 in D10 medium within T75 flasks (BD
Biosciences) and subcultured at 20-30% confluence to a ratio of 1:20 to prevent
differentiation. Terminal muscle differentiation was obtained by plating 2 × 104
cells/cm2 on Matrigel (0.1 mg/ml; BD Biosciences)-coated permanox (TPX) chamber
slides (Nunc™) in 5% horse serum (HS) in DMEM (Sigma) for 3-4 days.
2.3.3.2. H2K 2B4 murine myoblasts
This murine satellite cell-derived line was maintained in DMEM containing 100 U/ml
penicillin, 100 μg/ml streptomycin, 4 mM L-glutamine, 20% FBS and 2% chick
embryo extract with IFN-γ (20 U/ml) at 33°C in 10% CO2. Cells were passaged every
2-3 days at 40% confluence and plated at 5 × 104 cells per 175 cm2 gelatin (0.01%)-
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coated flask. To assess myogenicity, cells were plated on Matrigel-coated chamber
slides at 5.8 × 104/cm2, cultured in DMEM supplemented with 100 U/ml penicillin,
100 μg/ml streptomycin, 4 mM L-glutamine and 5% HS, and incubated at 37°C with
5% CO2. Myotubes began to be visible from day 2 of differentiation.
2.3.3.3. Human myoblasts
Normal skeletal muscle biopsy samples were obtained from patients undergoing
surgery for idiopathic scoliosis. Following informed consent, human adult MPCs were
prepared by sharp dissection into 1 mm3 pieces and disaggregated in solution
containing NaCl (7.6 mg/ml), 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
(HEPES, 7.2 mg/ml), glucose (2mg/ml), KCl (0.224 mg/ml), Phenol Red (1.1 µg/ml)
and 0.05% Trypsin-0.02% ethylenediaminetetraacetic acid (EDTA, Invitrogen) in
distilled water, 3 times at 37oC for 15 min in Wheaton flasks with vigorous stirring.
Cells were plated in non-coated plastic flasks at a density of 6 × 103 cells/cm2 and
cultured in skeletal muscle cell growth medium (Promocell) supplemented with 50
μg/ml gentamicin, 4 mM L-glutamine, 10% FBS (Invitrogen) and supplement mix
(Promocell) in 5% CO2 until differentiation.
Fetal MPCs were isolated by enzymatic disaggregation of leg muscles from a
human fetus at 14.3 weeks of gestation, obtained from the Medical Research Council
Fetal Tissue Bank (London). Human fetal muscle cells were similarly plated but on a
gelatin (0.01%; Sigma)-coated surface and in Ham’s F-10 medium (Invitrogen)
supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 4 mM L-glutamine
and 20% FBS (Invitrogen) in 5% CO2. Both adult and fetal MPCs were passaged at
subconfluence (20-30%).
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2.3.3.4. L6 rat skeletal myoblasts
The L6 myogenic line (Yaffe, 1968) (ATCC, CRL-1458) was used during co-culture
experiments. It was maintained in D10 medium at low plating density and subcultured
at 20-30% confluence with a subcultivation ratio of 1:20. Myogenesis was induced
over 3-4 days by differentiation medium of 2% HS in DMEM at a seeding density of
0.5 × 104/cm2 on 6-well plates (Nunc™).
2.4. Cell characterisation
2.4.1. Crystal violet
Crystal violet stain solution was freshly prepared by adding 10 mg crystal violet
powder (Sigma) per 1 ml distilled water. Medium was aspirated prior to cell staining
for 5 min which was followed by rinsing in distilled water to remove excess dye.
2.4.2. Growth kinetics
2.4.2.1. Nucleated cell counts
To determine the number of nucleated cells, 10 μl of cell suspension was diluted 10×
with 3% acetic acid with methylene blue (StemCell Technologies) and loaded into a
haemocytometer (Neubauer improved, VWR) for counting under an inverted
microscope (CK-40, Olympus). The cell concentration per ml was calculated as the
mean number of cells per quadrant × dilution factor × 104.
2.4.2.2. Doubling time and growth rate
The population doubling time of each passage was determined by noting the number
of adherent cells at the beginning and end of the passage and accounting for the time
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elapsed. Cells were plated at a density of 104/cm2 in a 10-cm dish and trypsinised
every 3-4 days at subconfluence. To estimate growth rate, 3,500 cells/cm2 were plated
in 6-well plate and the number of cells per well counted at 2-day intervals. Figure 2-
01 outlines the equation used to calculate growth kinetics.
t DT =
Log2 [ n / n0 ]
DT = doubling time
t = time in culture
n = final number of cells
n0 = initial number of cells
Log2 [ n / n0 ] = population doubling
Figure 2-01. Calculation of growth kinetics.
2.4.3. Differentiation
Differentiation experiments were carried out in early passage hfMSCs (<P5). All
differentiation reagents used were supplied by Sigma unless otherwise stated. During
this study, I had intended to use adult BM-MSCs as positive controls of differentiation.
However, I was unable to obtain these commercially as for the earlier studies from my
laboratory, since the cell bank had been flooded in New Orleans (Hurricane Katrina).
Therefore, I used hfMSC samples that had been previously characterised against adult
MSCs in our laboratory as the positive control.
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2.4.3.1. Osteogenic differentiation
For osteogenic differentiation, cells were seeded in 10-cm2 dishes at 3.1 × 103
cells/cm2 density in expanding medium overnight then changed to fresh medium
supplemented with 0 .2 mM ascorb ic acid , 1 0 mM β-glycerophosphate and 10-8 M
dexamethasone. Osteogenic medium was freshly prepared each time and replaced
twice weekly for 2-3 weeks. For Von Kossa staining, cells were fixed in 10%
formalin for 1 h, washed with distilled water and stained with 2% solution of silver
nitrate for up to 1 h and then photographed.
2.4.3.2. Adipogenic differentiation
For adipogenic differentiation, cells were plated at a density of 2.1 × 104/cm2 and
cultured in expanding medium supplemented with 5 μg/ml insulin, 10–6 M
dexamethasone and 60 μM indomethacin for 3 weeks with replacement of medium
every 3-4 days. Lipid vacuoles were stained with oil red O by fixation in formalin as
above, treating with 60% isopropanol (BDH Laboratory Supplies) in distilled water
and then stained with 0.6% solution of oil red O for 10 min to prepare for
photography.
2.4.3.3. Chondrogenic differentiation
To induce chondrogenic differentiation, 2.5 × 105 cells were pelleted in polypropylene
FACS tubes (BD Biosciences) by centrifuging twice at 150 ×g for 5 min in DMEM
with 0.05 U/ml penicillin, 0.05 μg/ml streptomycin (Invitrogen), 1% insulin-
transferrin-sodium selenite medium supplement (ITSS), 5.33 μg/ml linoleic acid, 1.25
mg/ml bovine serum albumin (BSA), 0.17 mM ascorbate-2-phosphate, 0.1 μM
dexamethasone, 1 mM Na pyruvate and 0.35 mM L-proline. Differentiation was
induced by supplementing the medium with 0.01 μg/ml TGF-ß3 (R&D Systems) and
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replacing with fresh medium every 2-3 days for 28 days. Pellets were washed with
PBS twice and fixed in 10% formalin for 1 h.
2.4.3.3.1. Wax embedding and microtome processing
Cartilage pellets were fixed overnight in neutral buffered formalin, dehydrated
through an ethanol series (70%, 90%, 100% × 3) for 1 h each, and incubated in Histo-
Clear™ (Fisher) for 3 h with change of fresh solution every hour. Wax embedding of
pellets occurred at 65°C following a 3-h incubation period, with an hourly change of
molten wax. Tissues were transferred into moulds filled with wax to make blocks
which were cooled on ice for 30 min then stored at 4°C until microtome processing.
Thin 4-μm sections were cut using a microtome and mounted on polylysine-coated
slides.
2.4.3.3.2. Alcian blue staining
Formalin-fixed, paraffin-mounted tissue slides were dewaxed by immersion twice in
Histo-Clear™ (Fisher) for 2 min each and rehydrated in an ethanol series (100% × 2
for 1 min each, 70% for 30 sec) followed by distilled water for 1 min. Sections were
stained for proteoglycans with 5% Alcian blue over 30 min and rinsed in distilled
water then Scott’s tap water and washed in distilled water again prior to mounting in
DPX mountant for photography.
2.5. Immunophenotyping
2.5.1. Flow cytometric analysis
Cells (1 × 106) were resuspended in 100 μl of FACS buffer (0.1% BSA and 0.1%
sodium azide in PBS, Sigma) and incubated at room temperature for 10 min with 5 μl
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mouse immunoglobulin G (IgG) to block non-specific binding. Cells were then
resuspended in 100 μl FACS buffer, incubated with 10 μl primary monoclonal
antibodies conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin
(PE) for 30 min at 40°C (BD Biosciences). Cells were resuspended with 2%
formaldehyde (Sigma) in 0.02% BSA following 2 washings in 2 ml FACS buffer until
flow cytometric analysis. The negative control was cells incubated with matched IgG
isotypes conjugated with the same fluorochrome as the primary antibodies.
Background fluorescence was determined by staining 1 × 106 cells with secondary
antibody only. Flow cytometric analysis was performed on a FACSCalibur analyser
(BD Biosciences) and CELLQuest software (BD Biosciences).
2.5.2. Immunostaining
2.5.2.1. Controls
Differentiating C2C12 myoblast cells were used as positive controls for Pax7, desmin,
MyoD, myogenin, and MF20 (Chapter 1.2.4) as well as negative controls for human-
specific lamins a/c and vimentin. Human fetal myoblasts acted as the positive control
for Gal-1, while SAOS-2 osteoblast cells (Fogh and Trempe, 1975) were the negative
control. SAOS-2 cells were also the negative control for desmin immunostaining.
Further negative controls were provided by HeLa cervical carcinoma cells for CD90
and 293T for laminin. Human fetal muscle sections were positive controls for
dystrophin and spectrin. Non-transplanted mouse muscle was used as negative
controls in animal transplantation work. Internal negative controls involved staining
without the primary or the secondary antibody.
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2.5.2.2. Immunocytochemistry
Cells were grown to 60% confluence on glass cover slips or TPX chamber slides,
fixed in 4% paraformaldehyde (PFA) in 125 mM HEPES (pH 7.6; 10 min, 4°C), re-
fixed in 8% PFA in HEPES (50 min, 4°C), and then permeabilised in 0.5% Triton X-
100 in PBS with gentle shaking for 30 min. Next, cells were blocked with PBS+ (PBS
containing 1% BSA, 0.2% fish gelatin, 0.1% casein at pH 7.6) for 1 h, incubated
overnight (4°C) with the appropriate primary antibody (Table 2-02) in PBS+, washed
for 1.5 h in PBS+, incubated with secondary antibodies in PBS+ for 1 h, washed
overnight (4°C) in PBS+, washed in PBS and then mounted in 4’,6 diamidino-2-
phenylindole (DAPI) Vectashield mounting medium (Vector Laboratories).
Secondary antibodies for immunofluorescence (Table 2-02) were donkey anti-mouse
IgG conjugated with FITC (Jackson ImmunoResearch Laboratories) and goat anti-
mouse or anti-rabbit IgG conjugated to either Alexafluor 488 or 594 (Molecular
Probes). Slides were viewed under fluorescence microscopy (Zeiss Axioscope I
microscope or Nikon Eclipse E600 microscope). Images were captured using a
photonics digital camera (Hamamatsu or Nikon) and processed by iPlab (Scanalytics)
or Lucia G (Laboratory Imaging) software.
90
Table 2-02. Primary antibody used for immunostaining and Western blotting.
Assay
method
Name Host
species
Human
specific
Dilution Manufacturer
Immunostain CD14 Mouse 1:50 Dako
CD19 Mouse 1:100 Dako
CD34 Mouse 1:50 Dako
CD45 Mouse 1:50 Dako
CD90 Mouse 1:10 Abcam
Desmin Mouse 1:200 Dako
Dystrophin
(P7)
Rabbit 1:1000 Gift from Dubowitz
Neuromuscular Centre
(Lu et al., 2005)
Galectin-1 Mouse 1:100 Novacastra
HLA-DR Mouse 1:50 Dako
Laminin Rabbit 1:50 Sigma
Lamins a/c Mouse + 1:100 Novacastra
MyoD Mouse 1:100 Dako
Myogenin Mouse 1:100 Dako
Myosin
(MF20) Mouse 1:50 DSHB
Pax7 Mouse 1:10 DSHB
SH2 Mouse 1:10 BD Biosciences
SH3 Mouse 1:10 BD Biosciences
Spectrin Mouse +/- 1:20 Novacastra
Vimentin Mouse + 1:50 Dako
Western blot Galectin-1 Rabbit 1:10000 Gift from A. Shiau
β-actin Rabbit 1:10000 Abcam
2.5.2.3. Immunohistochemistry
Muscle sections were air-dried and blocked with 10% goat serum in PBS (blocking
buffer) for 30 min, then with mouse on mouse block (M.O.M, Vector) for 1 h, and
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subsequently incubated with primary antibodies in blocking buffer for 1 h. Following
washes with PBS and 1 h incubation with secondary antibodies in PBS, slides were
washed in PBS and mounted onto cover slips in fluorescent mounting medium (Dako)
with DAPI (Sigma) for viewing under fluorescence microscopy. Where double
labelling was utilised, primary antibodies were from 2 different species (i.e. mouse
and rabbit) to allow co-detection using species-specific secondary antibodies
conjugated to different fluorochromes. For visualisation under epifluorescence
microscopy, fluorochrome-labelled secondary antibodies (Table 2-03) raised against
IgG from the species of the primary antibody were used (Molecular Probes).
Table 2-03. Secondary antibody used for immunostaining and Western blotting.
Assay
method
Name Host
species
Epitope Dilution Manufacturer
Immunostain FITC donkey
anti-mouse
Donkey Mouse 1:100 Jackson
ImmunoResearch
Laboratories
Alexafluor
488 goat anti-
mouse
Goat Mouse 1:200 Molecular Probes
Alexafluor
594 goat anti-
rabbit
Goat Rabbit 1:200 Molecular Probes
Western blot HRP goat
anti-rabbit
Goat Rabbit 1:10000 Dako
2.5.2.3.1. Analysis of muscle sections
Human cells were detected by human-specific lamins a/c antibody (Novacastra),
which stains the nucleus, is highly specific and gives a low false positive rate. As the
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mdx muscle lacks dystrophin, regenerated fibres derived from donor cells from
normal donors were demonstrated by positive dystrophin staining. However, as the P7
dystrophin antibody (Lu et al., 2005) detects both murine and human dystrophin,
revertant muscle fibres (Chapter 1.2.4.3.2) may lead to false positives. Therefore, a
human spectrin antibody was also utilised which detects this muscle protein on the
sarcolemmal membrane. These proteins have been used by many groups as markers
of human muscle differentiation in vivo within regenerating murine muscle (Brimah et
al., 2004; Chan et al., 2007; De Bari et al., 2003; Pye et al., 2004; Torrente et al.,
2004). Recently, the human spectrin antibody (Novacastra) was shown to cross-react
with newly-regenerating mouse muscle fibres, possibly cross-reacting with utrophin,
which is expressed in regenerating muscle fibres (J. Morgan, personal communication,
2006). Hence in this project, muscle fibres of human origin are confirmed by the
presence of all 3 proteins i.e. dystrophin- and spectrin-positive fibres with lamins a/c-
positive nuclei.
2.6. Muscle differentiation
2.6.1. Fibronectin coating
For myogenic differentiation experiments of hfMSCs, TPX chamber slides were pre-
treated with fibronectin (Sigma) at 4 μg/cm2 for 1 h and air-dried after excess solution
had been removed. Fibronectin is reported to retain cell adhesion to growth vessel
surfaces during differentiation (Garcia et al., 1997; Globus et al., 1998).
2.6.2. 5-azacytidine
HfMSCs were plated at different densities from 2-4 × 104/cm2 on glass chamber slides
(Nunc™) coated with fibronectin or on plastic dishes (Corning®) and after allowing
93
cell adherence overnight, exposed to different concentrations of a demethylating agent,
5-azacytidine (1-24 μM; Sigma), for 24-48 h in serum-free medium (SFM) defined as
DMEM (Sigma) with 2 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml
streptomycin (Invitrogen) supplemented with platelet-derived growth factor (PDGF)-
BB 10 ng/ml, epidermal growth factor (EGF) 10 ng/ml (Sigma) and ITS+ at final
concentrations of 6.25 μg/ml bovine insulin, 6.25 μg/ml transferrin, 6.25 μg/ml
selenious acid, 5.33 μg/ml linoleic acid, and 1.25 μg/ml bovine serum albumin
(Collaborative Biomedical Products). Cells were maintained in SFM for up to 14 days
following 5-azacytidine exposure.
2.6.3. Myoblast-conditioned medium
Conditioned medium was harvested from differentiated C2C12 cells by collecting the
supernatant after 72-h culture of 1 × 106 cells per T75 flask in D10 medium. Cell
debris was removed by passing supernatant through a 0.2-μm filter and the
conditioned medium was stored in aliquots of 5 or 10 ml at -20°C for future use.
Again, hfMSCs were plated at varying densities (2-4 × 104/cm2) and exposed to 33%
or 50% conditioned medium in SFM, which was replaced every 3 days. Cells were
maintained for up to 14 days to observe for any effects.
2.6.4. Galectin-1 conditioned medium
2.6.4.1. Synthesis of medium
Galectin-1 conditioned medium (GALM) was prepared using a published protocol
(Goldring et al., 2002b). Briefly, COS-1 cells (Gluzman, 1981) (ATCC, CRL-1650)
were seeded in 6-well plates at a density of 95,000 cells per well, grown overnight in
D10 medium and transfected with a rat Gal-1 expression plasmid (Figure 2-02) using
GeneJuice (Novagen). The expression plasmid was constructed by inserting rat Gal-1
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cDNA into HindIII and ApaI sites of pcDNA3 (Invitrogen), resulting in 5.9 kb
pcDNA3/rat galectin-1 under the control of the CMV (cytomegalovirus) promoter.
The supernatant was collected and filtered through a 0.2-μm syringe filter (Nalgene)
after 2 days, then stored at -20°C until use. Another set of COS-1 cells seeded in
parallel were transfected with pWPXL, a lentiviral vector encoding green fluorescent
protein (GFP) driven by the EF1 (cellular polypeptide chain elongation factor 1 alpha)
promoter (a gift from D. Trono, National Center for Competence in Research, École
Polytechnique Fédérale de Lausanne, Lausanne, Switzerland), for estimation of
transfection efficiency. Control COS-1 medium (COSM) was obtained by mock
transfection and collection of supernatant under identical conditions to GALM
production.
Figure 2-02. Map of pcDNA3/rat galectin-1 plasmid.
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2.6.4.2. Verification of GALM by Western blot analysis
After GALM was collected, COS-1 cells were washed in PBS, harvested by scraping
and then centrifuged (10,000 ×g for 30 sec). The cell pellet was resuspended in 100 μl
RIPA/SDS lysis buffer with 0.001% aprotinin (Sigma) and incubated on ice for 15
min. Following centrifugation at 10,000 ×g for 5 min in 4°C, supernatant was
collected for Western blot analysis. Protein concentrations were determined by
bicinchoninic acid (BCA) protein assay (Pierce). Samples were denatured by boiling
for 2 min, electrophoresed on a 10% SDS-PAGE (sodium dodecyl sulphate
polyacrylamide gel electrophoresis) gel (0.75 mm thickness) and transferred to
Hybond P membranes (Amersham). Membranes were blocked for 1 h with 5% non-
fat milk and 0.05% Tween 20 in PBS (PBS-T), then cut into upper and lower halves
to allow simultaneous probing with rabbit polyclonal anti-Gal-1 (gift from A. Shiau,
National Cheng Kung University, Tainan, Taiwan) or rabbit polyclonal β-actin
antibody (Abcam) (Table 2-01) overnight at 4°C. Membranes were washed in PBS-T,
incubated for 1 h with peroxidase-conjugated goat anti-rabbit IgG (Dako) (Table 2-
03), washed in PBS-T again, developed with chemiluminescence ECL Plus system
(Amersham) and then exposed to autoradiography films for 2 min at room
temperature. For analysis, blots were scanned, and densitometric signals determined
using Kodak 1D image analysis software. Data were expressed as arbitrary units after
normalisation to levels of β-actin.
Following thawing of frozen aliquots, GALM was concentrated by centrifugation
using Amicon Ultra-4 centrifugal filter units and/or immunopurified by magnetic cell
sorting (MACS) according to the manufacturer’s instructions (Miltenyi, 1999. MACS)
prior to analysis by Western blotting.
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2.6.4.3. GALM induced myogenesis
HfMSCs were plated at 2 × 104 cells/cm2 on fibronectin-coated (4 μg/cm2) chamber
slides with 1% GALM in SFM for 12 days. Control experiments were performed
using COSM, SFM and D10 (expansion medium for hfMSCs). Myogenic
differentiation was assessed by immunostaining for desmin and MF20 and counts
were made of the number of positive and negative nuclei in 6-10 random low-
powered fields. Level of myogenesis was expressed as a percentage of desmin- or
MF20-positive nuclei in total number of nuclei in all fields. Recombinant Gal-1 was
obtained from PeproTech and lactose was from Sigma.
2.6.5. Notch ICD induction
2.6.5.1. Verification of Notch ICD plasmid
Notch intracellular domain (NICD) plasmid (gift from M. Dezawa, Kyoto University,
Japan) was verified by restriction enzyme digestion (Dezawa et al., 2005) (Chapter
4.2.2). Before digestion with restriction enzymes, 300 ng of DNA was mixed with 6.6
μl diethylpyrocarbonate (DEPC)-treated water (Invitrogen), 1 μl 10× restriction
enzyme buffer and 0.1 μl 10 μg/μl BSA. DNA was digested with 2 U of EcoRI at
37°C for 2 h. In the double digests, 2 U of XbaI (Promega) was also added to the
reaction. Reaction was heat inactivated at 65°C for 15 min. The digested DNA was
loaded in a 1% agarose gel in Tris-borate-EDTA (TBE) electrophoresis buffer for
electrophoresis. DNA markers including 100 bp and 1 kb ladders (Promega) were
used as molecular weight standards.
2.6.5.2. Myogenic differentiation
I followed the published protocol of NICD induction of adult MSCs (Dezawa et al.,
97
2005) (Figure 2-03). Briefly, hfMSCs were plated on 6-well plates at 1,700-1,900
cells/cm2 and treated with bFGF (10 ng/ml, PeproTech), FSK (5 μM, Calbiochem),
neuregulin (200 ng/ml; R&D Systems) and PDGF (5 ng/ml, R&D Systems) in α-
MEM (Sigma) with 10% FBS, 2 mM L-glutamine and 100 mg/ml kanamycin (C-
MSCs). Cells were transfected with NICD using lipofectamine 2000 and selected by
G418 for 11 days according to the manufacturer’s protocols (Invitrogen) (CN-MSCs).
Cells recovered to 100% confluency over 18-19 days. Cells were then supplied with
filtered supernatant of hfMSC culture medium and observed for myogenesis over 14
days, with change of supernatant every 3-4 days. The supernatant was obtained by
plating hfMSCs at 20,000 cells/cm2, culturing with 10% human serum (Sigma) in α-
MEM and collecting the conditioned medium at 5 days. Conditioned medium was
filtered through a 0.2-μm syringe filter (Nalgene), then stored at -20°C until use.
Figure 2-03. Summary of NICD induction protocol.
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2.7. Generation of lentiviral vectors
All recombinant lentiviruses were produced by transient transfection of 293T cells
according to standard protocols (Zufferey et al., 1997). The plasmids used for
generation of lentiviral vectors were obtained from D. Trono (maps in Appendix 2).
