Compositions and methods for generation of human epithelial stem cells
View Patent ↗Compositions and methods for generating human epithelial stem cells from induced pluripotent stem cells are disclosed. Also disclosed are methods of using cells so generated in hair transplant procedures.
1. A method for differentiating human induced pluripotent stem cells (hiPSC) into hair follicles and interfollicular epidermis comprising the steps of:
(a) transducing isolated human dermal fibroblasts with nucleic acids encoding OCT3/4, SOX2 and KLF4 to obtain hiPSCs,
(b) culturing the hiPSCs of step (a) in a fresh medium comprising BMP4,
(c) culturing the cells of step (b) in a fresh medium comprising retinoic acid (RA),
(d) culturing the cells of step (c) in a fresh medium comprising RA, BMP4 and EGF,
(e) culturing the cells of step (d) in a fresh medium comprising BMP4 and EGF to obtain human epithelial stem cells (hEpSCs) which express CD200, ITGA6, and KRT15; and
(f) culturing the hEpSCs of step (e) under conditions to obtain hair follicles and interfollicular epidermis.
2. The method of claim 1 , wherein said hiPSCs are differentiated over a 25 day period and the BMP4 in step (b) is present at days 0-1, the RA in step (c) is present at days 1 to 11, the RA, BMP4 and EGF in step (d) are present from days 3-11 and the BMP4 and EGF in step (e) are present from days 11 to 25, and said method further comprises expanding said hEpSCs obtained in step (e) in a growth medium comprising EGF to obtain mature keratinocytes.
3. The method of claim 1 , wherein the hEpSCs in step (e) are cultured in a chamber with fibroblasts to obtain hair follicles.