Retinoic Acid and Bone Morphogenetic Protein Signaling Synergizeto Efficiently Direct Epithelial Differentiation of Human EmbryonicStem Cells

CHRISTIAN M. METALLO,a LIN JI,a,b JUAN J. DE PABLO,a SEAN P. PALECEKa,b

aDepartment of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA;bWiCell Research Institute, Madison, Wisconsin, USA

Key Words. Human embryonic stem cells • Ectodermal differentiation • Epithelial lineages • Retinoic acid • p63

ABSTRACT

Human embryonic stem cells (hESCs) can differentiate tovarious somatic lineages, including stratified squamous ep-ithelia, although the molecular mechanisms of epithelialspecification from hESCs currently remain undefined. Here,we demonstrate a novel, stage-specific effect of retinoic acid(RA) on epithelial differentiation of hESCs. RA stronglyupregulated expression of keratin 18 and the transcriptionfactor p63, which is involved in epidermal morphogenesisand ectodermal specification, while repressing early neural

marker transcription. RA-induced hESCs efficiently differ-entiated to keratin 14-expressing epithelial cells, althoughthis effect was dependent upon on the context of bone mor-phogenetic protein signaling. Furthermore, these hESC-de-rived keratinocytes could be subcultured to obtain relativelypure keratinocyte populations that retained the capacity toterminally differentiate. These findings suggest that RAplays an important role in epithelial differentiation ofhESCs. STEM CELLS 2008;26:372–380

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

INTRODUCTION

Human embryonic stem cells (hESCs) are pluripotent cells withan extensive proliferative capacity and the ability to differentiateinto the three embryonic germ layers [1]. Much excitement hascentered on their potential use in developmental biology re-search, diagnostic testing, and regenerative medicine; however,most applications will require relatively pure populations ofsomatic cell types, which, to date, have been difficult to pro-duce. To exploit the expansion potential of undifferentiatedhESCs, efficient differentiation processes must be developed togenerate lineage-restricted progenitors at a high purity whileeliminating pluripotent cells in culture.

Human ESCs can differentiate to the keratinocyte lineage[2], although current methods are inefficient and isolated kera-tinocytes have a reduced expansion potential [3, 4]. The hESCdifferentiation process toward keratinocytes is marked by ex-pression of p63, a transcription factor necessary for maintenanceof regenerative epithelia (i.e., the epidermis) [5–7], and keratin14 (K14), an intermediate filament present in the basal layer ofstratified squamous epithelia [8]. Studies using murine embry-onic stem cells (mESCs) have identified bone morphogeneticprotein 4 (BMP-4) as an inducer of epidermal differentiation [9],and BMP signaling plays a key role in early ectodermal fatechoices during development in multiple species [10–12]. Therole BMP-4 plays in human ectodermal development is cur-rently unclear, as this factor has been used to direct hESCs tononectodermal lineages [13, 14].

Retinoids are potent regulators of cell proliferation anddifferentiation and are highly involved in embryonic develop-

ment. Retinoic acid (RA) has been shown to inhibit the terminaldifferentiation of keratinocytes in vitro by modulating p63 iso-form expression [15] and is a caudalizing factor that directsembryonic stem (ES) cell-derived neuroepithelia to becomemotor neurons [16, 17]. Given the ethical problems associatedwith in vivo human embryo research, hESCs offer an attractivein vitro model system to study the effects of RA or otherchemicals on early human developmental processes.

In the present study, we used quantitative analysis of differen-tiated hESC populations to identify key signaling factors involvedin ectodermal lineage specification. RA applied to undifferentiatedhESCs efficiently mediated epithelial differentiation in conjunctionwith BMP signaling. Finally, we used this process to generaterelatively pure keratinocyte cultures capable of terminally differ-entiating and forming coherent epithelial sheets.

MATERIALS AND METHODS

Cell CultureH1 and H9 hESC lines were cultured on a layer of irradiated mouseembryonic fibroblasts (MEFs) in unconditioned medium (UM):Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 me-dium (F12) containing 20% Knockout Serum Replacer (Invitrogen,Carlsbad, CA, http://www.invitrogen.com), 1 � MEM nonessentialamino acids (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1 mM L-glutamine, 0.1 mM �-mercaptoethanol, and 4 ng/mlbasic fibroblast growth factor (bFGF). Alternatively, hESCs wereplated on Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com) in medium conditioned by MEFs and passagedevery 5–6 days using Dispase.

Correspondence: Sean P. Palecek, Ph.D., Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 EngineeringDrive, Madison, Wisconsin 53706, USA. Telephone: 608-262-8931; Fax: 608-262-5434; e-mail: palecek@engr.wisc.edu Received June 26,2007; accepted for publication October 18, 2007; first published online in STEM CELLS EXPRESS October 25, 2007. ©AlphaMed Press1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0501

EMBRYONIC STEM CELLS

STEM CELLS 2008;26:372–380 www.StemCells.com

EBs were formed via enzymatic detachment of hESC coloniesand cultured in UM without bFGF or N2 medium (N2): DMEM/F12containing 1 � N2 supplement and MEM nonessential amino acids.After 1–2 days, EBs were transferred to a new vessel to removeadherent MEFs, and treatment with medium changes every otherday was begun. Differentiated EBs were plated onto gelatin-coatedplates in defined keratinocyte serum-free medium (DSFM), chang-ing medium every other day (supplemental online Fig. 1A). Fordirect differentiation experiments, hESC colonies were grown for 6days on Matrigel in MEF-conditioned hESC medium before switch-ing to differentiation medium, which contained UM or N2 and acombination of DMSO, 1 �M all-trans RA (Sigma-Aldrich), 25ng/ml BMP-4 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), or 125 ng/ml Noggin (R&D Systems). After 6days, differentiated cells were detached with Dispase and culturedovernight before plating as described above (supplemental onlineFig. 1B).

Primary human keratinocytes (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) and hESC-derived keratinocytes were sub-cultured on collagen IV (Sigma-Aldrich)-coated plates in DSFM.All media and additives were obtained from Invitrogen unlessotherwise noted.

