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First published online October 25, 2007
Stem Cells Vol. 26 No. 2 February 2008, pp. 372 -380
doi:10.1634/stemcells.2007-0501; www.StemCells.com
© 2008 AlphaMed Press

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EMBRYONIC STEM CELLS

Retinoic Acid and Bone Morphogenetic Protein Signaling Synergize to Efficiently Direct Epithelial Differentiation of Human Embryonic Stem Cells

Christian M. Metalloa, Lin Jia,b, Juan J. de Pabloa, 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

Correspondence: Correspondence: Sean P. Palecek, Ph.D., Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706, USA. Telephone: 608-262-8931; Fax: 608-262-5434; e-mail: palecek{at}engr.wisc.edu

Received on June 26, 2007; accepted for publication on October 18, 2007.

First published online in STEM CELLS EXPRESS  October 25, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human embryonic stem cells (hESCs) can differentiate to various somatic lineages, including stratified squamous epithelia, although the molecular mechanisms of epithelial specification from hESCs currently remain undefined. Here, we demonstrate a novel, stage-specific effect of retinoic acid (RA) on epithelial differentiation of hESCs. RA strongly upregulated expression of keratin 18 and the transcription factor p63, which is involved in epidermal morphogenesis and ectodermal specification, while repressing early neural marker transcription. RA-induced hESCs efficiently differentiated to keratin 14-expressing epithelial cells, although this effect was dependent upon on the context of bone morphogenetic protein signaling. Furthermore, these hESC-derived keratinocytes could be subcultured to obtain relatively pure keratinocyte populations that retained the capacity to terminally differentiate. These findings suggest that RA plays an important role in epithelial differentiation of hESCs.

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


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Human embryonic stem cells (hESCs) are pluripotent cells with an extensive proliferative capacity and the ability to differentiate into the three embryonic germ layers [1]. Much excitement has centered on their potential use in developmental biology research, diagnostic testing, and regenerative medicine; however, most applications will require relatively pure populations of somatic cell types, which, to date, have been difficult to produce. To exploit the expansion potential of undifferentiated hESCs, efficient differentiation processes must be developed to generate lineage-restricted progenitors at a high purity while eliminating pluripotent cells in culture.

Human ESCs can differentiate to the keratinocyte lineage [2], although current methods are inefficient and isolated keratinocytes have a reduced expansion potential [3, 4]. The hESC differentiation process toward keratinocytes is marked by expression of p63, a transcription factor necessary for maintenance of regenerative epithelia (i.e., the epidermis) [5–7], and keratin 14 (K14), an intermediate filament present in the basal layer of stratified squamous epithelia [8]. Studies using murine embryonic stem cells (mESCs) have identified bone morphogenetic protein 4 (BMP-4) as an inducer of epidermal differentiation [9], and BMP signaling plays a key role in early ectodermal fate choices during development in multiple species [10–12]. The role BMP-4 plays in human ectodermal development is currently unclear, as this factor has been used to direct hESCs to nonectodermal lineages [13, 14].

Retinoids are potent regulators of cell proliferation and differentiation and are highly involved in embryonic development. Retinoic acid (RA) has been shown to inhibit the terminal differentiation of keratinocytes in vitro by modulating p63 isoform expression [15] and is a caudalizing factor that directs embryonic stem (ES) cell-derived neuroepithelia to become motor neurons [16, 17]. Given the ethical problems associated with in vivo human embryo research, hESCs offer an attractive in vitro model system to study the effects of RA or other chemicals on early human developmental processes.

In the present study, we used quantitative analysis of differentiated hESC populations to identify key signaling factors involved in ectodermal lineage specification. RA applied to undifferentiated hESCs efficiently mediated epithelial differentiation in conjunction with BMP signaling. Finally, we used this process to generate relatively pure keratinocyte cultures capable of terminally differentiating and forming coherent epithelial sheets.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
H1 and H9 hESC lines were cultured on a layer of irradiated mouse embryonic fibroblasts (MEFs) in unconditioned medium (UM): Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium (F12) containing 20% Knockout Serum Replacer (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 1 x MEM nonessential amino acids (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 4 ng/ml basic fibroblast growth factor (bFGF). Alternatively, hESCs were plated on Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com) in medium conditioned by MEFs and passaged every 5–6 days using Dispase.