Briefly, subconfluent 293T cells were co-transfected with 20 μg human Gal-1 plasmid
or pWPT-GFP, 15 μg of psPAX2 (packaging plasmid) and 5 μg of pMD2.G
(envelope plasmid) by calcium phosphate precipitation. Recombinant lentiviral
vectors were harvested at 48 and 72 h post-transfection, filtered through a 0.2-μm
syringe filter (Nalgene), and then stored at -80°C until use.
2.7.1. Lentiviral titres
293T cells (1.5 × 105 /well) were plated on a 6-well plate overnight in D10 medium.
Serial dilutions (2×, 10×, 100× and 1000×) of lentiviral vectors with 4 μg/ml
polybrene were used to transduce cells. After 12 h, the medium was replaced with
fresh D10 medium. Cells were harvested at 48 h to detect GFP expression by flow
cytometry. The viral titre is calculated as transduction units (TU)/ml = number of cells
upon infection × % GFP expression at FACS × dilution factor. Since the titre was 7.7
× 105 TU/ml, the amount of viral supernatant needed for transduction was calculated
as follows:
Number of cells for transduction × desired multiplicity of infection (MOI) = TU
needed
TU needed / viral titre = total ml of viral supernatant needed
2.7.2. Lentiviral transduction
HfMSCs were transduced at 70% confluence with 1:2 dilution (MOI = 3) of viral
supernatant to growth medium and 4 μg/ml polybrene (Sigma) over 16-20 h.
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Transduction efficiency of lentiviral vectors with Gal-1 transgene was estimated by
concurrent hfMSCs infection with lentiviral vectors expressing GFP.
2.8. Amplification of plasmids
2.8.1. Transformation of competent cells
Competent E.coli DH5α cells (100 μl) were thawed on ice and mixed with 10 μl of
plasmid DNA and incubated on ice for 30 min. Cells were heat shocked at 42°C for
30 sec followed by placing on ice for 2 min. One ml of Lysogeny broth (LB) was
added to cells and placed in 37°C shaker for 1 h. The culture broth (200 μl) was
spread on Luria agar plate containing appropriate antibiotics, which incubated
overnight at 37°C.
2.8.2. Plasmid maxiprep
A single colony of E. coli DH5α transformed with plasmid DNA was inoculated into
3 ml LB containing ampicillin (50 μg/ml), and shaken at 37°C for 8 h. The bacterial
culture (500 μl) was added to 500 ml LB with ampicillin (50 μg/ml) and cultured in a
37°C shaker for 14-16 h. Bacterial cells were harvested by centrifugation at 6,000 ×g
for 15 min at 4°C for plasmid DNA purification using Endofree Maxiprep (Qiagen)
according to manufacturer’s instructions.
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2.9. Polymerase chain reaction (PCR)
2.9.1. RNA extraction and reverse transcription PCR (RT-PCR)
2.9.1.1. Cells
Total RNA was extracted from hfMSC using a commercially available kit, RNeasy
(Qiagen), according to manufacturer’s instructions, and quantified
spectrophotometrically. Complementary DNA was synthesised as follows: 1 μg of
RNA was mixed with 1.2 μl of random primers (Promega) and DEPC-treated water
(Invitrogen) to a total volume of 9.3 μl, incubated at 75°C for 10 min, combined with
4 μl MgCl2, 2 μl 10× RT buffer and 2 μl 10 mM dNTP mix (Promega) for 10 min at
room temperature, and mixed with 0.5 μl RNAsin inhibitor and 1 μl avian
myeloblastosis virus reverse transcriptase (AMV RT) for 2 h at 42°C followed by
incubation at 75°C for 15 min.
PCR was carried out in 50 μl volumes where 1 μl of 137 ng/μl cDNA was added
to a PCR mixture of 37 μl DEPC-treated water, 5 μl reaction buffer, 15 mM MgCl2
(Bioline), 0.2 mM dNTP (Promega), 0.1 μM forward primer, 0.1 μM reverse primer
(Thermo Electron) and 2.5 U BioTaq DNA polymerase (Bioline). Negative controls
were either RT without AMV-RT enzyme or PCR with DEPC-treated water instead of
cDNA. The sequence of the primer and size of the PCR product are shown in Table 2-
04. After incubation at 94°C for 3 min in a Peltier thermal cycler (MJ Research Inc.),
amplification was carried out with 30 cycles of 1 min at 94°C, 1 min at 55°C, 40 sec
at 72°C and 5 min at 72°C. PCR products were electrophoresed in 1% agarose gels in
TBE electrophoresis buffer, stained with ethidium bromide and photographed under
ultraviolet (UV) light. DNA marker (1 kb ladder, Promega) was used to define
product size. PC3 human prostate adenocarcinoma cells (gift from C. Bevan, Imperial
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College London) that express high levels of Gal-1 were used as the positive control
(Van den Brule et al., 2001).
Primers for human Gal-1 were designed using Vector NTI Version 10 Program
(Informax Inc., Bethesda, MD, USA). The forward and reverse sequences are located
from 38 bp to 57 bp and from 360 bp to 341 bp of human Gal-1 cDNA (GeneBank
accession number NM 002305), respectively.
Table 2-04. Primers used for RT-PCR.
2.9.1.2. Tissues
To extract total RNA and genomic DNA from organs of mdx nude mice that
underwent intrauterine or neonatal transplantation, ~30 mg of partially thawed tissue
samples from spleen, bowel, heart, lung, kidney, stomach, and liver were
homogenised in lysis buffer provided by AllPrep RNA/DNA Mini kit (Qiagen) with a
TissueRuptor (Qiagen) for 30-60 sec at maximum speed. After homogenisation, RNA
and DNA were both extracted using AllPrep RNA/DNA Mini kit according to
manufacturer’s instructions and quantified spectrophotometrically. Thereafter, the
cDNA was synthesised. All primers were optimised using control DNA to achieve
maximum amplification of the sequence being probed.
Primer Forward 5’→ 3’ Reverse 5’→3’ Size
Gal-1 5’-AACCTGGAGAGTGCCTTC GA-3’ 5’-GTAGTTGATGGCCTCCAG GT-3’ 322 bp
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2.9.2. DNA extraction and PCR analysis
To detect whether human cells were present in mouse tissues after hfMSC
transplantation, genomic DNA from mouse tissues from different organs was isolated
using AllPrep RNA/DNA Mini kit. Primers specific for human HLA-DQα1 were used
to amplify human cells transplanted into mice, whereas mouse GAPDH primers
which amplify mouse but not human DNA sequences were used as loading controls
(Table 2-05). Genomic DNA from human lung cancer cells (CL1-5) (gift from A.
Shiau, Taiwan) and from tissues of non-injured mice without transplantation was used
as positive and negative controls, respectively. PCR was performed in a volume of 50
μl containing 0.3-0.5 μg DNA sample, 5 μl 10× PCR buffer, 0.2 mM of each dNTP,
0.4 μM forward primer, 0.4 μM reverse primer, and 1 U of Taq polymerase. After an
initial denaturation of 3 min at 94oC, 30 cycles of 1 min at 94oC, 30 sec at 50oC (for
human HLA-DQα1) or 57oC (for mouse GAPDH) and 30 sec at 72oC were carried out
followed by a final extension of 10 min at 72oC in a thermocycler. PCR products (10
μl from 50 μl) were separated by electrophoresis through 1.5% agarose gels in TBE
electrophoresis buffer, stained with ethidium bromide and photographed under UV
light. DNA marker (100 bp ladder, MBI) was used to define product size.
Primer sequences for human HLA-DQα1 were adapted from those described
previously with some modifications (Hillarby et al., 1993). The sequence of the
forward primer for human HLA-DQα1 includes 8 more nucleotides at the 5’ end,
whereas the sequence of its reverse primer contains 7 more nucleotides at the 5’ end
and 4 less nucleotides at the 3’ end, as compared to those described previously. The
forward and reverse sequences are located from 95 bp to 120 bp and from 334 bp to
313 bp of human HLA-DQα1 cDNA (GeneBank accession number XM 001129369),
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respectively. The primer sequences of mouse GAPDH used in this study have been
previously described (Ying et al., 2000).
Table 2-05. Primers used for PCR.
2.10. Co-cultures
2.10.1. Human myoblasts and murine myoblasts
Murine H2K 2B4 myoblasts (P20) were plated on Matrigel-coated 8-well chamber
slides at 2-5 × 104 cells/well and cultured in DMEM supplemented with 100 U/ml
penicillin, 100 μg/ml streptomycin, 4 mM L-glutamine and 5% HS (differentiation
medium). After incubation at 37°C with 5% CO2 for 4 h to allow adherence, human
myoblasts (fetal P9, adult P7) at various ratios (Table 5-01) were added to the wells
and mixed by gentle swirling for 30 sec. The triplicate co-culture wells were
incubated at 37°C and observed daily for formation of myotubes then fixed in PFA for
immunocytochemical examination at day 2. Control wells of murine myoblasts or
human myoblasts alone (seeded at 2-5 × 104 cells/well) in differentiation medium
were included.
Primer Forward 5’→ 3’ Reverse 5’→3’ Size
Human HLA-DQα1
5’-CCTCCTACGGTGTAAACTTGTACCAG-3’ 5’-CCTCATTGGTAGCAGCGGTAGA-3’ 230
Mouse GAPDH
5’ ACAGCCGCATCTTCTTGTGCAGTG-3’ 5’ GGCCTTGACTGTGCCGTTGAATTT-3’ 226
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2.10.2. HfMSCs and rat L6
Rat L6 myoblasts (P3) were seeded at a density of 0.5 × 104/cm2 on 6-well plates and
cultured in D10 medium. After 24 h, hfMSCs (P5) at different ratios (Table 5-02) in
triplicates were added to L6 myoblast cultures. Medium were changed to DMEM
supplemented with 2% HS the following day and the wells were observed daily for
myotube formation. At day 7 of co-culture, 0.5 ml of fresh medium was added to each
well. Control wells containing L6 cells or hfMSCs alone were also cultured in parallel.
In contrast to the previous experiment, rat L6 myoblasts were left for 24 h to
establish adherence instead of 4 h. This is because L6 cells could not grow in
differentiation medium and required D10 medium initially for cell proliferation.
2.10.3. HfMSCs and E15 myoblasts
Freshly isolated E15 myoblasts were plated on Matrigel-coated 8-well chamber slides
in differentiation medium and hfMSC were added to the culture 4 h or 24 h later. The
seeding ratios of the 2 cell types are shown in Table 5-03. Cells were fixed after 48 h
of co-culture as myotubes began to detach after this time point.
2.11. Animal work
2.11.1. Mdx
The dystrophin deficient mouse C57BL/10ScSn-Dmdmdx/J (Bulfield et al., 1984) was
housed under level II facilities in individually-vented cages. Time-mated mice at E14-
16 were used for intrauterine transplantations and neonatal transplantations were
administered in 3-day-old mice.
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2.11.2. Mdx/nude
An immunodeficient mdx mouse generated by crossing the mdx onto a nude
background (Flanagan, 1966; Pantelouris, 1968; Wortis et al., 1971) results in an
athymic dystrophic mouse model deficient in cell-mediated immunity (Partridge et al.,
1989) was housed under level II facilities in sterile individually-ventilated cages
(Chapter 1.2.3.1.1). Female mdx mice were crossed with male nu/nu mice to
eventually obtain a colony of mdx nu/nu and mdx nu/+ mice. To yield mice for
experiments, mdx nu/nu male were crossed with non-nude mdx nu/+ females to obtain
progeny which were half mdx nu/+ (hairy and immunocompetent) and half mdx nu/nu
(nude and immunodeficient). Mdx nu/+ females and nu/nu males were retained as the
basis of the breeding colony. Intramuscular transplantation in injured mice utilised
mice at 3-5 weeks of age. Neonatal transplantations were performed in 3-day-old
mice while time-mated mice at E14-16 were used for intrauterine transplantations.
2.11.3. Timed mating
Females were group-housed while males were individually housed at least 1 week
premating. Timed mating began from 6 weeks of age when males and females were
paired for a 3-day period. Signs of pregnancy were detectable by 12 days of gestation,
and the pregnant females were transferred into a clean cage prior to IUT. Sterile
bedding, shelter, nesting material and water were provided in all cages. Automatically
controlled photoperiods were 12 h light / 12 h dark (lights on from 0700 to 1900 h)
and temperature was maintained at 19-23°C.
2.11.4. Muscle injury protocols
Three types of muscle injury protocols were used to induce muscle regeneration in
mdx nude mice to examine the ability of transplanted cells to participate in muscle
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regeneration.
2.11.4.1. Notexin injection
Induction of anaesthesia was achieved with 4% isoflurane in 100% oxygen in an
induction chamber and then maintained at 2% isoflurane using a nose-cone. Notechis
scutatus scutatus notexin (10 μl of a 10 μg/ml solution, Latoxan), the Australian tiger
snake venom, was injected into the right TA of 3-week-old mice with a 26G
10-μl Hamilton syringe through a small superficial skin incision. Subcutaneous
buprenorphine (0.1 mg/kg body weight) was given as post-operative analgesia
followed by recovery in a heated box at 30°C. Cell transplantation was performed 24
h after notexin injection. Notexin causes muscle necrosis and regeneration by
specifically destroying muscle fibres and sparing other cells (Harris and Johnson,
1978).
2.11.4.2. Irradiation
Anaesthesia for restraint was achieved in mice at 3.5 weeks of age with 50 μl of
Hypnorm (0.79 mg/ml fentanyl citrate and 2.5 mg/ml fluanisone, Janssen) and
Hypnovel (1.25 mg/ml midazolam, Roche) administered subcutaneously. The right
lower limbs were irradiated in an IBL 637 cell irradiator (CIS Biointernational) with
18 Gy, delivered in 25 min with shielding of mouse bodies by 4 cm thick lead blocks
(Gross et al., 1999), leaving the left limbs as controls. Afterwards, the mice were
placed on a heated pad overnight. HfMSCs were transplanted 3 days after irradiation.
Irradiation of the muscle inhibits host muscle satellite cell regeneration and thus
allows donor-derived MPC and satellite cell to proliferate and regenerate muscle
fibres (Beauchamp et al., 1999; Collins et al., 2005; Morgan et al., 2002; Wakeford et
al., 1991).
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2.11.4.3. Cryodamage
Anaesthesia was achieved in 5-week-old mice with subcutaneous Hypnorm and
Hypnovel or inhalational anaesthesia as described above. The skin of the right legs
was opened to expose TA and a copper probe, chilled by immersion in liquid nitrogen,
was applied to proximal end of muscle for 10 sec and then the distal end for 10 sec
(Irintchev et al., 1997a). This procedure was repeated twice and the muscle allowed to
thaw between each application of the cryoprobe and before cell transplantation.
Following hfMSC transplantation, skin was closed with interrupted sutures of 6-0 silk.
Buprenorphine was given as post-operative analgesia and mice placed in heated box
at 30°C until fully recovered from anaesthetic. Cryodamage causes injury by
destroying mature muscle fibres as well as satellite cells (Irintchev et al., 1997a; Pye
and Watt, 2001).
2.11.5. HfMSC transplantation
Four different hfMSC samples were used for transplantation experiments and were all
relatively early passage cells (<P9).
2.11.5.1. Intramuscular injections
Following anaesthesia with isoflurane, a small incision was made in the skin
overlying the TA to transplant 5 × 105 hfMSC in 5 μl PBS with a 22G needle attached
to a 10-μl Hamilton microlitre syringe. Mice were recovered in a heated box as
described above. Animals were killed by cervical dislocation (schedule 1) and the
muscles harvested at 1 or 2 weeks after transplantation. This period will allow enough
time for donor cell-derived muscle regeneration (Brimah et al., 2004; Collins et al.,
2007; Morgan et al., 1990) and determining short-term graft survival, prior to
assessing the long-term fates of donor cells.
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2.11.5.2. Neonatal transplantation
Intraperitoneal injection of 1 × 106 hfMSCs in 40 μl PBS was performed without
anaesthesia using a 33G needle attached to a 100-μl Hamilton microlitre syringe. All
neonatal mice transplanted were at least 2 days old to allow time to establish bonding
with the mothers. Over the first 2 days after birth, the litters and their mothers and her
litters were not handled in order to prevent cannibalisation of the young.
2.11.5.3. Intrauterine transplantation
Under isoflurane anaesthesia, the gravid uterus was exposed through a full-depth
midline laparotomy. Through the translucent uterine wall, fetal abdomen was
visualised and 1 × 106 hfMSCs in 10 μl PBS were injected through the uterus to fetal
peritoneal cavity with a 33G, 100-μl Hamilton microlitre syringe. The laparotomy was
closed in two stages with 5-0 vicryl rapide (Ethicon), using continuous sutures for the
maternal peritoneum and interrupted stitches for skin. The procedure was performed
on a heat pad to prevent hypothermia and the uterus was kept hydrated with isotonic
saline. Postoperative buprenorphine was given subcutaneously and recovery from
anaesthetic took place in a temperature controlled environment over 1 h.
2.12. Tissue analysis
2.12.1. Optical bioluminescence imaging (BLI)
Animals were injected intraperitoneally with coelenterazine substrate (100 μl of 100
mg/ml in PBS, Promega) or D-luciferin (100 μl of 15 mg/ml, Xenogen). After 10-15
min, mice were anaesthetised with isoflurane and imaged with the Xenogen IVIS 100
system (Xenogen). The photon image was superimposed on a video image of mice
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with the Xenogen LIVINGIMAGE v2.50 (Xenogen) and IGOR Carbon v4.09A
(Wavemetrics) image analysis software.
2.12.2. Cryofixation and cryostat processing
Tissues from transplanted mice were harvested, attached onto cork discs with gum
tragacanth and frozen in liquid nitrogen-cooled isopentane (-80°C). Serial sections at
7 μm thickness were cut with a cryostat, collected onto polylysine-coated slides and
stored at -80°C until use for immunohistochemical analysis or haematoxylin and eosin
(H&E) staining. Organs from transplanted mice were transferred into
cryopreservation vials which were directly placed in liquid nitrogen for snap freezing.
DNA was extracted from these organs for PCR analysis.
2.12.3. Haematoxylin and eosin staining
For H&E staining, muscle cryosections were thawed, immersed in 10% formalin for 2
min, washed twice with PBS, fixed in ice-cold acetone for 1 min and again washed 2×
with PBS. They were then immersed in Erlich’s haematoxylin for 5 min, rinsed in
running water, dipped in acid ethanol for 1 min and washed in running tap water.
After 5 min in eosin, the slides were rinsed in tap water, dehydrated in an ethanol
series (70% for 20 sec, 100% for 30 sec and 100% for 30 sec), air-dried, and mounted
with coverslips in DPX mountant.
2.13. Statistical analysis
Parametric data were expressed as mean ± standard error of the mean (SEM).
Statistical significance between groups was assessed by unpaired Student’s t-test and
one-way ANOVA for parametric data. Where normal distribution could not be
110
assumed, non-parametric statistics were used and categoric data compared by Mann-
Whitney test and Kruskal-Wallis non-parametric ANOVA. Any p value <0.05 was
considered significant.
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CHAPTER 3
CELL CHARACTERISATION
112
3.1. Introduction
Stem cells are undifferentiated cells capable of proliferation, self-renewal and
differentiation. They can be classified according to tissue and stage of development of
origin: ESCs, embryonic germ cells, fetal stem cells and adult stem cells (Bongso and
Richards, 2004). ESCs have the advantage of pluripotency, yet ethical and safety
concerns directed at the destruction of the blastocyst during harvesting and embryonal
teratoma formation post-transplantation have led to much interest in multipotent adult
stem cells which are more accessible than ESCs. Though they present more limited
proliferative capacity and restricted plasticity, adult MSCs can differentiate into
mesoderm-derived tissues such as bone, fat and cartilage. HfMSCs are more primitive
and have greater differentiation potential than adult MSCs, but in contrast to ESCs, do
not form teratomas in vivo (Aguilar et al., 2007). In comparison with adult MSC,
hfMSCs self-renew faster in culture, senesce later, have greater differentiation
capacity and a higher level of telomerase activity, as well as express the purportedly
embryonic-specific pluripotency markers such as Oct-4, Nanog, SSEA-3, Rex-1, Tra-
1-81 and Tra-1-61 at protein level (Guillot et al., 2007).
Though more easily obtained, use of allogeneic hfMSCs raises ethical issues
associated with using tissues from termination of pregnancy, whereas autologous use
for ex vivo gene therapy is possible by utilising fetal blood or liver sampling which
allows sourcing from ongoing pregnancies and may be a more morally acceptable
option. Work in sheep suggested collection of second trimester FL carries a
substantial fetal loss rate (Surbek et al., 2002). On the other hand, relatively safe
techniques to access the fetal circulation have been developed over the last two
decades for fetal diagnosis and therapy after sixteen weeks, with a fetal loss rate of
approximately 1%. For collection of hfMSCs from early FB, prior to sixteen weeks
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gestation, procedures are limited by the small size of fetal vessels, the umbilical vein
for instance measuring only 2 mm in diameter at twelve weeks gestation. While
technically challenging, ultrasound-guided FB sampling has been applied at twelve
weeks gestation with <5% loss rate in ongoing pregnancies at risk of
haemoglobinopathies (Orlandi et al., 1990). Thin gauge embryo-fetoscopes also allow
early FB sampling from the umbilical vessels under direct visualisation (Surbek et al.,
2000). Ultrasound guided FB sampling maybe more appealing compared to the
fetoscopic approach, as it is simpler, less equipment-intensive and experience of this
technique for use at a later gestational age already exists in many specialist
fetomaternal centres. In our unit, the success rates of FB sampling were comparable in
fetoscopic (4/6) and ultrasound-guided (8/12) procedures in a recent technical study,
however procedural time was shorter with ultrasound-guidance (Chan et al., 2008). To
utilise hfMSC as a target cell type for ex vivo gene therapy, a sufficient volume of FB
is needed to isolate and expand hfMSC. However, the risk of inducing hypovolaemic
circulatory embarrassment in the fetus has also to be considered. Previous experience
in my laboratory suggests that at least 50 μl are needed for reliable hfMSC isolation,
but fetal bradycardia can occur post-FB sampling (1/3 using fetoscopy and 1/4 under
ultrasound-guidance) when >50 μl of FB are sampled in the first trimester (Chan et al.,
2008).
Fetal MSCs have also been isolated from placenta and AF obtained by CVS and
amniocentesis for prenatal diagnosis purposes. These present more accessible sources
of autologous MSCs using clinically validated methods (De Coppi et al., 2007;
Portmann-Lanz et al., 2006; Zhang et al., 2004b; Zhao et al., 2005).
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The aim of this part of my study was to characterise the hfMSCs used in later
transplantation experiments. This is necessary to ensure all work is done with a
consistent and well-characterised cell population throughout this thesis.
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3.2. Experimental design and results
3.2.1. Cell isolation
After personally obtaining patient consent, I collected 20 samples of first trimester
hfMSCs from FB (n=6), FL (n=4) and BM (n=10), expanded them to at least 2 × 107
cells per sample, and stored them in liquid nitrogen at early (P2-4) passage.
Following isolation of the total nucleated cell population in FB, FL or BM by
centrifugation or filtration, cells were plated in 10% batch-specific FBS in DMEM on
a 10-cm Corning® plastic tissue culture plate and cultured at 37°C in a humidified
atmosphere with 5% CO2. MSCs were selected by plastic adherence as non-adherent
mononuclear cells were washed away 72 h after initial plating to leave behind
colonies of spindle-shaped cells. Upon subconfluence, colonies were trypsinised and
re-plated at low density giving rise to a homogenous population exhibiting a
fibroblastic morphology (Figure 3-01 A and B). This method of MSC isolation has
been optimised by using different brand of tissue culture plates (Nunc™ versus
Corning®), varying duration of initial plating between 48 to 72 h, and changing the
number of PBS washes. At senescence, cell sizes enlarged and exhibited a flattened
broad stromal-like appearance along with a reduced cell number due to cell cycle
arrest (Figure 3-01 C and D). Table 3-01 shows the samples that underwent
characterisation for further use in this study.
