Direct Reprogramming Of Fibroblasts Toward Leydig-like Cells By Defined Small Molecular Compounds

Leydig Cell(LC)exists between the seminiferous tubules of male mammals,which is responsible for most androgens synthesis and secretion.Cell therapy is a safe and effective method for androgen deficiency treatment.However,Leydig cells are difficult to obtain and cannot proliferate in vitro,resulting in scarcity of seed cells.This study intended to induce the reprogramming of fibroblasts into functional Leydig-like cells using small molecule compounds.And the induced Leydig-like cells would be used for cell transplantation to treat androgen deficiency,which overcomes the limitations of seed cell source and immune rejection of Leydig cell,may have promising potential for the treatment of male hypogonadism while simultaneously preserving the HPG axis.In this study,a candidate small molecule compound library was established based on the mechanism of chemical reprogramming and the signal regulation pathway of Leydig cell development.Based on the expression level of key transcription factor gene Nr5a1 in Leydig cell development and the content of testosterone in the cell supernatant,we found the combination of Forskolin,20?-hydroxycholesterol,luteinizing hormone(LH),and SB431542 could significantly increase Nr5a1 expression and testosterone production.The small-molecule compounds were used to treat mouse embryonic fibroblasts in combination,and real-time quantitative PCR,immunofluorescence,Western blot,and radioimmunoassay were used to detect steroid-related genes,protein expression and testosterone production.After 14 days of induction,the induced cells expressed steroidogenesis genes and proteins,had a gene expression profile resemble to Leydig cells,and could synthesize and secrete androgens.In addition,this chemical cocktail could also induce human periodontal ligament fibroblasts(HPLFs)into Leydig-like cells,indicating that the method was versatile.Finally,to evaluate the biological function of Leydig-like cells in vivo,we transplanted these cells into the testicular stroma of a model rat with low androgen.At 21 days,implantation of induced Leydig like dramatically improved the recovery process and serum testosterone in Leydig like cell transplantation group nearly reached the normal control level.In summary,these results suggested that the combination of Forskolin,20?-hydroxycholesterol,luteinizing hormone(LH)and SB431542 could reprogram fibroblasts into functional Leydig-like cells.Unlike genetic manipulation,which may cause mutations,reprogramming fibroblast into Leydig cells by small molecules likely represent a safer approach which are much easier to be industrialized and applied in clinic.