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层细胞分化表型的形成是如何在滋养层祖细胞中协调的。令人惊讶的是，初步结果显示，来自一种新的恒河猴胚泡滋养层干细胞系的侵袭性滋养层细胞，而不是合体滋养层细胞，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在细胞滋养层细胞中持续表达。FoxD3通常被认为仅限于(小鼠)上胚层和滋养外胚层中的祖细胞以及发育中的神经脊细胞。我们的siRNA研究表明，FoxD3的表达对于具有迁移行为的滋养层细胞的形成是必需的。17-雌二醇改变FoxD3在细胞内的分布和表达，并诱导合体滋养层细胞的形成。基于这些和其他初步数据，我们假设滋养层细胞的分化和命运的指定取决于FoxD3的关键表达水平和亚细胞分布。该项目利用恒河猴胚泡来源的滋养层干细胞，以及其他体外滋养层细胞分化系统和胚泡植入模型，研究了FoxD3在恒河猴滋养层细胞中表达的调节机制。第一个目的是量化FoxD3的mRNA水平，并确定滋养层细胞分化过程中细胞质和细胞核中的FoxD3蛋白水平。第二个目的是研究FoxD3沉默和过表达对滋养层细胞分化和侵袭的影响。第三个目的是使用定点突变来确定磷酸化在调节FoxD3细胞内定位和功能中的作用。目的4研究172-雌二醇对FoxD3基因表达和功能的影响。最后一个目标将补充和扩展其他目标的研究，利用恒河猴胚泡植入模型表征FoxD3在滋养层分化中的作用。在完成这些目标后，我们将拥有调控来自不同滋养层祖细胞群体的侵袭性滋养层细胞分化的特征机制。我们还将发现转录因子FoxD3的一个新角色。此外，我们将提供一种新的滋养层干细胞系统的严格表征，这将有助于进一步的机制研究和再生技术的发展。胎盘是胎儿发育和成功妊娠所必需的。这项研究调查了恒河猴胚泡中的干细胞是如何形成胎盘细胞的。了解这些过程可以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.