Dermal Papillae (DP) is a unique population of mesenchymal cells that was shown to regulate hair follicle formation and growth cycle. During development most DP cells are derived from mesoderm, however, functionally equivalent DP cells of cephalic hairs originate from Neural Crest (NC). Here we directed human embryonic stem cells (hESCs) to generate first NC cells and then hair-inducing DP-like cells in culture. We showed that hESC-derived DP-like cells (hESC-DPs) express markers typically found in adult human DP cells (e.g. p-75, nestin, versican, SMA, alkaline phosphatase) and are able to induce hair follicle formation when transplanted under the skin of immunodeficient NUDE mice. Engineered to express GFP, hESC-derived DP-like cells incorporate into DP of newly formed hair follicles and express appropriate markers. We demonstrated that BMP signaling is critical for hESC-DP derivation since BMP inhibitor dorsomorphin completely eliminated hair-inducing activity from hESC-DP cultures. DP cells were proposed as the cell-based treatment for hair loss diseases. Unfortunately human DP cells are not suitable for this purpose because they cannot be obtained in necessary amounts and rapidly loose their ability to induce hair follicle formation when cultured. In this context derivation of functional hESC-DP cells capable of inducing a robust hair growth for the first time shown here can become an important finding for the biomedical science.

Funding: This work was supported by funds from Sanford Burnham Medical Research Institute to AT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Gnedeva et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Neural crest (NC) is a cell population that transiently arises from the dorsal neural tube in development and gives rise to multiple tissues including the peripheral neural system, adrenal medulla, melanocytes and various craniofacial mesenchymal tissues [ 19 ]. NC-specific (Wnt1-Cre) lineage tracing using Lox-STOP-Rosa26 or Z/EG reporter mice provided the genetic evidence of NC contribution to a large proportion of cephalic DP cells [ 20 , 21 , 22 ]. Human embryonic stem cells (hESCs) have been directed to various cell fates including hair follicle epidermal cells—keratinocytes [ 23 ], however, the derivation of DP cells have not been reported. Here we describe for the first time the derivation of functional DP-like cells from human embryonic stem cells.

It has long been suggested that in embryogenesis hair follicles are formed by reciprocal interactions between the epidermis and underlying mesoderm [ 1 , 2 , 3 , 4 ]. Dermal Papillae (DP) first arise as cell condensates in the dermis in response to epidermal placode formation. As hair follicles progress in development, epidermal cells in placodes proliferate actively and envelope the dermal condensates, now called dermal papillae, separating them from surrounding dermis [ 5 ]. Exposed to these new niche conditions, DP cells acquire the expression of BMP-4, its inhibitor noggin, and the surface markers N-CAM and p-75. Additionally, they secret specific extracellular matrix proteins (e.g. versican (VCAN)) and show high level of alkaline phosphatase activity (AP) [ 6 ]. Using double reporter Lef1-RFP / K14-H2BGFP mice, more recent studies identified detailed genetic signature of prospectively isolated mouse DP cells [ 7 ] and identified Wnt, BMP and FGF singling pathway as key requirement for murine DP maintenance and function [ 8 , 9 , 10 ]. DP cells play a critical role in hair growth and cycling [ 6 ] and determine hair size and hair type [ 11 , 12 ]. It has been long recognized that DP cells are able to induce hair follicle formation not only in embryogenesis but also postnatal. Vibrissae DP cells induced de novo hair formation when transplanted into the footpad of the adult rat, which is normally a non-haired skin area [ 13 ]. Human DP cells isolated from scalp skin contribute to hair formation when transplanted into rodents [ 14 , 15 ] and induce keratinocytes morphogenesis in cultures [ 16 ]. However, extensive amplification of DP cells in culture is not feasible as they quickly lose the hair-inducing potential with passaging [ 7 , 8 , 17 , 18 ]. This represents a practical roadblock for the use of prospectively isolated human DP cells to develop a cell-based treatment for hair loss diseases.

