Generation of hiPSCs from dermal fibroblasts

We generated hiPSCs from freshly isolated adult dermal fibroblasts using OCT3/4, SOX2 and KLF4 as previously described13,14,15,16. hiPSC clones exhibiting characteristic human embryonic stem cell (hESC) morphology were isolated ~45 days after transduction (Supplementary Fig. 1a). Similar to the H9 hESCs, our hiPSC lines showed high levels of alkaline phosphatase (also known as TRA2-49-6E) activity (Supplementary Fig. 1b) and expressed multiple pluripotency markers14,16, including nuclear transcription factors POU class 5 homeobox 1 (OCT3/4) and nanog homeobox (NANOG) as well as surface antigens SSEA3, SSEA4 and TRA-1-60 (Supplementary Fig. 1c). While a series of endogenous stemness genes, including OCT3/4, NANOG, SRY (sex determining region Y)-box 2 (SOX2), REX1 and telomerase reverse transcriptase (TERT), were activated in hiPSCs, as revealed by quantitative real-time PCR (qPCR) (Supplementary Fig. 2), and the three retroviral transgenes were silenced (Supplementary Fig. 3). Compared with the parental fibroblasts, hiPSCs displayed extensive demethylation of CpG dinucleotides in the OCT3/4 and NANOG promoters, as shown by bisulphite sequencing (Supplementary Fig. 4a). We also found that histone H3 lysine 4 was methylated and histone H3 was acetylated in the promoter regions of OCT3/4 and NANOG in hiPSCs (Supplementary Fig. 4b). Pluripotency of the hiPSC clones was confirmed in teratoma formation assays after injection of undifferentiated hiPSCs into immunocompromised NSG mice (Supplementary Fig. 5).

Generation of iPSC-derived EpSCs

CD200 and ITGA6 are known surface markers for hEpSCs within hair follicles8. To generate folliculogenic hEpSCs from hiPSCs, we first followed the prior keratinocyte differentiation protocols9,10,11,17. We monitored the temporal expression of CD200 and ITGA6 in differentiating hiPSCs using flow cytometric analysis and found that only a small percentage of cells expressing both CD200 and ITGA6 emerged after 11, 18 of 25 days of differentiation using these protocols (Fig. 1a,b). To generate a sufficient number of hEpSCs from hiPSCs, we tried different methods and found that timing of EGF in the culture medium was critical (Fig. 1b,c and Supplementary Fig. 6). Based on these results, we established a new sequential differentiation protocol that used retinoic acid to induce hiPSC to form ectodermal like cells (stage 1), which were then differentiated to form hEpSCs in the presence of BMP4 and EGF (stage 2), followed by the final expansion of the mature keratinocyte lineages in the presence of EGF alone (stage 3, Fig. 2a). The morphologies of the expected cell types at stages 2 and 3 were shown in Fig. 2b. Using this stage-defined differentiation protocol, we recapitulated the dynamic differentiation process from pluripotent stem cells to EpSCs and then to mature keratinocytes; thus capturing the EpSCs, a transient multipotent stem cell population in vitro. Using the sequential differentiation protocol, the CD200+/ITGA6+ cells emerged around day 11 after differentiation (Fig. 2f and Supplementary Fig. 6), and reached a maximum level of 26.8±3.0% around day 18 after differentiation (Fig. 2c,f and Supplementary Fig. 6). The appearance of KRT15-positive (KRT15+) cells, which reached 29.4%±4.0 by day 18 (Fig. 1d,g and Supplementary Fig. 7), closely paralleled the appearance of the CD200+/ITGA6+ population. We further analysed the percentage of CD200+/KRT15+, CD200+/keratin 14 (KRT14)-positive (KRT14+), KRT15+/ITGA6+, KRT14+/ITGA6+ and KRT15+/KRT14+ cell populations at day 18. The results demonstrated that these populations did not overlap completely (Supplementary Fig. 8). We analysed the percentage of KRT15+ and ITGB1+ expression among the CD200+/ITGA6+ cells. Similar to a prior study18, we found that only ~50% of CD200+/ITGA6+ cells expressed KRT15 (Supplementary Fig. 9). As expected from our prior experience with human EpSCs derived from hair follicles, the CD200+/ITGA6+ population was not stable in culture and their number decreased significantly after day 25 (Fig. 2f and Supplementary Fig. 6); whereas, KRT14+ mature keratinocytes increased steadily over time (Fig. 2e,h and Supplementary Fig. 10), indicating differentiation of hiPSC-derived CD200+/ITGA6+ cells to more mature keratinocytes. Correspondingly, flow cytometric analysis showed that the pluripotent stem cell (SSEA3+) population decreased progressively during differentiation (Supplementary Fig. 11).