EBs were formed via enzymatic detachment of hESC colonies and cultured in UM without bFGF or N2 medium (N2): DMEM/F12 containing 1 x N2 supplement and MEM nonessential amino acids. After 1–2 days, EBs were transferred to a new vessel to remove adherent MEFs, and treatment with medium changes every other day was begun. Differentiated EBs were plated onto gelatin-coated plates in defined keratinocyte serum-free medium (DSFM), changing medium every other day (supplemental online Fig. 1A). For direct differentiation experiments, hESC colonies were grown for 6 days on Matrigel in MEF-conditioned hESC medium before switching to differentiation medium, which contained UM or N2 and a combination of DMSO, 1 µM all-trans RA (Sigma-Aldrich), 25 ng/ml BMP-4 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com), or 125 ng/ml Noggin (R&D Systems). After 6 days, differentiated cells were detached with Dispase and cultured overnight before plating as described above (supplemental online Fig. 1B).

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

Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature before blocking and permeabilizing with 5% milk in phosphate-buffered saline (PBS) with 0.4% Triton X-100. Primary antibodies were incubated overnight at 4°C in blocking buffer and included mouse anti-p63, mouse anti-K14, rabbit anti-K14, mouse anti-K10 (Lab Vision, Fremont, CA, http://www.labvision.com), mouse anti-K3/K12 (Chemicon, Temecula, CA, http://www.chemicon.com), rabbit anti-βIII-tubulin (Sigma-Aldrich), mouse anti-Oct-4, mouse anti-nestin, goat anti-filaggrin, or goat anti-involucrin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). Cells were stained with the appropriate fluorophore-conjugated secondary antibody (Invitrogen) for 1 hour at room temperature and stained with Hoechst dye. Immunofluorescence images were observed on an Olympus IX70 microscope (Olympus, Tokyo, http://www.olympus-global.com; Leeds Precision Instruments, Minneapolis, MN, http://www.leedsmicro.com) using MetaVue imaging software (Molecular Devices, Sunnyvale, CA, http://www.moleculardevices.com).

Flow Cytometry
Cells were detached from culture plates using trypsin-EDTA and 2% chick serum, fixed in 1% paraformaldehyde for 10 minutes at 37°C, and permeabilized on ice in 90% methanol. Primary antibodies (described above) were incubated overnight in PBS with 2% fetal calf serum and 0.1% NaN3 at 1:100; control samples were included using isotype-specific or no primary antibody. After a 1-hour secondary stain, cells were analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences).

Semiquantitative and Quantitative Reverse Transcription-Polymerase Chain Reaction
RNA was harvested from EBs or cells using the RNeasy Mini kit (Qiagen, Valencia, CA, http://www1.qiagen.com), and cDNA was generated using Omniscript reverse transcriptase (RT) (Qiagen), 1 µg of RNA, and oligo(dT) primers. For semiquantitative analysis, polymerase chain reaction (PCR) was conducted using Platinum Taq with 1 µl of cDNA for 30 or 35 cycles. Quantitative PCR was conducted with Quantitect SYBR Green quantitative polymerase chain reaction (qPCR) kit (Qiagen) and 1 µl of cDNA on an iCycler (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Relative expression levels were calculated using the {Delta}CT method, normalizing to glyceraldehyde-3-phosphate dehydrogenase transcription. Primers (supplemental online Table 1) were designed such that they spanned introns or bound exon borders, and PCR products were verified by melting curve analysis and/or 2% agarose gel electrophoresis. No RT controls were conducted for primer sets that were able amplify genomic DNA.

Polyacrylamide Gel Electrophoresis and Western Blotting
Cellular protein was harvested using RIPA buffer (Santa Cruz Biotechnology) and quantified using a BCA protein assay (Pierce, Rockford, IL, http://www.piercenet.com). Equal amounts of protein were resolved on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% milk in PBS, membranes were probed with primary antibodies overnight and stained with horseradish peroxidase-conjugated antibodies for 1 hour. Protein levels were detected by chemiluminescence (Pierce), and protein loading was verified by probing against β-actin.

