Directed Differentiation of Human Embryonic Stem Cells Into Keratinocyte Progenitors in Vitro: An Attempt With Promise of Clinical Use

Objectives: To assess the effects of retinoic acid (RA), bone morphogenetic protein-4 (BMP4) and ascorbic acid (AA) on the differentiation of hESCs into keratinocyte progenitors in vitro.
Methods: The first media contained AA and BMP4; the second contained RA, AA and BMP4; and the third was commercial defined keratinocyte serum-free medium, which were used to differentiate H9 hESCs (direct approach) or embryoid bodies (EBs) (indirect approach) into keratinocyte progenitors in vitro Real-time RT-PCR, immunofluorescence and flow cytometry were used to characterize the differentiated cells.
Results: Cells induced by AA+BMP4+RA showed the typical epithelial morphology, while cells induced by AA+BMP4 showed multiple appearances. CK14 and p63 mRNA expressions in the AA+BMP4+RA-treated cells were higher than those of the AA+BMP4-treated cells (CK14: 22.4-fold; p63: 84.7-fold). Epithelial marker CK18, keratinocyte marker CK14, and transcription factor p63 mRNA expressions were higher in cells differentiated from hESCs compared with those differentiated from EBs (CK18: 1.52 ±0.11 vs. 0.08 ±0.05; CK14: 9.27±3.61 vs. 5.32±1.86; p63: 0.73±0.06 vs. 0.65±0.23). After hESCs indueced by AA+BMP4+RA, CK14 mRNA expression was upregulated after day 21 and reached a maximum by 35 days of differentiation.
Conclusions: Combined RA, BMP4 and AA could effectively induce differentiation of hESCs into keratinocyte progenitors in vitro. These keratinocytes could be used for oral mucosa and skin tissue engineering.