Results

Derivation of hESC-DP using NC cells intermediate Since the prior genetic evidence of NC contribution to DP cells in vivo [21,22], we thought to obtain human DP-like cells from human ESC via the NC intermediate (Fig. 1A). Previously we have described efficient differentiation of human ESC into the multipotent NC cells [24,25]. Identical protocol was used here to generate human ESC-derived Neural Crest cells (hESC-NC). hESC-NC cultures showed robust expression of the neural crest markers Sox10 and Foxd3 (Fig. 1B). Flow cytometry analysis confirmed that nearly 80% of cultured hESC-NC cells express the NC marker Integrin alpha 4 (ITGA4), the cognate receptor for fibronectin [26], and lack the expression of OCT4 and SSEA4 suggesting the absence of undifferentiated hESCs (Fig. 1C). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Differentiation of hESCs into DP-like cells via NC intermediate. (A) Schematic representation of the differentiation strategy. (B) Expression of migratory NC markers Sox 10 and Foxd3, in hESC-NC cultures. Immunofluorescent staining, DAPI in blue. (C) Flow cytometry analysis of NC marker integrin alpha-4 (ITGA4) and hESC markers OCT4 and SSEA4 in hESC-NC cultures. (D) Expression of p-75, Nestin, Versican, SMA and Alkaline Phosphatase (AP) in hESC-NC, hDP (from normal skin) and hESC-DP cultures. Immunofluorescent staining, DAPI in blue. (E) Q-PCR analysis of hDP markers p-75, Nexin-1, Versican, SMA and Vimentin in hESC-DP cultures during DP patterning. Day 0 = hESC-NC cells. Levels of gene expression, shown as log of fold change over hESC-NC levels, were normalized to 18S. For each gene the dashed line represents levels of gene expression in hDP cell cultures. Scale bars 100 μm. https://doi.org/10.1371/journal.pone.0116892.g001 Neural crest is a multipotent population of cells that give rise to precursors for various mesenchymal tissues [19]. The FACS analysis showed that hESC-NC cells on 14 days of differentiation expressed mesenchymal stem cell markers CD47 (99.95%), CD184 (20%), and CD44 (52.58%) (S1 Fig.). To generate DP-like cells, we further differentiated hESC-NCs in DMEM-F12 medium containing 10% FBS for two additional weeks (Fig. 1A). It has been shown that DP cells are somatic dermal stem cells [27] and that they also express mesenchymal stem cell (MSC) markers [28]. Therefore, we enriched differentiating hESC-NCs cultures for mesenchymal cells using preferential adherence to tissue culture plastic, a known property of MSC [29]. Routinely, about 20% of hESC-NC cultures adhered to plastic and were passaged in serum containing media giving rise to hESC-derived DP-like cells (hESC-DP). These results suggest hESC-derived NC cells contain the mesenchymal progenitor population of cells that can be enriched using isolation protocol and culture conditions for MSC and DP cells.

Characterization of hESC-DP cells The signature genes of mouse dorsal skin DP cells have been compiled [7], however very little is know about the gene expression in human cephalic dermal papillae cells. In order to characterize mesenchymal hESC-DP we used the markers shown to be present in both mouse and human DP cells. In agreement with previously reported data [21], expression of the common for DP and neural crest markers p-75 and Nestin was detected in hESC-NC cells, cultured human DP cells (hDP) (isolated from normal human skin) and hESC-DP cells (Fig. 1D). The expression of the three other well established human DP markers Versican, Smooth Muscle Actin (SMA) and Alkaline Phosphatase (AP) was undetectable in hESC-NC cells but was present in hDP and hESC-DP cultures (Fig. 1D). The specificity of staining for Nestin, Versican, SMA and AP in hDP was confirmed using human scalp skin sections (S2 Fig.). The expression of NC markers P-75 (~30%) and Nestin (~90%) in hESC-NC was similar to that in hESC-DP cultures (p-75 ~40%, Nestin ~90%) and showed a close pattern of expression in hDP cells (P-75 ~20%, Nestin ~90%). In contrast, hESC-NC cells were negative for Versican (<1%), SMA (<3%) and completely lacking AP activity, whereas the majority of hESC-DP expressed Versican (~70%), SMA (~70%) and showed high level of AP activity similar to that found in human DP cells, which were nearly 100% positive for Versican, SMA and AP. The overall cell morphology and sub-cellular localization of markers were similar between the cultures of human DP cells and hESC-DPs. To quantitatively evaluate the expression dynamic of DP markers during differentiation of hESC-NC into hESC-DP we used Q-PCR analysis. We found that the expression levels of all tested human DP markers tested (p-75, Nexin-1, Versican, SMA, and Vimentin) progressively increased during hESC-NC differentiation and after two weeks were comparable or higher then that found in human-DP cell cultures (Fig. 1E). Taken together, these results suggest the presence of human DP-like cells within hESC-DP cultures.

