signaling pathways controlling pluripotency and early cell fate decisions of human induced...

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Signaling Pathways Controlling Pluripotency and Early Cell Fate

Decisions of Human Induced Pluripotent Stem Cells

LUDOVIC VALLIER,aTHOMAS TOUBOUL,

a,bSTEPHANIE BROWN,

aCANDY CHO,

aBILADA BILICAN,

cMORGAN ALEXANDER,

a

JESSICA CEDERVALL,d SIDDHARTHAN CHANDRAN,c LARS AHRLUND-RICHTER,d ANNE WEBER,b ROGER A. PEDERSENa

aLaboratory for Regenerative Medicine, and cDepartment of Clinical Neurosciences, University of Cambridge,

Cambridge, United Kingdom; bLaboratoire de transfert de genes dans le foie: applications therapeutiques, Equipe

Mixte Inserm U804, Universite Paris, Le Kremlin Bicetre, France; dDepartment of Woman and Child Health,

Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Key Words. Induced pluripotent stem cells • Embryonic stem cells • Epiblast stem cells • Primitive endoderm • Trophectoderm •

Neuroectoderm • Mesendoderm • Mesoderm • Endoderm • Activin • Human • Mouse

ABSTRACT

Human pluripotent stem cells from embryonic origins andthose generated from reprogrammed somatic cells share

many characteristics, including indefinite proliferation anda sustained capacity to differentiate into a wide variety of

cell types. However, it remains to be demonstratedwhether both cell types rely on similar mechanisms tomaintain their pluripotent status and to control their dif-

ferentiation. Any differences in such mechanisms wouldsuggest that reprogramming of fibroblasts to generate

induced pluripotent stem cells (iPSCs) results in novelstates of pluripotency. In that event, current methods forexpanding and differentiating human embryonic stem cells

(ESCs) might not be directly applicable to human iPSCs.However, we show here that human iPSCs rely on activin/

nodal signaling to control Nanog expression and thereby

maintain pluripotency, thus revealing their mechanisticsimilarity to human ESCs. We also show that growth fac-

tors necessary and sufficient for achieving specification ofhuman ESCs into extraembryonic tissues, neuroectoderm,

and mesendoderm also drive differentiation of humaniPSCs into the same tissues. Importantly, these experi-ments were performed in fully chemically defined medium

devoid of factors that could obscure analysis of develop-mental mechanisms or render the resulting tissues incom-

patible with future clinical applications. Together thesedata reveal that human iPSCs rely on mechanisms similarto human ESCs to maintain their pluripotency and to con-

trol their differentiation, showing that these pluripotentcell types are functionally equivalent. STEM CELLS

2009;27:2655–2666

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Human embryonic stem cells (hESCs) are pluripotent stemcells generated from embryos at the blastocyst stage [1]. Theirembryonic origin confers upon them the capacity to prolifer-ate indefinitely in vitro while maintaining their ability to dif-ferentiate into a large number of cell types in vitro and invivo. These properties are shared by human induced pluripo-tent stem cells (hIPSCs) generated from fibroblasts that havebeen reprogrammed by overexpressing key transcription fac-tors (Oct-4, Nanog, Sox2, Klf-4, c-Myc, and other pluripo-tency-related genes) [2–4]. Despite these similarities, itremains to be demonstrated whether hESCs and hIPSCs relyon the same mechanisms to maintain their pluripotency and tocontrol their subsequent cell fate decisions. This is essential

for understanding whether pluripotent stem cells generatedfrom reprogrammed fibroblasts are functionally equivalent totheir embryonic counterparts. Moreover, such understandingis the key to using currently available methods for hESCs todrive differentiation of hIPSCs into diverse cell types.Although it has been clearly demonstrated that activin/nodalsignaling maintains the pluripotent status of hESCs [5–7], ithas proved more challenging to define the growth factors nec-essary and sufficient to control early events of hESC differen-tiation. Indeed, most of the current methods available forinducing differentiation of hESCs are based on culture mediacontaining factors (such as serum, stroma cells, and complexmatrices) that obscure analysis of developmental mechanismsor render the resulting tissues incompatible with future clini-cal applications [8, 9]. To address this issue, we recentlydeveloped an approach based on a fully chemically defined

Author contributions: L.V.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscriptwriting; T.T.: collection and/or assembly of data, data analysis and interpretation; S.B.: collection and/or assembly of data, data analysisand interpretation; C.C.: collection and/or assembly of data, data analysis and interpretation; B.B.: collection and/or assembly of data,provision of study material; M.A.: technical support; J.C.: collection and/or assembly of data; L.A.-R.: data analysis and interpretation;S.C.: provision of study material; A.W.: provision of study material; R.A.P.: data analysis and interpretation, manuscript writing.

Correspondence: Ludovic Vallier, PhD., Laboratory for Regenerative Medicine, West Forvie Building, Robinson Way, University ofCambridge, Cambridge, CB2 0SZ, United Kingdom. Telephone: 00 44 (0) 1223747489; Fax: þ44 (0) 1223 763350; e-mail: [email protected] Received April 7, 2009; accepted for publication August 6, 2009; first published online in STEM CELLS EXPRESS Month 00,2009. VC AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.199

STEM CELLS 2009;27:2655–2666 www.StemCells.com

medium to drive hESC differentiation into extraembryonic tis-sues, neuroectoderm, and mesendoderm [10], which representthe earliest cell types generated during mammalian embryonicdevelopment. Here, we analyzed the effect of these cultureconditions on hIPSCs and we observed that they workedequally well to promote their differentiation into similar tis-sues. In addition, hIPSCs could be generated and grown inchemically defined culture conditions supplemented with acti-vin and fibroblast growth factor 2 (FGF2), whereas inhibitionof activin/nodal/transforming growth factor b (TGFb) signalingprompted their differentiation, demonstrating that hIPSCs, likehESCs, rely on this signaling pathway to maintain their pluri-potency. These observations were reinforced by showing thatNanog expression is directly controlled by activin/nodal/TGFbsignaling in hIPSCs as in hESCs and that constitutive expres-sion of Nanog is sufficient to maintain pluripotency of hIPSCsgrown in the absence of activin. Together, these results demon-strate that human pluripotent stem cells of embryonic origin orthose generated from reprogrammed fibroblasts rely on thesame signaling pathways to control their pluripotency and theirdifferentiation. Importantly, the similarity of these responsesby hESCs and hIPSCs affirms their biologic equivalence anddistinguishes them from mouse ESCs (mESCs). Finally, theseresults show that pluripotent mammalian stem cells can beinduced to undergo early cell fate decisions in chemicallydefined medium supplemented with a minimal set of growthfactors, thereby providing a general approach for modeling thetransition from pluripotency to specification of the three germlayers that form during mammalian gastrulation.

