1α,25‐Dihydroxyvitamin D3 Modulates the Hair‐Inductive Capacity of Dermal Papilla Cells: Therapeutic Potential for Hair Regeneration

Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell‐based hair regrowth therapy. We previously reported that 1α,25‐dihydroxyvitamin D3 (VD3) upregulates expression of transforming growth factor (TGF)‐β2 and alkaline phosphatase (ALP) activity, both features of hair‐inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD3 actions on DPCs. VD3 suppressed hDPC proliferation in a dose‐dependent, noncytotoxic manner. Among the Wnt‐related genes investigated, Wnt10b expression was significantly upregulated by VD3 in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF‐β2, by VD3 was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all‐trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD3) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD3 upregulation of Wnt10b, ALPL, and TGF‐β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD3 significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD3 may promote functional differentiation of DPCs and be useful in preserving the hair follicle‐inductive capacity of cultured DPCs for hair regeneration therapies.


INTRODUCTION
Dermal papilla cells (DPCs) have follicle-inductive ability through interactions with neighboring epithelial components. Various trichogenic assays, using epithelial and mesenchymal cells, have indicated the therapeutic potential of folliculogenesis [1][2][3][4][5][6][7][8]. However, no successful clinical trials have yet been reported. One major reason for this is that human DPCs (hDPCs) lose their hairinductive capacity after long-term culture [5,9,10]. Optimization of culture media and related methods to efficiently expand hDPCs while maintaining their hair-inductive capacity has been a major challenge and is a prerequisite for future cell-based therapies for alopecia [11]. Findings reported in past literature suggest steps that need to be taken toward successful mitigation of the DPC expansion problem. One step is the use of conditioned medium obtained from epidermal keratinocyte culture (keratinocyte-condi-tioned medium [KCM]) [12]. KCM or coculture with epidermal keratinocytes has been also shown to stimulate proliferation of mesenchymal cells, including DPCs [12,13]. In addition, bone morphogenetic protein-6 [14] and basic fibroblast growth factor [15] were reported to aid in DPC expansion cultures for preserving the hair-inductive capacity.
The Wnt/␤-catenin signaling pathway leads to placode formation during embryogenesis [16] and activates downstream the transcription factor/lymphoid enhancer-binding factor 1 transcription complex. This complex is activated in both the epidermis and dermis during hair development [17]. Wnt-3a-treated DPCs have a higher capacity to induce hair formation in engraftment assays [18]. Although they were not used as an in vitro pretreatment for cultured DPCs, Wnt-7a and Wnt-10b have been shown to promote hair folliculogenesis in in vivo assays [19,20]. Other

TISSUE ENGINEERING AND REGENERATIVE MEDICINE
strategies to preserve the hair-inductive properties of DPCs include growing them in three-dimensional aggregates [15,21] and culturing them together with keratinocytes on extracellular matrix substrates [22] in order to mimic the in vivo microenvironment [11].
We previously examined the global gene signatures of hDPCs and human dermal fibroblasts (hDFs) by microarray analysis. This work revealed that transforming growth factor (TGF)-␤2, known to be required for folliculogenesis [23], was one of the genes specifically upregulated in hDPCs [24]. TGF-␤2 expression in hDPCs decreased after long-term culture but was elevated by KCM and was shown to correlate with the hair-inductive capacity of DPCs. We also found that among paracrine factors from keratinocytes, 1␣,25-dihydroxyvitamin D 3 (VD 3 ; also known as calcitriol) upregulated TGF-␤2 gene expression in hDPCs. VD 3 , likewise, upregulated alkaline phosphatase (ALP) activity [24], which is used as an index for the hair-inductive capacity of DPCs [14,25,26].
VD 3 , the most biologically active form of vitamin D, is a final synthetic product generated in kidney by 1␣-hydroxylase [27] and is systemically distributed in an endocrine manner (and thus sometimes classified as vitamin D hormone) [28,29]. Vitamin D is classically known to maintain calcium and bone homeostasis, but VD 3 also has been found to regulate, directly and/or indirectly, 0.8%-5% of all genes [30] and to be involved in various biological actions, such as apoptosis, suppression of cell growth, promotion of differentiation, and immunomodulation [28,31]. Recently, much attention has been paid to the preventive or regressive effects of VD 3 on cancers, including colorectal, breast, prostate, renal, ovarian, blood, and skin cancers [32,33].
Previous reports have described the effects of vitamin D on hair follicles. Topical pretreatment of VD 3 enhanced hair regrowth in a mouse model of chemotherapy-induced alopecia [34]. Nuclear vitamin D receptor (VDR)-null-mutant mice were reported to develop alopecia and poor whiskers, although they had normal hair until weaning after birth [35,36]. This suggests that such mice can develop a normal first coat of hair but cannot regulate postnatal hair cycles [30,37]. In humans, mutations in the VDR coding gene are known to cause hereditary vitamin Dresistant rickets with alopecia [38,39]. However, detailed effects of VD 3 on DPCs have not been elucidated. In this study, we focused on the biological effects of VD 3 on DPCs. Specifically, we tested whether VD 3 may be useful in preserving or promoting the hair-inductive capacity of DPCs in expansion cultures and, ultimately, in developing a reliable cell-based therapy for hair regeneration.

