Co-Culture of Spermatogonial Stem Cells with Sertoli Cellsin the Presence of Testosterone and FSH ImprovedDifferentiation via Up-Regulation of Post Meiotic Genes

Spermatogonial stem cells (sscs) maintain spermatogenesis throughout life in the male.maintenance of sscs and induction of spermiogenesis in vitro may provide a therapeutic strategy to treatmale infertility. this study investigated in vitro differentiation of mouse sscs in presence or absence ofsertoli cells, hormones and vitamins. spermatogonial populations were enriched from testes of 4-6 week oldmales by magnetic activated cell sorting and anti-thy-1 antibody. sertoli cells isolated from 6-8 week oldtestes were enriched using lectin-dsa-coated plates. isolated sscs were cultured in the presence ofleukemia inhibitory factor (lif) for 7 days in gelatin-coated dishes, then dissociated and cultured for 7 daysin media lacking lif in the presence or absence of sertoli cells, with or without fsh, testosterone andvitamins. after one week, the effects of sertoli cells ± supplementary media on ssc differentiation wasevaluated by microscopy and expression of meiotic and postmeiotic transcripts using rt-pcr. ssc colonieshad limited development after lif removal alone, exhibiting low expression of meiotic (scp3, th2b) but notpostmeiotic transcript, and loss of stra8 and dazl expression. sscs co-cultured with sertoli cells, hormonesand vitamins developed spermatid-like cells expressing postmeiotic markers (tp1, tp2, prm1) at levels over2-fold higher than sertoli cells or hormone/vitamins alone. our present ssc-sertoli co-culture providesconditions that may allow efficient in vitro differentiation of sscs for the treatment of male infertility.