MATERIALS AND METHODS

Generation of Human iPSCs

Human foreskin fibroblasts were obtained from American TypeCulture Collection (Manassas, VA, http://www.atcc.org)(CRL2429) and grown following the provider’s recommendedprotocol. Moloney murine leukemia virus-derived vectors eachcontaining the coding sequences of one of the four human genes,Oct-4, Sox2, c-Myc, and Klf4, and the corresponding viral par-

ticles were generated by Vectalys (Toulouse, France, http://www.vectalys.com). Importantly, the viral particles were purifiedby several rounds of tangential filtration to reach a titer of about109 transduction units/ml and to assure a high level of purity. Onday 1, 105 fibroblasts were plated in each well of 6-well plates inmedium containing fetal bovine serum (FBS, Biosera, EastSussex,U.K., http://www.biosera.com). The following day the cells weretransduced with the four viruses at a multiplicity of infection of10 (for each virus) for 24 hours. On day 3, the cells were washedthree times in phosphate-buffered saline (PBS, Gibco, GrandIsland, NY, http://www.invitrogen.com) and then grown in me-dium containing FBS for three additional days. On day 7, thecells were passaged on plastic plates containing irradiated mouseembryonic fibroblasts and then grown for 2 additional days inmedium containing FBS. After day 9, the cells were grown instandard hESC culture conditions (knockout [KSR], (Gibco) þFGF2 (4 ng/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com). The first hIPSC colonies appeared 10 days laterand they could be picked after 5 additional days of culture. Indi-vidual colonies were picked and either transferred into a singlewell of 12-well plates containing mouse feeders in KSR þ FGF2or directly transferred into a well of 12-well plates precoatedwith porcine gelatin (Sigma-Aldrich, St. Louis, http://www.sig-maaldrich.com) and FBS containing chemically defined medium(CDM) þ Activin (10 ng/ml, kindly provided by Marco Hyvo-nen) þ FGF2 (12 ng/ml). The resulting colonies were thenexpanded using enzymatic dissociation as described for hESCsgrown in similar culture conditions (as discussed in Growth ofhESCs and hIPSCs in Feeder-Free and Serum-Free Conditions).For derivation in chemically defined medium, the fibroblasts weregrown for 5 days in FBS-containing medium after transductionand then grown in CDM þ activin (10 ng/ml) þ FGF2 (12 ng/ml) for 36 additional days without splitting. hIPSC coloniesappeared on day 29 and individual colonies were picked 5 dayslater to be expanded in CDM þ activin (10 ng/ml) þ FGF2 (12ng/ml) as described above.

Growth of hESCs and hIPSCs in Feeder-Freeand Serum-Free Conditions

For feeder- and serum-free culture, H9 cells (WiCell ResearchInstitute, Madison, WI, http://www.wicell.org) and hIPSCs (Table1) were grown in CDM [5, 11], supplemented with activin

Table 1. Summary of hIPSC derivations

Experiment Culture condition No. of hIPSC colonies No. of hIPSC lines

1 (4 factors) SR þ FGF þ feeders 30 (Av. 7.5) 13Fibro CRL CDM activin þ FGF 23 (Av. 4.6) 10

2 (4 factors) SR þ FGF þ feeders 56 (Av. 18.7) 20Fibro CRL CDM activin þ FGF 6 (Av. 3) 2

3 (4 factors) SR þ FGF þ feeders 16 (Av. 8) 0Fibro CRL CDM activin þ FGF 4 (Av. 2) 0

4 (3 factors, no Myc) SR þ FGF þ feeders 8 (Av. 8) 8Fibro CRL CDM activin þ FGF ND 0

5 (4 factors) SR þ FGF þ feeders 24 (Av. 12) 10Fibro MRC5 CDM activin þ FGF ND 0

6 (4 factors) SR þ FGF þ feeders 34 (Av. 17) 10Fibro Bi CDM activin þ FGF ND 0

hIPSCs were generated from foreskin fibroblasts (CRL), fetal lung fibroblasts (MRC5), and adult dermal fibroblasts (Bi) as described inMaterials and Methods using four (Oct-4, Sox2, KLF-4, c-Myc) or three (Oct-4, Sox2, KLF-4) factors. hIPSCs were derived either onfeeders in the presence of knockout serum replacer (Invitrogen) supplemented with FGF2 or in CDM supplemented with activin and FGF2.For each experiment, the total number of hESC-like colonies generated is indicated along with the average number of individual hIPSCcolonies generated per plate. The number of hIPSC colonies picked and expanded is also indicated. In total, 22 hIPSC lines (15 generatedfrom CRL fibroblasts including 3 hIPSC lines generated without c-Myc, 2 hIPSC lines generated from MRC5 fibroblasts, and 2 from Bifibroblasts) were used to analyze the responsiveness of hIPSCs to culture conditions that were effective in maintaining the pluripotent statusand driving the differentiation of hESCs.Abbreviations: Av., average; CDM, chemically defined medium; FGF, fibroblast growth factor; hIPSC, human induced pluripotent stem cell;ND, not done; SR, serum replacer.

2656 Functional Comparison of Human Pluripotent Cells

Figure 1. hIPSCs can be grown in defined culture conditions developed for hESCs. (A): hIPSCs grown in CDM þ activin þ FGF2 maintainpluripotency markers. CRL-hIPSCs were grown for 10 passages (8 weeks) in CDM þ activin (10 ng/ml) þ FGF2 (12 ng/ml), and then immuno-staining was performed to detect expression of the pluripotency markers Oct4, Sox2, Nanog, and Tra-1-60. In addition, colorimetric assays wereperformed to detect alkaline phosphatase activity. Scale bar ¼ 100 lm. (B): Expression of pluripotency markers (Oct-4, Sox2, Nanog) in hIPSCsgrown in CDM þ activin þ FGF2. CRL-hIPSCs (lines 30, 35, 40) were grown for 4 days in these culture conditions and then RNAswere extracted and expression of the denoted genes was analyzed using quantitative polymerase chain reaction (PCR). hESCs grown in CDM þactivin þ FGF2 were used as positive controls. Normalized expression is shown as the mean � SD from three informative experiments. (C):hIPSCs grown in CDM þ activin þ FGF2 express homogeneously the pluripotency marker Tra-1-60, confirming the absence of spontaneous dif-ferentiation in these culture conditions. hIPSCs were grown for 10 passages in CDM þ activin þ FGF2, and then the fraction of cells expressingthe pluripotency marker Tra-1-60 was determined using fluorescence-activated cell sorting analyses. hESCs grown in CDM þ activin þ FGF2were used as positive controls. (D): Teratomas from hIPSCs cells grown in CDM þ Activin þ FGF2. Approximately 106 hIPSCs (line 40) grownfor 20 passages in CDM/AF were injected into the testis capsule of severe combined immunodeficient-beige mice. The resulting tumors were har-vested 2 months after injection. (E): Expression of exogenous and endogenous Oct-4, Sox2, Klf-4, and c-Myc in hIPSCs generated from foreskinfibroblasts. CRL-hIPSCs were grown for 4 days in CDM þ activin þ FGF2 and then expression of endogenous and exogenous Oct-4, Sox2, Klf-4, and C-Myc was determined using real-time PCR and specific primers. (F): Number of transgene copies in hIPSCs generated from foreskinfibroblasts. Copy number for each gene used to reprogram CRL foreskin fibroblasts was determined using real-time PCR. hESCs (H9 line) wereused as positive control. Abbreviations: AP, alkaline phosphatase; CDM, chemically defined medium; FGF2, fibroblast growth factor 2; hIPSC,human induced pluripotent stem cell.

