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).

Figure 1: Methods for hair follicle regeneration. (a) Schematic representation of the methods used for the generation and transplantation of a bioengineered 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 a natural vibrissa isolated from an adult mouse. Macroscopic and H & E staining of whole vibrissa is shown in left two panels. Dashed lines (red) via macro-morphological 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 of epithelium 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 the bulb 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 staining of 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 the bioengineered 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 the wound 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. Full size image

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.

Figure 2: Hair follicle regeneration via the intracutaneous transplantation of a bioengineered hair follicle germ. (a) Histological and immunohistochemical analyses of bioengineered pelage (upper) and vibrissa (middle) follicles. The boxed areas in the low-magnification H & E panels are shown at a higher 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 bulge-derived 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 morphological features (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, and 20 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. Full size image

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.

Figure 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 days in 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 the epithelial–mesenchymal boundary. Scale bars: 200 μm. (c) Analyses of EGFP fluorescence, enzymatic staining of ALP and H&E staining in adult vibrissa-derived 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) with various 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 hair shaft (lower) are shown. Cuticle layers (arrowheads) and dense medullary granules (arrow) are shown in the fine TEM photographs of the extremity and centre 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. Full size image

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 vivo42,43, were also detected in the DPs of the bioengineered pelage and vibrissa follicles (Fig. 4b).

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 shown as 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 areas in the left panel are shown at a higher magnification in the right panels. Nuclei were stained using Hoechst 33258 (Nuc, blue). Broken lines indicate the outermost 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 was reconstituted 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) and TEM (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 region of 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. Full size image

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).

Figure 5: Hair cycle analyses of bioengineered hair follicles. (a) Schematic representation of the hair cycle defined by histological and macro-morphological observations of the hair shaft. (b) Macro-morphological observations of the hair cycles in a bioengineered pelage (upper panels) and vibrissa (lower panels). In the bioengineered vibrissa, arrowheads (open or closed white, red, blue and green) indicate individual hairs. Closed white and red arrowheads are identical to hairs indicated as opened ones, which erupted from the respective follicles. Scale bars, 1.0 mm. (c) Assessment of 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. Full size image

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).

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 limit of 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 the hair shafts (black or white arrowheads in the left) moved after this treatment (centre) and merged (right). Scale bars, 1.0 mm. (c) Assessment of hair angle changes associated with the bioengineered pelage (light bars) and vibrissae (dark bars) before and after the administration of ACh, without or with atropine (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 (*) and P<0.05 (**) were defined as statistically significant, as analysed by t-test. Full size image

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).