Long-term expansion of directly reprogrammed keratinocyte-like cells and in vitro reconstitution of human skin

摘要

Direct reprogramming of human urine cells into iKCs. Schematic diagram of the induction of iKCs from human urine cells. Transcription factors (TFs). Morphological changes of urine cells during direct reprogramming. At passage 3, urine cells were infected with retroviruses encoding BN in urine proliferation medium. BN-infected urine cells were plated in 2% FKGM on a 3 T3-J2 feeder layer to form holoclones. At day 12, the holoclones were picked for further expansion and characterization. Scale bars = 500 μm. Immunostaining of the stem cell markers KRT15 and ITGA6 in BNK-, BN-, BK-, and NK-infected cells with specific antibodies at day 12 post-induction. Nuclei were counterstained with DAPI. The lower row shows magnified images of the boxed areas in the upper row. Scale bars = 200 μm (upper), 100 μm (lower). Number of ITGA6 KRT15 colonies formed by BNK-, BN-, BK-, NK-, B-, N-, and K-infected cells and urine cells at 12 days post-induction. Cells were seeded at a density of 2 × 10 cells per well in a 12-well plate. Data represent the mean ± SEM. ***  e qRT-PCR analysis of relative expression of stem cell markers in BNK-, BN-, BK-, NK-, B-, N-, and K-infected cells, urine cells, and pKCs at 12 days post-induction. Data represent the mean ± SEM. GAPDH was used as a loading control. B, BMI1; N, △NP63α; and K, KLF4