EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Signaling Pathways Controlling Pluripotency and Early Cell Fate Decisions of Human Induced Pluripotent Stem Cells
LUDOVIC VALLIER,a THOMAS TOUBOUL,a,b STEPHANIE BROWN,a CANDY CHO,a BILADA BILICAN,c MORGAN ALEXANDER,a JESSICA CEDERVALL,d SIDDHARTHAN CHANDRAN,c LARS AHRLUND-RICHTER,d ANNE WEBER,b ROGER A. PEDERSENa Laboratory 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 Neuroectoderm Mesendoderm Mesoderm Endoderm Activin Human Mouse

Primitive endoderm

Trophectoderm

ABSTRACT
Human pluripotent stem cells from embryonic origins and those generated from reprogrammed somatic cells share many characteristics, including indenite proliferation and a sustained capacity to differentiate into a wide variety of cell types. However, it remains to be demonstrated whether both cell types rely on similar mechanisms to maintain their pluripotent status and to control their differentiation. Any differences in such mechanisms would suggest that reprogramming of broblasts to generate induced pluripotent stem cells (iPSCs) results in novel states of pluripotency. In that event, current methods for expanding 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 mechanistic similarity to human ESCs. We also show that growth factors necessary and sufcient for achieving specication of human ESCs into extraembryonic tissues, neuroectoderm, and mesendoderm also drive differentiation of human iPSCs into the same tissues. Importantly, these experiments were performed in fully chemically dened medium devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Together these data reveal that human iPSCs rely on mechanisms similar to human ESCs to maintain their pluripotency and to control their differentiation, showing that these pluripotent cell types are functionally equivalent. STEM CELLS 2009;27:26552666

Disclosure of potential conicts of interest is found at the end of this article. for understanding whether pluripotent stem cells generated from reprogrammed broblasts are functionally equivalent to their embryonic counterparts. Moreover, such understanding is the key to using currently available methods for hESCs to drive differentiation of hIPSCs into diverse cell types. Although it has been clearly demonstrated that activin/nodal signaling maintains the pluripotent status of hESCs [57], it has proved more challenging to dene the growth factors necessary and sufcient to control early events of hESC differentiation. Indeed, most of the current methods available for inducing differentiation of hESCs are based on culture media containing factors (such as serum, stroma cells, and complex matrices) that obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications [8, 9]. To address this issue, we recently developed an approach based on a fully chemically dened

INTRODUCTION
Human embryonic stem cells (hESCs) are pluripotent stem cells generated from embryos at the blastocyst stage [1]. Their embryonic origin confers upon them the capacity to proliferate indenitely in vitro while maintaining their ability to differentiate into a large number of cell types in vitro and in vivo. These properties are shared by human induced pluripotent stem cells (hIPSCs) generated from broblasts that have been reprogrammed by overexpressing key transcription factors (Oct-4, Nanog, Sox2, Klf-4, c-Myc, and other pluripotency-related genes) [24]. Despite these similarities, it remains to be demonstrated whether hESCs and hIPSCs rely on the same mechanisms to maintain their pluripotency and to control their subsequent cell fate decisions. This is essential

Author contributions: L.V.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; T.T.: collection and/or assembly of data, data analysis and interpretation; S.B.: collection and/or assembly of data, data analysis and 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 of Cambridge, Cambridge, CB2 0SZ, United Kingdom. Telephone: 00 44 (0) 1223747489; Fax: 44 (0) 1223 763350; e-mail: lv225@ cam.ac.uk Received April 7, 2009; accepted for publication August 6, 2009; rst published online in STEM CELLS EXPRESS Month 00, C 2009. V AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.199

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Table 1. Summary of hIPSC derivations

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

1 (4 factors) Fibro CRL 2 (4 factors) Fibro CRL 3 (4 factors) Fibro CRL 4 (3 factors, no Myc) Fibro CRL 5 (4 factors) Fibro MRC5 6 (4 factors) Fibro Bi

SR FGF feeders CDM activin FGF SR FGF feeders CDM activin FGF SR FGF feeders CDM activin FGF SR FGF feeders CDM activin FGF SR FGF feeders CDM activin FGF SR FGF feeders CDM activin FGF

30 23 56 6 16 4 8

(Av. 7.5) (Av. 4.6) (Av. 18.7) (Av. 3) (Av. 8) (Av. 2) (Av. 8) ND 24 (Av. 12) ND 34 (Av. 17) ND

13 10 20 2 0 0 8 0 10 0 10 0

hIPSCs were generated from foreskin broblasts (CRL), fetal lung broblasts (MRC5), and adult dermal broblasts (Bi) as described in Materials and Methods using four (Oct-4, Sox2, KLF-4, c-Myc) or three (Oct-4, Sox2, KLF-4) factors. hIPSCs were derived either on feeders 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 hIPSC colonies generated per plate. The number of hIPSC colonies picked and expanded is also indicated. In total, 22 hIPSC lines (15 generated from CRL broblasts including 3 hIPSC lines generated without c-Myc, 2 hIPSC lines generated from MRC5 broblasts, and 2 from Bi broblasts) were used to analyze the responsiveness of hIPSCs to culture conditions that were effective in maintaining the pluripotent status and driving the differentiation of hESCs. Abbreviations: Av., average; CDM, chemically dened medium; FGF, broblast growth factor; hIPSC, human induced pluripotent stem cell; ND, not done; SR, serum replacer.