3.2.2. Growth kinetics
To ensure the samples I worked with had similar growth kinetics to that expected for
hfMSCs, growth rate, doubling time, and population doublings were assessed. The
growth rate was estimated by plating 3,500 cells/cm2 in 6-well plates and counting
cell numbers at 2-day intervals over a minimum period of 12 days. The rates were
116
Figure 3-01. Morphology of hfMSCs stained with crystal violet. Cells are spindle
shaped in culture (A and B) until senescence when the fibroblastic appearance is no
longer maintained (C and D). Original magnification 40× (A and C) or 100× (B and
D).
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Table 3-01. HfMSC samples. Fetal samples denoted by weeks (w) and days (d) of
gestation.
Fetal liver
Fetal blood
Fetal bone marrow
9w3d 9w4d 9w4d
10w4d 9w5d
10w6d 10w1d (a)
11w6d 10w1d (b)
10w3d
10w4d
11w2d
12w
12w5d
12w6d
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comparable in all BM samples tested during the first 192 h in culture, and depicted as
an initial lag phase of 48 h, followed by an accelerated phase while cells divided until
192 h to reach a plateau phase (Figure 3-02A). Furthermore, hfMSCs from FB and
BM from the same donor exhibited similar proliferation rates in vitro over the first
240 h (Figure 3-02B). These data confirmed that these hfMSCs are highly expandable
ex vivo and are similar to those previously described (Campagnoli et al., 2001; Guillot
et al., 2007).
The mean doubling time (discarding one outlier at late passage from the BM
curve) was 42.0 ± 4.0 h (Figure 3-03A), The outlier occurred at P18 of BM sample
and had a very long doubling time of 99.1 h, which is possibly due to cell senescence
at late passage. Cumulative population doublings progressively increased for hfMSCs
until senescence (Figure 3-03B). One BM sample achieved 37.3 doublings over 1,266
h, whereas samples FB 10w4d underwent 27.2 doublings over 1,150 h and FB 11w6d
had 19.3 doublings over 847 h.
The slope of the growth curve corresponded with that previously described
(Campagnoli et al., 2001), although the absolute numbers differed due to a different
initial plating density. However, the doubling time was longer than earlier reports,
being 42 h compared with 23 h (Guillot et al., 2007), though still significantly lower
than a doubling time of 80 h (Guillot et al., 2007) reported in our laboratory for adult
MSCs. Furthermore, my data are similar to a doubling time of 47 h in human FB-
MSCs obtained by a colleague (Moschidou, 2005) again in the same laboratory. In
examining different sources of hfMSCs, the doubling time is 34.5 ± 2.4 h for BM-
hfMSCs compared with 45.7 ± 2.4 h for FB-hfMSCs. This reflects the intrinsic
differences of hfMSCs from different donors. Unfortunately, the doubling time
experiment was done some time after assessing the growth rate, at which time viable
119
Figure 3-02. Growth rate of hfMSCs. (A) Cells from 4 different BM samples
exhibit similar growth rates during the first 192 h in culture (n=2). (B) Cells sourced
from BM and FB in the same donor show comparable growth rates during the first
240 h in culture (n=2).
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Figure 3-03. Doubling time and cumulative population doublings of hfMSCs. (A)
Separate samples show comparable doubling times prior to passage 10 (n=2). (B)
Cumulative population doublings progressively increased at similar rates for three
separate samples.
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hfMSC samples from different sources in the same donor were no longer available
following a nitrogen tank malfunction resulting in the loss of some cells I had
characterised for this project. The cumulative population doublings utilised the same
data set collected for doubling time and the trend for the BM sample is comparable to
published data (Guillot et al., 2007).
3.2.3. Immunophenotype
By immunofluorescence using monoclonal primary antibodies, cells were positive for
mesenchymal markers CD105 (SH2, endoglin) and CD73 (SH3), expressed
intracellular matrix proteins (vimentin, laminin), CD90 (Thy-1) and satellite cell
marker Pax7, were non-haematopoietic (CD14-/45-/34-) and did not express CD19 or
HLA-DR (representative samples shown in Figures 3-04, 3-05, 3-06 and 3-07).
Following staining with antibodies against surface molecules, FACS analysis further
confirmed hfMSCs expressed CD105 (SH2) and CD73 (SH4) and were negative for
CD14 and CD45 (Figure 3-08). These data corresponded with those previously
reported for hfMSCs (Campagnoli et al., 2001).
3.2.4. Differentiation
Multilineage differentiation potential was demonstrated in all samples characterised
with hfMSCs able to undergo osteogenic, adipogenic and chondrogenic differentiation.
Late passage (>P10) hfMSCs were used as negative controls except in chondrogenic
differentiation, where control hfMSCs were also grown in a 3-D environment in
parallel but without TGF-β supplementation.
3.2.4.1. Osteogenic differentiation
To induce osteogenic differentiation, hfMSCs were plated at high density in high
phosphate osteogenic medium for 2 weeks (Chapter 2.2.1). Optimisation experiments
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Figure 3-04. HfMSCs are positive for mesenchymal markers. Cells were analysed
by immunofluorescence with antibodies against SH2 (A) or SH3 (B) and by staining
for DNA with DAPI (C and D). The merged images represent the superposition of the
cells stained with DAPI and SH2 (E) or SH3 (F) to visualise co-localisation. Negative
controls shown in insets. Original magnification 400×.
123
Figure 3-05. HfMSCs express intracellular matrix proteins. Cells were analysed
by immunofluorescence with antibodies against vimentin (A) or laminin (B) and by
staining for DNA with DAPI (C and D). The merged images represent the
superposition of the cells stained with DAPI and vimentin (E) or laminin (F) to
visualise co-localisation. Negative controls shown in insets. Original magnification
400×.
124
Figure 3-06. HfMSCs are positive for CD90 and Pax7. Cells were analysed by
immunofluorescence with antibodies against CD90 (A) or Pax7 (B) and by staining
for DNA with DAPI (C and D). The merged image represents the superposition of the
cells stained with DAPI and CD90 (E) or Pax7 (F) to visualise co-localisation.
Negative controls shown in insets. Original magnification 400×.
125
Figure 3-07. HfMSCs are HLA-DR-negative, non-haematopoietic and do not
express B cell marker. Cells were analysed by immunofluorescence with antibodies
against HLA-DR (A), CD14 (C), CD34 (E), CD45 (G), or CD19 (I) and by staining
for DNA with DAPI (B, D, F, H and J). Note that cells were negative for all these
markers. Original magnification 400×. Positive controls were provided by M. Santos,
Imperial College London (data not shown).
126
Figure 3-08. Immunophenotype of hfMSCs by FACS analysis. Cells were positive
for mesenchymal markers SH2 (A) and SH4 (B), but negative for haematopoietic cell
markers CD14 (C) and CD45 (D). Isotype IgG1 was used as the negative control (E).
127
were carried out to ensure reproducibility of the medium as it was cytotoxic on a few
occasions due to variations within batches of ascorbic acid. Von Kossa staining
confirmed formation of extracellular calcium deposits, as the translucent silver nitrate
reagent turns black upon reduction in the presence of calcium ions and light (Figure 3-
09).
3.2.4.2. Adipogenic differentiation
HfMSCs differentiated into fat in the presence of insulin, dexamethasone and
indomethacin. Intracytoplasmic lipid vacuoles began to be visible under phase
contrast microscopy after 7 days in culture. With increased time in culture, the size
and number of vacuoles increased as well as coalesced with each other to form bigger
vacuoles, which were stained with oil red O. Different samples varied in their rate of
fat conversion, the slowest rate being 35 days (Figure 3-10).
3.2.4.3. Chondrogenic differentiation
Chondrogenic differentiation was induced by growing hfMSCs in a 3-D environment
containing TGF-β for at least one month (Chapter 2.2.3). Chondrocytes organised in
layers with lacunae surrounding cell aggregates, which are shown by Alcian blue
staining of proteoglycans (Figure 3-11).
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Figure 3-09. Osteogenic differentiation of hfMSCs shown by von Kossa staining.
Calcium deposits (shown in black) are evident in four samples of BM-derived
hfMSCs (A-D), one FL-derived sample (E) and one sample of FB-derived hfMSCs
(F). Original magnification 400×.
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Figure 3-10. Adipogenic differentiation of hfMSCs stained with oil red O. Lipid
vacuoles (shown in red) are evident in four samples of BM-derived hfMSCs (A-D),
one FL-derived sample (E) and one sample of FB-derived hfMSCs (F). Original
magnification 400×.
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Figure 3-11. Chondrogenic differentiation of hfMSCs stained with Alcian blue.
Cartilage differentiation is demonstrated by formation of lacunae (white arrow) in
three BM-derived cell samples shown in (A), (C) and (E) with corresponding controls
shown in (B), (D) and (F). Original magnification 200×.
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3.3. Discussion
Techniques of hfMSC isolation were optimised on fresh samples to provide a well
characterised collection of samples for later experimental evaluation. It is pertinent to
use cells at early passages to prevent the loss of differentiation and proliferation
capacity (P. Guillot, personal communication, 2006). These samples were verified in
terms of morphology, proliferation, immunophenotype and differentiation potential,
which confirmed that they were indeed hfMSCs. Specifically, hfMSCs from different
sources (FB and BM) but the same patient donor demonstrated consistent
characteristics.
There were some hurdles involved in isolation of hfMSC samples. The lower
numbers of samples from FB and FL sources reflected the fact that the patient is less
likely to consent to donating FB. In addition, the small size of the FL during first
trimester makes it difficult to collect the liver from fetal tissues. Fetal liver is
identified by its anatomical position under the diaphragm, which is difficult to
recognise in fetal tissues disrupted by the surgical procedure of termination. Second,
after isolation of cells, microbial and/or fungal contamination occurred in 14% of
cases, which was improved by careful cell culture techniques and repeated PBS
washings during initial isolation. Ideally, more and equal numbers of samples from
different sources should be characterised to ensure similar cell populations. However,
due to having characterised more BM-hfMSC samples than hfMSCs from other
sources and time constraints, I focused on using hfMSCs from BM for the remaining
parts of my project, with a view to allogeneic therapeutic uses.
One of the challenges in characterising MSCs is the lack of specific markers.
Therefore they are identified via the presence and/or absence of a number of antigens,
some of which are also expressed on other cells. Although there is a minimal set of
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criteria for identification of MSCs (Dominici et al., 2006), there is no consensus on
the myriad of newly identified MSC markers in the literature. Other hfMSC markers
such as CD68, CD29, CD44, CD106, CD31, von Willebrand factor, myoglobin,
collagen type I/II and prolyl-4-hydroxylase (Campagnoli et al., 2001) could be
evaluated using either FACS or immunocytochemical analyses in future studies.
However, I have used a standard accepted set of markers and demonstrated hfMSC
kinetics along with tri-lineage differentiation in my project.
3.3.1. Conclusion
During this study, hfMSCs were characterised according to published criteria for
MSCs by ISCT (Chapter 1.1.5.2.1.1) (Dominici et al., 2006). However in describing
new stem cell populations, a few studies have also used RT-PCR to ensure gene
expressions of osteogenic (osteopontin), adipogenic (PPARγ) and chondrogenic
(collagen) pathways or quantified the level of differentiation by calculating the level
of staining intensity on photographic images after von Kossa, oil O red or Alcian blue
staining. This has been done exhaustively in other studies on similar hfMSC
populations I collected for others in my laboratory. For the purpose of this project, it
is unlikely that these additional experiments would have added any more information,
as hfMSC is a well defined cell type which has been previously described and fully
characterised (Campagnoli et al., 2001). Overall, the cell characterisation performed
herein ensured that studies for this thesis were based on a consistent and robustly
characterised hfMSC population.
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CHAPTER 4
IN VITRO MYOGENENIC
DIFFERENTIATION
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4.1. Introduction
Some strategies have been described for in vitro differentiation of fetal MSCs down
the myogenic lineage, though most with inefficient results. Krupnick et al.
demonstrated that fetal sheep liver-derived MSCs fused to form multinucleated
myofibres and expressed myosin heavy chain protein, a marker of mature muscles,
upon exposure to either 5-azacytidine or dexamethasone (Krupnick et al., 2004).
Using term UCB-MSCs, Gang et al. showed the expression of muscle markers after
prolonged culture under low horse serum conditions (Gang et al., 2004).
A high rate (~89%) of myogenic conversion has been achieved in adult MSCs by
NICD gene transfer (Dezawa et al., 2005). The Notch gene encodes a 300 kDa single
transmembrane cell surface receptor protein which is activated by Delta/Serrate/Lag-1
ligands presented by neighbouring cells (Lundkvist and Lendahl, 2001). Upon ligand
binding, the intracellular portion of the Notch receptor is cleaved and enters the
nucleus, where it influences the expression of numerous transcription factors related
to maintenance of pool of lineage-dedicated adult muscle progenitor cells (Conboy
and Rando, 2002) and prevention of their premature differentiation (Nofziger et al.,
1999). NICD gene transfer followed by supplementation of certain trophic factors has
been used efficiently to induce in vitro and in vivo myogenesis in adult BM-MSCs
(Dezawa et al., 2005).
Previous work in my group’s laboratory has shown that hfMSCs are able to
undergo myogenesis efficiently in the presence of Gal-1, an abundant protein found in
myoblast-conditioned medium (Chapter 1.3.3.1). Naïve hfMSCs were thought not to
express desmin, an early marker of myogenesis. However, in the presence of Gal-1,
66% of hfMSCs started to express desmin over a 12-day period (Chan et al., 2006a).
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The aim of this part of my work was to optimise the protocol for in vitro
myogenesis of hfMSCs with a view to pre-differentiating hfMSCs prior to in vivo
transplantation in mdx/nude mice.
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4.2. Experimental design and results
4.2.1. 5-azacytidine and myoblast conditioned medium
Due to the encouraging pilot data showing high rates of myogenic conversion (Chan
et al., 2006a), my initial aim was to replicate previous work and follow this by further
refinement of the method, so as to enhance donor-derived muscle formation in vivo.
Four strategies were used: 5-azacytidine, C2C12 myoblast conditioned medium,
NICD induction and GALM, Gal-1 conditioned medium. In keeping with previous
work, 5-azacytidine was found to be cytotoxic and C2C12 conditioned medium led to
only 1-2% myogenic conversion (data not shown).
4.2.2. Notch intracellular domain induction
As gene transfer of NICD has been used successfully to induce adult MSCs towards
muscle differentiation (Dezawa et al., 2005), this approach was tested in hfMSCs. The
NICD expression plasmid, verified by restriction endonuclease digestion (Figure 4-
01), was transfected into hfMSCs as part of the muscle differentiation protocol. After
optimisation of the transfection and selection steps to obtain the highest rate of
hfMSC survival, the remainder of the previously described differentiation protocol
was followed (Chapter 2.6.5.2) and hfMSCs were tested in triplicates on three
separate occasions. However, hfMSCs did not form myotubes in culture, and did not
express markers of myogenesis on immunocytochemical examination.
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Figure 4-01. Verification of NICD plasmid. NICD plasmid (A) verified by enzyme
digestion (B). Lane 1: 100 bp ladder, 2: 1 kb ladder, 3: uncut control, 4-6: double
digest with EcoRI and XbaI in 3 clones, and 7-9: single digest with EcoRI in 3 clones.
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4.2.3. Verification of GALM
In view of the inefficient myogenesis achieved in the aforementioned experiments, I
concentrated my efforts on GALM, as it was shown previously to be a promising
inducer of myogenesis. Rat Gal-1 expression plasmid was obtained from the group
that originally reported GALM-induced myogenesis in skin fibroblasts (Goldring et
al., 2002a; Goldring et al., 2002b). The plasmid DNA, verified by auto-sequencing,
was prepared for use in subsequent experiments. Mouse Gal-1 has a high DNA
sequence homology to human (89%) and rat (94%) Gal-1 (Wilson et al., 1989).
GALM was produced using identical reagents and protocol to the original group.
Western blot analysis was used to quantify the amount of Gal-1 in GALM.
Higher levels of Gal-1 were present in lysate from GALM-producing COS-1 cells
compared with the lysate from mock-transfected COS-1 cells (Figure 4-02A).
However, Gal-1 protein could not be detected in neat GALM using Western blot nor
in concentrated GALM (30×), with or without immunopurification by MACS
(Miltenyi, 1999. MACS).
Indirect evidence for higher levels of Gal-1 in GALM compared to COSM,
COS-1 conditioned medium stems from the increased fusion index seen in the C2C12
cell line (Figure 4-02C), which corresponded with the original published findings
(Goldring et al., 2002b). In addition, to investigate whether the effect of GALM was
due to Gal-1, inhibition experiments were carried out using lactose, a classic inhibitor
via β-galactoside binding to the lectin (Vas et al., 2005). Lactose treatment
significantly reduced the fusion index in GALM treated C2C12 (Figure 4-02D). This
alternative method was used as a bioassay for quality control of different batches of
GALM.
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Figure 4-02. Verification of Gal-1 conditioned medium. (A) Western blot analysis
of COS-1 cell lysates from (1) rat Gal-1 transfected cells (producing GALM), (2)
human Gal-1 transfected cells and (3) mock-transfected cells (producing COSM). The
level of Gal-1 expression was normalised with that of β-actin expression. Ratios
between the intensity of the bands corresponding to Gal-1 and β-actin determined by
densitometric analysis are shown below the blot. (B) C2C12 myotubes stained with
desmin (green) and DAPI (blue). Original magnification 200×. (C) Effect of GALM
on C2C12 myotube formation quantified by fusion index (total number of nuclei in
myotubes divided by total number of nuclei in culture). (D) Comparison of the effects
of GALM in the presence or absence of lactose (100 mM) on C2C12 fusion index. *=
p<0.05, **= p<0.01 and ***= p<0.001 by unpaired t-test.
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4.2.4. Optimisation of GALM concentration for myogenesis
Next, the optimal dose for inducing myogenesis in hfMSC was determined by
comparing 1, 10 and 50% GALM in SFM and SFM alone for 12 days. Other control
groups were COSM with the same concentrations as above or 10% FBS (D10
expansion medium for hfMSC) alone. One percent GALM in SFM was found to be
the optimal condition compared to other dilutions of GALM, but there was no
significant difference to SFM. The concentration of Gal-1 in GALM had been
established as 18.5 µg/ml (Goldring et al., 2002b). As 1% of GALM was used in
subsequent experiments, this would have been equivalent to a Gal-1 concentration of
185 ng/ml of growth medium.
Myogenesis was quantified by staining with desmin (a marker of both MPCs and
myotubes) (Debus et al., 1983) and sarcomeric myosin, MF20 (a “late” myogenic
marker, present in myotubes, but not in MPCs) (Bader et al., 1982), proteins.
However, hfMSCs expressed desmin in the naïve non-differentiated state (24.5 ±
7.1%), the level of which did not change greatly over 12 days of culture in D10
expansion medium (23.6 ± 1.4%). Therefore, the increase in the level of desmin
expression following GALM treatment was comparatively very little (29.4 ± 1.8%
from 23.6 ± 1.4%), though significant (p<0.05) (Figure 4-03). Using appropriate
positive and negative controls (C2C12 murine myoblast cell line and SAOS human
osteosarcoma cell line), the finding of hfMSCs being desmin-positive in the naïve
state contrasts with previous data from our laboratory (Chan et al., 2006a), so I
arranged for naïve hfMSCs to be separately stained by two other members in our
group who both confirmed my result. Conversely, MF20 was not expressed in naïve
hfMSC (data not shown) and both GALM and SFM increased its expression to around
29% after treatment for 12 days.
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Figure 4-03. Desmin expression in hfMSCs following exposure to different
conditioned media. Merged images show desmin in red and DAPI in blue in hfMSCs
cultured in GALM (A), COSM (B), SFM (C), or DMEM with 10% FBS (D). Original
magnification 400×. (E) Conditioned media were used at various concentrations to
detect optimal level to induce desmin expression. *= p<0.05 and **= p<0.01 by
unpaired t-test.
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Although there was significantly higher expression of MF20 after GALM
treatment than after COSM or 10% FBS alone (p<0.001) (Figure 4-04), as GALM
was diluted in SFM, the SFM control group shows that COSM may in fact inhibit
myogenic marker expression (p<0.001) and that GALM only rescues it back to the
SFM level. While the difference between 1% GALM and COSM was significant
(p<0.001), it was only evident using the “late” myogenesis marker, MF20.
4.2.5. Effect of GALM on myogenesis
The ability of GALM to induce myogenesis was further tested on nine hfMSC
samples (Table 4-01). All were immunostained with desmin and some with MF20
markers. One single patient’s FB-derived hfMSCs showed no desmin expression
under all conditions and was excluded from further analysis. In all other samples,
GALM-treated hfMSCs had higher desmin expression than COSM-treated hfMSC.
However, this was only significant in 50% of samples. Expression of MF20 showed a
similar pattern with only one exception and the difference between GALM and
COSM was significant only in 29% of samples (p<0.05) (Figures 4-05 and 4-06).
Furthermore, SFM resulted in the highest desmin expression out of all medium
conditions in 50% of samples examined with the highest level being 17.6 ± 2.0%.
However, the same trend only occurred in 14% of samples stained for MF20
(p<0.001). Similar to the aforementioned optimisation experiment, GALM induced a
higher MF20 expression level than D10 expansion medium in 86% of samples.
Interestingly, the highest level of myogenesis achieved in all samples was from a
sample which showed the best result in GALM. For desmin expression, the level was
34.5 ± 3.7% compared with 25.2 ± 3.1% when cultured in 10% FBS. Based on the
previous dose titration experiment, I assumed that desmin expression did not change
after culture in D10 expansion medium for 12 days and therefore used this as another
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Figure 4-04. MF20 expression in hfMSCs following exposure to different
conditioned media. Merged images show MF20 in red and DAPI in blue in hfMSCs
cultured in GALM (A), COSM (B), SFM (C), or DMEM with 10% FBS (D). Original
magnification 400×. (E) Conditioned media were used at various concentrations to
detect optimal level to induce MF20 expression. ***= p<0.001 by unpaired t-test.
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Table 4-01. HfMSC samples examined. Samples named using weeks (w) and days
(d) in fetal gestation.
ID Sample Passage
A BM 10w1d (b) 5
B BM 10w3d 2
C BM 9w5d 4
D BM 12w5d 3
E BM 12w 6
F BM 10w4d 7
G BM 9w4d 4
H BM 12w6d 9
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Figure 4-05. Desmin expression in hfMSCs following treatment with 1% GALM
in SFM. Eight hfMSC samples from BM were tested (A-H). *= p<0.05, **= p<0.01
and ***= p<0.001 by unpaired t-test.
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Figure 4-06. MF20 expression in hfMSCs following treatment with 1% GALM in
SFM. Results are shown for eight hfMSC samples (A-H). *= p<0.05 and ***=
p<0.001 by unpaired t-test.
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control group. For MF20 expression, the highest level was 35.3 ± 4.7% compared to
4.2 ± 1.2% in 10% FBS (p<0.001). The discrepancy compared with the majority of
samples may be due to the particularly high basal level of desmin expression in this
particular sample. Sample H (BM 12w6d) is a sample that had been previously
characterised and shown to undergo GALM-induced myogenesis in our laboratory,
however its lack of MF20 expression under all culture conditions tested may be
explained by its late passage. With regards to the apparently ambiguous finding when
comparing desmin and MF20 expression in the same sample, it may be explained by
differences in marker expression within naïve hfMSCs, whereby they are MF20-
negative (data not shown) but in contrast to earlier findings (Chan et al., 2006a),
desmin-positive (~20-30%). Unfortunately, due to financial and time constraints, not
all samples tested were stained with MF20. Ideally, future myogenesis experiments
should be immunostained for not only desmin and MF20, but also MRF, such as
MyoD and myogenin, to elucidate further the mechanism, as MRF are crucial in
muscle development and regeneration (Sabourin and Rudnicki, 2000).
