Mechanisms of Rhesus Trophoblast Stem Cell Differentiation

描述（由申请人提供）：我们的总体目标是了解分化的滋养层表型的形成是如何在滋养层祖细胞中协调的。初步结果显示，令人惊讶的是，侵入性滋养层，而不是合胞体滋养层，来自一种新的恒河猴胚泡衍生的滋养层干细胞系表达叉头转录因子，FoxD 3，在其细胞核。对早期妊娠恒河猴胎盘组织的检查证实了细胞滋养层中FoxD 3表达的持续性。FoxD 3通常被认为局限于上胚层和滋养外胚层（在小鼠中）中的祖细胞以及发育中的神经嵴细胞。我们的siRNA研究表明，FoxD 3的表达是必需的滋养层细胞的迁移行为的形成。17-雌二醇改变FoxD 3的细胞内分布和表达并诱导合体滋养层形成。基于这些和其他初步数据，我们假设滋养层分化和命运的规范取决于一个关键的表达水平和亚细胞分布的FoxD 3。本项目研究使用恒河猴胚泡来源的滋养层干细胞以及其他体外滋养层分化系统和胚泡植入模型调节恒河猴滋养层中FoxD 3表达的机制。第一个目的是定量FoxD 3 mRNA水平，并确定FoxD 3蛋白水平在滋养层分化的细胞质和细胞核。第二个目的是表征FoxD 3沉默和过表达对滋养层分化和侵袭的影响。第三个目的是使用定点突变来确定磷酸化在调节FoxD 3细胞内定位和功能中的作用。目的4将表征172-雌二醇对FoxD 3表达和功能的影响。最后一个目标将补充和扩展的研究，在其他目标的特点，FoxD 3在滋养层分化的作用，恒河猴囊胚植入模型。在完成这些目标，我们将有特点的机制，从不同的滋养层祖细胞群的分化调节浸润性滋养层。我们还将发现转录因子FoxD 3的新作用。此外，我们将提供一个新的滋养层干细胞系统，应该是有用的进一步的机制研究和再生技术的发展严格的表征。胎盘对胎儿发育和成功怀孕至关重要。这项研究调查了恒河猴囊胚中的干细胞如何形成胎盘细胞。了解这些过程可能会导致预防某些妊娠疾病的策略，如流产和先兆子痫。

DESCRIPTION (provided by applicant): Our overall objective is to understand how the formation of differentiated trophoblast phenotypes is orchestrated in trophoblast progenitor cells. Preliminary results show, surprisingly, that invasive trophoblasts, but not syncytiotrophoblasts, derived from a novel rhesus blastocyst-derived trophoblast stem cell line express the forkhead transcription factor, FoxD3, in their nuclei. Examination of early gestation rhesus placental tissue confirms persistence of FoxD3 expression in cytotrophoblasts. FoxD3 is generally considered to be restricted to progenitor cells in the epiblast and trophectoderm (in the mouse) and to developing neural crest cells. Our siRNA studies suggest that FoxD3 expression is required for the formation of trophoblasts with migratory behavior. 17-estradiol alters the intracellular distribution and expression of FoxD3 and induces syncytiotrophoblast formation. Based on these and other preliminary data we hypothesize that trophoblast differentiation and fate specification depends on a critical expression level and subcellular distribution of FoxD3. This project investigates mechanisms that regulate FoxD3 expression in rhesus trophoblasts using rhesus blastocyst-derived trophoblast stem cells, as well as other in vitro trophoblast differentiation systems and a blastocyst implantation model. The first aim quantifies FoxD3 mRNA levels and determines FoxD3 protein levels in the cytoplasm and nucleus during trophoblast differentiation. The second aim characterizes the effects of FoxD3 silencing and overexpression on trophoblast differentiation and invasion. The third aim uses site-directed mutagenesis to determine the role of phosphorylation in regulating FoxD3 intracellular localization and function. Aim 4 will characterize the effects of 172- estradiol on FoxD3 expression and function. The last aim will complement and extend the studies in the other aims by characterizing the role of FoxD3 in trophoblast differentiation using a rhesus blastocyst implantation model. At the completion of these aims we will have characterized mechanisms which regulate the differentiation of invasive trophoblasts from different trophoblast progenitor cell populations. We will also have uncovered a new role for the transcription factor FoxD3. In addition, we will have provided a rigorous characterization of a novel trophoblast stem cell system that should be useful for further mechanistic studies and the development of regenerative techniques. The placenta is essential for fetal development and successful pregnancy. This research investigates how placental cells are formed from stem cells in the rhesus monkey blastocyst. Understanding these processes could lead to strategies for preventing certain disorders of pregnancy such as miscarriage and preeclampsia.