medium to drive hESC differentiation into extraembryonic tissues, neuroectoderm, and mesendoderm [10], which represent the earliest cell types generated during mammalian embryonic development. Here, we analyzed the effect of these culture conditions on hIPSCs and we observed that they worked equally well to promote their differentiation into similar tissues. In addition, hIPSCs could be generated and grown in chemically dened culture conditions supplemented with activin and broblast growth factor 2 (FGF2), whereas inhibition of activin/nodal/transforming growth factor b (TGFb) signaling prompted their differentiation, demonstrating that hIPSCs, like hESCs, rely on this signaling pathway to maintain their pluripotency. These observations were reinforced by showing that Nanog expression is directly controlled by activin/nodal/TGFb signaling in hIPSCs as in hESCs and that constitutive expression of Nanog is sufcient to maintain pluripotency of hIPSCs grown in the absence of activin. Together, these results demonstrate that human pluripotent stem cells of embryonic origin or those generated from reprogrammed broblasts rely on the same signaling pathways to control their pluripotency and their differentiation. Importantly, the similarity of these responses by hESCs and hIPSCs afrms their biologic equivalence and distinguishes them from mouse ESCs (mESCs). Finally, these results show that pluripotent mammalian stem cells can be induced to undergo early cell fate decisions in chemically dened medium supplemented with a minimal set of growth factors, thereby providing a general approach for modeling the transition from pluripotency to specication of the three germ layers that form during mammalian gastrulation.

MATERIALS

AND

METHODS

Generation of Human iPSCs

Human foreskin broblasts were obtained from American Type Culture Collection (Manassas, VA, http://www.atcc.org) (CRL2429) and grown following the providers recommended protocol. Moloney murine leukemia virus-derived vectors each containing 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 puried by several rounds of tangential ltration to reach a titer of about 109 transduction units/ml and to assure a high level of purity. On day 1, 105 broblasts were plated in each well of 6-well plates in medium containing fetal bovine serum (FBS, Biosera, EastSussex, U.K., http://www.biosera.com). The following day the cells were transduced with the four viruses at a multiplicity of infection of 10 (for each virus) for 24 hours. On day 3, the cells were washed three times in phosphate-buffered saline (PBS, Gibco, Grand Island, NY, http://www.invitrogen.com) and then grown in medium containing FBS for three additional days. On day 7, the cells were passaged on plastic plates containing irradiated mouse embryonic broblasts and then grown for 2 additional days in medium containing FBS. After day 9, the cells were grown in standard hESC culture conditions (knockout [KSR], (Gibco) FGF2 (4 ng/ml; R&D Systems Inc., Minneapolis, http://www. rndsystems.com). The rst hIPSC colonies appeared 10 days later and they could be picked after 5 additional days of culture. Individual colonies were picked and either transferred into a single well of 12-well plates containing mouse feeders in KSR FGF2 or directly transferred into a well of 12-well plates precoated with porcine gelatin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and FBS containing chemically dened medium (CDM) Activin (10 ng/ml, kindly provided by Marco Hyvonen) FGF2 (12 ng/ml). The resulting colonies were then expanded using enzymatic dissociation as described for hESCs grown in similar culture conditions (as discussed in Growth of hESCs and hIPSCs in Feeder-Free and Serum-Free Conditions). For derivation in chemically dened medium, the broblasts were grown for 5 days in FBS-containing medium after transduction and then grown in CDM activin (10 ng/ml) FGF2 (12 ng/ ml) for 36 additional days without splitting. hIPSC colonies appeared on day 29 and individual colonies were picked 5 days later to be expanded in CDM activin (10 ng/ml) FGF2 (12 ng/ml) as described above.

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

For feeder- and serum-free culture, H9 cells (WiCell Research Institute, Madison, WI, http://www.wicell.org) and hIPSCs (Table 1) were grown in CDM [5, 11], supplemented with activin

Figure 1. hIPSCs can be grown in dened culture conditions developed for hESCs. (A): hIPSCs grown in CDM activin FGF2 maintain pluripotency markers. CRL-hIPSCs were grown for 10 passages (8 weeks) in CDM activin (10 ng/ml) FGF2 (12 ng/ml), and then immunostaining was performed to detect expression of the pluripotency markers Oct4, Sox2, Nanog, and Tra-1-60. In addition, colorimetric assays were performed to detect alkaline phosphatase activity. Scale bar 100 lm. (B): Expression of pluripotency markers (Oct-4, Sox2, Nanog) in hIPSCs grown in CDM activin FGF2. CRL-hIPSCs (lines 30, 35, 40) were grown for 4 days in these culture conditions and then RNAs were extracted and expression of the denoted genes was analyzed using quantitative polymerase chain reaction (PCR). hESCs grown in CDM activin FGF2 were used as positive controls. Normalized expression is shown as the mean SD from three informative experiments. (C): hIPSCs grown in CDM activin FGF2 express homogeneously the pluripotency marker Tra-1-60, conrming the absence of spontaneous differentiation in these culture conditions. hIPSCs were grown for 10 passages in CDM activin FGF2, and then the fraction of cells expressing the pluripotency marker Tra-1-60 was determined using uorescence-activated cell sorting analyses. hESCs grown in CDM activin FGF2 were used as positive controls. (D): Teratomas from hIPSCs cells grown in CDM Activin FGF2. Approximately 106 hIPSCs (line 40) grown for 20 passages in CDM/AF were injected into the testis capsule of severe combined immunodecient-beige mice. The resulting tumors were harvested 2 months after injection. (E): Expression of exogenous and endogenous Oct-4, Sox2, Klf-4, and c-Myc in hIPSCs generated from foreskin broblasts. CRL-hIPSCs were grown for 4 days in CDM activin FGF2 and then expression of endogenous and exogenous Oct-4, Sox2, Klf4, and C-Myc was determined using real-time PCR and specic primers. (F): Number of transgene copies in hIPSCs generated from foreskin broblasts. Copy number for each gene used to reprogram CRL foreskin broblasts was determined using real-time PCR. hESCs (H9 line) were used as positive control. Abbreviations: AP, alkaline phosphatase; CDM, chemically dened medium; FGF2, broblast growth factor 2; hIPSC, human induced pluripotent stem cell.

