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: Jaenisch@wi.mit.edu (R.J.);

ttownes@uab.edu (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 (jaenisch@wi.mit.edu) or Tim Townes

(ttownes@uab.edu).

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

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