4.2.6. Different forms of galectin-1 for myogenic differentiation
Due to the small myogenic effect of GALM on hfMSCs, other forms of Gal-1 were
tested. Conditioned medium from human, rather than rat, Gal-1 plasmid transfected
COS-1 cells had similar effects on hfMSCs to GALM, where MF20 expression
following treatment was 13.43 ± 4.04% compared with 15.06 ± 4.18% in GALM-
treated hfMSCs, with the COSM control being 2 ± 2% (Figure 4-07A). Nevertheless,
GALM and human Gal-1 conditioned medium (H-GALM) both led to significantly
higher MF20 expression compared with COSM (p<0.05). Purified recombinant
human Gal-1 protein showed an inhibitory effect towards myogenesis in both C2C12
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Figure 4-07. Effect of H-GALM and recombinant Gal-1 on hfMSCs or C2C12
cells. (A) MF20 expression in hfMSCs (BM 10w1d (b), sample A) treated with H-
GALM was similar to those treated with GALM. (B) Desmin expression in hfMSCs
(BM 9w4d, sample G). Gal-1 was diluted in SFM at various concentrations. One-way
ANOVA was used to compare Gal-1 concentrations with 10% FBS. (C) Fusion index
in C2C12 cells. Gal-1 was diluted in 2% HS. *= p<0.05 and **= p<0.01 by unpaired
t-test.
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and hfMSC (Figure 4-07B and C), which is similar to previous findings in C2C12
cells (Cooper et al., 1991).
4.2.7. Effect of galectin-1 inhibitors on GALM-induced myogenesis
To investigate whether the effect of GALM, albeit small, was due to Gal-1, inhibition
experiments were carried out conventionally using lactose and also with IGF-1, a
possible inhibitor of Gal-1 but itself with myogenic properties (Andersen et al., 2003).
Lactose reduced both desmin and MF20 expression of hfMSCs stimulated with
GALM. However, IGF-1 did not affect GALM-treated cells, but instead increased
desmin expression in the control SFM group (Figure 4-08). This suggests that IGF-1
may inhibit Gal-1 via other non-lectin pathways and thus has no effect on the limited
myogenic ability of Gal-1. Furthermore, the mechanism may be more complex, as
IGF-1 itself is involved in muscle regeneration. This growth factor stimulates both
proliferation and differentiation of myoblasts. It plays roles in muscle hypertrophy via
increasing rate of protein synthesis and reducing rate of protein breakdown, as well as
contributing to increased recruitment and incorporation of circulating BM stem cells
at sites of muscle injury (De Arcangelis et al., 2003; Mourkioti and Rosenthal, 2005;
Musaro et al., 2004; Patel et al., 2005).
4.2.8. Galectin-1 expression in human fetal mesenchymal stem cells
By immunostaining 6 samples of naïve hfMSCs between passages 3 and 5 (BM:
9w5d, 10w1d (b), 12w, 12w5d; FB 11w6d; FL 9w3d), Gal-1 was found to be
constitutively-expressed in both the nucleus and cytoplasm. BM-derived hfMSCs had
higher basal expression of Gal-1 than FB- or FL-derived hfMSCs. Furthermore, Gal-1
was expressed in 100% of fetal myoblasts but the level reduced upon myotube
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Figure 4-08. Effects of Gal-1 inhibitors on Gal-1-induced hfMSC myogenesis.
HfMSC sample (BM 12w5d, sample D) was tested with lactose (100 or 10 mM) and
expressions of desmin (A) and MF20 (B) were measured. Another hfMSC sample
(BM 10w1d (b), sample A) was tested with IGF-1 (40 or 4 ng/ml) (C). Lactose or
IGF-1 were added to 1% GALM in SFM, 1% COSM in SFM, SFM or 10% FBS in
DMEM. *= p<0.05 by unpaired t-test.
151
formation (Figure 4-09). This suggests that Gal-1 may be present in the early stages of
myogenesis, but the level may be decreased upon mature muscle fibre formation.
4.2.9. Galectin-1 gene transduced human fetal mesenchymal stem cells
HfMSCs were lentivirally-transduced with human Gal-1 gene to increase the level of
Gal-1 production (Figure 4-10A). HfMSCs were simultaneously transduced with a
GFP expression plasmid to estimate transduction efficiency (>90%). RT-PCR showed
Gal-1 over-expression in transduced hfMSC (Figure 4-10B). In all three samples
cultured in 10% FBS, transduced hfMSC consistently showed higher expression of
myogenic markers by immunostaining compared with non-transduced controls (e.g.
for desmin, 45.5 ± 5.0% versus 19.5 ± 2.6%, p<0.01) (Figure 4-11). However, when
transduced hfMSCs were treated with GALM, no additional myogenesis was
observed (data not shown).
Galectin-1 transduced hfMSCs exhibited higher expression of myogenic markers
with the expected temporal relationship in muscle differentiation compared to non-
transduced hfMSCs. This further suggests that endogenous production rather than
exogenously added Gal-1 is advantageous in triggering myogenesis. However,
controlled over-expression may be required, as too much intracellular Gal-1 may not
be beneficial for myogenesis and excess protein may be excreted from the cell.
4.2.10. In vivo myogenesis of galectin-1 gene transduced human fetal
mesenchymal stem cells
Finally, cryodamaged TA muscles of mdx/nude mice (n=6) were injected with Gal-1
transduced hfMSCs (BM 12w), and their muscles were harvested after one week to
observe whether there was increased muscle regeneration in vivo compared to naïve
hfMSCs. There was evidence of more newly regenerated human muscle fibres
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Figure 4-09. Immunolabelling for Gal-1. Positive controls using human fetal
myoblasts (A) and differentiated human fetal myoblasts (D). Gal-1 is present in
hfMSCs (BM 12w5d, sample D) (G) and COS-1 cells (J). Negative control using
SAOS human osteosarcoma cell line (M). There is little background staining from
controls without primary antibody (P). Original magnification 400×. Gal-1 (red) was
localised in both nucleus and cytoplasm (A, D, G and J), and was not present in
negative control cells (M and P). DAPI staining (blue) is used to identify all nuclei (B,
E, H, K, N, and O). Merged images are shown in C, F, I, L, O, and R.
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Figure 4-10. Lentiviral transduction of hfMSCs with Gal-1 gene. (A) Lentiviral
vector expressing human Gal-1 under the transcriptional control of EF-1α promoter
(pWPXL/hGalectin-1) for transduction of hfMSCs. (B) Confirmation of Gal-1
expression in transduced hfMSCs by RT-PCR analysis showing 322 bp transcript of
Gal-1. Lane 1: 1 kb ladder, lane 2: positive control human prostate adenocarcinoma
PC3 cells expressing galactin-1, lane 3: hfMSCs transduced with pWPXL/hGalectin-1,
lane 4: untransduced hfMSCs served as negative control cells.
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Figure 4-11. Immunolabelling for myogenic markers in transduced hfMSCs.
Merged images show cytoplasmic staining of desmin (A) and MF20 (D) with original
magnification of 200× and nuclear staining of MyoD (B) and Myf4 (myogenin) (C)
with original magnification of 400×. Samples tested were BM 10w1d (b) (E), BM
10w3d (F), and BM 12w (G). Expression by immunolabelling of early and late
myogenesis markers in hfMSCs versus hfMSCs lentivirally transduced with human
Gal-1 (hfMSC-Gal) following 12-day culture in 10% FBS. *= p<0.05, **= p<0.01
and ***= p<0.001 by unpaired t-test.
155
(positive mouse and human dystrophin and human spectrin staining with positive
human lamins a/c nuclei) using Gal-1 transduced hfMSCs than non-transduced
hfMSCs. However, the numbers compared were very low (0.38 ± 0.11 versus 0.04 ±
0.04 fibres per muscle section, p=0.025) (Figure 4-12A and C). Interestingly, of the
cells that did not contribute to muscle differentiation, a smaller number of transduced
hfMSCs were present within the host muscle compared with non-transduced hfMSCs
(3.05 ± 0.86 versus 10.81 ± 3.08 cells per muscle section) (Figure 4-12B). Despite the
promising in vitro results with Gal-1 transduced hfMSCs, the level of in vivo muscle
regeneration after one week was only 0.02% of murine fibres on muscle sections
(assuming ~2,000 muscle fibres per section (Pagel and Partridge, 1999)), albeit 9.5
times greater than that in non-transduced controls.
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Figure 4-12. Transverse section of TA transplanted intramuscularly with
hfMSC-Gal. (A) Example of a human fibre, shown as a muscle fibre double stained
with human and mouse dystrophin (red fibre membrane, red arrow) and human
spectrin (green fibre membrane) with a human nucleus stained with lamins a/c (green
nucleus, white arrow) superimposed on DAPI staining (blue). Original magnification
400×. (B) Number of cells positive for lamins a/c and DAPI in transplanted muscle.
(C) Number of human fibres in TA from mdx/nude mice following intramuscular cell
transplant to cryodamaged muscle, quantified over 4 non-consecutive muscle
cryosections (~2,000 muscle fibres per section). The mean values are indicated by
horizontal bar, p=0.025 by Mann-Whitney test.
157
4.3. Discussion
4.3.1. Myoblast conditioned medium and notch intracellular domain
Consistent with previous work in our laboratory, hfMSCs differentiated along the
myogenic lineage when cultured with myoblast conditioned medium. This finding
suggests that the current cell bank of hfMSCs is similar to that previously employed.
After examining three hfMSC samples, NICD gene transfection of hfMSCs appeared
to be ineffective in stimulating myogenesis. Unfortunately, positive control
experiments using adult MSCs were not possible due to a shortage in commercial
adult MSC availability (Chapter 2.4.3). Therefore, it is difficult to ascertain whether
the lack of hfMSC muscle differentiation is due to inherent differences between
hfMSCs and adult BM-MSCs or a problematic protocol.
4.3.2. Galectin-1 conditioned medium
Batches of GALM were successfully produced and the presence of Gal-1 shown in
cell lysates, however, Gal-1 levels in GALM could not be directly measured using
Western blot analysis, a semi-quantitative method. Although COS-1 cells are known
to secrete Gal-1 endogenously and the molecular mechanism for Gal-1 secretion
remains elusive (Seelenmeyer et al., 2005), it is unlikely that the secretory system is
already at maximal capacity in the control cells and that Gal-1 transfection in fact, has
no additive effect. This is supported by my data showing a notable difference in levels
of myogenesis between GALM- and COSM-treated hfMSCs. Different batches of
GALM led to varying levels of myogenesis in hfMSCs, which corroborate with the
finding of cytotoxic batches (2 out of 4) in previous work (J. Chan, personal
communication, 2006). Therefore, after selecting for the batch that led to the most
myogenesis in an optimisation experiment, the same batch of GALM was used
158
throughout this work. A faster and more sensitive method to detect Gal-1 in GALM
and select for the best batch of GALM might have been employed such as an enzyme-
linked immunosorbent assay.
Verification of GALM was followed by determining the optimum dose for
induction of hfMSC myogenesis. The difference between 1% GALM and COSM was
only detectable using MF20, rather than desmin, as a marker of myogenesis. This may
suggest that myogenesis cannot be accurately determined until mature myofibre
formation, which may be a potential problem with regards to the overall aim of pre-
differentiating hfMSCs prior to IUT, as the fully differentiated hfMSCs may have lost
their ability to home and engraft to various muscle groups by then, thus negating any
potential benefits of IUT. To overcome this issue, one would have to induce
myogenesis, inject some cells in vivo first and keep an aliquot to determine in vitro
myogenicity later, which would run the risk of unnecessary experimentation given
that potential variation may exist between hfMSC samples. Although not all desmin-
positive cells are myogenic, one possible explanation for requiring the use of MF20 as
a marker may be that hfMSCs contain some desmin-positive muscle progenitor
fractions with varying degrees of myogenic potential, which are only distinguishable
at the point of myofibre maturation. Moreover, although GALM is more myogenic
than COSM, this may not be the case in comparison with SFM. Therefore, more
experiments were performed to further validate the findings of this initial experiment.
4.3.3. Recombinant human galectin-1
Although recombinant Gal-1 inhibited myogenesis, this is perhaps unsurprising.
Recombinant Gal-1 is known to decrease the fusion index in C2C12 (Cooper et al.,
1991) and inhibit proliferation in human BM-MSCs (Andersen et al., 2003). In
addition, Gal-1 exhibits β-galactoside-binding lectin activity only in a reduced form.
159
In oxidised form it promotes axonal growth in peripheral nerves and may act via other
non-lectin pathways (Inagaki et al., 2000). Since hfMSC myogenesis was inhibited by
lactose which blocks lectin activity (Figure 4-08A and B), it is likely that Gal-1 in the
non-oxidising form is involved. It is not known whether the recombinant Gal-1
protein used is preserved in oxidised or reducing form, which can influence the
outcome. Finally, there may be species-specific interactions, whereby using murine
recombinant Gal-1 protein may be more effective in the C2C12 cell line than on
human stem cells. A recent publication reported the use of recombinant Gal-1 to
induce myogenesis in foal UCB stem cells. Though some cells were desmin-positive,
they were unable to detect MyoD, myogenin, myosin heavy chain or troponin T (Reed
and Johnson, 2008).
There are some conflicting findings in the literature against the myogenic effect
of Gal-1. For instance, there was no desmin expression after GALM treatment in
mouse muscle fibroblasts (Goldring et al., 2002b). The concentration of Gal-1 appears
to be critical as it promotes apoptosis at high levels (μg range) and proliferation at low
levels (ng range) (Purkrabkova et al., 2003). Adding recombinant Gal-1 (25 μg/ml) to
C2C12 myoblasts inhibits myoblast adhesion and fusion (Vas et al., 2005) and
inhibits proliferation in 70% of human BM-MSCs (1,000 ng/ml) (Andersen et al.,
2003). It has been shown that differentiating myoblasts release Gal-1 and proliferating
ones do not (Camby et al., 2006). As C2C12 differentiate, there is an initial patchy
intracellular distribution of Gal-1, and then it is secreted by ectocytosis on myotube
formation (Cooper and Barondes, 1990; Harrison and Wilson, 1992). During
development in chick muscle, the highest level of Gal-1 occurs between days 8-16 of
development, the time of maximum myoblast fusion (Nowak et al., 1976). However,
in the 6-14 week human fetus, Gal-1 is present in skeletal muscle, fibroblasts, cardiac
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myocytes and smooth muscle, but at lower levels compared with adult (Van den Brule
et al., 1997), which is in keeping with the increased Gal-1 gene expression in aged
skeletal muscle (Giresi et al., 2005). Furthermore, a recent publication demonstrated
that Gal-1 levels increased during muscle degeneration in both mdx and wild type
mice, along with cellular co-localisation with infiltrating CD45+ leukocytes, followed
by decreased Gal-1 expression to baseline levels during the regenerative phase (Cerri
et al., 2008). Taken together, these results suggest that Gal-1 may play a complex role
in muscle homeostasis.
4.3.4. Human fetal mesenchymal stem cells express galectin-1
My finding that Gal-1 was present in naïve MSCs is in accordance with current
literature (Gotherstrom et al., 2005; Kadri et al., 2005; Purkrabkova et al., 2003; Vas
et al., 2005). The Gal-1 transcript has been detected by RT-PCR in human adult BM-
MSCs and is up to ten times more highly expressed than in skeletal muscle
(Panepucci et al., 2004; Silva et al., 2003). At the protein level in human adult MSCs,
nuclear presence of Gal-1 has been shown in 10% of the cell population, with Gal-1
representing 0.8% of total proteins extracted from the cells (Kadri et al., 2005;
Purkrabkova et al., 2003; Vas et al., 2005). This is further supported by work in the
C2C12 cell line, where intracellular Gal-1 is less abundant after mature muscle fibre
formation, and appears to be secreted by ectocytosis (Camby et al., 2006; Cooper and
Barondes, 1990; Harrison et al., 1989). Even though Gal-1 is intrinsically present in
naïve MSC, it may be at an insignificant concentration to drive myogenesis. It appears
that a certain level of intracellular Gal-1 triggers muscle differentiation, after which
myotube maturation leads to Gal-1 secretion.
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4.3.5. Galectin-1 gene transduced human fetal mesenchymal stem cells and their
in vivo differentiation
Galectin-1 transduced hfMSCs showed significantly higher expression of myogenic
markers compared to non-transduced hfMSCs, which suggests that endogenous
production rather than exogenously added Gal-1 can enhance myogenesis.
Transduced hfMSCs underwent greater levels of in vivo muscle regeneration than
naïve hfMSCs, though the level achieved was low (~0.02%) compared to the
published literature. Using retrovirally GFP-labelled human adult MSCs, Dezawa et al.
found 37.1 ± 9.9% out of 1,500 muscle fibres counted were GFP- and human
dystrophin-positive in cardiotoxin-treated mdx/nude gastrocnemius muscles 2 weeks
following intramuscular cell administration (Dezawa et al., 2005). In MF1 nude mice,
our collaborator (Prof. D. Watt) has previously shown that murine dermal fibroblasts
contributed to muscle regeneration in up to 12.0% of total fibres (115 donor and
regenerating fibres / 959 total fibres) in cryodamaged muscles after 3 weeks. Donor
cells were labelled with DAPI and regenerating fibres were stained with dystrophin
(Pye and Watt, 2001). In mdx mice that underwent neonatal irradiation followed by
transplantation of BM cells from GFP transgenic mice, there was no difference
detected in murine dystrophin expression to aged matched control up to 46 weeks
after implantation. However, when these mice underwent 4 fortnightly cycles of
intramuscular cardiotoxin damage from 9 weeks of age, more dystrophin-positive
fibres were found in mdx muscles with cardiotoxin damage (3.6 ± 1.7%) compared
with control (1.7 ± 0.9%) 6 weeks after the course of cardiotoxin treatment (Wernig et
al., 2005). On the other hand, these muscle fibres may have been revertant fibres
(Chapter 1.2.4.3.2). Yokota et al. showed that there is an expansion of revertant fibres
within degenerating mdx muscles which underwent irradiation followed by notexin
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injury 4 weeks later compared with those without further notexin treatment (Yokota et
al., 2006).
The discrepancy between my work and other groups’ may be attributable to
different cell types with a diverse range of in vitro culture conditions as well as
distinctive muscle injury protocols in mdx mice leading to sub-optimal environments
for engraftment and differentiation. HfMSCs may have been immunologically
rejected in this xenograft model. As discussed in Chapter 1.3.3.4, various muscle
injury paradigms cause different modes of muscle damage. For example, notexin
damages mature muscle fibres whereas cryodamage destroys both mature fibres and
satellite cells. If hfMSCs contribute to myogenesis mainly through a satellite cell
dependent mechanism, then their effects would not be as evident in cryodamaged-
compared with notexin damaged-muscles. It has been shown that donor cell
engraftment can occur without further in vivo muscle differentiation. Although over-
expression of MyoD in dermal fibroblasts from LacZ transgenic mice led to
myogenesis in vitro, these cells formed ß-galactosidase-positive but dystrophin-
negative fibres following transplantation into mdx mice (Huard et al., 1998). Though
GFP or LacZ are commonly used to mark donor cells, human-specific lamins a/c was
intentionally chosen in this project as I wanted to avoid further modification of
hfMSC with potential adverse effects on immunogenicity, trafficking, engraftment
and differentiation.
4.3.6. Conclusion
Although Gal-1 in pilot work by others had differentiated hfMSCs down the
myogenic lineage in vitro, this high level was not able to be reproduced in detailed
experiments, even after exhaustive repetition and technical optimisation. One possible
factor maybe the different hfMSC samples used, as only 50% of the samples tested
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were capable of muscle differentiation in previous work (Chan et al., 2006a). There
may have been subtle variations in the hfMSC isolation protocol due to operator
differences, which could have led to the selection of a slightly less myogenic cell
population than previously obtained. Another variant stems from the change of
GALM batch. To date, there have been no other reports in the literature reproducing
the original finding by our group that Gal-1 enhances the myogenicity in vitro of
hfMSCs. This raises the possibility of an unaccounted contaminant in the original
batch of GALM used. In the present work, GALM induced hfMSC myogenesis in
vitro to only a modest extent but in contrast recombinant Gal-1 inhibited myogenesis.
Gal-1 was found to be inherently expressed in naïve hfMSCs. Over-expression of
Gal-1 in cells via lentivirus-mediated transduction can enhance the degree of in vitro
muscle differentiation, however the level of in vivo myogenesis was low and unlikely
to be of clinical relevance.
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CHAPTER 5
CO-CULTURE SYSTEMS
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5.1. Introduction
It is important to determine how hfMSCs contribute to dystrophin-positive fibres after
IUT in the mdx model. A possible mechanism is cell fusion, as myoblast fusion is
essential to normal muscle development and repair (Horsley and Pavlath, 2004).
Transplanted stem cells should be able to fuse with each other or with growing or
regenerating myofibres upon de novo myogenesis. Another mechanism would be
through contribution to satellite cells, which have roles in muscle growth or
regeneration.
Cell fusion has been reported after co-culturing human BM-derived GFP-
labelled MSCs and C2C12 mouse myoblasts, as chimeric myotubes were formed
where myotubes positive for MSC nuclei also contained resident C2C12 cell nuclei
(Lee et al., 2005). In contrast, fresh murine adipose tissue stromal vascular fraction
(AT-SVF) cells have been shown to undergo myogenesis (0.2%) using TnT staining
when co-cultured with differentiating murine primary myoblasts including across
transwell filters, albeit at a tenfold lower efficiency. TnT is a component of the
troponin complex which binds to tropomyosin in the thin filament of striated muscle
and is integral to muscle contraction. Spontaneous formation of TnT-positive cells
was observed (0.001%) in the absence of inducing myogenic cells. In vivo, uncultured
AT-SVF engrafted in adductor muscles of wild type mice with hindlimb ischaemia,
and also formed dystrophin-positive fibres in mdx mice (Di Rocco et al., 2006).
However, there was no direct evidence for mosaicism and these dystrophin-positive
fibres are likely to be revertant fibres normally present in the mdx mice (Chapter
1.2.4.3.2).
In support of true transdifferentiation, co-culture of murine mesoangioblasts with
human primary muscle cells or with C2C12 myoblasts promotes activation of murine
166
MyoD mRNA in the mesoangioblasts. Moreover, mesoangioblasts co-cultured with
C2C12 myotubes increased their cell resting potential to that comparable to satellite
cells and increased their membrane capacitance by 150% (Grassi et al., 2004). It has
been shown previously that the capacitance of satellite cells increases by about 170%
during the pre-fusion steps of myogenic differentiation (Liu et al., 1998). Therefore,
upon co-culture with myogenic cells, mesoangioblasts appeared to acquire
characteristics similar to differentiating satellite cells.
Compared with grafting into adults, the mechanism underlying IUT is likely to
be different. Donor cells are more likely to fuse in utero with myoblasts or developing
muscle fibres than with existing muscle fibres. Promisingly, despite their different
stages of development, myoblasts from adult, fetal and embryonic chick and quail
muscles are all capable of forming muscle fibres in limb buds of chick embryos
(DiMario and Stockdale, 1995).
The technique of IUT has been optimised in the mouse embryo at E14-16 as
previous experience in my laboratory showed performing this procedure at earlier
stages resulted in a high rate of non-viability. In relation to embryonic skeletal muscle
development, primary muscle fibres appear in the limbs around E11-14 in mice and 6-
8 weeks of gestation in humans, whereas secondary fibres appear around E14-16 in
mice compared with 8-18 weeks in humans (Barbet et al., 1991; Ontell and Kozeka,
1984). Satellite cells can be found in mouse embryos after E17.5 and in human fetuses
around 10-14 weeks of gestation (Barbet et al., 1991; Seale and Rudnicki, 2000). In
other words, IUT of hfMSCs at E14-16 in mice would occur whilst secondary muscle
fibres are being formed but before satellite cells are present. Hence hfMSCs may give
rise to satellite cell progenitors.
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As the initial IUT experiements were unable to be reproduced en masse (Chapter
6), the aim of this chapter is to determine whether hfMSCs are capable of myoblast
fusion, which is a prerequisite for the successful regeneration of muscle in vivo after
IUT. This was tested by using mdx/nude fetal myoblasts at E15, as a model system of
IUT at E15 gestation.