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Figure 2.

Vallier, Touboul, Brown et al.

2659 www.taconic.com/) housed and maintained at 20 C24 C, 50% room humidi, in a 14- to 10-hour light-dark cycle with food and water libitum. The mice were sacriced after 60 days and then the injected testes were cut into equal pieces using a razor blade. The material was xed overnight in 4% neutral buffered formaldehyde, and dehydrated through a graded series of alcohols to xylene. The tissue was embedded in parafn, serially sectioned at 5 lm, followed by H&E staining and characterization. A human origin of the selected areas was veried by uorescent in situ hybridization (human-specic probes, CEP XY; Vysis Inc., Downers Grove, IL, http://www.vysis.com). The experiments were performed with permission from the Regional Committee for Animal Experimentation (Stockholm, Sweden; Dnr N107/06).

(10 ng/ml, kindly provided by Marco Hyvonen) and FGF2 (12 ng/ml, R&D Systems Inc.). The composition of CDM was 50% Iscoves modied Dulbeccos 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 of transferrin (Roche Diagnostics), 450 lM of monothioglycerol (Sigma-Aldrich), and 5 mg/ml bovine serum albumin fraction V (Europa Bioproducts, Cambridge, U.K., http://www.europabioproducts.com) or polyvinyl alcohol (CDM-PVA) (SigmaAldrich). Every 4 days, cells were harvested using 1 mg/ml dispase (Gibco) and then plated into dishes (Corning Costar Corp., Cambridge, MA, http://www.corning.com/index.aspx) that were precoated with 15 lg/ml of human bronectin (Chemicon) for 20 minutes at 37 C and then washed twice in PBS. Alternatively, dishes were precoated with porcine gelatin 1 mg/ml (SigmaAldrich) for 15 minutes to 1 hour and then precoated with fetal bovine serum-containing medium (5% in Dulbeccos modied Eagles medium) for 24 hours at 37 C.

Flow Cytometry and Cell Sorting

For detection of N-Cam, CXCR4, and platelet-derived growth factor a (PDGFa) receptor, adherent cells were washed twice in PBS and then incubated for 20 minutes at 37 C in cell dissociation buffer (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Cells were dissociated by gentle pipetting and resuspended at approximately 0.1-1 105 cells per milliliter in PBS 3% normal goat serum (NGS) containing 0.1% azide (Serotec Ltd., Oxford, U.K., http://www.serotec.com). Cells were incubated for 40 minutes at 4 C with uorescein isothiocyanate- or phycoerythrin-conjugated antibody to CXCR4 (1:50; BD Pharmingen, San Diego, http://www.bdbiosciences.com), N-Cam (1:200; BD Pharmingen), or PDGFa receptor (PDGFaR, 1:50; BD Pharmingen) or the corresponding isotype control (mouse IgG isotype control; BD Pharmingen). Subsequently, cells were resuspended in PBS 3% NGS for staining with 7-aminoactinomycin D (7-AAD) viability dye (Immunotech, Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp) at 20 ll/ml for 15 minutes at room temperature. Live cells identied by 7-AAD exclusion were analyzed for surface-marker expression using FACSCalibur (BD Biosciences, San Jose, California, USA, http:// www.bdbiosciences.com) or sorted using Dako Cytomation MoFlo high speed ow cytometer (Glostrup, Denmark, http:// www.dako.com).

Differentiation of hIPSCs in Chemically Dened Conditions

hIPSCs grown in feeder-free and serum-free conditions were harvested using 1 mg/ml dispase then split into plates precoated with bronectin or with FBS. hIPSCs were grown for the rst 2 days in CDM supplemented with recombinant activin and FGF2 (12 ng/ml; R&D Systems Inc.). To obtain extraembryonic tissue, hIPSCs were grown for 7 days in CDM in the presence of bone morphogenic protein 4 (BMP4, 10 ng/ml R&D Systems Inc.) and SB431542 (10 lM; Tocris Bioscience, Ellisville, MO, http:// www.tocris.com). To obtain neuroectoderm progenitors, hIPSCs were grown in CDM or in CDM-PVA in the presence of SB431542 (10 lM; Tocris Bioscience), FGF2 (12 ng/ml; R&D Systems Inc.), and Noggin (200 ng/ml R&D Systems Inc.) for 10 additional days. To obtain mesendoderm precursors, hIPSCs were grown for the 3 following days in CDM-PVA in the presence of BMP4 (10 ng/ml; R&D Systems Inc.), FGF2 (20 ng/ml; R&D Systems Inc.), activin (100 ng/ml; R&D Systems Inc.), and LY294002 (10 lM; Promega, Madison, WI, http://www. promega.com).

