Comparison of the efficacy of three concentrations of retinoic acid for transdifferentiation induction in sheep marrow‐derived mesenchymal stem cells into male germ cells

Summary Recent studies have shown the unique role of retinoic acid ( RA ) in the induction of transdifferentiation in mesenchymal stem cells ( MSC s) into germ cells ( GC s). This study is the first study that compares the efficacy of three different concentrations of RA for the production of male GC s in vitro . Male sheep marrow‐derived MSC s ( MMSC s) were treated with the following concentrations of RA : 1 μ m ( RA 1), 5 μ m ( RA 2) and 10 μ m ( RA 3) for a period of 21 days. The production of male GC s was evaluated by the assessment of expressions of GC ‐specific markers (by RT ‐ PCR , q RT ‐ PCR and immunocytochemistry), morphological characteristics and changes in alkaline phosphatase ( ALP ) activity. All three concentrations created male GC features. RA treatment upregulated the expressions of VASA and beta1 INTEGRIN and downregulated PIWIL2 and OCT4 . DAZL was not expressed by RA treatment. Interestingly, immunocytochemistry detected PGP 9.5 expression in all treatment groups, with the highest expression noted in the RA 3 group ( P  < 0.05). GC ‐like cells along with increased ALP activity were observed in all treated cultures, too. Finally, results showed that 10 μ m RA has the most efficiency for transdifferentiation induction in MMSC s and production of male GC s in vitro .