ARTICLE
Received 30 Aug 2011 | Accepted 12 Mar 2012 | Published 17 Apr 2012

DOI: 10.1038/ncomms1784

Fully functional hair follicle regeneration throughthe rearrangement of stem cells and their niches
Koh-ei Toyoshima1, Kyosuke Asakawa2, Naoko Ishibashi2, Hiroshi Toki1, Miho Ogawa3, Tomoko Hasegawa2,
Tarou Irié4, Tetsuhiko Tachikawa4, Akio Sato1,5,6, Akira Takeda1,7 & Takashi Tsuji1,2,3

Organ replacement regenerative therapy is purported to enable the replacement of organs
damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully
functional hair organ regeneration via the intracutaneous transplantation of a bioengineered
pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skinderived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered
hair follicle develops the correct structures and forms proper connections with surrounding
host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered
follicles also show restored hair cycles and piloerection through the rearrangement of follicular
stem cells and their niches. This study thus reveals the potential applications of adult tissuederived follicular stem cells as a bioengineered organ replacement therapy.

1 Research Institute for Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan. 2 Department of Biological Science andTechnology, Graduate School of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan. 3 Organ Technologies Inc.,Tokyo 101-0048, Japan. 4 Department of Oral Pathology, Showa University School of Dentistry, Tokyo 145-8515, Japan. 5 Tokyo Memorial Clinic, 151-0053,
Japan. 6 Department Regenerative Medicine, Plastic and Reconstructive Surgery, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374,
Japan. 7 Department of Plastic and Aesthetic Surgery, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan. Correspondence
and requests for materials should be addressed to T.T. (email: t-tsuji@rs.noda.tus.ac.jp).
nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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Onature communications | DOI: 10.1038/ncomms1784

rgan replacement regenerative therapy is expected to provide
novel therapeutic systems for donor organ transplantation,
which is an approach to treating patients who experienceorgan dysfunction as the result of disease, injury or aging1. Concepts in current regenerative therapy include stem cell transplantation and two-dimensional uniform cell sheet technologies, bothof which have the potential to restore partially lost tissue or organfunction2–4. The development of bioengineered ectodermal organs,such as teeth, salivary glands, or hair follicles may be achieved byreproducing the developmental processes that occur during organogenesis5–9. Ectodermal organs have essential physiological rolesand can greatly influence the quality of life by preventing the morbidity associated with afflictions such as caries and hypodontia inteeth10, hyposalivation in the salivary gland11, and androgenetic
alopecia, which affects the hair12. Recently, it has been proposedthat a bioengineered tooth can restore oral and physiological function through the transplantation of bioengineered tooth germ and abioengineered mature tooth unit, which would represent a successful organ-replacement regenerative therapy13.
The hair coat has important roles in thermoregulation, physical insulation, sensitivity to noxious stimuli, and social communication14. In the developing embryo, hair follicle morphogenesis
is regulated by reciprocal epithelial and mesenchymal interactions
that occur in almost all organs9,15,16. The hair follicle is divided into
a permanent upper region, which consists of the infundibulum andisthmus, and a variable lower region, which is the actual hair-shaft
factory that contains the hair matrix, differentiated epithelial cells
and dermal papilla (DP) cells15–17. DP cells are responsible for the
production of dermal-cell populations such as dermal sheath (DS)cells18, and they generate dermal fibroblasts and adipocytes19,20.
After morphogenesis, various stem cell types are maintained in certain regions of the follicle. For instance, follicle epithelial cells arefound in the follicle stem cell niche of the bulge region21,22; multipotent mesenchymal precursors are found in DP cells18,19; neuralcrest-derived melanocyte progenitors are located in the sub-bulge
region23–25, and follicle epithelial stem cells in the bulge region thatis connected to the arrector pili muscle15,26. The follicle variable
region mediates the hair cycle, which depends on the activation offollicle epithelial stem cells in the bulge stem cell niche during the
telogen-to-anagen transition27,28. This transition includes phasesof growth (anagen), apoptosis-driven regression (catagen)29 andrelative quiescence (telogen)17, whereas the organogenesis of mostorgans is induced only once during embryogenesis16.
To achieve hair follicle regeneration in the hair cycle, it isthought to be essential to regenerate the various stem cells and theirniches9,30. Many studies have attempted to develop technologies
to renew the variable lower region of the hair follicle31,32, toachieve de novo folliculogenesis via replacement with hair follicleinductive dermal cells33, and to direct the self-assembly of skinderived epithelial and mesenchymal cells34–39. We have also
reported that a bioengineered hair follicle germ, reconstituted fromembryonic follicle germ-derived epithelial and mesenchymal cells,
using our organ germ method, can generate a bioengineered hairfollicle and shaft7. However, it remains to be determined whether
the bioengineered hair follicle germ can generate a bioengineered
hair follicle and shaft by intracutaneous transplantation to provide
fully functional hair regeneration, including hair shaft elongation,
hair cycles, connections with surrounding tissues, and the regeneration of stem cells and their niches9,30.
Here we demonstrate fully functional orthotopic hair regeneration via the intracutaneous transplantation of bioengineered hairfollicle germ. The bioengineered hair has the correct structures of
the naturally occurring hair follicle and shaft, and it forms proper
connections with surrounding host tissues, such as the epidermis,
arrector pili muscle and nerve fibres. The bioengineered hair follicles
show full functionality, including the ability to undergo repeated

hair cycles through the rearrangement of various stem cell niches, aswell as responsiveness to the neurotransmitter acetylcholine (ACh).
Our current study thus demonstrates the potential for not only hair
regeneration therapy but also the realization of bioengineered organ
replacement using adult somatic stem cells.

Results
Hair follicle regeneration from a bioengineered follicle germ.
We first investigated whether bioengineered hair follicle germs,
reconstituted by our previously developed organ germ method,
could orthotopically erupt from a hair shaft with the proper tissue
structure and skin connections in the cutaneous environment of an
adult mouse (Fig. 1a). To regenerate a bioengineered pelage follicle
germ, the bioengineered pelage follicle germ was reconstituted
with E18 mouse embryonic back skin-derived epithelial andmesenchymal cells (Fig. 1b). The bioengineered vibrissa follicle
germ was regenerated using dissociated epithelial cells (1×104
cells) isolated from the adult vibrissa-derived bulge region, which
expressed CD49f and CD34 antigens (Fig. 1c), and primary cultured
DP cells (3×103 cells; Fig. 1d).
To prevent epithelial cyst formation and associations between theepithelium of the host skin and the bioengineered hair follicle germ,
we developed an inter-epithelial tissue-connecting plastic device
that used a nylon thread as a guide for the infundibulum direction via insertion into the bioengineered germ (Fig. 1b). Three days
after transplantation, the epithelial layer of the bioengineered germ
had extended along the guide to the host skin epithelium, and theresulting wound was allowed to heal by maintaining the eruptionof the guide, whereas the sample without a guide formed an epithelial cyst (Fig. 1e). At 14 days after the transplantation of the bioengineered germ, with or without the nylon thread into the dermal
layer, the eruption and growth of black hair shafts were observed
at a frequency of 94% or 38% (n = 78 or 8), respectively (Fig. 1e,f).
The bioengineered vibrissa follicle germ was also erupted using theinter-epithelial tissue-connecting plastic device at 20.5 ± 6.0 (n = 46)days, with a resulting frequency of 74% (n = 62; Fig. 1c). However,the bioengineered vibrissae produced unpigmented hairs with afrequency of 95.5% (n = 46; Fig. 1f).
Both the bioengineered pelage follicle and the vibrissa follicle
formed correct structures comprising an infundibulum and sebaceous gland in the proximal region as well as a hair matrix, hair shaft,inner root sheath, outer root sheath (ORS), DS and DP (Fig. 2a).
Each natural and bioengineered vibrissa follicle contained 500~1,000
DP cells (Fig. 2a). The bioengineered vibrissa follicle germs generated not only hair follicles in the variable region but also infundibulum and sebaceous gland structures in the permanent region, andin contrast to the bioengineered pelage follicle, the follicle-derived
cells did not distribute among the surrounding cutaneous tissues(Fig. 2a). DP cells in the bioengineered vibrissa follicle were also
found to express alkaline phosphatase (ALP) and versican, and thethin outermost dermal layer of the hair bulb was observed to express
α-smooth muscle actin (α-SMA; Fig. 2a). These results indicate thatthe bioengineered pelage and vibrissa follicle germs can regenerate
structurally correct follicles and hair shafts following intracutaneous transplantation.
Furthermore, the human bioengineered hair follicle germ, which
was composed of the dissociated bulge region-derived epithelial cells
and scalp hair follicle-derived intact DPs of an androgenetic alopecia
patient, grew a pigmented hair shaft in the transplantation area within
21 days after intracutaneous transplantation into the back skin of nude
mice (Fig. 2b). This bioengineered hair follicle formed the correct
structures, that is, an infundibulum and sebaceous gland in the proximal region as well as a hair matrix, hair shaft, inner root sheath, ORS,
DS, and a DP containing ~500 cells (Fig. 2b). By analysing the nuclear
morphology with Hoechst staining40, we confirmed that the cells inthe bioengineered hair follicle were of human origin (Fig. 2b).

