Modeling normal and abnormal trophoblasts

项目摘要 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，形成原始合胞体和细胞滋养层，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问题导致胎盘疾病和早期妊娠 损失现在已经很好地建立了人胚胎干细胞（ESC）和诱导多能干细胞（ipluripotent stem cells） 诱导多能干细胞（iPSC）可以通过将其暴露于BMP 4并抑制其信号传导而沿着滋养层谱系被沿着驱动。 维持多能表型的途径。总的前提是，这种体外系统是一种有价值的 模拟胎盘滋养层细胞的模型在妊娠早期形成， 容易受到许多在体内妊娠早期威胁体内妊娠的相同危险的影响。项目 将使用这种早期滋养层发育的干细胞模型来了解胎盘的发育， 妊娠的最早阶段和早发性先兆子痫（EOPE）的直接病理基础， 与胎盘形成浅和母体血液灌注不足有关的疾病。有三个目标： 1)检验孕早期绒毛结核代表一种过渡状态的假设 在植入时遇到的原始胎盘与第二和第三胎盘的成熟胎盘之间 三个月目的是验证从ESC和iPSC产生的“原始”TB是体外培养的TB的概念。 相当于早期胎盘结核病实验还将证实初步观察结果， 绒毛结核病与多能性结核病的原始结核病和绒毛结核病的分子特征有许多共同之处。 细胞和更成熟的胎盘结核从第二和第三个三月。2)采用干细胞模型 滋养层分化，以检验EOPE中应激反应途径已经异常的假设 滋养层形成初期的胎盘。目标是利用RNAseq分析和DNA甲基化 分析比较PE和CTL细胞在培养时相关的基因和基因网络变化 在正常和紧张的氧气条件下。3)检验假设， 滋养层出现将通过进行多能干细胞的分化来获得， 滋养层与培养的球状体，而不是在二维培养。该计划是使用这样一个系统来遵循 类器官内绒毛滋养层的出现。此外，将产生滋养层干细胞（TSC）， 并且还将这些沿着ESC/iPSC以产生嵌合类器官。最后， 当将这些类器官放置在乳腺脂肪垫和肾包膜下时， 免疫受损小鼠，以确定它们是否表现出生理和结构相互作用， 在宿主小鼠体内的侵入潜力。长期目标是确定PE和CTL是否 类器官在异位部位的侵袭性和与母体蜕膜化子宫内膜的相互作用方面不同。

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.