Human and Rat DPC and Dermal Fibroblast Cultures
Human scalp skin samples with hair and periauricular facial skin were obtained from patients undergoing facelift surgery. Informed consent was obtained from patients using a protocol approved by the ethical committee of the University of Tokyo School of Medicine. Dermal papillae were isolated from the hair follicles under a stereomicroscope using a needle and fine forceps. The isolated tissue was then attached to scratched scars made on a bottom of a plastic culture dish containing Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA, http:// www.invitrogen.com) supplemented with 10% fetal bovine serum (FBS). After 2 weeks of explant culture, hDPCs were subcul-tivated with the same medium, followed by cryopreservation at Ϫ80°C. DPCs at passages 3-4 were used for each experiment. For preparation of murine DPCs (mDPCs), whisker follicles of male Fisher 344 rats (6 weeks old) were used, and mDPCs were obtained with the same method as described for hDPCs.
For preparation of hDFs and murine dermal fibroblasts (mDFs), human facial skin and glabrous rat sole skin of F344 rats were excised with a 4-mm-diameter round scalpel. The round piece of skin was incubated in phosphate-buffered saline plus 1,000 U/ml dispase (Dispase II; EIDIA, Tokyo, Japan, http://www. eidia.co.jp/en) at 37°C for 20 minutes to separate the epidermis from the dermis. Then, the explant culture was performed by attaching the separated dermis to the bottom of a plastic culture dish. The remainder of the procedure was the same as described for hDPCs.

Real-Time Reverse Transcription-Polymerase Chain Reaction
After hDPCs were seeded at 3 ϫ 10 3 cells per cm 2 and cultured until subconfluence (5-7 days), total RNA was isolated from each sample using the RNeasy Mini Kit (Qiagen, Valencia, CA, http:// www.qiagen.com), followed by a reverse transcription reaction step. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed with the ABI 7700 sequence detection system and SYBR Green PCR master mix (both from Applied Biosystems, Foster City, CA, http://www. appliedbiosystems.com). Primer sequences used are shown in supplemental online Table 1. Expression levels were calculated by the comparative C T method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous reference gene, or are shown as a ratio compared with GAPDH.

Retroviral Short Hairpin RNA Against Endogenous VDR
Five sets of sense and antisense oligos targeting human VDR mRNA were designed and purchased from Operon Biotechnologies (Huntsville, AL, http://www.operon.com). Oligos with random sequences were used as negative controls. Each set of oligos was annealed to make double-stranded DNA and ligated into a multicloning site of the pSIREN retroQ ZsGreen vector (Clontech, Mountain View, CA, http://www.clontech.com). The sense sequences of six sets of oligos are shown in supplemental online Table 2. To prepare retroviral media, short hairpin RNA (shRNA) vectors were transfected into a platinum retrovirus expression system, amphotropic (Cell Biolabs, San Diego, CA, http://www. cellbiolabs.com), according to the manufacturer's instructions.
Human DPCs (passage 2) were infected in the viral media for 8 hours. After subcultivating, ZsGreen fluorescence-positive cells were sorted by a flow cytometric cell sorter (FACSAria; BD Biosciences) and further expanded for gene expression analyses.