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1784

ab
Embryonic
skin pelage

Epidermal
cells
Bulge
epithelial cells

Adultvibrissa

Dermal cells
Primary
DP cells

Intradermal

Epithelium

Epithelial
single cells
boiengineered
hair germ

Subrenal
capsuleMesenchymeceMacroscopy

ED18.5mouse back
skin

H&E

CD49f/Nuc.

Mesenchymal
single cells

Without guideDay 14

Day 0

With guideDay 3

Day 14

Day 0

Day 3

Day 14

Day 24

Day 0

Day 3

Day 25

Day 37

IF

H&E

IF

Bulge area
RW

CD34/Nuc.

1/2

GFP/Nuc.

RW

SB area

1/2

ALP/Nuc.

Phase contrast

ALP/Nuc.

Vibrissa

Pelagedf
Hair bulb

Figure 1 | Methods for hair follicle regeneration. (a) Schematic representation of the methods used for the generation and transplantation of abioengineered hair follicle germ. (b) Phase-contrast images of murine embryonic back skin, tissues, dissociated single cells and bioengineered hair follicle
germ, which was reconstituted using the organ germ method, with a nylon thread guide (arrow head). Scale bars, 200 µm. (c) Histological analyses of anatural vibrissa isolated from an adult mouse. Macroscopic and H & E staining of whole vibrissa is shown in left two panels. Dashed lines (red) via macromorphological observations (left) and H & E staining (right) indicate the interface of the bulge and SB region. Boxed area in the left panels that is H & E
stained to indicate the bulge, and SB area is shown at a higher magnifi cation in the right panel. The bulge region was immune-stained with anti-CD49f(red, left) and anti-CD34 (red, centre) antibodies and with Hoechst 33258 dye (blue). Black broken lines in high-magnified H & E indicate the interface ofepithelium of the hair follicle. IF, infundibulum; RW, ringwulst; 1/2, half portion of hair follicle. Scale bars, 100 µm. (d) Histological and ALP analyses of thebulb area of a natural vibrissa and of primary cultures of DP cells. Hair bulb (left two) and cultured DP cells (right two) were anlaysed via enzymatic stainingof ALP. The red dashed line indicates Auber’s line. Scale bars, 100 µm. (e) Longitudinal sections of bioengineered pelage during eruption and growth
processes mediated by an inter-epithelial tissue-connecting plastic device (with guide). The counterpart is shown as cyst formation with a bioengineered
pelage follicle at 14 days after intracutaneous transplantation (without guide). H & E staining (upper) and fluorescent microscopy (lower) of thebioengineered hair follicles at 0, 3 and 14 days after transplantation. Scale bars, 100 µm. (f) Macro-morphological observations of the bioengineered hairs
during the eruption and growth of bioengineered pelages (upper) and vibrissae (lower), including just after transplantation at day 0 (left), the healing of thewound at day 3 (centre), eruption of the hair shaft and growth at day 14 in the bioengineered pelage and 37 days in the vibrissa (right). Scale bars, 1.0 mm.

It is preferable for a clinically applicable hair regeneration therapy
to utilize autologous follicular unit transplantation (FUT), which is
the most popular treatment for androgenic alopecia41. To achieve
hair follicle regeneration at hair densities of 120 or 60–100 hairshafts cm − 2 of normal scalp or in FUT treatment41, respectively,
twenty-eight bioengineered hair germs were transplanted into acervical skin circle with a diameter of 1 cm. At 14 to 21 days after
transplantation, the bioengineered hairs were erupted at a highdensity of 124.0 ± 17.3 hair shafts cm − 2 (n = 3; Fig. 2c). These results
indicated that similar to FUT therapy, the transplantation of bioengineered hair follicle germs could be applied as a treatment for
androgenic alopecia.

Regulation of hair follicle number and hair shaft features. We
next analysed the formation of pelage follicles from the bioengineered follicle germ (Fig. 3a). These analyses demonstrated that the
bioengineered pelage follicles, which contain EGFP-labelled mousederived mesenchymal cells, could be induced to form autonomously
assembled cell aggregates with high-intensity green fluorescenceand alkaline phosphatase expression at the boundary between epidermal and mesenchymal cell layers after 2 days in an organ culture(Fig. 3a). After 3 days in an SRC, the bioengineered pelage follicle
germ faithfully reproduced the expression of required signalling
network genes, such as Shh, Wnt10b, Msx2, β-catenin, Versican,
Lef1, Bmp4 and Notch1, which have essential roles in early hairnature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1784

GFP/Nuc.

b

H&E

Macroscopy

H&E

Pelagea

Nuclei
Vibrissa

Human

ALP/GFP/Nuc.

α-SMA/GFP/
Nuc.

c

Bulb

Versican/GFP/
Nuc.

Mouse

Figure 2 | Hair follicle regeneration via the intracutaneous transplantation of a bioengineered hair follicle germ. (a) Histological and immunohisto­
chemical analyses of bioengineered pelage (upper) and vibrissa (middle) follicles. The boxed areas in the low-magnification H & E panels are shown at ahigher magnification in the right panels. The arrowhead indicates a sebaceous gland. Scale bars, 100 µm. The hair bulb of a bioengineered hair follicle was
immunostained with anti-versican (lower left) and α-SMA (arrowheads, lower right) antibodies and also enzymatically stained for ALP (lower centre).
Scale bars, 50 µm. (b) Bioengineered human hair produced by the transplantation of a human bioengineered hair follicle germ was reconstituted by bulgederived epithelial cells and the intact DPs of human scalp hair follicles. At 21 days after transplantation, the bioengineered human hair was photographed(microscopy) and analysed by H & E staining. The species identity of the bioengineered hair follicle was analysed, according to the nuclear morphologicalfeatures (right panels)40. The boxed areas are shown at a higher magnification in the insets. Scale bar, 500 µm for microscopy, 100 µm for H & E, and20 µm for nuclear staining. (c) High-density intracutaneous transplantation of the bioengineered follicle germs. A total of 28 independent bioengineered
hair follicle germs were transplanted into the cervical skin of nude mice, and they displayed high-density hair growth at 21 days after transplantation.
Scale bars, 5 mm.

