Fig. 2

Fates of Epi-SCs and SKPs in mixture in vitro and in vivo. a–f Epi-SCs and SKPs in skin organoid formation. Epi-SCs labeled with tdTomato (red) were cultured in monolayer (a) and SKPs derived from C57-EGFP mice were grown in spheroids (b). Single cells of the above were mixed evenly in Matrigel (c, d) to form a sphere, which was incubated at 37 °C for 24 h. Cross-sections of the sphere showed that the cells were repopulated into two compartments, with the Epi-SCs-tdTomato in the outer layer (red) and the SKPs-EGFP (green) in the inner compartment (e, f), resembling the structure of bilayer skin. g–r Fates of Epi-SCs and SKPs in vivo. A mixture of single Epi-SCs-tdTomato and SKPs-EGFP in Matrigel was implanted into an excisional wound in a nude mouse, and the graft was observed under two-photon microscope. In the first 3 days, Epi-SCs aggregated forming spheres (red, g and h). By day 5, Epi-SCs migrated upward and formed an epidermis-like layer over SKPs (i, j). Some Epi-SCs in the layer then moved downward into the SKPs forming a primary structure of the hair follicle by 12 days; images of graft surface (k), horizontal section (l), and vertical section (m) were shown. n–q Tissue sections of the wound at 14 days post transplantation showed that the Epi-SCs and SKPs formed de novo skin structures, where the DPs and dermal cells were derived from the GFP-expressing SKPs (n, q); the epidermis and the trunk of the hair follicle were formed by the Epi-SCs-tdTomato (o, q). r, s At the interface of DP and follicle germ was the matrix (r, s). HF, hair follicle; Epi, epidermis; DP, dermal papilla. Scale bar, 100 μm