Western Blotting
To examine which sequence interfered with mRNA most effectively, each shRNA vector and flag-tagged human VDR-expressing vector (pVDR; a kind gift from Dr. Eiji Ochiai, Teijin Pharma Ltd.) was cotransfected into the HEK293 cell line. Transfection efficiency of shRNA was evaluated by ZsGreen fluorescence. HEK293 cells were prepared in a 60-mm dish with DMEM/10% FBS and transiently transfected with both pVDR and shRNA vectors. Transfection was performed using Lipofectamine LTX and PLUS regent (Invitrogen, Carlsbad, CA, http://www.invitrogen. com). Three days after transfection, whole cell lysate was collected using radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com). Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, http://www.piercenet. com), and equal amounts of protein (10 g) were loaded into each sample lane of a sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The resolved proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, http://www.bio-rad.com), followed by immunostaining using rabbit anti-flag antibody (dilution: 1:1,000; Sigma-Aldrich) and horseradish peroxidase-conjugated goat anti-rabbit IgG (dilution 1:5,000; R&D Systems).

In Vivo Assay for De Novo Hair Folliculogenesis
All animal experiments were performed with approval from the Institutional Animal Care and Use Committee at the University of Tokyo. A transplantation method of DPCs termed the hemivascularized sandwich (HVS) method, which we recently reported [8], was used for de novo regeneration of hair follicles. Briefly, a circular split-thickness of rat's glabrous sole skin (8-week-old F344 rat) was cut off with a round scalpel and digested to separate the epidermis from the dermal compartment. The epidermis was replaced on the remaining deep dermis, in the donor hole, after a cultured DPC construct (a cell sheet fragment of rat DPCs) was grafted.
For preparation of the DPC construct, mDPCs (passage 2) derived from F344 rat whiskers were seeded at 4,000 cells per well in a 96-well plate and cultured in DMEM/10% FBS. After 7 days of culture, expanded mDPCs reached confluence. For preparation of the control construct, mDFs (passage 2) derived from F344 rat hairless sole skin were used. VD 3 (100 nM) was then supplemented for 0, 24, or 72 hours into the DPC culture before transplantation (n ϭ 8 for each group), whereas VD 3 was used for 0 or 72 hours in the dermal fibroblast (DF) culture. The mDPC and mDF sheets were scraped off the 96-well plate with a rubber scraper. One fragment of the mDPC or mDF sheet was used as a graft for the HVS method.

Histological Evaluation and Scoring of Regenerated Hair Follicles
Eight weeks after grafting the mDPC sheets or mDF sheets, rat sole skin was harvested and subjected to histological examination. Skin samples were embedded in O.C.T. compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan, http://www.sakura.com), frozen in liquid nitrogen, and cut into 10-m-thick sections. Every other section was processed for hematoxylin and eosin staining, and the remaining sections were stained with Hoechst 33342 (Dojindo). Stained sections were observed and photographed under a fluorescence microscope (BioZero; Keyence, Tokyo, Japan, http://www.keyence.com).
Hair follicle regeneration was evaluated histologically and classified into eight stages of developmental hair follicle maturation, according to a previously described method [40]. In addition, follicles were divided in each stage into regulated or dysregulated follicles, as we have previously reported [8] to classify regenerated follicles. In contrast to regenerated follicles, showing normal developmental stages, dysregulated regenerated follicles showed atypical morphology, including multiply fused follicles.

Statistical Analysis
Data were expressed as mean Ϯ SEM. The unpaired Student's t test was used to compare data between two groups. Comparisons of more than two groups were done by analysis of variance with the Bonferroni correction. A value of p Ͻ .05 was considered significant.

