Mouse Sertoli cells isolation by lineage tracing and sorting.,Molecular Reproduction and Development

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Mouse Sertoli cells isolation by lineage tracing and sorting.
Molecular Reproduction and Development ( IF 2.7 ) Pub Date : 2020-07-31 , DOI: 10.1002/mrd.23406
Helena D Zomer ₁ , Prabhakara P Reddi ₁

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Sertoli cells play a key role in spermatogenesis by supporting the germ cells throughout differentiation. The isolation of Sertoli cells is essential to study their functions. However, the close contact of Sertoli cells with other testicular cell types and the high proliferation of contaminating cells are obstacles to obtain pure primary cultures. Current rodent Sertoli cell isolation protocols result in enriched, rather than pure Sertoli cells. Therefore, novel approaches are necessary to improve the purity of Sertoli cell primary cultures. The goal of this study is to obtain pure mouse Sertoli cells using lineage tracing and fluorescence‐activated cell sorting (FACS). We bred the Amh‐Cre mouse line with tdTomato line to generate mice constitutively expressing red fluorescence specifically in Sertoli cells. Primary cultures of Sertoli cells isolated from prepubertal mice showed that 79% of cells expressed tdTomato, as evaluated by fluorescence microscopy and flow cytometry; however, nearly all adherent cells were positive for vimentin. Most of the tomato‐negative cells expressed α‐smooth muscle actin (α‐SMA), a peritubular myoid cell marker, but double‐negative populations were also present. These findings suggest that vimentin lacks Sertoli cell‐specificity and that α‐SMA is not adequate to identify all of the contaminating cells. Upon FACS sorting; however, virtually 100% of the cells were tdTomato positive, expressed vimentin, but not α‐SMA. Prepubertal mice yielded a higher number of Sertoli cells compared to adults, but both could be adequately sorted. In conclusion, our study shows that lineage tracing and sorting is an efficient strategy for acquiring pure populations of murine Sertoli cells.

中文翻译：

通过谱系追踪和分类分离小鼠支持细胞。

通过在整个分化过程中支持生殖细胞，支持细胞在精子发生中起关键作用。Sertoli细胞的分离对于研究其功能至关重要。然而，Sertoli细胞与其他睾丸细胞类型的紧密接触以及污染细胞的高增殖是获得纯原代培养物的障碍。当前的啮齿动物支持细胞分离方案导致富集而不是纯的支持细胞。因此，需要新的方法来提高Sertoli细胞原代培养物的纯度。这项研究的目的是通过沿袭谱系和荧光激活细胞分选术（FACS）获得纯小鼠Sertoli细胞。我们将thTomato系与Amh-Cre小鼠系进行了繁殖，以生成在Sertoli细胞中组成型表达红色荧光的小鼠。从青春期前小鼠分离的Sertoli细胞的原代培养表明，通过荧光显微镜和流式细胞术评估，有79％的细胞表达tdTomato。但是，几乎所有粘附细胞的波形蛋白均为阳性。大部分番茄阴性细胞表达α-平滑肌肌动蛋白（α-SMA），一种肾小管周围肌细胞标记，但也存在双重阴性菌群。这些发现表明波形蛋白缺乏Sertoli细胞特异性，α-SMA不足以鉴定所有污染细胞。根据FACS排序；然而，实际上100％的细胞是tdTomato阳性，表达波形蛋白，但不表达α-SMA。与成年小鼠相比，青春期前小鼠的Sertoli细胞数量更高，但是可以对它们进行充分的分类。结论，

更新日期：2020-08-28

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