Workflow Optimization for Identification of Female Germline or Oogonial Stem Cells in Human Ovarian Cortex Using Single-Cell RNA Sequence Analysis.
Workflow Optimization for Identification of Female Germline or Oogonial Stem Cells in Human Ovarian Cortex Using Single-Cell RNA Sequence Analysis.
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DOI：
10.1093/stmcls/sxac015
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发表时间：
2022-05-27
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2004年，雌性种系或卵原干细胞（OSC）的鉴定可以支持成年小鼠卵巢产后卵子发生，引发了生殖生物学的重大范式转变。尽管这些发现已经过独立验证，并进一步扩展到包括从猪和牛到非人灵长类动物和人类等许多物种的成年卵巢中 OSC 的鉴定，但最近一项基于成人卵巢皮质组织的单细胞 RNA 序列分析 (scRNA-seq) 的研究声称 OSC 不存在，并且其他研究组在通过磁辅助或磁力辅助或磁力辅助分离后研究 OSC。 荧光激活细胞分选将血管周围细胞 (PVC) 误认为是生殖细胞。在这里，我们报告说，在优化分析工作流程参数后，可以通过 scRNA-seq 在成人卵巢皮质组织中鉴定出基因表达谱与 OSC 匹配但与其他细胞（包括卵母细胞和 PVC）不同的稀有生殖谱系细胞。更深入的逐个细胞表达谱还发现了成人卵巢中生殖细胞经历减数分裂 I 的证据。最后，我们表明，如果控制不当，由于固有的高细胞自发荧光，在设计用于分选 OSC 的流式细胞术方案期间可能会无意中分离 PVC。然而，由于基因表达谱不重叠，人类 PVC 和人类生殖细胞在 scRNA-seq 后分离成不同的簇，这将排除在功能表征研究期间将 PVC 错误地识别和用作 OSC。
In 2004, the identification of female germline or oogonial stem cells (OSCs) that can support post–natal oogenesis in ovaries of adult mice sparked a major paradigm shift in reproductive biology. Although these findings have been independently verified, and further extended to include identification of OSCs in adult ovaries of many species ranging from pigs and cows to non–human primates and humans, a recent study rooted in single–cell RNA sequence analysis (scRNA-seq) of adult human ovarian cortical tissue claimed that OSCs do not exist, and that other groups working with OSCs following isolation by magnetic-assisted or fluorescence-activated cell sorting have mistaken perivascular cells (PVCs) for germ cells. Here we report that rare germ lineage cells with a gene expression profile matched to OSCs but distinct from that of other cells, including oocytes and PVCs, can be identified in adult human ovarian cortical tissue by scRNA-seq after optimization of analytical workflow parameters. Deeper cell-by-cell expression profiling also uncovered evidence of germ cells undergoing meiosis-I in adult human ovaries. Lastly, we show that, if not properly controlled for, PVCs can be inadvertently isolated during flow cytometry protocols designed to sort OSCs because of inherently high cellular autofluorescence. However, human PVCs and human germ cells segregate into distinct clusters following scRNA-seq due to non–overlapping gene expression profiles, which would preclude the mistaken identification and use of PVCs as OSCs during functional characterization studies.
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