follicle development (Fig. 3a,b)15,17. After the isolation of thebioengineered pelage follicle germ with different numbers ofcondensed dermal cell aggregates, we found that the number ofbioengineered pelage follicles was linearly correlated with thatof the condensed dermal cell aggregates (Fig. 3a). These results
also indicated that the condensed dermal cells and surrounding
epithelial cells recapitulated hair follicle morphogenesis during
embryonic organogenesis.
We further observed the development of the bioengineered
vibrissa follicle germ. The DP cells in the follicle germ were allobserved to express ALP and form autonomous cell aggregates, andepithelial cyst formation was not detected after two days of culture(Fig. 3c). These results indicate that all of the adult vibrissa-derived
bulge region epithelial cells and cultured DP cells can participate
in bioengineered follicle formation. Furthermore, the number ofthe bioengineered vibrissa follicles also correlated with the numberof bulge-derived epithelial and primary-cultured DP cells in the
preparation of bioengineered follicle germs (Fig. 3c). These results
suggest that the number of bioengineered vibrissa follicles depends
on the quantity of follicle-forming cells in bulge-derived epithelial
and primary-cultured DP cells, and they show that this procedure
can provide a precise transplantation technology for future hair
regeneration therapy.
The distinct hair follicle types, which are classified into awl/
auchene, guard, zigzag and vibrissa hair shafts in murine skin basedon properties such as length, thickness, kinks and hardness, arethought to be specified by DP cell types through the communication between DP cells and overlying epithelial cells34,42,43. The bioengineered pelage follicle germs were found to produce all types ofpelage hairs in accordance with their follicle fate as determined during embryonic development (Fig. 3d). The bioengineered vibrissa
follicle germ regenerated a vibrissa-type hair shaft (Fig. 3d). Thebioengineered hair shafts had the correct morphological and histological structures, including the hair medulla, cortex and cuticle,
as observed by scanning electron microscopy (SEM; Fig. 3d). The
pelage-type hair shafts were distinguishable from the vibrissa-type
shafts by transmission electron microscopic analysis (TEM) of thecross-sectional shapes (Fig. 3e). The natural and bioengineered
pelage hairs both had a central cavity corresponding to a ladderlike structure observed by light microscopy44, although these structures were not observed in the vibrissae (Fig. 3e). Dense medullary
and melanin granules were present in the central cavity of naturaland bioengineered pelages (Fig. 3e)43,44. The outermost layer of thenatural and bioengineered pelage-type hair shafts were surrounded
by a thin cuticle comprising two to four layers (Fig. 3e). The bioengineered vibrissae, which had a six-layer-thick cuticle, were similar
to natural vibrissae (Fig. 3e). Melanin granules were found in thepigmented natural hairs. However, the unpigmented bioengineered
vibrissa hair had no detectable melanin granules (Fig. 3e). These
results indicate that our bioengineering method for hair follicle
regeneration can reproduce all hair types with correct hair structuresin accordance with the fate of their follicle, which is determined
during embryonic development.
Hair follicle stem cell niches in the hair follicle. We next investigated whether various stem/progenitor cells and their niches werereconstructed in the bioengineered hair follicles. The bioengineered
hair follicles were identified by their EGFP expression (Fig. 2a) and/or hair features such as black hair colour (Fig. 4a). These follicles
were also identified based on the size of the hair bulbs of the bioengineered vibrissa follicles, which had a mean diameter of 251 ± 32 µm(n = 10) in contrast to the diameter of 84 ± 19 µm (n = 10; Fig. 4a)for the host pelages of nude mice. The epithelial stem cell markers,
CD34 and CD49f, were observed in the bulge region of the bioengi-

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1784

Cell number (×104 cells)
E 1.0 / F 0.3 E 1.0 / D 0.3 E 2.0 / D 0.6 E 3.0 / D 0.9

NaturalBioengineered pelage
pelageAwl/Auchene Awl/Auchene
Zigzag

8

**

3 DC

*

Notch16
4
2
0

Vibrissa
Natural

Bmp4

BioengineeredeNaturalBioengineered pelage
pelageAwl/Auchene Awl/Auchene
ZigzagVibrissa
NaturalBioengineered

SE

Centre

LM

d

**

10

Lef1

Natural
E18.5

At 2 weeks
in SRC

Mesenchymal
Versican

Bio

Organ
culture 40 h

Bio

1

Whole

ALP

*

Extremity

GFP

Number of bio-vibrissa

At day 2 in organ culture

β-Catenin20cEpithelial
Wnt10bMsx2

Natural
E18.5

3

*

TEM

3 DCs

*

Shh

2 DC

2 DCs

4

E 3.0/D 0.9

1 DC

b

At day 3
in SRC

1 DC

None dermalcluster (DS)Organculture 40 h

0 DC

Cyst

ALP
Cyst

E 1.0/F 0.3

GFP

E 1.0 / D 0.3
E 2.0/D 0.6

At day 2 in organ culture

Number of bio-pelageaFigure 3 | Control of the number and features of the bioengineered hairs. (a) Analysis of EGFP fluorescence (green), ALP activity (red) and H&E staining
in bioengineered pelage follicles reconstituted from normal epithelial cells and EGFP-labelled mesenchymal cells derived from embryonic skin at 2 daysin organ culture (upper left) and subsequently transplanted into the subrenal capsule (SRC, upper left). Dermal cell clusters (DCs) in the bioengineered
pelage germ were detected by EGFP fluorescence under confocal imaging (lower left). The number of bioengineered pelage follicles regenerated in vivo
from the bioengineered hair follicle germ is shown in the lower left panels. The data are shown as the mean ± s.d. (n>9). P < 0.01 (*) and P < 0.05 (**),
analysed by t-test. Scale bars: 200 µm for H&E, 50 µm for immunofluorescent microscopy images, 1 mm for macroscopic images. (b) In situ hybridization
analysis of the expression of regulatory genes during folliculogenesis in the natural and bioengineered pelage follicle in vivo. Dotted lines indicate theepithelial–mesenchymal boundary. Scale bars: 200 µm. (c) Analyses of EGFP fluorescence, enzymatic staining of ALP and H&E staining in adult vibrissaderived bioengineered hair germ after 2 days in organ culture (upper left). Bioengineered vibrissa follicles regenerated in SRC for 2 weeks were also
analysed using H&E staining. The bioengineered vibrissa germs were reconstituted using bulge epithelial cells (E) and primary culture DP cells (D) withvarious cell numbers (lower left), and murine sole skin dermis-derived fibroblasts (F) were used as a control for DP cells. The dotted lines in the lower
left panels indicate the epithelial–mesenchymal boundary. The number of bioengineered vibrissa follicles regenerated from the in vivo transplantation of
bioengineered hair follicle germ reconstituted under various conditions is described in the lower left. Data are shown as the mean ± s.d. (n>4). P < 0.01 (*)
and P < 0.05 (**) analysed by t-test. Scale bars, 100 µm. (d and e) Microscopic observation of the bioengineered pelage and vibrissa shafts. The hair shafts
were analysed by using light microscopy (LM), SEM and TEM. Whole (TEM, upper), high-magnification extremity (middle), and central regions of the hairshaft (lower) are shown. Cuticle layers (arrowheads) and dense medullary granules (arrow) are shown in the fine TEM photographs of the extremity andcentre regions. Scale bars, 1 mm for low magnification (LM), 20 µm for high magnification of LM and SEM, 10 µm for whole TEM and 2 µm for fine TEM.

neered pelage and vibrissa follicles (Fig. 4a). Nephronectin (NPNT),
which is also expressed in follicle epithelial stem cells in the bulge
region and provides the microenvironment for arrector pili muscle development by binding with the α8β1 integrin of mesenchymal progenitor cells26, was detected in the bulge region-epithelial
stem cells of the bioengineered pelage follicle. In contrast, NPNT
was not detected in the bulge region of the bioengineered vibrissae(Fig. 4a). The α8 integrin was distributed in the dermal cells inthe bulge ORS layer of the bioengineered pelage and vibrissa fol-licles (Fig. 4a). Sox2-positive DP cells (Sox2 is a mesenchymal stem
cell marker), which retain multilineage differentiation potential
in vivo42,43, were also detected in the DPs of the bioengineered
pelage and vibrissa follicles (Fig. 4b).
Hair shaft pigmentation is provided by neural crest-derived
melanocyte progenitor cells associated with the hair follicle pigmentary unit in the sub-bulge region of vibrissa hair follicles atanagen phases23,24. The white hair shafts of the bioengineered vibrissae could be caused by the loss of neural crest-derived melanocyte

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1784

a

H&E

CD34

CD49f

α8 Integrin

NPNT

Vibrissa

PelageMacroscopybcMacroscopy

PelageCentrePelagePigmented hair

Bioengineered
pigmented hair

Naturalpigmented hair

Vibrissa
Bioengineered
unpigmented hair

Bioengineered
pigmented hair

30

0

+SB

+SB

+PHM

0

None

20

e
60

+PHM

40

90

None

Ratio of Pigmented hairs withnormal morphological feature (%)