ImmunocytochemistryCells were fixed in 4% paraformaldehyde for 15 minutes at roomtemperature before blocking and permeabilizing with 5% milk inphosphate-buffered saline (PBS) with 0.4% Triton X-100. Primaryantibodies were incubated overnight at 4°C in blocking buffer andincluded mouse anti-p63, mouse anti-K14, rabbit anti-K14, mouseanti-K10 (Lab Vision, Fremont, CA, http://www.labvision.com),mouse anti-K3/K12 (Chemicon, Temecula, CA, http://www.chemicon.com), rabbit anti-�III-tubulin (Sigma-Aldrich), mouseanti-Oct-4, mouse anti-nestin, goat anti-filaggrin, or goat anti-in-volucrin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). Cells were stained with the appropriate fluoro-phore-conjugated secondary antibody (Invitrogen) for 1 hour atroom temperature and stained with Hoechst dye. Immunofluores-cence images were observed on an Olympus IX70 microscope(Olympus, Tokyo, http://www.olympus-global.com; Leeds Preci-sion Instruments, Minneapolis, MN, http://www.leedsmicro.com)using MetaVue imaging software (Molecular Devices, Sunnyvale,CA, http://www.moleculardevices.com).

Flow CytometryCells were detached from culture plates using trypsin-EDTA and2% chick serum, fixed in 1% paraformaldehyde for 10 minutes at37°C, and permeabilized on ice in 90% methanol. Primary antibod-ies (described above) were incubated overnight in PBS with 2%fetal calf serum and 0.1% NaN3 at 1:100; control samples wereincluded using isotype-specific or no primary antibody. After a1-hour secondary stain, cells were analyzed on a FACSCalibur flowcytometer using CellQuest software (BD Biosciences).

Semiquantitative and Quantitative ReverseTranscription-Polymerase Chain ReactionRNA was harvested from EBs or cells using the RNeasy Mini kit(Qiagen, Valencia, CA, http://www1.qiagen.com), and cDNA wasgenerated using Omniscript reverse transcriptase (RT) (Qiagen), 1�g of RNA, and oligo(dT) primers. For semiquantitative analysis,polymerase chain reaction (PCR) was conducted using PlatinumTaq with 1 �l of cDNA for 30 or 35 cycles. Quantitative PCR wasconducted with Quantitect SYBR Green quantitative polymerasechain reaction (qPCR) kit (Qiagen) and 1 �l of cDNA on an iCycler(Bio-Rad, Hercules, CA, http://www.bio-rad.com). Relative expres-sion levels were calculated using the �CT method, normalizing toglyceraldehyde-3-phosphate dehydrogenase transcription. Primers(supplemental online Table 1) were designed such that they spannedintrons or bound exon borders, and PCR products were verified bymelting curve analysis and/or 2% agarose gel electrophoresis. NoRT controls were conducted for primer sets that were able amplifygenomic DNA.

Polyacrylamide Gel Electrophoresis and WesternBlottingCellular protein was harvested using RIPA buffer (Santa CruzBiotechnology) and quantified using a BCA protein assay (Pierce,Rockford, IL, http://www.piercenet.com). Equal amounts of proteinwere resolved on a 10% polyacrylamide gel and transferred to a

Figure 1. RA induces epithelial gene expression in human ESC(hESC)-derived EBs. Morphology of day 6 H1 EBs cultured in UM with(A) or without (B) RA for 5 days. (C–F): Quantitative polymerase chainreaction (PCR) of cDNA harvested from H1 EBs differentiated in thepresence or absence of RA for 2 or 8 days. (C): Oct-4 expression wassignificantly reduced upon RA addition; �, p � .05, UM versus RA. (D):K18 transcription was significantly upregulated; �, p � .02, UM versusRA. (E): Nestin expression was decreased in RA cultures. (F): Pax6 wasdetected at significant levels only in untreated EBs. Error bars indicateSEM for duplicate PCRs. (G): Semiquantitative PCR of RA-treated/untreated H1 EBs. p63 isoforms were detected only in treated EBs,whereas Sox1 was expressed more highly in untreated EBs at day 8.(H–K): Phase-contrast (H, J) and p63 immunocytochemistry (I, K)images of control (H, I) and RA-treated (J, K) H1 EBs plated on gelatinand cultured for 1 week in keratinocyte medium. Scale bars � 50 �m.Similar observations were made in at least three separate experimentsusing either H1 or H9 hESC lines. Statistical analysis was performedusing Student’s t test. Abbreviations: GAPDH, glyceraldehyde-3-phos-phate dehydrogenase; K18, keratin 18; RA, retinoic acid; UM, uncon-ditioned medium.

373Metallo, Ji, de Pablo et al.

www.StemCells.com

nitrocellulose membrane. After blocking with 5% milk in PBS,membranes were probed with primary antibodies overnight andstained with horseradish peroxidase-conjugated antibodies for 1hour. Protein levels were detected by chemiluminescence (Pierce),and protein loading was verified by probing against �-actin.

Colony Forming AssayhESC-derived keratinocytes were trypsinized and plated on colla-gen I-coated six-well plates (BD Biosciences) in DSFM at a densityof 5,000 cells per well. Cells were cultured with medium changesevery other day for 2 weeks before being fixed with 4% parafor-maldehyde and stained with 0.5% rhodamine B for 30 minutes.After a brief wash, the plates were dried, and colonies were countedin triplicate wells.

