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滋养细胞祖细胞中分化的滋养细胞表型是如何形成的。初步结果显示，令人惊讶的是，来自一种新的恒河胚泡来源的滋养细胞干细胞系的侵袭性滋养细胞，而不是合胞滋养细胞，在其细胞核中表达叉头转录因子FoxD3。对妊娠早期恒河猴胎盘组织的检查证实FoxD3在细胞滋养细胞中持续表达。FoxD3通常被认为局限于外胚层和滋养外胚层的祖细胞（小鼠）和发育中的神经嵴细胞。我们的siRNA研究表明FoxD3的表达是形成具有迁移行为的滋养细胞所必需的。17-雌二醇改变细胞内FoxD3的分布和表达，诱导合体滋养细胞的形成。基于这些和其他初步数据，我们假设滋养细胞分化和命运规范取决于FoxD3的临界表达水平和亚细胞分布。本项目利用恒河胚泡来源的滋养细胞干细胞，以及其他体外滋养细胞分化系统和囊胚植入模型，研究了FoxD3在恒河滋养细胞中的表达调控机制。第一个目的是定量FoxD3 mRNA水平，并测定滋养细胞分化过程中细胞质和细胞核中FoxD3蛋白水平。第二个目的是表征FoxD3沉默和过表达对滋养细胞分化和侵袭的影响。第三个目的是使用位点定向诱变来确定磷酸化在调节FoxD3细胞内定位和功能中的作用。目的4将描述172-雌二醇对FoxD3表达和功能的影响。最后一个目标将通过利用恒河囊胚植入模型表征FoxD3在滋养细胞分化中的作用来补充和扩展其他目标的研究。在完成这些目标后，我们将描述从不同滋养细胞祖细胞群中调节侵袭性滋养细胞分化的机制。我们还将发现转录因子FoxD3的新作用。此外，我们将提供一种新的滋养细胞干细胞系统的严格特征，这将有助于进一步的机制研究和再生技术的发展。胎盘对胎儿发育和成功怀孕至关重要。本研究探讨了恒河猴胚泡干细胞是如何形成胎盘细胞的。了解这些过程可能会导致预防某些怀孕疾病的策略，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.