Poultry spermatogonial stem cells (SSCs) have the potential to serve as a model for studying the basic biology of SSC and they can also be used for biotechnological purposes. However, the small number of SSCs and the presence of the testicular somatic cells with SSCs have limited their applications. Therefore, this study was undertaken for the first time to investigate the effect of a serum-free medium supplemented with a combination of specific growth factors and B27 on the proliferation and enrichment of newborn chicken SSCs in vitro. Newborn chicken testicular cells were cultured in a serum-free DMEM, supplemented with GDNF, bFGF, LIF, and EGF growth factors and also B27 as an alternative for FBS. Presence and maintenance of the SSCs in the enriched cultures were evaluated by detection of alkaline phosphatase (AP) activity and ASZ1, POU5F1, CVH and GPR125 gene expression. A small number of clusters and colonies were emerged in testicular cell cultures before treatment with the enriched cell culture medium. Enrichment of the DMEM with the above indicated factors strongly promoted the proliferation of the chicken SSCs. Moreover, this culture condition declined attachment and maintenance of the testicular somatic cells and thus they decreased gradually in the cultures. The enriched SSCs were positive for AP activity and with detectable levels of ASZ1, POU5F1, CVH and GPR125 gene expression. This study shows that serum-free medium supplemented with a combination of B27 and the above indicated growth factors induces proliferation and enrichment of chicken SSCs in vitro in a short period of time.

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