60

+PHM

120

80
Ratio of pigmented hairs (%)

Extremity+SB

Vibrissa

d

TEM

SEM

Unpigmented
hair

Sox2

Figure 4 | Rearrangement of follicular stem cells and their niches in the bioengineered pelage and vibrissa follicles. (a) Immunohistochemical
analysis of the bulge region in a bioengineered pelage (upper) and vibrissa (lower) follicle. Boxed areas in the left panels (H&E staining) are shownas immunofluorescent microscopy images in the right panels. The bulge region was analysed by immunostaining with specific antibodies for CD34,
CD49f, NPNT and α8 integrin. The nuclei were stained using Hoechst 33258 (Nuc, blue). Broken lines indicate the outermost part of the hair follicle.
Arrowheads indicate positively stained areas. Scale bars, 200 µm for H&E, 50 µm for immunofluorescent microscopy images, 1 mm for macroscopy.
(b) Immunohistochemical analysis of the DP in a bioengineered pelage (upper) and vibrissa (lower) hair follicle with anti-Sox2 antibodies. Boxed areasin the left panel are shown at a higher magnification in the right panels. Nuclei were stained using Hoechst 33258 (Nuc, blue). Broken lines indicate theoutermost limit of the hair follicle. Arrows indicate positively stained nuclei. Scale bars, 50 µm. (c) Analyses of hair pigmentation of the bioengineered
vibrissae by various combinations with the bioengineered vibrissa follicle cell components. The bioengineered vibrissa follicle germ, which wasreconstituted between the bulge region-derived epithelial stem cells and the primary cultured DP cells (upper), was combined with the sub-bulge ( + SB;
middle) or proximal region of the hair matrix ( + PHM; lower). These bioengineered hair shafts were analysed by macroscopic (left), SEM (centre) andTEM (right 2 panels) observations. Scale bars, 1 mm for macroscopy, 20 µm for SEM and 2 µm for TEM. (d) The frequencies of pigmented hair shafts(left) and pigmented hairs with normal morphological features (right) were compared. The data are presented as the mean ± s.e. from three individual
experiments; n = 5–12 and n = 3–8 per experiment in the + SB and + PHM conditions, respectively. (e) In situ hybridization analysis of the sub-bulge regionof natural and bioengineered hair follicles, using a specific antisense probe for Dct mRNA. The nuclei were stained using Hoechst 33258 (Nuc, blue).
The arrowheads indicate Dct mRNA expression in the cells. The broken lines indicate the outermost limit of the hair follicle. Scale bars, 50 µm.

stem/progenitor cells (Figs 1c, d, f and 4c). The addition of cells fromthe sub-bulge (SB) region (Fig. 1d)23,24, which contains melanoblasts
in the ORS, caused the bioengineered vibrissae to be pigmentedwith a black colour at a frequency of 68.3% (n = 26; Fig. 4c,d) at
3 weeks after transplantation. The shape, however, was normal witha frequency of 27.0% (n = 17; Fig. 4c,d). In contrast, the bioengi-

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1784

neered vibrissae derived from regenerated follicles with proximal
hair matrix (PHM) region-derived cells (Fig. 2c), which containimmature melanocytes that are the progeny of melanoblasts23–25,
were pigmented black with a frequency of 40.1% (n = 19; Fig. 4c,d)and had the structural properties of natural vibrissae, as assessedby electron microscopy (83.3%, n = 6; Fig. 4c,d). The fine structureof the bioengineered vibrissa harbouring PHM region cells was
similar to that of natural vibrissae, but the hair that was bioengineered with SB region cells had an abnormal shape withregard to the structure of the cuticle layer and the cortex region(Fig. 4c). Dopachrome tautomerase (Dct) messenger RNA-positive
cells, which are neural crest-derived melanocyte stem/progenitor
cells23–25, were also detected in the SB region of pigmented bioengineered pelage and vibrissa follicles, but not the unpigmented
bioengineered vibrissa follicle, by in situ hybridization (n = 10,
Fig. 4e). These results indicated that various follicle stem cells
and their niches were successfully rearranged in the bioengineered pelage and vibrissa follicles using appropriate epithelial andmesenchymal cell populations.
Bioengineered hair cycles. We further analysed the hair cycles,that is, the alternative growth (hair-growing) and regression (nongrowing) phases (Fig. 5a), of the bioengineered pelage and vibrissa
shafts that had erupted from bioengineered follicles for 80 days
(Fig. 5b,c). The bioengineered pelage and vibrissa follicles repeatedthe hair cycle at least 3 times during the 80-day period (Fig. 5b,c).
For natural pelage follicles transplanted into the back skin of nude
mice, the average growth and regression periods were 10.7 ± 2.0
and 10.5 ± 1.6 days, respectively (Fig. 5c,d). For the bioengineered
pelage follicles, these periods were 9.3 ± 2.5 and 12.4 ± 2.8 days, ascalculated from 3 hair cycle periods (Fig. 5c,d). The growth andregression phases of the natural vibrissa follicles were 10.6 ± 3.4 and
5.5 ± 3.9 days, respectively, and those of the bioengineered vibrissa
follicles were 10.7 ± 4.3 and 5.4 ± 3.3 days, respectively (Fig. 5c,d).
No significant differences in the hair cycle periods were foundbetween the natural and bioengineered follicles (both pelage andvibrissae; Fig. 5d).
To demonstrate that hair growth and regression are dependent
on the hair cycle, we performed hair cycle analysis by immunohistochemical detection of Ki67 and TUNEL assays29. To synchronize
the hair cycle via hair depilation29, we examined the relationships
between the growth and regression phases determined by the observations of hair shafts and hair cycles. The phases observed from hairshaft conditions were conventionally divided into ‘Growth start’,‘Growth’ and ‘Growth end’ (Fig. 5a). In early anagen phases I-IV,
tips of newly growing hair shafts were observed to grow in theskin layer and enter the hair canals. The newly growing hairs erupt
and grow through the skin until the end of hair growth in anagen
IV–VI. During the anagen phase, proliferation, but not apoptosis,
was detected in cells of the hair matrix region in the hair bulb via theexpression of Ki67 (Supplementary Fig. S1). In contrast, apoptosis
was immediately detected in cells of the hair matrix region during
the catagen phase (Supplementary Fig. S1). The resulting retractionsof the variable portion of the hair follicle were observed histologically via the appearance of TUNEL-positive cells and the reductionof the proximal tip (Supplementary Fig. S1). During the catagen to
telogen phase, hair shafts are not produced and are lost from thehair follicle gradually. It has been indicated that the hair growth andregression phases of the bioengineered hairs were dependent on thehistological changing of the bioengineered hair follicles according
to the hair cycling (Supplementary Fig. S1). Thus, these results indicated that the bioengineered hair follicle could represent proper haircycles according to the cell types of origin. It has been suggested
that the bioengineered pelage and vibrissa follicles could reproduce
these hair cycles, which are maintained by stem cells and provide astem cell niche.

Piloerection ability of the regenerated hair follicle. To achieve
functional hair follicle regeneration, it is essential that the engraftedfollicle be able to connect to the arrector pili muscle and nerve,
and that the follicle has a piloerection capability45. The possibilityof reconstituting the niche for arrector pili muscle development, inwhich the cells express NPNT, was demonstrated in the bulge regionof the bioengineered pelage (Fig. 4a). The bulge region of a natural pelage follicle is connected to the arrector pili muscle, which iscomposed of calponin-positive smooth muscle, but not troponinpositive striated muscle, and is innervated through the development of neuromuscular junctions, which surround the arrector pilimuscle with sympathetic nerve fibres that extend from a deep dermal nerve plexus (Fig. 6a). All bioengineered pelage follicles wereobserved to connect to the calponin-positive arrector pili muscle,
but not the troponin-positive striated muscle, and to connect tothe nerve fibres at the bulge region. These connections were also
observed in the natural pelage follicle (n = 10; Fig. 6a). The bioengineered vibrissa follicles were connected to the troponin-positive
striated muscle cells at the hair bulb regions but not to calponinpositive smooth muscle, in any other areas, which is similar to thestructure of the natural vibrissa follicle (Fig. 6a). The bioengineered
vibrissa follicles were also connected to nerve fibres, and they
formed neuron-follicular junctions in an ORS layer of the bulge
region (Fig. 6a).
Finally, to investigate whether the bioengineered pelage andvibrissa follicles possessed the capacity for piloerection, 1 µg of ACh
was administered intradermally in the vicinity of the engrafted follicles, and the angles of the hair shafts before and after this treatment
were calculated (Fig. 6b,c). ACh exposure significantly increased theangle of piloerection in the bioengineered pelage and vibrissae compared with a control (Fig. 6b,c). In contrast, an anti-cholinergic agent,atropine (AT), inhibited this effect (Fig. 6c). These results indicatethat the bioengineered hair follicles have a piloerection ability thatis comparable to that of natural follicles45. These findings indicatedthat our bioengineered follicles could induce selective connections
with the appropriate types of muscles and nerve fibres, and they
showed that these follicles were able to achieve piloerection throughthe rearrangement of stem cells and their niches (Fig. 7).

