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

项目摘要/摘要 人类胎盘在维持胎儿生长的适当环境中起着重要作用，但仍然是 最鲜为人知的器官。这个项目的目的是大大提高我们对这一重要的 通过系统、详细地研究人体器官的早期发展，然后结合这一点 了解再生医学的最新技术，以便为患者开发体外模型 对正常和异常胎盘发育的研究。具体地说，我们的目标是了解 建立和维持多潜能人滋养层干细胞的机制， 一种可以产生所有其他亚型滋养细胞的细胞，即胎盘上皮细胞。在过去的时间里 几年来，我们已经确定了一条关键途径，由p53相关蛋白p63指导，这是 早期胎盘中未分化的细胞滋养细胞(CTB)干细胞的维持。最近，我们已经 注意到这些CTB的一个子集共表达CDX2，CDX2是维持TS细胞所需的转录因子 并假设这些CDX2+/p63+CTB是多潜能的人类TS细胞。我们会 使用FACS分类和分化分析相结合的方法，进一步表征这一亚群 以及批量和单细胞转录组分析。在这种基于发现的方法中，我们将把我们的 表征该细胞的转录因子和细胞表面标志物的鉴定分析 人类早期胎盘中的人口。同时，我们将采取更加注重基因的方法，探索 P63和CDX2在早孕CTB增殖分化中的特异性作用(S) 下游目标。最后，我们将把这一知识应用于人类多能干细胞(HPSCs)--两者 胚胎干细胞(HESCs)和诱导多能干细胞(HiPSCs)-为了建立体外模型 人滋养层细胞谱系特征及分化的研究。我们已经建立了一部小说 HPSCs逐步分化的分化方案，首先分化成CTB，然后分化成hCG- 分泌合体滋养细胞(STB)和人类白细胞抗原G+绒毛外滋养细胞(EVT)。使用这个协议，我们有 发现21三体IPSC长时间处于CTB干细胞状态，并表现出钝化 分化为功能性STB，与从胎盘分离的原发CTB表型相同 21三体。这些令人兴奋的初步数据表明，hPSCs可能有助于建立滋养层细胞的模型。 分化缺陷，这是胎盘功能障碍的基础。我们将比较hPSC派生的 植入前和植入后组织中滋养层细胞与原代滋养层细胞的关系 它们最像是。这一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类TS细胞，并建立基于hPSC的模型 胎盘疾病，从而为诊断标记物的发现和 以这个重要的人体器官为靶点的治疗将是可能的。

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.