The properties of self-renewal and divisionin spermatogonial stem cells (SSCs) support spermatogenesis. There are a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and self-renewal ofgermline stem cells (GSCs) is of tremendous benefit for clinical trials, experimental research andas potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system.
Materials and methods
In this system, testicular cells from neonate mice have been cultured in agarose coated plates in the presence of the DMEM (CTRL group) medium, 10% FBS (10% group) and growth factor (G group), containing 2% FBS, GDNF, EGF, and FGF. Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, RT-PCR and flow cytometry against the germ cell markers (α6, β1, c-Kit, Thy-1, C-Ret, Plzf and Oct4). The statistical significance between mean values in different groups was determined by one-way analysis of variance (ANOVA).
Spermatogonial stem-like colonies were established in both G and 10% groups, but not in the CTRL group. Immunocytochemistry, flow cytometry and RT-PCR confirmed the expressions of germ cells markers in these spermatogonial stem-like cells. In the spermatogonial stem-like cells, we observed a significant expression (p<0.05) of germ cell markers in G and 10% groups versus the testis cells (T). Their proliferative and apoptotic activities were examined by Ki67 and PI/Annexin V-FITC. An Alkaline phosphatase assay showed that mouse spermatogonial stem-like colonies were partially positive.
According to these results,a non-adherent culture system could provide a favorable methodfor in vitro short term culture of spermatogonial stem-like cell colonies.