Colony Forming Assay
hESC-derived keratinocytes were trypsinized and plated on collagen I-coated six-well plates (BD Biosciences) in DSFM at a density of 5,000 cells per well. Cells were cultured with medium changes every other day for 2 weeks before being fixed with 4% paraformaldehyde and stained with 0.5% rhodamine B for 30 minutes. After a brief wash, the plates were dried, and colonies were counted in triplicate wells.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
RA Induces Epithelial Gene Expression in EBs
RA is a common differentiation agent used to direct hESCs to various lineages. Addition of RA to newly formed embryoid body (EB) cultures induced the formation of translucent, spherical outgrowths (Fig. 1A), whereas control EBs retained a more uniform morphology (Fig. 1B). Quantitative PCR analysis demonstrated a pronounced decrease in Oct-4 transcription in the presence of RA (Fig. 1C), and the lack of detectable mesoderm and endoderm markers (Brachyury and FoxA2) led us to focus on the ectodermal fates of cells in RA-treated EBs (not shown). The first markers specific to neuroectoderm expressed in differentiating hESCs are Pax6 and Sox1 [18]; transcription of nestin (Fig. 1E), Pax6 (Fig. 1F), and Sox1 (Fig. 1G) was diminished or undetectable in RA-treated EB cultures compared with controls. K18, a simple epithelial marker, was significantly upregulated upon RA addition (Fig. 1D), and both trans-activating and dominant-negative ({Delta}N) p63 isoforms were only detected in RA cultures at the tested time points, with {Delta}Np63 isoforms expressed at the highest levels (Fig. 1G; data not shown). Upon adhesion to gelatin-coated plates and culture in keratinocyte medium lacking RA, virtually all cells previously treated with RA expressed p63 (Fig. 1J, 1K), whereas control cultures contained few p63+ cells (Fig. 1H, 1I). Cells in distinct regions of control cultures were very heterogeneous in size and morphology (not shown).


Figure 1
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  		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 chain reaction (PCR) of cDNA harvested from H1 EBs differentiated in the presence or absence of RA for 2 or 8 days. (C): Oct-4 expression was significantly reduced upon RA addition; *, p < .05, UM versus RA. (D): K18 transcription was significantly upregulated; *, p < .02, UM versus RA. (E): Nestin expression was decreased in RA cultures. (F): Pax6 was detected at significant levels only in untreated EBs. Error bars indicate SEM 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 gelatin and cultured for 1 week in keratinocyte medium. Scale bars = 50 µm. Similar observations were made in at least three separate experiments using either H1 or H9 hESC lines. Statistical analysis was performed using Student's t test. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; K18, keratin 18; RA, retinoic acid; UM, unconditioned medium.

 
The distinct phenotype of RA-induced EBs was also evident upon quantification of overall cell expansion. While cells proliferated at similar rates in treated and untreated EB cultures, the efficiency at which control EBs adhered to gelatin was significantly lower (Fig. 2A). In fact, adhesion of untreated EBs was highly variable both within and between experiments, whereas RA-treated EBs adhered uniformly to gelatin-coated plastic. Taking into account the gene expression results described above, this result is not surprising given the demonstrated role of p63 in regulating epithelial cell adhesion [19].


Figure 2
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  		Figure 2. Generation of keratinocytes from RA-treated/untreated EBs. (A): Normalized cell number of H9 human ESCs (hESCs) differentiated as EBs 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 cell populations. 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 of Oct-4+; **, p ≤ .001, UM versus UM + RA percentage of K14+. Error bars indicate SEM (n = 3). Statistical analysis was performed using Student's t test. (E–H): Phase contrast and immunocytochemistry of RA-treated H1 EBs differentiated for 20 days. (F): K14+ (red) cells migrating from a differentiated colony containing many p63+ (green) cells. (H): Two colonies of differentiated cells, one containing K14+ (red) cells and the other expressing nestin (green). Nuclei are blue. Scale bars = 50 µm. (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.

 
RA-Induced EBs Readily Differentiate into Keratinocytes
Basal cells of stratified squamous epithelia express K14, and cultivation of adherent RA-treated EBs in keratinocyte medium generated significant populations of K14+ cells, as determined by flow cytometry and immunostaining. The percentage of K14+ cells continued to rise for approximately 30 days after plating and was always significantly greater than that observed in untreated EB cultures (Fig. 2B). This enhancement in keratinocyte generation was observed in both H1 and H9 hESC lines, which produced similar quantities of K14+ cells after plating (Fig. 2C). RA-treated EBs formed monolayer colonies upon adhesion to gelatin, whereas untreated EBs attached as large cellular aggregates, often containing undifferentiated hESCs expressing Oct-4 (Fig. 2D). Immunocytochemistry demonstrated 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 as nestin-positive, indicating these cells were a common contaminating cell type in both RA cultures (Fig. 2H, 2K) and untreated controls (Fig. 2J).

