1/29
Date received: 16/10/2008
Date revised: 05/01/2009, 04/02/2009
Date accepted: 23/02/2009
OA / JCMM-10-2008-061.R2
Title: TGF-β2 is specifically expressed in human dermal papilla cells and modulates hairfolliculogenesis
Authors:
Keita Inoue, M.D.1, Noriyuki Aoi, M.D.1, Yuji Yamauchi, M.S.2, Takahiro Sato, M.S.2,
Hirotaka Suga, M.D.1, Hitomi Eto, M.D.1, Harunosuke Kato, M.D.1, Yasuhiko Tabata, Ph.D.,
D.Med.Sci., D.Pharm.3, and Kotaro Yoshimura, M.D.1*
Affiliations:
Department of Plastic Surgery, University of Tokyo School of Medicine
7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan.
2
Division of Research and Development, Biomaster, Inc.
Yokohama Excellent III Bldg. 8F, 3-35, Minami-nakadori, Naka-ku, Yokohama, Kanagawa
231-0006, Japan.
3
Department of Biomaterials, Institute for Frontier Medical Sciences, Kyoto University
53 Kawara-cho Seigoin, Sakyo-ku, Kyoto 606-8507, Japan.
1

Short running head: TGF-β2 and human hair folliculogenesis
Keywords: TGF-β2, dermal papilla cell, hair regrowth, hair regeneration, tissue engineering,cell therapy, vitamin D3*Correspondence:
Kotaro Yoshimura, M.D.
Department of Plastic Surgery, University of Tokyo School of Medicine,
7-3-1, Hongo, Bunkyo-Ku, Tokyo 113-8655, Japan.
TEL: +813-5800-8948
FAX: +813-5800-8947
e-mail: yoshimura-pla@h.u-tokyo.ac.jp
Conflict of interests:
The authors declare that they have no competing financial interests.
Abbreviation
AR: androgen receptor
BMP: bone morphogenetic protein
DMEM: Dulbecco's modified Eagle's medium
ELISA: enzyme-linked immunosorbent assay
ENA: epithelial cell-derived neutrophil-activating peptide
FGF: fibroblast growth factor
GMB: gelatin hydrogel microsphere bead
GRO: growth-related oncogene
HB-EGF: heparin-binding EGF-like growth factor
This is an Accepted Work that has been peer-reviewed andapproved for publication in the Journal of Cellular and Molecular Medicine, but
has yet to undergo copy-editing and proof correction. See
http://www.blackwell-synergy.com/loi/jcmm for details.
Please cite this article as a “Postprint”;10.1111/j.1582-4934.2009.00739.x

2/29
hDPC: human dermal papilla cell
hDF: human dermal fibroblast
HGF: hepatocyte growth factor
IGF: insulin-like growth factor
IGFBP: insulin-like growth factor binding protein
KCM: keratinocyte conditioned media
KGF: keratinocyte growth factor
MCP: monocyte chemotactic protein
MDC: macrophage-derived chemokine
MIP: macrophage inflammatory protein
NGF: nerve growth factor
PAI-1: plasminogen activator inhibitor 1
PDGF: platelet-derived growth factor
pSMAD2: phosorylated SMAD2RANTES: regulated upon activation, normal T-cell expressed and secreted
RT-PCR: reverse transcription polymerase chain reaction
TIMP: tissue inhibitor of metalloproteinase
TGF: transforming growth factor
VDR: vitamin D receptor
VEGF: vascular endothelial growth factor

3/29
Abstract
Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hairfollicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hairis theoretically possible by expanding autologous human DPCs and transplanting them intobald skin, though much remains to be overcome before clinical success. In this study, we
compared gene signatures of human DPCs at different passages and human dermal
fibroblasts, and found TGF-β2 to be highly expressed in cultured human DPCs. Keratinocyte
conditioned medium, which is known to help preserve the hair inducing capacity of hDPCs,
upregulated TGF-β2 expression of human DPCs and also enhanced their alkaline phosphataseactivity, a known index for hair inductive capacity. Through screening of components
secreted from keratinocytes, the vitamin D3 analogue was found to promote TGF-β2
expression and alkaline phosphatase activity of human DPCs. In animal hair folliculogenesis
models using rat epidermis and expanded human DPCs, inhibition of TGF-β2 signaling at theligand or receptor level significantly impaired hair folliculogenesis and maturation. These
results suggest an important role for TGF-β2 in hair follicle morphogenesis and provide
insights into the establishment of future cell therapies for hair regrowth by transplanting
expanded DPCs.

4/29
INTRODUCTION
The mammalian hair follicle is a complex mini-organ that consists of different lineages ofcells, including epithelial, mesenchymal and pigmented cells. The dermal papilla, consideredas the most important mesenchymal component, plays versatile roles in hair follicle
morphogenesis and hair cycling via epithelial-mesenchymal interactions [1-4]. Since cultured
dermal papilla cells (DPCs) as well as organ dermal papilla were found to have hair inductive
capacity [5-7], many attempts have been made to regenerate hair follicles by transplanting
expanded DPCs, sometimes together with epithelial stem cells. However, challenges indeveloping regeneration strategies have arisen, as the hair inductive ability of DPCs is lost
upon culture and the molecules and mechanisms responsible for the hair inducing capacity are
not yet fully elucidated [8].
There are six major morphogenetic molecular family systems in hair follicle development andcycling: fibroblast growth factor (FGF), transforming growth factor (TGF)-β, sonic hedgehog,
Wingless or Wnt pathway, neurotrophins, and homeobox gene families [4, 9, 10]. In eachmorphogenetic stage, all function as responsible molecules for the reciprocal signaling
between hair follicle epithelial and dermal components. In the context of hair follicle“neogenesis”, however, it is unclear which signaling molecule(s) among these pathways
function in hair induction in transplanted DPCs. Thus far, specific signaling molecules, suchas bone morphogentic protein (BMP)-6 [11], have been shown to enhance mouse hairfolliculogenesis. Wnt3a signaling from epithelial component is also required to maintain the
inductive capacity of DPCs and to generate hair follicles [12]. These factors have been
determined as candidates for hair inducing activity by employing sophisticated transgenic
approaches such as specific knockout or over-expression in vivo. In humans, however, thedifficulty in applying transgenic approaches has hampered such studies for specific in vivo
gene function [13]. Therefore, although various biomarkers specifically expressed in human
DPCs have been reported [14, 15], their functions remain to be clarified.
Conditioned media obtained from epidermal keratinocyte culture (keratinocyte conditioned
media, or KCM) is known to maintain DPC capacity to proliferate and induce hair follicles
for a longer period than control media [16], suggesting that cultured keratinocytes release key
factors for DPCs to maintain hair inducing capacity. Keratinocytes produce a vast variety ofsoluble factors including growth factors, hormones and chemokines [17, 18]. Screening of
biologically active components in KCM may identify the substances that stimulate DPCs tomaintain their hair inducing capability and provide an efficient method for in vitro expansionof hair inductive DPCs.
We hypothesized that specific genes relating to hair inducing capacity are upregulated inhuman DPCs (hDPCs) and that expression is promoted by particular components contained inhuman KCM. In this study, the global gene signatures of hDPCs at early and later passages
and human dermal fibroblasts (hDFs) with no hair inducing capacity were compared by
microarray analysis. Our results showed that the TGF-β2 gene was specifically expressed in
hDPCs and its expression was upregulated by KCM. We further investigated potential roles of
TGF-β2 in hair induction by hDPCs and sought to identify keratinocyte-derived componentsthat can affect the hair inducing capacity of hDPCs.

5/29
MATERIALS AND METHODS
Human DPC and DF culture
Scalp and facial skin with hair were obtained from facelift operations performed at two
institutions; informed consent was obtained using protocols approved by institutional review
boards from each individual institution. Dermal papillae were isolated from the hair follicles
under a microscope, and placed onto a culture dish containing Dulbecco's modified Eagle's
medium (Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (10% DMEM).
After two weeks of explant culture, expanded hDPCs were subcultured with the same
medium. Human DFs were obtained from the explant culture of facial skin dermis of the same
individuals and cultured in 10% DMEM.
Human epidermal keratinocyte culture and preparation of the conditioned culture media
Human facial skin was cut into 3 mm square pieces and incubated in 10% DMEM
supplemented with 1,000 unit/ml DispaseTM II (Sankyo, Tokyo, Japan) at 4°C for 15-18
hours. The epidermis was carefully peeled off from the dermis and incubated in phosphatebuffered saline (PBS) supplemented with 0.25% trypsin and EDTA mixture at 37°C for 20
minutes to obtain fresh keratinocyte cell suspension. Keratinoctyes were cultured in serum
free media, DKSFMTM (Gibco), for 7-10 days up to 60-80% confluence; afterwards, theculture medium was switched to 10% DMEM. The culture supernatant was collected after one
week, centrifuged at 3,000 g for 30 minutes, and filtrated through a 0.22 μm membrane filter(Micropore, Madison, NJ). The supernatant was mixed with fresh 10%DMEM at a 1:1 ratio tomake KCM for hDPC culture.
Reagents
Reagents supplemented to hDPC culture were as follows: acidic FGF (Peprotech, Rocky Hill,
NJ); basic FGF (Peprotech); BMP2 (Wako, Osaka, Japan); interleukin (IL)-1β (Endogen,
Rockford, IL); IL-6 (Peprotech); IL-8 (Wako); vascular endothelial growth factor (VEGF)(Wako); platelet-derived growth factor (PDGF)-BB (Wako); nerve growth factor (NGF)(Sigma-Aldrich, Louis, MO); heparin-binding EGF-like growth factor (HB-EGF) (Peprotech);macrophage inflammatory protein (MIP) 3α (R&D systems, Minneapolis, MN); monocyte
chemotactic protein (MCP)-1 (R&D systems); insulin-like growth factor (IGF)-1 (SigmaAldrich); epithelial cell-derived neutrophil-activating peptide (ENA)-78 (Wako); growthrelated oncogene (GRO)-α (Wako); 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (LKT
laboratories, St. Paul, MN); cholesterol sulfate (Sigma-Aldrich); all-trans retinoic acid
(Biomol, Hamburg, Germany); 17β-estradiol (Cayman Chemical, Ann Arbor, MI); anddihydrotestosterone (Biomol). All reagents were diluted in PBS or ethanol to 1,000 fold of the
final working concentrations indicated in manufacturer’s instructions and stored in aliquots at-20°C.
Real-time reverse transcription polymerase chain reaction (RT-PCR)
RNA was isolated from cultured hDPCs or hDFs using an RNeasyTM Mini Kit (Qiagen,
Valencia, CA), followed by reverse transcription. PCR amplification of cDNA was performed
in a 50 μl reaction consisting of 1x TaqManTM Universal Master Mix (Applied Biosystems,
Foster City, CA) with the ABI 7700 sequence detection system. Gene expression of various
hair follicle-related genes was quantified based on measurement of the cycle threshold using
the following TaqManTM pre-designed primers and probes (Applied Biosystems): TGF-β2(Hs00236092_m1); TGF-β1 (Hs00998129_m1); BMP2 (Hs00154192_m1); syndecan1

