Pluripotent human stem cells as models for normal and diseased trophoblast

多能人类干细胞作为正常和患病滋养层的模型

• 批准号: 8028598
• 负责人: R. MICHAEL ROBERTS
• 金额: $ 34.36万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-16 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8028598
• 关键词: 2-methoxyestradiol; Abbreviations; Activins; Affect; Angiogenesis Inhibitors; Apoptosis; BMP4; Blood Vessels; Blood flow; Cell Culture Techniques; Cells; Cessation of life; Characteristics; Child; Child health care; Chorionic villi; Collagen Type I; Decidua; Development; Disease; Edema; Embryo; Event; Exhibits; Extracellular Matrix; Failure; Fetus; Fibroblasts; First Pregnancy Trimester; Glycine; Goals; Growth; HIF1A gene; HLA G antigen; Homologous Gene; Human; Hypertension; In Vitro; Infant; Inflammatory Response; Invaded; Lead; Life; Matrix Metalloproteinases; Minor; Mission; Modeling; Mothers; Mus; National Institute of Child Health and Human Development; Natural Killer Cells; Nodal; Oxygen; Pathology; Perfusion; Phenotype; Placenta; Pluripotent Stem Cells; Population; Pre-Eclampsia; Pregnancy; Production; Property; Proteins; Proteinuria; Reaction; Signal Transduction; Spiral Artery of the Endometrium; Stress; Study models; Symptoms; Syncytiotrophoblast; Syndrome; Term Birth; Time; Transformed Cell Line; Umbilical cord structure; Up-Regulation; Uterus; Vascular Diseases; Villous; Woman; Women' s Health; base; blood perfusion; bone morphogenic protein; cell type; crosslink; cytokine; cytotrophoblast; design; early onset; human embryonic stem cell; human stem cells; immune resistance; in vitro Model; induced pluripotent stem cell; inhibitor/antagonist; innovation; matrigel; migration; myometrium; novel strategies; obesity risk; stem; transcription factor; trend; trophoblast; vascular inflammation

DESCRIPTION (provided by applicant): The main goal of this proposal is to generate extravillous trophoblast (EVT) from human embryonic stem cells (hESC) after they have been treated with BMP4 (BMP-hESC) and use them as a model to study EVT emergence, migration, and invasiveness, as well as intrinsic and extrinsic factors that influence these properties. This project has high significance because EVT ultimately become the invasive trophoblast (TR) cells of human placenta that penetrate maternal blood vessels and enhance blood flow. While invasion of the uterine decidua and the proximate inner third of the myometrium by EVT is a characteristic feature of a healthy pregnancy, shallow invasion is associated with placental pathologies, among them pre-eclampsia (PE) and intra-uterine growth retardation (IUGR). PE is a pregnancy-specific syndrome that affects ~ 3 % of pregnancies and is responsible for 15 % of pre-term births and an estimated 50,000 deaths per year worldwide. It is characterized by maternal hypertension, edema, and proteinuria, conditions that in the more serious, early onset disease can manifest as soon as 20 weeks of pregnancy. Aim 1 will establish and characterize a hESC-derived cell culture model for the study of EVT. This aim is based on the hypothesis that the sub-population of HLA-G positive BMP-hESC that migrates through a Matrigel-coated matrix under high oxygen (O2) conditions are EVT homologs. HLA-G positive cells that remain associated with the BMP-hESC colonies under low O2 conditions represent a precursor population of non- invasive EVT whose properties will be distinct from the invasive population captured under high O2 conditions. Aim 2 will examine the ability of the EVT derived from BMP-hESC to invade through cross-linked, relatively stiff matrices, such as Type I collagen, and to interact with microcapillaries cultured within such matrices. Aim 3 will examine a possible model for PE in which up-regulation of stable HIF1 alpha (HIF1A) protein is hypothesized to counteract the effects of high O2 during in vitro development of BMP-hESC and retard development into invasive EVT. If time permits or the HIF1A hypothesis proves incorrect, we would plan to study the effects of knockdown of at least one transcription factor that has been implicated in EVT emergence and differentiation, namely ASCL2. Aim 4 will attempt to re-create EVT from infants born to mothers with PE by generating induced pluripotent stem cells (iPSC) from discarded umbilical cord and converting these pluripotent cells to CT, EVT and other lineages by the BMP4 approach. The properties of these cells can then be compared with EVT generated from cord cells from pregnancies not complicated by PE. The hypothesis underpinning this aim is that some forms of PE are initiated by genetically-based placental pathology, and these abnormalities will be manifested in the phenotype of EVT generated from umbilical cord-derived iPSC. Together, these aims will bring innovative and new approaches to the study of human TR cells and to examining the basis of placental pathologies, especially PE. They will create the first in vitro model that makes possible the study of both the emergence and invasiveness of EVT cells, and the first model to study these early events directly in TR from pregnancies complicated by PE. PUBLIC HEALTH RELEVANCE: This project is designed to establish a new model for studying extravillous trophoblast (EVT), a placental cell type that invades the wall of the womb during the first trimester of pregnancy and whose failure to invade properly can lead to serious consequences for mother and child, including a condition called pre-eclampsia. Instead of isolating the cells from placentae, the goal is to generate EVT from human pluripotent stem cells, thereby allowing us to study factors that control their invasiveness. Also by generating such pluripotent cells from umbilical cords of babies born to mothers that developed pre-eclampsia, we hope to recreate the cell type that caused the disease in the first place.