Concentration and Temporal Dependence of RA Differentiation
In contrast with our results, various differentiation strategies have used RA to direct ESCs to neural rather than epithelial fates [16, 17, 20]. We therefore hypothesized that modulating the concentration or temporal presentation of RA might regulate epithelial lineage specification. EBs were induced with various RA concentrations (9 days) and subsequently cultured on gelatin in DSFM (19 days); differentiated cell populations were analyzed for K14 and nestin expression. Significant keratinocyte induction occurred only when EBs were cultured with 1 µM RA (Fig. 3A), whereas higher concentrations (10 µM) proved to be toxic to undifferentiated hESCs (not shown). We also observed a concomitant decrease in nestin+ populations as the RA concentration increased (Fig. 3B), decreasing from as high as 70% in control cultures to as low as 20% when EBs were treated with 1 µM RA.


Figure 3
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  		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 were significantly 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 was added to H9 hESCs during the EB phase (+/–) and/or the adherent culture phase (+/+, –/+) or not at all (–/–). (C): Flow cytometric analysis of 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 yield of K14+ cells (***, p < .001, +/– vs. +/+). (D): Nestin+ populations were reduced when RA was added during the EB phase, especially when RA application continued during adherent culture (*, p < .005, –/+ vs. +/+). (E): Quantitative PCR of cDNA from day 17 adherent cultures normalized to glyceraldehyde-3-phosphate dehydrogenase. Nestin expression was highest with late but not early RA application. RA decreased Sox1 expression at all times of application. {Delta}Np63 transcription was highest in cultures from EBs treated with RA, whereas K14 was expressed most in the early but not late RA exposure. Error bars indicate SEM (n = 3). Statistical analysis was performed using Student's t test. Abbreviations: K14, keratin 14; RA, retinoic acid.

 
To understand the temporal effects of RA-induced differentiation, we added RA to hESC cultures at various stages of differentiation (during either EB induction or adherent culture in DSFM). RA exposure for 4–8 days during the EB phase, but not after plating on gelatin, generated substantial numbers of K14+ cells. Addition of RA during adherent culture 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 RA enriched nestin+ populations, as evidenced by flow cytometry (Fig. 3D) and qPCR analysis (Fig. 3E). Cultures that were never exposed to RA expressed the highest levels of the early neural marker Sox1. RA therefore exhibited differential effects on cell phenotype depending on the stage of application. Although nestin expression alone is not sufficient to designate neural precursor populations, these findings provide indirect evidence for the importance of temporal RA application with respect to neural versus epithelial specification. In fact, Pankratz et al. made similar observations in their study of neural specification of hESCs, as early RA application generated unidentified non-neural lineages, and caudalization with RA could only be accomplished after first obtaining primitive anterior neuroepithelia [18]. However, more extensive studies using neurogenic substrates and culture media must be completed to definitively identify whether neural precursor populations are enriched in these control cultures.

Differentiation of hESCs in Defined Media
EB differentiation in the experiments described above used medium containing undefined components, including Knockout Serum Replacer, which can activate the BMP signaling pathway [21]. To better understand the mechanisms involved in keratinocyte differentiation, we identified a defined, N2 supplement-containing medium, which permitted keratinocyte generation to a similar extent as UM. As before, RA enhanced epithelial gene expression ({Delta}Np63 and K18) and reduced 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 early neural differentiation or directed hESCs to more terminal neural fates, we analyzed differentiated cell populations at various times for transcription of neuronal (Synaptophysin) and astrocytic (glial fibrillary acidic protein [GFAP]) markers. Throughout differentiation, Synaptophysin expression in cultures derived from RA-treated EBs was similar to or less than that observed in control cultures (Fig. 4A). GFAP expression was undetectable via RT-PCR (Fig. 4A) and immunocytochemistry (not shown) as well. Although the lack of definitive neural markers expressed in our system may have resulted from our use of epithelial substrates and media, these results provide evidence that early RA application does not reduce subsequent nestin+ populations by inducing neural precursors to terminally differentiate.