VD 3 Effects on Proliferation of Cultured hDPCs
During the 7 days of culture, the number of expanded hDPCs was smaller in VD 3 -supplemented media (1, 10, and 100 nM) than in the control media, and the difference was statistically significant on day 7 (Fig. 1A). BrdU incorporation assay confirmed the suppression of hDPC proliferation by VD 3 , with significant suppression observed at 10 and 100 nM (Fig. 1B). To assess whether the proliferation-suppressive effect of VD 3 resulted from a cytotoxic effect on hDPCs, immunocytochemical staining for apoptosis (Hoechstϩ/Annexinϩ/PIϪ) and necrosis (Hoechstϩ/Annexinϩ/PIϩ) was performed (Fig. 1C,  1D). Representative images of an apoptotic cell and a necrotic cell are shown in Figure 1C by the arrow and arrowhead, respectively. As expected, serum-free medium induced significantly higher percentages of apoptotic and necrotic cells compared with VD 3 -free serum-containing medium. Importantly, there were no significant differences among apoptotic (or necrotic) cells in VD 3 -free medium versus VD 3 -containing media, regardless of VD 3 concentration. Thus, the proliferation-suppressive effect of VD 3 did not result from a cytotoxic effects of VD 3 on hDPCs.

VD 3 Effects on Wnt-Related Gene Expression in Cultured hDPCs
We investigated VD 3 effects on expression of various Wnt-related genes (Wnt3a, Wnt5a, Wnt7a, Wnt10a, Wnt10b, E-cadherin, snail, and slug). Only Wnt10b was significantly upregulated by VD 3 treatment compared with the control treatment ( Fig. 2A). Kinetic analysis showed that Wnt10b mRNA was upregulated after 24 hours of culture (Fig. 2B) and in a dose-dependent manner up to 100 nM (Fig. 2C). Immunocytochemical staining after 3 days of treatment with VD 3 confirmed that Wnt10b protein expression in hDPCs was also upregulated in a dose-dependent manner (Fig. 2D).  VD 3 is catabolized into an inactive form, calcitric acid, by 24hydroxylase (24(OH)ase). The promoter of human 24(OH)ase has a vitamin D-responsive element (VDRE), and its expression is directly upregulated via the VDR pathway in an autocrine manner [27,30]. Through this negative feedback system, vitamin D limits its own biologic effects. In our study, we confirmed that gene expression of 24(OH)ase in hDPCs was upregulated exponentially by VD 3 during the 48 hours of observation (supplemental online Fig. 1A). Likewise, gene expression of ALPL and TGF-␤2, both related to the hair-inductive capacity of hDPCs [24], was upregulated by VD 3 treatment at 24 -48 hours (supplemental online Fig. 1B, 1C). This suggested that VD 3 indirectly upregulated ALPL and TGF-␤2 mRNA expression. We further examined whether the VD 3 -induced upregulation of ALPL and TGF-␤2 gene expression in hDPCs was mediated by Wnt signals. Inhibition of Wnt signals by Wif-1 did not affect the upregulation of ALPL and TGF-␤2 gene expression, suggesting that the effects of VD 3 are independent of Wnt signaling (supplemental online Fig. 2).

Comparison of VD 3 Effects on Wnt10b, ALPL, and TGF-␤2 Gene Expression Between hDPCs and hDFs
We further investigated the difference in the influential effects of VD 3 on the gene expression of Wnt10b, ALPL, and TGF-␤2 between hDPCs and hDFs. At baseline, Wnt10b, ALPL, and TGF-␤2 mRNAs were expressed in hDPCs, approximately 6, 350, and 15 times as high as in hDFs, respectively (Fig. 3). After a 48-hour incubation with VD 3 , the gene expression of Wnt10b, ALPL, and TGF-␤2 was significantly upregulated in hDPCs, whereas VD 3 upregulated only ALPL gene expression in hDFs. Thus, the effects of VD 3 on the gene expression of Wnt10b and TGF-␤2 appeared to be specific to hDPCs, and this fact may characterize hDPCs that have hair-inductive ability.