Figure 1: Generation of human EpSCs from hiPSCs. (a) Flow cytometric ananlysis of the percentage of CD200+/ITGA6+ cell population at day 11, 18 and 25 after hiPSCs were induced with BMP4 and without EGF. (b) Flow cytometric analysis of the percentage of CD200+/ITGA6+ cell population at day 11, 18 and 25 after hiPSCs were induced with BMP4 and EGF, while EGF was added 1 day after induction with BMP4. (c) Flow cytometric analysis of the percentage of CD200+/ITGA6+ cell population at day 11, 18 and 25 after hiPSCs were induced with BMP4 and EGF, while EGF was added 2 days after induction with BMP4. Full size image

Figure 2: Staged differentiation of hiPSCs into human EpSCs. (a) An outline of the protocol used to differentiate hiPSCs to EpSCs and then mature keratinocytes. (b) Morphologies of hiPSCs, hiPSC-derived EpSCs (hiPSC-EpSCs, obtained at day 18 after differentiation) and hiPSC-derived mature keratinocytes (hiPSC-keratinocytes, obtained at day 45 after differentiation). Scale bar, 100 μm. (c–e) Flow cytometric analysis of CD200+/ITGA6+, KRT15+ and KRT14+ cells at day 0 and 18 during the differentiation. (f–h) Quantification of CD200+/ITGA6+, KRT15+ and KRT14+ cells by flow cytometric analysis. Data shown are mean±s.d. of cell percentage from three independent experiments. (i) qPCR analysis of OCT3/4, NANOG, KRT5, KRT8, KRT14, KRT15, LamB3, involucrin and filaggrin expression in hiPSC-derived cells at different stages of differentiation. Samples collected at day 0, day 11, day 18, day 25 and day 30 after differentiation were used for qRT–PCR analysis. Data shown are mean±s.d. of the expression from three independent experiments. Full size image

We also analysed the temporal gene expression profile of the differentiating cultures and the results demonstrated a step-wise progression from embryonic immature cells (OCT3/4+ and NANOG+) to epithelial lineages, characterized by keratin 5 (KRT5) and keratin 8 (KRT8) expression, around 11 days after induction (Fig. 2i and Supplementary Fig. 12). The expression of keratinocyte-specific genes in the differentiating culture derived from hiPSCs increased over time, with concomitant decrease in OCT3/4 and NANOG expression (Fig. 2i and Supplementary Fig. 12). Consistent with flow cytometric analysis results, the expression of KRT15, began around day 11, peaked around day 18 and then decreased significantly by day 30 (Fig. 2i and Supplementary Fig. 12).

Characterization of iPSC-derived EpSCs in vitro

EpSCs are known to have high colony-forming efficiency3. We compared the colony-forming efficiency of unfractionated cells, CD200+/ITGA6+ cells, CD200−/ITGA6+ cells and CD200+/ITGA6− cells derived from hiPSCs at day 18 after differentiation; mature keratinocytes were derived from hiPSCs and normal keratinocytes from adult skin as previously described3. We found that CD200+/ITGA6+ cells derived from hiPSCs had the highest colony-forming efficiency among all the epithelial cells, as demonstrated by the higher number and larger size of colonies, compared with the other epithelial cell populations, after 3 weeks in culture (Fig. 3a and Supplementary Fig. 13). Nevertheless, the unfractionated cells formed the most colonies among all the cells tested and hiPSC-derived CD200+/ITGA6− cells formed a similar number of colonies to hiPSC-derived CD200+/ITGA6+ cells (Supplementary Fig. 13). CD200 has been recently reported to be expressed in hiPSCs and hESCs19. Our data confirmed the expression of CD200 in hiPSCs, and we found its expression later retained in a subpopulation of ITGA6+ cells as hiPSCs differentiated. Pluripotent stem cell markers, such as OCT3/4, NANOG and REX1, were barely detected in cells isolated at day 18 from the CD200+/ITGA6+ population (Fig. 3b), confirming that the hiPSC-derived CD200+/ITGA6+ cells contain few undifferentiated hiPSCs. However, hiPSC-derived CD200+/ITGA6− cells retained expression of ESC markers, including OCT3/4 and NANOG (Fig. 3b) with little expression of keratinocyte markers (Fig. 3c). The colony morphology formed by CD200+/ITGA6− cells was distinctively different from the colony morphology formed by CD200+/ITGA6+ cells (Fig. 3d) and unfractionated cells formed colonies with variable morphologies, supporting that CD200+/ITGA6− cells are non-epithelial cells.

