Isolation, Characterization, and Culture of Human Spermatogonia
Isolation, Characterization, and Culture of Human Spermatogonia
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DOI：
10.1095/biolreprod.109.078550
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发表时间：
2010-02-01
影响因子：
3.6
通讯作者：
Dym, Martin
中科院分区：
文献类型：
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作者：
He, Zuping;Kokkinaki, Maria;Dym, Martin
本研究旨在分离、鉴定和培养人精原细胞。使用免疫组化小管部分，我们本地化GPR 125的质膜的一个子集的精原细胞。免疫组化还显示MAGEA 4在所有精原细胞（A型（深色）、A型（浅色）和B型）和可能的细线期前精母细胞中表达。值得注意的是，KIT在晚期精母细胞和圆形精子细胞中表达，但显然不在人精原细胞中表达。UCHL 1在精原细胞的细胞质中被发现，而POU 5 F1在任何人生殖细胞中均未检测到。GFRA 1和ITGA 6定位于精原细胞的质膜。接下来，我们分离GPR 125阳性精原细胞从成人睾丸使用两步酶消化，然后磁激活细胞分选。分离的GPR 125阳性细胞共表达GPR 125、ITGA 6、THY 1和GFRA 1，并且它们可以培养短时间，并且如增殖测定所示，显示出细胞数量的显著增加。假定的干细胞基因在培养2周后的免疫细胞化学显示，细胞维持在未分化状态。与新鲜分离的细胞相比，GPR 125阳性精原细胞培养2周后MAPK 1/3磷酸化增加。总之，这些结果表明，人类精原细胞共享一些，但不是所有的表型与精原干细胞（SSCs）和其他物种的祖细胞。GPR 125阳性精原细胞是表型上推定的人类精原干细胞，并在体外保持未分化状态。这项研究为人类精原干细胞和/或祖细胞的分子特征、分离和培养提供了新的见解，并表明MAPK 1/3通路参与了它们的增殖。
This study was designed to isolate, characterize, and culture human spermatogonia. Using immunohistochemistry on tubule sections, we localized GPR125 to the plasma membrane of a subset of the spermatogonia. Immunohistochemistry also showed that MAGEA4 was expressed in all spermatogonia (A(dark), A(pale), and type B) and possibly preleptotene spermatocytes. Notably, KIT was expressed in late spermatocytes and round spermatids, but apparently not in human spermatogonia. UCHL1 was found in the cytoplasm of spermatogonia, whereas POU5F1 was not detected in any of the human germ cells. GFRA1 and ITGA6 were localized to the plasma membrane of the spermatogonia. Next, we isolated GPR125-positive spermatogonia from adult human testes using a two-step enzymatic digestion followed by magnetic-activated cell sorting. The isolated GPR125-positive cells coexpressed GPR125, ITGA6, THY1, and GFRA1, and they could be cultured for short periods of time and exhibited a marked increase in cell numbers as shown by a proliferation assay. Immunocytochemistry of putative stem cell genes after 2 wk in culture revealed that the cells were maintained in an undifferentiated state. MAPK1/3 phosphorylation was increased after 2 wk of culture of the GPR125-positive spermatogonia compared to the freshly isolated cells. Taken together, these results indicate that human spermatogonia share some but not all phenotypes with spermatogonial stem cells (SSCs) and progenitors from other species. GPR125-positive spermatogonia are phenotypically putative human SSCs and retain an undifferentiated status in vitro. This study provides novel insights into the molecular characteristics, isolation, and culture of human SSCs and/or progenitors and suggests that the MAPK1/3 pathway is involved in their proliferation.