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5.2. Experimental design and results
5.2.1. Human myoblasts fused with murine myoblasts
To determine whether cross species fusion can occur between human and murine
MPCs, a xenogeneic co-culture system was set up. In vivo conditions at the time of
IUT were simulated by plating conditionally-immortal mouse myoblasts (H2K 2B4
satellite cell-derived line) in chamber slide wells under myogenic conditions followed
by the addition of either human adult or fetal myoblasts 4 h later (Chapter 2.3.3.2,
2.3.3.3 and 2.10.1). Earlier experience in my laboratory has shown that the optimal
cell concentration for H2K 2B4 to undergo differentiation is at 4 × 104 per well in 8-
well chamber slides. Myotubes were observed on day 2 in control wells with murine
cells only, at which time all cells (both experimental and control groups) were fixed
for immunostaining as at later time points the myotubes began to detach from wells.
Both human adult and fetal myoblasts fused with murine myoblasts but only at a
plating ratio of 1:1 and with murine cell density being 4 × 104 per well (n=3; Table 5-
01). As shown in Figures 5-01 and 5-02, human nuclei identified by an antibody
specific for human lamins a/c, an intermediate filament type protein, were
incorporated into differentiated mouse myotubes (visible under phase contrast
microscope and positive staining for desmin, a muscle protein). Figure 5-01E shows
one nucleus derived from an human adult myoblast (white arrow) fused with three
murine MPCs, though not all human cells fused to form chimeric myotubes (yellow
arrowhead). In Figure 5-02E, one nucleus derived from a human fetal myoblast (white
arrow) fused with two murine MPCs.
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Table 5-01. Co-culture conditions of human myoblasts with mouse myoblasts.
Mouse cells / well Human cells / well Ratio of mouse to
human cells
2 × 104 2.0 × 104 1:1
2 × 104 0.6 × 104 3:1
2 × 104 0.4 × 104 5:1
4 × 104 4.0 × 104 1:1
4 × 104 1.3 × 104 3:1
4 × 104 0.8 × 104 5:1
5 × 104 5.0 × 104 1:1
5 × 104 1.6 × 104 3:1
5 × 104 1.0 × 104 5:1
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Figure 5-01. Human adult myoblasts fused with mouse myoblasts in co-culture.
(A) Human cells were detected by human-specific lamins a/c antibody (red).
Myotubes were detected under bright-field microscopy (B) and fluorescence
microscopy for desmin expression (green) (C). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged to show co-localisation of a human
cell in a myotube, with phase-contrast microscopy. Yellow arrowhead depicts a
human cell that has not fused in a myotube. (F) Merged image shown without phase
contrast for clarity of immunostaining. White arrow indicates the same cell. Original
magnification 600×.
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Figure 5-02. Human fetal myoblasts fused with mouse myoblasts in co-culture.
(A) Human cells were detected by human-specific lamins a/c antibody (red).
Myotubes were detected under bright-field microscopy (B) and fluorescence
microscopy for desmin expression (green) (C). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged to show co-localisation of a human
cell in a myotube, with phase-contrast microscopy. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell.
Original magnification 600×.
172
The main challenge was the varying length of time it took for human and murine
myogenic cells to differentiate, which occurred at 7 and 2 days, respectively, as
evidenced in the control wells. Promisingly, this set of experiments shows that human
myoblasts differentiate faster in the presence of murine myoblasts through fusion,
compared with human myoblasts cultured alone.
5.2.2. Human fetal mesenchymal stem cells fused with rat myoblasts
Having established that human myoblasts can fuse with mouse myoblasts from a
conditionally immortal cell line, my next step was to determine the optimal ratio of
myogenic cells to hfMSCs. A rat myoblast cell line (L6) was utilised for initial
optimisation experiments instead of using myoblasts from mouse fetuses at E15 for all
experiments in order to reduce the total number of animals used. Rat myoblasts are
known to differentiate at a cell density of 0.5 × 104/cm2 after 4 days in differentiation
medium (Chapter 2.3.3.4) and thus longer than conditionally-immortal mouse MPCs.
After 24 h under differentiating conditions, hfMSCs were added to L6 myoblast
cultures at varying ratios in triplicate sets of 6-well plates (Chapter 2.10.2; Table 5-
02). Myotubes were seen at day 9 of co-culture and cells were fixed for
immunostaining at days 9, 10 and 11. After this time point, most of the myotubes had
detached from the plates and the experiment was therefore terminated.
Rat L6 myoblasts fused with hfMSCs both at a ratio of 3:1 and 5:1, with the
most chimeric myotubes being seen at day 10 of co-culture. Two interpretations can
be offered for the myotube consisting of only lamins a/c-positive nuclei shown in
Figure 5-03D. The myotube may be human in origin suggesting a myogenic
environment can trigger autologous fusion of hfMSCs, despite the different rate of
myogenesis for L6 cells and hfMSCs, being 4 and 12 days, respectively. Alternatively,
the formation of a mosaic myotube is supported by the weaker staining for lamins a/c
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Table 5-02. Co-culture conditions of hfMSCs with rat L6 myoblasts.
L6 myoblasts / cm2 HfMSCs / cm2 Ratio of rat to human
cells
0.5 × 104 0.50 × 104 1:1
0.5 × 104 0.16 × 104 3:1
0.5 × 104 0.10 × 104 5:1
0.5 × 104 0.05 × 104 10:1
Figure 5-03. HfMSCs fused with L6 rat myoblasts in co-culture. (A) Human cells
were detected by human-specific lamins a/c antibody (red). (B) Myotubes were
positive for desmin expression (green). (C) Cell nuclei were identified by DAPI
staining (blue). (D) All images were merged to show co-localisation of human cells in
a myotube. White arrow indicates same cell nucleus. Original magnification 400×.
174
in the arrowed, possibly L6, nucleus (Figure 5-03D; white arrow) as lamins a/c
protein synthesized in cytoplasm could have been transported to a rat nucleus during
fusion. Blaveri and colleagues have previously described a similar staining issue
(Blaveri et al., 1999) (Chapter 5.3.3).
5.2.3. E15 myoblasts
5.2.3.1. E15 myoblasts are positive for desmin, MyoD and myogenin
Myoblasts from E15 mdx/nude mouse fetuses were obtained by enzymatic digestion
of fetal muscle (Chapter 2.3.2). A mixed cell population, which would have also
contained blood, connective tissue, endothelial and other cell types, was obtained due
to the crude nature of cell isolation. Figure 5-04A shows a multinucleated myotube
(black arrow) amongst flattened, stellate cells with fibroblastic morphology. However,
mouse MPCs with their typical small and round morphology were not seen. The
number of cells harvested per fetus was 1.20 ± 0.44 × 105 (n=33). They stopped
proliferating after only 1 passage, with a doubling time of 72 h. The myogenic
fraction of these cells was positive for desmin (“early” myogenesis marker) as well as
MyoD and myogenin (both intermediate MRF), but negative for Pax7 (satellite cell
marker) as detected by immunostaining (Figure 5-05). However, they became
senescent in culture, as indicated by the presence of binucleate cells (Serrano et al.,
1997; Yablonka-Reuveni and Nameroff, 1987). Satellite cells have been shown to
adopt divergent fates where a proportion becomes Pax7-negative during proliferation
but a small quiescent fraction stays Pax7-positive. Therefore, though E15 myoblasts
were Pax7-negative, they may have been proliferating at the time of culture (Zammit
et al., 2004b).
175
Figure 5-04. Morphology of E15 myoblasts. Phase contrast images showing a
myotube (black arrow) and a binucleate cell (red arrow) amongst a mixed cell
population with varied sizes and appearances. Original magnification 200×.
176
Figure 5-05. E15 myoblasts express desmin (A), MyoD (C) and myogenin (E), but
not Pax7 (G). Corresponding DAPI staining shown in B, D, F and H. White arrow
indicates same cell nucleus. Original magnification 400×.
177
5.2.3.2. E15 myoblasts form myotubes under permissive conditions
Conditions determining differentiation in cell culture can be split into the physical
matrix microenvironment (Engler et al., 2006), soluble inducers in the growth
medium and cell-to-cell contact. In terms of myogenesis, Matrigel, low HS condition
and high cell plating density have been used in murine myoblast cell lines with
success (Balogh et al., 1996; Langen et al., 2003). Therefore I chose to use these
conditions to study whether E15 myoblasts could differentiate into myotubes.
Thirteen cell densities between 8,333 and 50,000 cells per well were tested in 8-well
chamber slides. After 48 h, cells initially plated at densities of 25,000, 37,500 and
50,000 cells per well began to fuse with each other and myotubes started to detach by
72 h. Immunostaining with desmin confirmed these findings (Figure 5-06).
5.2.3.3. HfMSCs undergo myogenic conversion with or without fusion to E15
myoblasts
HfMSCs were co-cultured with E15 myoblast to mimic the in vivo situation at IUT
where hfMSCs will be injected into mdx/nude fetuses at E15 days old. E15 myoblasts
were plated to allow adherence to 8-well chamber slide wells, followed by the
addition of hfMSCs either 4 h or 24 h later (Chapter 2.10.3, Table 5-03). Cells were
fixed after 48 h of co-culture.
HfMSC fusion occurred when the initial plating of E15 myoblasts was 37,500
cells per well, co-cultured with hfMSCs 4 h later, at mouse to human cell ratios of 1:2,
1:1 and 3:1. Human nuclei (lamins a/c-positive) could be seen in murine myotubes
positive for desmin on immunostaining (Figures 5-07 and 5-08). However, its
incidence was low, as not all hfMSCs underwent fusion to form chimeric myotubes.
Figure 5-09E shows a lamins a/c- and desmin-positive hfMSC nucleus (white arrow)
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Figure 5-06. E15 myoblasts form myotubes. Myotubes were detected under bright-
field microscopy (A) and fluorescence microscopy for desmin expression (green) (B).
(C) Cell nuclei were identified by DAPI staining (blue). (D) All images were merged
to show myotubes, with phase-contrast microscopy. (E) Merged image shown without
phase contrast for clarity of immunostaining. Original magnification 400×.
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Table 5-03. Co-culture conditions of hfMSCs with E15 mouse myoblasts.
Total number of
cells / well
Myoblasts / well HfMSCs / well Ratio of mouse to
human cells
75,000 25,000 50,000 1:2
50,000 25,000 25,000 1:1
33,333 25,000 8,333 3:1
30,000 25,000 5,000 5:1
112,500 37,500 75,000 1:2
75,000 37,500 37,500 1:1
50,000 37,500 12,500 3:1
45,000 37,500 7,500 5:1
60,000 50,000 10,000 1:2
100,000 50,000 50,000 1:1
66,666 50,000 16,666 3:1
60,000 50,000 10,000 5:1
25,000 8,333 16,666 1:2
25,000 12,500 12,500 1:1
25,000 18,750 6,250 3:1
25,000 20,833 4,166 5:1
37,500 12,500 25,000 1:2
37,500 18,750 18,750 1:1
37,500 28,125 9,375 3:1
37,500 31,250 6,250 5:1
50,000 16,666 33,333 1:2
50,000 25,000 25,000 1:1
50,000 37,500 12,500 3:1
50,000 41,666 8,333 5:1
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Figure 5-07. HfMSCs can fuse with E15 mouse myoblasts in co-culture, example
1. (A) Human cells were detected by human-specific lamins a/c antibody (red).
Myotubes were detected under bright-field microscopy (B) and fluorescence
microscopy for desmin expression (green) (C). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged to show co-localisation of human
cells in myotubes, with phase-contrast microscopy. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell.
Original magnification 600×.
181
Figure 5-08. HfMSCs can fuse with E15 mouse myoblasts in co-culture, example
2. (A) Human cells were detected by human-specific lamins a/c antibody (red).
Myotubes were detected under bright-field microscopy (B) and fluorescence
microscopy for desmin expression (green) (C). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged to show co-localisation of human
cells in a myotube, with phase-contrast microscopy. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell.
Original magnification 600×.
182
Figure 5-09. HfMSCs do not always fuse with E15 mouse myoblasts in co-culture.
(A) Human cells were detected by human-specific lamins a/c antibody (red).
Myotubes were detected under bright-field microscopy (B) and fluorescence
microscopy for desmin expression (green) (C). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell.
Original magnification 600×.
183
separate from a murine myotube. Encouragingly, hfMSCs differentiated in the
presence of E15 myoblasts without fusion, as shown by double staining of co-cultures
for lamins a/c and MyoD (Figures 5-10 and 5-11) or myogenin (Figure 5-12). In
Figure 5-12, there are two areas of false positive lamins a/c staining as they are DAPI-
negative, which is possibly due to under-blocking of the slide.
Unfortunately, I was unable to conclusively demonstrate fusion of hfMSCs into
chimeric myotubes using myogenin and MyoD staining, despite having shown this
using desmin staining. It could be due to the very low numbers of chimeric myotubes
in the first place which was further compounded by detachment from the chamber
slide either while in culture or during washing steps of immunostaining, thus resulting
in unsatisfactory images. On the other hand, the autonomous myogenic activity of
hfMSCs without fusion could not be unequivocally determined using desmin staining
as a fraction of hfMSC is intrinsically desmin-positive.
184
Figure 5-10. HfMSCs differentiate in the presence of E15 mouse myoblasts in co-
culture, example 1. (A) Human cells were detected by human-specific lamins a/c
antibody (red). (B) Phase contrast image in the same field as A. (C) Myogenesis was
revealed by positive MyoD expression (green). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell that
co-express lamins a/c and MyoD. Original magnification 600×.
185
Figure 5-11. HfMSCs differentiate in the presence of E15 mouse myoblasts in co-
culture, example 2. (A) Human cells were detected by human-specific lamins a/c
antibody (red). (B) Phase contrast image in the same field as A. (C) Myogenesis was
revealed by positive MyoD expression (green). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell that
co-express lamins a/c and MyoD. Original magnification 600×.
186
Figure 5-12. HfMSCs differentiate in the presence of E15 mouse myoblasts in co-
culture, example 3. (A) Human cells were detected by human-specific lamins a/c
antibody (red). (B) Phase contrast image in the same field as A. (C) Myogenesis was
revealed by positive myogenin expression (green). (D) Cell nuclei were identified by
DAPI staining (blue). (E) All images were merged. (F) Merged image shown without
phase contrast for clarity of immunostaining. White arrow indicates the same cell that
co-express lamins a/c and myogenin. Original magnification 600×.
187
5.3. Discussion
5.3.1. Fusion of murine myoblasts with human myobalsts or hfMSCs
Positive control experiments co-culturing adult and fetal human MPCs with
conditionally-immortal mouse MPCs showed that human cells can fuse with mouse
cells at a ratio of 1:1. For initial experiments, the L6 rat myoblast cell line was co-
cultured with hfMSCs at varying ratios of myogenic cells to hfMSCs, which showed
the optimal ratios were 3:1 and 5:1.
5.3.2. E15 myoblasts
Mouse myoblasts were harvested from E15 fetuses by adapting a methodology used
for isolating murine satellite cells (Pinset and Montarras, 1998). Despite being a
mixed cell population with low proliferative capacity, a myogenic fraction was
identifiable by its immunophenotype. Under myogenic culture conditions, E15
myoblasts were able to undergo terminal differentiation into multinucleated myotubes.
The imprecise method of isolating myoblasts resulted in inconsistent cell
numbers at each harvest and a mixed cell population. It is known that primary
myoblast cultures contain both myoblasts and fibroblasts and much research has
focused on obtaining a pure population of myoblasts (Jones et al., 1990; Morgan,
1988; Rando and Blau, 1994). Furthermore, within the myogenic fraction itself there
is a mixture of MPCs including satellite cells (Cossu et al., 1987; Mauro, 1961), SP
cells (Gussoni et al., 1999) and muscle-derived stem cells (Qu-Petersen et al., 2002).
Future work may involve further characterisation of the E15 myoblast population
isolated to identify the ratio of myogenic to non-myogenic fractions to ensure
comparable data. On the other hand, the heterogeneous population realistically
188
reflects the in vivo milieu at the time of IUT. Nevertheless, the limitation of using an
in vitro model to simulate the in vivo environment cannot be overlooked.
HfMSCs fused with E15 myoblasts at ratios of 2:1, 1:3 and 1:5, with a faster rate
of myogenesis than hfMSCs cultured alone. This suggests a possible mechanism for
therapeutic viability of IUT in mdx/nude mice. However, a very high concentration of
hfMSCs will be required to be administered in vivo in order to mimic the required
ratios I identified in vitro.
5.3.3. Staining
A potential technical problem may arise from expression of human lamins a/c via
diffusion within the myofibre, resulting in the over-estimation of lamins a/c-positive
nuclei. It has been shown that within the syncytial multinucleate muscle fibre,
nuclear-localizing β-gal is not totally restricted to the nucleus encoding it (Yang et al.,
1997), both mRNA and the protein translated from this transcript being subject to
diffusion within the sarcoplasm, with the result that it may localize in host myonuclei
neighbouring the donor myonucleus that encoded it (Blaveri et al., 1999). However,
even if this was the case in the chimeric myotubes, there would need to be at least one
lamins a/c-positive nucleus within the fibre before diffusion of lamins a/c can take
place.
5.3.4. Conclusion
Cell fusion has been well described as a possible mechanism of transdifferentiation in
vivo, where within a regenerative muscle setting, stem cells do not undergo
prespecification to a myogenic fate, but stochastically become incorporated into
muscle fibre (Camargo et al., 2003; Kirillova et al., 2007; Schulze et al., 2005). Upon
fusion, myogenic transcription factors expressed in trans from neighbouring nuclei
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activate a myogenic program in stem cell nuclei. In future work, it would be useful to
examine dystrophin expression in the chimeric myotubes to determine functionality.
However, this will require at least a 10-day culture of myotubes to express dystrophin
for a Western blot analysis, as mouse myotubes cannot be reliably immunostained
with dystrophin.
Developmentally, multinucleated myotubes are formed by fusion of myoblasts.
MPCs have been shown to graft into growing or regenerating skeletal muscle fibres of
mice with inherited myopathies leading to improvements at genetic and protein levels.
However, this occurred at low efficiency where mosaic fibres with myonuclei of
donor origin were detected in only 5-10% of muscles examined from mice injected
with normal mouse MPCs (Morgan et al., 1988; Watt et al., 1984). Rat satellite cell
progenies on single myofibres can form myotubes by fusion among themselves in
vitro, even when the single mature myofibres are killed (Bischoff, 1979, 1980). On
the other hand, satellite cell fusion to adjacent myofibres was not enhanced by
inhibiting proliferation of satellite cells in the hope of driving them towards
differentiation, while rat embryonic myoblasts are able to fuse with nucleated sprouts
growing from the ends of mature myofibres (Bischoff, 1990). Though my data
demonstrated hfMSCs fuse with mouse embryonic myoblasts, these MPCs are likely
to be undergoing myotube formation in vivo at E15. Thus after transplantation in
utero, hfMSCs are more likely to directly contribute to developing myofibres. As
hfMSCs are positive for satellite cell marker Pax7 (Chapter 3.2.3), they may give rise
to satellite cells first which themselves form myotubes.
Myoblast fusion follows a sequence of cellular interactions where myoblasts
recognise and adhere to each other via protein and calcium dependent processes,
followed by alignment through apposition of myoblast membranes to allow
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membrane union (Rash and Fambrough, 1973; Wakelam, 1985). However, the
underlying molecular mechanism is not clearly understood. The first phase of
myoblast fusion forms nascent myotubes with a limited number of nuclei and is
thought to involve factors including integrins (VCAM-1, VLA-4, ß1-integrin),
prostaglandin E1, extracellular calcium and calmodulin (Bar-Sagi and Prives, 1983;
David and Higginbotham, 1981; Rosen et al., 1992; Rossi et al., 1989; Schwander et
al., 2003). Nascent myotubes group to form mature myotubes and molecules such as
interleukin-4, prostaglandin F2α and nuclear factor of activated T cell family of
transcription factors have been implicated (Horsley et al., 2001; Horsley et al., 2003;
Horsley and Pavlath, 2003). As hfMSCs express VCAM-1, VLA-4 and ß1-integrin
(Campagnoli et al., 2001), these integrins may serve to regulate hfMSC fusion to the
E15 myoblasts.
Overall, though my findings show that IUT could contribute to therapeutic
myogenesis in mdx/nude mice, the low incidence of chimeric myotube formation and
autonomous myogenic differentiation is discouraging.
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CHAPTER 6
CELL TRANSPLANTATION
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6.1. Introduction
Transplantation during the neonatal period is thought to be better than in adult life, as
neonatal injection of allogeneic cells is known to induce tolerance to alloantigens of
the donor cells, which aids in establishing a chimeric state (Billingham et al., 1953;
Streilein, 1979). When donor cells engraft in adult mice, signs of de novo
differentiation into muscle can be absent (Wang et al., 2005a), whereas in the neonatal
muscle microenvironment there is rapid growth and development, and neonatal
therapy may contribute more towards muscle regeneration. In the mdx model, a
modified AAV vector with dystrophin was injected into TA muscles of neonatal or
juvenile (4-6-week-old) mice and more biochemical and functional improvements
were found in the neonatally treated group (Gilbert et al., 2003). Moreover, neonatally
injected mouse BM or FL cells are recruited to cardiotoxin-treated mdx muscles
where they participate in regeneration (Fukada et al., 2002). When murine MSCs
were administered intracranially into newborn and adult mice, though a small
percentage of injected MSCs survived in both neonatal and adult recipients, only the
cells which engrafted in neonatal mice expanded in number post-transplantation
(Phinney et al., 2006). Young and colleagues have shown that marrow-derived
endothelial progenitor cells engraft into vasculature of non-ablated newborn mice but
not adult mice (Young et al., 2002). After first inducing tolerance to alloantigens by
neonatal allogenic BM cell transplantation in rats and demonstrating 3% BM cell
chimerism at ten weeks, Butler et al. transplanted allogenic limb tissues. They
demonstrated marked improvements in tissue graft viability compared to those
without initial neonatal tolerance induction (Butler et al., 2000).
On the other hand, following neonatal injection of murine MSCs through the
superficial temporal vein in B6C3Fe a/a mice, the cells were detected in lung, liver
193
and bone at early time points, but only in lungs by day 150 (Niyibizi et al., 2004). The
lung trapping occurs not only because this is the first organ encountered by the cells
after intravenous injection, but also cells are trapped by the small capillary lumen
which restricts cell migration. Although neonatal intramuscular injections could
prevent cells being trapped by lungs, the potential to treat the wide range of muscle
groups involved in DMD would be lost.
Most of the arguments for neonatal transplantation could be used similarly in
favour of IUT, if not even more compelling as pulmonary trapping has not been
reported in IUT. A relatively novel approach of serial transplantation of hfMSCs
utilises the immunological tolerance induced by IUT, which allows further cell
transplantations to occur without eliciting any immune response. BLI can be utilised
for cell tracking in this protocol, as it can detect a minimum of 200 cells (Troy et al.,
2004) and has been successfully used not only in adult MSCs (Love et al., 2007), but
also to track hfMSCs in vivo (Guillot et al., 2008a; Guillot et al., 2008b). One
difficulty in assessing stem cell transplantation in animal models to date has been the
need for histological assessment as the primary means of quantifying engraftment and
differentiation, which allows only a single analysis on post-mortem tissue. The
development of BLI technique allows in vivo detection and quantification of light
emitted from bioluminescent reporters (Doyle et al., 2004), which provides repeated
non-invasive longitudinal analysis of cell engraftment or trafficking. To distinguish
between in utero transplanted and neonatally transplanted cells, one set can be
labelled with renilla luciferase (R-Luc) reporter, while the other with firefly luciferase
(F-Luc) reporter. These separate cell populations can then be identified using different
substrates: coelenterazine for R-Luc and D-luciferin for F-Luc. To allow further
distinction at immunohistochemistry, the cells can be identified by the presence or
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absence of GFP marking. However, limitations to bioluminescence include
attenuation of signal as light passes through pigmented and deep tissues which absorb
light in the visible region of the spectrum. Therefore, in mouse strains with black fur
such as the mdx, shaving of animal fur is necessary (Dickson et al., 2007). A further
potential problem lies in quantifying cell engraftment between separate tissues due to
differences in emitted spectra depending on the depth of the cells within the tissue
(Rice et al., 2001). Recently, Sacco et al. successfully used BLI to track grafted
satellite cells (Sacco et al., 2008). However, donor cells were only evaluated in the
TA muscle compartment of albino NOD/scid mice recipients.