Figure 2.


(10 ng/ml, kindly provided by Marco Hyvonen) and FGF2 (12ng/ml, R&D Systems Inc.). The composition of CDM was 50%Iscove’s modified Dulbecco’s medium (Gibco, Grand Island, NY,http://www.invitrogen.com) plus 50% F12 NUT-MIX (Gibco),supplemented with 7 lg/ml of insulin (Roche Diagnostics, Basel,Switzerland, http://www.roche-applied-science.com), 15 lg/ml oftransferrin (Roche Diagnostics), 450 lM of monothioglycerol(Sigma-Aldrich), and 5 mg/ml bovine serum albumin fraction V(Europa Bioproducts, Cambridge, U.K., http://www.europa-bioproducts.com) or polyvinyl alcohol (CDM-PVA) (Sigma-Aldrich). Every 4 days, cells were harvested using 1 mg/ml dis-pase (Gibco) and then plated into dishes (Corning Costar Corp.,Cambridge, MA, http://www.corning.com/index.aspx) that wereprecoated with 15 lg/ml of human fibronectin (Chemicon) for 20minutes at 37�C and then washed twice in PBS. Alternatively,dishes were precoated with porcine gelatin 1 mg/ml (Sigma-Aldrich) for 15 minutes to 1 hour and then precoated with fetalbovine serum-containing medium (5% in Dulbecco’s modifiedEagle’s medium) for 24 hours at 37�C.

Differentiation of hIPSCs in ChemicallyDefined Conditions

hIPSCs grown in feeder-free and serum-free conditions were har-vested using 1 mg/ml dispase then split into plates precoated withfibronectin or with FBS. hIPSCs were grown for the first 2 daysin CDM supplemented with recombinant activin and FGF2 (12ng/ml; R&D Systems Inc.). To obtain extraembryonic tissue,hIPSCs were grown for 7 days in CDM in the presence of bonemorphogenic protein 4 (BMP4, 10 ng/ml R&D Systems Inc.) andSB431542 (10 lM; Tocris Bioscience, Ellisville, MO, http://www.tocris.com). To obtain neuroectoderm progenitors, hIPSCswere grown in CDM or in CDM-PVA in the presence ofSB431542 (10 lM; Tocris Bioscience), FGF2 (12 ng/ml; R&DSystems Inc.), and Noggin (200 ng/ml R&D Systems Inc.) for 10additional days. To obtain mesendoderm precursors, hIPSCs weregrown for the 3 following days in CDM-PVA in the presence ofBMP4 (10 ng/ml; R&D Systems Inc.), FGF2 (20 ng/ml; R&DSystems Inc.), activin (100 ng/ml; R&D Systems Inc.), andLY294002 (10 lM; Promega, Madison, WI, http://www.promega.com).

Teratomas

hIPSCs were harvested mechanically immediately prior to im-plantation, and approximately 106 cells were inoculated beneaththe testicular capsule [12] of 8-week-old C.B.-17/GbmsTac-scid-bgDF N7 male mice (Taconic M&B, ejby, Denmark, http://

www.taconic.com/) housed and maintained at 20�C–24�C, 50%room humidi, in a 14- to 10-hour light-dark cycle with food andwater libitum. The mice were sacrificed after 60 days and thenthe injected testes were cut into equal pieces using a razor blade.The material was fixed overnight in 4% neutral buffered formal-dehyde, and dehydrated through a graded series of alcohols to xy-lene. The tissue was embedded in paraffin, serially sectioned at 5lm, followed by H&E staining and characterization. A human or-igin of the selected areas was verified by fluorescent in situhybridization (human-specific probes, CEP XY; Vysis Inc.,Downers Grove, IL, http://www.vysis.com).

The experiments were performed with permission from theRegional Committee for Animal Experimentation (Stockholm,Sweden; Dnr N107/06).

Flow Cytometry and Cell Sorting

For detection of N-Cam, CXCR4, and platelet-derived growthfactor a (PDGFa) receptor, adherent cells were washed twice inPBS and then incubated for 20 minutes at 37�C in cell dissocia-tion buffer (Invitrogen, Carlsbad, CA, http://www.invitrogen.-com). Cells were dissociated by gentle pipetting and resuspendedat approximately 0.1-1 � 105 cells per milliliter in PBS þ 3%normal goat serum (NGS) containing 0.1% azide (Serotec Ltd.,Oxford, U.K., http://www.serotec.com). Cells were incubated for40 minutes at 4�C with fluorescein isothiocyanate- or phycoery-thrin-conjugated antibody to CXCR4 (1:50; BD Pharmingen, SanDiego, http://www.bdbiosciences.com), N-Cam (1:200; BD Phar-mingen), or PDGFa receptor (PDGFaR, 1:50; BD Pharmingen) orthe corresponding isotype control (mouse IgG isotype control;BD Pharmingen). Subsequently, cells were resuspended in PBS þ3% NGS for staining with 7-aminoactinomycin D (7-AAD) via-bility dye (Immunotech, Luminy, France, http://www.beckman-coulter.com/products/pr_immunology.asp) at 20 ll/ml for 15minutes at room temperature. Live cells identified by 7-AADexclusion were analyzed for surface-marker expression usingFACSCalibur (BD Biosciences, San Jose, California, USA, http://www.bdbiosciences.com) or sorted using Dako CytomationMoFlo high speed flow cytometer (Glostrup, Denmark, http://www.dako.com).