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

RNA Extraction and Real-Time Polymerase Chain Reaction

Total RNAs were extracted from hIPSCs or differentiated progenitors using the RNeasy Mini Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Each sample was treated with RNaseFree 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 signaling induces differentiation of hIPSCs into extraembryonic tissues and neuroectoderm. hIPSCs were grown for 12 days in chemically dened medium (CDM) SB431542 (10 lM) FGF2 (12 ng/ml), and then immunostaining analyses were performed to detect the expression of the pluripotency marker Oct-4, the extraembryonic markers Sox7, GATA4, and GATA6, the trophectoderm markers Cdx2 and Eomes, and the neuroectoderm markers Sox2, Sox1, Pax6, NCam, and Nestin. Scale bar 100 lm. (B): Genomic regions of the Nanog gene bound by Nanog and Smad2/3 proteins. Chromatin immunoprecipitation assays were performed using antibodies directed against Smad2/3 or Nanog. The immunoprecipitated DNA was then amplied using quantitative polymerase chain reaction (PCR) and specic primers to detect enrichment in the denoted genomic regions. Results were normalized against a control region (6237 6414) and are expressed as standard derivation from three experiments. (C): Mutation of a putative Smad2/3 binding site in the Nanog promoter inhibits the transcriptional activation induced by activin/nodal signaling. Luciferase reporter genes containing the promoter of the human Nanog gene (379 18) with or without mutated Smad2/3 bindingsites were cotransfected into hIPSCs along with Renilla expression vector (control for normalization) in the presence of activin and FGF2 or in the presence of SB431542 (negative control). Relative rey luciferase activity (normalized to Renilla activity) is expressed as mean standard deviation from three independent experiments. (D): Constitutive expression of Nanog prevents differentiation of hIPSCs induced by inhibition of activin/nodal signaling. CRL-hIPSCs and Nanog-CRL-hIPSCs subline 2 (Nanog) were grown for 14 days in the presence of SB431542 FGF2 and then immunostaining analyses were performed to detect the expression of Oct4 and Nanog. Scale bar 100 lm. (E): Nanog overexpression blocks neuroectoderm differentiation of hIPSCs grown in the absence of activin/nodal signaling. Nanog-overexpressing hIPSCs (Nanog) were grown for 14 days in CDM SB431542 FGF2 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 activin FGF2 were used as negative and CRL-hIPSCs grown in CDM SB431542 FGF2 were used as positive control (WT). Abbreviations: A F, activin and FGF2; FGF2, broblast 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 Superscript II Reverse Transcriptase (Invitrogen). Real-time polymerase chain reaction (PCR) reaction mixtures were prepared as described (SensiMiX protocol; Quantace, London, http:// www.quantace.com) then denatured at 94 C for 5 minutes and cycled at 94 C for 30 seconds, 60 C for 30 seconds, and 72 C for 30 seconds, followed by a nal extension at 72 C for 10 minutes after completion of 40 cycles. Primer sequences are described elsewhere [8, 13]. Real-time PCR reactions were performed using a Stratagene Mx3005P (La Jolla, CA, http:// www.stratagene.com) in triplicate and normalized to porphobilinogen deaminase (PBGD) in the same run.

Immunouorescence
hIPSCs or their differentiated progeny were xed for 20 minutes at 4 C in 4% paraformaldehyde and then washed three times in PBS. Cells were incubated for 20 minutes at room temperature in PBS containing 10% donkey serum (Serotec Ltd.) and subsequently incubated overnight at 4 C with primary antibody diluted in 1% donkey serum in PBS as follows: Oct-4 (1:100; Abcam ab18976 [Cambridge, U.K., http://www.abcam.com] or Santa Cruz Biotechnology Inc. [Santa Cruz, CA, http://www.scbt.com]), Sox2 (1:100; Abcam ab15830), Brachyury (1:100; Abcam ab20680 or R&D Systems Inc.), Sox17 (R&D Systems Inc.), FoxA2 (1:50; Abcam ab5074), GATA4 (1:250; Santa Cruz Biotechnology Inc.), GATA6 (1:200; Abcam ab22600 or Santa Cruz Biotechnology Inc.), CXCR4 (1:100; R&D Systems Inc. or BD Pharmingen), Nestin (1:200; Abcam ab5968), NCAM (1:100; Abcam ab8077), and N-Cadherin (1:100; Abcam ab18203). Cells were then washed three times in PBS and incubated with Texas Red or uorescein isothiocyanate-conjugated anti-mouse IgG (Sigma-Aldrich; 1:200 in 1% donkey serum in PBS) or rabbit IgG (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 in PBS. Hoechst 33258 was added to the rst wash (Sigma-Aldrich; 1:10,000).

RESULTS
Derivation of hIPSCs in Chemically Dened Culture Conditions
Several groups have recently reported that human broblasts can be reprogrammed into induced pluripotent stem cells (iPSCs) by overexpressing the four transcription factors Oct4, Klf-4, Sox2, and c-Myc or other factors [24]. Using such an approach with four factors as described by Takahashi et al. [2], we generated hIPSC lines from three sources of broblasts: neonatal foreskin broblasts (CRL), embryonic lung broblasts (MRC5), and adult dermal broblasts from a 28year-old female (Bi) (Table 1). Importantly, we also obtained similar results using only three factors, conrming that c-Myc is not necessary for hIPSC derivation [3, 14]. The resulting lines were initially grown on mouse feeder cells in media containing serum supplemented with FGF2. After one passage, hIPSC colonies were transferred to a chemically dened culture system (CDM activin FGF2), which is currently used in our laboratory to grow hESCs and also epiblast stem cells (EpiSCs) derived from the epiblast layer of postimplantation mouse embryos [11]. The resulting hIPSC colonies were grown for 20 passages without noticeable differentiation while maintaining the expression of endogenous pluripotency marker genes Oct-4, Sox2, Nanog, Tra-1-60, and alkaline phosphatase (Fig. 1A, 1B). In addition, uorescence-activated cell sorting (FACS) analyses showed that 90% of the cells