The initial part of this work focuses on intramuscular transplantation of hfMSCs
into adult mdx/nude mice to assess their ability to engraft and differentiate into
skeletal muscle in vivo and whether this ability is affected by the mode of muscle
injury. The level of in vivo myogenesis by hfMSCs was further investigated using a
neonatal transplantation model as well as an IUT model.
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6.2. Experimental design and results
Naïve hfMSCs rather than pre-differentiated hfMSCs were used as the pre-
differentiation strategies were inefficient in vitro (Chapter 4). The same sample of
hfMSC (BM 10w1d (a)) was used for all experiments except in the pilot intrauterine
transplantations (Chapter 6.2.3.1).
6.2.1. Adult transplantation
Muscle injuries have been used in mdx mice to mimic muscle damage seen in DMD
patients, as mdx mice continue to regenerate well throughout life. Standard muscle
injury protocols used in mdx research include notexin (Harris and Johnson, 1978),
irradiation (Wakeford et al., 1991) and cryodamage (Irintchev et al., 1997a). These
cause muscle injury in the hope of enhancing donor-derived muscle regeneration.
Notexin injury was used during in vivo work discussed in Chapter 3, whereas the aim
of this part of my study was to investigate whether different injury protocols would
affect the degree of hfMSC engraftment and muscle differentiation in vivo.
HfMSCs (0.5 × 106) were injected into bilateral TA muscles of adult mdx/nude
mice with one injured and one non-injured leg (n=21) (Table 6-01). The right legs
underwent injury by notexin, irradiation or cryodamage prior to cell grafting as
described in Chapter 2.11.4, and both TA muscles were harvested two weeks after
hfMSC injection. This time point was chosen because I have shown that hfMSCs can
engraft and lead to muscle regeneration at one week following intramuscular
transplantation (Chapter 4.2.10) and I wished to investigate whether the contribution
to regenerated fibres increased with time. Experiments performed by a previous
colleague demonstrated that hfMSCs engraft but do not contribute to regenerated
muscle fibres in non-injured mdx/nude TA muscles (Chan, 2006). Human cells on
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Table 6-01. HfMSC transplantation to adult mdx/nude with TA muscle injury.
Type of injury Injury time No. of nu/nu mice
injected
No. of nu/+ mice
injected
Notexin P22 2 4
Irradiation P22 3 4
Cryodamage P34 5 3
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muscle cryosections were identified using human-specific lamins a/c antibody shown
as nuclear staining (red nuclei in Figure 6-01B). Human fetal muscles provided
positive controls of antibody co-staining with dystrophin (Figure 6-01A), spectrin and
lamins a/c (red fibre membranes and red nuclei in Figure 6-01B). In terms of internal
control, the mdx nu/+ mice were immunocompetent and were used as the control
group to the immunodeficient mdx nu/nu mice (Chapter 2.11.2).
Figure 6-02D illustrates the trend of enhanced hfMSC engraftment in injured but
not in non-injured muscles of nu/nu hosts except in the case of notexin injury, while
no cells engrafted in the immunocompetent mdx nu/+ controls (data not shown). The
highest level of hfMSC engraftment reached 2.0 ± 1.2 donor (human) cells per muscle
section in cryodamaged TA. As one TA muscle section contains approximately 2,000
fibres (Pagel and Partridge, 1999), this engraftment level equated to ~0.001 cells per
fibre which was very low.
6.2.2. Neonatal transplantation
As the immune system is not yet fully developed in the neonatal period, neonatal cell
transplantations offer an opportunity for early systemic intervention of disease.
HfMSCs (1 × 106) were administered intraperitoneally at postnatal day 3 to mdx nu/nu
(n=14) and mdx nu/+ (n=10) mice, and twenty of these mice underwent injury
protocols (notexin, irradiation or cryodamage) aged between 3-5 weeks and tissue
harvesting at 2 weeks post-injury comparable with the adult transplantation
experiments (Figure 6-03, Table 6-02). Three muscles were examined; the skeletal
muscles TA and diaphragm which tend to exhibit early pathological features, as well
as cardiac muscle (Figure 6-04).
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Figure 6-01. Positive and negative controls for antibodies used. Human fetal
muscles were used as the positive control. Co-staining of dystrophin (green fibre
membrane) (A), spectrin (red fibre membrane) and nuclear lamins a/c (red nuclei) (B),
and DAPI (blue nuclei) (C) is shown on a merged image (D). Original magnification
400×. Negative control muscle section from non-injected and non-injured mdx nu/+
mice. No staining for human-specific lamins a/c antibody with DAPI nuclei stain
shown in inset (blue) in muscle cryosection from TA (E). Original magnification
200×.
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Figure 6-02. Intramuscular injection of hfMSCs engrafted to a greater extent in
mdx nu/nu TA muscles damaged by notexin, irradiation or cryoinjury than in
non-injured muscles. Human cell (white arrow) was detected by human-specific
lamins a/c antibody (red) (A) and DAPI staining (blue) showed all nuclei present (B).
Merged image show co-localisation of a human cell in a representative mouse TA
muscle cryosection with cryodamage (C). Original magnification 400×. Numbers of
human nuclei per cryosection from TA muscles are compared between non-injured
and injured legs (D). The mean values are indicated by horizontal bars.
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Figure 6-03. Experimental design of neonatal hfMSC transplantation in
mdx/nude mice followed by subsequent TA muscle injury. Neonatal mice
underwent hfMSC transplantation at postnatal day 3. The right TA muscles of
engrafted mice were injured by notexin, irradiation or cryoinjury between postnatal
days 22-34. Tissues were harvested 2 weeks post-injury.
Table 6-02. Neonatal hfMSC transplantation followed by TA injury in adult life.
Type of injury Injury
time
No. of nu/nu
mice injected
No. of nu/+
mice injected
Harvest
age
Notexin P22 3 3 P37
Irradiation P24 4 3 P41
Cryodamage P34 4 3 P48
Injected, non-injured --- 3 1 P48
Non-injected, non-injured --- 1 2 P48
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Figure 6-04. H&E staining of non-injured and non-hfMSC-injected mdx nu/+
muscles showing variations in fibre size (black arrow) and peripheral nucleation
(red arrow). TA muscle with original magnification 400× (A), cardiac (B) and
diaphragm (C). Original magnification 200×.
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Figure 6-05 demonstrates hfMSC engraftment in TA muscle. Similar to adult
injury models, cryodamage in immunocompromised mdx nu/nu mice appeared to
induce the highest level of hfMSC engraftment at 1.17 ± 0.67 cells per section
(p=0.04, Kruskal-Wallis non-parametric ANOVA) (Figure 6-06C), though myofibres
of human origin were still not observed. At the time of TA muscle harvesting,
cryodamaged muscles exhibited an inflamed appearance macroscopically compared to
TA muscles that underwent other forms of injury. The least number of hfMSCs
engrafted after irradiation injury (0.08 ± 0.04 cells per section, n=3) compared to
notexin injury (0.4 ± 0.2 cells per section, n=3) and cryodamage (1.17 ± 0.67 cells per
section, n=3). Muscle injuries using cryodamage or irradiation, but not notexin, led to
an increase in cell engraftment compared to non-injury, which corresponds with my
findings in adult transplantation (Chapter 6.2.1). In fact, when the control legs of the
notexin group is excluded from analysis, the numbers of donor cells in all non-injured
control legs of the experiment (both mdx nu/nu and mdx nu/+) were similar with a
mean value of 0.05 ± 0.02 cells per section. Graft survival was better in mdx nu/nu
than in mdx nu/+ with the exception of the irradiated subgroup. However, there was a
wide data spread in the mdx nu/+ irradiated (0.12 ± 0.07 cells) compared with mdx
nu/nu irradiated group (0.08 ± 0.04 cells) resulting in statistically non-significant data.
HfMSC engraftment in diaphragm and cardiac muscles were inconsistent with
the trend in TA muscle. This may be because the tissues examined were distant from
the injured TA muscle. In diaphragm, the site of early mdx pathology (Louboutin et
al., 1993; Stedman et al., 1991), hfMSC engrafted most in mdx nu/nu mice with TA
notexin injury (2.0 ± 1.2 cells per section) compared to other modes of injury (0.08 ±
0.04 cells per section) and non-injured control groups (0.1 ± 0 cells per section)
(Figure 6-06E). Surprisingly, in the relatively disease-free young mdx/nude cardiac
203
Figure 6-05. Neonatally administered hfMSCs engrafted in injured mdx nu/nu
and nu/+ TA muscles. Nuclei of human origin (white arrow) were detected by
human-specific lamins a/c antibody (red) (A) and DAPI staining (blue) showed all
nuclei present (B). Merged image show co-localisation of human cells in a
representative mouse TA muscle cryosection that had been injured by cryodamage 2
weeks previously (C). Original magnification 400×.
204
Figure 6-06. Neonatally administered hfMSCs engrafted in mdx nu/nu and nu/+
TA muscles, diaphragm and heart in mice that underwent subsequent TA muscle
injury. Numbers of hfMSCs engrafted per cryosection from TA muscles are
205
compared between non-injured and injured legs by notexin (A), irradiation (B) and
cryodamage (C). Negative control group was provided by TA from
immunocompetent (nu/+) and immunodeficient nude (nu/nu) mice that underwent
neonatal hfMSC transplantation without injury to legs (D). Numbers of hfMSCs
engrafted per cryosection from diaphragm (E) and heart (F) are compared between
mice that underwent neonatal hfMSC transplantation in subgroups with injured and
non-injured legs as well as immunocompetent (nu/+) and immunodeficient (nu/nu)
mice subgroups. The mean values are indicated by horizontal bar. Kruskal-Wallis
non-parametric ANOVA used where ** = p<0.01.
206
muscle, there was a significant improvement (p=0.006) in hfMSC survival by TA
cryodamage (2.0 ± 0.2) in mdx nu/nu and by TA irradiation (2.1 ± 1.0) in mdx nu/+
compared to all other groups (mdx nu/nu control = 0.2 ± 0.1; mdx nu/nu irradiation =
0.08 ± 0.04; remaining groups = 0) (Figure 6-06F). Overall, hfMSCs engrafted better
in mdx nu/nu mice (diaphragm = 2.0 ± 1.2; heart = 2.0 ± 0.2) than in mdx nu/+ mice
(diaphragm / heart = 0 ± 0) apart from two instances: diaphragm from mdx nu/+ non-
injured controls and heart from mdx nu/+ mice in which hindlimbs had been irradiated.
This curious finding of increased donor cell engraftment to diaphragm and heart
subsequent to injury to a far-distant TA muscle may be analogous to the abscopal
effect in cancer biology, which is defined as an action at a distance from the irradiated
volume but within the same organism (Kaminski et al., 2005; Mole, 1953). Following
total body irradiation in NOD/scid mice, the use of additional local irradiation to right
posterior legs has been shown to further increase the engraftment of injected human
adult MSCs not only in the injured leg, but also in the brain, a site distant to the
muscle injury. In the same study, a similar abscopal effect was also found after
additional irradiation to the abdomen, whereby a greater level of engraftment was
found in the irradiated abdominal tissues as well as lungs and BM (Francois et al.,
2006).
Despite hfMSC engraftment, they did not contribute to skeletal muscle fibres in
TA, diaphragm and cardiac muscles in neonatally transplanted mice. This may be
either due to the low level of hfMSC engraftment being inadequate for in vivo
myogenesis or a low sensitivity in detecting human spectrin. If any human fibres were
formed, human spectrin protein may have spread along the fibre which could make it
difficult to detect on the muscle sections sampled (Blaveri et al., 1999). Negative
207
control muscle sections from non-injected and non-injured mdx nu/+ mice are shown
in Figure 6-01E which demonstrate the absence of human lamins a/c nuclei.
PCR of organs harvested from these neonatally injected mice was not sensitive
enough to detect the low levels of engrafted human cells (Figure 6-07) and is further
demonstrated by a PCR with DNA dilutions (Figure 6-08). This experiment was
designed to determine the sensitivity of the PCR to amplify human DNA sequence in
the mouse tissue. An equal amount of DNA extracted from the liver tissue of a
negative control (non-injected, non-injured mdx nu/+) mouse was mixed with 10-fold
serial dilutions of genomic DNA from human lung cancer cell line CL1-5, ranging
from 0.00246-24.6 ng. Regardless of the presence of mouse tissue, DNA sequence
corresponding to human HLA-DQα1 could be detected up to 2.46 ng by PCR analysis
(Figure 6-08A). Genomic DNA (93 μg) was purified from 2 × 106 aneuploid CL1-5
lung cancer cells, which is equivalent to 0.0465 ng DNA per cell. Therefore, the PCR
sensitivity of 2.46 ng DNA approximates to 50 cells. Figure 6-08B shows the
amplification of mouse GAPDH sequence from the DNA samples containing mouse
liver tissue (lanes 1-5). As expected, mouse DNA sequence was not detectable in any
human CL1-5 DNA samples tested (lanes 6-10).
6.2.3. Intrauterine transplantation
Due to the lack of hfMSC contribution to skeletal muscle regeneration in vivo in adult
and neonatal mice, an even earlier time point for cell transplantation was utilised
(Figure 6-09). Injection of 1 × 106 hfMSCs into the peritoneal cavity of fetal E14-16
mdx nu/nu and nu/+ mice was used (Chapter 2.11.2, 2.11.5.3). As mentioned in
Chapter 5, this is the time of secondary muscle fibre formation but prior to satellite
cell development. The murine pregnancies continued until littering down at E20, and
these fetally-transplanted animals were harvested at P9 or P19 as described below.
208
Figure 6-07. PCR to detect human cells in mouse organs from neonatally
transplanted mice. (A): Human DNA was not detected in neonatally transplanted
mice which were injured in adulthood. Lanes 1: spleen, 2: bowel, 3: heart, 4: lung, 5:
kidneys, 6: stomach, 7: liver, M: marker with bands at every 100 bp between 100-
1000 bp, then 1200 bp, 1500 bp, 2000 bp, and 3000 bp, 8: CL1-5 human lung cancer
cell line (0.5 μg), 9: nu/nu non-transplanted mouse, 10: nu/+ non-transplanted mouse.
(B): Corresponding loading controls using mouse GAPDH.
209
Figure 6-08. Sensitivity of PCR in amplifying human DNA sequence in the mouse
tissue. Equal amounts of DNA extracted from the liver tissue of a negative control
mouse (non-injured and non-injected with hfMSCs) admixed with various
concentrations of genomic DNA from human lung cancer cell line CL1-5. (A):
Detection of DNA sequence corresponding to human HLA-DQα1. Lanes 1: mouse
DNA (512 ng) + human DNA (24.6 ng), 2: mouse DNA (512 ng) + human DNA
(2.46 ng), 3: mouse DNA (512 ng) + human DNA (0.246 ng), 4: mouse DNA (512 ng)
+ human DNA (0.0246 ng), 5: mouse DNA (512 ng) + human DNA (0.00246 ng), M:
100 bp DNA ladder, 6: human DNA (24.6 ng), 7: human DNA (2.46 ng), 8: human
DNA (0.246 ng), 9: human DNA (0.0246 ng), 10: human DNA (0.00246 ng). (B):
Corresponding loading controls using mouse GAPDH in lanes 1-5.
Figure 6-09. Intrauterine transplantation model. E14-16 fetuses are approximately
1 cm in length (A) and hfMSCs were transplanted via a 50-μl Hamilton syringe and a
33G needle through the translucent uterine wall into fetal peritoneal cavity (B).
210
6.2.3.1. Pilot experiments
For the initial pilot experiments to validate the technique of IUT in the mdx and
mdx/nude colonies, I recruited the assistance of Dr. P. Guillot, who was experienced
with this procedure (Guillot et al., 2008a; Guillot et al., 2008b).
6.2.3.1.1. Intrauterine transplantation and postnatal muscle injury in mdx/nude mice
HfMSCs (sample BM 12w5d) were transplanted in utero into 6 pups of 1 pregnant
mdx/nude mouse, but only 2 were live born. These 2 mice (1 mdx nu/+ and 1 mdx
nu/nu) then underwent notexin injury to right TA muscle at postnatal day 19 and were
harvested 6 days following the injury to allow time for muscle regeneration (Figure 6-
10). Gross et al. showed that day 6 or 7 after notexin injury is the optimal time to
detect newly regenerated muscle fibres of donor origin (Gross and Morgan, 1999).
Notexin damage was chosen because it has been used to investigate the contribution
of donor cells to functional stem cells that can participate in muscle regeneration
(Collins et al., 2005; Collins et al., 2007; Gross and Morgan, 1999). The level of
hfMSC engraftment in injured TA muscle of mdx nu/nu was 4.7 ± 2.0 cells per
section (p=0.04) and gave rise to 1.8 ± 0.8 myofibres positive for both dystrophin and
lamins a/c per muscle section of the injured TA compared to 0.5 ± 0.2 cells per
section hfMSC engraftment with 0.4 ± 0.2 human myofibre regeneration per muscle
section of the non-injured leg (Figures 6-11 and 6-12). In the injured leg of mdx nu/+
mouse, 2.7 ± 1.2 cells per section engrafted, but did not form muscle fibres.
Immunostaining evidence of myogenesis is shown in Figure 6-11.
211
Figure 6-10. Experimental design of intrauterine transplantation and postnatal
muscle injury. Fetal mice underwent IUT of hfMSCs at E15 gestation. After birth,
mice underwent notexin injury to right TA muscles at postnatal day 19. Tissues were
harvested at postnatal day 25.
212
Figure 6-11. HfMSCs transplanted in utero underwent myogenesis in notexin-
damaged TA muscles. Human cells (white arrow) were detected by human-specific
lamins a/c antibody (red nuclei, A). Dystrophin-positive fibre (green muscle fibre
membrane, B) is also positive for spectrin (red muscle fibre membrane, A). DAPI
staining (blue) showed all nuclei present (C). Merged image shows co-localisation of
human nuclei with a myofibre of human origin in a TA cryosection (D). Original
magnification 200×.
213
Figure 6-12. HfMSCs transplanted in utero engrafted and underwent myogenesis
in notexin-damaged TA muscles. A greater number of hfMSCs engrafted (A) and
generated human myofibres (B) in injured than non-injured TA muscles and in TA
muscles from mdx nu/nu than those from mdx nu/+ mice. Kruskal-Wallis non-
parametric ANOVA used where * = p<0.05.
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6.2.3.1.2. Double transplantation in mdx mice
IUT of hfMSCs (sample FB 11w1d) marked with GFP and R-Luc were performed in
7 mdx pups in 1 pregnant female with 4 being live-born which underwent BLI at 2
days of age. The following day, 3 of the 4 mice that had undergone IUT and imaging
were transplanted intraperitoneally with a further 1 × 106 F-Luc-labelled hfMSCs. All
mice had repeat BLI at postnatal day 9 and their tissues harvested after imaging
(Figure 6-13). Figure 6-13B shows the presence of hfMSCs on BLI in limbs and tail 7
days post-IUT, while Figure 6-13E shows some presence of neonatally injected
hfMSCs 1 week following neonatal transplantation. On histological analysis, the level
of hfMSC engraftment was 1.1 ± 0.1 cells per section with no in vivo myogenesis in
the IUT only mouse, compared with 2.0 ± 0.2 hfMSC engraftment per section and 2.0
± 0.5 human fibres per section in the IUT followed by neonatal transplantation group
(Figures 6-14 and 6-15).
6.2.3.2. Optimisation
With the encouraging data of the pilot experiments, I set out to optimise the IUT
technique using the mdx/nude model. A total of 28 dams were transplanted over a 7-
month period, of which 6 dams were transplanted by Dr. P. Guillot and 5 dams
operated on by myself while under direct supervision of Dr. P. Guillot (Table 6-03).
Intrauterine transplantation procedures are known to be challenging with a steep
learning curve, and only a few groups reporting positive results. Although IUT has
been successfully performed and published with our group, mdx mice have yielded far
poorer results than wild type hosts, and in earlier work we observed only a 12% live
birth rate and a 7% survival-to-weaning rate (Chan et al., 2007). Many factors were
considered and experimented with in terms of reducing peri-operative mortality of the
transplanted fetuses which are summarised in Table 6-04 (Deacon, 2006).
Figure 6-13. Experimental design and bioluminescence imaging results of double
transplantation. Fetal mice underwent IUT of hfMSCs with R-Luc and GFP
reporters at E15 gestation (A). Two days after birth, mice were imaged and showed
presence of hfMSCs near limbs and tail (B). Neonatal transplantation of hfMSCs with
216
F-luc reporters was performed in 3 mice at postnatal day 3. All mice were imaged at
postnatal day 9. The presence of hfMSC-F-luc in the upper limb was evident (E).
Mice injected with PBS and hfMSCs subcutaneously an hour before imaging were
used as negative (Neg) (C, F) and positive (Pos) (D,G) controls, respectively. Mouse
tissues were harvested after imaging at postnatal day 9.
217
Figure 6-14. HfMSCs transplanted in utero and neonatally engrafted underwent
myogenesis in TA muscles. Human cells (white arrow) were detected by human-
specific lamins a/c antibody (red nuclei, A). Dystrophin-positive fibre (green fibre
membrane, B) is also positive for spectrin (red fibre membrane, A). DAPI staining
(blue) showed all nuclei present (C). Merged image shows co-localisation of
dystrophin and human spectrin in a myofibre in a TA cryosection (D). Original
magnification 200×.
218
Figure 6-15. Doubly transplanted hfMSCs engrafted and underwent myogenesis
in TA muscles. A greater number of hfMSCs engrafted (A) and generated human
myofibres (B) in TA muscles from mice transplanted twice than in utero only.
219
Table 6-03. Intrauterine transplantation experiment. Abbreviations: LB, litters
born; NVS, named veterinary surgeon; PG, Pascale Guillot; PW, Pensee Wu.
Dam Operator
(Supervisor)
Strain Gestation Fetus
injected
LB Outcome
1 PG Mdx E13 ------ ---- Wrong gestation
Not injected
2 PG Mdx E16 9 0 Wound dehisced
Died day 1
3 PW
(PG)
Mdx E16 8 0 Wound dehisced
Died day 1
4 PW
(PG)
Mdx E15 12 0 Wound dehisced
Culled day 1
5 PW
(PG)
Mdx Not
pregnant
------ ---- Culled
6 PW
(PG)
Mdx E14 8 0 Wound dehisced
Died day 1
7 PW
(PG)
Mdx E14 11 0 Wound dehisced
Died day 1
8 PW Mdx E15 7 0 No LB
9 PG Mdx E15 6 6 2 died at P13
10 PG Mdx/nude E15 2 0 No LB
11 PG Mdx/nude E15 6 2 1 nu/nu, 1 nu/+
12 PW
(PG)
Mdx/nude E15 8 0 Wound dehisced
Died day 3
13 PW Mdx E15 11 0 No LB
14 PW Mdx E15 9 0 No LB
15 PW Mdx E15 7 0 No LB
16 PW Mdx/nude E15 ------ ---- Anaesthetic
complications
Culled
17 PW Mdx/nude Not ------ ---- Culled
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pregnant
18 PW Mdx/nude E15 9 0 No LB
19 PG Mdx/nude E15 5 5 4 nu/nu, 1 nu/+
20 PG Mdx/nude E15 4 3 All nu/nu
21 PW Mdx/nude E15 11 0 No LB
22 PW Mdx/nude E15 9 0 No LB
23 PW Mdx/nude E15 12 0 No LB
24 PW Mdx/nude E15 13 0 No LB
25 PW Mdx/nude E15 11 0 No LB
26 PW Mdx/nude E15 8 0 No LB
27 PW Mdx/nude E16 10 0 No LB
28 PW Mdx/nude E15 9 0 No LB
29 PW Mdx/nude E18 12 0 Wound dehisced
Died day 2
30 PW Mdx/nude E11 ------ ---- Wrong gestation
Not injected
31 PW Mdx/nude E19 ------ ---- Wrong gestation
Not injected
32 PW Mdx/nude E15 ------ ---- Fetal resorption
Not injected
33 PW
(NVS)
Mdx/nude E15 12 0 Harvested day 7
Empty uterus
34 PW
(NVS)
Mdx/nude E16 8 0 Harvested day 2
Died in utero
35 PW
(NVS)
Mdx/nude E14 11 0 Harvested day 7
Empty uterus
Total 248 16
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Table 6-04. Summary of strategies for the improvement of live birth rate.
