Characterization and in vitro culture of putative spermatogonial stem cells derived from feline testicular tissue.

Spermatogonial stem cells (SSCs) function to regulate the balance of self-renewal and differentiation of male gametes. SSCs have been successfully isolated and cultured in vitro in several species, but not in feline. Therefore, in this study, we aimed to culture and characterize feline SSCs. In experiment 1, testes (n=5) from different pubertal domestic cats were cryosectioned and fluorescently immunolabeled to examine the expression of SSC (GFRα-1), differentiated spermatogonium (c-kit) and germ cell (DDX-4) markers. In experiments 2 and 3, testicular cells were digested and subsequently cultured in vitro. The resultant presumptive SSC colonies were then collected for SSC identification (experiment 2), or further cultured in vitro on feeder cells (experiment 3). Morphology, gene expression and immunofluorescence were used to identify the SSCs. Experiment 1 demonstrated that varying types of spermatogenic cells existed and expressed different germ cell/SSC markers. A rare population of putative SSCs located at the basement membrane of the seminiferous tubules was specifically identified by co-expression of GFRα-1 and DDX-4. Following enzymatic digestion, grape-like colonies formed by 13-15 days of culture. These colonies expressed GFRA1 and ZBTB16, but did not express KIT. Although we successfully isolated and cultured feline SSCs in vitro, the SSCs could only be maintained for 57 days. In conclusion, this study demonstrates, for the first time, that putative SSCs from testes of pubertal domestic cats can be isolated and cultured in vitro. These cells exhibited SSC morphology and expressed SSC-specific genes. However, long-term culture of these putative SSCs was compromised.