RNA Extraction and Real-Time PolymeraseChain Reaction

Total RNAs were extracted from hIPSCs or differentiated progen-itors using the RNeasy Mini Kit (Qiagen, Hilden, Germany,http://www1.qiagen.com). Each sample was treated with RNase-Free DNase (Qiagen) to avoid DNA contamination. For each

Figure 2. Activin/nodal signaling maintains pluripotency of hIPSCs by controlling Nanog expression in hIPSCs. (A): Inhibition of activin sig-naling induces differentiation of hIPSCs into extraembryonic tissues and neuroectoderm. hIPSCs were grown for 12 days in chemically definedmedium (CDM) þ SB431542 (10 lM) þ FGF2 (12 ng/ml), and then immunostaining analyses were performed to detect the expression of thepluripotency marker Oct-4, the extraembryonic markers Sox7, GATA4, and GATA6, the trophectoderm markers Cdx2 and Eomes, and the neuro-ectoderm markers Sox2, Sox1, Pax6, NCam, and Nestin. Scale bar ¼ 100 lm. (B): Genomic regions of the Nanog gene bound by Nanog andSmad2/3 proteins. Chromatin immunoprecipitation assays were performed using antibodies directed against Smad2/3 or Nanog. The immunopre-cipitated DNA was then amplified using quantitative polymerase chain reaction (PCR) and specific primers to detect enrichment in the denotedgenomic regions. Results were normalized against a control region (�6237 þ6414) and are expressed as � standard derivation from three experi-ments. (C): Mutation of a putative Smad2/3 binding site in the Nanog promoter inhibits the transcriptional activation induced by activin/nodalsignaling. Luciferase reporter genes containing the promoter of the human Nanog gene (�379 þ18) with or without mutated Smad2/3 binding-sites were cotransfected into hIPSCs along with Renilla expression vector (control for normalization) in the presence of activin and FGF2 or inthe presence of SB431542 (negative control). Relative firefly luciferase activity (normalized to Renilla activity) is expressed as mean � standarddeviation from three independent experiments. (D): Constitutive expression of Nanog prevents differentiation of hIPSCs induced by inhibition ofactivin/nodal signaling. CRL-hIPSCs and Nanog-CRL-hIPSCs subline 2 (Nanog) were grown for 14 days in the presence of SB431542 þ FGF2and then immunostaining analyses were performed to detect the expression of Oct4 and Nanog. Scale bar ¼ 100 lm. (E): Nanog overexpressionblocks neuroectoderm differentiation of hIPSCs grown in the absence of activin/nodal signaling. Nanog-overexpressing hIPSCs (Nanog) weregrown for 14 days in CDM þ SB431542 þ FGF2 þ Nanog, and then the expression of the denoted genes was analyzed using real-time PCR.Normalized expression is shown as the mean � SD from three informative experiments. CRL-hIPSCs grown in CDM þ activin þ FGF2 wereused as negative and CRL-hIPSCs grown in CDM þ SB431542 þ FGF2 were used as positive control (WT). Abbreviations: A þ F, activin andFGF2; FGF2, fibroblast growth factor 2; hIPSC, human induced pluripotent stem cell; SB, SB431542; WT, wild type.

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sample 0.6 lg of total RNA was reverse transcribed using Super-script II Reverse Transcriptase (Invitrogen). Real-time polymerasechain reaction (PCR) reaction mixtures were prepared asdescribed (SensiMiX protocol; Quantace, London, http://www.quantace.com) then denatured at 94�C for 5 minutes andcycled at 94�C for 30 seconds, 60�C for 30 seconds, and 72�Cfor 30 seconds, followed by a final extension at 72�C for 10minutes after completion of 40 cycles. Primer sequences aredescribed elsewhere [8, 13]. Real-time PCR reactions were per-formed using a Stratagene Mx3005P (La Jolla, CA, http://www.stratagene.com) in triplicate and normalized to porphobili-nogen deaminase (PBGD) in the same run.

Immunofluorescence

hIPSCs or their differentiated progeny were fixed for 20 minutesat 4�C in 4% paraformaldehyde and then washed three times inPBS. Cells were incubated for 20 minutes at room temperature inPBS containing 10% donkey serum (Serotec Ltd.) and subse-quently incubated overnight at 4�C with primary antibody dilutedin 1% donkey serum in PBS as follows: Oct-4 (1:100; Abcamab18976 [Cambridge, U.K., http://www.abcam.com] or SantaCruz Biotechnology Inc. [Santa Cruz, CA, http://www.scbt.com]),Sox2 (1:100; Abcam ab15830), Brachyury (1:100; Abcamab20680 or R&D Systems Inc.), Sox17 (R&D Systems Inc.),FoxA2 (1:50; Abcam ab5074), GATA4 (1:250; Santa Cruz Bio-technology Inc.), GATA6 (1:200; Abcam ab22600 or Santa CruzBiotechnology Inc.), CXCR4 (1:100; R&D Systems Inc. or BDPharmingen), Nestin (1:200; Abcam ab5968), NCAM (1:100;Abcam ab8077), and N-Cadherin (1:100; Abcam ab18203). Cellswere then washed three times in PBS and incubated with TexasRed or fluorescein isothiocyanate-conjugated anti-mouse IgG(Sigma-Aldrich; 1:200 in 1% donkey serum in PBS) or rabbitIgG (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org;1:400 in donkey serum in PBS) or goat IgG (Jackson Laboratory;1:400 in donkey serum in PBS) for 2 hours at room temperature.Unbound secondary antibody was removed by three washes inPBS. Hoechst 33258 was added to the first wash (Sigma-Aldrich;1:10,000).

