Mouse Sertoli cells isolation by lineage tracing and sorting.
Mouse Sertoli cells isolation by lineage tracing and sorting.
复制标题
通过谱系追踪和分选分离小鼠支持细胞。
DOI：
10.1002/mrd.23406
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发表时间：
2020
影响因子：
2.5
通讯作者：
Reddi,PrabhakaraP
中科院分区：
文献类型：
--
作者：
Zomer,HelenaD;Reddi,PrabhakaraP
支持细胞通过支持生殖细胞的整个分化过程，在精子发生过程中起着关键作用。支持细胞的分离是研究其功能的必要条件。然而，支持细胞与其他睾丸细胞类型的密切接触以及污染细胞的高增殖是获得纯原代培养的障碍。目前的啮齿动物支持细胞分离方案导致富集，而不是纯粹的支持细胞。因此，需要新的方法来提高支持细胞原代培养的纯度。本研究的目的是利用谱系追踪和荧光激活细胞分选（FACS）获得纯小鼠支持细胞。我们将Amh - Cre小鼠系与tdTomato系杂交，生成了在Sertoli细胞中特异性表达红色荧光的小鼠。从青春期前小鼠分离的支持细胞原代培养显示，荧光显微镜和流式细胞术检测显示，79%的细胞表达tdTomato；然而，几乎所有贴壁细胞都呈波形蛋白阳性。大多数番茄阴性细胞表达α‐平滑肌肌动蛋白（α‐SMA），这是一种管周肌样细胞标志物，但双阴性群体也存在。这些发现表明，vimentin缺乏支持细胞特异性，α - SMA不足以识别所有污染细胞。FACS分类；然而，几乎100%的细胞tdTomato阳性，表达vimentin，但不表达α‐SMA。与成年小鼠相比，青春期前小鼠产生了更多的支持细胞，但两者都可以充分分选。总之，我们的研究表明，谱系追踪和分选是获得小鼠支持细胞纯群体的有效策略。
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.