RESULTS

RA Induces Epithelial Gene Expression in EBsRA is a common differentiation agent used to direct hESCs tovarious lineages. Addition of RA to newly formed embryoidbody (EB) cultures induced the formation of translucent, spher-ical outgrowths (Fig. 1A), whereas control EBs retained a moreuniform morphology (Fig. 1B). Quantitative PCR analysis dem-onstrated a pronounced decrease in Oct-4 transcription in thepresence of RA (Fig. 1C), and the lack of detectable mesodermand endoderm markers (Brachyury and FoxA2) led us to focuson the ectodermal fates of cells in RA-treated EBs (not shown).The first markers specific to neuroectoderm expressed in differ-entiating hESCs are Pax6 and Sox1 [18]; transcription of nestin

(Fig. 1E), Pax6 (Fig. 1F), and Sox1 (Fig. 1G) was diminished orundetectable in RA-treated EB cultures compared with controls.K18, a simple epithelial marker, was significantly upregulatedupon RA addition (Fig. 1D), and both trans-activating anddominant-negative (�N) p63 isoforms were only detected in RAcultures at the tested time points, with �Np63 isoforms ex-pressed at the highest levels (Fig. 1G; data not shown). Uponadhesion to gelatin-coated plates and culture in keratinocytemedium lacking RA, virtually all cells previously treated withRA expressed p63 (Fig. 1J, 1K), whereas control cultures con-tained few p63� cells (Fig. 1H, 1I). Cells in distinct regions ofcontrol cultures were very heterogeneous in size and morphol-ogy (not shown).

The distinct phenotype of RA-induced EBs was also evidentupon quantification of overall cell expansion. While cells pro-liferated at similar rates in treated and untreated EB cultures, theefficiency at which control EBs adhered to gelatin was signifi-cantly lower (Fig. 2A). In fact, adhesion of untreated EBs washighly variable both within and between experiments, whereasRA-treated EBs adhered uniformly to gelatin-coated plastic.Taking into account the gene expression results describedabove, this result is not surprising given the demonstrated role ofp63 in regulating epithelial cell adhesion [19].

RA-Induced EBs Readily Differentiate intoKeratinocytesBasal cells of stratified squamous epithelia express K14, andcultivation of adherent RA-treated EBs in keratinocyte mediumgenerated significant populations of K14� cells, as determined

Figure 2. Generation of keratinocytes from RA-treated/untreated EBs. (A): Normalized cell number of H9 human ESCs (hESCs) differentiated asEBs for 6 days in UM or UM � RA and subsequently cultured in keratinocyte medium. (B): Flow cytometric analysis of the percentage of K14�cells corresponding to various points in (A) (�, p � .0005, RA� vs. RA�). (C): Flow cytometric analysis of K14� cells in differentiated cellpopulations. Each pair of data points represents a separate differentiation experiment using the indicated hESC line; �, p � .05, UM versus UM �RA. (D): Flow cytometric analysis of Oct-4 and K14 expression in H9 EBs plated for 10 days; �, p � .01, UM versus UM � RA percentage ofOct-4�; ��, p � .001, UM versus UM � RA percentage of K14�. Error bars indicate SEM (n � 3). Statistical analysis was performed using Student’st test. (E–H): Phase contrast and immunocytochemistry of RA-treated H1 EBs differentiated for 20 days. (F): K14� (red) cells migrating from adifferentiated colony containing many p63� (green) cells. (H): Two colonies of differentiated cells, one containing K14� (red) cells and the otherexpressing nestin (green). Nuclei are blue. Scale bars � 50 �m. (I, J, K): Flow cytometry analysis of nestin� and K14� populations in UM-treated(J) and UM � RA-treated (K) H1 EBs subcultured for 21 days. Abbreviations: K14, keratin 14; RA, retinoic acid; UM, unconditioned medium.

374 RA Mediates Epithelial Differentiation of hESCs

by flow cytometry and immunostaining. The percentage ofK14� cells continued to rise for approximately 30 days afterplating and was always significantly greater than that observedin untreated EB cultures (Fig. 2B). This enhancement in kera-tinocyte generation was observed in both H1 and H9 hESClines, which produced similar quantities of K14� cells afterplating (Fig. 2C). RA-treated EBs formed monolayer coloniesupon adhesion to gelatin, whereas untreated EBs attached aslarge cellular aggregates, often containing undifferentiatedhESCs expressing Oct-4 (Fig. 2D). Immunocytochemistry dem-onstrated that virtually all RA-treated colonies contained p63�/K14� cells proliferating and migrating at the periphery (Fig.2E, 2F). However, further analysis identified many colonies asnestin-positive, indicating these cells were a common contam-inating cell type in both RA cultures (Fig. 2H, 2K) and untreatedcontrols (Fig. 2J).

Concentration and Temporal Dependence of RADifferentiationIn contrast with our results, various differentiation strategieshave used RA to direct ESCs to neural rather than epithelialfates [16, 17, 20]. We therefore hypothesized that modulatingthe concentration or temporal presentation of RA might regulateepithelial lineage specification. EBs were induced with variousRA concentrations (9 days) and subsequently cultured on gelatinin DSFM (19 days); differentiated cell populations were ana-lyzed for K14 and nestin expression. Significant keratinocyteinduction occurred only when EBs were cultured with 1 �M RA

(Fig. 3A), whereas higher concentrations (10 �M) proved to betoxic to undifferentiated hESCs (not shown). We also observeda concomitant decrease in nestin� populations as the RA con-centration increased (Fig. 3B), decreasing from as high as 70%in control cultures to as low as 20% when EBs were treated with1 �M RA.

To understand the temporal effects of RA-induced differ-entiation, we added RA to hESC cultures at various stages ofdifferentiation (during either EB induction or adherent cul-ture in DSFM). RA exposure for 4 – 8 days during the EBphase, but not after plating on gelatin, generated substantialnumbers of K14� cells. Addition of RA during adherentculture decreased or eliminated keratinocyte differentiation,depending on the presence of RA during EB culture (Fig. 3C,3E). Exposure of plated cells from untreated EBs to RAenriched nestin� populations, as evidenced by flow cytom-etry (Fig. 3D) and qPCR analysis (Fig. 3E). Cultures thatwere never exposed to RA expressed the highest levels of theearly neural marker Sox1. RA therefore exhibited differentialeffects on cell phenotype depending on the stage of applica-tion. Although nestin expression alone is not sufficient todesignate neural precursor populations, these findings pro-vide indirect evidence for the importance of temporal RAapplication with respect to neural versus epithelial specifica-tion. In fact, Pankratz et al. made similar observations in theirstudy of neural specification of hESCs, as early RA applica-tion generated unidentified non-neural lineages, and caudal-ization with RA could only be accomplished after first ob-taining primitive anterior neuroepithelia [18]. However, more

