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Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes.

Abstract：

PURPOSE: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. METHODS: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. RESULTS: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. CONCLUSIONS:We successfully developed a novel high-yield technique to isolate SSCs from porcine testes to facilitate future porcine SSC-related research.

journal_name

J Assist Reprod Genet

journal_title

Journal of assisted reproduction and genetics

authors

Park MH,Park JE,Kim MS,Lee KY,Park HJ,Yun JI,Choi JH,Lee Es,Lee ST

doi

10.1007/s10815-014-0271-7

subject

Has Abstract

pub_date

2014-08-01 00:00:00

pages

983-91

issue

eissn

1058-0468

issn

1573-7330

journal_volume

pub_type

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Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes.