Effect of poly-D-lysine on proliferation and senescence of caprine spermatogonial stem cells in vitro

Abstract

Spermatogonial stem cells (SSCs) have the unique ability of both self-renewing and to produce progeny that undergoes differentiation to spermatozoa. As SSCs exist in very low numbers, therefore efficient in vitro expansion of SSCs is important prior to their clinical applications. In this study, we tried to improve the functionality of putative SSCs (pSSCs) during culture using poly-D-lysine (PDL) coating. For this, plates were coated with 0.01% PDL with different coating time interim treatments (5, 30 and 60 min) while control remained uncoated. The adequate amount of pSSCs of the goat was isolated and enriched using two-step enzymatic digestion and differential plating methods. Further, the functionality of pSSCs was evaluated by cell growth analysis, cell proliferation, senescence, and the presence of pluripotency (alkaline phosphatase, OCT-4) and SSC related (PGP-9.5) markers. The number and size of pSSCs colonies in 0.01% PDL coating groups were significantly (P <0.05) higher than in the control group. Similarly, pSSCs on uncoated plates expressed significantly (P <0.05) higher 𝛽-galactosidase expression indicating higher senescence of pSSCs on uncoated plates than PDL-coated plates. The results suggest that PDL provides a favourable microenvironment for pSSCs culture as indicated by early attachment, proliferation, and decreased senescence. This strategy may significantly contribute to the efficient culture of pSSCs for further studies of the regulatory mechanism of pSSCs proliferation and differentiation.