Reprogramming Of Human Fibroblasts To Leydig-like Cells By Small Molecules And SF1

Objective To study the feasibility of reprogramming human foreskin fibroblasts(HFFs)to functional Leydig-like cells by SF1 associating with small molecules.Methods HFFs were carefully isolated and cultured,then transfected by SF1 overexpressing virus at passage 2-3.4 days after the transfection,cells were cultured by medium containing different combinations of A 83-01,BIX 01294,CHIR99021,forskolin,DAPT,purmorphamine and SU 16f(“ABCFDPS”)when the expression of SF1 became steady.The culture medium were collected every other day to measure the concentration of testosterone.Screening was performed according secretion function to find the optimal set.Specific markers of Leydig-like cells(STAR,CYP11A1,CYP17A1,HSD3B1,HSD17B3)were detected at the RNA level by qRT-PCR and protein level by immunofluorescence and western blotting.Results Cells induced by ABCFDPS and SF1 could secrete testosterone.The cocktail of forskolin,DAPT,purmorphamine was identified as the optimal set for the reprogramming of HFFs toward functional Leydig-like cells with SF1.RNA expression profiling by qRT-PCR showed that all Leydig cell specific markers were highly expressed.Immunofluorescence and western blotting analysis confirmed the expression of Leydig cell specific markers at the protein level.Conclusion HFFs could be reprogrammed into functional Leydig-like cells by SF1 associating with small molecules.