RESULTS

Derivation of hIPSCs in Chemically DefinedCulture Conditions

Several groups have recently reported that human fibroblastscan be reprogrammed into induced pluripotent stem cells(iPSCs) by overexpressing the four transcription factors Oct-4, Klf-4, Sox2, and c-Myc or other factors [2–4]. Using suchan approach with four factors as described by Takahashi et al.[2], we generated hIPSC lines from three sources of fibro-blasts: neonatal foreskin fibroblasts (CRL), embryonic lungfibroblasts (MRC5), and adult dermal fibroblasts from a 28-year-old female (Bi) (Table 1). Importantly, we also obtainedsimilar results using only three factors, confirming that c-Mycis not necessary for hIPSC derivation [3, 14]. The resultinglines were initially grown on mouse feeder cells in mediacontaining serum supplemented with FGF2. After one pas-sage, hIPSC colonies were transferred to a chemically definedculture system (CDM þ activin þ FGF2), which is currentlyused in our laboratory to grow hESCs and also epiblast stemcells (EpiSCs) derived from the epiblast layer of postimplan-tation mouse embryos [11]. The resulting hIPSC colonieswere grown for 20 passages without noticeable differentiationwhile maintaining the expression of endogenous pluripotencymarker genes Oct-4, Sox2, Nanog, Tra-1-60, and alkalinephosphatase (Fig. 1A, 1B). In addition, fluorescence-activatedcell sorting (FACS) analyses showed that 90% of the cells

grown in these culture conditions expressed the pluripotencymarker Tra-1-60, demonstrating the absence of background ofspontaneous differentiation (Fig. 1C). We then derivedhIPSCs directly in CDM þ activin þ FGF2 in the total ab-sence of feeders or serum (Table 1). Using this approach, wegenerated 33 hIPSC lines expressing similar levels of the en-dogenous pluripotency factors Oct-4, Sox2, Nanog, and Tra-1-60 (Fig. 1A–1C, hIPSC line 40) compared with hIPSC linesderived on feeders in the presence of serum (Fig. 1A–1C,hIPSC line 30 and 35). In addition, hIPSC lines derived inchemically defined conditions showed the same capacity fordifferentiation as hIPSCs derived on feeders in serum contain-ing medium (see below and Figs. 3-5, hIPSC line 40) andthey had the capacity to form teratomas containing derivativesof the three germ layers when injected in the testis capsule ofimmunodeficient mice (Fig. 1D).

We further characterized the hIPSCs lines generated andgrown in CDM by defining the expression of exogenous trans-genes and endogenous genes using real-time PCR. These analy-ses showed that the exogenous transgenes were stronglysilenced in fully reprogrammed cells (Fig. 1E), with the excep-tion of Klf-4. However, we observed that levels of KLF-4 tran-scripts were very low in hESCs (change in cycle threshold,>32), suggesting that KLF-4 was not expressed in hESCs and,thus, that exogenous expression of KLF-4 remained limited.This was confirmed by the unperturbed self-renewal and differ-entiation behavior of hIPSCs with KLF4 residual expression(see below). These results apparently contradict recent publica-tions suggesting that constitutive expression of exogenoustransgene can be an issue in hIPSCs and thus that transgeneexcision is necessary to establish robust hIPSCs [15]. This dif-ference might be explained by our use of retroviruses, whichare known to be strongly silenced in pluripotent cells [16] incontrast to lentiviruses, whose expression may persist. How-ever, we cannot exclude that our culture conditions could alsohave an impact on transgene expression. Finally, we determinedthe number of transgene copies inserted in the genome of fivehIPSC lines, showing that each line contains between 3 and 10copies for each transgene (Fig. 1F), which is in the range of pre-vious studies [2–4].

Taken together these results demonstrate that a chemicallydefined medium containing activin and FGF2 is sufficient togenerate and grow hIPSCs, suggesting that hESCs and hIPSCsrely on the same signaling pathways to maintain their pluripo-tent status.

Human Induced Pluripotent Stem Cells Rely onActivin/Nodal Signaling to Maintain TheirPluripotent Status

To examine the importance of activin/nodal signaling inhIPSCs, we analyzed the effect of SB431542, a chemical in-hibitor of activin receptor activity, on their pluripotency.hIPSCs generated from three different fibroblast lines weregrown for 10 days in CDM þ FGF2 þ SB431542 or in CDMþ FGF2 (omitting activin) and then the expression of pluripo-tency markers was analyzed using immunostaining (Figs. 2A,6; supporting information Fig. 1). Absence of activin/nodalsignaling systematically induced differentiation of hIPSCs, asshown by the loss in pluripotency markers (Fig. 2A; support-ing information Fig. 1), thus confirming that activin/nodal sig-naling is necessary to maintain pluripotency in hIPSCs. Wethen analyzed the expression of specific markers for the threegerm layers and extraembryonic tissues to determine the na-ture of the cells generated by inhibiting activin signaling inhESCs (Figs. 2A, 6; supporting information Fig. 1) and weobserved that cells generated in these culture conditions


expressed mainly markers of extraembryonic lineages (primi-tive endoderm: Sox7, GATA4, and GATA6; trophectoderm:CDX2 and Eomes) and neuroectoderm (Sox2, Sox1, Pax6,Nestin, and NCam). These results suggest that activin signal-ing blocks extraembryonic and neuroectoderm differentiationof hIPSCs similarly to the effects of activin/nodal signalingseen in hESCs and they reinforced recent studies showingsimilar outcomes using serum-containing media [17, 18].Importantly, however the inhibitory effect of activin/nodalsignaling on extraembryonic differentiation is specific tohIPSCs since hESCs grown in the absence of activin/nodal

signaling homogenously differentiate into neuroectoderm[10].

Activin/Nodal Signaling Maintains Nanog Expressionin hIPSCs

The observations that hIPSCs can be grown and derived (Ta-ble 1) in CDM supplemented with activin and FGF2 and thatinhibition of activin/nodal signaling induces their differentia-tion suggest that hESCs and hIPSCs rely on the same signalingpathways to maintain their pluripotent status. We examined

Figure 3. BMP4 drives differentiation of hIPSCs into extraembryonic tissues. (A): hIPSCs grown in the presence of BMP4 (and in the absenceof activin) differentiate into extraembryonic tissues. CRL-hIPSCs were grown for 12 days in chemically defined medium (CDM) þ BMP4(10 ng/ml) þ SB431542 (10 lM), and then immunostaining analyses were performed to detect expression of pluripotency markers Oct4 andNanog, primitive endoderm markers Sox7, GATA4, and GATA6, and trophectoderm markers CDX2 and Eomes. Scale bar ¼ 100 lm. (B): Ab-sence of pluripotency markers (Oct-4, Sox2, Nanog) and induction of extraembryonic markers (CDX2, Hand1, Sox7) in hIPSCs grown inCDM þ BMP4 þ SB431542. CRL-hIPSCs (lines 30, 35, 40) and hESCs (H9) were grown for 10 days in these culture conditions, and thenRNAs were extracted and expression of the denoted genes was analyzed using quantitative polymerase chain reaction. Normalized expression isshown as the mean � SD from three informative experiments. hESCs grown in CDM þ activin þ fibroblast growth factor 2 (FGF2) were usedas negative controls. (C): Fluorescence-activated cell sorting (FACS) analysis of the fraction of cells expressing Tra-1-60 after extraembryonicdifferentiation confirmed homogenous differentiation of hIPSCs. CRL-hIPSCs (lines 30, 35, 40) were induced to differentiate for 10 days intoextraembryonic tissues using the culture conditions described above, and then the fraction of cells expressing Tra-1-60 was determined usingFACS. hESCs grown in CDM þ activin þ FGF2 were used as positive control (hESCs). Abbreviations: BMP4, bone morphogenic protein 4;hIPSC, human induced pluripotent stem cell; SB, SB431542; Undiff, undifferentiated negative controls.