Figure 3. RA effects on human ESC (hESC) differentiation depend on concentration and time of application/stage of differentiation. (A, B):Flow cytometric analysis of adherent H1 cultures differentiated with various RA concentrations as EBs. (A): K14� populations weresignificantly induced only when EBs were differentiated with 10�6 M RA (�, p � .02, 10�6 M vs. all others). (B): Generation of nestin�populations was also dependent upon RA concentration (�, p � .03, 10�6 M vs. all others; ��, p � .03, 10�7 M vs. 10�8 M). (C–E): RA wasadded to H9 hESCs during the EB phase (�/�) and/or the adherent culture phase (�/�, �/�) or not at all (�/�). (C): Flow cytometric analysisof K14� cells in adherent cultures. RA addition during the EB phase significantly induced K14 differentiation, when RA was absent (�, p �.0001, �/� vs. �/�) or present (��, p � .02, �/� vs. �/�) during adherent culture. RA addition during adherent culture reduced the yieldof K14� cells (���, p � .001, �/� vs. �/�). (D): Nestin� populations were reduced when RA was added during the EB phase, especiallywhen RA application continued during adherent culture (�, p � .005, �/� vs. �/�). (E): Quantitative PCR of cDNA from day 17 adherentcultures normalized to glyceraldehyde-3-phosphate dehydrogenase. Nestin expression was highest with late but not early RA application. RAdecreased Sox1 expression at all times of application. �Np63 transcription was highest in cultures from EBs treated with RA, whereas K14was expressed most in the early but not late RA exposure. Error bars indicate SEM (n � 3). Statistical analysis was performed using Student’st test. Abbreviations: K14, keratin 14; RA, retinoic acid.

375Metallo, Ji, de Pablo et al.

www.StemCells.com

extensive studies using neurogenic substrates and culturemedia must be completed to definitively identify whetherneural precursor populations are enriched in these controlcultures.

Differentiation of hESCs in Defined MediaEB differentiation in the experiments described above usedmedium containing undefined components, including Knock-out Serum Replacer, which can activate the BMP signalingpathway [21]. To better understand the mechanisms involvedin keratinocyte differentiation, we identified a defined, N2supplement-containing medium, which permitted keratino-cyte generation to a similar extent as UM. As before, RAenhanced epithelial gene expression (�Np63 and K18) andreduced expression of early neural markers such as Sox1,Otx2, and nestin in EBs cultured in N2 medium for 6 days(Fig. 4A– 4C). To assess whether RA treatment blocked earlyneural differentiation or directed hESCs to more terminalneural fates, we analyzed differentiated cell populations atvarious times for transcription of neuronal (Synaptophysin)and astrocytic (glial fibrillary acidic protein [GFAP]) mark-ers. Throughout differentiation, Synaptophysin expression incultures derived from RA-treated EBs was similar to or lessthan that observed in control cultures (Fig. 4A). GFAP ex-pression was undetectable via RT-PCR (Fig. 4A) and immu-nocytochemistry (not shown) as well. Although the lack ofdefinitive neural markers expressed in our system may haveresulted from our use of epithelial substrates and media, theseresults provide evidence that early RA application does notreduce subsequent nestin� populations by inducing neuralprecursors to terminally differentiate.

Cultivation of differentiated EBs in DSFM resulted in ker-atinocyte induction similar to that observed using UM �/� RA(Fig. 4D). In addition, cells derived from RA-treated EBs in N2medium proliferated well after plating in DSFM (Fig. 4E), withthe percentage of K14� cells increasing steadily with time (Fig.

4F) as well. In contrast to EBs differentiated in UM, hESCsdifferentiated in N2 and DSFM for extended periods (30 days)generated occasional �III-tubulin� neurons within large, EB-derived colonies. Neuronal differentiation was observed sporad-ically from both control EBs (Fig. 4G) and RA-treated EBs (Fig.4H) at similar frequencies. Given that UM has previously beenshown to activate the BMP signaling pathway [21] and thatBMP activity can block early neural differentiation during de-velopment [12], the appearance of neurons in N2 differentiatedEBs provided evidence for a role of BMP signaling in thedifferentiation process.

Direct Differentiation in Two-Dimensional CultureTo provide a more uniform microenvironment during differ-entiation we also eliminated EB formation and directly in-duced differentiation of hESC colonies cultivated on Matri-gel (supplemental online Fig. 1B). After 6 days of treatmentwith RA or carrier, epithelial proteins such as K18 and p63were detected at elevated levels in RA-treated cells (Fig. 5A).Interestingly, N2-supplemented medium promoted greaterdifferentiation than unconditioned hESC medium, presum-ably because of the absence of Knockout Serum Replacer,which can induce transforming growth factor-�/Activin/Nodal signaling. Activation of this pathway has been shownto maintain hESC self-renewal [21] and block neuroectoder-mal differentiation [22]. Subculture of differentiated coloniesin keratinocyte medium yielded K14� populations of higherpurity than the EB process. After 5 weeks of differentiation,87% of cells expressed K14 using the direct method versusonly 15% K14� cells in the EB method, demonstrating asignificant enhancement in keratinocyte differentiation (Fig.5B, 5C). Furthermore, keratinocyte differentiation resultsfrom H1 and H9 hESC lines were similar using this process(supplemental online Fig. 2). The uniformity and definedconditions provided by the above modifications then permit-

