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首页> 外文期刊>The international journal of developmental biology >Heterogeneity of planarian stem cells in the S/G2/M phase
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Heterogeneity of planarian stem cells in the S/G2/M phase

机译：平面虫干细胞在S / G2 / M期的异质性

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The planarian adult stem cell (pASC) population has a specific molecular signature and can be easily visualized and isolated by flow cytometry. However, the lack of antibodies against specific surface markers for planarian cells prevents a deeper analysis of specific cell populations. Here, if we describe the results of the immunoscreening of pASC plasma membrane proteins (PMPs). A novel papain-based method for planarian cell dissociation enabling both high yield and improved cell viability was used to generate single cell preparations for PMP purification. PMPs were used for intraperitoneal immunization of mice and thus about 1000 hybridoma clones were generated and screened. Supernatants collected from the hybridoma clones were first screened by ELISA and then by live immuno-staining. About half of these supernatants stained all the planarian cells, whereas the other half specifically labeled a subfraction thereof. A detailed analysis of two hybridoma supernatants revealed that large subfractions of the X1, X2 and Xin populations differentially express specific membrane markers. Quantitative PCR data disclosed a correlation between the immunostaining results and the expression of markers of the early and late progeny, also for those pASCs in the S/G2/M phase of the cell cycle (X1 population). Thus, about two thirds of the cycling pASCs showed a specific membrane signature coupled with the expression of markers hitherto considered to be restricted to differentiating, post-mitotic progeny. In summary, a library of 66 monoclonal antibodies against planarian PMPs was generated. The analysis of two of the clones generated revealed that a subset of cells of the X1 population expresses early and late progeny markers, which might indicate that these cells are committed while still proliferating. The findings demonstrate the usefulness of our PMP antibody library for planarian research.
机译：涡虫成体干细胞（pASC）群体具有特定的分子特征，可以通过流式细胞术轻松观察和分离。但是，缺乏针对涡虫细胞的特定表面标记的抗体，无法对特定细胞群进行更深入的分析。在这里，如果我们描述pASC质膜蛋白（PMPs）的免疫筛选结果。一种新颖的基于木瓜蛋白酶的平面蛋白解离方法，可实现高产量和改善的细胞活力，可用于生成用于PMP纯化的单细胞制剂。 PMP用于小鼠的腹膜内免疫，因此产生并筛选了约1000个杂交瘤克隆。首先通过ELISA筛选从杂交瘤克隆中收集的上清液，然后再进行活体免疫染色。这些上清液中约有一半将所有涡虫细胞染色，而另一半则专门标记了其亚组分。对两个杂交瘤上清液的详细分析显示，X1，X2和Xin群体的大亚部分差异表达特定的膜标记物。定量PCR数据揭示了免疫染色结果与早期和晚期子代标志物表达之间的相关性，也针对细胞周期（X1群体）的S / G2 / M期的那些pASC。因此，约三分之二的循环pASC显示出特定的膜特征以及迄今为止被认为仅限于分化的有丝分裂后代的标志物的表达。总之，生成了针对平面虫PMP的66种单克隆抗体文库。对产生的两个克隆的分析表明，X1群体的一部分细胞表达了早期和晚期子代标记，这可能表明这些细胞在增殖时仍然存在。这些发现证明了我们的PMP抗体文库对于平面研究的有用性。

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