6/29(Hs00896423_m1); integrin-β1 (Hs00559595_m1); keratinocyte growth factor (KGF)(Hs00940253_m1); VEGF (Hs00900054_m1); IGF-1 (Hs01547656_m1); HGF(Hs00300159_m1); PDGF (Hs00234042_m1); steroid 5α-reductase II (Hs01399057_m1);
versican (Hs00171642_m1); ephrin-A3 (Hs00191913_m1); and androgen receptor(Hs00171172_m1). We used GAPDH (Hs99999905_m1) as an endogenous reference gene.
Microarray generation and analysis
To identify genes differentially expressed in hDPCs responding to the substances secreted
from keratinocytes, gene expressions of hDPCs and hDFs of the same individual werecompared. tRNA of hDPCs cultured in KCM (passage 2 and 8) and hDFs (passage 2) hDFs in
10% DMEM were isolated using an RNeasyTM Mini Kit. The quality of each sample wasassessed by rRNA 28S/18S ratio and RIN (RNA Integrity Number) using Agilent 2100
BioanalyzerTM (Agilent Technology, Palo Alto, CA). cDNA was obtained from 5 μg of tRNAby one-cycle of reverse transcription. The biotin-labeled cRNAs were purified, fragmented,
and hybridized to the GeneChipTM Human Genome U133 Plus 2.0 Array (Affymetrix, Santa
Clara, CA), which was then scanned by the GeneChipTM 3000 Scanner (Affymetrix)
following the manufacturer’s protocol. The numerical data of the signal intensity was
analyzed using GeneChipTM Command ConsoleTM Software (Affymetrix) and Microsoft
ExcelTM.
Cytokine array analysis
The supernatant of human keratinocyte culture was collected at one or two weeks after the
medium switch from DKSFM to 10% DMEM by the methods described above. Expression
levels of multiple cytokines were assayed in each sample using the Human Cytokine Array VI(Ray Biotech, Norcross, GA) following the manufacturer’s instruction. The image of eacharray was captured by a digital camera (Nikon, Tokyo, Japan) and converted to the binary
image format. The signal intensity was calculated using the image processing software,
ScionTM Image (Scion Corp., Frederick, MD).
ELISA for TGF-β2
The influence of KCM and other keratinocyte-derived factors on TGF-β2 protein production
in hDPCs was assessed by sandwich ELISA, QuantikineTM human ELISA for TGF-β2 (R&D
systems). Reagents supplemented to the hDPC culture were as described above. The
supernatant of hDPC culture at passage 2 was collected after 96 hours of incubation, andprocessed with ELISA following the manufacturer’s instructions.
Alkaline phosphatase activity assay
The influence of KCM and other keratinocyte-derived factors on hDPC alkaline phosphatase(ALP) activity was assessed by fluorescent based ELISA, SensolyteTM FDP Alkaline
Phosphatase Assay Kit (AnaSpec, San Jose, CA). Reagents supplemented to the hDPCs
culture were as described above. Human DPCs were seeded on a 24-well plate and cell lysates
were collected after 48 hours of incubation. The fluorescence intensity was measured using a
microplate fluorescence reader, BTX-880 (Beckman-Coulter, Brea, CA). The fluorescencereading was normalized with the cell number at time of harvest.
MTT proliferation assay
The influence of KCM and other keratinocyte-derived factors on hDPC proliferation wasassessed by a MTT cell proliferation assay kit (Roche, Basel, Switzerland). Human DPCs

7/29
were seeded on a 96-well plate and cell lysates were collected after 96 hours of incubation.
The fluorescence intensity was measured using a microplate fluorescence reader, BTX-880(Beckman-Coulter), and cell number was calculated following the manufacturer’s instruction.
Animal assays for hair folliculogenesis
We generated rat-human chimeric hair follicles in nude mice using a previously-describedsandwich method [16, 19, 20] with some modifications. Briefly, hDPCs were cultured as
described above, and the hDPC sheet was scraped off, cut into 1-mm square pieces, and usedas transplanted constructs. The follicular foot pad skin of 8-week-old F344 rats was cut into 3mm square pieces and incubated in 10% DMEM and 1,000 U/ml DispaseTM II at 37°C for 20
minutes to separate the epidermis and dermis. The DPC construct was placed between theepidermis and dermis of the foot pad and transplanted to the subcutis of a 6-week-old Balb/cnude mouse (Supplemental Fig. 1). The transplants were harvested 4 weeks later andprocessed for histological evaluation of the number and maturation stage of generated hairfollicles. The maturation stage was categorized into 8 stages (S1 to S8) according to apreviously described method [21] (Supplemental Fig. 2).
A chamber grafting method was also employed according to previously reported methods [11,20, 22, 23] with some modifications (Supplemental Fig. 1). A combination of cultured hDPCs(P3) and cultured neonatal B6 mouse keratinocytes was employed to reconstitute humanmouse chimeric hair follicles. In another experiment, a combination of freshly isolated fetal
dermal cells and fetal keratinocytes isolated from BL6 mouse embryos was also utilized. For
preparation of cultured mKC, the dorsal skin of newborn mice was incubated in 1,000 U/ml
DispaseTM II at 4°C for 15-18 hours, and the epidermis was consequently separated from thedermis. The epidermis was digested with a 0.05% Trypsin and 0.2 mM EDTA mixture at
37°C for 20 minutes to obtain single cells. The resultant cell suspension was filtrated througha 40-μm cell strainer and cultured in DKSFMTM for 4 days. For preparation of mouse fetal
dermal cells and keratinocytes, the dorsal skin of E17.5 BL6 embryos was digested as
described above. A dome-shaped polypropylene chamber made from a PCR tube lid (Greiner
Bio-One, Frickenhausen, Germany) was transplanted onto the back of a nude mouse 5 days
before cell transfer. The number of mesenchymal cells (cultured hDPCs or mouse fresh fetal
dermal cells) and epithelial cells (cultured mouse neonatal keratinocytes or fresh fetal
keratinocytes) transplanted into each chamber was 106 (2 × 106 in total). Skin samples ofrecipient nude mice were harvested 4 weeks after cell transplantation. Each group consisted of
4 chambers on 4 mice.
TGF-β signal inhibition in vivo
TGF-β signal inhibitors were administered to the recipient nude mice. A selective kinase
inhibitor for the TGF-β type I receptor, SB431542 (10 μM; Sigma-Aldrich), or an equivalentamount of vehicle was administrated continuously (0.25 μl/hour) by AlzetTM osmotic pumps(Durect, Cupertino, CA), and transplanted subcutaneously beside the foot pad construct in the
sandwich model or the chamber in the chamber model. A specific antibody for human TGFβ2 (BioVision, Mountain View, France) or TGF-β1/2/3 (R&D systems) was used to neutralize
TGF-β2 or all three isoforms (TGF-β1/2/3) of TGF-β ligand. One μg of neutralizing or
negative control IgG (R&D systems) was administered every second day by a local injection
to the subcutis. Signal inhibition in both models was performed for 4 weeks.
Immunohistochemical staining8/29
After harvest, the foot pad transplant was embedded in the OCT compound (Sakura Finetek,
Tokyo, Japan), frozen in liquid nitrogen, and stored at -80°C until sectioning. Frozen sections(10 μm) were placed on slides, air dried at room temperature for 1 hour, fixed inparaformaldehyde (4% in PBS) for 1 minute, and washed in PBS for 5 minutes. Every otherslide was stained with hematoxylin and eosin by the standard protocol, and the number andmaturation of generated hair follicles were evaluated. The other slides were processed byimmunohistochemical staining. Briefly, the sections were incubated with 5% goat serum atroom temperature for 30 minutes, followed by incubation with mouse anti-human TGF-β2(1:100, Neo Markers, Fremont, CA), mouse anti-human TGF-β1 (1:100, Lab Vision,Fremont, CA), rabbit anti-rat phospho-SMAD2 (1:100, Millipore, Billerica, MA), rabbit antirat SMAD7 (1;100, Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-rat PAI(plasminogen activator inhibitor)-1 (1:100, Innovative Research, Novi, MI) antibodies atroom temperature for 60 minutes. AlexaTM546-conjugated goat anti-mouse IgG (1:100,
Molecular Probes, Eugene, CA) or AlexaTM 488 conjugated goat anti-rabbit IgG (1:100,
Molecular Probes) was used as a secondary antibody to detect the primary antibodies. Counterstaining was performed using Hoechst33342 (Dojindo, Kumamoto, Japan).
Statistical analysis
Data were presented as mean ± standard error. To test the significance of quantitative data, theunpaired Student’s t-test was applied.