Figure 4. Epithelial differentiation of human ESCs (hESCs) in defined medium. (A): Reverse transcription-polymerase chain reaction (PCR) analysisof RNA isolated from H9 hESCs differentiated as EBs in N2 �/� RA for 6 days and cultured on gelatin in defined keratinocyte serum-free medium.All PCRs except that for GAPDH (25 cycles) were run for 30 cycles along with PC from hESCs, human neural progenitors, or primary humankeratinocytes. (B, C): Quantitative PCR analysis of K18 and nestin mRNA expression from day 6 EBs cultured in N2. (D): Flow cytometric analysisof K14� cell populations (day 27) derived from H1 EBs differentiated in UM or N2 �/� RA (�, p � .02, RA� vs. RA�). (E): Normalized cellnumber of H9 hESCs differentiated as EBs for 6 days in N2 or N2 � RA and subsequently cultured in keratinocyte medium. (F): Flow cytometricanalysis of the percentage of K14� cells corresponding to various points in (E) (�, p � .0005, RA� vs. RA�). Error bars indicate SEM. Statisticalanalysis was performed using Student’s t test. (G, H): Day 27 keratinocyte cultures derived from EBs cultured in N2 (G) or N2 � RA (H) stainedfor �III-tubulin (red) and K14 (green). Nuclei are blue. Scale bars � 100 �m. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase;GFAP, glial fibrillary acidic protein; K14, keratin 14; K18, keratin 18; N2, N2 medium; PC, positive control cDNA; RA, retinoic acid; UM,unconditioned medium.

376 RA Mediates Epithelial Differentiation of hESCs

ted us to conduct a more detailed analysis of the signalingpathways involved in epithelial specification.

Molecular Mechanisms of Epithelial Differentiationfrom hESCsPrevious studies of mESCs [9] and Xenopus [11] identifiedBMP signaling as a key inducer of epidermal fates duringdevelopment. To elucidate the molecular mechanisms of epithe-lial lineage specification in hESC cultures, we directly differ-entiated colonies in N2 medium containing RA, BMP-4, or theBMP inhibitor Noggin. RNA was isolated from cultures differ-entiated for 6 days and analyzed for expression of epithelial andneuroectodermal transcripts (Fig. 5D). �Np63 transcription waselevated in all RA-treated cell samples, regardless of the statusof BMP signaling, whereas BMP-4 and RA synergisticallyinduced K18 expression. Neuroepithelial gene expression(Pax6, Sox1) was effectively downregulated in RA-treated cul-

tures, although this effect was mitigated by inhibition of BMPsignaling.

Subsequent cultivation of differentiated hESCs in keratino-cyte medium further demonstrated that RA-mediated differen-tiation outcomes were dependent upon the status of BMP sig-naling. Addition of BMP-4 to RA�/� cultures had only a slighteffect on keratinocyte yields, whereas inhibition of BMP sig-naling by Noggin significantly decreased epithelial differentia-tion (Fig. 5E). On the other hand, early treatment with RA in thepresence of endogenous or exogenous levels of BMPs elimi-nated virtually all nestin� cells detected in day 18 subcultures,whereas Noggin-mediated inhibition of BMP signaling gener-ated large populations of nestin� cells (Fig. 5F). Similar trendswere observed in RA-treated EBs upon treatment with BMP-4and Noggin, although the level of keratinocyte induction waslower compared with direct differentiation (supplemental onlineFig. 3). Taken together, these data demonstrate that BMP andRA signaling play synergistic but distinct roles in hESC ecto-

Figure 5. Synergism of RA and BMP signaling during directed differentiation of human ESCs (hESCs). (A): SDS-polyacrylamide gel electro-phoresis of protein extracts from H9 hESCs differentiated for 6 days in various media. RA induces p63 and K18 expression while downregulatingOct-4. (B, C): Flow cytometry histograms of K14� populations derived using EB differentiation (B) or direct differentiation (C) methods for H9differentiation. (D): Quantitative PCR analysis of cDNA derived from H9 hESCs directly differentiated for 6 days in the presence of RA, BMP-4,and/or Noggin (normalized to glyceraldehyde-3-phosphate dehydrogenase). RA reduced expression of Pax6 in differentiated cells, and BMP-4 andRA both reduced Sox1 expression, although Noggin mitigated the effects of both factors. RA and BMP-4 synergistically induced expression of �Np63and K18 transcription. (E): Flow cytometric analysis of K14� populations in differentiated H9 hESCs subcultured for 18 days. Inhibition of BMPsignaling by Noggin significantly reduced keratinocyte generation; �, p � .05, Noggin only versus negative or BMP-4 only. Addition of RA andBMP-4 significantly enhanced keratinocyte generation; ��, p � .02, RA only versus all others except �/�/�; ���, p � .001 BMP-4�RA versus allothers except �/�/�. (F): Flow cytometric analysis of nestin� populations. Addition of Noggin significantly enhanced the percentage of nestin�cells, whether or not RA was present during differentiation; �, p � .05 versus all others except �/�/�. Differentiation with BMP-4 and/or RArepressed neural differentiation; ��, p � .02 versus all except negative and �/�/�. Error bars indicate SEM (n � 3). Statistical analysis wasperformed using Student’s t test. Similar trends were observed in at least three separate experiments. Abbreviations: BMP, bone morphogenetic protein4; DMSO, dimethyl sulfoxide; K14, keratin 14; K18, keratin 18; N2, N2 medium; RA, retinoic acid; UM, unconditioned medium.

377Metallo, Ji, de Pablo et al.

www.StemCells.com

dermal differentiation. Active BMP signaling (endogenous orexogenous) reduced presumptive neural differentiation, as evi-denced by relative Sox1 expression levels and subsequent nes-tin� cell populations. Under these conditions, RA effectivelyinduced keratinocyte differentiation (K14� populations). How-ever, Noggin treatment alone significantly reduced keratinocytedifferentiation, regardless of the whether RA was administeredduring early differentiation. Inhibition of BMP signaling pre-sumably resulted in survival of highly proliferative nestin�cells, ultimately decreasing K14� populations in our differen-tiated cultures.