Possible factors Actions taken to minimise risks
Pre-operative Husbandry condition Sterile caging for immuno-
compromised strains
Environmental enrichment with
cardboard mouse shelter and soft
cloth nesting material
Transfer of animals into clean cages 2
days before IUT to allow time to
familiarise with environment
Environmental stress Handling by myself only from onset
of breeding
No handling of pregnant dams from
date of IUT to 1 week postpartum
Paired housing to allow social contact
Intra-operative Anaesthetic risks Separate anaesthetist monitoring
mouse vital signs during operation
Supported head tilt to maintain
patency of airway
Left tilt of body to alleviate pressure
of pregnant uterus on circulation
Infection Use of aseptic technique and
prophylactic enrofloxacin antibiotic
Surgical technique Live births achieved after sham
operation
Dye injection confirmed correct route
was followed after intra-peritoneal
administration
Hypothermia Use of intra-operative warming pads
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Dehydration Rehydration with sterile isotonic
saline to uterus and subcutaneous
injection intra- and post-operatively
Post-operative Analgesia Adequate vetergesic administered
intra- and post-operatively
Recovery 1 hour recovery under temperature
controlled environment
Wet diet given on floor of cage to
avoid need for excessive movements
to feed or drink
Cannibalism of the
young
Use of experienced ex-breeder
females
223
Initially, loss of the entire litter was presumed to be due to cannibalism of the
young. As the pregnant mice and any litters thus received enriched nesting and were
not touched until 1 week postpartum to allow maternal bonding, it was problematic to
see if there had, or had not been any live births without disturbing the mice. Advice
was sought from the named veterinary surgeon (NVS) of the animal facility and
cross-fostering at birth or following Caesarean section was thought to be the
necessary next step. However, as no live litters had been directly observed,
miscarriage with or without intrauterine death may have occurred post-operatively.
Hence, events following the IUT procedure were carefully scrutinised. One animal
was sacrificed at 7 days after IUT on the expected date of delivery in order to conduct
a post-mortem examination. The animal was healthy and demonstrated adequate
wound healing (Figure 6-16A). On entering the abdominal cavity, the uterus was
empty (Figures 6-16B and C) and there was no neonatal body remnants in the
stomach contents, which suggested cannibalism was unlikely. Following this
discovery, the next animal was examined 2 days after IUT. Again, there was evidence
of good wound healing (Figure 6-17A) but the uterus contained fetuses undergoing
maceration or shrinkage (Figure 6-17B and C).
A sham operation was performed where the abdomen was opened without
injecting fetuses intraperitoneally which yielded 8 live pups. However, this animal
died unexpectedly 1 month after IUT with minimal findings at the autopsy (Figure 6-
18A) except for a distended bladder. Cultures of swabs taken from the peritoneal
cavity grew coliform bacteria, Micrococcus and Enterococcus faecalis. Septicaemia
could not be presumed as cause of death since these organisms are commensals. The
cause of death is unlikely to have been due to the IUT procedure, as operative
mortality is defined as death within 30 days of an operation (Jacobs et al., 2006).
224
The route of fetal intraperitoneal injection was assessed using methylene blue
dye. This demonstrated correct intraperitoneal positioning of the dye in 6 out of 7
(86%) fetuses, and off target intra-thoracic placement of the substrate in only 1 out of
7 (14%) fetuses (Figure 6-18B). The low percentage suggests this is an unlikely cause
of in utero death. The introduction of using aseptic technique and prophylactic
antibiotics eliminated the growth of Enterococcus faecalis from post-mortem
intrauterine swab cultures, though coagulase-negative Staphylococcus continued to be
prevalent.
225
Figure 6-16. Gross dissection of mdx nu/+ mouse 7 days following intrauterine
transplantation procedure. There is adequate wound healing (A) but empty uterus in
situ (B) and magnified (C).
226
Figure 6-17. Gross dissection of mdx nu/+ mouse 2 days following intrauterine
transplantation procedure. There is adequate wound healing (A) and the uterus (B)
contains macerated or shrinking fetuses (C).
Figure 6-18. Post-mortem examination of mice following intrauterine injection.
(A) Mdx nu/+ mouse with unexpected mortality 1 month after IUT showing a
distended bladder and macerated organs. (B) Fetuses following intrauterine dye
injection to illustrate correct intra-peritoneal positioning of dye in 6 from 7 cases.
227
6.3. Discussion
6.3.1. Adult and neonatal transplantation
HfMSC engraftment was obtained following intramuscular transplantation into the
TA muscle of adult mdx/nude mice, albeit at limited levels. This could be improved
by injury paradigms, in particular using cryodamage which achieved 2.0 ± 1.2 cells
per section. By administering hfMSCs earlier in life during the neonatal period,
engraftment was 1.2 ± 0.7 cells in the cryodamage group, even after systemic
administration.
In these adult and neonatal transplantation experiments, cryodamage induced the
highest level of hfMSC engraftment, which can be explained by the inherent
differences in methods of muscle injury. Among the three protocols, cryodamage
caused the greatest amount of injury as it destroys both mature muscle fibres and
satellite cells, whereas irradiation only inhibits satellite cell regeneration and thus
yielded the lowest level of hfMSC engraftment. HfMSCs may stimulate the activation
of host satellite cell via paracrine mechanisms, or may replenish satellite cell or MPC
pools. Human adult synovial membrane MSCs have been shown to contribute to
functional satellite cells in vivo (De Bari et al., 2003) Theoretically, after MPC
transplantation into irradiated mouse muscle, if any of the implanted MPCs remained
within the host muscle as undifferentiated myogenic cells able to give rise to new
muscle at later times, they would presumably be activated only when and where
necrosis occurs. Necrosis is, however, sporadic and focal in mdx muscles (Carnwath
and Shotton, 1987; Coulton et al., 1988). By additional notexin injury to cause whole
muscle degeneration in irradiated muscles, the level of de novo myogenesis was
increased possibly by increasing the likelihood of activating rare reserve myogenic
cells (Gross and Morgan, 1999) or satellite cells of donor origin (Collins et al., 2005).
228
Therefore, the same argument could be used for cryodamage which causes a similar
extent of injury to that from a combination of irradiation and notexin. Surprisingly,
the highest rate of hfMSC engraftment occurred in cardiac muscle compared with
diaphragm and TA muscle, though minimal pathological changes exist in the mdx
heart except in old age (Quinlan et al., 2004). A possible explanation is that hfMSCs
engraft better in cardiac muscles than in skeletal muscles. It is well documented that
adult and fetal MSCs can engraft in both skeletal and cardiac muscles following
systemic administration, though the engraftment efficiency in different uninjured
tissues has not been reported (Chan et al., 2007; Mackenzie and Flake, 2001;
Mackenzie et al., 2002).
The low level of cell engraftment contributed to the lack of in vivo myogenesis.
As the same cell sample was used in all animal work except for the two pilot IUT
experiments, the properties of this particular sample may not be particularly
favourable for animal work in terms of migration and immunogenicity. As hfMSCs
have not been screened for infectious agents (e.g. hepatitis B and C), this sample
might even be toxic in the immunotolerant fetal milieu, leading to the fetal deaths
seen following IUT during the final part of this work, despite giving no adverse
outcomes in the neonatal and adult periods. This highlights the inherent variation
between hfMSC samples and suggests that in vitro characterisation of samples cannot
fully predict their behaviour in vivo. More research is needed to develop novel
methods to correlate in vitro cell properties with those in vivo.
6.3.2. Intrauterine transplantation
HfMSCs transplanted in utero alone achieved in vivo muscle differentiation of modest
degree. IUT of hfMSCs followed by postnatal notexin injury in mdx/nude mice
achieved an engraftment rate of 4.7 ± 2.0 nuclei per section and gave rise to 2.9 ± 1.4
229
fibres per section, while double transplantation in mdx mice attained a level of 2.0 ±
0.2 hfMSC engraftment per section and 2.0 ± 0.5 newly regenerated fibres per section.
In comparison with published data on hfMSC engraftment in TA muscle
following IUT, my result of 2.7 ± 1.2 cells per section at postnatal day 25 in notexin-
injured mdx nu/+ muscle is lower than 7.05 ± 1.80 cells per section shown in non-
injured mdx muscles between postnatal 6-18 weeks (Chan et al., 2007). However, due
to the aforementioned reasons and high loss rate detailed herein, this result was
available for one animal only, and increased numbers would be needed for more
meaningful interpretation. Furthermore, the differences in mouse models (mdx
compared to mdx nu/+ and nu/nu) may account for the discrepancies between my
work and that of Chan et al. (Chan et al., 2007).
In terms of efficiency in inducing in vivo myogenesis, double transplantation of
hfMSCs appeared better than IUT, which itself was more effective than neonatal
transplantation alone. My finding is in agreement with Kim et al. who demonstrated
that IUT is superior to neonatal transplantation in terms of donor HSC chimerism in
an allogeneic setting (Kim et al., 1998). Moreover, Milner et al. conducted serial HSC
transplantations in congenic neonatal mice that had undergone fetal transplantations
whilst in utero and showed a significant increase in donor cell engraftment to those
that had undergone IUT only (Milner et al., 1999). Postnatal muscle injury enhanced
the number of hfMSCs that contributed towards muscle regeneration, as the injury
may have encouraged hfMSCs to proliferate and participate in tissue repair. Tissue
damage facilitates engraftment and differentiation. In non-injury models, human
MSCs home and engraft in tissues at only very low levels, but can be recruited from
remote storage sites to areas of wound healing (Liechty et al., 2000; Mackenzie and
Flake, 2001). In my neonatal transplantation experiments, hfMSC detection was 24-
230
fold times greater in TA muscle of mice which underwent cryodamage (1.2 ± 0.7)
compared to the control (0.5 ± 0.02). Using a murine model of OI with in utero bone
fractures, Guillot et al. achieved a significant amelioration of the oim phenotype even
with a relatively low level of donor hfMSC chimerism (~5%) following IUT (Guillot
et al., 2008a), which is in keeping with clinical case reports on phenotypic
improvement with low engraftment (2-7%) in patients with OI (Horwitz et al., 1999;
Horwitz et al., 2001; Le Blanc et al., 2005). They also found much higher hfMSC
engraftment at fracture sites where active bone formation and repair occur, which is
consistent with donor cell mobilisation from their reservoirs, such as BM, to the sites
of acquired tissue injury (Guillot et al., 2008a). In contrast, the mdx model lacks overt
pathology at the time of IUT treatment.
6.3.3. Mdx animal model
Past experience in our laboratory has shown that mdx mice are not as robust compared
to wild type in terms of survival post-IUT, with a 12% live birth rate and a 7%
survival-to-weaning rate (Chan et al., 2007). Furthermore, the mdx/nude appears to be
an even more delicate strain for IUT and there have been no reports of successful IUT
in this model within the literature. Breeding colonies are more difficult to establish in
mdx/nude mice compared with mdx mice (E. England, personal communication, 2007)
and even in the hands of an experienced operator, there was a high fetal loss rate
(Table 6-03). Female nude mice have lower serum levels of oestradiol, progesterone
and thyroxine compared to the wild type, which leads to their poor fertility state
(Kopf-Maier and Mboneko, 1990).
231
6.3.4. Antibody
Problems were encountered with some of the primary antibodies used in
immunohistochemistry. As seen in the positive control figures (Figure 6-01A-D), the
lamins a/c antibody was not very sensitive in identifying all human nuclei, though it
had high specificity. This antibody did not produce a false positive result so continued
to be used in all immunostaining work and indeed has been used extensively by others
to detect human nuclei in mouse xenografts (Brimah et al., 2004; Chan et al., 2007;
Cooper et al., 2003; Silva-Barbosa et al., 2005). In hindsight, perhaps an alternative
antibody, such as human-specific vimentin, could have been chosen to better detect
the human cells (Figure 3-05A-C) (Dekel et al., 2006; Mirmalek-Sani et al., 2006).
However, vimentin staining is cytoplasmic rather than nuclear, and could lead to
identification problems with double antibody stains with spectrin or dystrophin, as
they stain around the muscle fibres. In the double transplantation experiment, anti-
GFP antibody was utilised to avoid the potential confusion between skeletal muscle
autofluorescence and GFP fluorescence (Jackson et al., 2004). The anti-GFP antibody
was found to stain poorly in muscle cryosections as the sections were not immediately
fixed after cutting and the GFP leached out rendering it difficult to detect in muscle
fibres (data not shown). Therefore, future work may preferentially utilise the β-
galactosidase gene as the reporter instead of GFP since it does not have the
confounding problem of autofluorescence, and in addition has been well validated
(Cousins et al., 2004; De Bari et al., 2003; Ferrari et al., 1998; Zammit et al., 2004a) .
6.3.5. Conclusion
Overall, hfMSCs engrafted in mdx/nude model, which is improved by earlier (in utero
> postnatal > adult) cell transplantation. Engraftment and differentiation was further
enhanced by injury paradigms and double transplantation. The mdx/nude model
232
proved to be a challenging strain to establish IUT. In the future, if the IUT procedure
can be reproducibly established in the mdx/nude colony, further research would
involve IUT followed by postnatal cryodamage and serial transplantations in adult life
in addition to IUT and neonatal transplantations as well as refinements in the use of
BLI technique.
233
CHAPTER 7
GENERAL DISCUSSION
234
7.1. Implications
With its unrelenting and progressive pathology, the search for a cure in DMD
continues. Many therapeutic strategies for DMD have been investigated, including
cell therapy, gene therapy, or a combination of both. The self-renewing and
multilineage differentiation properties of stem cells may contribute to the
development and maintenance of muscle tissues during postnatal turnover and their
regeneration following damage. To be able to treat DMD efficiently, stem cells must
either repair existing or form new muscle fibres with sufficient dystrophin expression.
While endogenous satellite cells can regenerate muscle fibres, they lose function with
time. Therefore, donor stem cells must also replenish the host satellite cell
compartment with functional stem cells. In the past, many stem cell populations have
been investigated in terms of their potential to treat DMD, mostly with limited success
(Price et al., 2007). A relatively new cell source, hfMSC, has been further explored in
my study following encouraging pilot data (Chan et al., 2006a; Chan et al., 2007).
This work confirms that hfMSCs can be harvested and expanded to a large scale
for cell transplantation, and is supported by other literature (Chan et al., 2007; Guillot
et al., 2008a; Guillot et al., 2007). Their ability to contribute to satellite cells is
evidenced by the expression of Pax7 protein in hfMSCs in vitro (Chapter 3.2.3).
However, their ability to undergo Gal-1-induced myogenesis is more modest than
previously suggested (Chan et al., 2006a). The role of Gal-1 in myogenesis is
intriguing, as differentiating myoblasts release Gal-1 upon myotube formation, while
proliferating ones do not (Camby et al., 2006; Cooper and Barondes, 1990; Harrison
and Wilson, 1992). Gal-1 is amply present in adult skeletal muscles, mainly in
activated satellite cells, after muscle injury (Kami and Senba, 2005). The abundant
level of intracellular Gal-1 may contribute to the ability of hfMSCs to undergo muscle
235
differentiation. While exogenously supplemented Gal-1 did not promote further
myogenesis, instead successful differentiation was achieved by perturbing intrinsic
Gal-1 expression in hfMSCs. For the purpose of cell therapy, both exogenous Gal-1
supplementation and Gal-1 transduction can be ruled out as potential inducers of
significant myogenesis in hfMSCs, due to their low efficiency in achieving hfMSC
myogenic conversion. Further identification of key regulators of myogenic
commitment in hfMSCs such as MyoD, myogenin and myf5 (Chan et al., 2006a) may
allow their manipulation to increase their ability to participate in muscle regeneration
or contribute to the pool of muscle satellite cells. MyoD over-expression has been
used to induce fibroblasts down the myogenic lineage (Qin et al., 2007).
My co-culture experiments have shown a possible mechanism for muscle repair
by in utero transplanted hfMSCs. Myoblasts harvested from fetal mdx/nude mice at
E15 gestation were able to fuse with hfMSCs and form chimeric myotubes. In other
words, hfMSCs were capable of fusing with MPCs that would have been present
within the fetal muscles at the time of hfMSC IUT. Although most studies have
shown that the ability to regenerate muscle after injury is either equivalent or superior
in mdx compared with non-dystrophic hindlimb muscles, at least in terms of histology
(Itagaki et al., 1995; Mechalchuk and Bressler, 1992; Pastoret and Sebille, 1995),
there are some discrepancies in the literature (Irintchev et al., 1997b). In mdx masseter
muscle after degeneration, the regenerated muscle fibres acquire characteristics
entirely different from their normal counterparts. Mdx mice did not express myosin
heavy chain-2b (fast-type isoform) as strongly as in the control mice at the
transcriptional level but strongly expressed slow-type myosin heavy chain-1 which
was rarely observed in the control mice but did not allow rapid chewing (Lee et al.,
2006c). Therefore, hfMSCs may modify regenerated muscles upon fusing with mdx
236
myoblasts. For example, hfMSCs may serve to strengthen the myotubes formed as
hfMSCs are harvested from disease-free individuals and should therefore produce
dystrophin. Research has shown that after injection of normal MPCs into irradiated
mdx TA muscles, there was a prolonged survival of dystrophin-positive myotubes
compared to dystrophin-negative ones (Morgan et al., 1993). On the other hand,
hfMSCs could fuse into multinucleated myotubes, yet not produce dystrophin, as has
been shown for human MPCs (Gussoni et al., 1997). BM-derived SP stem cells have
been shown to incorporate into both skeletal and cardiac muscle upon transplantation
into mice without δ-sarcoglycan, a model of cardiomyopathy and muscular dystrophy,
but were unable to express sarcoglycan and repair tissue (Lapidos et al., 2004). In
autologous applications, hfMSCs may exert their effects via the paracrine system or
provide a supportive niche for regeneration akin to adult MSCs (Chen et al., 2008;
Kinnaird et al., 2004).
My in vivo studies suggest that earlier (in utero > postnatal > adult) cell
transplantation improves engraftment and differentiation, which supports the
rationales of increased tolerance to foreign antigens in utero and pre-emptive
treatment to prevent morbidity. IUT has been shown to lead to more HSC engraftment
than neonatal transplantation in an allogeneic mouse model (Kim et al., 1998). The
double transplantation experiment in this thesis consolidates the hypothesis of
inducing tolerance to foreign antigens while in utero to allow a high-dose cell
injection during the neonatal period. This implies that further repeated injections
during adult life may be also beneficial as is the case with serial HSC transplantations
(Milner et al., 1999).
Though the low levels of engraftment observed are unlikely to furnish a
therapeutic effect in the mdx mouse, this system may benefit other inherited diseases
237
such as the mucopolysaccharidoses and haemophilias, since low levels of enzymes or
clotting factors, even in the order of 1-5%, may allow attenuation of disease
pathology (Birkenmeier et al., 1991; Kaufman, 1999; Marechal et al., 1993; Moullier
et al., 1993; Wolfe et al., 1992).
Engraftment and in vivo differentiation were greater with IUT compared with
transplantation at other times, while postnatal injury further enhanced the effect of
IUT. Although hfMSCs were not detected in non-muscle tissues due to the low
sensitivity of PCR, it is still possible that hfMSCs may be attracted and migrate
towards sites of injury in a similar fashion to adult MSCs which are capable of
homing to BM as well as migrating and engrafting into various tissues following
systemic infusion (Devine et al., 2001; Devine et al., 2003; Koc et al., 2000). In rat
models, rat MSCs injected intravenously or intra-arterially migrate into neuronal
tissue and reduce functional deficits after stroke (Chen et al., 2001), whilst they
migrated and engrafted into ischaemic myocardium following intravenous delivery
after myocardial infarction (Barbash et al., 2003). Intra-articular delivery of
autologous goat MSCs can engraft in and repair damaged meniscus and cartilage of
goat osteoarthritic knee joints (Murphy et al., 2003). The chemotactic ability of
human adult MSCs has been attributed to their wide chemokine receptor expression
profile, which includes SDF-1 (Lee et al., 2006c). MSCs secrete SDF-1 and the
interaction of SDF-1 with its receptor, CXCR4, increases survival of progenitor cells
(Zhang et al., 2007b). For example, the naturally occurring regenerative process in
post-infarcted myocardium may be enhanced through the over-expression of SDF-1
within the myocardium (Ringe et al., 2007).
The successful use of BLI for cell tracking in live animals can be utilised to
investigate the homing and migration routes of injected hfMSCs in vivo. BLI has good
238
signal intensity due to stable transfection of cells with a light producing enzyme and
continuous expression of the reporter gene, hence only viable and surviving cells are
detected. It has been used to detect tumour growth (Beilhack et al., 2005; Cao et al.,
2005; Contag and Ross, 2002) as well as for tracking stem cells following
transplantation in cell therapy (Cao et al., 2006; Guillot et al., 2008a; Guillot et al.,
2008b; Olivo et al., 2008; Toegel et al., 2008). My study has shown that by using
different chemoluminescence reporters (F-Luc and R-Luc), separate cell populations
and their interaction with each other could be examined with different substrates. This
is supported by Bhaumik et al. who transfected C6 rat glioma cells with either F-Luc
or R-Luc, transplanted these two cell populations in contralateral sites of same mouse
and showed distinct kinetics from the reporter proteins which distinguished between
the separate populations (Bhaumik and Gambhir, 2002). Dual BLI has also been used
to monitor the effects of R-Luc expressing therapeutic proteins in F-Luc expressing
gliomas (Shah et al., 2003). However, BLI is unlikely to be useful in quantitative
research in the foreseeable future due to confounding factors of signal attenuation
between different tissues (Rice et al., 2001), postsurgical wound healing affecting
overlying tissue thickness, animal positioning, light source location, site of cell or
reporter substrate (D-luciferin or coelenterazine) administration (Virostko et al., 2004).
Many stem cell sources have been investigated as candidates for DMD cell
therapy (Chapter 1.2.4.4). Compared to adult MSC, the enhanced proliferation and
plasticity of hfMSC make it a valuable cell type. With the advent of improved FB
sampling techniques and the isolation of MSCs from placenta (Battula et al., 2007;
Portmann-Lanz et al., 2006) and AF (In't Anker et al., 2003b; Kaviani et al., 2003;
Tsai et al., 2004), hfMSCs are likely to represent an autologous cell source for therapy.
Further research is now warranted to understand the signalling pathways underlying
239
the generation of MPCs or satellite cells from hfMSCs so that their potential in
muscular dystrophy therapy can be fully evaluated.
7.2. Limitations
It took considerable time to build up and fully characterise my hfMSC cell bank with
representative samples from different gestational age and sources. This was hampered
by a nitrogen tank malfunction half-way through the project, such that further samples
needed to be re-collected and re-characterised, which contributed to the restricted cell
profiles investigated in the latter part. First trimester FB samples were particularly
difficult to collect due to patient reluctance to consent, as it involved an additional
invasive procedure to termination of pregnancy, which women understandably often
decline during a period of personal turmoil.
