Human Trophoblast Stem Cells: the In Vivo Niche and Relationship to Pluripotent Stem Cells

项目总结/摘要 人类胎盘在维持胎儿生长的适当环境方面起着重要作用，但仍然是 最不了解的器官。该项目旨在大大提高我们对这一重要问题的认识。 通过系统、详细地研究人体器官早期发育，然后将这一联合收割机 知识与再生医学的最新技术，以开发体外模型， 研究胎盘正常和异常发育。具体来说，我们的目标是了解 建立和维持多能人滋养层干（TS）细胞的潜在机制， 一种可以产生所有其他滋养层亚型，胎盘的上皮细胞。过去 几年来，我们已经确定了一个关键的途径，由p53相关蛋白p63指导，这是必需的， 在早期胎盘中维持未分化的细胞滋养层（CTB）干细胞。最近，我们有 注意到这些CTB的一个子集共表达CDX 2，CDX 2是维持TS细胞所需的转录因子 在小鼠中，并假设这些CDX 2 +/p63+ CTB是多能的人TS细胞。我们将 使用流式细胞分选，随后进行分化测定的组合，进一步表征该亚群 以及整体和单细胞转录组分析。在这种基于发现的方法中，我们将重点关注 鉴定表征该细胞的转录因子和细胞表面标志物的分析 早期人类胎盘中的一种。与此同时，我们将采取一种更注重基因的方法， p63和CDX 2在早期妊娠CTB增殖和分化中的特定作用，包括它们的 下游目标最后，我们将把这些知识应用于人类多能干细胞（hPSC）， 胚胎干细胞（hESC）和诱导多能干细胞（hiPSC）-为了开发用于 人滋养层细胞谱系特化和分化研究。我们建立了一个小说 - 用于hPSC逐步分化的分化方案，首先分化成CTB，随后分化成hCG。 分泌合胞体滋养层细胞（STB）和HLA-G+绒毛外滋养层细胞（EVT）。使用该协议，我们有 发现Trisomy 21 iPSC在CTB干细胞状态中花费较长时间，并显示钝化， 分化为功能性STB，与从胎盘分离的原发性CTB的表型相同， 21号染色体三体。这些令人兴奋的初步数据表明，hPSC可能有助于模拟滋养层细胞， 分化缺陷，这是胎盘功能障碍的基础。我们将比较hPSC衍生的 从植入前和植入后组织的滋养层到初级滋养层，以确定 他们最像。该项目的成功完成有可能改变人类的领域 胎盘生物学，通过鉴定胎盘内的人TS细胞和建立基于hPSC的模型 胎盘疾病，从而构建了一个坚实的基础上，诊断标志物的发现和 这一重要的人体器官的治疗靶向将是可能的。

Project Summary/Abstract The human placenta plays a major role in maintaining the proper environment for fetal growth, but remains as the most poorly understood organ. This project aims to substantially improve our knowledge of this important human organ by studying its early development in a systematic, detailed manner, then to combine this knowledge with the latest technologies in regenerative medicine in order to develop in vitro models for the study of both normal and abnormal placental development. Specifically, we aim to understand the mechanisms underlying the establishment and maintenance of a multipotent human trophoblast stem (TS) cell, one which can give rise to all other subtypes of trophoblast, the epithelial cells of the placenta. Over the past few years, we have identified a key pathway, directed by the p53-related protein, p63, which is required for maintenance of undifferentiated cytotrophoblast (CTB) stem cells in the early placenta. More recently, we have noted that a subset of these CTB co-express CDX2, a transcription factor required for maintenance of TS cells in mice, and hypothesize that these CDX2+/p63+ CTB are multipotent human TS cells. We will characterize this subpopulation further, using a combination of FACS sorting, followed by differentiation assays and both bulk and single cell transcriptome profiling. In this discovery-based approach, we will focus our analysis on identification of transcription factors and cell surface markers, which characterize this cell population in the early human placenta. At the same time, we will take a more gene-focused approach, probing the specific role(s) of p63 and CDX2 in first trimester CTB proliferation and differentiation, including their downstream targets. Finally, we will apply this knowledge to human pluripotent stem cells (hPSCs)--both embryonic (hESCs) and induced pluripotent stem cells (hiPSCs)—in order to develop in vitro models for the study of human trophoblast lineage specification and differentiation. We have established a novel differentiation protocol for step-wise differentiation of hPSCs, first into CTB, and subsequently into hCG- secreting syncytiotrophoblast (STB) and HLA-G+ extravillous trophoblast (EVT). Using this protocol, we have found that Trisomy 21 iPSC spend a prolonged period in the CTB stem cell state, and show blunted differentiation into functional STB, identical to the phenotype of primary CTB isolated from placentas with Trisomy 21. These exciting preliminary data suggest that hPSCs may be useful for modeling trophoblast differentiation defects, which are the basis for placental dysfunction. We will compare hPSC-derived trophoblast to primary trophoblast from both pre- and post-implantation tissues in order to determine which they most resemble. The successful completion of this project has the potential to transform the field of human placental biology, by both identifying human TS cells within the placenta, and establishing hPSC-based models of placental disease, thereby constructing a firm foundation on which diagnostic marker discovery and therapeutic targeting of this important human organ would be possible.