Organ replacement regenerative therapy is expected to provide novel therapeutic systems
for donor organ transplantation, which is an approach to treating patients who experience
organ 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, both of which have the potential to restore partially lost
tissue or organ function2
3
4. The development of bioengineered ectodermal organs, such as teeth, salivary glands,
or hair follicles may be achieved by reproducing the developmental processes that
occur during organogenesis5
6
7
8
9. Ectodermal organs have essential physiological roles and can greatly influence
the quality of life by preventing the morbidity associated with afflictions such as
caries and hypodontia in teeth10, hyposalivation in the salivary gland11, and androgenetic
alopecia, which affects the hair12. Recently, it has been proposed that a bioengineered
tooth can restore oral and physiological function through the transplantation of bioengineered
tooth germ and a bioengineered 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 and isthmus, 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
16
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 are found in the follicle
stem cell niche of the bulge region21
22; multipotent mesenchymal precursors are found in DP cells18
19; neural crest-derived melanocyte progenitors are located in the sub-bulge region23
24
25, and follicle epithelial stem cells in the bulge region that is connected to the
arrector pili muscle15
26. The follicle variable region mediates the hair cycle, which depends on the activation
of follicle epithelial stem cells in the bulge stem cell niche during the telogen-to-anagen
transition27
28. This transition includes phases of growth (anagen), apoptosis-driven regression
(catagen)29 and relative quiescence (telogen)17, whereas the organogenesis of most
organs is induced only once during embryogenesis16.
To achieve hair follicle regeneration in the hair cycle, it is thought to be essential
to regenerate the various stem cells and their niches9
30. Many studies have attempted to develop technologies to renew the variable lower
region of the hair follicle31
32, to achieve de novo folliculogenesis via replacement with hair follicle-inductive
dermal cells33, and to direct the self-assembly of skin-derived epithelial and mesenchymal
cells34
35
36
37
38
39. We have also reported that a bioengineered hair follicle germ, reconstituted from
embryonic follicle germ-derived epithelial and mesenchymal cells, using our organ
germ method, can generate a bioengineered hair follicle 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 hair follicle 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, as well 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 and mesenchymal 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 the epithelium 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 the resulting wound was allowed to heal by maintaining the
eruption of 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 the inter-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 a frequency 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, and in 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 the thin outermost dermal layer of the hair bulb was observed
to express α-smooth muscle actin (α-SMA; Fig. 2a). These results indicate that the
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 in the bioengineered hair follicle were of human origin
(Fig. 2b).
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 hair shafts cm−2 of normal scalp or in FUT treatment41, respectively,
twenty-eight bioengineered hair germs were transplanted into a cervical skin circle
with a diameter of 1 cm. At 14 to 21 days after transplantation, the bioengineered
hairs were erupted at a high density 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 mouse-derived mesenchymal cells, could be induced to form
autonomously assembled cell aggregates with high-intensity green fluorescence and
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 hair follicle development (Fig. 3a,b)15
17. After the isolation of the bioengineered pelage follicle germ with different numbers
of condensed dermal cell aggregates, we found that the number of bioengineered pelage
follicles was linearly correlated with that of 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 all observed to express ALP and form autonomous
cell aggregates, and epithelial 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 of the bioengineered vibrissa follicles also correlated
with the number of 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 based on properties such as length, thickness,
kinks and hardness, are thought 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 of pelage
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 the cross-sectional shapes (Fig. 3e). The natural and bioengineered pelage hairs
both had a central cavity corresponding to a ladder-like 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 natural
and bioengineered pelages (Fig. 3e)43
44. The outermost layer of the natural 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 the pigmented 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 structures in 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 were reconstructed
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 bioengineered 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 in the bulge ORS layer of the bioengineered
pelage and vibrissa follicles (Fig. 4a). Sox2-positive DP cells (Sox2 is a mesenchymal
stem cell marker), which retain multilineage differentiation potential in vivo
42
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 at anagen phases23
24. The white hair shafts of the bioengineered vibrissae could be caused by the loss
of neural crest-derived melanocyte stem/progenitor cells (Figs 1c, d, f and 4c). The
addition of cells from the 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 with a frequency of 27.0% (n=17;
Fig. 4c,d). In contrast, the bioengineered vibrissae derived from regenerated follicles
with proximal hair matrix (PHM) region-derived cells (Fig. 2c), which contain immature
melanocytes that are the progeny of melanoblasts23
24
25, were pigmented black with a frequency of 40.1% (n=19; Fig. 4c,d) and had the structural
properties of natural vibrissae, as assessed by electron microscopy (83.3%, n=6; Fig.
4c,d). The fine structure of 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 with regard 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
24
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 and mesenchymal cell populations.
Bioengineered hair cycles
We further analysed the hair cycles, that is, the alternative growth (hair-growing)
and regression (non-growing) 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 repeated the 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, as calculated from 3 hair cycle periods
(Fig. 5c,d). The growth and regression 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 found between the natural and bioengineered follicles
(both pelage and vibrissae; 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 hair shaft 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 the skin 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 the expression 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 retractions of the variable portion of the hair follicle were
observed histologically via the appearance of TUNEL-positive cells and the reduction
of the proximal tip (Supplementary Fig. S1). During the catagen to telogen phase,
hair shafts are not produced and are lost from the hair follicle gradually. It has
been indicated that the hair growth and regression phases of the bioengineered hairs
were dependent on the histological 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 hair cycles 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 a stem cell niche.
