Culture and characterization of spermatogonial stem cells (SSCs) in post-pubertal mice

Abstract

Spermatogonial Stem Cells (SSCs) represent only a small percentage of cells in the adult testis.This present study is aimed to carry out isolation, culture and characterization of Spermatogonial stem cells in post-pubertal mice. Isolation of SSCs was done by two-step enzymatic digestion method and the isolated cells were seeded in a six-well plate. In primary culture (P0), colony forming unit was observed on day four post-incubation. Mouse spermatogonial stem cell (mSSC) adherence was observed on day eight post incubation. On twelve day post
incubation, the cells were subcultured for further expansion. In the subsequent passages (P1 and P2) though a heterogenous morphology was observed, most of the cells showed spindle shaped structure. mSSCs were positive for Alkaline phosphatase activity at passage 1 and passage 2 (P1 and P2) and showed positive expression for the transcription factors viz., Oct4 and Sox2 which was indicated by intranuclear green fluorescence. Flowcytometric analysis revealed that the mSSCs showed positive expression for the SSEA1 marker.