Figure 8: Functional analysis of induced human melanocytes in vivo.

(a) Skin reconstitution assays showed pigmented hair follicles using hiMels. hiMels, hMels or fetal hFs combined with neonatal mouse dermal fibroblasts and epithelial cells derived from BALB/c (albino) mouse skin. Cells were injected into the back skin of an immunodeficient mouse. After 3 weeks, grafts were photographed from the underside of the skin. Pigmented hair follicles were observed in hiMels or hMels groups in the skin reconstitution assays, whereas pigmented hair follicles were not observed in the group containing fetal hFs. Scale bar, 5 mm. n=5. (b) Human-specific Alu staining (green nuclei) confirmed human origin of iMels (left, upper panel) and H&E staining of consecutive section showed a cyst with hair follicle formation (right, upper panel). Scale bar, 200 μm. Human cells located in the bulb region of a hair follicle (left, lower panel) and basal layer of epidermis (right, lower panel). Scale bar, 50 μm. (c,d) Immunostaining of the xenografts from the patch assays using antibodies against human DCT (c) and TYRP1 (d). DCT⁺ cells were present in the interfollicular dermis and bulb region of hair follicles. TYRP1⁺ cells were observed in both hair follicles and the epidermis. Scale bar, 20 μm. (e) Immunohistochemical staining of the xenografts using the antibody against human S100. S100⁺-positive cells were located in epidermis, hair follicle and the dermis. Scale bar, 20 μm. (f) Fontana–Masson staining of the xenografts showed no pigments in xenografts formed by fetal hFs and mouse cells (left), whereas abundant melanin pigment was evident in the epidermis and follicular epithelium when hiMels were included in the assays (right). Scale bar, 20 μm.