Discussion
In this study, we successfully demonstrate fully functional bioengineered hair follicle regeneration that produces follicles that canrepeat the hair cycle, connect properly with surrounding skin tissues and achieve piloerection. This regeneration occurs throughthe rearrangement of various follicular stem cells and their niches.
These findings significantly advance the technological developmentof bioengineered hair follicle regenerative therapy.
Hair regeneration methods that rely on the reproduction of epithelial–mesenchymal interactions have been attempted in several
previous studies31–39. It was also reported that the aggregation ofdissociated epithelial and mesenchymal cells induced to form hairfollicle self-assemblies during embryonic hair follicle development could be reproduced34–38. The replacement of hair-inductive
mesenchymal cells in an amputated hair follicle19,20 or in glabrous
skin33 has also been reported as an analogy to the regenerationof the anagen phase in the adult hair cycle. In hair growth after
birth, the position of the infundibular opening and hair shaft eruption are determined by the connection between the hair follicle andthe interfollicular epidermis, and this position is maintained as afollicle-invariable region, during hair cycling16. Our bioengineered
vibrissa follicle germ that was reconstituted using adult folliclederived stem cells successfully regenerated the hair follicle following
intracutaneous transplantation. Hair shafts also erupted from thisbioengineered germ with the proper connections to the surrounding host tissues. Hence, our findings indicate that it is possible to not
only restore a hair follicle but also to re-establish successful connec-

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

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nature communications | DOI: 10.1038/ncomms1784

ac

Pelage20

Regression

Phase ofhair cycle

GrowthRegression

AnagenCata/Telo

GrowthAnagen

Ki67

–

+

+

+

+

–

+

+

+

+

TUNEL

+

–

–

–

–

+

–

–

–

–

16

16

12

12

8
4
0

Time

b

1st hair cycle
Day 19Day 37

2nd hair cycle
Day 44

Day 56

3rd hair cycle
Day 65

Days

Hair shaft

Days

Changes ofHair follicledDay 7520

Vibrissa

8
4

1st 2nd 3rdNatural bioengineered0

1st 2nd 3rdNatural bioengineered

25

10

0

Natural5
Bioengineered

3rd hair cycle
Day 53
Day 55Natural

2nd hair cycle
Day 41
Day 46

Vibrissa

1st hair cycle
Day 27
Day 31

15

Bioengineered

Days

Pelage20

Pelage Vibrissa

Figure 5 | Hair cycle analyses of bioengineered hair follicles. (a) Schematic representation of the hair cycle defined by histological and macromorphological observations of the hair shaft. (b) Macro-morphological observations of the hair cycles in a bioengineered pelage (upper panels) andvibrissa (lower panels). In the bioengineered vibrissa, arrowheads (open or closed white, red, blue and green) indicate individual hairs. Closed whiteand red arrowheads are identical to hairs indicated as opened ones, which erupted from the respective follicles. Scale bars, 1.0 mm. (c) Assessmentof the hair growth (open circles) and regression (closed circles) phases of the bioengineered pelage and vibrissa up to 80 days after transplantation.
Natural pelages and vibrissae that were transplanted onto the back skin of Balb/c nu/nu mice were also followed as a control. The data are presented
as the mean ± s.e.; n = 5 for natural pelages, n = 10 for the bioengineered pelages, n = 6 for natural vibrissae, and n = 8 for the bioengineered vibrissae.
(d) Cycles of hair growth (dark bars) and regression (light bars) of a natural pelage and vibrissa (Natural), and of a bioengineered pelage and vibrissa(Bioengineered), up to 80 days after transplantation. The data are presented as the mean ± s.e.; n = 6 for natural pelages, n = 7 for bioengineered pelages,
n = 8 for natural vibrissae, and n = 9 for bioengineered vibrissae.

tions with the recipient skin by intracutaneous transplantation of
the bioengineered follicle germ.
Stem cells with organ-inductive potential exist only during
embryonic organogenesis and are maintained as tissue stem cells
in the stem cell niche of each organ to enable tissue repair after
birth17,46. It is well known that the hair follicle organ-inductive
epithelial and mesenchymal stem cells provide a source of differentiated hair follicle cells that enable hair cycling to occur over the lifetime of a mammal16,47. Thus, it is logical for hair follicle regenerative
therapy to utilize organ-inductive stem cells of epithelial and mesenchymal origin that are isolated from adult tissues34–39,48–50. It is alsoessential to rearrange these various stem cells and their niches inthe bioengineered follicle to reproduce enduring hair cycles15,30,47.
The sub-bulge, bulge and the bulge-to-sebaceous gland regions provide niches for epithelial stem cells and melanoblast stem cells21–25.
In contrast, stem/progenitor cell populations that can differentiate
into DS cells, adipocytes, cartilage cells and fibroblasts are foundamong the DP cells18–20,42. Our bioengineered hair follicle canregenerate and sustain hair cycles according to the origin of its
cells (that is, pelage or vibrissa), which suggests that bioengineered
follicles have the potential to maintain stem cells through thereconstitution of niches for epithelial stem cells and DP cells. Theseobservations also provide insight into the formation and maintenance of stem cell niches in their microenvironments9,30,47.
For hair regenerative therapy, it is critical to consider whether
bioengineered hair follicles can regenerate normal inherent traits41,
physiological functions such as hair shaft types43, qualities30, andcooperation with host cutaneous tissues including the arrector pilimuscle and nerve system26,51,52. Properties of the hair shaft, whichreflect the function of the hair in the body region14–16, are regulatedby hair follicle mesenchymal DP and DS cells and are also modulated by the expression of various genes in the epidermis15–17. It isalso thought that hair pigmentation is controlled by melanocyte
differentiation and the proliferation of melanocyte-lineage stem
cells below the bulge region23–25. Thus, hair properties can be controlled by the arrangement of cell types during the regeneration of
the bioengineered hair follicle germ34,30,47. We provide evidence
for this arrangement by showing that bioengineered hair with aproper shape and colour can be generated through the appropriate cell populations, such as bulge-derived epithelial cells, DP
cells, and the PHM region-derived cells, but not sebaceous glandregion-derived cells. Our findings thus provide new insights into
the regulation of hair properties and strongly suggest that thesecharacteristics could be properly restored by cell processing fororgan regeneration and by the transplantation of bioengineered
hair follicle germ.
The peripheral nervous system has essential roles in organfunction and the perception of noxious stimuli, such as pain andmechanical stress52,53. The restoration of the nervous system is thus
a critical issue to be addressed by organ replacement regenerative
therapy13,53. Previously, we demonstrated, in the successful regeneration of a tooth, the proper perception of noxious stimuli mediatednature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1784

a
Natural

CNN/NF/Nuc.

TNN/NF/Nuc.

ddp

ddp

Pelagedm

dm

Bioengineered

CNN/NF/GFP/Nuc. CNN/NF/Nuc.

dm

TNN/NF/GFP/Nuc.

TNN/NF/Nuc.

ddp
ddp
dm
CNN/NF/Nuc.

TNN/NF/Nuc.

Naturalmm

Vibrissam
CNN/NF/GFP/Nuc. CNN/NF/Nuc.

TNN/NF/Nuc.

Bioengineered

TNN/NF/GFP/Nuc.

dm

c 0.6

*

0.4
0.3
0.2

PBS(–)

Vibrissa

Pelage0Pelage0.1
Vibrissa

Vibrissa**

0.5PelageMerge

Vibrissa

After dose

PelageBefore admin

Changes of hair angle (rad)b

ACh AT+ACh

Figure 6 | Analyses of the connections to surrounding tissues and the piloerection capability of bioengineered hair follicles. (a) The connections of
the follicles of a natural and EGFP-labelled bioengineered pelage (upper two lines) and natural and EGFP-labelled bioengineered vibrissa (lower two lines)
to arrector pili muscles and nerve fibres were analysed by immunohistochemical staining using specific antibodies against calponin for smooth muscle(CNN, red in the left three panels), troponin for striated muscle (TNN, red in the right three panels) and neurofilament H (NF, white). The nuclei were
stained using Hoechst 33258 (Nuc, blue). The boxed areas in the left panel in each data set are shown at a higher magnification in the right two panels.
The arrows and arrowheads indicate the muscle and nerve fibres connected with pelage follicles, respectively. Broken lines indicate the outermost limitof the hair follicle. Scale bars, 100 µm in low-magnification and 50 µm in high-magnification photographs. ddp, deep dermal plexus; dm, dermal muscle
layer; m, muscle. (b) Analysis of the piloerection capability of bioengineered hair follicles following an intradermal injection of ACh. The positions of thehair shafts (black or white arrowheads in the left) moved after this treatment (centre) and merged (right). Scale bars, 1.0 mm. (c) Assessment of hairangle changes associated with the bioengineered pelage (light bars) and vibrissae (dark bars) before and after the administration of ACh, without or withatropine (AT + ACh). The data are shown as the mean ± s.e.; n = 10 for the bioengineered pelage and n = 6 for the bioengineered vibrissa. P < 0.01 (*) andP < 0.05 (**) were defined as statistically significant, as analysed by t-test.

by trigeminal innervation13,53. In the hair follicle, the follicles, pelage
and vibrissae achieve piloerection using the surrounding arrector
pili muscle through the activation of the sympathetic nerves26. The
rodent vibrissa also functions as a sensory organ26,45,51,52. We also
demonstrate that the nerve fibres and muscles can connect autonomously to the pelage and vibrissa follicle, and that the bioengineered

follicles exhibit ACh-induced piloerection. Our findings suggest thatthe transplantation of a bioengineered hair follicle germ can restore
natural hair function and re-establish the cooperation between thefollicle and the surrounding recipient muscles and nerve fibres. Thus,
the transplantation of bioengineered hair follicle germ is potentiallyapplicable to the future surgical treatment of alopecia.