RESULTSTGF-β2 gene is specifically detected in hDPCs by microarray analysis
We performed comparative microarray analysis of the molecular gene signatures of cultured
hDPCs and hDFs. We hypothesized that gene(s) related to hair inducing pathways arecontained in DPs, but not in DFs, and that expression of the gene(s) decreases upon passage.
We did not use the same culture conditions for hDPCs and hDFs in this experiment; we used
KCM for culturing hDPCs to maximize their hair inducing capacity but used DMEM (basal
medium of KCM) for hDFs not to provide this support. In each comparison, out of 54613
genes, we first identified genes with signal intensity of at least 100. When we comparedcultured hDPCs with hDFs of the same individual at the earlier passage (P2), we found 567
upregulated and 498 downregulated genes (“early-DPC genes”) in hDPCs, of which the folddifference from hDFs was at least 1 or at most -1 in log ratio. At passage 8, the number of
upregulated and downregulated genes with the same features decreased to 143 and 174 genes,
respectively (“late-DPC genes”). We found 34 overlapping upregulated genes (Supplemental
Table 1) and 48 overlapping downregulated genes (Supplemental Table 2) in both early-DPC
and late-DPC genes. When limited to genes whose expression changed significantly frompassage 2 to passage 8, only 11 upregulated and 5 downregulated genes were listed (Table 1).
TGF-β2 was included among the 11 upregulated genes, suggesting its putative function in hairinducing capacity.
TGF-β2 gene is preferentially expressed in cultured hDPCs
Since cultured DPCs have been shown to contain hair inducing capacity but lose it upon
culture [6, 22], the gene expression profile of cells at early passages of culture wasdetermined. We selected a set of genes previously reported to be related to DPC function [14,24-36], and examined which genes were upregulated in hDPC cultured in 10%DMEM at9/29
passage 2 in comparison with expression in hDFs cultured in 10%DMEM; the set of genes
included TGF-β1, TGF-β2, BMP2, syndecan1, integrin-β1, KGF, VEGF, HGF, PDGF, 5αreductase II (5αRII), versican, ephrin-A3, and androgen receptor (AR). Quantitative real-time
PCR revealed that TGF-β2 was significantly upregulated in cultured hDPCs compared to
hDFs (Fig. 1A). We also examined hDPCs at later passages. At passage 8, hDPCs were viable
enough to keep proliferating and showed no sign of apoptosis or growth arrest. TGF-β2 gene
expression was still upregulated in hDPCs at passage 8 compared to hDFs, and was slightly
lower compared to hDPCs at passage 2 (Fig. 1B).
TGF-β2 gene expression in cultured hDPCs is enhanced by epidermal keratinocyte
conditioned media
It is well documented that rodent DPCs obtained from the vibrissa hair follicles maintain their
proliferative and hair inducing capacities when they are cultured in KCM [16]. To assess
whether hDPCs would exhibit similar properties, we first tested the proliferative effect ofhuman KCM on cultured hDPCs. KCM showed a marked effect on promoting hDPC
proliferation to the extent of an approximate 1,000 fold increase in cell number within 8
passages (60-70 days) (Fig. 2A). Although KCM did not cause apparent alterations in themorphology of hDPCs, TGF-β2 mRNA expression was significantly upregulated in hDPCs
cultured in KCM compared to control media or other kind of commercially available growth
media (AmniomaxTM II) (Fig. 2B).
Human DPCs respond to soluble factors from epidermal keratinocytes
We next sought to identify possible KCM components that enhance TGF-β2 expression in
hDPCs. We first examined the cytokine expression profiles of human KCM using a cytokine
antibody array. Among 79 cytokines examined, IL-8, IL-6, IL-1β, MCP-1, Gro, MIP3α,tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, ENA-78, macrophage-derived
chemokine (MDC), PDGF-BB, VEGF, insulin-like growth factor binding protein (IFGBP)-2
and regulated upon activation, normal T-cell expressed and secreted (RANTES) were highly
expressed in KCM compared with control media (Fig. 3A). In addition to these cytokines,
soluble factors considered to have biological activity on the hair follicles [17, 18, 37-46]
were tested for induction of TGF-β2 expression in hDPCs cultured in 10% DMEM. Among
21 factors tested, a biologically active metabolite of vitamin D3, 1,25(OH)2D3, and IL-1β, aswell as KCM, showed a marked effect on promoting TGF-β2 mRNA expression in hDPCs(Fig. 3B). The promotion of TGF-β2 mRNA expression by 1,25(OH)2D3 or IL-1β was
independent of serum supplementation (Supplemental Fig. 3). ELISA for TGF-β2 protein
revealed that TGF-β2 secretion from hDPCs was highly elevated upon supplementation with
1,25(OH)2D3 or KCM, while no effect was observed with the other factors (Fig. 3C).
Furthermore, ALP activity, a well-established marker for DPCs and hair inducing property in
hDPCs [11, 47], was significantly higher in the presence of 1,25(OH)2D3 as well as KCM(Fig. 3D). In contrast, MTT assay revealed that hDPC proliferation was impaired by
1,25(OH)2D3, while enhanced by KCM (Fig. 3E).
Active form of vitamin D promotes TGF-β2 gene expression of human DPCs
Real-time RT-PCR revealed that supplementation of 10-1000 nM of 1,25(OH)2D3
significantly upregulated TGF-β2 mRNA expression in hDPCs after 24 or 48 hours of
incubation (Fig. 4A), and this upregulation of TGF-β2 expression was seen as early as 8 hours(Fig. 4B). ELISA analysis of secreted TGF-β2 protein showed that 1,25(OH)2D3 significantly
promoted TGF-β2 secretion from hDPCs in a dose-dependent manner (Fig. 4C).

10/29Inhibition of TGF-β2 signaling at either the receptor or ligand level suppresses hairfolliculogenesis in vivo
The functional capacity of TGF-β2 in contributing to hair induction was assessed in an animalmodel for hair folliculogenesis. Rat-human chimeric hair follicles were generated in nude
mice using the sandwich method described above. In control mice, the chimeric hair follicles
were generated in 3-4 weeks after grafting of cultured hDPCs and showed histologicalfeatures at a variety of developmental stages (Supplemental Fig. 2), mimicking those of fetal
hair follicle morphogenesis as described previously [20, 48]. Immunohistchemistry confirmedthat TGF-β2, but not TGF-β1, was expressed in the dermal sheath of newly developed hairfollicles (Fig. 5A).
To evaluate the dependency of TGF-β signaling in hair folliculogenesis in the above animalmodels, signal transduction was inhibited by a continuous infiltration of SB431542, a highly
selective kinase inhibitor for TGF-β type I receptor [49] via osmotic pump. Histologicalevaluation revealed that SB431542 suppressed both the frequency and maturation of hairfollicle development. In control mice, well-maturated hair follicles were observed that could
be categorized into high maturation stages, with structures such as the hair shaft and thesebaceous gland (Fig. 5B). On the other hand, in the SB431542-administered mice, signs of
impaired maturation, such as pseudo-keratosis of the inner root sheath, were seen (Fig. 5B).
The total number of generated hair follicles was significantly decreased in the SB431542administered mice (Fig. 5C). Furthermore, a morphometric categorization of maturation
stages of generated hair follicles demonstrated the suppressive effects of SB431542 on
folliculogenesis (Fig. 5D); follicles of control mice were observed to be in well-maturated
stages (Stage 5 to 7), while follicles of SB431542-administered mice were classified as
poorly-maturated stages (Stages 2 to 4).
Two neutralizing antibodies were employed for the TGF-β ligand-neutralizing approach andadministered via local injections: a neutralizing antibody specific for TGF-β2 with no effecton TGF-β1 or TGF-β3 and a pan TGF-β neutralizing antibody that inhibits TGF-β1, TGF-β2,
and TGF-β3 activity. Although histological analysis did not reveal any significant differences
in maturation stages of generated hair follicles among the groups, the number of inducted hairfollicles was significantly decreased in the antibody-administered mice compared with thenonspecific IgG-administered mice (Fig. 6A–C).
In addition, a hair reconstitution assay with or without signal inhibition by anti-TGF-β2
antibody was also performed using the chamber method. The number of generated hairfollicles was significantly smaller in mice treated with anti-TGF-β2 antibody compared tocontrol mice (Fig. 7A, 7B). In chamber models using mouse DPCs (fetal dermal cells),
SB431542 and anti-TGF-β2 antibody substantially decreased the average number ofgenerated hair follicles, though the differences did not reach statistical significance(Supplemental Figure 4).
To further assess effects of augmentation of TGF-β2 signal on hair follicle induction, TGFβ2-releasing gelatin hydrogel microsphere beads (GMBs) were incorporated into thesandwich models; no positive effect of TGF-β2 signal augmentation was observed with thisapproach (Supplemental Fig. 5).

11/29Immunohistological analysis of TGF-β signal transduction in animal models
To analyze TGF-β2 signal transduction in hair folliculogenesis in the sandwich models,
phosphorylation of SMAD2 and expression of two SMAD2 target genes (SMAD7 and PAI-1)[50] were evaluated by immunohistology (Fig. 8). In control mice, phosphorylated SMAD2(pSMAD2) translocation to nuclei, and positive signals of SMAD7 and PAI-1 were observedin the epithelium of generated hair follicles. In regions in which no hair follicle development
was observed despite the presence of DiI-labeled hDPCs beneath the basal lamina, signs of
TGF-β2 signaling activation were not observed in either SB431542- or anti-TGF-β2 antibodytreated mice. In SB431542- and anti-TGF-β2 antibody-treated mice, generated hair follicles
expressed pSMAD2, SMAD7 or PAI-1 in the epithelium, especially when the follicles were
well matured, though expression levels were not as strong compared to the control.

