Modeling Human Placentation via Single Cell RNA-Sequencing

摘要 胎盘功能障碍会导致妊娠相关疾病，包括胎儿宫内生长受限 胎儿宫内发育迟缓(IUGR)和先兆子痫，也是导致出生后和成人疾病的发育原因。通常， 胎盘形成过程中导致缺陷妊娠的原因改变发生在早期。 怀孕了。然而，由于道德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采集的样本进行的，因此，限制了我们的 了解胎盘在整个孕期的发育和功能的能力。幸运的是，最近的 单核RNA测序(SnRNA-seq)策略的进展及其在获得真人中的成功 从绒毛细胞滋养层细胞(CTB)获得的滋养层干细胞(TSCs)开辟了新的研究领域 直接评估早期人类胎盘形成过程的策略。 绒毛取样(CVS)是一种在晚期进行的标准保健程序。 怀孕前三个月的情况。通过CVS获得的多余组织是一个有价值的来源 对滋养层细胞进行实验分析，从而为盘问早期发育提供了一个独特的机会 胎盘。 利用基因敲除小鼠模型，我们发现GATA转录家族的耗尽 滋养层祖细胞中的GATA2和GATA3因子导致妊娠或胎儿生长丧失 限制。作为我们确定这些发现对人类胎盘的重要性的努力的一部分， 我们已经成功地对人绒毛样本进行了scRNA-seq研究，并发现， 在妊娠早期胎盘中，GATA2和GATA3在不同的细胞类型中广泛表达，包括 CTB、合体滋养细胞和绒毛外滋养细胞。此外，我们还 能够从CTB中建立人TSCs，从绒毛中分离，并发现GATA2的缺失 在人TSCs中的表达损害了EVT和SynTB的分化。这些观察将我们带到了 这一假设的中心假设是单细胞基因表达模式，包括GATA的表达 妊娠早期胎盘中的因素可预测晚期妊娠相关疾病 怀孕了。为了验证这一假设，我们将使用CVS来执行snRNA-seq并建立特定于患者的序列 用于后续功能研究的人类TSCs。我们还将监测这些怀孕的情况- 相关疾病，包括宫内生长受限和先兆子痫和收集足月胎盘 用于后续分析的样品。总之，我们提议的研究，在现代技术的支持下，将 阐明先前未知的途径，这些途径是胎盘在单细胞水平上适应的基础 正常妊娠和病理性妊娠。

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.