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1784

PelageBioengineered hair

Vibrissa
Natural hairAPM
(donor/host)

Bioengineered hair

CollagesheathBulge(donor, –NPNT)

Nerve (host)Striated
muscleBulge(donor, +NPNT)

Striated muscle(donor/host)
Nerve

Nerve
(host)

Figure 7 | Model of the autonomous connections of the bioengineered hair follicles with surrounding tissues in adult skin. The bioengineered pelage
and vibrissa follicles were connected with other tissues such as nerve fibres, arrector pili muscles and striated muscle, which were derived from host and/or donor cells. The bioengineered pelage follicle autonomously connected with smooth muscle as a result of the reproduction of the NPNT-expressing
bulge region in a manner similar to that of the natural pelage. Neither NPNT expression nor smooth muscle connection was detected in bulge regions of
the natural and bioengineered vibrissa follicles.

In conclusion, this study provides novel evidence of fully functional hair follicle regeneration through the rearrangement of various stem cells and their niches in bioengineered hair follicles. Our
study provides a substantial contribution to the development of
bioengineering technologies that will enable future regenerative
therapy for hair loss caused by injury or by diseases such as alopecia and androgenic alopecia. Further studies on the optimizationof human hair follicle-derived stem cell sources for clinical applications and further investigations of stem cell niches will contribute to
the development of hair regenerative therapy as a prominent class oforgan replacement regenerative therapy in the future.

Methods

Animals. C57BL/6 and Balb/c nu/nu mice were purchased from Japan SLC(Shizuoka, Japan). C57BL/6-TgN (act-EGFP) OsbC14-Y01-FM131 mice wereobtained from Japan SLC and the RIKEN Bioresource Center (Tsukuba, Japan).
The mouse care and handling conformed to NIH guidelines, and the requirementsof the Tokyo University of Science Animal Care and Use Committee.
Isolation and dissociation of embryonic skin. E18 mouse embryonic back skinspecimens were aseptically isolated in DMEM10/HEPES medium, which was
prepared with DMEM (WAKO, Osaka, Japan) containing 10% fetal calf serum(GIBCO, Grand Island, NY, USA), 100 U ml − 1 penicillin (Sigma, St. Louis, MO,
USA), 100 mg ml − 1 streptomycin (Sigma), and 20 mM HEPES (Invitrogen,
Carlsbad, CA, USA) on ice, treated with 4.8 U ml − 1 dispase (BD, Franklin Lakes,
NJ, USA) in PBS at 4 °C for 1 h, and then divided into epithelial and mesenchymal layers. To prepare the epithelial cells, the epithelial layer was treated with
100 U ml − 1 collagenase (Worthington, Lakewood, NJ, USA) for 40 min at 37 °C,
washed with PBS, and treated with 0.25% trypsin (Invitrogen) at 37 °C for 10 min.
To prepare the mesenchymal cells, the dermal layer was treated with 10,000 U ml − 1collagenase (Worthington) for 1 h at 37 °C with shaking, followed by filtering
through a cell strainer (35 µm mesh, BD).
Preparation of various cell populations from adult vibrissae. Adult vibrissa
follicles of a1-5, b1-5 and E-H, as defined previously54, were removed from 7- or
8-week-old mice. The DP and other epithelial components were isolated from adultvibrissa follicles from which the collagen sheath was removed. DPs were isolated
from anagen I–IV vibrissa follicles, explanted onto a plastic culture dish (BD), andmaintained as primary cultures for 9 days, as described previously55. The cultured
DP cells were collected with 0.05% trypsin (Invitrogen), and single cells wereisolated. The bulge region, which was defined as an area from below the sebaceous
gland to above the ringwurst23,24, and the sub-bulge region (Fig. 2b), which was
defined as the area from below the ringwurst to above the middle of the hairfollicle23,24, were then treated for 4 min at 37 °C with PBS containing 4.8 U ml − 1
dispase II (BD) and 100 U ml − 1 collagenase (Worthington). The epithelial tissue
was then treated for 1 hour with 0.05% trypsin (Invitrogen), and single cells
were isolated using a cell strainer (BD) with a 35 µm pore. The SB region-derived
epithelial tissue, which was dissected at the end of the bulge region in the middleof the hair follicle, was also treated for 4 min, at 37 °C, in the dispase II/collagenase(Worthington) solution, as described above. The PHM region, which is located10

under the Auber’s line and surrounds the papillary stalks, was isolated, as previously described56. The SB and PHM region-derived tissues were also treated for
1 hour with 0.05% trypsin (Invitrogen), and single cells were then isolated.
Reconstitution of bioengineered murine hair follicle germ. The bioengineeredmurine hair follicle germs were reconstituted using mouse epithelial and dermal
cells, according to the organ germ method reported previously7. The bioengineered
pelage follicle germs were reconstituted using embryonic skin epithelial andmesenchymal cells (7.5×103 of each cell type). To analyse the follicle formation in
the bioengineered pelage follicle, dermal cell aggregates with high-intensity green
fluorescence, in the bioengineered pelage follicle germ, were dissected after 2 daysin an organ culture. The bioengineered vibrissa follicle germs were regenerated
from 7- to 8-week-old vibrissa-derived bulge region epithelial (1×104 cells) andprimary cultured DP cells (3×103 cells). These bioengineered hair germs were thenplaced onto a cell culture insert (0.4 µm pore diameter; BD) and incubated at 37 °C,
for two days, in DMEM10 medium. To investigate the formation of bioengineered
vibrissae in the bioengineered germ, the bioengineered germs for the assay were
regenerated with various numbers of epithelial and mesenchymal cells. To form
inter-epithelial tissue connections between the host skin and the bioengineered
hair follicle, a nylon thread guide (8–0 nylon surgical suture; Natsume, Tokyo,
Japan) was appended into a bioengineered hair germ through the thrustingepithelial and mesenchymal portions.
Hair pigmentation assay with bioengineered vibrissa. The SB23,24 or PHM56
regions were isolated and trypsinized, as described above. For each of theseregions, 200 cells, isolated from the region, were added to 104 bulge-derived
epithelial cells and used to reconstitute the bioengineered vibrissa follicle germ,which consisted of 3,000 cultured DP cells. This preparation was intracutaneouslytransplanted into the backs of nude mice, as described above.
Engraftment of bioengineered hair follicle germ. For hair follicle regeneration,
the bioengineered hair germs were ectopically engrafted into the subrenal
capsules of 8-week-old C57BL/6 mice, as previously described7,13, or they wereintracutaneously transplanted onto the back skin of 6-week-old Balb/c nu/nu
mice. Shallow stab wounds, which were nearly parallel to the skin surface and~0.4 mm vertical and 2.8 mm horizontal from the needle-stick point, were madein the back skin of nude mice with a 20-G Ophthalmic V-Lance (Alcon Japan,
Tokyo, Japan). The bioengineered hair follicle germ, which contained a nylon
guide, was arranged in the epithelial portion and above, and it was intradermallytransplanted into the wounds. The nylon guide was held so that it protruded fromthe skin surface. The transplantation sites were then covered with surgical bandage
tape (Nichiban, Tokyo, Japan). To examine the engraftment of the transplants
and to study the hair cycles of the bioengineered hairs, all transplanted sites wereobserved using a SteREO Lumar V12 and AxioCam fluorescent stereoscopic
microscope system (Carl Zeiss, Oberkochen, Germany). Observations weremade every 2–3 days.
Bioengineering of human hair follicle germ. This procedure was performed withthe approval of the ethics board of the Tokyo University of Science and Tokyo
Memorial Clinic. Small pieces (1.0–2.0 cm2) of occipital scalp skin were obtained
from 39- and 63-year-old male donors, who provided informed consent in accordance with the Helsinki Declaration. The scalp samples were treated with povidoneiodine and 70% ethanol for 10 s, twice successively. The hair follicles in the anagen

