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www.sciencemag.org/cgi/content/full/1152092/DC1
Supporting Online Material for
Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin
Jacob Hanna, Marius Wernig, Styliani Markoulaki, Chiao-Wang Sun,
Alexander Meissner, John P. Cassady, Caroline Beard, Tobias Brambrink, Li-Chen Wu, Tim M. Townes,* Rudolf Jaenisch*
*To whom correspondence should be addressed. E-mail: [email protected] (R.J.);
[email protected] (T.M.T.)
Published 6 December 2007 on Science Express DOI: 10.1126/science.1152092
This PDF file includes:
Materials and Methods
SOM Text
Figs. S1 to S8
Table S1
References
i
Supplementary Materials
Treatment of Sickle Cell Anemia Mouse Model with iPS Cells
Generated from Autologous Skin
Jacob Hanna1, Marius Wernig1, Styliani Markoulaki1, Chiao-Wang Sun2, Alexander
Meissner1, John Cassady1,3, Caroline Beard1, Li-Chen Wu2, Tobias Brambrink1, Tim
M. Townes2 & Rudolf Jaenisch1,3.
1 The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142. 2 UAB, Department of Biochemistry and Molecular Genetics, Schools of Dentistry and Medicine, University of Alabama, Birmingham, Alabama 35294. 3 MIT Department of Biology, Cambridge, Massachusetts 02142.
Correspondence should be addressed to Rudolf Jaenisch ([email protected]) or Tim Townes
ii
Materials and Methods
Cell culture and viral infections.
ES and iPS cells were cultivated on irradiated MEFs in DME containing 15% FCS,
leukemia inhibiting factor (LIF), penicillin/streptomycin, L-glutamine, beta-
mercaptoethanol and nonessential amino acids. For the generation of ITT4 and ITTO26
iPS lines, 2X10^5 fibroblasts at passage 3–4 were infected overnight with pooled viral
supernatant generated by transfection of HEK293T cells (Fugene, Roche) with the
Moloney-based retroviral vectors pLIB (Clontech) encoding for cDNAs of Oct4, Sox2,
Klf4 and c-Myc together with the packaging plasmid pCL-Eco (1). MSCV HOXB4-GFP
virus was generated with the same later packaging (2). For the generation of iPS cells
from tail tip fibroblasts from hBS/hBS mice, 3X10^5 cells were initially infected in a
10cm culture dish with Moloney viruses encoding for Oct4, Sox2, and Klf4. 24 hours
after infection, cells were split and plated in 2 6-well plates and infected with a lentivirus
encoding a 2-lox c-Myc cDNA. At day 16 colonies were picked from the 2 wells that
received the lowest lentviral titer but still generated colonies. The 2-lox c-Myc virus was
generated as follows: an oligo containing a loxP site was inserted into the BspEI site of
the 3'LTR of FUGW (3). The c-myc cDNA was cloned into the EcoRI sites of the
resulting FUGW-loxP vector. Adenovirus encoding for Cre-recombinase was purchased
from the University of Iowa gene transfer vector core (http://www.uiowa.edu/~gene/)
iii
Gene correction in humanized knock in mouse model of sickle cell anemia.
The production of a novel, knock-out/knock-in mouse model of sickle cell disease
and the targeting construct for repair Sickle mutation were previously described (4).
Briefly, a knock-in mouse model of sickle cell disease was generated by replacing the
mouse α globin genes with a human α-globin gene (hα/hα) and by replacing the mouse
β-globin genes with human Aγ- and βS-globin genes (–1400 γ-βS/–1400 γ-βS) (4). The –
383 γ-βA DNA construct was linearized by NotI digestion and electroporated into the iPS
cells (hα/hα, –1400 -βS/–1400 -βS). Positive/negative selection in hygromycin (135
µg/mL) and gancyclovir (2 µM) for 2 weeks was used to enrich for homologous
recombinants. DNA isolated from individual iPS cell colonies was analyzed by
polymerase chain reaction (PCR). Homologous recombinants were identified with primer
1 and primer 2 to identify correct 5' sequences (primer 1 is outside of the vector
homology region) and with primers 5 and 6 to identify correct 3' sequences (primer 6 is
outside of the vector homology region). PCR with primers 3 and 4 followed by Bsu36I
digestion was used to distinguish S and A alleles. Primer sequences are as follows:
primer 1, 5'-CTCCTGACTCGGTATCCTGC-3'; primer 2, 5'-
GAAGTTCTCAGGATCCACATGC-3'; primer 3, 5' GATATATCTTAGAGGAGGGC-
3'; primer 4, 5'-CCAACTTCATCCACGTTCAC-3'; primer 5, 5'-
CAGAGCTTGGTTGACGGCAATTTCG-3'; and primer 6, 5'-
TGAGCTCCGGAGGTACCCAGG-3'. Bsu36I digestion of PCR fragments derived with
primers 3 and 4 was used to genotype mice and determine β globin allele in mice. βA
fragments are digested, but βS fragments are resistant to digestion.
