Modeling normal and abnormal trophoblasts

项目总结 人类胎盘在妊娠一至五周期间的发育情况知之甚少。 当滋养细胞侵入子宫时，形成原始合体和细胞滋养细胞，然后形成初级绒毛。 因此，需要模型来研究控制早期人类胎盘的分子和细胞机制。 发育以及这些过程中可能出现的错误导致胎盘疾病和早期受孕 损失。现已证实，人类胚胎干细胞(ESC)和诱导的多能干细胞 (IPSC)可以通过使滋养层细胞暴露于BMP4并抑制信号转导而沿着滋养层细胞谱系驱动 维持多能性表型的途径。总的前提是这个体外系统是一个有价值的 胎盘滋养层细胞在妊娠早期形成的模型 容易受到威胁体内妊娠早期存在的许多相同危险的影响。该项目 将使用这个早期滋养细胞发育的干细胞模型来了解胎盘的发育 早发性子痫前期(EOPE)的最早妊娠阶段和直接病理基础 与胎盘浅和母体血液灌流不足有关的疾病。有三个目标： 1)检验一种假设，即妊娠前半期的绒毛结核代表过渡状态 在植入时遇到的原始胎盘和第二和第三个成熟胎盘之间 三个月。其目的是验证ESC和IPSC产生的“原始”结核病是体外培养的 相当于早期胎盘结核。实验还将证实初步观察到，怀孕前三个月 绒毛结核与其分子特征有许多相似之处，既有源于多能性的原始结核 妊娠中期和晚期的细胞和较成熟的胎盘结核。2)采用干细胞模型 滋养层细胞分化以验证应激反应通路在EOPE中已经异常的假设 滋养层细胞初始形成时的胎盘。目标是使用RNAseq分析和DNA甲基化 比较PE和CTL细胞培养时基因和基因网络的变化 在正常和紧张的氧气条件下。3)检验以下假设：更好地表示 通过将多能干细胞分化为 用培养的球体培养的滋养层细胞而不是2D培养的滋养层细胞。我们的计划是使用这样的系统来遵循 器官内出现绒毛滋养细胞。此外，还将产生滋养层干细胞(TSC) 来自ESC和IPSC，并与ESC/IPSC一起使用来创建嵌合有机化合物。最终的命运是 当这些有机化合物被放置在乳房脂肪垫和肾胶囊下时，将进行检查 以确定免疫低下的小鼠是否表现出生理和结构上的相互作用 宿主小鼠体内的侵袭潜力。更长期的目标将是确定PE和CTL是否 有机物在异位部位的侵袭性以及与母体蜕膜化子宫内膜的相互作用方面有所不同。

PROJECT SUMMARY Very little is known of human placental development in the period between one and five weeks of gestation when trophoblasts invade the uterus, form the primitive syncytium and cytotrophoblast, and then primary villi. Thus, models are needed to study the molecular and cellular mechanisms controlling early human placental development and what can go wrong with these processes to cause placental disease and early conceptus loss. It is now well established that human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) can be driven along the trophoblast lineage by exposing them to BMP4 and inhibiting the signaling pathways that maintain the pluripotent phenotype. The overall premise is that this in vitro system is a valuable model for mimicking placental trophoblast formed early in the first trimester of pregnancy when it is most vulnerable to many of the same hazards that threaten an in vivo pregnancy early in its existence. The project will use this stem cell model of early trophoblast development to understand the development of the placenta in the earliest stages of pregnancy and the immediate pathological basis of early onset preeclampsia (EOPE), a disease linked to shallow placentation and insufficient perfusion with maternal blood. There are three aims: 1) Test the hypothesis that villous TB from the first half of the first trimester represents a transitional state between the primitive placenta encountered at implantation and the mature placenta of the second and third trimester. The goal is to validate the notion that the “primitive” TB generated from ESC and iPSC is an in vitro equivalent of early placental TB. Experiments will also confirm preliminary observations that first trimester villous TB shares many features of its molecular signature with both this primitive TB derived from pluripotent cells and more mature placental TB from the second and third trimesters. 2) Employ the stem cell model of trophoblast differentiation to test the hypothesis that stress response pathways are already aberrant in EOPE placentas upon initial formation of trophoblast. The goal is to employ RNAseq analysis and DNA methylation profiling to compare gene and gene network changes associated with PE and CTL cells when they are cultured under normal and stressful oxygen conditions. 3) Test the hypothesis that a better representation of trophoblast emergence will be gained by conducting the differentiation of the pluripotent stem cells to trophoblast with cultured spheroids rather than in 2D-cultures. The plan is to use such a system to follow the emergence of villous trophoblast within organoids. Additionally, trophoblast stem cells (TSC) will be generated from ESC and iPSC and also use these along with ESC/iPSC to create chimeric organoids. Finally the fate of these organoids will be examined when they are placed under the mammary fat pads and kidney capsules of immunocompromised mice to determine whether they exhibit physiologic and structural interactions and invasive potential within the host mouse. A longer term goal will be to determine whether PE and CTL organoids differ in invasiveness at ectopic sites and in interactions with maternal decidualized endometrium.