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

ARTICLE

nature communications | DOI: 10.1038/ncomms1784
phase were then dissected at the upper end of the hair bulb, using a surgical knife.
The intact DPs and bulge-derived epithelial cells were isolated as described above.
The bioengineered hair follicles were then reconstituted using human intact DPs
and the bulge region-derived epithelial cells, by using the organ germ method7.
At 21 days after transplantation with the inter-epithelial tissue-connecting
plastic device, the growth of pigmented hair shafts was observed underneaththe epithelium of the host skin. The hair shafts were recovered by cutting into
the host epithelium.
Immunohistochemistry. Paraffin sections (5 µm) were stained with haematoxylin
and eosin, and observed, using an Axioimager A1 (Carl Zeiss) microscope and
AxioCAM MRc5 (Carl Zeiss) camera. For fluorescent immunohistochemistry,frozen sections (10 and 100 µm) were prepared and immunostained, as previously
described7,45,53. The primary antibodies were as follows: versican (1:100, rabbit,
Millipore, Billerica, MA, USA); α-SMA (1:500, rabbit, Epitomics, Burlingame,
CA, USA); integrin α6 (1:500, CD49f; rat, Abcam, Cambridge, MA, USA);
CD34 (1:100, rat, eBioscience, California, USA); integrin α8 (1:50, goat, R&D
systems, Minneapolis, MN, USA); Sox2 (1:250, Cell Signaling Technology Japan,
Tokyo, Japan); NPNT (1:50 rabbit, Trans Genic, Kobe, Japan) neurofilament-H(1:500, rat, Chemicon, California, USA); calponin (1:250, rabbit, Abcam); andtroponin (1:100, rabbit, Abcam), in blocking solution. The primary antibodies
were detected using highly cross-adsorbed Alexa Fluor 594 goat anti-mouse
IgG (H + L) (1:500, Invitrogen) for 1 hour at room temperature, together
with Hoechst 33258 dye (1:500, Dojindo, Kumamoto, Japan). All fluorescencemicroscopy images were captured under a confocal microscope: the LSM 780(Carl Zeiss).
Detection of human cells by nuclear staining. The human bioengineered hairfollicles were dissected, fixed in 10% formalin, processed for standard paraffin
embedding, and sectioned at 5 µm. Discrimination between mouse and humannuclei was performed as described previously40.
In situ hybridization. In situ hybridizations were performed using 10-µm frozen
sections. Digoxigenin-labelled probes for specific transcripts were prepared by
PCR, with primers designed using published sequences (Supplementary Table S1).
The mRNA-expression patterns were visualized by immunoreactivity with antidigoxigenin alkaline phosphatase-conjugated Fab-fragments (Roche, Basel,
Switzerland), according to the manufacturer’s instructions.
Electron microscopy. For SEM observations, the hair shafts were dehydrated
in 100% ethanol. After coating with platinum, the samples were examined witha Hitachi S-4700 SEM (Hitachi High-Tech, Tokyo, Japan) at 10–15 kV. SEM
observations were performed mainly for the surfaces of the lower portion of thehair shafts57. For TEM analysis, the samples were embedded and fixed, as previously described58. Ultrathin sections were prepared, as previously described58,
and examined using a Hitachi H-7600 transmission electron microscope (Hitachi)with an accelerating voltage of 75 kV.
Piloerection of bioengineered hair. To investigate whether bioengineered
hairs could reproduce the piloerection ability, the effects of a neurotransmitter
agent, 10 mg ml − 1 acetylcholine (Sigma), and of a piloerection inhibitor, 1 µl of
a 100 mg ml − 1 atropine sulphate solution (Sigma), were examined, as described
previously45,59,60. The piloerection angle was measured by microscopic image
analysis and compared with the pre-injection angle as the reference point.

References

1. Sharpe, P. T. & Young, C. S. Test-tube teeth. Sci. Am. 293, 34–41 (2005).
2. González, F., Boué, S. & Belmonte, J. C. Methods for making induced
pluripotent stem cells: reprogramming à la carte. Nat. Rev. Genet. 12, 231–242(2011).
3. Priya, S. G., Jungvid, H. & Kumar, A. Skin tissue engineering for tissue repair
and regeneration. Tissue Eng. Part B Rev. 14, 105–118 (2008).
4. Nishida, K. et al. Functional bioengineered corneal epithelial sheet grafts from
corneal stem cells expanded ex vivo on a temperature-responsive cell culture
surface. Transplantation 77, 379–385 (2004).
5. Pispa, J. & Thesleff, I. Mechanisms of ectodermal organogenesis. Dev. Biol. 262,
195–205 (2003).
6. Bianchini, C. et al. Use of autologous dermal graft in the treatment ofparotid surgery wounds for prevention of neck scars: preliminary results.
J. Otolaryngol. Head Neck Surg. 37, 174–178 (2008).
7. Nakao, K. et al. The development of a bioengineered organ germ method.
Nat. Methods 4, 227–230 (2007).
8. Hoffman, R. M. The pluripotency of hair follicle stem cells. Cell Cycle. 5,
232–233 (2006).
9. Chuong, C. M., Wu, P., Plikus, M., Jiang, T. X. & Bruce Widelitz, R. Engineeringstem cells into organs: topobiological transformations demonstrated by beak,feather, and other ectodermal organ morphogenesis. Curr. Top. Dev. Biol. 72,
237–274 (2006).

