Fig. 1: Cultured Tp63-expressing epithelial stem cells have hairy skin competence.

a Schematic representation of the experimental strategy. Various epithelial cells were obtained from EGFP rats, cultured and serially transplanted into the skin of newborn wild type mice. A typical experiment, from the initial biopsy to the end of the 2nd transplantation, usually ran for more than a year. b Typical appearance of colonies initiated by esophageal epithelial cells (10 independent experiments). Left: phase contrast appearance of a 7 days old progressively growing colony; bar: 100 μm. Right: appearance of 10 days old Rhodamine B stained colonies (red). c Immunostaining showing that clonogenic epithelial cells cultured from whisker, bladder and trachea expressed ∆Np63 (red nuclear staining). Nuclei (blue) were counterstained with Hoechst 33342. d Serial transplantation of Tp63-expressing epithelial stem cells from whisker, bladder, and trachea into the skin of newborn wild type mice; 34 mice were transplanted in the first round of transplantation out of which 21 displayed a long-term EGFP transplant; 23 mice were transplanted in the second round of transplantation out of which 12 displayed a long-term EGFP transplant. Representative microphotographs showing that transplanted EGFP cells from whisker and bladder formed epidermis, hair follicles, and sebaceous glands whereas tracheal cells only formed an epidermis-like epithelium. EGFP expression in the transplanted cells was revealed by immunohistochemistry. First transplantation: upper and middle panels; Upper panel: whisker: 236 days old transplant; bladder (dome): 238 days old transplant; trachea: 122 days old transplant. Middle panel: high magnification showing that the EGFP cells contributed to the differentiated layers of epidermis and hair follicles. Whisker: 100 days old transplant; bladder (dome and trigone): 238 days old transplants; trachea: 122 days old transplant. Second transplantation: lower panel; Whisker: 117 days old transplant; bladder (trigone): day 84 transplant; trachea: 100 days old transplant. Nuclei (blue) were counterstained with Hoechst 33342; bars: 100 μm. e Dot plot showing the expression of Tp63, ΔNp63, and TAp63 isoforms determined by qPCR analysis before and after the 1st round of transplantation. ΔCt results were normalized against housekeeping genes (TBP, SDHA, and Tubb). Mean of 3–9 technical replicates with individual data represented by empty circles.