Figure 3: Colony formation capacity of hiPSC-derived EpSCs. (a) Colony formation assays. Representative dishes of hiPSC-derived mature keratinocytes (hiPSC-keratinocytes), hiPSC-derived CD200+/ITGA6+ cells (hiPSC-EpSCs), passaged normal keratinocytes from adult skin (passage 3) and hiPSC-derived CD200−/ITGA6+ cells (hiPSC-CD200−/ITGA6+), cultured for 3 weeks on 3T3 fibroblast feeder cells. The dishes were stained with H&E. Representative images from three independent experiments. (b) qPCR analysis of pluripotent stem cell markers in the hiPSC-derived CD200+/ITGA6+, CD200+/ITGA6− and CD200−/ITGA6+ cells. The pluripotent stem cell markers used are OCT3/4, NANOG and REX1. Data shown are mean±s.d. of the expression from three independent experiments. (c) qPCR analysis of the keratinocyte-specific genes in hiPSC-derived CD200+/ITGA6+ and CD200+/ITGA6− cells. Data shown are mean±s.d. of the expression from three independent experiments. (d) Morphology of colonies formed by CD200+/ITGA6+ and CD200+/ITGA6− cells. Scale bar in upper panel, 100 μm; scale bar in lower panel, 20 μm. Full size image

Next, transcriptional analysis by qPCR showed that EpSC-specific network genes such as LGR5 (ref. 20), LGR6 (ref. 21), FZD2, TCF4, DKK3, CTNNB1, LEF1 and LHX2 (ref. 22) were activated in CD200+/ITGA6+ cells derived from hiPSCs and the expression levels were similar to hair follicles EpSCs (Fig. 4a). We also analysed these EpSC markers in iPSC-derived KRT14+/KRT15+/ITGA6+ cells, and the results showed that the levels of EpSC marker expression in these cells were similar to CD200+/ITGA6+ cells (Supplementary Fig. 14). Immunocytochemical staining analysis further confirmed the expression of KRT1, KRT10, KRT15 and ITGB1 expression in the hiPSC-derived CD200+/ITGA6+ cells (Fig. 4b–e), respectively; short-term culture of CD200+/ITGA6+ EpSCs isolated from human hair follicles was used as a control. To further compare the gene expression signature of EpSCs derived from hiPSCs to EpSCs derived from human hair follicles, we analysed global gene expression patterns of CD200+/ITGA6+ cells isolated from human fetal hair follicles, CD200+/ITGA6+ cells derived from hiPSCs and the parental hiPSCs. The results showed that hiPSC-derived CD200+/ITGA6+ cells clustered with CD200+/ITGA6+ cells isolated from fetal hair follicles, and were distinctively different from the parental hiPSCs as illustrated by unsupervised hierarchical clustering analysis (Fig. 4f,g). Notably, there was considerable overlap of genes between hiPSC-derived and hair follicle-derived CD200+/ITGA6+ cells (Fig. 4h), with many representative genes of epithelial lineages, such as KRT1, KRT8, KRT10, KRT15, ITGB1 and ITGA6 being highly enriched in the CD200+/ITGA6+ population compared with the parental hiPSCs and their expression levels were similar to EpSCs isolated from human hair follicles (Supplementary Fig. 15). Conversely, expression of the markers of ESCs, such as SOX2 and NANOG, decreased in the hiPSC-derived CD200+/ITGA6+ cell population (Fig. 4i). These findings indicate that the hiPSC-derived CD200+/ITGA6+ cells share similar molecular signatures with human EpSCs isolated from hair follicles.