Influence of Paracrine or Endocrine Factors on Wnt10b mRNA Expression in hDPCs
To examine whether other skin-related paracrine or endocrine factors affect Wnt10b mRNA expression in hDPCs, 20 such factors were examined in culture, with or without serum, in an effort to find an optimized culture medium for DPC expansion. Results from serum-free medium cultures showed that VD 3 alone remarkably promoted Wnt10b mRNA expression at 48 hours of culture, whereas atRA upregulated its expression at 2 hours (Fig. 4A, 4B). Here, atRA may have directly, but temporarily, promoted Wnt10b. In serum-containing media, almost the same result was obtained at 2 hours of culture (Fig. 4C), whereas not only VD 3 but also atRA showed a promoting effect on Wnt10b mRNA expression at 48 hours (Fig. 4D). These results suggested that atRA and serum synergistically influence Wnt10b mRNA expression.

Combined Effects of VD 3 and atRA, KCZ, or TEI on Wnt10b mRNA Expression in hDPCs
We further examined the possible interactions between VD 3 and atRA, KCZ, or TEI, because these three agents were previously reported to interact with VD 3 . atRA significantly upregulated Wnt10b gene expression at 4 -6 hours but not at 48 hours (supplemental online Fig. 3A), whereas a synergistic enhancement with atRA and VD 3 (100 nM) was not clearly detected. KCZ neither affected Wnt10b mRNA expression in hDPCs nor suppressed VD 3 -induced upregulation of Wnt10b in vitro (supplemental online Fig. 3B), although it was previously reported that VD 3 signaling in hDPCs is stimulated by KCZ in vivo [41]. TEI is a vitamin D 3 analog that acts as an agonist of VDR-mediated genomic actions in rodent cells and as an antagonist of them in human cells [42,43]. The extent of the antagonism has been shown to depend on the cell taxonomy and culture conditions [44]. Our quantitative RT-PCR results using VD 3 and TEI showed that TEI neither enhanced nor

Knockdown Assay of VDR with Short Hairpin RNAs
To examine whether VD 3 -induced upregulation of Wnt10b, ALPL, and TGF-␤2 genes was mediated through a VDR-VDRE transduction pathway, five shRNA vectors for knocking down VDR were designed and prepared (supplemental online Table 1). Western blotting revealed that shRNAs 1, 3, 4, and 5 successfully suppressed human VDR protein expression, although vector 2 did not (Fig. 5A). Both shRNAs 1 and 3, as well as a negative control shRNA, were selected for further use. Since the transfection efficacy by lipofection was quite low in hDPCs (data not shown), retroviral infection was used for inserting shRNA vectors into hDPCs. After retroviral transfection, hDPCs expressing Zs-Green were sorted by flow cytometry, and infected cells were collected (supplemental online Fig. 4). Suppression of VDR-mediated signaling by shRNAs 1 and 3 was confirmed by quantitative RT-PCR for VDR-signal target gene 24(OH)ase (supplemental online Fig. 5).
Treatment with shRNA 1 significantly suppressed VD 3 -mediated upregulation of Wnt10b mRNA (Fig. 5B), which was previously observed at 48 hours (Fig. 2B). In addition, upregulated expression of ALPL and TGF-␤2 mRNA by VD 3 was also significantly suppressed by treatment with shRNA 1 (Fig. 5C,  5D). These results were also confirmed by independent assays using shRNA 3 (data not shown), suggesting that upregulated gene expression of Wnt10b, ALPL, and TGF-␤2 by VD 3 was mediated by a VDR genomic signaling pathway.