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this hypothesis at the molecular level by analyzing Nanog reg-ulation. It has been recently shown that activin/nodal/TGFbsignaling maintains pluripotency of hESCs and mouse EpiSCs(mEpiSCs) by controlling the expression of Nanog through theTGFb effectors Smad2/3 [7, 19, 20]. To determine whethersimilar mechanisms occur in hIPSCs, chromatin immunopreci-pitation (ChIP) assays were performed to identify genomicregions bound by Smad2/3 in the Nanog promoter (Fig. 2B).This showed that Smad2/3 binds the same genomic regionidentified in hESCs using ChIP. Interestingly, this region con-tains binding sites for Oct4, Sox2, and Nanog [21, 22] andalso two consensus Smad2/3 binding sites (S2/3-(1) and S2/3-(2), Fig. 2B)). Luciferase assays revealed that mutations inthese Smad2/3 binding sites abolished transcriptional activityinduced by activin/nodal signaling (Fig. 2C), strongly suggest-ing that they are functionally important in Nanog regulation.Importantly, similar results were obtained using hESCs andtwo independent hIPSC lines generated from foreskin fibro-blasts and adult fibroblasts (supporting information Fig. 2),suggesting this molecular mechanism is common to hIPSClines [19]. Finally, we stably overexpressed Nanog in hIPSCsand then grew the resulting sublines (Nanog-hIPSCs) in CDMsupplemented with SB431542 and FGF2. Wild-type hIPSCs orGFP-expressing hIPSCs grown in these culture conditionsquickly differentiated into neuroectoderm progenitors as

shown by the decrease in the pluripotency markers Oct-4 andNanog and in the increase in the neuroectoderm markers Gbx2and Sox1 (Fig. 2D, 2E). On the other hand, Nanog-hIPSCsmaintained a pluripotent morphology and sustained expressionof Oct-4, Sox2, and Nanog without expression of neuroecto-derm markers (Fig. 2D, 2E). This suggests that Nanog is suffi-cient to substitute for activin/nodal signaling. Together, thesefindings demonstrate that activin/nodal signaling maintainspluripotency of hIPSCs by directly controlling the expressionof Nanog, as it does in hESCs and mEpiSCs [19], therebystrikingly mimicking the mechanisms maintaining pluripo-tency of embryo-derived pluripotent stem cells.

hIPSCs Are Similar to hESCs and mEpiSCs inTheir Response to Culture Conditions ControllingDifferentiation

We then analyzed the effect of three fully defined culture con-ditions recently developed to drive differentiation of hESCsand mEpiSCs into extraembryonic tissues, neuroectoderm,and mesendoderm [10]. Addition of BMP4 in the absence ofactivin and FGF2 is sufficient to drive differentiation ofhESCs and mEpiSCs into primitive endoderm and trophecto-derm. hIPSCs grown in similar culture conditions differenti-ated into a mixed population expressing markers for primitive

Figure 4. Inhibition of activin/nodal signaling and bone morphogenic protein (BMP) signaling in the presence of FGF2-induced neuroectodermdifferentiation of hIPSCs. (A): hIPSCs grown in the absence of activin/nodal and BMP4 signaling differentiate into neuroectoderm. CRL-hIPSCswere grown for 12 days in chemically defined medium (CDM) þ FGF2 (12 ng/ml) þ SB431542 (10 lM) þ Noggin (200 ng/ml), and then im-munostaining analyses were performed to detect expression of the pluripotency markers Oct4 and Nanog and the neuroectoderm markers Sox2,Pax6, NCam, Nestin, and bIII Tubulin. Scale bar ¼ 100 lm. (B): Absence of pluripotency markers (Oct-4, Nanog) and induction of neuroecto-derm markers (Sox2, Sox1, Gbx2) in hIPSCs grown in CDM þ SB431542 þ FGF2. CRL-hIPSCs (lines 30, 35, 40) and hESCs (H9) were grownfor 10 days in these culture conditions, and then RNAs were extracted and expression of the denoted genes was analyzed using quantitativepolymerase chain reaction. Normalized expression is shown as the mean � SD from three informative experiments. hESCs grown in CDM þactivin þ FGF2 were used as control. (C): Fluorescence-activated cell sorting (FACS) analysis for the fraction of cells expressing NCam showinghomogenous formation of neuroectoderm. CRL-hIPSCs (lines 30, 35, 40) were differentiated for 10 days using the culture conditions describedabove, and then the fraction of cells expressing N-Cam was determined using FACS. hESCs grown in CDM þ activin þ FGF2 were used as neg-ative control (hESCs). Abbreviations: FGF2, fibroblast growth factor 2; hIPSC, human induced pluripotent stem cell; SB, SB431542; Undiff, un-differentiated negative controls.


endoderm (GATA4, GATA6, Sox7; supporting informationFig. 3) and trophectoderm (CDX2; supporting informationFig. 3). However, the decrease in expression of pluripotencyfactors Oct-4 and Nanog was observed only after 12 dayswith hIPSCs compared with 7 days with hESCs, suggestingthat BMP4 effects might be less potent with hIPSCs (support-ing information Fig. 3). However, we also observed that theeffect of BMP4 was strongly increased by adding the activin/nodal receptor inhibitor SB431542 (Fig. 3A–3C), suggestingthat endogenous activin/nodal signaling is enough to delayextraembryonic differentiation of hIPSCs, reflecting a compet-itive interaction between these two signaling pathways [18].Together, these results demonstrate that hIPSCs have thecapacity to differentiate into extraembryonic tissues, includingcells expressing trophectoderm markers, a characteristic spe-cific to hESCs and mEpiSCs but not mESCs.