Figure 4: Molecular characterization of hiPSC-derived EpSCs. (a) qPCR analysis of known EpSC markers, including LGR5, LGR6, CD200, KRT15, ITGA6, TCF4, FZD2, DKK3, CTNNB1, LEF1 and LHX2 in hiPSC-EpSCs compared with control CD200+/ITGA6+ cells isolated from fetal hair follicles (hEpSCs) and parental hiPSCs. Data shown are mean±s.d. of the expression from three independent experiments. (b–e) Immunocytochemical analysis of KRT15, KRT1, KRT10 and ITGB1 in hiPSC-EpSCs and hEpSCs culture. Secondary antibodies are conjugated with FITC. Antibody against ITGB1 is conjugated with PE. Scale bar, 50 μm. (f) Hierarchical clustering among the three cell populations analysed. (g) Heat map of genes differentially expressed in RNA-microarray analysis performed on hiPSCs, hiPSC-EpSCs and hEpSCs. (h,i) Scatter plots show that epithelial markers are expressed in hiPSC-EpSCs, whereas iPSCs markers are silenced. Full size image

Characterization of iPSC-derived EpSCs in vivo

EpSCs located in the bulge of the hair follicle have been documented to play a crucial role in hair follicle growth and cycling3,5. Although CD200 and ITGA6 are surface markers for human EpSCs8, it is unknown whether human CD200+/ITGA6+ cells isolated from adult scalp can form hair follicles in skin reconstitution assays. To determine whether hiPSC-derived EpSCs are multipotent and capable of generating all of the epithelial lineages within the skin, we first performed patch assays23,24 for skin reconstitution using day 18 hiPSC-derived CD200+/ITGA6+ cells, which were enriched by fluorescence-activated cell sorting (FACS) or magnetic bead selection. We found that the magnetic bead approach enabled isolation of large numbers of cells required for in vivo studies with minimal damage to the cells25. To minimize contamination of undifferentiated pluripotent stem cells and potential tumorigenesis26,27, we depleted potential remaining undifferentiated hiPSCs in the CD200+/ITGA6+ population using magnetic beads conjugated with antibody against the hiPSC membrane marker SSEA3. Neonatal foreskin keratinocytes were used as a positive control, whereas parental hiPSCs, hiPSC-derived CD200−/ITGA6+ cells at day 18, hiPSC-derived mature keratinocytes or mature keratinocytes isolated from hair bearing normal human skin were used as controls for comparison. The hiPSC-derived CD200+/ITGA6+ cells or control cells were combined with neonatal mouse dermal cells and injected subcutaneously into the back skin of immune-deficient nude mice. Two and half weeks later the skins of the mice were then excised and examined under a dissecting microscope. Hair follicle-like structures were observed from the underside of the skin (Fig. 5a and Supplementary Fig. 16). Histological analysis revealed that the injected epithelial cells aggregated to form small cystic spheres in the host subcutis. The cysts consisted of both basal keratinocytes and stratified epidermis (Fig. 5b). Hair follicles growing outward from the cyst were evident (Fig. 5b). This phenomenon was similar to what was observed previously in skin reconstitution assays using mouse EpSCs and neonatal dermal cells3,23,24. We also sorted out CD200+/ITGA6+ cells from day 11 and 25 cultures and these cells were also capable of forming hair follicles. The human origin of the epithelial cells in the new hair follicle structures and interfollicular epidermis was confirmed by DNA in situ hybridization with a human-specific Alu-repeat sequence probe (Fig. 5c). Double DNA in situ hybridization with human and mouse pan-centromeric probes further confirmed that the hair follicle epithelium and interfollicular epidermis were of human origin and the surrounding mesenchymal components were composed of mouse cells (Fig. 5d,e). The neonatal foreskin keratinocytes also formed hair follicles and we found that these cells contained distinct populations of CD200+/ITGA6+ or KRT15+ cells (Supplementary Fig. 17a). The qPCR results further confirmed that neonatal foreskin keratinocytes expressed EpSC markers but at a lower level compared with that of hiPSC-derived EpSCs (Supplementary Fig. 17b). The other controls including hiPSC-derived mature keratinocytes did not form any hair follicles and flow cytometric analysis showed that few CD200+/ITGA6+ or KRT15+ cells in the hiPSC-derived mature keratinocytes (Supplementary Fig. 17a). These data indicate that EpSCs are required for hair follicle formation.

