A defined culture system for in vitro studies on trophoblast differentiation

项目摘要/摘要 囊胚期胚胎的滋养外胚层是所有滋养层细胞类型的前体。 在胎盘中-绒毛细胞滋养细胞(VCTB)、合体滋养细胞(STB)和绒毛外滋养细胞 (EVTS)包括柱状细胞滋养细胞(CCTB)和侵袭性细胞滋养细胞(ICTB)。中的异常现象 结核病的发展会导致胎盘病变，并与妊娠期间的不良结局有关。然而， 尽管结核病对母婴健康至关重要，但人类结核病的发展 人们对此仍然知之甚少。人类结核病发展的机制研究因挑战而变得困难 在对人类胚胎的研究中，以及从怀孕早期获得的胎盘样本有限。此外， 人类胎盘样本与异质性有关，含有多种结核和非结核细胞类型，以及 很可能已经适应了母体的环境。为了克服这些限制，我们建议 以人胚胎干细胞来源的结核作为研究人类结核的模型系统 发展。然而，在人类胚胎干细胞中获得结核病归宿的分子机制 目前仍不清楚。这种缺乏理解是广泛使用hESC来源的结核病作为 一种早期人类结核病发展的模式系统。在这方面的一个主要挑战是缺乏明确的 人胚胎干细胞分化结核和/或将人胚胎干细胞来源的结核分化为 特化的结核病亚型。因此，我们在这里建议验证一个完全定义的文化系统 用于hESCs的TB分化，包括选择性地分化为STB或iCTB。 因此，在目标1中，我们将验证基于完全定义的E6/7/8培养基的培养系统， 和玻璃连结素包被的组织培养板。具体地说，我们将调查一组 使用这些条件可以获得确认真正的结核病亚型的生物标记物。在目标2中，我们将 调查人类胚胎干细胞是否可以在特定条件下分化为STB和iCTB 不添加外源性骨形态发生蛋白(BMP)，或其中BMP信号通过Smad1/5/8 信号通路受到抑制。最后，在目标3中，我们将测试Rho/ROCK信号的假设 在所提出的培养体系中，S1P激活的下游介导了hESCs的TB分化。 我们工作的意义在于它与我们对早期人类结核病的理解有关 发展。确定的人类胚胎干细胞结核分化培养条件的验证将使机械化 人类胚胎干细胞的结核病分化研究，以及人类胚胎干细胞来源的结核病分化为特化结核亚型的研究。这 这反过来将使人们能够常规接受使用hESC衍生的结核病，这是早期结核病发展的模式系统。这样的一个 模型系统将成为进一步了解人类早期胎盘发育的有力工具。

PROJECT SUMMARY/ABSTRACT The trophectoderm layer of the blastocyst stage embryo is the precursor for all trophoblast (TB) cell types in the placenta – the villous cytotrophoblasts (vCTBs), syncytiotrophoblast (STB), and extravillous trophoblasts (EVTs) comprising column cytotrophoblasts (cCTBs) and invasive cytotrophoblasts (iCTBs). Abnormalities in TB development result in placental pathology, and are associated with adverse outcomes during pregnancy. Yet, despite being critically important to maternal and fetal health, human TB development remains poorly understood. Mechanistic studies on human TB development are difficult due to challenges in research with human embryos, and limited availability of placental samples from early gestation. Further, human placental samples are associated with heterogeneity, containing multiple TB and non-TB cell types, and have likely undergone adaptation to the maternal environment. To overcome these limitations, we propose to use TB derived from human embryonic stem cells (hESCs) as a model system for studies on human TB development. However, the molecular mechanisms underlying the acquisition of TB fate in hESCs remain unclear. This lack of understanding is a major impediment for widespread use of hESC-derived TB as a model system for early human TB development. A major challenge in this context is the lack of defined culture conditions for initiating TB differentiation of hESCs and/or differentiation of hESC-derived TB to specialized TB subtypes. Therefore, here we propose to validate a completely defined culture system for TB differentiation of hESCs, including selective differentiation to STB or iCTBs. Accordingly, in Aim 1, we will validate a culture system based on the completely defined E6/7/8 medium, and vitronectin-coated tissue culture plates. Specifically, we will investigate the expression of a panel of biomarkers to confirm that bona fide TB sub-types can be obtained using these conditions. In Aim 2, we will investigate whether TB differentiation of hESCs to STB and iCTBs can occur in defined conditions where exogenous bone morphogenetic protein (BMP) is not added, or where BMP signaling through the SMAD1/5/8 signaling pathway is inhibited. Finally, in Aim 3, we will test the hypothesis that Rho/ROCK signaling downstream of S1P activation mediates TB differentiation of hESCs in the proposed culture system. The significance of our work relates to its relevance to our understanding of early human TB development. Validation of defined culture conditions for TB differentiation of hESCs will enable mechanistic studies on TB differentiation of hESCs, and differentiation of hESC-derived TB to specialized TB sub-types. This in turn will enable routine acceptance use of hESC-derived TB a model system for early TB development. Such a model system will be a powerful tool for advancing understanding of early human placental development.