CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development [RNA-seq]

The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout (KO) screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas. Overall design: We performed RNA-Seq of four WT, two DLX3 KO, two GCM1 KO, and two genetically unmodified clones of hTSCs. To generate DLX3 KO and GCM1 KO hTSC clones, we employed CRISPR/Cas9 with sgRNAs that were designed to remove the DNA binding domains of DLX3 and GCM1.

胎盘作为母体与胎儿之间的界面，介导二者独立血液循环系统间的气体与营养物质交换。胎盘滋养层细胞在这一过程中发挥核心作用。目前我们对哺乳动物滋养层发育的认知，很大程度上依赖于小鼠模型。然而，鉴于哺乳动物胎盘的多样性，小鼠胎盘的研究结果无法直接外推至包括人类在内的其他哺乳动物物种。为填补这一认知空白，本研究在人类滋养层干细胞（human trophoblast stem cells, hTSCs）中开展了CRISPR敲除（CRISPR knockout, KO）筛选。本研究靶向了小鼠胎盘发育所必需的基因，最终在人类hTSCs与小鼠胎盘中均鉴定出百余个关键调控基因。其中，我们对两个转录因子DLX3与GCM1进行了深入表征，并揭示了二者在hTSC分化中的核心功能。此外，基于基因功能对人类与小鼠滋养层亚型进行的比较分析显示，二者的关联与此前基于组织定位或细胞功能得出的推测存在显著差异。值得注意的是，本研究数据表明，人类hTSCs可能并不类似于小鼠TSCs或其体内TSCs所在的胚外外胚层（extraembryonic ectoderm, ExE）。相反，人类hTSCs可能更类似于小鼠胎盘锥与绒毛膜中的祖细胞。这一发现与人类胎盘发育过程中不存在类ExE结构的现象相符。本研究数据不仅加深了我们对人类滋养层发育的认知，同时也为哺乳动物胎盘的跨物种比较研究提供了助力。
整体实验设计：我们对4份野生型（wild type, WT）、2份DLX3 KO、2份GCM1 KO以及2份基因未修饰的hTSCs克隆进行了RNA测序（RNA-Seq）。为构建DLX3 KO与GCM1 KO的hTSC克隆，本研究使用了携带靶向敲除DLX3与GCM1 DNA结合结构域的sgRNAs的CRISPR/Cas9系统。