In vitro propagation of adult SSCs. SSCs are derived from the adult testis using somatic feeders and media containing diverse growth factors such as GDNF and FGF-2. In the case of human SSCs, a preenrichment sorting step (FACS or MACS) using previously identified surface marker, is critical for the successful expansion of SSCs. Mouse cultures established in such way can be maintained for over 1 year. Long-term expansion of SSCs in vitro is confirmed by analyzing the expression of molecular markers of spermatogonia. Furthermore, the number of stem cells expanded in the culture must be validated and quantified by an in vivo functional assay consisting of transplantation of SSCs into busulfan-treated recipient mouse testis. The fluorescent image corresponds to seminiferous tubules repopulated with donor GFP-positive cells. Potential clinical applications of SSCs include restoration of male fertility and/or in vitro correction of mutated alleles prior to transplant. Furthermore, in vitro SSCs can spontaneously reprogram to embryonic-like stem cells and could be used for regenerative therapy.