Transplantation of Cultured mDPCs or mDFs Pretreated with VD 3 for Hair Folliculogenesis in Mice
We further examined the therapeutic effect of VD 3 on the hair-inductive capacity of cultured mDPCs in vivo. Since VD 3 not only promoted expression of hair induction-related genes (supplemental online Figs. 1B, 1C, 2B) but also suppressed proliferation of DPCs (Fig. 1A, 1B), we performed VD 3 treatment after cultured DPCs reached confluence. A cell sheet of mDPCs was incubated with VD 3 (100 nM) for a short period of time (0, 24, or 72 hours) just before transplantation. As a control, a cell sheet of mDFs was incubated with VD 3 (100 nM) for 0 or 72 hours (Fig. 6). The cultured mDPC and mDF sheet fragments were grafted into rat sole skin using the HVS method [8]. In this method, the DPC construct is expected to interact with epithelial stem cells in the epidermis, harvested from a nonhairy area for neogenesis of hair follicles (Fig. 6A). Macroscopic and histological evaluations of regenerated hair follicles are summarized in supplemental online Table 3, and representative histological images are shown in Figure 7. Hair folliculogenesis was observed in all samples from the three groups of DPCs. In contrast, the DF constructs induced a very small number of hair follicles, and the number and maturity were not enhanced by VD 3 incubation time. High-stage hair development, such as stage 7 or 8, was seen in each group of DPCs, and a hair shaft and/or sebaceous glands were detected in well-matured follicles (Fig. 7). The total number of DPCinduced hair follicles increased with increased VD 3 incubation time, although the difference did not reach statistical significance (Fig. 6B, top left). Regenerated follicles of stages 6 -8 (matured follicles) from DPCs significantly increased in number when pretreated with VD 3 (Fig. 6B, top right), whereas DF constructs induced no matured hair follicles. Moreover, the number of outgrowing hairs, defining the ultimate maturation of the regenerated follicle, induced by DPCs was significantly greater in the 72-hour pretreatment group: nine regenerated hairs in a total of eight samples, versus four hairs in the 24hour pretreatment group and two hairs in the no-pretreatment group (Fig. 6B, bottom). These results suggested that VD 3 , which upregulates the gene expression of Wnt10b as well as ALPL and TGF-␤2 in DPCs, may be useful in expansion cultures of DPCs targeting a cell-based therapy for hair regeneration.