Terminal Differentiation of hESC-DerivedKeratinocyteshESC-derived keratinocytes could be subcultured onto gelatinor collagen IV substrates, and these secondary cultures yieldedrelatively pure keratinocyte populations (K14�) compared withprimary foreskin keratinocytes. Flow cytometry analysis of K14expression measured 90% K14� cells in a feeder-free primaryculture (Fig. 6A), whereas hESC-derived keratinocytes con-tained 96% K14� cells (Fig. 6B). This result was verified withtwo anti-K14 antibodies (not shown) and qualitatively via im-munocytochemistry (Fig. 6C). As has previously been described[3], these epithelial cultures exhibited a lower expansion poten-tial than primary cultures; cryopreserved hESC-derived keratin-ocytes could be propagated for approximately 10 populationdoublings (Fig. 6D). However, we were able to obtain colony-forming keratinocytes at a frequency of at least 0.003 fromdifferentiated hESCs plated, a sixfold increase over previouslyreported methods that used implantation into immunocompro-mised mice [3]. It must also be noted that our growth analysisused feeder-free culture in serum-free medium, and it isunclear how these results might translate to more routinelyused protocols.

We also examined the capacity of hESC-derived keratin-ocytes produced via RA induction to express terminal differ-entiation markers in high-density and Ca2�-containing cul-tures. Small areas of stratification appeared in extendeddifferentiation cultures with or without Ca2�; the top layersexpressed filaggrin (Fig. 6E), involucrin (Fig. 6F), and, insome cases, the epidermal-specific keratin K10 (Fig. 6F).Interestingly, we also observed some remaining cells thatexpressed K18 (Fig. 6G) and many cells positive for thecorneal epithelial marker K3/K12 (Fig. 6H). Others havedemonstrated generation of corneal epithelial-like cells fromhESCs using limbal fibroblast-conditioned medium [23]rather than the defined conditions described here, so it ispossible that retinoid secretion mediated their observed con-ditioning effects. In addition, confluent keratinocyte culturesin Ca2�-containing, defined keratinocyte medium could bedetached from the substrate as an intact epithelial sheet usingDispase, contracting accordingly (Fig. 6I). RA-inducedhESCs produced relatively pure keratinocyte cultures thatexpressed terminal differentiation markers and formed coher-ent epithelial sheets under the appropriate conditions; thehigh-efficiency differentiation process coupled with the self-renewal capacity of undifferentiated hESCs provides a meansof generating large quantities of nontransformed keratino-cytes.

DISCUSSION

A common challenge in hESC biology is the identification ofefficient differentiation processes to generate functional somaticcell lineages. Various growth factors and signaling molecules

induce hESC differentiation. However, the specific responsedepends upon the time, concentration, and combination of ap-

Figure 6. Retinoic acid-mediated H9 human ESC (hESC) differen-tiation yields relatively pure keratinocyte cultures capable of re-sponding to terminal differentiation stimuli. (A, B): Flow cytometrichistograms of K14� expression of primary foreskin keratinocyte (A)and H9 hESC-derived keratinocyte (B) cultures. (C): Immunocyto-chemistry of K14 (red) and p63 (green) in a confluent subculture ofhESC-derived keratinocytes. (D): Subculture of cryopreservedhESC-derived keratinocytes over multiple passages. Approximately10 population doublings were observed before cells became senes-cent. (E, F): Extended culture at confluence induced terminal dif-ferentiation marker expression in stratified cell layers. (E): K14 (red)staining was evident in monolayers, whereas Filaggrin� (green)cells were localized to multilayered areas. (F): Involucrin (red) andK10 (green) expression were also localized to stratified regions. (G):K14 (red) and K18 (green) staining of a subculture with nuclearHoechst stain (blue). (H): K14 (red) and K3/K12 (green) staining ofa confluent culture with nuclear Hoechst stain (blue). Yellow denotesgreen/red colocalization. Scale bars � 50 �m. (I): Human embryonicstem cell-derived keratinocytes were cultured on a collagen IV-coated dish in defined keratinocyte serum-free medium containing 1mM Ca2� for 8 days. After Dispase treatment for 1 hour, an intactepithelial sheet detached from the surface. Abbreviation: hESC,human ESC.

378 RA Mediates Epithelial Differentiation of hESCs

plied factors, and pure populations of lineage-specific progeni-tors are difficult to obtain [24]. RA signaling is involved inmany developmental processes during embryogenesis, includ-ing pattern formation and limb development [25, 26]. In addi-tion, RA is commonly used as a caudalizing agent to specifymotor neuron differentiation from ES cells; however, theseprotocols require establishment of neuroectodermal fates priorto RA administration [16, 17]. Here, we describe a novel,stage-specific effect of RA on early hESC differentiation, inwhich ectodermal derivatives acquire epithelial fates in responseto RA.

RA mediates cellular responses by binding to nuclearretinoic acid receptors (RARs), which in turn modulate tran-scription through several pathways. Undifferentiated hESCs,which express RAR� [27], initiated p63 transcription within48 hours of RA administration, with �Np63 isoforms ex-pressed at the highest levels. Mice expressing dominant-negative forms of RAR� under the K14 promoter exhibitreduced p63 expression and severe defects similar to thoseobserved in p63 knockouts [28, 29]. Furthermore, RA hasbeen shown to induce �Np63 expression in RAR��-nullkeratinocytes in the presence of elevated RAR� levels (butnot RAR�) [28], so RA may regulate p63 transcription,directly or indirectly, in hESCs because of differential ex-pression of RAR subtypes. Given its demonstrated role inepithelial commitment [5, 7, 10, 30], cell adhesion [19], anddifferentiation [31], p63 is likely a primary driver of theepithelial cell enrichment observed in our process.

