Enrichment of CD9 spermatogonial stem cells from goat (Capra aegagrus hircus) testis using magnetic microbeads

The well documented source for adult multipotent stem cells is spermatogonial stem cells (SSCs) of mammalian testis. It is foundation of spermatogenesis in the testis throughout adult life by balancing self-renewal and differentiation. SSCs isolation from mammalian testis is difficult because of their scarcity and the lack of well characterized cell surface markers. Thus, the isolation of SSCs is of great interest for exploration of spermatogonial physiology and therapeutic approaches for fertility preservation. CD9 is a surface marker expressed in mouse and rat male germline stem cells. In this study, CD9 positive SSCs were successfully isolated from the goat testis using enzymatic digestion followed by three step purification: Differential plating, percoll discontinuous density gradient followed by magnetic activated cell sorting (MACS). Percoll discontinuous density gradient showed significant differences in the percentage of CD9 SSCs across individual fraction. The fraction 36% and 40% gave the highest percentage of CD9 SSCs i.e. 82% ± 1.2% and 9.2% ± 1.3% respectively. Magnetic activated cell sorting of CD9 cells in the magnetic fraction of goat testes was in the range of 15% 18% which is upto threefolds. CD9 SSCs were further recovered with appreciable efficiency after immunomagnetic isolation by using various bead: cells ratio in which 4:1 ratio gave the highest yield of 69.06 × 10 with 18% of CD9 SSCs. Magnetic activated cell sorting using anti-CD9 antibodies provides an efficient and fast approach as compared to conventional approaches such as differential plating and percoll discontinuous density gradient for enrichment strategy for spermatogonial stem cells from goat testes for undertaking research on basic and applied reproductive biology.