The prevailing problem during in vitro myogenesis of hfMSCs (Chapter 3) was
the inability to reproduce earlier results by a previous worker in the same group (Chan
et al., 2006a). There might have been subtle changes in the protocol due to operator
difference. It was not possible to compare the protocol on the earlier hfMSC samples
previously used successfully due to the same nitrogen tank accident which left only
one such sample at a very late passage. Essentially, the level of myogenic conversion
using Gal-1 was considerably less than previously achieved in the same laboratory in
terms of positive desmin staining (34% versus 66%), though the percentage of
samples capable of muscle differentiation was similar (50%) (Chan et al., 2006a).
This finding might be due to the different hfMSC samples used, which could give rise
to heterogeneous cell populations. However, these issues are relevant to potential
clinical treatment, as it is important to account for cell or operator differences in order
to develop robust and reproducible therapeutic protocols.
240
During the IUT experiments, the mdx/nude colony had low fecundity and it was
a challenge to establish a steady supply of pregnant females over duration of the study,
as breeding colonies are more difficult to establish in mdx/nude mice compared with
mdx mice (E. England, personal communication, 2007). The 19 successfully pregnant
females used in my IUT experiments were from a total of 79 littermates (males and
females) produced over 4 months by 11 mothers as a result of mating 30 females.
Pregnant mdx/nude females produced nu/+ (immunocompetent) and nu/nu
(immunocompromised) offspring in a ratio approximating to 50:50. However, only
the nu/+ female offspring (roughly a quarter of each litter) can be utilised in adult life
as pregnant dams for IUT since nude females are not robust mothers and do not
lactate as well (Nagasawa and Yanai, 1977). This is further compounded by the well
known technical difficulties of IUT. In wild type mice, the rate of survival post-IUT
has been reported as between 30-70% (Carrier et al., 1995; Chan et al., 2007; Gregory
et al., 2004; Kim et al., 1998; Pixley et al., 1998), while the survival rate in mdx mice
is reduced to 12% (Chan et al., 2007) or <10% (T. Mackenzie, personal
communication, 2004). Therefore, it is perhaps unsurprising that it proved a challenge
to establish the IUT procedure in the mdx/nude, a more delicate strain compared with
the mdx, as evidenced by the paucity of publications on this subject.
In terms of in vivo differentiation, human spectrin antibody cross-reacts with
regenerating murine tissue (J. Morgan, personal communication, 2006). This may
explain the apparently high level of myogenesis achieved in our group previously,
where GALM pre-differentiated hfMSCs formed spectrin-positive fibres in mdx/scid
mice (32.8 ± 20.2 per section) compared to none formed using naïve hfMSC. Another
possibility is cell fusion, where donor cells fuse with recipient cells, adopt their
phenotype without genetic reprogramming but is mistaken for transdifferentiation
241
(Terada et al., 2002; Wang et al., 2003; Ying et al., 2002). For example, Krause et al.
showed a single BM cell could give rise to multiple cell types, from gut to lung to
skin (Krause et al., 2001), but another group was unable to obtain such a wide range
of cell types from one BM cell and refuted the notion of transdifferentiation (Wagers
et al., 2002). The physical integration of BM cells to myotubes is well documented
(Bittner et al., 1999; Ferrari et al., 1998; Gussoni et al., 1999), which occurs to a
greater extent in MSCs than HSCs (Shi et al., 2004) and may be an essential step in de
novo muscle regeneration (Lee et al., 2005).
No single isolation method of MSC is regarded as the gold standard in this field.
Human adult MSCs exhibit some variation in their pattern of expressed genes among
different donor preparations using the same isolation protocols, and larger variations
occur as sparse cultures become confluent and are expanded by serial passage while
approaching senescence (Gregory et al., 2005). MSCs from different donors exhibited
different responses to osteogenic differentiation induced by dexamethasone, such that
osteogenic gene expressions varied on real-time PCR analysis (Siddappa et al., 2007).
In murine MSCs, the increase in proliferative response to serum stimulation was
greater in cultures from younger than older subjects (Bergman et al., 1996). Apart
from donor variation, differences also exist between early and late cultures from the
same donor. In day 2 cultures of human BM-MSCs, upregulated genes were mainly
involved in cell division, whereas genes upregulated on day 7 primarily affected
development, morphogenesis and physiological processes, according to microarray
and real-time PCR data (Larson et al., 2008). Furthermore, variations are present in
the colony forming efficiency, proliferative capacity and osteogenesis depending on
the method of harvest (Phinney et al., 1999).
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Many publications have overlooked these subtleties and assumed homogeneous
cell populations were present in high density and confluent MSC cultures. Similarly,
there is an inherent variation in hfMSCs collected from different individuals,
demonstrated herein by a disparity in efficiency of bone, fat and cartilage
differentiation. This challenge may be overcome by using a well characterised clonal
population at early passage, to prove that the observed results are not due to
contamination by rare populations of more multipotent cells. On the other hand, it is
important to ensure hfMSC populations have broadly similar properties in terms of
the development of autologous hfMSC applications, as individual patient-tailored
treatment precludes the use of a ready-characterised clone. HfMSC colonies do not
grow well from single cells with or without mitomycin-treated feeder layers of
hfMSCs (Kennea, 2007). A single-cell-derived-clone was obtained by growing a
GFP-expressing male cell on a feeder layer of senescent female cells, though this was
achieved over a period of six weeks (Kennea, 2007). Due to the inefficiency in
isolation and expansion of a clonal population at present, it would be impractical to
contemplate characterising each clone prior to autologous transplantation.
Failure to reproduce earlier in vivo work is not only not uncommon but also
topical in stem cell biology. For example, mouse neural stem cells were thought to
transdifferentiate into haematopoietic cells (Bjornson et al., 1999). However, this was
not replicable despite 2.5 years of work by a separate group (Morshead et al., 2002).
Another example is multipotent adult progenitor cells (MAPCs) which were shown to
contribute promisingly to all the major cell types of chimeric mice, including brain,
heart, BM, skin, blood and lung when injected into developing mouse embryos (Jiang
et al., 2002). Notwithstanding this, many other groups (Serafini et al., 2007) have
failed in trying to reproduce the MAPC work.
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7.3. Future research directions
Although many properties of hfMSCs are known, there remains a need to define the
characteristics of this cell type that are critical for their stem cell properties. As
hfMSC is a relatively novel cell population, it may be prudent to compare data with
adult MSCs, due to the greater literature and thus knowledge base in the adult field.
The properties of hfMSCs from different gestations need to be critically evaluated to
determine the optimal gestation of cell harvest. Varying sources of hfMSCs, e.g. FL,
BM and FB, may exhibit distinct characteristics and preferences towards site of
migration and engraftment according to the site of harvest. Other factors to consider
are the effects of increased time in culture on proliferation and plasticity, and the
development of serum-free medium to standardise culture conditions, which has been
used successfully in adult MSCs (Meuleman et al., 2006). There remains a critical
need to identify specific markers of hfMSCs, as for adult-derived MSCs, to ensure
homogeneous cell populations have been studied and compared.
In terms of pre-differentiation of hfMSCs down muscle lineage, protocols which
have been used in other cell types can be explored. For example, the use of Wnt
proteins, which are known to play an important role in muscle differentiation
pathways (Polesskaya et al., 2003), or biophysical methods such as altering
consistency of culture matrices (Engler et al., 2006) can be investigated. The
mechanism of in vitro myogenic differentiation may be explored with
immunolabelling for not only desmin and MF20, but also the intermediate MRF such
as MyoD and myogenin, which are important in myoblast determination and myotube
formation. Once a robust method of myogenesis has been determined which is
efficient in the majority of hfMSC populations, then a clonal population could be
selected for further in vivo work. Different muscle injury protocols following
244
transplantation can be examined, where factors including timing of injury, repeated
injury, combination of injury protocols and timing of tissue harvesting require further
consideration. Also cardiomyopathy is a major cause of mortality in DMD, so it
would be pertinent additionally to investigate ways of differentiating hfMSCs into
cardiomyocytes. To date there has been only limited success in differentiating adult
BM-MSCs into cardiomyocytes using 5-azacytidine. However, past experience in my
group has demonstrated cytotoxic effects of 5-azacytidine on hfMSCs. Other
published strategies used in adult BM-MSCs include co-culture with murine
cardiomyocytes (Wang et al., 2006) and use of differentiation medium containing
basic fibroblast growth factor and activin A (Zhao et al., 2005).
An additional co-culture experiment of primary mouse satellite cells and hfMSCs
may be of interest. Though satellite cells would not have been present at the time of
IUT, it would elucidate whether hfMSCs have any effects on the proliferation and
differentiation of satellite cells, such as by contributing to the satellite cell pool
themselves or by promoting mouse satellite cell quiescence, proliferation and
differentiation or a combination of the above. In terms of clinical feasibility, hfMSCs
co-cultured with human fetal myoblasts may determine whether hfMSCs could
differentiate into and/or fuse with human muscle in the fetal milieu.
Given the wide variation between different animals, in vivo data should be based
on work in a large number of animals in order to establish a statistically significant
trend (Collins et al., 2005). More robust methods to determine in vivo differentiation
in disease models are needed as some benefits of adult MSCs appear to be from
paracrine effects or cell fusion by providing a niche, akin to the embryological role of
MSCs in supporting HSCs, rather than from differentiation.
245
The mdx mouse is a genetic and biochemical homologue with a lack of
dystrophin but the phenotype is less severe than human DMD (Chapter 1.2.3.1). There
is little impairment of muscle strength and mechanical function in mdx mice and
cardiomyopathy, muscle damage and fibrosis are only present in aged animals
(Bulfield et al., 1984; Collins and Morgan, 2003). Therefore, the selective advantage
for engrafted normal cells expressing dystrophin, would be expected to be greater in a
model that closely reflects the severity seen in the human disease, for example the
utrophin and dystrophin DKO mouse model (Chapter 1.3.3.2) or the GRMD dog
model (Sampaolesi et al., 2006).
My data suggest that double transplantation improves cell engraftment. Future
experiments using a higher number of animals undergoing repeat cell transplants in
adult life can be used to validate whether a dose-response effect exists. Serial HSC
transplantation was effective in wild type mouse models (Hayashi et al., 2004;
Hayashi et al., 2002). However, when this strategy was used in four human fetuses
with inborn errors of metabolism or haematological disease, who were transplanted
with fetal cells before the maturation of their own thymus, though tolerance was
obtained, the persistence of donor cells was lower than that in patients with
immunodeficiency disease and was not sufficient for full tolerance (Touraine et al.,
2005).
The effects of IUT on mice throughout life can provide insight to whether
hfMSCs have the ability to survive without rejection and provide therapeutic benefits
on a long term basis, such as 1-2 years. The pathological features mimicking DMD
such as cardiomyopathy are more prominent in older mdx mice (Muller et al., 2001;
Quinlan et al., 2004). Organs such as diaphragm and skeletal muscle could be
investigated to detect whether the pattern of increased engraftment in heart compared
246
to other tissues persists into old age. Further experiments to examine muscle strength
and function in transplanted mice would provide clinically relevant data.
However, any benefits conferred by transplanted hfMSCs may be affected by the
aging niche. Aged skeletal muscle regenerate poorly and muscles are replaced by
fibrous connective tissue and adipose tissue (Conboy and Rando, 2005; Grounds,
1998; Sadeh, 1988). When old muscle is exposed to a youthful environment by means
of parabolic pairing of young and aged mice sharing a common circulation, the aged
muscle showed improved regenerative responses (Conboy et al., 2005) where Wnt
inhibition is thought to play a role (Brack et al., 2007). On the other hand, in
experiments conducted using satellite cells from aged mouse myofibres, a subset of
satellite cells survived the effects of aging and were able to regenerate and self-renew
as effectively as those from young mouse myofibres (Collins et al., 2007). HfMSCs
may possess similar characteristics to satellite cells in the aged environment, and
research is needed to explore this avenue further.
IUT is a promising treatment approach for different disease models such as gene
or cell therapy for haemophilia (Waddington et al., 2003) and various enzyme
deficiency syndromes such as the mucopolysaccharidoses (Casal and Wolfe, 2001)
and lysosomal storage diseases (Barker et al., 2001; Shen et al., 2004). The
identification of hfMSC raises the possibility of utilising these cells for IUT in
inherited diseases of mesenchymal origin. HfMSCs, in contrast to HSCs, have
extensive ability to self-renew and expand readily into clinically useful numbers for
cell therapy. In addition, they circulate in the first trimester of gestation and thus may
be amenable to isolation in ongoing pregnancies, ex vivo genetic manipulation and
reinfusion into fetuses with inherited mesenchymal disorders. Optimisation of
amniocentesis and CVS techniques for the harvest of AF-derived and placenta-
247
derived MSCs from ongoing pregnancies would allow the use of fetal MSCs in
autologous cell therapy with very few ethical concerns. Further research could address
unresolved questions, such as the optimal time point of transplantation, dose of
injected cells, role of immune effector cells in the host as well as intrauterine intra-
muscular cell injections.
In terms of cell tracking and using BLI, alternative methods of marking hfMSCs
could be used instead of a lentiviral vector. Although integrating vectors, such as
onco-retrovirus or lentivirus, have been favoured as a method of gene delivery
because they confer long-term delivery of the desired transgene to target cells, there
are considerable risks associated with the viral approach. Insertional mutagenesis, a
rare but possible consequence of the disruption of genetic sequences within the
genome, may occur via the inactivation of a tumour suppressor gene or activation of
an oncogene within the genome. The occurrence of leukaemia in four of ten children
treated in the French trial (Kohn et al., 2003) and in one of ten children from the
London trial (Thrasher, 2007) due to insertion of the transgene near a proto-oncogene
has raised serious concerns. Several lines of investigation are currently being pursued
to reduce this risk, such as the use of insertional site screening prior to re-infusion, the
use of safer and/or site-specific vectors and the use of regulable vectors which could
be switched off should an adverse event arise. Furthermore, the developments of
integrating non-viral vectors such as meganucleases or transposons are being studied
for use in hfMSCs.
7.4. Conclusion
Human fetal mesenchymal stem cells are an attractive class of stem cells for therapy
because of their reduced immunogenicity and greater differentiation potential
248
compared with adult cells, yet they do not cause tumour formation. Gene therapy
using transduced fetal stem cells utilises their self-renewing properties to avoid
repeated gene vector administration, and if combined with IUT, provides the exciting
prospect of prenatally treating genetic diseases using autologous cells.
My project demonstrated that hfMSCs are easily isolated and expandable with
the ability to undergo myogenesis. Co-culture experiments provided an in vitro model
for the underlying mechanism for muscle differentiation of hfMSC in vivo following
IUT. Engraftment and differentiation of hfMSCs were enhanced using strategies such
as postnatal injury and double transplantations. This provides the background for
further studies to optimise engraftment and differentiation after cell transplantation to
underpin future clinical applications for fetal stem cells.
Intrauterine stem cell therapy offers the possibility of cure for many devastating
congenital diseases that have been diagnosed prenatally. The complexity of hfMSC
transplantation demands the close collaboration of different branches of medicine.
The concept is attractive but is associated with complicated ethical issues. At present,
the outcome after IUT in animal models is uncertain for all models except OI. Based
on the animal work and the human experience to date with fetuses suffering from OI,
OI would appear to comprise a more interesting and translatable candidate group in
the short term for IUT than muscular dystrophy.
249
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APPENDICES
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Appendix 1 – Patient information sheets and consent forms
(Version for patients undergoing multifetal pregnancy reduction)
Information Sheet for Research Participants You will be given a copy of this Information Sheet Study title: Biology of fetal stem cells We would like to invite you to participate in a research study. If you decide to take part, please let us know beforehand if you have been involved in any study during the last year. Ask us if there is anything that is not clear or if you would like more information. Take time to decide whether or not you wish to take part. Thank you for reading this. What is the purpose of the study? Stem cells are underdeveloped cells that have the potential to repair damaged tissues or treat inherited diseases. We are trying to see what type of stem cells are present in fetal blood or tissues, and how they compare with other sources of stem cells. Why have I been chosen? You are being asked because you have a multiple pregnancy and are due to undergo a selective reduction of one or more of the fetuses. We would like permission to use any fetal blood collected at that time. Do I have to take part? It is up to you to decide whether or not to take part. If you do decide to take part you will be given this information sheet to keep and be asked to sign a consent form. If you decide to take part you are still free to withdraw at any time and without giving a reason. A decision to withdraw at any time, or a decision not to take part, will not affect the standard of care you receive. What will happen to me if I take part? Unless you request otherwise, the few drops of fetal blood that would be taken at the time of the needling procedure would normally be disposed of after the operation. We ask your permission instead to allow fetal blood taken at the time of termination to be used for research. The use of this tissue will be strictly controlled according to official guidelines and restricted to uses for which fetal tissue is necessary for medical benefit. The stem cells we derive will then be thoroughly tested and evaluated in the laboratory. Occasionally some cells will be introduced into experimental animal models to test their stem cell properties. If you are unhappy about this, please tell us and your cells would then be used only for test tube studies in the laboratory. Some cells may be stored or banked for future research. What are the possible disadvantages and risks of taking part? The small volume of blood would otherwise be discarded. What are the possible benefits of taking part? There is no benefit to you from taking part. You will not receive any extra information on the health of you or your pregnancy. The information we get from this study will help develop better treatment methods in future for babies before and after birth. These include treating
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brain damage from oxygen shortage, and developing cures for serious blood disorders, and handicapping hereditary conditions. What if something goes wrong? This is highly unlikely. However, in the event of your suffering any adverse effects as a consequence of your participation in this study, you will be compensated through the Imperial College School of Medicine’s “No Fault” Compensation Scheme. Will my taking part in this study be kept confidential? Yes. The samples collected will be anonymised and cannot be traced back to you. What will happen to the results of the research study? The work using your sample may be published in the medical and scientific literature. Who is organising and funding the research? The work is funded by a variety of bodies, including Wellcome and the Medical Research Council. No member of staff is being paid for including you in this study. Who has reviewed the study? The research has been approved by the joint Queen/Queen Charlotte’s & Chelsea and Acton Hospitals’ Research Ethics Committee. It complies with government guidelines on the use of Fetal Tissue for Research Purposes. A research ethics committee would also approve any future research on stored tissues or cells. Contact for Further Information Further information can be obtained from Dr Wu (0207-594 2121) or Dr Kumar (0208-383 3998). Thank you for taking part in this study. You will be given a copy of the information sheet and a signed consent form to keep.
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(Version for patients undergoing termination of pregnancy) Information Sheet for Research Participants You will be given a copy of this Information Sheet Study title: Biology of fetal stem cells We would like to invite you to participate in a research study. If you decide to take part, please let us know beforehand if you have been involved in any study during the last year. Ask us if there is anything that is not clear or if you would like more information. Take time to decide whether or not you wish to take part. Thank you for reading this. What is the purpose of the study? Stem cells are underdeveloped cells that have the potential to repair damaged tissues or treat inherited diseases. Fetal blood or tissues appear to have high concentrations of stem cells. We are trying to see what type of stem cells are present, and how they compare with other sources of stem cells. Why have I been chosen? We are asking you because you are due to have a termination of pregnancy under general anaesthetic. You may also have agreed to undergo a training procedure. We would like permission to use any fetal blood or tissue collected at that time. Do I have to take part? It is up to you to decide whether or not to take part. If you do decide to take part you will be given this information sheet to keep and be asked to sign a consent form. If you decide to take part you are still free to withdraw at any time and without giving a reason. A decision to withdraw at any time, or a decision not to take part, will not affect the standard of care you receive. What will happen to me if I take part? Unless you request otherwise, tissues from the operation would normally be disposed of after it is over. We ask your permission instead to allow fetal blood or tissue taken at the time of termination to be used for research. The use of this tissue will be strictly controlled according to official guidelines and restricted to uses for which fetal tissue is necessary for medical benefit. The stem cells we derive will then be thoroughly tested and evaluated in the laboratory. Occasionally some cells will be introduced into experimental animal models to test their stem cell properties. If you are unhappy about this, please tell us and your cells would then be used only for test tube studies in the laboratory. Some cells may be stored or banked for future research. What are the possible disadvantages and risks of taking part? There is no risk to you or your baby. What are the possible benefits of taking part? There is no benefit to you from taking part. The information we get from this study will help develop better treatment methods in future for babies before and after birth. These include treating brain damage from oxygen shortage, and developing cures for serious blood disorders, and handicapping hereditary conditions.
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What if something goes wrong? This is highly unlikely. However, in the event of your suffering any adverse effects as a consequence of your participation in this study, you will be compensated through the Imperial College School of Medicine’s “No Fault” Compensation Scheme. Will my taking part in this study be kept confidential? Yes. The samples collected will be anonymised and cannot be traced back to you. What will happen to the results of the research study? The work using your sample may be published in the medical and scientific literature. Who is organising and funding the research? The work is funded by a variety of bodies, including Wellcome and the Medical Research Council. No member of staff is being paid for including you in this study. Who has reviewed the study? The research has been approved by the joint Queen/Queen Charlotte’s & Chelsea and Acton Hospitals’ Research Ethics Committee. It complies with government guidelines on the use of Fetal Tissue for Research Purposes. A research ethics committee would also approve any future research on stored tissues or cells. Contact for Further Information Further information can be obtained from Dr Wu (0207-594 2121) or Dr Kumar (0208-383 3998). Thank you for taking part in this study. You will be given a copy of the information sheet and a signed consent form to keep.
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Participant Consent Form Registration No: 2001\6234 Title of project:
Biology of fetal stem cells The participant should complete the whole of this sheet herself.
(please tick each statement if it applies to you)
I have read the Information Sheet for Patients and Healthy Volunteers. I have been given the opportunity to ask questions and discuss this study. I have received satisfactory answers to all my questions. I have received enough information about the study. The study has been explained to me by: Prof/Dr/Mr/Mrs/Ms_______________________________________ I understand that I am free to withdraw from the study at any time, without having to give a reason for withdrawing and without affecting my future medical care. I agree to take part in this study. Signed........................................................................Date................................. (NAME IN BLOCK CAPITALS).......................................................…………….. Investigator’s signature.........................................…..Date…............................. (NAME IN BLOCK CAPITALS)..........................................….…………………...
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FOR INFORMATION
Directorate of Women’s and Children’s Services Queen Charlotte’s Hospital
Hammersmith Hospital Trust
Training Procedures at Termination of Pregnancy
Diagnostic tests on the baby are often done through a small needle put into the womb through the mother’s abdomen. A small sample of fluid (amniocentesis), placenta (chorionic villus sampling) or blood (fetal blood sampling) is then collected with a syringe. These tests require considerable expertise and specialists in Fetal medicine who perform these tests need to practice to become or stay skilled in these procedures. We would like to ask you your permission for one of our Fetal Medicine doctors to perform one of these tests while you are asleep under general anaesthetic just before the pregnancy is terminated. This will involve no extra risk to you but you may notice a small pin prick on the lower part of your abdomen after you wake up from the anaesthetic. If you decide not to participate, your medical treatment will not be affected. I ………………………………………… (Hospital identification sticker) consent to either an amniocentesis, chorionic villus sampling or fetal blood sampling before the termination of pregnancy. This has been fully explained to me by Dr ………………………….. ………………………………………. (signature of patient) ………………………………………. (signature of doctor)
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Participant Consent Form Registration No: 2002\6482 Title of project:
First trimester fetal blood sampling The participant should complete the whole of this sheet herself.
(please tick each statement if it applies to you)
I have read the Information Sheet for Patients and Healthy Volunteers. I have been given the opportunity to ask questions and discuss this study. I have received satisfactory answers to all my questions. I have received enough information about the study. The study has been explained to me by: Prof/Dr/Mr/Mrs/Ms_______________________________________ I understand that I am free to withdraw from the study at any time, without having to give a reason for withdrawing and without affecting my future medical care. I agree to take part in this study. Signed........................................................................Date................................. (NAME IN BLOCK CAPITALS).......................................................…………….. Investigator’s signature.........................................…..Date................................. (NAME IN BLOCK CAPITALS)..........................................….…………………...
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Appendix 2 – Plasmids used to generate lentiviral vectors
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