Modulation of �Np63 expression in zebrafish embryosdemonstrated its role in early dorsal/ventral ectodermal pat-terning as well as neural/epidermal specification [10, 30].�Np63 was identified as a target of Smad-mediated BMPsignaling, and its overexpression in embryos lacking BMPactivity rescued non-neural ectoderm formation, implyingthat p63 may mediate the neural repression described in theneural default model [10]. In our system, we observed anearly RA-induced upregulation of �Np63 transcription andsubsequent reduction in Pax6 and Sox1 expression. Pax6 andSox1 have recently been described as early markers of prim-itive anterior and definitive neuroepithelia, respectively, inhESCs [18]. That study used RA as a caudalizing agent toinduce formation of posterior neuroepithelia; interestingly,the authors found that early RA application induced uniden-tified non-neural cell fates [18]. It is therefore conceivablethat these unidentified cells were p63� primitive epitheliacapable of differentiating into keratinocytes. Recently,BMP-4 has been shown to induce keratinocyte differentiationof mESCs [9] as well as induce apoptosis of neural precursorsthrough the Smad pathway [12]. We observed a furtherdecrease in neural gene expression when BMP-4 was addedto cultures during RA treatment. Conversely, the BMP an-tagonist Noggin was able to mitigate RA-mediated repressionof neuroepithelial gene transcription and increase the per-centage of nestin� cells in subcultures grown in keratinocytemedium. This finding highlights the distinct roles of RA andBMP signaling in epithelial differentiation, with BMP actingto block neural differentiation and RA directing cells to anepithelial fate (presumably through induction of �Np63 ex-pression).

RA mediation of p63 expression may also be of clinicalrelevance, as vitamin A and other retinoids are known to beteratogens. High vitamin A intake can induce musculoskele-tal and cranial-neural crest defects in particular, with anincreased prevalence when taken shortly before or after con-ception [32]. Heterozygous mutations in p63 are a primarycause of ectrodactyly, ectodermal dysplasia, cleft lip/palatesyndrome, in which patients exhibit congenital craniofacial,

limb, and other abnormalities [33]. Given the similarity indevelopmental abnormalities associated with retinoid-in-duced birth defects and p63 mutations, it is conceivable thatRA-mediated dysregulation of p63 expression could be in-volved in the teratogenicity of retinoids. However, moredetailed biochemical studies are necessary to definitivelyunderstand how RA regulates p63 expression, as no RAresponse elements have been identified in the p63 promoter[28].

In addition to providing insight into the molecular mech-anisms of epidermal commitment, RA-induced differentia-tion of hESCs offers an efficient means of generating kera-tinocytes. Previous studies have demonstrated thatspontaneous keratinocyte differentiation in EBs occurs inef-ficiently [2, 4]. These hESC derivatives exhibited a lowerproliferative capacity compared with primary keratinocytes,and immortalization was required to extend the lifespan ofthese cultures [3]. Although RA-induced hESC cultures pro-duced keratinocytes with a replicative potential similar to thatof controls, the high efficiency of this process provides analternative to transformation, which can interfere with bio-logical functions and complicates clinical use. Furthermore,application of RA rapidly eliminated Oct-4� undifferentiatedhESCs from culture and allowed for differentiation in definedmedium, whereas other methods have used coculture [2, 34]and/or undefined medium [9, 23]. Gene therapy of tissue-engineered skin may be used in clinical, diagnostic, and basicresearch applications [35]; presumably, keratinocyte differ-entiation can be accomplished using genetically modified,clonally derived hESCs, allowing for the generation of non-transformed human keratinocytes and/or skin constructs withspecific genetic backgrounds.

Although hESC-derived keratinocytes can express terminaldifferentiation markers and form epithelial sheets, the “burdenof proof” regarding in vivo functionality must still be met.Organotypic culture of keratinocytes at an air-to-liquid interfaceprovides a better means of analyzing terminal differentiation,but long-term engraftment studies in the proper niche must beconducted to gauge the self-renewal capacity of these cells aftertransplantation. Epithelial grafting may also be informative, asembryonic epithelia (or ES cell-derived epithelia) may retaingreater plasticity in response to tissue-specific mesenchymethan adult cells [36].

Here, we have demonstrated a novel, developmental stage-specific effect of RA on hESC differentiation to the keratinocytelineage using two distinct cell lines. Although recent evidencesuggests that the propensity of individual cell lines to differen-tiate toward certain lineages may differ [37, 38], it seemsunlikely that the signaling mechanisms regulating differentia-tion to particular lineages are different. Thus, our findings intwo cell hESC lines should be applicable to other lines, althoughrelative levels of differentiation may vary. Although the pleio-tropic nature of RA signaling complicates the identification ofspecific mechanisms, the induction of p63 expression and de-pendence on BMP signaling provide insight into the molecularmechanisms of human epithelial development. Finally, this pro-cess provides an effective means of generating large quantitiesof keratinocytes from hESCs under feeder-free, defined condi-tions.

ACKNOWLEDGMENTS

We acknowledge Kathy Schell and staff at the University ofWisconsin Comprehensive Cancer Center Flow Cytometry fa-cility for technical assistance. Funding for this research was

379Metallo, Ji, de Pablo et al.

www.StemCells.com

provided by the National Science Foundation-sponsored Uni-versity of Wisconsin Materials Research Science and Engineer-ing Center and the NIH-funded University of Wisconsin Bio-technology Training Program (to C.M.M.). C.M.M. and L.J.contributed equally to this work.

DISCLOSURE OF POTENTIAL CONFLICTS

OF INTEREST

The authors indicate no potential conflicts of interest.

REFERENCES

1 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem celllines derived from human blastocysts. Science 1998;282:1145–1147.

2 Green H, Easley K, Iuchi S. Marker succession during the developmentof keratinocytes from cultured human embryonic stem cells. Proc NatlAcad Sci U S A 2003;100:15625–15630.

3 Iuchi S, Dabelsteen S, Easley K et al. Immortalized keratinocyte linesderived from human embryonic stem cells. Proc Natl Acad Sci U S A2006;103:1792–1797.

4 Ji L, Allen-Hoffmann BL, de Pablo JJ et al. Generation and differenti-ation of human embryonic stem cell-derived keratinocyte precursors.Tissue Eng 2006;12:665–679.

5 Mills AA, Zheng B, Wang XJ et al. p63 is a p53 homologue required forlimb and epidermal morphogenesis. Nature 1999;398:708–713.

6 Yang A, Kaghad M, Wang Y et al. p63, a p53 homolog at 3q27–29,encodes multiple products with transactivating, death-inducing, anddominant-negative activities. Mol Cell 1998;2:305–316.