DISCUSSION
Attempts over the last several decades to regenerate hair follicles by transplanting expanded
hDPCs have been hampered by the lack of knowledge of the signal and mechanism in hDPCs
to induce hair folliculogenesis. How the hair inducing capacity of hDPCs can be maintained
upon expansion culture also remains unclear. Here we sought to identify a gene(s) in cultured
hDPCs responsible for or contributing to hair inductive capability. In addition, we tried to
optimize the cultured method to help preserving hair inducing capacity of hDPCs. By
comparison of the gene expression profiles of hDPCs and hDFs, along with additional gene
analysis of DPC biomarkers, TGF-β2 was identified as a factor specifically expressed bycultured hDPCs. Our results showed that TGF-β2 expression slightly decreased over culture
time, as was the hair inductive property of hDPCs reported in the literature [8, 12, 16].
For developing an expansion method of hDPCs while maintaining the hair inductive activity,
one piece of evidence may provide some insight: a previous observation that KCM showed apositive effect in maintaining the proliferative and hair inductive ability of rodent DPCs [16].
Our results demonstrated that TGF-β2 gene expression was upregulated in KCM-treated
hDPCs compared to non-treated hDPCs. ELISA further revealed that KCM promoted TGF-β2
protein secretion from hDPCs. In addition, a concomitant elevation of ALP activity in KCMtreated hDPCs suggested KCM-mediated effects on the hair inductive ability of hDPCs.
These effects of KCM indicated the possibility that KCM contains a key component tomaintain hair inductive property of hDPCs, and the key component may also stimulate
cultured hDPCs to express TGF-β2. Since hair follicle epithelium and dermal papilla are incontact with each other and send reciprocal signals to induce hair folliculogenesis andmaintain hair cycles [1-3], it is not surprising that KCM contains a key component in thisprocess. Cytokine array analysis of KCM detected inflammatory cytokines such as IL-1β, IL6, IL-8, MCP-1, RANTES, ENA-78 and Gro, which are known to be secreted fromkeratinocytes as an acute or late phase response to inflammation [17, 18]. Our results also
revealed that KCM contained known mitotic growth factors for DPCs, such as PDGF-BB [40]or VEGF [39, 43], suggesting that the proliferative effect of KCM is attributed to such growth
factors.
Screening analysis of components in KCM detected an unexpected function of vitamin D3:
promoting effects on TGF-β2 expression and ALP activity of hDPCs. Cultured hDPCs

12/29express vitamin D receptor (VDR) (data not shown), and thus 1,25(OH)2D3-induced TGF-β2mRNA upregulation may be mediated via VDR. However, TGF-β2 mRNA expression
increases over time up to 48 hours and remains high for 5 to 7 days (data not shown), which
may suggest the involvement of other signal pathways. A similar observation was made in
hDFs: 1,25(OH)2D3 specifically induces TGF-β2 mRNA expression in hDFs in the earlyphase of signal transduction, followed by induction of all TGF-β isoforms (TGF-β1, β2, andβ3 mRNA) in an autocrine manner [51]. Thus, TGF-β2 mRNA induction by 1,25(OH)2D3
shows a monophasic increase with time [51]. Other studies showed that TGF-β signal
positively regulates the vitamin D signaling pathway by formation of the Smad3-VDR
complex [52], and vitamin D3 induces strong activation of Smad2/Smad3 within 24 hours in
HL-60 cells [53]. These data indicate a direct interplay between TGF-β and vitamin D
signaling pathways mediated by VDR and Smad proteins. Taken together, there appears to bea prolonged activation of both TGF-β and vitamin D signaling pathways in cultured hDPCs as
the result of a close interplay and positive feedback loop, although further investigation
should be performed.
Ablation of VDR in mice [54, 55] and mutations of VDR in humans result in thedevelopment of alopecia [56, 57]. VDR is expressed in the two major cell components thatmake up hair follicles: the mesenchymal component, dermal papilla, and the epithelial
component, outer root sheath keratinocytes [58]. Recently, VDR expression in follicular
keratinocytes was shown to be essential in maintaining hair follicle homeostasis [59, 60].
However, less is known of the effects of vitamin D3 signal on dermal papilla thus far. The
biologically active metabolite of vitamin D3, 1,25(OH)2D3, is mainly produced in kidneys[61], but is also produced and secreted by keratinocytes in the presence of endogenous 1αhydroxylase [37, 38, 62]. Thus, it is suggested that 1,25(OH)2D3 secreted by keratinocytes
likely works as a signaling molecule to stimulate DPCs to secret TGF-β2 and initiate thevitamin D3 and TGF-β signaling loop.
In most types of cells, the fundamental functions of TGF-β isoforms are growth inhibition anddeposition of extracellular matrix [63]. Especially during fetal development, TGF-βs arefound in a broad range of organs, such as epithelium, myocardium, cartilage and bone ofextremities, and in the nervous system, suggesting its critical functions in organogenesis. In
hair follicle physiology, TGF-βs have been shown to exert unique multidirectional effects [4],i.e., both positive and suppressive effects on hair growth. TGF-β1 blocks anagen and induces
catagen [64, 65] and inhibits hair growth [66]. TGF-β1 and TGF-β2 stimulate proliferation ofouter root sheath keratinocytes [67, 68]. TGF-β2 induces premature hair follicle regression in
adult hair cycling [35, 69], while TGF-β2 was also shown to be required for hairfolliculogensis [31, 70].
In the context of adult hair cycle, TGF-β2 is synthesized in the dermal papilla by thestimulation of dihydrotestosterone at the initiation of catagen, triggering the intrinsic caspase
network and subsequent apoptotic cell death of hair follicle epithelial cells [35, 69]. In
contrast, during hair development, TGF-β2 receptors are focally expressed initially in theplacode and subsequently in the outer root sheath [71], and TGF-β2 exerts its morphogenetic
function [31] through transient induction of the transcriptional factor Snail in the hair bud[70]. These highly elaborate spatio-temporal manners of expression suggest critical roles of
the TGF-β ligand-receptor system in hair folliculogenesis. In our study, phosphorylation of
SMAD2 and expression of SMAD2 targeted gene products (SMAD7 and PAI-1) were seen in13/29the epithelium of generated follicles but not in epithelium in which folliculogenesis was not
induced despite the adjacent localization of transplanted hDPCs; together this suggests that
the SMAD2-mediated signal may be required for generated hair follicle maturation.
The hair folliculogenesis in our animal models depends on the epithelial-mesenchymal
interaction, mimicking fetal hair follicle morphogenesis, not transition to anagen in the adulthair cycle. Suppression of hair folliculogenesis in this study by inhibition of TGF-β2 signaltransduction both at the receptor and ligand level may reflects TGF-β signaling function
observed in fetal hair follicle morphogenesis [31]. Although careful considerations should begiven to differences between our animal models and normal physiological conditions [72],
our findings may draw attention to the underestimated TGF-β2 function in hairfolliculogenesis and provide insights into clinical hair regeneration with expanded hDPCs.
In conclusion, TGF-β2 was specifically expressed in hDPCs at higher levels compared to
hDFs, and inhibition of TGF-β2 signal at either the ligand or receptor level impaired hairfolliculogenesis in an hDPC transplantation animal model. The vitamin D3 analogue promoted
TGF-β2 expression and ALP activity in hDPCs and may be a critical functional factor in
KCM in the enhancement and preservation of the hair inducing capacity of cultured hDPCs,
suggesting its potential use for treatment of alopecia with expanded hDPC transplantation.
The results of this study suggest a critical role for TGF-β2 and vitamin D3 signaling pathways
in hair folliculogenesis.

Acknowledgments
We gratefully acknowledge Prof. Kohei Miyazono (Department of Molecular Pathology,
University of Tokyo Graduate School of Medicine) for valuable suggestions on the analysis
of TGF-β signal transduction in vivo. Contract grant sponsor: Grants-in-Aid by the Japanese
Ministry of Education, Culture, Sports, Science, and Technology (MEXT); contract grant
numbers: B2-1730474 and B2-18591964.

14/29Downregulated

Upregulated

Table 1. Selected genes up- or down-regulated in human dermal papilla cells (hDPCs).
Signal intensities of gene expression in hDPCs were compared with those of human dermal
fibroblasts of the same individual, and the fold changes were expressed as log ratio values in
the right two columns (P2 and P8). Among the upregulated 34 genes in Supplemental Table 1,
11 genes showed a fold change of at least 2 (log ratio), with a decrease of the fold change
from P2 to P8 (“Upregulated”, upper panel). Among the downregulated 44 genes in
Supplemental Table 2, 5 genes showed a difference of at most -2 (log ratio)(“Downregulated”, lower panel).
Symbol
CCL2
MGC5618
G0S2
TFPI2
HNT
TGFB2
PRG1
HLA-C

Gene namechemokine (C-C motif) ligand 2
hypothetical protein MGC5618
G0/G1 switch 2
tissue factor pathway inhibitor 2
neurotrimintransforming growth factor, beta 2proteoglycan 1, secretory granulemajor histocompatibility complex, class I, C
fibroblast growth factor 7 (keratinocyte growth
FGF7
factor)tumor necrosis factor receptor superfamily,
TNFRSF10B member 10b
pentraxin-related gene, rapidly induced by IL-1
PTX3
beta
EGR1
early growth response 1
TK1
thymidine kinase 1, soluble
SGK
serum/glucocorticoid regulated kinase
DOK5
docking protein 5
CDC20 cell division cycle 20 homolog (S.
CDC20
cerevisiae)

