Germ cell differentiation of bone marrow mesenchymal stem cells.

Abstract

Bone marrow mesenchymal stem cells (BM-MSCs) were first cultured under induction of retinoic acid (RA), Sertoli cells conditioned medium and RA + con (conditioned medium) as treatment groups. The presence of Sertoli cells was confirmed by immunocytochemistry of follicle-stimulating hormone receptor in Sertoli cells and flow cytometry by anti-Gata4 antibody. Cell viability and morphology of nucleus and cytoplasm of BM-MSCs were evaluated by MTT test and DAPI staining respectively. The expression of Oct4, Plzf, Scp3, Caspases 8, 9 and 3 genes was evaluated by RT-PCR. For increasing the accuracy of experiment, the expression of Vasa and SCP3 genes was investigated quantitatively by real-time PCR after 0, 5, 10, 15days of culture. The results showed that the number of apoptotic cells increased in RA group. The expression of apoptosis genes (Caspases 3, 8 and 9) was also observed in this group all days of culture. Measurement of Vasa and Scp3 genes by RT-PCR confirmed the positive effects of retinoic acid on increasing of genes expression. So, in this study, a group with maximum expression of differentiation genes and minimum expression of apoptotic genes was RA + conditioned medium group. DNA fragmentation was not observed in all groups.