In the second set of culture conditions SB435132 wasused to drive differentiation of hESCs into neuroectoderm byinhibiting activin/nodal signaling in the presence of FGF2. In-hibition or absence of activin/nodal signaling in hIPSCsresulted in loss of the pluripotency markers Oct-4 and Nanog(Fig. 2A; supporting information Fig. 1). Immunostaininganalyses revealed that the differentiated cells resulting fromthis treatment expressed neuroectoderm markers (Sox2, Sox1,Pax6, and Nestin, Fig. 2A) and extraembryonic markers(GATA6, GATA4, and CDX2, Fig. 2A). Thus, inhibition ofactivin signaling appeared to drive differentiation of hIPSCsinto a mixed population of neuroectoderm and extraembryoniccells. Interestingly, a combination of SB431542 and the BMPinhibitor Noggin (200 ng/ml) strongly reduced the extraem-bryonic differentiation induced by the inhibition of activin/nodal signaling, resulting in a nearly homogeneous population

Figure 5. A combination of activin (high-dose), BMP4, and FGF2 drives differentiation of hIPSCs into mesendoderm. (A): Immunostaininganalyses for the expression of mesendoderm markers in hIPSCs grown in culture conditions capable of inducing mesendoderm differentiation ofhESCs. CRL-hIPSCs were grown for 4 days in chemically defined medium (CDM) þ activin (100 ng/ml) þ BMP4 (10 ng/ml) þ FGF2 (20 ng/ml) þ LY294002 (10 lM), and then immunostaining analyses were performed to detect expression of pluripotency markers Oct4 and Nanog, themesendoderm marker Brachyury, and the definitive endoderm markers FoxA2, Sox17, N-Cadherin (NCad), and CXCR4. Scale bar ¼ 100 lm.(B): Absence of pluripotency markers (Oct-4, Nanog, Sox2) and extraembryonic marker (Sox7) and induction of mesendoderm markers (Bra-chyury, Mixl1, GSG, Sox17) in hIPSCs grown in CDM activin þ BMP4 þ FGF2 þ LY294002. CRL-hIPSCs (lines 30, 35, 40) and hESCs (H9)were grown for 10 days in these culture conditions, and then RNAs were extracted and expression of the denoted genes was analyzed using quan-titative polymerase chain reaction. hESCs grown in CDM þ activin þ FGF2 were used as a negative control. Normalized expression is shown asthe mean � SD from three informative experiments. (C): Fluorescence-activated cell sorting (FACS) analysis of the fraction of cells expressingCXCR4 and PDGFaR after mesendoderm differentiation confirming homogenous differentiation of hIPSCs. CRL-hIPSCs (lines 30, 35, 40) cellswere differentiated for 3 days into mesendoderm progenitors using the culture conditions described above, and then the fraction of cells express-ing CXCR4 or PDGFaR was determined using FACS. hESCs grown in CDM þ activin þ FGF2 were used as a negative control. Abbreviations:BMP4, bone morphogenic protein 4; FGF2, fibroblast growth factor 2; hIPSC, human induced pluripotent stem cell; Mesendo, mesendoderm;PDGFaR, platelet-derived growth factor a receptor; Undiff, undifferentiated negative controls.


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of cells expressing neuroectoderm markers (Sox2, Sox1,Pax6, Nestin, NCam, and bIII Tubulin, Fig. 4A–4C). Togetherthese results demonstrate that similarly to hESCs, inhibitionof activin/nodal signaling is sufficient to drive differentiationof hIPSCs into neuroectoderm, with the difference that an en-dogenous BMP-like activity in hIPSCs apparently interfereswith their neuroectoderm differentiation, which therebyrequires inhibition of both activin/nodal and BMP pathways.

In the third of these chemically defined conditions, athree-step protocol is used to drive differentiation into mesen-doderm and then into endoderm [10]. In the first step, pluripo-tent stem cells grown initially in CDM þ activin þ FGF2 areplated for 48 hours on fibronectin in CDM-PVA medium con-taining activin (10 ng/ml) and FGF2 (12 ng/ml). In the secondstep, the FGF receptor inhibitor SU5402 (10 lM) and a lowerdose of activin (5 ng/ml) is added for 3 days. Addition ofSU5402 reduces adhesion of the colonies, which begin toform compact aggregates of cells expressing Oct4 and lowlevels of the mesendoderm marker Brachyury but not Sox17.In the third step, a combination of BMP4 (10 ng/ml), FGF2(20 ng/ml), and activin (30 or 100 ng/ml) is added, drivingpluripotent colonies to differentiate into homogenous popula-tion of endoderm cells expressing Sox17, CXCR4, GSC,FoxA2, and Mixl1 [10]. When hIPSCs were subjected to thisthree-step protocol, they strongly differentiated at the secondstep (inhibition of the FGF signaling pathway), generatingextraembryonic cells (supporting information Fig. 4), suggest-ing that FGF signaling is strictly necessary to maintain hIPSCpluripotency. We then analyzed the effect of the third step inthis protocol (combination of activin [high-dose, 100 ng/ml],FGF2 [20 ng/ml], and BMP4 [10 ng/ml]). hIPSCs grown inthese conditions differentiated into heterogeneous populationsof cells expressing mesoderm (Brachyury), endoderm(Sox17), and pluripotency (Oct-4) markers (supporting infor-mation Fig. 5). Interestingly addition of the PI3Kinase inhibi-tor LY294002 increased the expression of mesendoderm (Bra-chyury) and endoderm (Sox17, FoxA2, NCad, and CXCR4)markers, but decreased the expression of pluripotency markers(Oct-4, Nanog, Sox2) (Fig. 5A–5C) without inducing theexpression of the extraembryonic endoderm marker Sox7,thus extending to hIPSCs the recent observation thatLY294002 can improve endoderm differentiation of hESCs[23]. In addition, FACS analyses revealed that almost 70% ofthe cells generated under these conditions expressed the defin-itive endoderm marker CXCR4, whereas the remaining cellsexpressed the mesendoderm marker PDGFaR, confirming thathIPSCs differentiated entirely toward the mesendoderm/endo-derm pathway. Importantly, hESCs grown in the same cultureconditions (combination of activin [high-dose], FGF2, BMP4,and LY294002) differentiated homogenously into definitiveendoderm cells (T. Touboul et al., manuscript in preparation).Therefore, the combination of a high-dose of activin, BMP4,and FGF2 together with inhibition of PI3Kinase is sufficientto drive differentiation of hIPSCs and hESCs into nearly ho-mogenous endoderm population. Taken together with ourresults using the other chemically defined conditionsdescribed here (CDM þ BMP4 � activin/nodal, CDM þSB431542 þ FGF2 þ Noggin), these findings confirm thathIPSCs and hESCs are similarly responsive to growth factorscontrolling their differentiation into extraembryonic and em-bryonic lineages.