Figure 4
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  		Figure 4. Epithelial differentiation of human ESCs (hESCs) in defined medium. (A): Reverse transcription-polymerase chain reaction (PCR) analysis of 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 human keratinocytes. (B, C): Quantitative PCR analysis of K18 and nestin mRNA expression from day 6 EBs cultured in N2. (D): Flow cytometric analysis of K14+ cell populations (day 27) derived from H1 EBs differentiated in UM or N2 +/– RA (*, p < .02, RA+ vs. RA–). (E): Normalized cell number of H9 hESCs differentiated as EBs for 6 days in N2 or N2 + RA and subsequently cultured in keratinocyte medium. (F): Flow cytometric analysis of the percentage of K14+ cells corresponding to various points in (E) (*, p < .0005, RA+ vs. RA–). Error bars indicate SEM. Statistical analysis was performed using Student's t test. (G, H): Day 27 keratinocyte cultures derived from EBs cultured in N2 (G) or N2 + RA (H) stained for βIII-tubulin (red) and K14 (green). Nuclei are blue. Scale bars = 100 µm. 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.

 
Cultivation of differentiated EBs in DSFM resulted in keratinocyte induction similar to that observed using UM +/– RA (Fig. 4D). In addition, cells derived from RA-treated EBs in N2 medium proliferated well after plating in DSFM (Fig. 4E), with the percentage of K14+ cells increasing steadily with time (Fig. 4F) as well. In contrast to EBs differentiated in UM, hESCs differentiated in N2 and DSFM for extended periods (30 days) generated occasional βIII-tubulin+ neurons within large, EB-derived colonies. Neuronal differentiation was observed sporadically from both control EBs (Fig. 4G) and RA-treated EBs (Fig. 4H) at similar frequencies. Given that UM has previously been shown to activate the BMP signaling pathway [21] and that BMP activity can block early neural differentiation during development [12], the appearance of neurons in N2 differentiated EBs provided evidence for a role of BMP signaling in the differentiation process.

Direct Differentiation in Two-Dimensional Culture
To provide a more uniform microenvironment during differentiation we also eliminated EB formation and directly induced differentiation of hESC colonies cultivated on Matrigel (supplemental online Fig. 1B). After 6 days of treatment with RA or carrier, epithelial proteins such as K18 and p63 were detected at elevated levels in RA-treated cells (Fig. 5A). Interestingly, N2-supplemented medium promoted greater differentiation than unconditioned hESC medium, presumably because of the absence of Knockout Serum Replacer, which can induce transforming growth factor-β/Activin/Nodal signaling. Activation of this pathway has been shown to maintain hESC self-renewal [21] and block neuroectodermal differentiation [22]. Subculture of differentiated colonies in keratinocyte medium yielded K14+ populations of higher purity than the EB process. After 5 weeks of differentiation, 87% of cells expressed K14 using the direct method versus only 15% K14+ cells in the EB method, demonstrating a significant enhancement in keratinocyte differentiation (Fig. 5B, 5C). Furthermore, keratinocyte differentiation results from H1 and H9 hESC lines were similar using this process (supplemental online Fig. 2). The uniformity and defined conditions provided by the above modifications then permitted us to conduct a more detailed analysis of the signaling pathways involved in epithelial specification.


Figure 5
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  		Figure 5. Synergism of RA and BMP signaling during directed differentiation of human ESCs (hESCs). (A): SDS-polyacrylamide gel electrophoresis of protein extracts from H9 hESCs differentiated for 6 days in various media. RA induces p63 and K18 expression while downregulating Oct-4. (B, C): Flow cytometry histograms of K14+ populations derived using EB differentiation (B) or direct differentiation (C) methods for H9 differentiation. (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 and RA both reduced Sox1 expression, although Noggin mitigated the effects of both factors. RA and BMP-4 synergistically induced expression of {Delta}Np63 and K18 transcription. (E): Flow cytometric analysis of K14+ populations in differentiated H9 hESCs subcultured for 18 days. Inhibition of BMP signaling by Noggin significantly reduced keratinocyte generation; *, p < .05, Noggin only versus negative or BMP-4 only. Addition of RA and BMP-4 significantly enhanced keratinocyte generation; **, p < .02, RA only versus all others except +/+/+; ***, p < .001 BMP-4+RA versus all others 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 RA repressed neural differentiation; **, p < .02 versus all except negative and +/+/+. Error bars indicate SEM (n = 3). Statistical analysis was performed using Student's t test. Similar trends were observed in at least three separate experiments. Abbreviations: BMP, bone morphogenetic protein 4; DMSO, dimethyl sulfoxide; K14, keratin 14; K18, keratin 18; N2, N2 medium; RA, retinoic acid; UM, unconditioned medium.