iv
In vitro derivation of hematopoietic progenitors from iPS and ES cells.
Differentiation protocol was performed as previously described (2, 5). Briefly, a
confluent flask of ITT026 iPS, ITT4 iPS and V6.5 ES cells (all of C57black6/129sv F1
genetic background) were trypsinized and resuspended in 5 mL of embryoid body (EB)
differentiation media (IMDM (Invitrogen), 200microg/ml Holo-transferrin (Sigma), 1%
pen/strep/glutamine, 15% serum (Stem Cell technologies, catalog #06952), 50microg/ml
ascorbic acid (sigma) and transferred to a non-gelatin coated T25 flask. Cells were
incubated at 37°C for 45 minutes and centrifuged to collect feeder-depleted cells.
Subsequently cells were resuspended in EB differentiation media at 333,333 cells/50 mL
and plated on five 15 cm petri plates (18-22) rows of drops per plate. Plates were gently
flipped to invert drops and incubated at 37°C/5% CO2 for 48 hours. EB drops were
pooled by gently swirling plates and transferred to 15 mL conical tube. Pooled EBs
settled by gravity (about 10 minutes) and were resuspended in 10 mL EB differentiation
media and transferred to 10 cm non-adherent petri plate and incubated for 4 more days.
On day six of differentiation, EBs were collected and dissociated by adding 250
µL of dissociation enzyme mix (500 mg Collagenase IV (Invitrogen), 1 gr
Hyaluronidase (Sigma), 40,000U Dnase (Sigma) in 50ml DMEM) and 1 mL PBS.
Incubation was done in 37°C water bath for 20 minutes and by occasionally swirling tube
to mix enzymes and EBs. Afterwards we added 8 mL enzyme-free dissociation buffer
(Invitrogen). Mixtures were triturated using 5 mL pipette until EBs were fully dissociated
and cells were collected by centrifugation.
v
Resuspended EB-derived cells at 100,000 cells/2 mL 10% IMDM were infected with
viral supernatant of MSCV HOXB4-iresGFP (A kind gift by K. Humphries and G.
Sauvageau). Polibrene was added to a final concentration of 8 µg/mL. 2 mL EB/viral
supernatant mix was plated per well of four 6 well plates pre-plated with OP9 stroma
cells (ATCC). Cells were centrifuged at 900 rpm at room temperature for 60 minutes and
then transferred to incubator at 37°C 5% CO2. After 12-16 hours, supernatant was
harvested from plates to collect any potential cells remaining in suspension via
centrifugation. Pellet was resuspended in 48 mL 10% IMDM+cytokines (100 ng/mL
Flt3L, 100 ng/mL SCF, 40 ng/mL TPO, 40 ng/mL VEGF, 10 ng/ml IFNγ (Peprotech)).
Cells were plated in original four 6-well plates in which infection was performed. Plates
were incubated at 37°C 5%CO2 for seven days. On day seven post-infection, cells were
trypsinized collected and pooled from all wells by treatment with trypsin. Cells were
either used for further in vitro analysis or further expanded (20 mL fresh 10%
IMDM+cytokines distributed into four T75 flasks). Methylcellulose colony forming
assay was performed as previously described (6).
