Stem cell: a cell capable of 1) tissue plasticity - make different cell types 2) infinite self renewal through asymmetric division

skin

muscle

nerve

stem cellstem cell

Properties of STEM cellsProperties of STEM cells

Plasticity

Self renewal

STEM CELLS

1. Source

2. Cloning

3. Plasticity

CLASSIC EXAMPLES of STEM CELLS

• Embryonic stem cells (ESC)

• Bone marrow derived stem cells

Blastocyst

•Fluid-filled cavity termed

blastocoele

ICM

Trophoblast• Blastocyst implants in

uterine wall

• Two regions identifiable

- outer trophoblast

- inner cell mass (ICM)

ICM = embryonic stem cells

Origin of vertebrate stem cells

USES of EMBRYONIC STEM CELLS

1. Source of different types of human cells

for Transplantation: for Cell Therapy or Tissue Engineering (organs). Merit and ethics are controversial

2. Cloning:

3. Somatic nuclear transfer (therapeutic cloning):

USES of EMBRYONIC STEM CELLS

1. Source of different types of human cells for transplantation: for Cell Therapy or Tissue Engineering. Merit and ethics are controversial

2. Cloning: to make ‘genetically identical’ new individuals Achieved for animals – unlikely for humans.

3. Somatic nuclear transfer (therapeutic cloning):

to generate autologous cells for transplantation Avoids problems of immune rejection of non-self

CLONING

1962 John Gurdon in the UK took (diploid) nucleus from tissue of adult frog and implanted this into an unfertilised egg that had the nucleus removed. The special influence of the maternal cytoplasm caused the ‘differentiated’ adult nucleus to give rise to a complete new frogFIRST EXAMPLE OF ADULT CLONING. Shows mature nucleus has capacity to revert to ‘equivalent’ ESC.

1996 Over 30 years later Dolly the sheep was cloned in Scotland. Then cattle, pigs, cats pets – humans??

ISSUES• Ethics (especially for humans)• Genes vs environment• Status/quality of ‘aged’ DNA• Role of maternal cytoplasmic factors and mtDNA

Movies like Boys from Brazil and Jurassic park

Variation on this is THERAPEUTIC CLONING (NOT make a new adult) . Perhaps a better name to avoid ethical issues is somatic nuclear transfer.

USES of EMBRYONIC STEM CELLS

1. Source of different types of human cells for transplantation: for Cell Therapy or Tissue Engineering. Merit and ethics are controversial

2. Cloning: to make ‘genetically identical’ new individuals Achieved for animals – unlikely for humans.

3. Somatic nuclear transfer (therapeutic cloning):

to generate autologous cells for transplantation Avoids problems of immune rejection of non-self

ADULT STEM CELLS

Bone marrow derived stem cell

classic source

Haematopoiesis

Stem Cell (HSC)

adult

embryo

ES cells

EG cells

Somatic Stem cells

Multiple paths to new cell identities

Fluorescent Activated Cell Sorting (FACS)

to isolate stem cells

based on many cell surface markers Sca1, CD34 etc

Blastocyst

Embryo/Fetal

Post-Natal Tissues

• Bone marrow (HSC)• Blood vessels (ESC)• Interstitial connective tissue (MSC)• Other tissues

Germ cellsFetal tissues

ES cells

Umbilical cord blood (UCB)Supporting tissues (MSC)

Umbilical Cord

UBC

ES Cells

Stem cells can be derived from tissues throughout development

STEM CELLS

1. Source

2. Cloning

3.Plasticity

Myogenic Stem Cells

Satellite cellTerry Partridge

myotubesmyofibre(only part is shown)

myoblasts

1 satellite cell3 myonucleus

2 multipotential/stem cell

Sources of myoblasts within skeletal muscle

Plasticity

Resident C/T cellsResident C/T cells skeletal cardiac pluripotent STEM cells (multi)

progenitor cells VascularVascular

endothelial smooth muscle ()

pericytes ?