grown in these culture conditions expressed the pluripotency marker Tra-1-60, demonstrating the absence of background of spontaneous differentiation (Fig. 1C). We then derived hIPSCs directly in CDM activin FGF2 in the total absence of feeders or serum (Table 1). Using this approach, we generated 33 hIPSC lines expressing similar levels of the endogenous pluripotency factors Oct-4, Sox2, Nanog, and Tra1-60 (Fig. 1A1C, hIPSC line 40) compared with hIPSC lines derived on feeders in the presence of serum (Fig. 1A1C, hIPSC line 30 and 35). In addition, hIPSC lines derived in chemically dened conditions showed the same capacity for differentiation as hIPSCs derived on feeders in serum containing medium (see below and Figs. 3-5, hIPSC line 40) and they had the capacity to form teratomas containing derivatives of the three germ layers when injected in the testis capsule of immunodecient mice (Fig. 1D). We further characterized the hIPSCs lines generated and grown in CDM by dening the expression of exogenous transgenes and endogenous genes using real-time PCR. These analyses showed that the exogenous transgenes were strongly silenced in fully reprogrammed cells (Fig. 1E), with the exception of Klf-4. However, we observed that levels of KLF-4 transcripts 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 conrmed by the unperturbed self-renewal and differentiation behavior of hIPSCs with KLF4 residual expression (see below). These results apparently contradict recent publications suggesting that constitutive expression of exogenous transgene can be an issue in hIPSCs and thus that transgene excision is necessary to establish robust hIPSCs [15]. This difference might be explained by our use of retroviruses, which are known to be strongly silenced in pluripotent cells [16] in contrast to lentiviruses, whose expression may persist. However, we cannot exclude that our culture conditions could also have an impact on transgene expression. Finally, we determined the number of transgene copies inserted in the genome of ve hIPSC lines, showing that each line contains between 3 and 10 copies for each transgene (Fig. 1F), which is in the range of previous studies [24]. Taken together these results demonstrate that a chemically dened medium containing activin and FGF2 is sufcient to generate and grow hIPSCs, suggesting that hESCs and hIPSCs rely on the same signaling pathways to maintain their pluripotent status.

Human Induced Pluripotent Stem Cells Rely on Activin/Nodal Signaling to Maintain Their Pluripotent Status
To examine the importance of activin/nodal signaling in hIPSCs, we analyzed the effect of SB431542, a chemical inhibitor of activin receptor activity, on their pluripotency. hIPSCs generated from three different broblast lines were grown for 10 days in CDM FGF2 SB431542 or in CDM FGF2 (omitting activin) and then the expression of pluripotency markers was analyzed using immunostaining (Figs. 2A, 6; supporting information Fig. 1). Absence of activin/nodal signaling systematically induced differentiation of hIPSCs, as shown by the loss in pluripotency markers (Fig. 2A; supporting information Fig. 1), thus conrming that activin/nodal signaling is necessary to maintain pluripotency in hIPSCs. We then analyzed the expression of specic markers for the three germ layers and extraembryonic tissues to determine the nature of the cells generated by inhibiting activin signaling in hESCs (Figs. 2A, 6; supporting information Fig. 1) and we observed that cells generated in these culture conditions

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Figure 3. BMP4 drives differentiation of hIPSCs into extraembryonic tissues. (A): hIPSCs grown in the presence of BMP4 (and in the absence of activin) differentiate into extraembryonic tissues. CRL-hIPSCs were grown for 12 days in chemically dened medium (CDM) BMP4 (10 ng/ml) SB431542 (10 lM), and then immunostaining analyses were performed to detect expression of pluripotency markers Oct4 and Nanog, primitive endoderm markers Sox7, GATA4, and GATA6, and trophectoderm markers CDX2 and Eomes. Scale bar 100 lm. (B): Absence of pluripotency markers (Oct-4, Sox2, Nanog) and induction of extraembryonic markers (CDX2, Hand1, Sox7) in hIPSCs grown in CDM BMP4 SB431542. 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 quantitative polymerase chain reaction. Normalized expression is shown as the mean SD from three informative experiments. hESCs grown in CDM activin broblast growth factor 2 (FGF2) were used as negative controls. (C): Fluorescence-activated cell sorting (FACS) analysis of the fraction of cells expressing Tra-1-60 after extraembryonic differentiation conrmed homogenous differentiation of hIPSCs. CRL-hIPSCs (lines 30, 35, 40) were induced to differentiate for 10 days into extraembryonic tissues using the culture conditions described above, and then the fraction of cells expressing Tra-1-60 was determined using FACS. hESCs grown in CDM activin 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.

expressed mainly markers of extraembryonic lineages (primitive endoderm: Sox7, GATA4, and GATA6; trophectoderm: CDX2 and Eomes) and neuroectoderm (Sox2, Sox1, Pax6, Nestin, and NCam). These results suggest that activin signaling blocks extraembryonic and neuroectoderm differentiation of hIPSCs similarly to the effects of activin/nodal signaling seen in hESCs and they reinforced recent studies showing similar outcomes using serum-containing media [17, 18]. Importantly, however the inhibitory effect of activin/nodal signaling on extraembryonic differentiation is specic to hIPSCs since hESCs grown in the absence of activin/nodal www.StemCells.com

signaling homogenously differentiate into neuroectoderm [10].