For bone marrow transplantation, cells were collected after 14-16 days of culture on
OP9 cells by trypsinization and we sorted samples for GFP+ SSEA1- cells (FacsAria, BD
Biosciences). Purified cells (1*10^6 cells per animal) were delivered via retro-orbital
injection to 4-6 weeks old C57black/129sv F1 male mice (weighing between 15-20
grams) subjected to a two doses of 600Rads separated by 4 hours. 5 week old male
hBS/hBS mice (weighing 14-15gr) were subjected to two irradiation doses of 550 Rad
separated by 4 hours. Mice (transplanted and control groups) were transiently subjected
vi
to intraperitoneal injections (25-50 microliters per injection/mouse) with anti-asialo GM1
(rabbit) antibody (Wako Biosystems) (-8, -1, +2 days, +1, +2 and +3 weeks after
transplantation) to deplete host NK cells and enhance engraftment of the transplanted
cells. Transplanted and control mice were maintained under sterile conditions for the first
4 weeks after transplant. Experiments were carried out with Institutional Animal Care
and Use Committee approval.
Immunofluorescence stainings.
Cells were fixed in 4% paraformaldehyde for 20 minutes at 25 °C, washed 3 times
with PBS and blocked for 15 min with 5% FBS in PBS containing 0.1% Triton-X. After
incubation with primary antibodies against Nanog (polyclonal rabbit, Bethyl) and SSEA1
(monoclonal mouse, Developmental Studies Hybridoma Bank) for 1 h in 1% FBS in PBS
containing 0.1% Triton-X, cells were washed 3 times with PBS and incubated with
fluorophore-labeled appropriate secondary antibodies purchased from Jackson
Immunoresearch. Specimens were analyzed on an Olympus Fluorescence microscope and
images were acquired with a Zeiss Axiocam camera.
vii
Viral integrations.
Genomic DNA was digested with PvuII restriction enzyme overnight, followed by
electrophoresis and transfer. The blots were hybridized to the radioactively labeled c-myc
cDNA.
Blastocyst injections.
Diploid blastocysts (94–98 h after HCG injection) were placed in a drop of
DMEM with 15% FCS under mineral oil. A flat tip microinjection pipette with an
internal diameter of 12–15 mm was used for hBS/hBS IPS #3.3 cell injections. After
injection, blastocysts were returned to potassium simplex optimization medium and
placed at 37 °C until transferred to recipient females. Ten to twenty injected blastocysts
were transferred to each uterine horn of 2.5-d-postcoitum-pseudopregnant B6D2F1
females. Pups were recovered at day 19.5 and fostered to lactating BALB/c mothers if
necessary.
Hematological indices measurements.
Blood was collected from anesthetized animals into Microtainer EDTA collection
tubes (Becton Dickinson, Franklin Lakes, NJ). RBC count was measured on a HemaVet
1700 (CDC Technology, Oxford, CT) hematology analyzer. Hemoglobin concentration
was determined spectrophotometrically after conversion to cyanmethemoglobin with
viii
Drabkin reagent (Sigma, St Louis, MO). Before determining the hemoglobin
concentration, red cell membranes were pelleted at 16,000g for 5 minutes in an
Eppendorf microfuge. Packed cell volume (PCV) was measured with a JorVet J503
(Jorgenson Laboratories Systems, Loveland, CO) microhematocrit centrifuge.
Reticulocyte counts were determined by flow cytometry after staining with thiazole
orange. Urine osmolality was measured with the Wescor Vapor Pressure Osmometer
5100 (Logan, UT) after food and water were withheld from the mice for 16 hours.
Automated electrophoretic analysis of B hemoglobin was performed on 250 microliter
blood samples of mice collected in EDTA tubes by using SPIFE 2000 system (Helena
Laboratories, Texas).
FACS staining.
Peripheral blood lymphocytes (PBLs) were treated with red blood cell lysis buffer
(Sigma) and further analyzed by FACS. Antibodies used were APC labeled anti-IgM,
anti-CD4, anti-CD8, anti-Gr1, anti-Mac1 and anti-c-Kit and PE labeled anti-CD41. All
antibodies were purchased from Ebiosciences. APC labeled anti-mSSEA1 antibody was
purchase from RnD systems. FACS analyses were performed on a Becton-Dickinson cell
sorter. Isolation and staining of bone marrow erythroid cells was performed with Bead
enrichment for Ter-119+ cells and subsequent staining (Miltenyi biotec) according to
manufacturer’s instructions.