Piloerection ability of the regenerated hair follicle
To achieve functional hair follicle regeneration, it is essential that the engrafted
follicle be able to connect to the arrector pili muscle and nerve, and that the follicle
has a piloerection capability45. The possibility of reconstituting the niche for arrector
pili muscle development, in which the cells express NPNT, was demonstrated in the
bulge region of the bioengineered pelage (Fig. 4a). The bulge region of a natural
pelage follicle is connected to the arrector pili muscle, which is composed of calponin-positive
smooth muscle, but not troponin-positive striated muscle, and is innervated through
the development of neuromuscular junctions, which surround the arrector pili muscle
with sympathetic nerve fibres that extend from a deep dermal nerve plexus (Fig. 6a).
All bioengineered pelage follicles were observed to connect to the calponin-positive
arrector pili muscle, but not the troponin-positive striated muscle, and to connect
to the 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 calponin-positive smooth muscle, in any other areas, which is similar to
the structure 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 and vibrissa 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 the angle
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 indicate that the bioengineered hair follicles have
a piloerection ability that is comparable to that of natural follicles45. These findings
indicated that 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 through the 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 can repeat the hair cycle, connect properly
with surrounding skin tissues and achieve piloerection. This regeneration occurs through
the rearrangement of various follicular stem cells and their niches. These findings
significantly advance the technological development of bioengineered hair follicle
regenerative therapy.
Hair regeneration methods that rely on the reproduction of epithelial–mesenchymal
interactions have been attempted in several previous studies31
32
33
34
35
36
37
38
39. It was also reported that the aggregation of dissociated epithelial and mesenchymal
cells induced to form hair follicle self-assemblies during embryonic hair follicle
development could be reproduced34
35
36
37
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 regeneration
of 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 and the interfollicular epidermis, and this position is
maintained as a follicle-invariable region, during hair cycling16. Our bioengineered
vibrissa follicle germ that was reconstituted using adult follicle-derived stem cells
successfully regenerated the hair follicle following intracutaneous transplantation.
Hair shafts also erupted from this bioengineered 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 connections
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
35
36
37
38
39
48
49
50. It is also essential to rearrange these various stem cells and their niches in
the 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
22
23
24
25. In contrast, stem/progenitor cell populations that can differentiate into DS cells,
adipocytes, cartilage cells and fibroblasts are found among the DP cells18
19
20
42. Our bioengineered hair follicle can regenerate 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 the reconstitution of
niches for epithelial stem cells and DP cells. These observations 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, and cooperation with host cutaneous tissues including
the arrector pili muscle and nerve system26
51
52. Properties of the hair shaft, which reflect the function of the hair in the body
region14
15
16, are regulated by hair follicle mesenchymal DP and DS cells and are also modulated
by the expression of various genes in the epidermis15
16
17. It is also thought that hair pigmentation is controlled by melanocyte differentiation
and the proliferation of melanocyte-lineage stem cells below the bulge region23
24
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
a proper 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 gland region-derived cells. Our findings thus provide new insights
into the regulation of hair properties and strongly suggest that these characteristics
could be properly restored by cell processing for organ regeneration and by the transplantation
of bioengineered hair follicle germ.
The peripheral nervous system has essential roles in organ function and the perception
of noxious stimuli, such as pain and mechanical 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 mediated 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 that the transplantation of a bioengineered
hair follicle germ can restore natural hair function and re-establish the cooperation
between the follicle and the surrounding recipient muscles and nerve fibres. Thus,
the transplantation of bioengineered hair follicle germ is potentially applicable
to the future surgical treatment of alopecia.
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 optimization of 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 of organ 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 were obtained from Japan SLC and the RIKEN Bioresource
Center (Tsukuba, Japan). The mouse care and handling conformed to NIH guidelines,
and the requirements of the Tokyo University of Science Animal Care and Use Committee.
Isolation and dissociation of embryonic skin
E18 mouse embryonic back skin specimens 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−1 collagenase (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
adult vibrissa follicles from which the collagen sheath was removed. DPs were isolated
from anagen I–IV vibrissa follicles, explanted onto a plastic culture dish (BD), and
maintained as primary cultures for 9 days, as described previously55. The cultured
DP cells were collected with 0.05% trypsin (Invitrogen), and single cells were isolated.
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 hair follicle23
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 middle of 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 located 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 bioengineered murine 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 and mesenchymal
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 days in an organ culture.
The bioengineered vibrissa follicle germs were regenerated from 7- to 8-week-old vibrissa-derived
bulge region epithelial (1×104 cells) and primary cultured DP cells (3×103 cells).
These bioengineered hair germs were then placed 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 thrusting epithelial 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
these regions, 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 intracutaneously
transplanted 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 were intracutaneously 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 made in 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 intradermally transplanted into the
wounds. The nylon guide was held so that it protruded from the 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 were observed using a SteREO Lumar V12 and AxioCam fluorescent
stereoscopic microscope system (Carl Zeiss, Oberkochen, Germany). Observations were
made every 2–3 days.
Bioengineering of human hair follicle germ
This procedure was performed with the 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 povidone-iodine
and 70% ethanol for 10 s, twice successively. The hair follicles in the anagen 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 underneath the 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); and troponin (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 fluorescence microscopy images were captured
under a confocal microscope: the LSM 780 (Carl Zeiss).
Detection of human cells by nuclear staining
The human bioengineered hair follicles were dissected, fixed in 10% formalin, processed
for standard paraffin embedding, and sectioned at 5 μm. Discrimination between mouse
and human nuclei 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 anti-digoxigenin 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 with a 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 the hair 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.
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.
Additional information
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).
Supplementary Material
Supplementary Information
Supplementary Figure S1 and Supplementary Table S1