 
Molecular Mechanisms of Epithelial Differentiation from hESCs
Previous studies of mESCs [9] and Xenopus [11] identified BMP signaling as a key inducer of epidermal fates during development. To elucidate the molecular mechanisms of epithelial lineage specification in hESC cultures, we directly differentiated colonies in N2 medium containing RA, BMP-4, or the BMP inhibitor Noggin. RNA was isolated from cultures differentiated for 6 days and analyzed for expression of epithelial and neuroectodermal transcripts (Fig. 5D). {Delta}Np63 transcription was elevated in all RA-treated cell samples, regardless of the status of BMP signaling, whereas BMP-4 and RA synergistically induced K18 expression. Neuroepithelial gene expression (Pax6, Sox1) was effectively downregulated in RA-treated cultures, although this effect was mitigated by inhibition of BMP signaling.

Subsequent cultivation of differentiated hESCs in keratinocyte medium further demonstrated that RA-mediated differentiation outcomes were dependent upon the status of BMP signaling. Addition of BMP-4 to RA+/– cultures had only a slight effect on keratinocyte yields, whereas inhibition of BMP signaling by Noggin significantly decreased epithelial differentiation (Fig. 5E). On the other hand, early treatment with RA in the presence of endogenous or exogenous levels of BMPs eliminated virtually all nestin+ cells detected in day 18 subcultures, whereas Noggin-mediated inhibition of BMP signaling generated large populations of nestin+ cells (Fig. 5F). Similar trends were observed in RA-treated EBs upon treatment with BMP-4 and Noggin, although the level of keratinocyte induction was lower compared with direct differentiation (supplemental online Fig. 3). Taken together, these data demonstrate that BMP and RA signaling play synergistic but distinct roles in hESC ectodermal differentiation. Active BMP signaling (endogenous or exogenous) reduced presumptive neural differentiation, as evidenced by relative Sox1 expression levels and subsequent nestin+ cell populations. Under these conditions, RA effectively induced keratinocyte differentiation (K14+ populations). However, Noggin treatment alone significantly reduced keratinocyte differentiation, regardless of the whether RA was administered during early differentiation. Inhibition of BMP signaling presumably resulted in survival of highly proliferative nestin+ cells, ultimately decreasing K14+ populations in our differentiated cultures.

Terminal Differentiation of hESC-Derived Keratinocytes
hESC-derived keratinocytes could be subcultured onto gelatin or collagen IV substrates, and these secondary cultures yielded relatively pure keratinocyte populations (K14+) compared with primary foreskin keratinocytes. Flow cytometry analysis of K14 expression measured 90% K14+ cells in a feeder-free primary culture (Fig. 6A), whereas hESC-derived keratinocytes contained 96% K14+ cells (Fig. 6B). This result was verified with two anti-K14 antibodies (not shown) and qualitatively via immunocytochemistry (Fig. 6C). As has previously been described [3], these epithelial cultures exhibited a lower expansion potential than primary cultures; cryopreserved hESC-derived keratinocytes could be propagated for approximately 10 population doublings (Fig. 6D). However, we were able to obtain colony-forming keratinocytes at a frequency of at least 0.003 from differentiated hESCs plated, a sixfold increase over previously reported methods that used implantation into immunocompromised mice [3]. It must also be noted that our growth analysis used feeder-free culture in serum-free medium, and it is unclear how these results might translate to more routinely used protocols.


