Present study aims to describe a simple and robust protocol to delineate the presence of pluripotent, very small embryonic-like stem cells (VSELs) in addition to spermatogonial stem cells (SSCs) in adult mouse testes. Testicular seminiferous tubules were subjected to enzymatic dissociation to obtain single cells suspension. Stem cells were enriched by spinning at different speeds wherein majority of somatic cells were pelleted at 1000 rpm (250 g, Pellet A) and putative stem cells by spinning the supernatant (obtained after separating Pellet A) at 3000 rpm (1000 g, Pellet B). Viable (7AAD-ve), 2–6 μm, LIN-CD45-SCA-1+ VSELs were studied after doublets exclusion by flow cytometry in both Pellets A & B. Almost ten-fold enrichment of VSELs was obtained in Pellet B (0.27 + 0.05%) compared to Pellet A (0.03 + 0.003%). SCA-1 expressing SSCs (>6 μm, 0.18 + 0.06%) were clearly distinguished from VSELs (2–6 μm, 0.07 + 0.003%) by flow cytometry studies on total testicular cells suspension collected by spinning at 3000 rpm. Enriched stem cells from Pellet B were used to study expression of OCT-4, NANOG, SCA-1, SSEA-1, LIFR, GFRa, c-KIT, ERα and ERβ. Cells in Pellet B were also subjected to RT-PCR to study pluripotent (Oct-4a, Sox2, Nanog), primordial germ cells (Stella, Fragilis), SSCs (Oct-4) and estrogen receptors (ERα and ERβ) specific transcripts. qRT-PCR analysis showed >2 folds up-regulation of stem cell markers in Pellet B (Oct-4A, Oct-4, Sox2, Nanog) compared to Pellet A. To conclude, spinning at higher speed led to successful enrichment of pluripotent VSELs from testes which have remained ignored till now. Expression of ERα & β on VSELs/SSCs makes them vulnerable to endocrine disruption.

中文翻译：

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在成年小鼠睾丸中建立两个干细胞群体（包括VSEL和SSC）存在的其他证据。

本研究旨在描述一种简单而健壮的方案，以描述成年小鼠睾丸中除精原干细胞（SSC）外还存在多能，非常小的胚胎样干细胞（VSEL）。睾丸生精小管进行酶解以获得单细胞悬液。通过以不同的速度旋转来富集干细胞，其中大多数体细胞以1000 rpm（250 g，颗粒A）沉淀，推定的干细胞通过以3000 rpm（1000 g，颗粒B分离）上清液（分离A分离后获得）沉淀。 ）。通过流式细胞术排除了A和B颗粒中的双链体后，研究了存活的（7AAD-ve），2–6μmLIN-CD45-SCA-1 + VSEL。在B颗粒中，VSEL的富集几乎达到了十倍（0.27  +  0.05％），而药丸A（0.03 +  0.003％）。 通过以3000 rpm旋转收集的总睾丸细胞悬浮液的流式细胞术研究，表达SCA-1的SSC（> 6μm，0.18  +  0.06％）与VSEL（2–6μm，0.07 + 0.003％）明显不同 。使用来自沉淀B的富集的干细胞来研究OCT-4，NANOG，SCA-1，SSEA-1，LIFR，GFRa，c-KIT，ERα和ERβ的表达。颗粒B中的细胞也经过RT-PCR研究多能性（Oct-4a，Sox2，Nanog），原始生殖细胞（Stella，Fragillis），SSC（Oct-4）和雌激素受体（ERα和ERβ））特定的成绩单。qRT-PCR分析显示，与小球A相比，小球B（Oct-4A，Oct-4，Sox2，Nanog）中干细胞标志物的上调> 2倍。总而言之，以更高的速度旋转可成功富集多能VSEL。直到今天仍然被忽略的睾丸。ERα和β在VSEL / SSC上的表达使其易受内分泌干扰。