ix
Discussion
HOXB4 is one of the most attractive tools to expand hematopoietic stem cells in vitro
and in vivo and to promote the formation of hematopoietic cells from in vitro
differentiated embryonic stem cells and iPS cells as we suggest. The use of HOXB4
ectopic expression to facilitate iPS-derived hematopoietic engraftment is based on a
number of earlier studies with conventional mouse ESC (2, 5). The Humphries lab (7-11)
and the Ostertag groups (12-14) have demonstrated that ectopic HOXB4 expression in
ES-derived and bone marrow hematopoietic stem cells, resulted in a concentration-
dependent perturbation of lineage differentiation. Myeloid development was enforced
and T and B lymphoid development suppressed over a wide range of expression levels,
whereas only high expression levels of the transcription factor were detrimental for
erythroid development. However, the expression levels compatible with the favorable
effect of enhanced self-renewal without perturbing differentiation, in vivo, remain to be
determined. It will ultimately be necessary to define the "therapeutic width" of HOXB4
expression. It also important to emphasize that extrapolation from mouse to man in non-
human primate transplant model, Zhang et al. (11, 15) demonstrated that HOXB4
overexpression in CD34+ cells has a dramatic effect on expansion and engraftment of
short-term repopulating cells and a significant, but less pronounced, effect on long-term
repopulating cells. Moreover, Bhatias group (16) showed that HOXB4 is unable to
induce hematopoietic repopulating capacity from hESCs, underscoring the notion that
single genes, such as HOXB4, are unlikely to represent a master gene capable of
x
conferring engraftment potential to human ES derivedd hematopoietic cells. Thus,
developing a robust and relevantly safe in vitro differentiation protocol of hematopoietic
progenitors from human ES cells will be an additional required important step for any
potential future applications in humans.
DAPI
DAPI
DAPI Nanog
SSEA1
AP DAPI
DAPI
DAPI Nanog
SSEA1
AP
Figure S1. Characterization of ITT026 and ITT4 iPS cell lines.Immuno-staining was performed on (a) ITTO26 and (b) ITT4 iPSlines for alkaline phosphatase (AP), SSEA1 and Nanog markers.
(a) (b)
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 1.55
98.40
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 3.51
96.50
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 6.83
93.20
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 1.31
98.70
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 1.24
98.80
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 16.8
83.20
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 0.018
1000
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 0.19
99.80
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 0.12
99.90
0 200 400 600 800 1000100
101
102
103
104
FSC-HFL
2-H
0 2.57
97.40
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 10.3
89.70
0 200 400 600 800 1000100
101
102
103
104
FSC-H
FL2-
H
0 1.32
98.70
V6.5 EScells
ITTO26 iPScells
ITT4 iPScells
Isotypecontrol CD41 Ter-119 Gr-1
Figure S2. Characterization of hematopoietic markers on invitro differentiated iPS cells.Surface antigen expression of ES and IPS derived infected cellswith HoxB4 grown on OP9 stroma for 7 days.
Figure S3. In vitro differentiation of iPS cells into maturehematopoietic cells.
Morphology of typical CFU-GMM and CFU-GEMM colonies from V6.5 ES, ITT026and ITT4 iPS cell lines are shown.
CFU-GM
CFU-GEMM CFU-GEMM
CFU-GMCFU-GM
CFU-GEMM
V6.5 ES ITTO26 iPS ITT4 iPS
100 101 102 103 104100
101
102
103
104
100 101 102 103 104100
101
102
103
104
100 101 102 103 104100
101
102
103
104
100 101 102 103 104100
101
102
103
104
20.2% 7.8%
CD4/
CD8
IgM
100 101 102 103 104100
101
102
103
104
100 101 102 103 104100
101
102
103
104
61.2% 73.5%
Gr-1
MAC
-1
Control(not irradiated,
not transplanted)
GFP
100
101
102
103
104
100
101
102
103
104
73.1%
Ter-1
19
Isot
ype
cont
rol
Isot
ype
cont
rol
0.2% 72.8%
Transplanted mouse
Figure S4. Representative engraftment analysis in transplantedmice.
FACS analysis of peripheral blood samples from representative ITT4 IPS derived cellsamples at 20 weeks for myeloid (Gr-1, MAC-1) and lymphoid (IgM, CD4/CD8)markers. Ter-119 staining was performed on bone marrow derived fraction enrichedfor erythroid cells. Percentages in right upper quadrants represent the fraction of GFP+donor cells that express a given differentiation antigen out of total cells that express thesame antigen. Percentages indicated in lower right quadrant of first two panels indicateGFP+ cell percentage in representative control or transplanted mice.
Figure S5. Characterization of iPS #3.3 cell line.