P2
4.93
4.31
3.93
2.85
2.84
2.47
2.31
2.23

P8
1.98
1.20
2.05
1.40
1.01
1.49
2.19
1.29

2.10

1.50

2.10

1.08

2.00
-3.46
-2.46
-2.27
-2.09

1.12
-1.62
-2.57
-1.23
-1.14

-2.02 -2.86

15/29Figure legends
Figure 1
TGF-β2 mRNA is preferentially expressed in cultured human dermal papilla cells (hDPCs).
(A) Relative mRNA expression of hair follicle-related genes in cultured hDPCs. mRNA was
isolated from cultured hDPCs and hDFs of the same individual (passage 2: P2) and expression
levels were quantified by real-time PCR. The expression levels were normalized to GAPDHmRNA expression and shown as fold changes (hDPC/hDF). N = 3. (B) TGF-β2 mRNA
relative expression in cultured hDPCs and hDFs of the same individual at passage 2 (P2) or 8
(P8). mRNA expression levels were normalized to GAPDH mRNA expression and foldchanges to hDF (P2) were shown. N = 3. *Significant difference from hDFs (p < 0.01).
Figure 2
Effects of keratinocyte conditioned medium (KCM) on proliferation and TGF-β2 gene
expression in human dermal papilla cells (hDPCs).
(A) Integral cell numbers of hDPCs cultured in 10% DMEM or KCM. Human DPCs and DFs
from two different individuals (#1 and #2) were cultured in 10% DMEM or KCM up topassage 8. Each plot indicates the integral cell number (shown as fold increase from initial
seeding) at the time of each subculture. (B) TGF-β2 mRNA relative expression in hDPCs andhDFs (passage 2) cultured in 10% DMEM, KCM or AmniomaxTM II (AM). mRNA
expression levels were normalized to GAPDH mRNA expression and fold changes to hDF
were shown. N = 4. *Significant difference from hDFs (p < 0.01). **Significant difference
from hDPCs cultured in 10% DMEM (p < 0.05).
Figure 3
Influences of keratinocyte conditioned media (KCM) components and other factors on humandermal papilla cells (hDPCs) in vitro.
(A) Cytokine expression in human KCM. Each panel shows expression of each indicatedcytokine released by hDPCs cultured in serum-free DMEM (SF-DMEM), DMEM with 10%
fetal bovine serum (10% DMEM), and KCM harvested one or two weeks after the medium
switch (KCM-1w or KCM-2w). (B) TGF-β2 mRNA expression of hDPCs 48 hours after
supplementation of each factor. The data is shown as fold change compared to control media(10% DMEM). (C) TGF-β2 protein in hDPCs 48 hours after supplementation of each factor.
The data is shown as fold change compared to control media (10% DMEM). N = 3. (D)Alkaline phosphatase activity of cultured hDPCs 48 hours after supplementation of eachfactor. The data is shown in 10-14 g/cell equivalent. N = 3. (E) Relative cell number of hDPCs
measured by the MTT assay. The data is shown as fold change compared to control media(10% DMEM). N = 3.
Supplements and concentrations used in (B), (C), (D) and (E) were as follows: acidic FGF(aFGF, 100 ng/ml), basic FGF (bFGF, 100 ng/ml). BMP2 (100 ng/ml), IL-1β (100 ng/ml), IL6 (100 ng/ml), IL-8 (100 ng/ml), VEGF (100 ng/ml), PDGF-BB (100 ng/ml), NGF (100ng/ml), HB-EGF (100 ng/ml), MIP3α (100 ng/ml), MCP1 (100 ng/ml), IGF-1 (100 ng/ml),
ENA-78 (100 ng/ml), GRO-α (100 ng/ml), 1,25(OH)2D3 (VD, 100 nM), ascorbic acid (VC,
100 μM), cholesterol sulfate (CS, 100 μM), all-trans retinoic acid (ATRA, 10 nM), 17βestradiol (10 nM), and dihydrotestosterone (DHT, 10 nM).
Figure 4

16/29
1,25-dihydroxyvitamin D3 (1,25(OH)2D3) upregulates TGF-β2 expression in human dermal
papilla cells (hDPCs).
(A) Quantitative real-time PCR detection of TGF-β2 mRNA expression in hDPCs (passage 2)
cultured for 24 or 48 hours in the presence of various concentrations (0, 0.1, 1, 10, 100, 1000nM) of 1,25(OH)2D3. The data are shown as fold changes to the base-line expression (at 0nM). N = 5. *Significant difference from base-line expression (p < 0.05). (B) Quantitative
real-time PCR detection of TGF-β2 mRNA expression in hDPCs (passage 2) at various times
of incubation (0.5, 2, 4, 8, 24, 48) in the presence of 100 nM of 1,25(OH)2D3. The data areshown as the fold changes to the base-line expression (at 0 hour). N = 3. *Significant
difference between each pair (p < 0.05). **Significant difference from base-line (p < 0.05).
(C) Quantitative detection by ELISA of TGF-β2 protein secretion from cultured hDPCs(passage 2, normalized to 106 cells equivalent, 3 days incubation) in the presence of various
concentrations of 1,25(OH)2D3 (0, 1, 10, 100 nM). *Significant difference from 0 nM (p <0.05).
Figure 5
Histological analysis of generated chimeric hair follicles in sandwich models with or without
TGF-β signal inhibition.
Cell sheets of human dermal papilla cells (hDPCs) were inserted between the dermis andepidermis of rat foot pad skin. The sandwiched construct was transplanted into the subcutis ofnude mice (see Fig. 1), and harvested 4 weeks after transplantation. (A) The bulb region of agenerated hair follicle. Left: Hematoxylin and eosin staining (HE staining), middle and right:
immunohistochemical stainings for TGF-β1 and TGF-β2. Nuclear staining was performed
using Hoechst33342. Bars = 20 μm. (B) Histology of generated hair follicles with or without
SB431542-mediated inhibition of TGF-β signal. Generated hair follicles and boundaries areindicated with black arrowheads and dashed lines. The maturation stage is indicated besideeach follicle, e.g. ‘S2’, ‘S4’, or ‘S7’, and was evaluated according to the classification
proposed by Paus et al [21] after modification. (See Supplemental Table 3 and Supplemental
Figure 2 for details). Hematoxylin and eosin staining. Bars = 50 μm. (C) Number of generated
hair follicles per transplant. Hair follicle morphogenesis was significantly smaller in numberin the SB431542-administered mice compared to control mice. Four recipient mice were used
for each group; three hDPC sheet fragments were sandwiched for each mouse. *Significant
difference from the control mice (p < 0.05). (D) Maturation stage data of generated hairfollicles with or without SB431542-mediated inhibition of TGF-β signal. Note the decreased
distribution of well-maturated hair follicles (S5 to S7) in the SB431542-administered mice.
Figure 6
Neutralizing antibodies against TGF-βs impair hair folliculogenesis in sandwich models.
(A) Histology of generated hair follicles with or without inhibition of TGF-β signal byneutralizing antibody against TGF-β2 or TGF-β1/2/3. Generated hair follicles and theirboundaries are indicated with black arrowheads and dashed lines. The maturation stage isindicated beside each follicle, e.g. ‘S2’, ‘S4’, or ‘S7’, and was evaluated according to theclassification proposed by Paus et al [21] after modification. (See Supplemental Table 3 andSupplemental Figure 2 for details). Hematoxylin and eosin staining. Bars = 50 μm. (B)
Number of generated hair follicles per transplant. Hair follicle morphogenesis was
significantly smaller in number in mice treated with anti-TGF-β2 antibody or anti-TGF-β1/2/3
antibody than in those treated with control IgG. Five recipient mice were used for each group;
three hDPC sheet fragments were sandwiched for each mouse. *Significant difference from

17/29the control mice (p < 0.05). (C) Maturation stage data of hair follicles generated in mice withadministration of anti-TGF-β2 or anti-TGF-β1/2/3 neutralizing antibody or nonspecificcontrol IgG.
Figure 7
Histological analysis of hair folliculogenesis in chamber models with or without TGF-β2
signal inhibition.
Cultured human dermal papilla cells (hDPCs) and cultured keratinocytes derived fromnewborn BL6 mice (mKC) were mixed as a cell suspension and implanted into the chamberon the back of nude mice. Skin samples were harvested 4 weeks after cell transfer; hairfolliculogenesis was evaluated with histological sections. Four chambers were prepared onfour mice per group. (A) Histology (HE staining) and macroscopic views (inset). Recipientnude mice were treated with a neutralizing antibody against TGF-β2 (top) or a negative
control IgG (bottom). Bars = 200 μm (HE staining) or 2 mm (inset). (B) Average number ofgenerated hair follicles per section (N = 4). Hair follicle morphogenesis was significantly
smaller in number in mice treated with anti-TGF-β2 antibody than those treated with control
IgG. *Significant difference from control mice (p < 0.05).
Figure 8
Immunohistochemical staining for TGF-β signal transduction related factors in sandwichmodels.
Cell sheet fragments of human dermal papilla cells (hDPCs) were placed between the dermis
and epidermis of rat foot pad skin; the sandwiched transplant was then inserted into thesubcutis of a nude mouse and harvested 4 weeks after transplantation. SB431542, anti-TGFβ2 neutralizing antibody, or vehicle was administered to the nude mice during the four weeks.
Each sample was serially sectioned and stained for HE or immunostained against
phosphorylated SMAD2 (pSMAD2) and two SMAD2 target genes (SMAD7 and PAI-1). Hairfollicle maturation stages were indicated in the HE images as “S2” or “S6”. Representative
images are also shown in which hDPCs were present but folliculogenesis was absent (shown
as “No follicle”). Immunostaining for pSMAD2, SMAD7 and PAI-1 were visualized in green
fluorescence, red color indicates DiI-labeled hDPCs, and Hoechst33342 was used for nuclear
staining. In vehicle-administered control mice, positive pSMAD2 signal was detected
predominantly in the nuclei and positive SMAD7 signals were located in the nuclei and/or
cytoplasm of epithelial cells (arrowheads). PAI-1 signal was detected in the cytoplasm ofepithelial cells and/or interstitial spaces in generated hair follicles (arrowheads). Similarfindings were also observed in well-matured generated follicles, but no signals for pSMAD2,
SMAD7 and PAI-1 were found in the “No follicle” area. White dotted lines indicate theboundary of hair follicles and asterisks (*) indicate non-specific fluorescence in the stratum
corneum. Insets are single-immunostained images for pSMAD2, SMAD7, or PAI-1. Whitebar = 50 μm, yellow bar = 10 μm.