10. Nieminen, P. Genetic basis of tooth agenesis. J. Exp. Zool. B. Mol. Dev. Evol.
312, 320–342 (2009).
11. Trotti, A. & Eisbruch, A. Reducing xerostomia through advanced technology.
Lancet Oncol. 12, 110–111 (2011).
12. Randall, V. A. et al. Hormones and hair growth: variations in androgen
receptor content of darmal papilla cells cultured from human and red
deer (Cervus elaphus) hair follicles. J. Invest. Dermatol. 101, 114S–120S(1993).
13. Oshima, M. et al. Functional tooth regeneration using a bioengineered tooth
unit as a mature organ replacement regenerative therapy. PLoS One. 6, e21531(2011).
14. Chuong, C. M. Molecular Basis of Epithelial Appendage Morphogenesis(ed. Chuong C. M.) (R.G. Landes Company: Austin, Texas, 1998).
15. Fuchs, E. Scratching the surface of skin development. Nature. 445, 834–842(2007).
16. Hardy, M. H The secret life of the hair follicle. Trends Genet. 8, 55–61 (1992).
17. Stenn, K. S. & Paus, R. Controls of hair follicle cycling. Physiol. Rev. 81,
449–494 (2001).
18. Yamao, M. et al. Contact between dermal papilla cells and dermal sheath cells
enhances the ability of DPCs to induce hair growth. J. Invest. Dermatol. 130,
2707–2718 (2010).
19. Jahoda, C. A., Whitehouse, J., Reynolds, A. J. & Hole, N. Hair follicle dermal
cells differentiate into adipogenic and osteogenic lineages. Exp. Dermatol. 12,
849–859 (2003).
20. Jahoda, C. A. & Reynolds, A. J. Hair follicle dermal sheath cells: unsung
participants in wound healing. Lancet 358, 1445–1448 (2001).
21. Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K. & Barrandon, Y.
Morphogenesis and renewal of hair follicles from adult multipotent stem cells.
Cell 104, 233–245 (2001).
22. Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal,
multipotency, and the existence of two cell populations within an epithelial
stem cell niche. Cell 118, 635–648 (2004).
23. Nishimura, E. K. et al. Dominant role of the niche in melanocyte stem-cell fate
determination. Nature 416, 854–860 (2002).
24. Nishimura, E. K., Granter, S. R. & Fisher, D. E. Mechanisms of hair graying:incomplete melanocyte stem cell maintenance in the niche. Science. 4, 720–724(2005).
25. Kunisada, T. et al. Transgene expression of steel factor in the basal layer of
epidermis promotes survival proliferation differentiation and migration ofmelanocyte precursors. Development. 125, 2915–2923 (1998).
26. Fujiwara, H. et al. The basement membrane of hair follicle stem cells is a muscle
cell niche. Cell 144, 577–589 (2011).
27. Rendl, M., Polak, L. & Fuchs, E. BMP signaling in dermal papilla cells is required
for their hair follicle-inductive properties. Genes Dev.. 22, 543–557 (2008).
28. Greco, V. et al. A two-step mechanism for stem cell activation during hair
regeneration. Cell Stem Cell 4, 155–169 (2009).
29. Lindner, G. et al. Analysis of apoptosis during hair follicle regression (catagen).
Am. J. Pathol. 151, 1601–1617 (1997).
30. Chuong, C. M., Cotsarelis, G. & Stenn, K. Defining hair follicles in the age ofstem cell bioengineering. J. Invest. Dermatol. 127, 2098–2100 (2007).
31. Jahoda, C. A., Horne, K. A. & Oliver, R. F. Induction of hair growth by
implantation of cultured dermal papilla cells. Nature 311, 560–562 (1984).
32. Jahoda, C. A., Oliver, R. F., Reynolds, A. J., Forrester, J. C. & Horne, K. A.
Human hair follicle regeneration following amputation and grafting into thenude mouse. J. Invest. Dermatol. 107, 804–807 (1996).
33. Stenn, K. et al. Bioengineering the hair follicle. Organogenesis 3, 6–13 (2007).
34. Weinberg, W. C. et al. Reconstitution of hair follicle development in vivo:
determination of follicle formation, hair growth, and hair quality by dermal
cells. J. Invest. Dermatol. 100, 229–236 (1993).
35. Kishimoto, J. et al. Selective activation of the versican promoter by epithelialmesenchymal interactions during hair follicle development. Proc. Natl Acad.
Sci. USA 96, 7336–7341 (1999).
36. Lichti, U., Anders, J. & Yuspa, S. H. Isolation and short-term culture of primary
keratinocytes, hair follicle populations and dermal cells from newborn miceand keratinocytes from adult mice for in vitro analysis and for grafting toimmunodeficient mice. Nat. Protoc. 3, 799–810 (2008).
37. Zheng, Y. et al. Organogenesis from dissociated cells: generation of mature
cycling hair follicles from skin-derived cells. J. Invest. Dermatol. 124, 867–876(2005).
38. Gilmour, S. K., Teti, K. A., Wu, K. Q. & Morris, R. J. A simple in vivo system forstudying epithelialization, hair follicle formation, and invasion using primaryepidermal cells from wild-type and transgenic ornithine decarboxylaseoverexpressing mouse skin. J. Invest. Dermatol. 117, 1674–1676 (2001).
39. Mannik, J., Alzayady, K. & Ghazizadeh, S. Regeneration of multilineage skinepithelia by differentiated keratinocytes. J. Invest. Dermatol. 130, 388–397(2010).
40. Ferraris, C., Bernard, B. A. & Dhouailly, D. Adult epidermal keratinocytes areendowed with pilosebaceous forming abilities. J. Invest. Dermatol. 3, 491–498(1997).

nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.

11

ARTICLE

nature communications | DOI: 10.1038/ncomms1784

41. Unger, W., Shapiro, R., Unger, M. A. & Unger, U. Hair Transplantation 5th edn(2010).
42. Biernaskie, J. et al. SKPs derive from hair follicle precursors and exhibit
properties of adult dermal stem cells. Cell Stem Cell 5, 610–623 (2009).
43. Driskell, R. R., Giangreco, A., Jensen, K. B., Mulder, K. W. & Watt, F. M. Sox2positive dermal papilla cells specify hair follicle type in mammalian epidermis.
Development 136, 2815–2823 (2009).
44. Hojiro, O. Fine structure of the mouse hair follicle. J. Electron Microsc. 21,
127–138 (1972).
45. Sato, A. et al. Single follicular unit transplantation reconstructs arrector pilimuscle- and nerve-connections and restores functional hair follicle piloerectionin preparing,. J. Dermatol. 39, 1–6 (2012).
46. Jensen, K. B. et al. Lrig1 expression defines a distinct multipotent stem cell
population in mammalian epidermis. Cell Stem Cell 4, 427–439 (2009).
47. Lee, L. F. & Chuong, C. M. Building complex tissues: high-throughput
screening for molecules required in hair engineering. J. Invest. Dermatol. 129,815–817 (2009).
48. Claudinot, S., Nicolas, M., Oshima, H., Rochat, A. & Barrandon, Y. Long-term
renewal of hair follicles from clonogenic multipotent stem cells. Proc. Natl
Acad. Sci. U.S.A. 102, 14677–14682 (2005).
49. Morris, R. J. et al. Capturing and profiling adult hair follicle stem cells.
Nat. Biotechnol. 22, 411–417 (2004).
50. Bonfanti, P. et al. Microenvironmental reprogramming of thymic epithelial
cells to skin multipotent stem cells. Nature 466, 978–982 (2010).
51. Grant, R. A., Mitchinson, B., Fox, C. W. & Prescott, T. J. Active touch sensing
in the rat: anticipatory and regulatory control of whisker movements during
surface exploration. J. Neurophysiol. 101, 862–874 (2009).
52. Peters, E. M., Botchkarev, V. A., Botchkareva, N. V., Tobin, D. J. & Paus, R.
Hair-cycle-associated remodeling of the peptidergic innervation of murine
skin, and hair growth modulation by neuropeptides. J. Invest. Dermatol. 116,
236–245 (2001).
53. Ikeda, E et al. Fully functional bioengineered tooth replacement as an organ
replacement therapy. Proc. Natl Acad. Sci. USA 106, 13475–13480 (2009).
54. Ibrahim, L. & Wright, E. The growth of rats and mice vibrissae under normal
and some abnormal conditions. J. Embryol. Exp. Morpho. 33, 831–844 (1975).
55. Osada, A., Iwabuchi, T., Kishimoto, J., Hamazaki, T. S. & Okochi, H. Longterm culture of mouse vibrissal dermal papilla cells and de novo hair follicle
induction. Tissue Eng. 13, 975–982 (2007).
56. Reynolds, A. J. & Jahoda, C. A. Hair matrix germinative epidermal cells confer
follicle-inducing capabilities on dermal sheath and high passage papilla cells.
Development 122, 3085–3094 (1996).

12

57. Eaton, S., Wepf, R. & Simon, K. Roles for Rac1 and Cdc42 in planar
polarization and hair outgrowth in the wing of drosophila. J. Cell Biol. 135,
1277–1289 (1996).
58. Irie, T. et al. Intracellular transport of basement menbrane-type heparansulphate proteoglycan in adenoid cystic carcinoma cells of salivary glandorigin: an immunoelectron microscopic study. Virchows Arch. 433, 41–48(1998).
59. Hellmann, K. The isolated pilomotor muscle as an in vitro preparation.
J. Physiol. 169, 603–620 (1963).
60. Vanhouttec, P. M. Inhibition by acetylcholine of adrenergic neurotransmission
in vascular smooth muscle. Circ. Res. 34, 317–326 (1974).

Acknowledgements

We thank M. Okabe (Osaka University) for providing the C57BL/6-TgN (act-EGFP)
OsbC14-Y01-FM131 mice. We are grateful to Dr. Y Ishii (Skin clinic, Omiya, Japan)
for advice about surgical techniques. We are also grateful to K. Isumi, C. Gotoh,
T. Kanayama, F. Tobe, A. Iwadate, S. Noguchi and Y. Yuge for technical assistance.

Author contributions

T. Tsuji and K.T. designed the research plan; K.T., K.A., H.T., N.I., T.H., M.O. and K.N.
performed the experiments; T.I., and T. Tachikawa performed electron microscopic
analyses; K.T., A.S., A.T. and T. Tsuji discussed the results; and K.T. and T. Tsuji wrote
the paper.

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How to cite this article: Toyoshima, K.-e. et al. Fully functional hair follicle regeneration
through the rearrangement of stem cells and their niches. Nat. Commun. 3:784
doi: 10.1038/ncomms1784 (2012).
License: This work is licensed under a Creative Commons Attribution-NonCommercialShare Alike 3.0 Unported License. To view a copy of this license, visit http://
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nature communications | 3:784 | DOI: 10.1038/ncomms1784 | www.nature.com/naturecommunications© 2012 Macmillan Publishers Limited. All rights reserved.