PMC

(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.

(A) Hair growth is strongly reduced in β-catenin cKO mice compared to WT. Note that guard hairs are absent from the lower backskin. (B) H&E sections showed thinner skin with fewer follicles in cKO. (C) Overall length of hair shafts from all three waves is shorter in cKO. Note that 2^nd wave auchene hairs were missing. (D) Morphological analysis of hair shafts from all hair types. Awl hair contained two different subtypes in cKO; auchene hair was absent. (E) Quantification of all hair shaft types. Guard hairs were strongly reduced; auchene hairs were absent in cKO, but awl hair numbers were increased.

Mouse harmonin b fused with EGFP on the C-terminus (harmonin-EGFP) and frog PMCA2 fused with EGFP on the C-terminus (PMCA2-EGFP) were transfected into frog and chick hair cells using the modified setup. Harmonin-EGFP and PMCA2-EGFP are pseudo-colored green, and phalloidin-stained actin is shown in red, except in panel K, in which co-transfected mCherry-actin is shown in red. A–D, Frog sacculus hair cells transfected with harmonin-EGFP. E, Frog sacculus hair cells labeled with anti-harmonin antibody H3. Structured-illumination image. F, Chick utricle hair cell transfected with harmonin-EGFP; G, Higher digital gain image of E, showing clusters of harmonin-EGFP along the stereocilia (arrows). H–I, chick cochlea hair cells transfected with harmonin-EGFP. J, Frog sacculus hair cells labeled with anti-harmonin antibody H3. K, Live-cell imaging of a chick utricle hair cell transfected with PMCA2-EGFP and mCherry-actin (red). L, Frog sacculus hair cell transfected with PMCA2-EGFP. M, Frog sacculus hair cell labeled with anti-PMCA2a antibody F2a. Panels are 18.4 µm wide.

(A) Hair follicloids after 10 days of culture. Whole-mounted hair follicloids were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed using a confocal laser microscope (i) and scanning electron microscope (SEM) (ii). The whole-mounted hair follicloids were stained with oil red O and observed using a stereomicroscope (iv). The hair follicloids were sectioned and stained with HE (iii), a fluorescent antibody against α-SMA (v), Sox9 (vi), CD34 (vi), K5 (vii), Ki67 (viii), versican (ix), gp100 (x), TRP1 (xi), and c-Kit (xii). The inserted images show magnified views of the box area in the images. BF, bright field. (B) Hair cuticles of generated hair shafts. The hair shaft was observed after 10 days of culture using a scanning electron microscope. (C) Hair bulb of generated hair follicle after 10 days of culture. Hair follicloids were observed using a stereomicroscope. (D) Microstructures of generated and native hair shafts. Cross sections were observed via transmission electron microscopy. White arrowheads indicate melanin granules, and black arrowheads indicate hair cuticles.