Comparison of colonization of co-culture of neonate mouse spermatogonial stem cells with sertoli cells in the presence and absence soft agar

Background: To investigate the effects of soft agar on in vitro proliferation of mouse spermatogonial stem cells (SSCs) were co-cultured with sertoli cells obtained from 3 to 6- day old mice.Methods: SSCs and sertoli cells were isolated from the testes of neonate male mice, and their purities were evaluated by flow cytometry technique using PLZF antibody. The isolated testicular cells were cultured in the absence (control group) or presence of soft agar-coated dishes (experimental group) along with leukemia inhibitory factor (LIF) and Glia cell line– derived neurotrophic factor (GDNF) for two weeks. Colony formation after the two weeks of culture was confirmed by alkaline phosphatase staining and the expression of the undifferentiated genes ID-4 and Plzf using real-time PCR on the 14th day . The number and diameter of the colonies of SSCs were assessed by ImageJ software Result: The number and diameter of colonies and the level of expression of ID-4 and Plzf (undifferentiated gene) increased in the experimental group compared with the control group. However, the expression of c-kit (differentiated gene) decreased in the experiment than in the control group.Conclusion: Our findings show that culture in soft agar-coated dishes can have a positive effect on the enhancement of the proliferation of SSCs.

کلیدواژه ها:

Colonization ، Mouse ، Proliferation ، Soft agar ، Spermatogonial stem cell