Figure 5: Folliculogenic capacity of EpSCs derived from hiPSCs tested in two different types of reconstitution assays. (a) hiPSC-derived CD200+/ITGA6+/SSEA3− cells form hair follicles in a patch reconstitution assay. hiPSC-derived CD200+/ITGA6+/SSEA3− cells were combined with mouse neonatal dermal cells and injected into the dermis of an immunodeficient mouse. After 3 weeks, hair follicles and hair follicle-like structures were observed at the site of injection photographed from the underside of the skin. Dotted short lines outline a hair follicle. An arrowhead points to the pigmented bulb region of a hair follicle. Representative image from seven independent experiments. Scale bar, 500 μm. (b) H&E staining of an epidermal cyst with attached hair follicles formed from hiPSC-EpSCs. An arrowhead points to a hair follicle. Scale bar, 200 μm. (c) Human-specific Alu probe staining (green nuclei) confirms human origin of follicular epithelium and epidermal cyst lining, which were generated by hiPSC-derived EpSCs. An arrowhead points to a hair follicle. Scale bar, 200 μm. (d,e) In situ hybridization using pan-centromeric probes specific for human (red) and mouse (green) respectively show human origin of follicular epithelium (d) and epidermal lining (e). Scale bar, 30 μm. (f) Hair follicles form from hiPSC-derived CD200+/ITGA6+/SSEA3− cells and mixed with mouse neonatal dermal cells in a reconstitution assay using a silicone chamber. hiPSC-EpSCs and mouse neonatal dermal fibroblasts were mixed together and placed in a chamber transplanted onto the back skin of a nude mouse29. After 3 weeks, skin and hair follicles formed in the chamber. Pigmented human-like hair shafts were thicker than the surrounding mouse hair shafts. Arrowheads point to the pigmented hair shafts. Scale bar, 1 mm. (g) H&E staining of a reconstituted hair follicle (H&E stain). An arrowhead points to the hair shaft. Scale bar, 150 μm. (h) A human-like multilayered epidermis was formed (H&E stain). Scale bar, 100 μm. (i,j) Human-specific Alu probe staining of human hair follicles (i) and epidermis (j). Human cells were stained by human-specific Alu probe labelled with FITC. An arrowhead points to the hair shaft (i). Scale bar, 150 μm (i), 100 μm (j). Representative images from three independent experiments. Full size image

To further confirm the folliculogenic capacity of hiPSCs-derived EpSCs, we performed a chamber-based skin reconstitution assay28,29 using hiPSC-derived EpSCs and neonatal mouse dermal cells. The hiPSC-derived EpSCs were mixed with C57BL/6 neonatal dermal fibroblasts in a chamber and transplanted onto mouse back skin. Three weeks later hair follicle-like structures were observed in the grafts (Fig. 5f and Supplementary Fig. 16). Histological examination showed large human-like hair follicles with hair shafts, which were distinctively different from mouse hair shafts in the grafts (Fig. 5f,g). Human-like multilayered epidermis was also formed in the grafts (Fig. 5h). The newly formed epidermis was impermeable to toluidine blue dye, indicating a functional skin barrier (Supplementary Fig. 18). Human origin of the epithelial cells in the hair follicles and epidermis was confirmed by DNA in situ hybridization with a human-specific Alu-repeat sequence probe (Fig. 5i,j).

Next, we performed immunostaining to characterize the hair follicles and epidermis originating from hiPSC-derived EpSCs. We found that KRT15 was expressed in the bulge region of the chimeric hair follicles (Fig. 6a, right panel), similar to hair follicles formed by neonatal foreskin keratinocytes (Fig. 6a, left panel). Other keratinocyte markers, such as KRT14, were detected in the outer root sheath of the chimeric hair follicles (Fig. 6b). H&E staining showed that the innermost regions of the chimeric hair follicle structures resembled the hair cortex and the medulla of mature human hair follicles (Fig. 6c–e). The formation of such structures were further confirmed by immunohistochemical staining for AE13, a marker for hair follicle cortex, and AE15, a marker for inner root sheath and medulla, in a pattern that is similar to normal human hair follicles (Fig. 6c,d and Supplementary Fig. 19a,b). We also found K75 expressed specifically in the companion layer of the chimeric hair follicle (Fig. 6e and Supplementary Fig. 19c). The hiPSC-derived CD200+/ITGA6+ cells not only reconstituted the epithelial components of the hair follicle but also the interfollicular epidermis. The multilayered interfollicular epidermis expressed KRT10 and involucrin (Fig. 6f,g). Our findings indicate that hiPSC-derived CD200+/ITGA6+ cells are capable of generating functional epidermis and they respond to inductive dermal signals to generate the epithelial component of hair follicles.