Activin/Nodal Signaling Maintains Nanog Expression in hIPSCs

The observations that hIPSCs can be grown and derived (Table 1) in CDM supplemented with activin and FGF2 and that inhibition of activin/nodal signaling induces their differentiation suggest that hESCs and hIPSCs rely on the same signaling pathways to maintain their pluripotent status. We examined

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Figure 4. Inhibition of activin/nodal signaling and bone morphogenic protein (BMP) signaling in the presence of FGF2-induced neuroectoderm differentiation of hIPSCs. (A): hIPSCs grown in the absence of activin/nodal and BMP4 signaling differentiate into neuroectoderm. CRL-hIPSCs were grown for 12 days in chemically dened medium (CDM) FGF2 (12 ng/ml) SB431542 (10 lM) Noggin (200 ng/ml), and then immunostaining 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 100 lm. (B): Absence of pluripotency markers (Oct-4, Nanog) and induction of neuroectoderm markers (Sox2, Sox1, Gbx2) in hIPSCs grown in CDM SB431542 FGF2. 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 quantitative polymerase chain reaction. Normalized expression is shown as the mean SD from three informative experiments. hESCs grown in CDM activin FGF2 were used as control. (C): Fluorescence-activated cell sorting (FACS) analysis for the fraction of cells expressing NCam showing homogenous formation of neuroectoderm. CRL-hIPSCs (lines 30, 35, 40) were differentiated for 10 days using the culture conditions described above, and then the fraction of cells expressing N-Cam was determined using FACS. hESCs grown in CDM activin FGF2 were used as negative control (hESCs). Abbreviations: FGF2, broblast growth factor 2; hIPSC, human induced pluripotent stem cell; SB, SB431542; Undiff, undifferentiated negative controls.

this hypothesis at the molecular level by analyzing Nanog regulation. It has been recently shown that activin/nodal/TGFb signaling maintains pluripotency of hESCs and mouse EpiSCs (mEpiSCs) by controlling the expression of Nanog through the TGFb effectors Smad2/3 [7, 19, 20]. To determine whether similar mechanisms occur in hIPSCs, chromatin immunoprecipitation (ChIP) assays were performed to identify genomic regions bound by Smad2/3 in the Nanog promoter (Fig. 2B). This showed that Smad2/3 binds the same genomic region identied in hESCs using ChIP. Interestingly, this region contains binding sites for Oct4, Sox2, and Nanog [21, 22] and also two consensus Smad2/3 binding sites (S2/3-(1) and S2/3(2), Fig. 2B)). Luciferase assays revealed that mutations in these Smad2/3 binding sites abolished transcriptional activity induced by activin/nodal signaling (Fig. 2C), strongly suggesting that they are functionally important in Nanog regulation. Importantly, similar results were obtained using hESCs and two independent hIPSC lines generated from foreskin broblasts and adult broblasts (supporting information Fig. 2), suggesting this molecular mechanism is common to hIPSC lines [19]. Finally, we stably overexpressed Nanog in hIPSCs and then grew the resulting sublines (Nanog-hIPSCs) in CDM supplemented with SB431542 and FGF2. Wild-type hIPSCs or GFP-expressing hIPSCs grown in these culture conditions quickly differentiated into neuroectoderm progenitors as

shown by the decrease in the pluripotency markers Oct-4 and Nanog and in the increase in the neuroectoderm markers Gbx2 and Sox1 (Fig. 2D, 2E). On the other hand, Nanog-hIPSCs maintained a pluripotent morphology and sustained expression of Oct-4, Sox2, and Nanog without expression of neuroectoderm markers (Fig. 2D, 2E). This suggests that Nanog is sufcient to substitute for activin/nodal signaling. Together, these ndings demonstrate that activin/nodal signaling maintains pluripotency of hIPSCs by directly controlling the expression of Nanog, as it does in hESCs and mEpiSCs [19], thereby strikingly mimicking the mechanisms maintaining pluripotency of embryo-derived pluripotent stem cells.

hIPSCs Are Similar to hESCs and mEpiSCs in Their Response to Culture Conditions Controlling Differentiation
We then analyzed the effect of three fully dened culture conditions recently developed to drive differentiation of hESCs and mEpiSCs into extraembryonic tissues, neuroectoderm, and mesendoderm [10]. Addition of BMP4 in the absence of activin and FGF2 is sufcient to drive differentiation of hESCs and mEpiSCs into primitive endoderm and trophectoderm. hIPSCs grown in similar culture conditions differentiated into a mixed population expressing markers for primitive

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

endoderm (GATA4, GATA6, Sox7; supporting information Fig. 3) and trophectoderm (CDX2; supporting information Fig. 3). However, the decrease in expression of pluripotency factors Oct-4 and Nanog was observed only after 12 days with hIPSCs compared with 7 days with hESCs, suggesting that BMP4 effects might be less potent with hIPSCs (supporting information Fig. 3). However, we also observed that the effect of BMP4 was strongly increased by adding the activin/ nodal receptor inhibitor SB431542 (Fig. 3A3C), suggesting that endogenous activin/nodal signaling is enough to delay extraembryonic differentiation of hIPSCs, reecting a competitive interaction between these two signaling pathways [18]. Together, these results demonstrate that hIPSCs have the capacity to differentiate into extraembryonic tissues, including cells expressing trophectoderm markers, a characteristic specic to hESCs and mEpiSCs but not mESCs. www.StemCells.com

