Direct Conversion Of Human Fibroblasts Into Functional Leydig-like Cells By GATA4,SF-1 And NGFI-B

Background Androgen deficiency is increasingly becoming a major issue affecting men's health.Traditional hormone replacement therapy has many drawbacks.At present,studies have shown that differentiation of stem cells into Leydig-like cells for transplantation is better,but stem cells are less sourced and the induction efficiency is low.However,attempts to reprogramm fibroblasts into Leydig-like cells with testosterone secretion founction are expected to become a new therepy.Objective To investigate the feasibility of reprogramming human foreskin fibroblasts into Leydig-like cells with testosterone secretion in vitro.Methods Enzymatic digestion were used to obtain fibroblasts from children's foreskin.Four key transcription factors(GATA4,SF-1,NGFI-B and COUP TF2)during Leydig-like cell development were transfected into foreskin fibroblasts using GFP-tagged lentiviral infection.One week after transfection,GFP positive cells were selected by flow cytometry for further culture.Specific markers of fibroblasts(E-Cadherin)and Leydig-like cells(CYP11A1,CYP17A1,HSD3B1,StAR and HSD17B3)were detected at mRNA level at 1 week,2 weeks,3 weeks and 4 weeks after transfection.The cell transformation efficiency was determined by detecting the levels of CYP11A1,CYP17A1 and HSD3B1 at the protein level.The supernatant fluid of cultured cells was collected at 1 week,2weeks,3 weeks and 4 weeks after transfection,estinating testosterone levels by chemiluminescence assay.Results qRT-PCR showed that Leydig cell-specific markers in experimental group(CYP11A1,CYP17A1,HSD3B1,StAR and HSD17B3)were significantly higher than those in control group(P <0.05)while no significant difference existed in fibroblast markers(P> 0.05),consistent with the results of immunofluorescence.Although Western blotting sugested that the expression of CYP11A1 and CYP17A1 in experimental group was higher than that in control group at 3 weeks and 4 weeks after transfection,a decreasing trend occured.Experimental group began to secrete testosterone 3 days after transfection,reaching a peak of 3.69 ng /ml(P <0.05)on the 12th day after transfection,and finally decreased to0.147 ng / ml 4 weeks after transfection(P <0.05).In addition,We found that GATA4 and SF-1 were particularly critical for Leydig-specific markers expression and that GATA4,SF-1 and NGFI-B were necessary to generate functional iLCs that secreted testosterone.Conclusion Human fibroblasts can be reprogrammed into induced Leydig-like cells by overexpression of three transcription factors(GATA4,SF-1,NGFI-B)in vitro and may provide insight into potential therapies to treat testosterone deficiency.