Finally, similar results were obtained with hIPSC lines(n ¼ 15) generated from foreskin fibroblasts (CRL-hIPSC)(including three generated without c-Myc) and with hIPSClines generated from MRC5 fibroblasts (n ¼ 2) and Bi fibro-blasts (n ¼ 2) (Fig. 6), suggesting that our findings are inde-pendent of the hIPSC line used. Taken together, these obser-

vations demonstrate that hIPSCs respond robustly tochemically defined culture conditions that have been devel-oped and used to differentiate hESCs into early progenitorpopulations and therefore that similar signaling pathways cancontrol differentiation of both hIPSCs and hESCs.

Figure 6. Human induced pluripotent stem cells (hIPSCs) generatedfrom diverse fibroblast lines are similarly responsive to culture condi-tions controlling differentiation of hESCs. Expression of pluripotency(Oct-4, Sox2, Nanog), extraembryonic (Cdx2, Sox7, GATA4), neuro-ectoderm (Sox2, Sox1, Pax6), and mesendoderm (Brachyury, FoxA2,Sox17) markers during differentiation of hIPSCs generated fromMRC5 fibroblasts (line 5) and Bi fibroblasts (line 11). hIPSCs weregrown in chemically defined medium þ activin þ FGF2 and theninduced to differentiate following the protocols described above forhESCs and CRL-hIPSCs (BMP4 þ SB431542 for extraembryonic tis-sues, SB431542 þ FGF2 þ Noggin for neuroectoderm, activin þFGF2 þ BMP4 þ LY294002 for mesendoderm). Immunostaininganalyses were performed at 5 days (pluripotent cells and mesendo-derm) or 10 days (extraembryonic and neuroectoderm) after plating.Scale bar ¼ 100 lm. Abbreviations: BMP4, bone morphogenic pro-tein 4; Bra, Brachyury; FGF2, fibroblast growth factor 2; SB,SB431542.


CONCLUSION

Taken together, our observations reinforce the similaritybetween hESCs and hIPSCs, showing that both cell types relyon similar signaling pathways to maintain their pluripotencyand to control their differentiation. However, our study alsoreveals some differences between these two pluripotent celltypes, mainly in the interaction between the activin signalingpathway and the BMP signaling pathway. Indeed, inhibitionof BMP signaling is required in addition to inhibition of acti-vin/nodal signaling to drive differentiation of hIPSCs into ahomogenous population of neuroectoderm cells. Moreover, in-hibition of activin/nodal signaling is necessary in addition totreatment with BMP4 to induce extraembryonic differentiationof hIPSCs. A potential explanation for this difference betweenhESCs and hIPSCs is that hIPSCs display higher endogenouslevels of BMP4 and activin/nodal activity, which in turnrequires their respective inhibitors to promote differentiationinto neuroectoderm and extraembryonic pathways, respec-tively. By contrast, the hESC lines used in our study did notdisplay significant BMP4 signaling activity, as shown by theabsence of Smad1/5/8 phosphorylation in hESCs grown inCDM [5]. Interestingly, mEpiSCs also require inhibition ofBMP signaling to differentiate homogenously into neuroecto-derm and activin signaling to differentiate into extraem-bryonic tissues.

Despite such differences, our results emphasize the overallsimilarity between hESCs, hIPSCs, and mEpiSCs. Indeed, allthree pluripotent cell types rely on activin to control Nanogexpression and to maintain their pluripotency. In addition,they all rely on similar pathways to induce differentiation intoextraembryonic and embryonic lineages. Specifically, inhibi-tion of activin in the presence of FGF2 is essential for induc-ing neuroectoderm differentiation in the three cell types,whereas BMP4 drives their differentiation into extraem-bryonic tissues including cells expressing markers of trophec-toderm or of primitive endoderm. Finally, the combination ofa high dose of activin plus BMP4 and FGF2 and inhibition ofPI3K are sufficient to generate a near homogenous populationof definitive endoderm. These observations demonstrate thathESCs, hIPSCs, and mEpiSCs are similarly responsive to keysignaling pathways controlling their early cell fate decisions.Accordingly, these three pluripotent cell types appear to sharea common pluripotent state that defines their capacity to reactto exogenous growth factors. Importantly, this shared pluripo-tent state is also associated with a specific embryonic identitycorresponding to the epiblast of postimplantation stage mam-malian embryos [11, 24] from which mEpiSCs are derived.Therefore, our results demonstrate for the first time the extentto which hESCs and hIPSCs are functionally similar, repre-senting a pluripotent status equivalent to that of the late epi-blast of the pregastrulation mouse embryo (EpiSCs). Thisfinding led to the conclusion that the technical and conceptualknowledge accumulated during the past decade using hESCs

(and more recently using EpiSCs) should be readily transfera-ble to hIPSCs.

Finally, we describe experiments showing that hIPSCscan be derived and directed to differentiate into extraem-bryonic tissues, neuroectoderm, or mesendoderm, in fullychemically defined culture conditions. Because previouslyavailable conditions for derivation and differentiation ofhIPSCs along these lineages were based on media containingundefined components (serum) and animal products (feedercells and matrices) [2–4], our findings represent a first steptoward the potential use of hIPSCs in cell-based therapies.However, the use of animal products to establish fibroblastsfrom skin and other biopsies and the high number of viralintegrations preclude the clinical use of the lines described inthis study. Nevertheless, skin fibroblasts can be generated ingood manufacturing practice conditions using xeno-free con-ditions and recent studies have described the derivation ofhIPSCs without stable genetic modification [25–28]. There-fore, our culture system could be advantageously used incombination with such approaches to derive hIPSCs in condi-tions fully compatible with clinical applications. In addition,the establishment of chemically defined protocols and charac-terization of outcomes for in vitro differentiation of hIPSCsprovide unique in vitro systems to validate the capacity ofhIPSCs to differentiate into mature progeny of the threegerm layers. Such studies could potentially replace in vivoanalyses such as teratoma assays, which are resource con-suming and nonquantitative. Finally, our findings could becrucial in defining ‘‘universal’’ protocols of differentiation ca-pable of yielding particular differentiated cell types that applyto a wide range of pluripotent lines including patient-specifichIPSC lines.

ACKNOWLEDGMENTS

We thank Glyn Stacey and the U.K. Stem Cell Bank for theMRC5 fibroblasts. This work was supported by an MRCresearch grant (R.A.P), by association Francaise pour l’etude dufoie (T.T.), by Agence National de la Recherche (ANR GrantANR-05-BLAN-006-02) (T.T. and A.W.), by the Juvenile Dia-betes Research Fundation (R.A.P., and L.V.), by the EvelynTrust (R.A.P, M.A.), by anMRC/Diabetes U.K. Career Develop-ment fellowship (L.V.), by a MRC senior non-clincal fellowship(S.B.) and by National Institute for Health Research CambridgeBiomedical Research Centre.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

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