In the second set of culture conditions SB435132 was used to drive differentiation of hESCs into neuroectoderm by inhibiting activin/nodal signaling in the presence of FGF2. Inhibition or absence of activin/nodal signaling in hIPSCs resulted in loss of the pluripotency markers Oct-4 and Nanog (Fig. 2A; supporting information Fig. 1). Immunostaining analyses revealed that the differentiated cells resulting from this treatment expressed neuroectoderm markers (Sox2, Sox1, Pax6, and Nestin, Fig. 2A) and extraembryonic markers (GATA6, GATA4, and CDX2, Fig. 2A). Thus, inhibition of activin signaling appeared to drive differentiation of hIPSCs into a mixed population of neuroectoderm and extraembryonic cells. Interestingly, a combination of SB431542 and the BMP inhibitor Noggin (200 ng/ml) strongly reduced the extraembryonic differentiation induced by the inhibition of activin/ nodal signaling, resulting in a nearly homogeneous population

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of cells expressing neuroectoderm markers (Sox2, Sox1, Pax6, Nestin, NCam, and bIII Tubulin, Fig. 4A4C). Together these results demonstrate that similarly to hESCs, inhibition of activin/nodal signaling is sufcient to drive differentiation of hIPSCs into neuroectoderm, with the difference that an endogenous BMP-like activity in hIPSCs apparently interferes with their neuroectoderm differentiation, which thereby requires inhibition of both activin/nodal and BMP pathways. In the third of these chemically dened conditions, a three-step protocol is used to drive differentiation into mesendoderm and then into endoderm [10]. In the rst step, pluripotent stem cells grown initially in CDM activin FGF2 are plated for 48 hours on bronectin in CDM-PVA medium containing activin (10 ng/ml) and FGF2 (12 ng/ml). In the second step, the FGF receptor inhibitor SU5402 (10 lM) and a lower dose of activin (5 ng/ml) is added for 3 days. Addition of SU5402 reduces adhesion of the colonies, which begin to form compact aggregates of cells expressing Oct4 and low levels 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, driving pluripotent colonies to differentiate into homogenous population of endoderm cells expressing Sox17, CXCR4, GSC, FoxA2, and Mixl1 [10]. When hIPSCs were subjected to this three-step protocol, they strongly differentiated at the second step (inhibition of the FGF signaling pathway), generating extraembryonic cells (supporting information Fig. 4), suggesting that FGF signaling is strictly necessary to maintain hIPSC pluripotency. We then analyzed the effect of the third step in this protocol (combination of activin [high-dose, 100 ng/ml], FGF2 [20 ng/ml], and BMP4 [10 ng/ml]). hIPSCs grown in these conditions differentiated into heterogeneous populations of cells expressing mesoderm (Brachyury), endoderm (Sox17), and pluripotency (Oct-4) markers (supporting information Fig. 5). Interestingly addition of the PI3Kinase inhibitor LY294002 increased the expression of mesendoderm (Brachyury) and endoderm (Sox17, FoxA2, NCad, and CXCR4) markers, but decreased the expression of pluripotency markers (Oct-4, Nanog, Sox2) (Fig. 5A5C) without inducing the expression of the extraembryonic endoderm marker Sox7, thus extending to hIPSCs the recent observation that LY294002 can improve endoderm differentiation of hESCs [23]. In addition, FACS analyses revealed that almost 70% of the cells generated under these conditions expressed the denitive endoderm marker CXCR4, whereas the remaining cells expressed the mesendoderm marker PDGFaR, conrming that hIPSCs differentiated entirely toward the mesendoderm/endoderm pathway. Importantly, hESCs grown in the same culture conditions (combination of activin [high-dose], FGF2, BMP4, and LY294002) differentiated homogenously into denitive endoderm 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 sufcient to drive differentiation of hIPSCs and hESCs into nearly homogenous endoderm population. Taken together with our results using the other chemically dened conditions described here (CDM BMP4 activin/nodal, CDM SB431542 FGF2 Noggin), these ndings conrm that hIPSCs and hESCs are similarly responsive to growth factors controlling their differentiation into extraembryonic and embryonic lineages. Finally, similar results were obtained with hIPSC lines (n 15) generated from foreskin broblasts (CRL-hIPSC) (including three generated without c-Myc) and with hIPSC lines generated from MRC5 broblasts (n 2) and Bi broblasts (n 2) (Fig. 6), suggesting that our ndings are independent of the hIPSC line used. Taken together, these obser-

Figure 6. Human induced pluripotent stem cells (hIPSCs) generated from diverse broblast lines are similarly responsive to culture conditions controlling differentiation of hESCs. Expression of pluripotency (Oct-4, Sox2, Nanog), extraembryonic (Cdx2, Sox7, GATA4), neuroectoderm (Sox2, Sox1, Pax6), and mesendoderm (Brachyury, FoxA2, Sox17) markers during differentiation of hIPSCs generated from MRC5 broblasts (line 5) and Bi broblasts (line 11). hIPSCs were grown in chemically dened medium activin FGF2 and then induced to differentiate following the protocols described above for hESCs and CRL-hIPSCs (BMP4 SB431542 for extraembryonic tissues, SB431542 FGF2 Noggin for neuroectoderm, activin FGF2 BMP4 LY294002 for mesendoderm). Immunostaining analyses were performed at 5 days (pluripotent cells and mesendoderm) or 10 days (extraembryonic and neuroectoderm) after plating. Scale bar 100 lm. Abbreviations: BMP4, bone morphogenic protein 4; Bra, Brachyury; FGF2, broblast growth factor 2; SB, SB431542.

vations demonstrate that hIPSCs respond robustly to chemically dened culture conditions that have been developed and used to differentiate hESCs into early progenitor populations and therefore that similar signaling pathways can control differentiation of both hIPSCs and hESCs.

