Modeling Human Placentation via Single Cell RNA-Sequencing

摘要 胎盘功能障碍导致妊娠相关疾病，包括宫内生长受限 胎儿生长迟缓（IUGR）和先兆子痫，也是产后和成人疾病的发育原因。通常情况下， 胎盘形成过程中的因果变化，导致有缺陷的怀孕，发生在早期， 怀孕然而，由于伦理障碍和实验系统的困难，大多数 对人类胎盘功能的研究是用足月时获得的样本进行的，因此，限制了我们的研究。 了解整个妊娠期胎盘发育和功能的能力。幸运的是，最近 单核RNA测序（snRNA-seq）策略的进展和成功推导真人类 来自绒毛细胞滋养层细胞（CTB）的滋养层干细胞（人TSCs）已经开辟了新的 直接评估早期人类胎盘形成过程的策略。 绒毛膜绒毛取样（CVS）是一个标准的卫生保健程序进行后期阶段 在怀孕的前三个月。通过CVS获得的多余组织代表了一种有价值的来源， 滋养层细胞的实验分析，因此，一个独特的机会，询问早期发展中国家， 胎盘 利用基因敲除小鼠模型，我们发现转录加塔家族的缺失， 滋养层祖细胞中的GATA 2和GATA 3因子导致妊娠失败或胎儿生长 限制.作为我们追求确定这些发现对人类胎盘形成的重要性的一部分， 我们已经成功地对人绒毛膜绒毛样品进行了scRNA-seq研究并发现， 在妊娠早期胎盘中，GATA 2和GATA 3在不同的细胞类型中广泛表达，包括 CTB、合胞体滋养层细胞（SynTs）和绒毛外滋养层细胞（EVT）。此外，我们还 我们能够从分离自绒毛膜绒毛的CTB中建立人TSC，并发现GATA 2的缺失 在人TSC中的表达损害EVT和SynTB分化。这些观察使我们得出 该建议的中心假设是单细胞基因表达模式，包括加塔的表达 因素，在妊娠早期胎盘可以预测妊娠相关疾病在晚期 怀孕为了验证这一假设，我们将使用CVS进行snRNA-seq，并建立患者特异性 用于后续功能研究的人TSC。我们也会对这些孕妇进行监测- 相关疾病，包括宫内生长受限和先兆子痫，并收集足月胎盘 用于后续分析的样品。总之，我们提出的研究，由现代技术支持，将 阐明以前未知的途径，这些途径是单细胞水平上胎盘适应的基础 正常妊娠和病理妊娠。

Abstract Placental dysfunction leads to pregnancy-associated disorders, including intrauterine growth restriction (IUGR) and preeclampsia, and also serves as a developmental cause for postnatal and adult diseases. Often, the causal alterations in the placentation process, which lead to defective pregnancies, occur early in gestation. However, due to ethical barriers and difficulties with the experimental systems, most of the studies on human placental function are performed with samples obtained at term, thereby, limiting our ability to understand placental development and function throughout gestation. Fortunately, the recent advancement of single-nucleus RNA-sequencing (snRNA-seq) strategy and success in deriving true human trophoblast stem cells (human TSCs) from villous cytotrophoblast cells (CTBs) have opened up new strategies for direct assessment of early human placentation process. Chorionic villus sampling (CVS) is a standard health care procedure performed during latter stages of the first trimester of pregnancy. Surplus tissue obtained via CVS represents a valuable source of trophoblast cells for experimental analysis and thus, a unique opportunity to interrogate the early developing placenta. Using gene knockout mouse models, we discovered that depletion of GATA family of transcription factors, GATA2 and GATA3 in trophoblast progenitor cells leads to either loss of pregnancy or fetal growth restriction. As part of our pursuit to define importance of these findings with respect to human placentation, we have successfully performed scRNA-seq study with human chorionic villous samples and found that, within a first-trimester placenta, GATA2 and GATA3 are broadly expressed in distinct cell types, including CTBs, syncytioytrophoblasts (SynTs) and extravillous trophoblasts (EVTs). Furthermore, we have also been able to establish human TSCs from CTBs, isolated from chorionic villi, and found that loss of GATA2 expression in human TSCs impairs both EVT and SynTB differentiation. These observations led us to the central hypothesis of this proposal that single cell gene expression patterns, including expression of GATA factors, in a first-trimester placenta could be predictive of pregnancy associated disorders during late gestation. To test this hypothesis, we will use CVS to perform snRNA-seq and to establish patient-specific human TSCs for subsequent functional studies. We will also monitor those pregnancies for pregnancy- associated disorders, including intrauterine growth restriction and preeclampsia and collect term placental samples for follow up analyses. Together, our proposed study, bolstered by modern technologies, will illuminate previously unknown pathways, which underlie placental adaptation at a single cell level during normative and pathological pregnancies.