Figure 6
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  		Figure 6. Retinoic acid-mediated H9 human ESC (hESC) differentiation yields relatively pure keratinocyte cultures capable of responding to terminal differentiation stimuli. (A, B): Flow cytometric histograms of K14+ expression of primary foreskin keratinocyte (A) and H9 hESC-derived keratinocyte (B) cultures. (C): Immunocytochemistry of K14 (red) and p63 (green) in a confluent subculture of hESC-derived keratinocytes. (D): Subculture of cryopreserved hESC-derived keratinocytes over multiple passages. Approximately 10 population doublings were observed before cells became senescent. (E, F): Extended culture at confluence induced terminal differentiation 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) and K10 (green) expression were also localized to stratified regions. (G): K14 (red) and K18 (green) staining of a subculture with nuclear Hoechst stain (blue). (H): K14 (red) and K3/K12 (green) staining of a confluent culture with nuclear Hoechst stain (blue). Yellow denotes green/red colocalization. Scale bars = 50 µm. (I): Human embryonic stem cell-derived keratinocytes were cultured on a collagen IV-coated dish in defined keratinocyte serum-free medium containing 1 mM Ca2+ for 8 days. After Dispase treatment for 1 hour, an intact epithelial sheet detached from the surface. Abbreviation: hESC, human ESC.

 
We also examined the capacity of hESC-derived keratinocytes produced via RA induction to express terminal differentiation markers in high-density and Ca2+-containing cultures. Small areas of stratification appeared in extended differentiation cultures with or without Ca2+; the top layers expressed filaggrin (Fig. 6E), involucrin (Fig. 6F), and, in some cases, the epidermal-specific keratin K10 (Fig. 6F). Interestingly, we also observed some remaining cells that expressed K18 (Fig. 6G) and many cells positive for the corneal epithelial marker K3/K12 (Fig. 6H). Others have demonstrated generation of corneal epithelial-like cells from hESCs using limbal fibroblast-conditioned medium [23] rather than the defined conditions described here, so it is possible that retinoid secretion mediated their observed conditioning effects. In addition, confluent keratinocyte cultures in Ca2+-containing, defined keratinocyte medium could be detached from the substrate as an intact epithelial sheet using Dispase, contracting accordingly (Fig. 6I). RA-induced hESCs produced relatively pure keratinocyte cultures that expressed terminal differentiation markers and formed coherent epithelial sheets under the appropriate conditions; the high-efficiency differentiation process coupled with the self-renewal capacity of undifferentiated hESCs provides a means of generating large quantities of nontransformed keratinocytes.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
A common challenge in hESC biology is the identification of efficient differentiation processes to generate functional somatic cell lineages. Various growth factors and signaling molecules induce hESC differentiation. However, the specific response depends upon the time, concentration, and combination of applied factors, and pure populations of lineage-specific progenitors are difficult to obtain [24]. RA signaling is involved in many developmental processes during embryogenesis, including pattern formation and limb development [25, 26]. In addition, RA is commonly used as a caudalizing agent to specify motor neuron differentiation from ES cells; however, these protocols require establishment of neuroectodermal fates prior to RA administration [16, 17]. Here, we describe a novel, stage-specific effect of RA on early hESC differentiation, in which ectodermal derivatives acquire epithelial fates in response to RA.

RA mediates cellular responses by binding to nuclear retinoic acid receptors (RARs), which in turn modulate transcription through several pathways. Undifferentiated hESCs, which express RAR{alpha} [27], initiated p63 transcription within 48 hours of RA administration, with {Delta}Np63 isoforms expressed at the highest levels. Mice expressing dominant-negative forms of RAR{alpha} under the K14 promoter exhibit reduced p63 expression and severe defects similar to those observed in p63 knockouts [28, 29]. Furthermore, RA has been shown to induce {Delta}Np63 expression in RAR{alpha}{gamma}-null keratinocytes in the presence of elevated RAR{alpha} levels (but not RAR{gamma}) [28], so RA may regulate p63 transcription, directly or indirectly, in hESCs because of differential expression of RAR subtypes. Given its demonstrated role in epithelial commitment [5, 7, 10, 30], cell adhesion [19], and differentiation [31], p63 is likely a primary driver of the epithelial cell enrichment observed in our process.

