Mechanisms of Rhesus Trophoblast Stem Cell Differentiation

恒河猴滋养层干细胞分化机制

• 批准号: 7568230
• 负责人: GORDON C DOUGLAS
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7568230
• 关键词: 3-Dimensional; Amino Acid Substitution; Behavior; Biochemical; Biological Assay; Cell Line; Cell Nucleus; Cells; Coculture Techniques; Complement; Confocal Microscopy; Cytoplasm; Data; Development; Differentiation Antigens; Dose; Environment; Epiblast; Epithelial; Estradiol; Estrogen Receptors; Event; FGF2 gene; Fetal Development; Immunoprecipitation; In Vitro; Incubated; Laser Scanning Cytometry; Lead; Macaca mulatta; Measures; Messenger RNA; Modeling; Mus; Neural Crest Cell; Phenotype; Phosphorylation; Phosphorylation Site; Placenta; Population; Pre-Eclampsia; Pregnancy; Process; Proteins; Regulation; Reporter; Research; Role; Site-Directed Mutagenesis; Small Interfering RNA; Spontaneous abortion; Stem cells; Syncytiotrophoblast; System; Techniques; Testing; Time; Tissues; Western Blotting; base; blastocyst; cytotrophoblast; expression vector; forkhead protein; implantation; monolayer; natural Blastocyst Implantation; novel; overexpression; pregnancy disorder; prevent; promoter; regenerative; response; stem cell differentiation; transcription factor; trophoblast

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.

描述（由申请人提供）：我们的总体目标是了解如何在滋养细胞祖细胞中精心策划分化的滋养细胞表型的形成。令人惊讶的是，初步结果表明，侵入性的滋养层细胞，但不是源自新型的源自芽孢杆菌源细胞源细胞干细胞系在其核中表达出叉头的转录因子FOXD3的颗粒型滋养细胞。早期妊娠颗粒组织的检查证实了细胞增多质细胞中FOXD3表达的持久性。 FOXD3通常被认为仅限于肉毒细胞和滋养剂中的祖细胞（在小鼠中）和发展神经rest细胞。我们的siRNA研究表明，形成具有迁移行为的滋养细胞需要FOXD3表达。 17-雌二醇改变了FOXD3的细胞内分布和表达并诱导合成肌细胞形成。基于这些和其他初步数据，我们假设滋养细胞的分化和命运规范取决于FOXD3的临界表达水平和亚细胞分布。该项目研究了使用恒河-囊泡细胞衍生的滋养细胞干细胞以及其他体外滋养细胞分化系统和胚泡植入模型来调节恒河-滋养细胞中FOXD3表达的机制。第一个目标量化了FOXD3 mRNA水平，并确定滋养细胞分化过程中细胞质和核中FOXD3蛋白水平。第二个目标是FOXD3沉默和过表达对滋养细胞分化和入侵的影响。第三目的使用位置定向的诱变来确定磷酸化在调节FOXD3细胞内定位和功能中的作用。 AIM 4将表征172-雌二醇对FOXD3表达和功能的影响。最后一个目标将通过使用恒河猴胚泡植入模型来表征FOXD3在滋养细胞分化中的作用来补充和扩展其他目的的研究。在完成这些目标时，我们将具有特征的机制，这些机制调节了不同滋养细胞祖细胞群体的侵入性滋养细胞的分化。我们还将发现转录因子FOXD3的新角色。此外，我们还将提供一种新型滋养细胞干细胞系统的严格特征，该系统应有助于进一步的机械研究和再生技术的发展。胎盘对于胎儿发育和成功怀孕至关重要。这项研究调查了如何由恒河猴胚泡中的干细胞形成的胎盘细胞。了解这些过程可能会导致预防某些怀孕疾病（例如流产和先兆子痫）的策略。