Unlocking the vital role of host cells in hair follicle reconstruction by semi-permeable capsules

Abstract

Organ regeneration is becoming a promising choice for many patients; however, many details about the mechanisms underlying organ regeneration remain unknown. As regenerative organs, hair follicles offer a good model to study the mechanisms associated with regenerative medicine. The relevant studies have mainly focused on donor cells, and there are no systematic studies involving the effect of host factors on hair follicle reconstruction. Thus, we intend to explore the effect of host cells on hair follicle reconstruction. Epidermal and dermal cells from red fluorescent protein (RFP) transgenic newborn mice were injected into green fluorescent protein (GFP) transgenic mice. In addition, we wrapped the mixed dermal and epidermal cells from GFP transgenic and RFP transgenic mice by the Cell-in-a-Box kit to form "capsules," so that the cells within would be isolated from host cells. These capsules were cultured in vitro and transplanted in vivo. Fully developed reconstructed hair follicles were observed after the injection of mixed cells. These reconstructed follicles mainly consisted of donor cells, as well as a small number of host cells. The encapsulated cells gradually aggregated into cell spheres in vitro without apparent differentiation towards hair follicles. With respect to the transplanted capsules, concentric circle structures were observed, but no hair follicles or hair shafts formed. When the concentric circle structures were transplanted in vivo, mature hair follicles were observed 30 days later. Host cells were found in the reconstructed hair follicles. Thus, we conclude that host cells participate in the process of hair follicle reconstruction, and they play a vital role in the process, especially for the maturation of reconstructed hair follicles. Furthermore, we established a special hair follicle reconstruction system with the help of capsules: transplant cells were isolated from host, but other factors from host could exchange with cells inside.

Fig 1. The hair regeneration of cells from RFP mice when injected into GFP mice.

(a) Abundant hair follicles were reconstructed when dermal and epidermal cells were injected together, but no hair follicles were reconstructed when epidermal cells (b) or dermal cells (c) were injected alone. (d) Fourteen days after the injection of dermal and epidermal cells: HE sections, arrows indicate the regenerated HFs (green) and the panniculus carnosus (black). (e, f) Frozen sections of reconstructed hair follicles observed under a fluorescence microscope, red indicates donor cells, green indicates host cells. (g) DAPI staining of reconstructed hair follicle. (h) Synthesis, arrows indicate the green fluorescent cells (white). Scale bars = 1 mm in a, b, and c; 100 μm in d, e, f, g, and h.

Fig 2. The cultivation of capsules in vitro.

Dermal and epidermal cells were encapsulated in capsules (a). Cells in the capsules gradually aggregated into small multicellular aggregates (b). Dermal cells (red) and epidermal cells (green) were observed under an inverted fluorescence microscope (c, d). Four days later, multicellular aggregates merged into hybrid spheroids (e). Seven days later, the morphology of hybrid spheroids became stable (f, g [10 d after encapsulation]). Twenty days after capsules were cultured in vitro, cell spheroids were taken out of capsules (h). HE sections of cell spheres were made; neither hair follicles nor concentric circles were observed (i). Cell spheroids attached to the wall when reseeded in culture dishes; cells inside then migrated from the spheroids (j–l, 0 d, 2 d, and 7 d after reseeding, respectively). The capsules were cultured in vitro for 30 d, no apparent change was observed (m). Confocal micrographs taken at 7d (n–p). These images showed dermal (green) and epidermal cells (red) in cell spheroids. The z reconstituted image clearly showed that dermal cells were located in the center and epidermal cells were sorted to the surface (q). Scale bars = 1 mm in a, b, c, d, e, f, g, h, j, l, and m; 200 μm in k; 100 μm in i, n, o, p, and q.

Fig 3. Capsules transplanted in vivo.

(a) Capsules were taken out. (b–d) Observed under the microscope, arrows indicate cell spheroids (red). (e) Thirty days after the capsules were transplanted subcutaneously into nude mice, HE sections, and cells formed concentric circles. Scale bars = 1 mm in b, c, and d; 50 μm in e.

Fig 4. Hair reconstruction of the cell spheroids inside the capsules 10 d after the transplantation.

(a) Cell spheres inside the capsules were acquired for single transplantation into nude mice. Thirty days after transplantation, mature hair follicles were reconstructed successfully. (b) Red fluorescence of the reconstructed hair follicle. (c) Green fluorescence figure of the reconstructed hair follicle. (d) DAPI staining of the reconstructed hair follicle. (e) Synthesis, arrows indicate GFP cells from host mice (red). Scale bars = 50 μm in a; and 100 μm in b, c, d, and e.