Figure 6: Characterization of hair follicles and interfollicular epidermis formed by hiPSC-EpSCs. (a,b) Reconstitution of stem cell niche in hair follicles by EpSCs derived from hiPSCs. CD200+/ITGA6+/SSEA3− cells derived from hiPSCs were combined with mouse neonatal dermal cells and injected into the dermis of an immunodeficient mouse. Neonatal foreskin keratinocytes (Fk) were used as a positive control. Immunostaining of reconstituted hair follicles formed by neonatal foreskin keratinocytes or hiPSC-EpSCs was performed using antibodies against KRT15 (a) or KRT14 (b), respectively. Scale bar, 100 μm. hiPSC-hair follicle represents the hair follicle derived from hiPSC-EpSCs. FK hair follicle represents the hair follicle formed by neonatal foreskin keratinocytes. (c–e) Immunostaining of hair follicles formed by hiPSC-EpSCs using hair differentiation markers AE13 (c), AE15 (d) and K75 (e). Arrowheads point to the positive areas. Fetal scalp tissue was sectioned and stained with antibodies against AE13, AE15 and K75, respectively as positive controls. AE13 marks hair follicle cortex and AE15 marks inner root sheath and medulla. K75 marks companion layer of a hair follicle. Scale bar, 100 μm. (f,g) Immunostaining of interfollicular epidermal lining of cyst formed by hiPSC-EpSCs was performed using antibodies against KRT10 (f) or Involucrin (g). Scale bar, 50 μm. Full size image

Sebocytes and mature keratinocytes from iPSC-derived EpSCs

To further define the differentiation capacity of hiPSC-derived CD200+/ITGA6+ cells, we cultured these cells under sebocyte differentiation conditions as previous described30. Three weeks after differentiation, some of the cells acquired abundant cytoplasm with oil droplets that were positive for oil red staining (Supplementary Fig. 20a). The sebocyte-like cells also expressed sebocyte markers, such as keratin 7 (KRT 7), peroxisome proliferator-activated receptor alpha (PPAR-α or PPARA) and lipoprotein lipase (LPL), as shown by qPCR (Supplementary Fig. 20b). To the contrary, hiPSC-derived mature keratinocytes could not be differentiated into sebocytes under the same condition. These results indicate that hiPSC-derived CD200+/ITGA6+ cells are capable of sebocyte differentiation. In order to generate mature keratinocytes from hiPSC-derived CD200+/ITGA6+ cells, the hiPSC-derived EpSCs were cultured for an additional 25 days in the presence of EGF until the KRT14+ keratinocytes population reached 98.8% purity as shown by flow cytometric analysis, whereas CD200+/ITGA6− cells cultured under the same conditions resulted in generating only 18.0% KRT14+ keratinocytes (Fig. 7a). Transcriptional analysis by qPCR showed that KRT14+ keratinocytes derived from hiPSC expressed a panel of epidermal genes similar to those of normal skin keratinocytes (Fig. 7b). Immunostaining analysis showed that most cells expressed mature squamous cell markers, such as keratin proteins (KRT14), p63, integrins (ITGA6 and ITGB4), pan-cytokeratin and E-cadherin (also known as CDH1) (Fig. 7c–g). We next generated 3D skin equivalents using keratinocytes from foreskin and mature keratinocytes from hiPSC-derived CD200+/ITGA6+ cells. The 3D skin equivalents exhibited a multilayered epidermis, expressing squamous differentiation markers such as KRT14 and KRT5, and also epithelial markers Pan-cytokeratin (Pan-CK) (Fig. 7h), in a pattern similar to skin equivalents formed by foreskin keratinocytes. These data support that hiPSC-derived CD200+/ITGA6+ cells are multipotent. However, surprisingly, we were not able to identify human sebaceous glands derived from the hiPSC-derived CD200+/ITGA6+ cells or neonatal foreskin keratinocytes in the skin reconstitution assays. It is well known that mouse EpSCs (CD34+/ITGA6+ or KRT15high/ITGA6+) are capable of regenerating both follicular epithelium and sebaceous units in the skin reconstitute assays3,31. These results suggest that hiPSC-derived CD200+/ITGA6+ cells, although folliculogenic and sharing molecular signatures similar to those of human EpSCs, are likely more restricted in their lineage potential compared with EpSCs or they may require additional signals for sebocyte differentiation in vivo.