DISCUSSION
This study showed that VD 3 suppressed proliferation of hDPCs in a dose-dependent manner, without inducing cytotoxicity. Among various Wnt-related genes investigated, VD 3 significantly upregulated hDPC Wnt10b gene expression, in an indirect manner. VD 3 also significantly upregulated hDPC gene expression of two other folliculogenesis-related genes, ALPL and TGF-␤2. The dermal papilla and lower dermal sheath around the end bulb of a follicle specifically express ALP activity and have the potential capacity to restore hair growth [25]. TGF-␤2 is involved in promotion of the hair placode [16], contributes to anagen induction [45], is highly expressed in hDPCs compared with hDFs, and is suggested to mediate the hair-inductive capacity of DPCs [24]. Wnt10b is known to play crucial roles in fetal development of hair follicles. Specifically, Wnt10b is first expressed strongly in the placode of the epidermis at embryonic days 13.5-14.5 in mice [46,47], suggesting its possible contribution to the initiation of folliculogenesis. Activation of the Wnt pathway is thought to be the first mesenchymal signal involved in the epithelialmesenchymal interaction of folliculogenesis [16,48]. A chamber assay (mix of dissociated competent epithelial and mesenchymal cells) with Wnt10b-producing cells promoted hair folliculogenesis [20]. Intriguingly, hair follicle neogenesis was induced in a Wnt signal-dependent manner in normal mice after skin wounding [19]. This suggested that the activation of Wnt signaling and subsequent stabilization of ␤-catenin serve as a trigger for the initiation of folliculogenesis and may determine whether activated stem cells in damaged skin differentiate into epidermis or hair. Furthermore, Wnt10b was also shown to enhance hair shaft growth in organ culture of hair follicles [49]. The upregulated expression of Wnt10b in hDPCs induced by VD 3 may mean terminal and functional differentiation of proliferating hDPCs, although it has not been verified by this study.
atRA upregulated Wnt10b gene expression in hDPCs directly and temporarily in serum-free medium and prolonged the upregulation in serum-containing medium. It has previously been reported that VDR signaling can be stimulated by the presence of a retinoid X receptor (RXR) ligand (such as 9-cis retinoic acid) in human skin in vivo [41]. A major part of the effects of vitamin D are mediated through its nuclear receptor, VDR [50], which forms a heterodimeric complex with RXR [28,51]. atRA is one of the natural ligands of RXR and may interfere with the VDR-VDRE Hair regeneration experiment using transplantation of a cultured mDPC construct in F344 rats. Cultured cell sheets of mDPCs (or mDFs as a control) were prepared and treated with VD 3 for 0, 24, or 72 hours. After VD 3 pretreatment, the mDPC (or mDF) sheet was fragmented, and the small fragment was transplanted into the nonhairy sole skin of F344 rats, using the hemivascularized sandwich method. Split-thickness skin (150 -300 m in thickness) was sliced off and digested with dispase, to separate the epidermis from the dermal compartment. The dermal compartment was discarded, and the epidermis was replaced on the remaining deep dermis after a cultured mDPC or mDF sheet fragment was grafted. Eight weeks after the transplantation, hair folliculogenesis was evaluated. (B): Hair regeneration induced by mDPC or mDF transplantation. Hair folliculogenesis of each sample was histologically evaluated (detailed data are summarized in supplemental online Table 3). Regenerated hair follicles were classified by maturity into stages 1-8. The numbers of total regenerated hair follicles (top left), matured (stage 6 -8) follicles (top right), and outgrowing hairs (bottom) are shown. Significant differences among groups are shown as ‫ء‬ (p Ͻ .05) or ‫ءء‬ (p Ͻ .01). Abbreviations: DF, dermal fibroblast; DPC, dermal papilla cell; NS, not significant; VD 3 , 1␣,25-dihydroxyvitamin D 3 .
www.StemCellsTM.com signaling pathway. Since VD 3 is converted from 25-hydroxyvitamin D 3 (25(OH)D 3 ) in the kidney, and serum concentration of VD 3 is maintained at 1/1,000 to 1/500 of that of 25(OH)D 3 (less than 0.4 nM), which is found at 25-80 ng/ml in healthy subjects [52], the synergistic response of Wnt10b by atRA and serum may possibly be due to serum-contained VD 3 . KCZ is an imidazole that inhibits cytochrome P-450 enzymatic activity, such as 24(OH)ase [53], and enhances the biological effect of VD 3 in human skin [41]. However, synergistic upregulation of Wnt10b was not detected in cultured hDPCs.
Although most physiologic activities of VD 3 are mediated via the VDR-VDRE signaling pathway, vitamin D is also known to act via nongenomic pathways, such as transmembrane ion channels [54,55], and other intracellular signaling pathways [56]. By using RNA interference against VDR, we examined whether upregulation of Wnt10b, ALPL, and TGF-␤2 in hDPCs, induced by VD 3 , was mediated through VDR, and we confirmed that all of these up-regulations were exerted via the genomic VDR pathway in hD-PCs.
Finally, we examined the potential of VD 3 in a cell-based therapy targeting hair follicle regeneration, where cultured DPCs were pretreated with VD 3 before transplantation. VD 3 pretreatment augmented therapeutic efficiency. Specifically, a greater number of regenerated outgrowing hair shafts were macroscopically detected, and histological evaluation confirmed that the maturation of regenerated follicles in VD 3 -treated groups was significantly superior to that in the untreated group. Thus, the results suggested that pretreatment with VD 3 , in expanding cultures, enhances the hair-inductive capacity of DPCs. It is probably important to administer VD 3 to DPCs after sufficient confluence has been reached, because VD 3 showed a suppressive effect on DPC proliferation. VD 3 may enhance functionality of cultured DPCs by promoting its differentiation.

CONCLUSION
VD 3 was shown to upregulate gene expression related to hair inductive capacity of DPCs such as Wnt10b, ALPL, and TGF-␤2, although VD 3 did not promote proliferation of DPCs. An animal study indicated that pretreatment with VD 3 , in expanding culture, enhanced the hair regeneration by DPC transplantation to the nonhairy skin. Our results suggest that VD 3 may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies, although the method of usage should be carefully designed.