Hair-inducing properties of hESC-DP upon transplantation Next, we investigated whether hESC-DP cells are competent to induce the formation of hair follicles upon transplantation in athymic nu/nu (Nude) mice. We took advantage of the patch method of cell transplantation previously used to demonstrate the hair-inducing potential of mouse skin-derived dermal precursors [27]. Briefly, cells of interest were combined with mouse epidermal cells (keratinocytes) isolated from the newborn animals and transplanted subcutaneously into the Nude mice as a thick cell suspension. Because Nude mice have the BALB/c (albino) genetic background, we were able to distinguish between newly formed and preexisting hairs by using the epidermal cells from dark haired C57BL/6 mice for transplantation. Hair-inducing capacity was measured as the number of hairs formed per transplant. Patch method does not allow newly formed hairs to enter the skin surface that perturbs hair follicle morphology on the advanced stages of morphogenesis. Therefore the analysis was carried out at 14 days post transplantation when hair follicles were formed, but not fully developed. Consistent with the previously published experiments, transplantation of epidermal cells alone resulted in minimal hair induction, likely due to the presence of endogenous DP cells (Fig. 2A, 2B). The mouse dermal cells (mDC), used as the positive control, induced robust hair growth (P = 0.0282) with efficiency similar to that reported previously for same transplantation model [27](Fig. 2A, 2B). As expected, cultured human DP cells isolated from adult scalp skin didn’t induce a significant number of hairs compare to the negative control (keratinocytes alone) (Fig. 2A, 2B). Indeed, human DP cells have been shown to contribute in trans-species reformation of single hairs [14] but the robust hair-inducing capability of human DP cells in the mouse model has not been reported [18]. In contrast, we observed significant (P = 0.0002) hair-induction by hESC-DPs similar to that of mDC (Fig. 2A, 2B). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Subcutaneous cell transplantations into Nude mice. (A) Stereo images of the whole mounts of keratinocytes transplanted alone or in combination with mouse neonatal Dermal Cells (mDC), hDP, hESC-DP, hESC differentiated in serum for 14 days (hESC-serum), human hESC-derived Neural Crest cells (hESC-NC) and hESC-NC differentiated to DP for 7 days (hESC-DP 7 days). (B) Quantification hairs induced by keratinocytes transplanted alone or in combination with mDC, hDP or hESC-DP. (C) Dynamics of hair inductive capability of ESC-DP cells with time of differentiation from hESC-NC (day 0) shown as number of hairs formed per transplantation (trend visualized by the red line) or hESC differentiated in presence of serum (blue diamond) in comparison with keratinocytes alone (visualized by the dashed line). All data are represented as mean ± SEM and were analyzed with one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison post test). *, P ≤ 0.05; **, P ≤ 0,001. Scale bars 1 mm. https://doi.org/10.1371/journal.pone.0116892.g002 To monitor the dynamics of hair inducing properties during the differentiation of hESCs towards DP-like cells we transplanted hESC-DPs and several related cell populations, namely, hESC differentiated for two weeks in DP medium skipping the intermediate step of NC induction (hESC), hESC-NC and partially differentiated hESC-DPs (7 days of differentiation) (Fig. 2C). Surprisingly, the transplantation of the hESC differentiated in serum conditions as well as hESC-NC resulted in significant inhibition of hair growth compared to negative control (Fig. 2C). Therefore, the differentiation of hESC-NC to hESC-DPs cells resulted in nearly 100-fold increase in hair-inducing ability (Fig. 2C). The number of hairs formed in partially differentiated hESC-NCs transplants was not significantly different than in the negative control (Fig. 2C). These results suggest that hESC-DP cells described here have a robust hair-inducing capacity similar to that of neonatal mouse dermal cells and that hESC-NCs acquire this capacity along the differentiation procedure.

hESC-DP incorporate into DP of de novo formed hair follicles Transplanted hESC-DP could either recruit / activate the endogenous mouse DP cells or directly mediate the observed induction of hair follicle formation. To address this question we engineered hESC-DPs to express GFP and analyzed newly formed hair follicles in situ (Fig. 3). Fourteen days after transplantation under the skin of nude mice using the patch method we observed a de novo hair formation that can be determined by the black pigmentation of the hair shafts. Stereoscopic observation of hESC-DP transplants, suggested that the majority of DPs and dermal capsules of the newly formed hairs were composed of GFP-positive cells (Fig. 3A). Confocal microscopy of the whole mount hairs isolated from hESC-DP transplants showed the presents of GFP-positive DP cells within newly induced hairs (Fig. 3C, 3D, 3E). We confirmed that GFP-positive DPs of these hairs stained positive for specific markers, namely Versican (Fig. 3C) and AP (Fig. 3D). In addition to GFP-positive DPs, we observed the presence of GFP-positive cells in the outer and the inner root sheaths area (Fig. 3E). We also observed the presence of melanin granules in the cytoplasm of GFP-positive cells in the hair matrix (Fig. 3F). Although further analysis is required to characterize the GFP-positive cells found in different compartments of hair follicles, these data suggest that transplanted hESC-DP can acquire the heir-inducing function of DP cells. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Subcutaneous transplantations of GFP-labeled hESC-DPs and hIPSC-DPs. (A) Stereoscopic observation of the whole mount transplants identified GFP-positive hESC-DP cells in positions of DP (arrows heads) and dermal capsule (arrows) in the newly formed hairs; insets show 2x enlargements of the DP regions. (B) GFP-labeled hIPSC-DPs can be found in DP and dermal capsule of the hairs: whole mount transplants (GFP/bright field) and 8um sections (bright field). Inset, fluorescence image of GFP-positive cells in the DP area of hair follicle (2x enlargement of the white square of DP area in the bright field image). (C, D) GFP-positive DPs of newly formed hairs (GFP/bright field, confocal microscopy) are positive for Versican (Versican, confocal microscopy) and Alkaline Phosphatase (AP, bright field)). (E) Rarely (~1% of newly formed hairs) NC-derived GFP-positive cells were detected in the outer root sheath area (arrows) as well as GFP-positive DP (outlined). Confocal image in the GFP panel. (F) NC-derived GFP-positive cells were found in hair matrix in transplants (confocal microscopy). Inset shows 2x enlargement of GFP-positive cells; GFP in white. Note multiple melanin granules (in black) present throughout GFP-positive cells. Scale bars 250μm for A; 50 μm for B-F. https://doi.org/10.1371/journal.pone.0116892.g003

Derivation and characterization of hIPSC-DP In addition to H9 line of human ESC we used 3 previously characterized human iPSC lines generated from normal human BJ fibroblasts [30]. The hIPSC-NC cells were generated following previously described protocol and analyzed for the presence of neuroephitelial markers Sox2, Sox9 and nestin. We observed that hIPSC-NC obtained from all three lines showed a similar pattern of expression. Only about 50% of hIPSC-NC cells expressed Sox2 and Sox9, additionally nestin staining revealed morphological differences when compared to hESC-NC cells (S4 Fig., Fig. 1D). hIPSC-NC cells were differentiated to obtain hIPSC-DP using the protocol described above. The immunostaining for DP markers SMA, p-75 and nestin as well as Q-PCR analysis of Versican, Nexin-1, p-75, Vimentin and SMA showed that only one IPSC line (BJ16) gave rise to cells with some expression of DP markers when compared to hESC-DP cells (S4 Fig. vs Fig. 1A, the levels of gene expression in both hIPSC-DP and hESC-DP are shown relative to hESC-NC cells). BJ16 IPSC-DP cells were further characterized by patch transplantation. This cell population did not induce significant number of hairs when compared to negative control (data is not shown). However, the transplantation of GFP-positive BJ16 IPSC-DP cells resulted in formation of hairs with GFP-positive dermal papillae and dermal capsules albeit with much lower frequencies (1 hair out of 50) then in case of hESC-DP cells. The presence of GFP-positive cells within DP of these hairs was confirmed in sections (Fig. 3B). Noteworthy, the integration of transplanted cells into the papillae and capsule area of newly formed hairs was observed only in the case of hESC-DP and hIPSC-DP cells. Although transplanted human DP cells engineered to express GFP were present in the dermis, these cells were never found in the DP of neighboring hair follicles (S3 Fig.). This results suggest that although human pluripotent cell-derived DP-like cells share the expression of some specific markers with DP cells isolated from adult human skin their hair-inducing capacity is higher that can enable the application of this cells for the cell-based treatment for hair loss diseases.