Park Min Hee, Park Ji Eun, Kim Min Seong, Lee Kwon Young, Park Hye Jin, Yun Jung Im, Choi Jung Hoon, Lee Eun song, Lee Seung Tae
Department of Animal Life Science, Kangwon National University, Chuncheon, 200-701, South Korea.
J Assist Reprod Genet. 2014 Aug;31(8):983-91. doi: 10.1007/s10815-014-0271-7. Epub 2014 Jun 18.
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.
迄今为止，从猪睾丸细胞中分离精原干细胞（SSCs）的现有方法细胞分离效率较低。因此，我们试图开发一种具有高产细胞分离能力的新型分离技术，以从猪睾丸中分离SSCs。
我们通过测量新生猪睾丸来源的睾丸细胞中的碱性磷酸酶（AP）活性和SSC特异性基因表达来确认SSCs的存在。随后，使用以下不同技术从睾丸细胞中分离SSCs：差异铺板（DP）、双重DP、DP后培养皿铺板、磁激活细胞分选（MACS）以及DP后MACS。采用AP阳性染色来评估和比较每种方法的分离效率。
DP后培养皿铺板产生了最高的分离效率。然后，通过分析SSC特异性基因和相关蛋白以及生殖细胞特异性基因的表达，对使用该方法分离的假定SSCs进行进一步表征。OCT4、NANOG、EPCAM、THY1和UCHL1在转录水平表达，而OCT4、NANOG、SOX2、TRA-1-60、TRA-1-81和PLZF在86%的分离SSCs中在翻译水平表达。相比之下，在SSCs和阴性对照细胞之间，表达黄体生成素受体（LHR，一种睾丸间质细胞特异性蛋白）或GATA4（一种支持细胞特异性蛋白）的细胞百分比没有差异。此外，分别未检测到原始生殖细胞特异性标志物VASA的转录表达，而检测到减数分裂前生殖细胞特异性标志物DAZL的转录表达。
我们成功开发了一种从猪睾丸中分离SSCs的新型高产技术，以促进未来与猪SSC相关的研究。
