Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes
- 451 Downloads
To date, the methods available for isolating spermatogonial stem cells (SSCs) from porcine testicular cells have a low efficiency of cell separating. Therefore, we tried to develop a novel isolation technique with a high-yield cell separating ability to isolate SSCs from porcine testes.
We confirmed the presence of SSCs by measuring alkaline phosphatase (AP) activity and SSC-specific gene expression in neonatal porcine testis-derived testicular cells. Subsequently, the isolation of SSCs from testicular cells was performed using different techniques as follows: differential plating (DP), double DP, Petri dish plating post-DP, magnetic-activated cell sorting (MACS), and MACS post-DP. Positive AP staining was used to assess and compare the isolation efficiency of each method.
Petri dish plating post-DP resulted in the highest isolation efficiency. The putative SSCs isolated using this method was then further characterized by analyzing the expression of SSC-specific genes and -related proteins, and germ cell-specific genes. OCT4, NANOG, EPCAM, THY1, and UCHL1 were expressed transcriptionally, and OCT4, NANOG, SOX2, TRA-1-60, TRA-1-81, and PLZF were expressed translationally in 86 % of the isolated SSCs. In contrast, no difference was observed in the percentage of cells expressing luteinizing hormone receptor (LHR), a Leydig cell-specific protein, or GATA4, a Sertoli cell-specific protein, between SSCs and negative control cells. In addition, transcriptional expression of VASA, a primordial germ cell-specific marker, and DAZL, a premeiotic germ cell-specific marker, wasn’t and was detected, respectively.
We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.
KeywordsPorcine Spermatogonial stem cells Testis Cell sorting
This research was supported by Agricultural Biotechnology Development Program (IPET112015-4), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.
- 15.Kubota H, Brinster RL. Technology insight: in vitro culture of spermatogonial stem cells and their potential therapeutic uses. Nature. 2006;2:99–108.Google Scholar
- 16.Vlajković S, Cukuranović R, Bjelaković MD, Stefanović V. Possible therapeutic use of spermatogonial stem cells in the treatment of male infertility: a brief overview. Sci World J. 2012;2012:374151.Google Scholar
- 23.Han SY, Gupta MK, Lee SJ, Uhm HT. Isolation and in vitro culture of pig spermatogonial stem cell. AJAS. 2009;22:187–93.Google Scholar