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CONCLUSION
Taken together, our observations reinforce the similarity between hESCs and hIPSCs, showing that both cell types rely on similar signaling pathways to maintain their pluripotency and to control their differentiation. However, our study also reveals some differences between these two pluripotent cell types, mainly in the interaction between the activin signaling pathway and the BMP signaling pathway. Indeed, inhibition of BMP signaling is required in addition to inhibition of activin/nodal signaling to drive differentiation of hIPSCs into a homogenous population of neuroectoderm cells. Moreover, inhibition of activin/nodal signaling is necessary in addition to treatment with BMP4 to induce extraembryonic differentiation of hIPSCs. A potential explanation for this difference between hESCs and hIPSCs is that hIPSCs display higher endogenous levels of BMP4 and activin/nodal activity, which in turn requires their respective inhibitors to promote differentiation into neuroectoderm and extraembryonic pathways, respectively. By contrast, the hESC lines used in our study did not display signicant BMP4 signaling activity, as shown by the absence of Smad1/5/8 phosphorylation in hESCs grown in CDM [5]. Interestingly, mEpiSCs also require inhibition of BMP signaling to differentiate homogenously into neuroectoderm and activin signaling to differentiate into extraembryonic tissues. Despite such differences, our results emphasize the overall similarity between hESCs, hIPSCs, and mEpiSCs. Indeed, all three pluripotent cell types rely on activin to control Nanog expression and to maintain their pluripotency. In addition, they all rely on similar pathways to induce differentiation into extraembryonic and embryonic lineages. Specically, inhibition of activin in the presence of FGF2 is essential for inducing neuroectoderm differentiation in the three cell types, whereas BMP4 drives their differentiation into extraembryonic tissues including cells expressing markers of trophectoderm or of primitive endoderm. Finally, the combination of a high dose of activin plus BMP4 and FGF2 and inhibition of PI3K are sufcient to generate a near homogenous population of denitive endoderm. These observations demonstrate that hESCs, hIPSCs, and mEpiSCs are similarly responsive to key signaling pathways controlling their early cell fate decisions. Accordingly, these three pluripotent cell types appear to share a common pluripotent state that denes their capacity to react to exogenous growth factors. Importantly, this shared pluripotent state is also associated with a specic embryonic identity corresponding to the epiblast of postimplantation stage mammalian embryos [11, 24] from which mEpiSCs are derived. Therefore, our results demonstrate for the rst time the extent to which hESCs and hIPSCs are functionally similar, representing a pluripotent status equivalent to that of the late epiblast of the pregastrulation mouse embryo (EpiSCs). This nding led to the conclusion that the technical and conceptual knowledge accumulated during the past decade using hESCs

(and more recently using EpiSCs) should be readily transferable to hIPSCs. Finally, we describe experiments showing that hIPSCs can be derived and directed to differentiate into extraembryonic tissues, neuroectoderm, or mesendoderm, in fully chemically dened culture conditions. Because previously available conditions for derivation and differentiation of hIPSCs along these lineages were based on media containing undened components (serum) and animal products (feeder cells and matrices) [24], our ndings represent a rst step toward the potential use of hIPSCs in cell-based therapies. However, the use of animal products to establish broblasts from skin and other biopsies and the high number of viral integrations preclude the clinical use of the lines described in this study. Nevertheless, skin broblasts can be generated in good manufacturing practice conditions using xeno-free conditions and recent studies have described the derivation of hIPSCs without stable genetic modication [2528]. Therefore, our culture system could be advantageously used in combination with such approaches to derive hIPSCs in conditions fully compatible with clinical applications. In addition, the establishment of chemically dened protocols and characterization of outcomes for in vitro differentiation of hIPSCs provide unique in vitro systems to validate the capacity of hIPSCs to differentiate into mature progeny of the three germ layers. Such studies could potentially replace in vivo analyses such as teratoma assays, which are resource consuming and nonquantitative. Finally, our ndings could be crucial in dening universal protocols of differentiation capable of yielding particular differentiated cell types that apply to a wide range of pluripotent lines including patient-specic hIPSC lines.

ACKNOWLEDGMENTS
We thank Glyn Stacey and the U.K. Stem Cell Bank for the MRC5 broblasts. This work was supported by an MRC research grant (R.A.P), by association Francaise pour letude du foie (T.T.), by Agence National de la Recherche (ANR Grant ANR-05-BLAN-006-02) (T.T. and A.W.), by the Juvenile Diabetes Research Fundation (R.A.P., and L.V.), by the Evelyn Trust (R.A.P, M.A.), by an MRC/Diabetes U.K. Career Development fellowship (L.V.), by a MRC senior non-clincal fellowship (S.B.) and by National Institute for Health Research Cambridge Biomedical Research Centre.

DISCLOSURE

OF OF

POTENTIAL CONFLICTS INTEREST

The authors indicate no potential conicts of interest.

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