Expression profiling of stemness markers in testicular Germline Stem Cells from neonatal and adult Swiss albino mice during their transdifferentiation in vitro

Abstract

Abstract Background. Spermatogonial stem cells (SSCs) were considered to be stem cells with limited potencies due to their existence in adult organisms. However, the production of spermatogonial stem cell colonies with broader differentiation capabilities in primary germ cell cultures from mice of select genetic backgrounds (C57BL6/Tg14, ddY, FVB and 129/Ola) indicated that SSCs from these strains were pluripotent. Methods. We established primary cultures of SSCs from neonatal and adult Swiss 3T3 Albino mice. Stemness of SSC colonies were evaluated by performing real-time PCR and immunofluorescence analysis for a panel of chosen stemness markers. Differentiation potentials of SSCs were examined by attempting the generation of embryoid bodies and evaluating the expression of ectodermal, mesodermal and endodermal markers using immunofluorescence and real-time PCR analysis. Results. Spermatogonial stem cells from neonatal and mature mice testis colonised in vitroand formed compact spermatogonial stem cell colonies in culture. Alkaline phosphatase positivity and the presence of stem cell marker Oct-4 indicated stemness in these colonies. The differentiation potential of these SSC colonies was demonstrated by their transformation into embryoid bodies upon withdrawal of growth factors from the culture medium. SSC colonies and embryoid bodies formed were evaluated using immunofluorescence and real-time PCR analysis. Embryoid body like structures derived from both neonatal and adult mouse testis were quite similar in terms of the expression of germ layer markers. Conclusion. These results strongly suggest that SSC-derived EB-like structures could be used for further differentiation into cells of interest in cell-based therapeutics.