Modulation of {Delta}Np63 expression in zebrafish embryos demonstrated its role in early dorsal/ventral ectodermal patterning as well as neural/epidermal specification [10, 30]. {Delta}Np63 was identified as a target of Smad-mediated BMP signaling, and its overexpression in embryos lacking BMP activity rescued non-neural ectoderm formation, implying that p63 may mediate the neural repression described in the neural default model [10]. In our system, we observed an early RA-induced upregulation of {Delta}Np63 transcription and subsequent reduction in Pax6 and Sox1 expression. Pax6 and Sox1 have recently been described as early markers of primitive anterior and definitive neuroepithelia, respectively, in hESCs [18]. That study used RA as a caudalizing agent to induce formation of posterior neuroepithelia; interestingly, the authors found that early RA application induced unidentified non-neural cell fates [18]. It is therefore conceivable that these unidentified cells were p63+ primitive epithelia capable of differentiating into keratinocytes. Recently, BMP-4 has been shown to induce keratinocyte differentiation of mESCs [9] as well as induce apoptosis of neural precursors through the Smad pathway [12]. We observed a further decrease in neural gene expression when BMP-4 was added to cultures during RA treatment. Conversely, the BMP antagonist Noggin was able to mitigate RA-mediated repression of neuroepithelial gene transcription and increase the percentage of nestin+ cells in subcultures grown in keratinocyte medium. This finding highlights the distinct roles of RA and BMP signaling in epithelial differentiation, with BMP acting to block neural differentiation and RA directing cells to an epithelial fate (presumably through induction of {Delta}Np63 expression).

RA mediation of p63 expression may also be of clinical relevance, as vitamin A and other retinoids are known to be teratogens. High vitamin A intake can induce musculoskeletal and cranial-neural crest defects in particular, with an increased prevalence when taken shortly before or after conception [32]. Heterozygous mutations in p63 are a primary cause of ectrodactyly, ectodermal dysplasia, cleft lip/palate syndrome, in which patients exhibit congenital craniofacial, limb, and other abnormalities [33]. Given the similarity in developmental abnormalities associated with retinoid-induced birth defects and p63 mutations, it is conceivable that RA-mediated dysregulation of p63 expression could be involved in the teratogenicity of retinoids. However, more detailed biochemical studies are necessary to definitively understand how RA regulates p63 expression, as no RA response elements have been identified in the p63 promoter [28].

In addition to providing insight into the molecular mechanisms of epidermal commitment, RA-induced differentiation of hESCs offers an efficient means of generating keratinocytes. Previous studies have demonstrated that spontaneous keratinocyte differentiation in EBs occurs inefficiently [2, 4]. These hESC derivatives exhibited a lower proliferative capacity compared with primary keratinocytes, and immortalization was required to extend the lifespan of these cultures [3]. Although RA-induced hESC cultures produced keratinocytes with a replicative potential similar to that of controls, the high efficiency of this process provides an alternative to transformation, which can interfere with biological functions and complicates clinical use. Furthermore, application of RA rapidly eliminated Oct-4+ undifferentiated hESCs from culture and allowed for differentiation in defined medium, 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 basic research applications [35]; presumably, keratinocyte differentiation can be accomplished using genetically modified, clonally derived hESCs, allowing for the generation of nontransformed human keratinocytes and/or skin constructs with specific genetic backgrounds.

Although hESC-derived keratinocytes can express terminal differentiation markers and form epithelial sheets, the "burden of proof" regarding in vivo functionality must still be met. Organotypic culture of keratinocytes at an air-to-liquid interface provides a better means of analyzing terminal differentiation, but long-term engraftment studies in the proper niche must be conducted to gauge the self-renewal capacity of these cells after transplantation. Epithelial grafting may also be informative, as embryonic epithelia (or ES cell-derived epithelia) may retain greater plasticity in response to tissue-specific mesenchyme than adult cells [36].

Here, we have demonstrated a novel, developmental stage-specific effect of RA on hESC differentiation to the keratinocyte lineage using two distinct cell lines. Although recent evidence suggests that the propensity of individual cell lines to differentiate toward certain lineages may differ [37, 38], it seems unlikely that the signaling mechanisms regulating differentiation to particular lineages are different. Thus, our findings in two cell hESC lines should be applicable to other lines, although relative levels of differentiation may vary. Although the pleiotropic nature of RA signaling complicates the identification of specific mechanisms, the induction of p63 expression and dependence on BMP signaling provide insight into the molecular mechanisms of human epithelial development. Finally, this process provides an effective means of generating large quantities of keratinocytes from hESCs under feeder-free, defined conditions.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We acknowledge Kathy Schell and staff at the University of Wisconsin Comprehensive Cancer Center Flow Cytometry facility for technical assistance. Funding for this research was provided by the National Science Foundation-sponsored University of Wisconsin Materials Research Science and Engineering Center and the NIH-funded University of Wisconsin Biotechnology Training Program (to C.M.M.). C.M.M. and L.J. contributed equally to this work.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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