18/29Supplemental contents
Supplemental Materials and Methods
TGF-β2 releasing gelatin hydrogel microsphere beads
Gelatin hydrogel microsphere beads (GMBs) containing 1 μg TGF-β2 in 40 μl of PBS or PBS
alone were prepared according to the previously reported method [Hiraoka, et al. Tissue Eng.
2006; 12:1475-87]. hDPC sheet fragments were inserted between the epidermis and dermis of
rat foot pad with 2 μl of GMB solution on top of a hDPC sheet. The sandwich transplants
were transferred to the subcutis of nude mice, harvested 4 weeks after transplantation andprocessed for histological examination of hair folliculogenesis.
Supplemental Figure 1
Animal assays for hair folliculogenesis.
(A) Sandwich model. Human dermal papilla cells (hDPCs) cultured at full confluence were
scraped off and cut into pieces. The follicular foot pad skin of a F344 rat was cut and digested
with a medium supplemented with DispaseTM to separate the epidermis from the dermis. After
placement of the hDPC sheet between the epidermis and dermis, the sandwiched transplant
was inserted into the subcutis of a Balb/c nude mouse. (B) Chamber model. The chamber wastransplanted on the back of a Balb/c nude mouse prior to cell transfer. Human DPCs andkeratinocytes derived from newborn BL6 mice (mKC) were cultured, combined in a cellsuspension and transplanted into the chamber.
Supplemental Figure 2
Representative examples of generated hair follicles at various stages.
Maturation of generated hair follicles was classified according to the scale (8 stages from S1
to S8) proposed by Paus et al. [21] after modification, as listed in Supplemental Table 3.
Hematoxylin and eosin staining. Bars = 50 μm.
DP: dermal papilla; HS: hair shaft; IRS: inner root sheath; SG: sebaceous gland.
Supplemental Figure 3
Influence of serum supplementation on TGF-β2 mRNA expression in cultured human dermal
papilla cells (hDPCs)
mRNA was isolated from hDPCs cultured for 48 hours in the presence of 1,25dihydroxyvitamin D3 (1,25(OH)2D3, 100 nM) or IL-1β (100 ng/ml) with or without 10%
serum (fetal bovine serum). TGF-β2 mRNA expression was assessed by real-time PCR andrelative expression levels normalized by GAPDH expression are shown. Serumsupplementation did not influence TGF-β2 mRNA upregulation by 1,25(OH)2D3 or IL-1β. N= 4. *p < 0.05, **p < 0.01.
Supplemental Figure 4
Histological analysis of generated murine hair follicles in chamber models with or without
TGF-β signal inhibition.
Fresh dermal cells and fresh keratinocytes isolated from fetal BL6 mice were mixed as a cellsuspension and implanted into the chamber on the back of a nude mouse. Skin samples were
harvested 4 weeks after cell transfer, and hair folliculogenesis was evaluated with histologicalsections. Four chambers were prepared on four mice per group. (A) Histology (HE staining)and macroscopic views (inset) of samples treated with SB431542 (top) or vehicle (bottom).

19/29
Bars = 200 μm (HE staining) or 2 mm (inset). (B) Average number of generated hair follicles
per section. N = 4. SB431542 substantially decreased the average number of regenerated
follicles, though the difference between mice treated with SB431542 and the control mice was
not significant (p = 0.075). (C) Histology (HE staining) and macroscopic views (inset) of
samples treated with a neutralizing antibody against TGF-β2 (top) or a negative control IgG(bottom). (D) Average number of generated hair follicles per section. N = 4. TGF-β2
decreased the average number of regenerated follicles, though the difference between micetreated with TGF-β2 and the control mice was not significant (p = 0.117). Bars = 200 μm (HEstaining) or 2 mm (inset).
Supplemental Figure 5
Effects of augmented TGF-β2 signal on hair folliculogenesis.
TGF-β2 signal was augmented by implanting TGF-β2 releasing gelatin hydrogel microspherebeads (GMBs) together with sheets of DiI-labeled cultured human dermal papilla cells(hDPCs) in our sandwich models; after placement of the hDPC sheet between the epidermis
and dermis of rat foot pad skin, the sandwiched transplant was inserted into the subcutis of a
Balb/c nude mouse and harvested 4 weeks after transplantation. GMBs with PBS alone wereused as a negative control. (A) Histology of sandwich model samples with or without TGF-βsignal augmentation. Serial sections were stained for HE (Hematoxilin and eosin) or
Hoechst33342. GMBs and DiI-labeled hDPCs were observed in yellow (due to non-specificfluorescence) and red under a fluorescent microscope, respectively. Asterisks (*) indicate
GMBs. Bar = 50 μm. (B, C) Maturation stage (B) and number per sample (C) of generated
hair follicles (TGF-β2-GMBs: n=9, PBS-GMBs, n=10). No significant difference inmaturation or number of generated hair follicles between the two groups (p = 0.283).

Supplemental Table 1. Genes upregulated in human dermal papilla cells (hDPCs) both atpassage 2 (P2) and passage 8 (P8) in comparison with human dermal fibroblasts (hDFs).
Signal intensities of gene expression in hDPCs were compared with those in hDFs of thesame individual, and the fold changes were expressed as log ratio values in the right twocolumns of P2 and P8. There were 567 upregulated genes in hDPCs at P2 with a difference ofat least 1 (log ratio), and 143 genes in hDPCs at P8. Thirty-four genes in P2 and P8
overlapped and are shown in order of the fold change at P2.
Supplemental Table 2. Genes downregulated in human dermal papilla cells (hDPCs) both atpassage 2 (P2) and passage 8 (P8) in comparison with human dermal fibroblasts (hDFs).
Signal intensities of gene expression in hDPCs were compared with those in hDFs of thesame individual, and the fold changes were expressed as log ratio values in the right twocolumns of P2 and P8. There were 498 downregulated genes in hDPCs at P2 with a differenceof at most -1 (log ratio), and 174 genes in hDPCs at P8. Forty-four genes in P2 and P8
overlapped and are shown in order of the fold change at P2.
Supplemental Table 3. Classification scale of generated hair follicles.
The classification was originally proposed by Paus et al [21]. Maturation stage is classified
into 8 categories (S1-S8) according to histological features. See Supplemental Figure 1 forrepresentative histological views.

20/29References
1. Millar SE. Molecular mechanisms regulating hair follicle development. J Invest Dermatol.
2002; 118:216-25.
2. Cotsarelis G. Epithelial stem cells: a folliculocentric view. J Invest Dermatol. 2006; 126:1459-68.
3. Fuchs, E. Scratching the surface of skin development. Nature. 2007; 445: 834-42.
4. Stenn KS, Paus R. Controls of hair follicle cycling. Physiol Rev 2001; 81: 449-94.
5. Cohen J. The transplantation of individual rat and guinea pig whisker papillae. J Embryol
Exp Morphol. 1961; 9: 117-27.
6. Jahoda CAB, Horne KA, Oliver RF. Induction of hair growth by implantation of cultured
dermal papilla cells. Nature. 1984; 311: 560-2.
7. Jahoda CAB. Induction of follicle formation and hair growth by vibrissa dermal papillae
implanted into rat ear wounds: vibrissa-type fibres are specified. Development. 1992; 115:
1103-9.
8. Stenn KS, Cotsarelis G. Bioengineering the hair follicle: fringe benefits of stem cell
technology. Curr Opin Biotechnol. 2005; 16: 493-7.
9. Philpott M, Paus R. Principles of hair follicle morphogenesis. In: Chuong CM, editor.
Molecular Basis of Epithelial Appendage Morphogenesis. Austin: Landes; 1998. p. 75-110.
10. Oro AE, Scott MP. Splitting hairs: dissecting roles of signaling systems in epidermal
development. Cell.1998; 95: 575-8.
11. Rendl M, Polak L, Fuchs E. BMP signaling in dermal papilla cells is required for their
hair follicle-inductive properties. Genes Dev. 2008; 22: 543-57.
12. Kishimoto J, Burgeson RE, Morgan BA. Wnt signaling maintains the hair-inducing
activity of the dermal papilla. Genes Dev. 2000; 14: 1181-5.
13. Randall VA, Sundberg JP, Philpott MP. Animal and in vitro models for the study ofhair follicles. J Invest Dermatol Sym Proc. 2003; 8: 39-45.
14. Midorikawa T, Chikazawa T, Yoshino T, Takada K, Arase S. Different gene
expression profile observed in dermal papilla cells related to androgenic alopecia by DNA
macroarray analysis. J Dermatol Sci. 2004; 36: 25-32.
15. Rutberg SE, Kolpak ML, Gourley JA, Tan G, Henry JP, Shander D. Differences in
expression of specific biomarkers distinguish human beard from scalp dermal papilla cells. J
Invest Dermatol. 2006; 126: 2583-95.

