Hair organ regeneration via the bioengineered hair follicular unit transplantation

Abstract

Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia.

Figure 1. Ectopic regeneration of a hair follicle via transplantation of a bioengineered hair follicle germ.

(a) A schematic representation of the method for regenerating the hair follicle via transplantation into the sub-renal capsule (SRC). (b) Morphologic and histological analysis of the bioengineered hair follicle germ. Phase contrast photographs of a hair follicle germ at 0 hr and 2 days after cultivation (left two panels). The dotted lines indicate a boundary between the epithelial and mesenchymal cells in the OC (organ culture) at 0 hr and day 2. Macro-morphological observations of the bioengineered hairs at 14 days after transplantation into the sub-renal capsule (right panel). (c) Histological analysis of the bioengineered hair follicles. The boxed area shows high-magnification in the right two panels. ad, adipocyte; cys, cyst; dp, dermal papilla; hs, hair shaft; irs, inner root sheath; ors, outer root sheath; sg, sebaceous gland. (d) Microscopic observations of the ectopically bioengineered hair shafts classified as awl, zigzag or unclassified hairs. The percentage of regenerated hair types (right). Bars represent standard deviations. (e) A comparison of the length of the hair follicle and the bulb between the natural and regenerated awl hairs.

Figure 2. Hair follicle regeneration via intracutaneous transplantation of regenerated bioengineered hair follicles.

(a) Macroscopic observations of the transplantation procedure of the regenerated hair follicles. A slit is made using a surgical knife (left), the regenerated hair follicle is transplanted using forceps (middle), and a surgical tape bandage is used to cover the transplanted hair follicle (right). (b) Macroscopic observations of a transplanted regenerated hair follicle at day 16 (left panels) and day 22 (right panels). (c) Histological analyses of the transplanted regenerated hair follicles. H&E-staining (upper panels) and fluorescent microscopy (lower panels) of the transplanted regenerated hair follicle of a serial section is shown. The boxed areas in the left panels are shown at a higher magnification in the right panels. Broken lines indicate the outermost of the hair follicle. (d) Immunohistochemical analysis of the bulge region in a transplanted regenerated hair follicle. The bulge region was analyzed by immunostaining with specific antibodies for CD34 (upper center) and CD49f (upper right), and the sebaseous gland which was located upper region of the bulge was stained with oil red (upper left). The hair bulb was analyzed by immunostaining with anti-versican (lower left) and α-SMA (arrowheads, lower right) antibodies and by enzymatic staining for ALP (lower center). The nuclei were stained using Hoechst 33258 (Nuc, blue). The arrowheads show positively staining areas.

Figure 3. Hair cycles of transplanted regenerated hair follicle ectopically.

(a) The macroscopic analyses of various phases of the hair cycles. The bioengineered hair follicles were observed at 13 days, 17 days, 27 days and 39 days after transplantation. (b) Cycles of hair growth (dark bars) and regression (light bars) phases. The left panel shows the term of growth and regression phases of the individual regenerated hairs until 60 days after transplantation. The right panel shows the hair cycle term of natural hair (Natural hair) and the transplanted regenerated hair (Bio-hair) in the 1st and 2nd hair cycles. The error bars indicate the s.e.m. (n = 8 in natural hair, n = 5 in transplanted regenerated hair). (c) Histological and immunohistochemical analyses of the transplanted regenerated hair follicles at day 30 and day 42. H&E-staining (upper panels) and immunostaining with anti-GFP (lower panels) of a transplanted regenerated hair follicle of a serial section is shown. The boxed areas in each panel are shown at a higher magnification in the right panels. (d) Macroscopic observations of transplanted regenerated Awl hair (upper, at 1st and 2nd growth term) and zigzag hair (lower, at 1st and 2nd growth term). The right panels show macro-morphological images of each hair follicle at day 43 and day 49.

Figure 4. Piloerection ability of ectopically transplanted regenerated hair follicles.

(a) Immunohistochemical analysis of the regenerated hair follicles in the sub-renal capsule at day 14 (center) and 22 days after transplantation into the back skin (right) and natural back skin (left). Specific antibodies against calponin (Cal, to detect smooth muscle, red) and neurofilament H (NF, white) were used. The boxed areas in each panel are shown at a higher magnification in the lower panels. The nuclei were stained using Hoechst 33258 (Nuc, blue). The arrows and arrowheads show positively stained calponin and NF-H areas. (b) Analyses of the piloerection ability of the transplanted regenerated hair follicles following an intradermal injection of ACh. The positions of the hair shafts (black arrowheads in left) moved after this treatment (white arrowheads in center) and merged (right). (c) Assessment of hair angle changes associated with regenerated hair (light bars) and natural hair (dark bars) before (PBS(-)) and after the administration of ACh. The error bars represent the standard deviation (n = 6). P<0.05 (*) was regarded as statistically significant.