MyofibroblastsMyofibroblasts ()

Ectopic cells Ectopic cells (chickens)(chickens) Thymus Thymus (myoid cells) NeuralNeural ((multimulti)) ()

DermisDermis Circulating bone-marrow Circulating bone-marrow **((multimulti)) **

Issues of immune rejection

of foreign.

2. Separation of

HOST specific stem

cell type

3. Correction or replacement

of DONOR

bone marrowstem cells

Healthy Healthy donordonor bone-marrow bone-marrow derived stem cells to repopulate derived stem cells to repopulate

diseased diseased host tissueshost tissues

Issues of immune rejection of foreign cells can be reduced by using closely matched donor and host cells.

Inject healthy donor stem cells derived from another person. These circulating donor stem cells may repopulate any damaged host tissue e.g heart

Bone-marrow stem cell

(1) Muscle precursor cell with limited proliferation

1

2

Conversion

Conversion

Asymmetric Cell division

AA

BB

Possibilities to explain presence of bone-marrow derived (donor) nucleus (cell) within a (host) cell or tissue. Illustrated for muscle

(2) Ideal scenario = Muscle Stem cellwith capacity to formmany (cardio)myoblasts

+

1

2

3

Bone-marrow stem cell

Conversion

Conversion

Fusion

(1) Muscle precursor cell with limited proliferation

(2) Muscle Stem cell

Asymetric Cell division

(3) Fusion of 2 cells

capacity to form many (cardio)myoblasts

Hybrid stem-muscle cell with 2 or more nuclei

AA

BB

Stem Stem cellcell

XX

CC

The stem cell has NOT become a muscle cell

Autograft of genetically corrected stem cells: delivered through the circulation

1. Remove patient’s bone-marrow

2. Separation of specific type of host stem cell

3. Correction or replacement of defective gene in host stem cell

4. Infusion of host’s corrected stem cells to replace or supplement defective host cells

Use of own cells avoids immune problemsand rejection

Two studies show that few of the bone-marrow derived nuclei located within muscle cells actually express muscle-specific genes:

indicating fusion without lineage conversion

• Beth McNally (normal male) bone marrow (b/m) reconstitution of female sarcoglycan (SG) deficient host mice: The rare male b/m-derived nuclei within some myofibres and heart muscle cells, showed NO expression of SGLapidos KA et al (2004)

• Anton Wernig male/GFP b/m reconstitution of female mdx (dystrophin deficient) mice: ~80% of male b/m myonuclei showed NO expression of skeletal muscle specific genesWernig G et al (2005) 3 labels: Y-probe, GFP, dystrophin

1, 2: SERIAL SECTIONS

Donor nucleus (Y-FISH) without dystrophin expression

1

WERNIG G et al (2005)

Current interest in blood vessel derived circulating

STEM CELLS: mesangioblasts.Sampaolesi M…. Cossu J (2006). Mesoangioblast stem cells ameliorate

muscle function in dystrophic dogs. Nature Nov 15.

Major problems in data interpretation due to lack of fundamental controlsDogs injected with immunosuppressants alone (without stem cells) were not included. Yet….

Cyclosporine alone reduces severity of muscular dystrophyPrecise source of stem cells? Poor correlation between increased dystrophin immunostaining (derived from the circulating stem cells) and

improved muscle function. Potential issues with digital imaging and image enhancementConfounded by high biological variation

Causes major confusion and distress for families of boys with DMD who are seeking a cure/treatment

Davies K, Grounds MD (2006) Treating muscular dystrophy with stem cells? Cell.  Dec 29.

Grounds MD, Davies K (2007) The allure of stem cell therapy for muscular dystrophy. Neuromuscular Disorders March.

KEY issues for researchKEY issues for research

•INDUCERS to recruit/convert stem cells into specific lineages: critical effects of environment (Plasticity)

•EXPANSION of cell numbers(proliferation and stem cell renewal)

•Stem cell isolation/identification

Properties of STEM cellsProperties of STEM cells

Plasticity

Self renewal