21/2916. Inamatsu M, Matsuzaki T, Iwanari H, Yoshizato K. Establishment of rat dermal
papilla cell lines that sustain potency to induce hair follicles from afollicular skin. J Invest
Dermatol. 1998; 111: 767-75.
17. Uchi H, Terao H, Koga T, Furue M. Cytokines and chemokines in the epidermis. J
Dermatol Sci. 2000; 24: S29-38.
18. Sebastiani S, Albanesi C, De Pita O, Puddu P, Cavani A, Girolomoni G. The role ofchemokines in allergic contact dermatitis. Arch Dermatol Res. 2002; 293: 552-9.
19. Reynolds AJ, Jahoda CAB. Cultured dermal papilla cells induce follicle formation andhair growth by transdifferentiation of an adult epidermis. Development. 1992; 115: 587-93.
20. Inoue K, Kato H, Sato T, Osada A, Aoi N, Suga H, Eto H, Gonda K, Yoshimura K.
Evaluation animal models for the hair inducing capacity of cultured human dermal papillacells. Cells Tissues Organs, in press.
21. Paus R, Muller-Rover S, van der Veen C, Maurer M, Eichmuller S, Ling G,
Hofmann U, Foitzik K, Mecklenburg L, Handjiski B. A comprehensive guide for therecognition and classification of distinct stages of hair follicle morphogenesis. J Invest
Dermatol. 1999; 113: 523-32.
22. Weinberg WC, Goodman LV, George C, Morgan DL, Ledbetter S, Yuspa SH, Lichti
U. Reconstitution of hair follicle development in vivo: determination of follicle formation,
hair growth, hair quality by dermal cells. J Invest Dermatol. 1993; 100: 229-36.
23. Kishimoto J, Ehama R, Wu L, Jiang S, Jiang N, Burgeson RE. Selective activation of
the versican promoter by epithelial-mesenchymal interactions during hair follicle
development. Proc Natl Acad Sci USA. 1999; 96: 7336-41.
24. Itami S, Kurata S, Sonoda T, Takayasu S. Characterization of 5α-reductase in cultured
human dermal papilla cells from beard and occipital scalp hair. J Invest Dermatol. 1991; 96:
57-60.
25. Trautman MS, Kimelman J, Bernfield M. Developmental expression of syndecan, an
integral membrane proteoglycan, correlates with cell differentiation. Development. 1991; 111:
213-20.
26. Couchman JR. Hair follicle proteoglycans. J Invest Dermatol. 1993; 101: S60-4.
27. Shimaoka S, Tsuboi R, Jindo T, Imai R, Takamori K, Rubin JS, Ogawa H.
Hepatocyte growth factor/scatter factor expressed in follicular papilla cells stimulates humanhair growth in vitro. J Cell Physiol. 1995; 165: 333-8.
28. Rosenquist TA, Martin GR. Fibroblast growth factor signaling in the hair growth cycle:
expression of the fibroblast growth factor receptor and ligand genes in the murine hairfollicle. Dev Dyn. 1996; 205: 379-86.

22/2929. Almond-Roesler B, Schön, M, Schön MP, Blume-Peytavi U, Sommer C, Löster K,
Orfanos CE. Cultured dermal papilla cells of the rat vibrissa follicle. Proliferative activity,
adhesion properties and reorganization of the extracellular matrix in vitro. Arch Dermatol
Res. 1997; 289: 698-704.
30. Kozlowska U, Blume-Peytavi U, Kodelja V, Sommer C, Goerdt S, Majewski S,
Jablonska S, Orfanos CE. Expression of vascular endothelial growth factor (VEGF) in
various compartments of the human hair follicle. Arch Dermatol Res. 1998; 290: 661-8.
31. Foitzik K, Paus R, Doetschman T, Dotto GP. The TGF-β2 isoform is both a required
and sufficient inducer of murine hair follicle morphogenesis. Dev Biol. 1999; 212: 278-89.
32. Inui S, Fukuzato Y, Nakajima T, Yoshikawa K, Itami S. Androgen-inducible TGF-β1
from balding dermal papilla cells inhibits epithelial cell growth: a clue to understandparadoxical effects of androgen on human hair growth. FASEB J. 2002; 16: 1967-9.
33. Bayer-Garner IB, Sanderson RD, Smoller BR. Syndecan-1 is strongly expressed in theanagen hair follicle outer root sheath and in the dermal papilla but expression diminishes withinvolution of the hair follicle. Am J Dermatopathol. 2002; 24: 484-9.
34. Kamp H, Geilen CC, Sommer C, Blume-Peytavi U. Regulation of PDGF and PDGF
receptor in cultured dermal papilla cells and follicular keratinocytes of the human hair follicle.
Exp Dermatol. 2003; 12: 662-72.
35. Foitzik K, Spexard T, Nakamura M, Halsner U, Paus P. Towards dissecting thepathogenesis of retinoid-induced hair loss: all-trans retinoic acid induces premature hairfollicle regression (catagen) by upregulation of transforming growth factor-β2 in the dermal
papilla. J Invest Dermatol. 2005; 124:1119-26.
36. Iino M, Ehama R, Nakazawa Y, Iwabuchi T, Ogo M, Tajima M, Arase S. Adenosine
stimulates fibroblast growth factor-7 gene expression via adenosine A2b receptor signaling indermal papilla cells. J Invest Dermatol. 2007; 127: 1318-25.
37. Bikle DD, Nemanic MK, Gee E, Elias P. 1,25-Dihydroxyvitamin D3 production byhuman keratinocytes kinetics and regulation J Clin Invest. 1986; 78: 557-66.
38. Pillai S, Bikle DD, Elias PM. 1,25-Dihydroxyvitamin D production and receptor bindingin human keratinocytes varies with differentiation. J Biol Chem. 1988; 15: 5390-5.
39. Lachgar S, Moukadiri H, Jonca F, Charveron M, Bouhaddioui N, Gall Y, Bonafe JL,Plouët J. Vascular endothelial growth factor is an autocrine growth factor for hair dermal
papilla cells. J Invest Dermatol. 1996; 106: 17-23.
40. Goodman LV, Ledbetter SR. Secretion of stromelysin by cultured dermal papilla cells:
differential regulation by growth factors and functional role in mitogen-induced cellproliferation. J Cell Physiol. 1992; 151: 41-9.

23/2941. Dooley TP. Molecular biology of the human cytosolic sulfotransferase gene superfamily
implicated in the bioactivation of minoxidil and cholesterol in skin. Exp Dermatol. 1999; 8:
328-9.
42. Randall VA, Hibberts NA, Thornton MJ, Hamada K, Merrick AE, Kato S, Jenner
TJ, De Oliveira I, Messenger AG. The hair follicle: a paradoxical androgen target organ.
Horm Res. 2000; 54: 243-50.
43. Yano K, Brown LF, Detmar M. Control of hair growth and follicle size by VEGFmediated angiogenesis. J Clin Invest. 2001; 107: 409-17.
44. Edmondson SR, Thumiger SP, Werther GA, Wraight CJ. Epidermal homeostasis: therole of the growth hormone and insulin-like growth factor systems. Endocr Rev. 2003; 24:
737-64.
45. Osada A, Iwabuchi T, Kishimoto J, Hamazaki TS, Okochi H. Long-term culture ofmouse vibrissal dermal papilla cells and de novo hair follicle induction. Tissue Eng. 2007; 13:975-82.
46. Paus R, Arck P, Tiede S. (Neuro-)endocrinology of epithelial hair follicle stem cells.
Mol Cell Endocrinol. 2008; 288: 38-51.
47. Iida M, Ihara S, Matsuzaki T. Hair cycle-dependent change of alkaline phosphataseactivity in the mesenchyme and epithelium in mouse vibrissal follicles. Develop GrowthDiffer. 2007; 49: 185-95.
48. Hardy MH. The secret life follicle of the hair. Trends Genet. 1992; 8: 55-61.
49. Laping NJ, Grygielko E, Mathur A, Butter S, Bomberger J, Tweed C, Martin W,
Fornwald J, Lehr RHJ, Gaster L, Callahan JF, Olsen BA. Inhibition of transforming
growth factor (TGF)-β1-induced extracellular matrix with a novel inhibitor of the TGF-β type
I receptor kinase activity: SB-431542. Mol Pharmacol. 2002; 62: 58-64.
50. Miyazono K, ten Dijke P, Hedlin C-H. TGF-β signaling by SMAD proteins. Adv.
Immunol. 2000; 75: 115-57.
51. Oyama N, Iwatsuki K, Satoh M, Akiba H, Kaneko F. Dermal fibroblasts are one of thetherapeutic targets for topical application of 1α,25-dihydroxyvitamin D3: the possible
involvement of transforming growth factor-β induction. Brit J Dermatol. 2000; 143: 1140-8.
52. Yanagi Y, Suzawa M, Kawabata M, Miyazono K, Yanagisawa J, Kato S. Positive andnegative modulation of vitamin D receptor function by transforming growth factor-β signaling
through Smad proteins. J Biol Chem. 1999; 274: 12971-4.
53. Cao Z, Flanders KC, Bertolette D, Lyakh LA, Wurthner JU, Parks WT, Letterio JJ,
Ruscetti FW, Roberts AB. Levels of phospho-Smad2/3 are sensors of the interplay between
effects of TGF-β and retinoic acid on monocytic and granulocytic differentiation of HL-60
cells. Blood. 2003; 101: 498-507.

24/2954. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB. Targeted
ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II
with alopecia. Proc Natl Acad Sci U.S.A. 1997; 94: 9831-5.
55. Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T,
Alioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, Kato S. Micelacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia andgrowth retardation after weaning. Nat Genet. 1997; 16: 391-6.
56. Malloy PJ, Hochberg Z, Tiosano D, Pike JW, Hughes MR, Feldman D. The molecularbasis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families. J Clin
Invest. 1990; 86: 2071-9.
57. Arita K, Nanda A, Wessagowit V, Akiyama M, Alsaleh QA, McGrath JA. A novel
mutation in the VDR gene in hereditary vitamin D-resistant rickets. Br J Dermatol. 2008;
158: 168-71.
58. Reichrath J, Schilli M, Kerber A, Bahmer FA, Czarnetzki BM, Paus R. Hair follicle
expression of 1,25-dihydroxyvitamin D3 receptors during the murine hair cycle. Br J
Dermatol. 1994; 131: 477-82.
59. Skorija K, Cox M, Sisk JM, Dowd DR, MacDonald DP, Thompson CC, Demay MB.
Ligand-independent actions of the vitamin D receptor maintain hair follicle homeostasis. Mol
Endocrinol. 2005; 19: 855-62.
60. Sakai Y, Kishimoto J, Demay MB. Evaluation of keratinocyte proliferation anddifferentiation in vitamin D receptor knockout mice. Endocrinology. 2000; 141: 2043-9.
61. Sutton ALM, MacDonald PN. Vitamin D: More than a “bone-a-fide” hormone. Mol
Endocrinol. 2003; 17: 777-91.
62. Reichrath J. Vitamin D and the skin: an ancient friend, revisited. Exp Dermatol. 2007;
16: 618-25.
63. Massagué J. How cells read TGF-beta signals. Nat Rev Mol Cell Biol. 2000; 1: 169-78.
64. Schmid P, Cox D, Bilbe G, Maier R, McMaster GK. Differential expression of TGF β1,
β2, β3 genes during mouse embryogenesis. Development. 1991; 111: 117-30.
65. Millan FA, Denhez F, Kondaiah P, Akhurst RJ. Embryonic gene expression patterns of
TGFβ1, β2 and β3 suggest different developmental functions in vivo. Development. 1991;
111: 131-43.
66. Foitzik K, Lindner G, Muller-Rover S, Maurer B, Botchkareva N, Botchkarev V,
Handjiski B, Metz M, Hibino T, Soma T, Dotto GP, Paus R. Control of murine hairfollicle regression (catagen) by TFG-β1 in vivo. FASEB J. 2000; 14: 752-60.