7 Yang A, Schweitzer R, Sun D et al. p63 is essential for regenerativeproliferation in limb, craniofacial and epithelial development. Nature1999;398:714–718.

8 Moll R, Franke WW, Schiller DL et al. The catalog of human cytoker-atins: Patterns of expression in normal epithelia, tumors and culturedcells. Cell 1982;31:11–24.

9 Coraux C, Hilmi C, Rouleau M et al. Reconstituted skin from murineembryonic stem cells. Curr Biol 2003;13:849–853.

10 Bakkers J, Hild M, Kramer C et al. Zebrafish DeltaNp63 is a direct targetof Bmp signaling and encodes a transcriptional repressor blocking neuralspecification in the ventral ectoderm. Dev Cell 2002;2:617–627.

11 Wilson PA, Hemmati-Brivanlou A. Induction of epidermis and inhibitionof neural fate by Bmp-4. Nature 1995;376:331–333.

12 Gambaro K, Aberdam E, Virolle T et al. BMP-4 induces a Smad-dependent apoptotic cell death of mouse embryonic stem cell-derivedneural precursors. Cell Death Differ 2006;13:1075–1087.

13 Xu RH, Chen X, Li DS et al. BMP4 initiates human embryonic stem celldifferentiation to trophoblast. Nat Biotechnol 2002;20:1261–1264.

14 Chadwick K, Wang L, Li L et al. Cytokines and BMP-4 promotehematopoietic differentiation of human embryonic stem cells. Blood2003;102:906–915.

15 Bamberger C, Pollet D, Schmale H. Retinoic acid inhibits downregula-tion of DeltaNp63alpha expression during terminal differentiation ofhuman primary keratinocytes. J Invest Dermatol 2002;118:133–138.

16 Li XJ, Du ZW, Zarnowska ED et al. Specification of motoneurons fromhuman embryonic stem cells. Nat Biotechnol 2005;23:215–221.

17 Wichterle H, Lieberam I, Porter JA et al. Directed differentiation ofembryonic stem cells into motor neurons. Cell 2002;110:385–397.

18 Pankratz MT, Li XJ, Lavaute TM et al. Directed neural differentiation ofhuman embryonic stem cells via an obligated primitive anterior stage.STEM CELLS 2007;25:1511–1520.

19 Carroll DK, Carroll JS, Leong CO et al. p63 regulates an adhesionprogramme and cell survival in epithelial cells. Nat Cell Biol 2006;8:551–561.

20 Okada Y, Shimazaki T, Sobue G et al. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentia-tion of mouse embryonic stem cells. Dev Biol 2004;275:124–142.

21 James D, Levine AJ, Besser D et al. TGFbeta/activin/nodal signaling isnecessary for the maintenance of pluripotency in human embryonic stemcells. Development 2005;132:1273–1282.

22 Vallier L, Reynolds D, Pedersen RA. Nodal inhibits differentiation ofhuman embryonic stem cells along the neuroectodermal default pathway.Dev Biol 2004;275:403–421.

23 Ahmad S, Stewart R, Yung S et al. Differentiation of human embryonicstem cells into corneal epithelial-like cells by in vitro replication of thecorneal epithelial stem cell niche. STEM CELLS 2007;25:1145–1155.

24 Metallo CM, Mohr JC, Detzel CJ et al. Engineering the stem cellmicroenvironment. Biotechnol Prog 2007;23:18–23.

25 Kawakami Y, Raya A, Raya RM et al. Retinoic acid signalling linksleft-right asymmetric patterning and bilaterally symmetric somitogenesisin the zebrafish embryo. Nature 2005;435:165–171.

26 Thaller C, Eichele G. Identification and spatial distribution of retinoids inthe developing chick limb bud. Nature 1987;327:625–628.

27 Schuldiner M, Yanuka O, Itskovitz-Eldor J et al. Effects of eight growthfactors on the differentiation of cells derived from human embryonicstem cells. Proc Natl Acad Sci U S A 2000;97:11307–11312.

28 Chen CF, Lohnes D. Dominant-negative retinoic acid receptors elicitepidermal defects through a non-canonical pathway. J Biol Chem 2005;280:3012–3021.

29 Saitou M, Sugai S, Tanaka T et al. Inhibition of skin development bytargeted expression of a dominant-negative retinoic acid receptor. Nature1995;374:159–162.

30 Lee H, Kimelman D. A dominant-negative form of p63 is requiredfor epidermal proliferation in zebrafish. Developmental Cell 2002;2:607– 616.

31 Koster MI, Kim S, Mills AA et al. p63 is the molecular switch forinitiation of an epithelial stratification program. Genes Dev 2004;18:126–131.

32 Rothman KJ, Moore LL, Singer MR et al. Teratogenicity of high vitaminA intake. N Engl J Med 1995;333:1369–1373.

33 Celli J, Duijf P, Hamel BC et al. Heterozygous germline mutations inthe p53 homolog p63 are the cause of EEC syndrome. Cell 1999;99:143–153.

34 Troy TC, Turksen K. Derivation of epidermal colony-forming pro-genitors from embryonic stem cell cultures. Methods Mol Biol 2006;330:93–104.

35 Andreadis ST. Gene-modified tissue-engineered skin: The next gen-eration of skin substitutes. Adv Biochem Eng Biotechnol 2007;103:241–274.

36 Fuchs E. Scratching the surface of skin development. Nature 2007;445:834–842.

37 Wu H, Xu J, Pang ZP et al. Integrative genomic and functional analysesreveal neuronal subtype differentiation bias in human embryonic stemcell lines. Proc Natl Acad Sci U S A 2007;104:13821–13826.

38 Kim SE, Kim BK, Gil JE et al. Comparative analysis of the develop-mental competence of three human embryonic stem cell lines in vitro.Mol Cells 2007;23:49–56.

See www.StemCells.com for supplemental material available online.

380 RA Mediates Epithelial Differentiation of hESCs