Staining of hβS/hβS iPS#3.3 cell line for ES markers alkaline phosphatase(AP), SSEA1 and NANOG protein.
DAPI
DAPI
DAPI NANOG
SSEA1
AP
Figure S6. Correction of human mutated sickle allele by genespecific targeting.
Schematic representation of knock-in locus in mice used for this study and thereplacement vector used for gene correction. Homologous recombinants were identifiedby PCR with primers 1 and 2 to identify correct 5' sequences (primer 1 is outside of thevector homology region) and with primers 5 and 6 to identify correct 3' sequences(primer 6 is outside of the vector homology region). PCR with primers 3 and 4 followedby Bsu36I digestion was used to distinguish hβS and hβA alleles
Figure S7. Engraftment follow up in treated hβS/hβS mice.Representative FACS staining for detection of GFP-positive iPSderived cells in the peripheral blood of non transplanted andtransplanted hβS/hβS recipients 9 weeks post-transplantation.
0 200 400 600 800 1000
10 0
10 1
10 2
10 3
10 4
0 200 400 600 800 1000
10 0
10 1
10 2
10 3
10 4
0 200 400 600 800 1000
10 0
10 1
10 2
10 3
10 4
0 200 400 600 800 1000
10 0
10 1
10 2
10 3
10 4
GFP
62.3% 73.2%69.8%
0.1%
0 200 400 600 800 1000
10 0
10 1
10 2
10 3
10 4
0.4%
Untreated hβS/hβS #2
Untreated hβS/hβs #1
Treated hβS/hβS #2
Treated hβS/hβS #1
Treated hβS/hβS #3
FSC
HbA HbS
Per
cent
age
of β
glo
bin
isof
orm
out
of t
otal
β g
lobi
n pr
otei
nUnt
reate
d hβ
A/hβA
Untre
ated
hβS/hβ
S
Untre
ated
hβS/hβ
ATr
eated
hβS
/hβS
(4 w
eeks
) Tr
eated
hβS
/hβS
(8 w
eeks
)
P<0.001P<0.01
Figure S8. Hemoglobin electrophoresis follow up in treatedhβS/hβS mice. Mean values for wild type ß globin A (HbA) andsicle ß globin (HbS) content out of total human ß globin in bloodsamples of different mouse groups. (n=3) in each mousesubgroup.
xi
Table S1. Summary of blastocyst injections.
Cell Line Injected Blastocysts
End point of analyses
Live Chimeras
% Chimerisma
20 embryonic day13.5 3 ND b Sickle cells
iPS #3.3 100 full term 7c ~50-80% a Estimated by coat color b Chimerism confirmed by specific PCR detection of human B globin allele. c Three pups were cannibalized by their foster moms 2 days after birth.
xii
References 1. M. Wernig et al., Nature 448, 318 (2007). 2. S. Eckardt et al., Genes Dev 21, 409 (2007). 3. C. Lois, E. J. Hong, S. Pease, E. J. Brown, D. Baltimore, Science 295, 868 (2002). 4. L. C. Wu et al., Blood 108, 1183 (2006). 5. M. Kyba, R. C. Perlingeiro, G. Q. Daley, Cell 109, 29 (2002). 6. Y. Wang, F. Yates, O. Naveiras, P. Ernst, G. Q. Daley, Proc Natl Acad Sci U S A
102, 19081 (2005). 7. C. Abramovich, N. Pineault, H. Ohta, R. K. Humphries, Ann N Y Acad Sci 1044,
109 (2005). 8. J. Antonchuk, G. Sauvageau, R. K. Humphries, Cell 109, 39 (2002). 9. J. Krosl, N. Beslu, N. Mayotte, R. K. Humphries, G. Sauvageau, Immunity 18,
561 (2003). 10. M. D. Milsom et al., Leukemia 19, 148 (2005). 11. X. B. Zhang, J. L. Schwartz, R. K. Humphries, H. P. Kiem, Stem Cells 25, 2074
(2007). 12. S. Pilat et al., Proc Natl Acad Sci U S A 102, 12101 (2005). 13. B. Schiedlmeier et al., Blood 101, 1759 (2003). 14. B. Schiedlmeier et al., Proc Natl Acad Sci U S A 104, 16952 (2007). 15. X. B. Zhang et al., PLoS Med 3, e173 (2006). 16. L. Wang et al., J Exp Med 201, 1603 (2005).