25/29
67. Philpott MP, Green MR, Kealey T. Human hair growth in vitro. J Cell Sci. 1990; 97:463-71.
68. Weinberg WC, Brown PD, Stetler-Stevenson WG, Yuspa SH. Growth factors
specifically alter hair follicle cell proliferation and collagenolytic activity alone or in
combination. Differentiation. 1990; 45: 168-78.
69. Hibino T, Nishiyama T. Role of TGF-β2 in the human hair cycle. J Dermatol Sci. 2004;
35: 9-18.
70. Jamora C, Lee P, Kocieniewski P, Azhar M, Hosokawa R, Chai Y, Fuchs E. A
signaling pathway involving TGF-β2 and snail in hair follicle morphogenesis. PLoS Biol.
2004; 3: e11.
71. Paus R, Foitzik K, Welker P, Bulfone-Paus S, Eichmuller S. Transforming growth
factor-β, receptor type I and type II expression during murine hair follicle development andcycling. J. Invest. Dermatol. 1997; 109: 518-26.
72. Inamatsu M, Tochio T, Makabe A, Endo T, Oomizu S, Kobayashi E, Yoshizato K.
Embryonic dermal condensation and adult dermal papilla induce hair follicles in adultglabrous epidermis through different mechanisms. Develop Growth Differ. 2006; 48: 73-86.

26/29Supplemental contents
Supplemental Table 1.
Symbol
CCL2
MGC5618
G0S2
TFPI2
HNT
TGFB2
PRG1
HLA-C
FGF7
TNFRSF10B
PTX3
CCPG1
NTN4
HLA-G
ANTXR2EDIL3FBN2IGFBP7
GCLM
RPL37
HIST1H2BK
FLJ11259
C9orf5
GNPTAB
ATP10D
SEMA3CH2BFS
KLF3ADAMTS5
DPP4
LAMA2
RSNL2
BACH1
TGM2

Gene namechemokine (C-C motif) ligand 2
hypothetical protein MGC5618
G0/G1 switch 2
tissue factor pathway inhibitor 2
neurotrimintransforming growth factor, beta 2proteoglycan 1, secretory granulemajor histocompatibility complex, class I, C
fibroblast growth factor 7 (keratinocyte growth factor)
tumor necrosis factor receptor superfamily, member 10b
pentraxin-related gene, rapidly induced by IL-1 beta
cell cycle progression 1
netrin 4
HLA-G histocompatibility antigen, class I, G
anthrax toxin receptor 2
EGF-like repeats and discoidin I-like domains 3
fibrillin 2 (congenital contractural arachnodactyly)
insulin-like growth factor binding protein 7
glutamate-cysteine ligase, modifier subunit
ribosomal protein L37
histone 1, H2bk
hypothetical protein FLJ11259
chromosome 9 open reading frame 5
N-acetylglucosamine-1-phosphate transferase, alpha and beta
subunits
ATPase, Class V, type 10Dsema domain, immunoglobulin domain (Ig), short basic domain,
secreted, (semaphorin) 3C
H2B histone family, member S
Kruppel-like factor 3 (basic)
ADAM metallopeptidase with thrombospondin type 1 motif, 5(aggrecanase-2)
dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing
protein 2)
laminin, alpha 2 (merosin, congenital muscular dystrophy)
restin-like 2
BTB and CNC homology 1, basic leucine zipper transcription
factor 1
transglutaminase 2 (C polypeptide, protein-glutamine-gammaglutamyltransferase)

P2
4.93
4.31
3.93
2.85
2.84
2.47
2.31
2.23
2.10
2.10
2.00
1.97
1.86
1.85
1.80
1.66
1.63
1.62
1.58
1.58
1.57
1.56
1.35

P8
1.98
1.20
2.05
1.40
1.01
1.49
2.19
1.29
1.50
1.08
1.12
1.40
2.18
1.04
1.15
1.58
3.14
1.16
1.15
1.13
1.40
1.71
1.07

1.32

1.35

1.23

1.41

1.23

1.65

1.23
1.22

1.23
1.13

1.16

1.20

1.09

1.01

1.08
1.07

1.02
1.37

1.03

1.05

1.03

1.41

27/29Supplemental Table 2.
Symbol
EGR1
TK1
SGK
DOK5
CDC20
LBR
CDKN3
CDCA3
CCNB1
MCM5
LRRC15CCNA2TPX2LOC283824
GPSM2
KIF2C
CEP55
BM039
UHRF1
ASPM
BIRC5
TOP2A
DTYMK
CCNB2
RRM2
TYMS
MCM6
DDX39
ANLN
U2AF2
ANP32EWHSC1ZWILCH
PRC1
C6orf173
H2AFX
NUSAP1
HMGB2SUV39H2
KIAA0101DNM3
SHCBP1

Gene nameearly growth response 1
thymidine kinase 1, soluble
serum/glucocorticoid regulated kinase
docking protein 5
CDC20 cell division cycle 20 homolog (S. cerevisiae)
lamin B receptor
cyclin-dependent kinase inhibitor 3 (CDK2-associated dual
specificity phosphatase)cell division cycle associated 3
cyclin B1
MCM5 minichromosome maintenance deficient 5, cell division
cycle 46 (S. cerevisiae)
leucine rich repeat containing 15
cyclin A2
TPX2, microtubule-associated, homolog (Xenopus laevis)
hypothetical protein LOC283824
G-protein signalling modulator 2 (AGS3-like, C. elegans)kinesin family member 2C
centrosomal protein 55kDa
uncharacterized bone marrow protein BM039
ubiquitin-like, containing PHD and RING finger domains, 1
asp (abnormal spindle)-like, microcephaly associated(Drosophila)baculoviral IAP repeat-containing 5 (survivin)topoisomerase (DNA) II alpha 170kDa
deoxythymidylate kinase (thymidylate kinase)cyclin B2
ribonucleotide reductase M2 polypeptide
thymidylate synthetase
MCM6 minichromosome maintenance deficient 6 (MIS5homolog, S. pombe) (S. cerevisiae)
DEAD (Asp-Glu-Ala-Asp) box polypeptide 39
anillin, actin binding protein (scraps homolog, Drosophila)
U2 (RNU2) small nuclear RNA auxiliary factor 2
acidic (leucine-rich) nuclear phosphoprotein 32 family,
member E
Wolf-Hirschhorn syndrome candidate 1
Zwilch, kinetochore associated, homolog (Drosophila)
protein regulator of cytokinesis 1
chromosome 6 open reading frame 173
H2A histone family, member X
nucleolar and spindle associated protein 1
high-mobility group box 2
suppressor of variegation 3-9 homolog 2 (Drosophila)
KIAA0101
dynamin 3
SHC SH2-domain binding protein 1

P2
-3.46
-2.46
-2.27
-2.09
-2.02
-1.99

P8
-1.62
-2.57
-1.23
-1.14
-2.86
-1.31

-1.94 -2.18-1.76 -2.23-1.54 -2.15-1.53 -2.26
-1.48
-1.47
-1.44
-1.44
-1.40
-1.39
-1.38
-1.37
-1.35

-2.52
-1.84
-1.64
-1.10
-1.52
-2.21
-2.14
-1.84
-2.04

-1.30 -2.24
-1.30
-1.29
-1.29
-1.27
-1.27
-1.27

-2.11
-2.51
-1.54
-2.15
-2.44
-1.97

-1.27 -1.03-1.23 -1.59-1.22 -2.00-1.22 -1.11-1.22 -1.50
-1.18
-1.18
-1.17
-1.17
-1.16
-1.16
-1.15
-1.15
-1.13
-1.12
-1.11

-1.53
-1.22
-2.07
-1.54
-1.54
-2.16
-1.61
-1.04
-2.08
-1.06
-2.00

28/29CSTF3NOLC1RACGAP1
THOC4
TMPO
RPA3

cleavage stimulation factor, 3' pre-RNA, subunit 3, 77kDa
nucleolar and coiled-body phosphoprotein 1
Rac GTPase activating protein 1
THO complex 4
thymopoietin
replication protein A3, 14kDa

-1.09
-1.09
-1.07
-1.03
-1.03
-1.02

-1.32
-1.00
-1.91
-1.03
-1.19
-1.52

29/29Supplemental Table 3.
Maturation stage
S1
S2
S3
S4
S5
S6
S7
S8

Histological feature
Epidermal thickening and dermal condensation
Downward elongation of hair-germ without dermal papillaformation
Formation of hair-peg-like structure with concaved basal bordersurrounding rounded dermal papilla
Formation of bulb in the proximal end of hair germ andformation of corn-shaped inner root sheath distal to the hairmatrixDistal elongation of internal root sheath and pseudokeratinization of inner root sheath
Formation of hair shaft and differentiation of sebocytes
Distal elongation of hair shaft into hair canal and formation of
sebaceous grandElongation of hair shaft through epidermis and proximal growthof hair follicle to subcutaneous layer