描述（由申请人提供）：本提案的主要目标是从人胚胎干细胞（hESC）经BMP 4处理后产生绒毛外滋养层（EVT）（BMP-hESC），并将其用作研究EVT出现、迁移和侵袭性以及影响这些特性的内在和外在因素的模型。该项目具有重要意义，因为EVT最终成为人类胎盘的侵入性滋养层（TR）细胞，穿透母体血管并增强血流。虽然EVT对子宫蜕膜和子宫肌层近内三分之一的侵入是健康妊娠的特征，但浅侵入与胎盘病理学相关，其中包括先兆子痫（PE）和宫内生长迟缓（IUGR）。PE是一种妊娠特异性综合征，影响约3%的妊娠，造成15%的早产，全球每年估计有50，000人死亡。它的特征是母体高血压、水肿和蛋白尿，在更严重的情况下，早发型疾病可以在怀孕20周内表现出来。目的1建立人胚胎干细胞体外培养模型，为EVT的研究奠定基础。这一目的是基于这样的假设，即在高氧（O2）条件下迁移通过Matrigel涂覆的基质的HLA-G阳性BMP-hESC的亚群是EVT同源物。在低O2条件下保持与BMP-hESC集落结合的HLA-G阳性细胞代表非侵入性EVT的前体群体，其性质将不同于在高O2条件下捕获的侵入性群体。目的2将检查来自BMP-hESC的EVT通过交联的、相对刚性的基质（如I型胶原）侵入的能力，以及与在这样的基质内培养的微毛细管相互作用的能力。目的3将研究一种可能的PE模型，其中稳定的HIF 1 α（HIF 1A）蛋白的上调被假设为抵消高O2在体外BMP-hESC发育过程中的作用，并延缓发展为侵袭性EVT。如果时间允许或HIF 1A假说被证明是不正确的，我们将计划研究至少一种与EVT出现和分化有关的转录因子（即ASCL 2）的敲低效应。目标4将尝试通过从丢弃的脐带中产生诱导多能干细胞（iPSC）并通过BMP 4方法将这些多能细胞转化为CT、EVT和其他谱系，从患有PE的母亲所生的婴儿中重建EVT。这些细胞的特性可以与来自未并发PE的妊娠的脐带细胞产生的EVT进行比较。支持这一目标的假设是，某些形式的PE是由基于遗传的胎盘病理学引发的，并且这些异常将表现在由脐带来源的iPSC产生的EVT的表型中。总之，这些目标将为人类TR细胞的研究带来创新和新的方法，并检查胎盘病理学的基础，特别是PE。他们将创建第一个体外模型，使研究EVT细胞的出现和侵袭性成为可能，并且是第一个直接研究这些早期事件的模型。 公共卫生相关性：该项目旨在建立一种新的模型来研究绒毛外滋养层（EVT），这是一种胎盘细胞类型，在怀孕的前三个月侵入子宫壁，如果不能正确侵入，可能会对母亲和孩子造成严重后果，包括一种称为先兆子痫的疾病。我们的目标不是从胎盘中分离细胞，而是从人类多能干细胞中产生EVT，从而使我们能够研究控制其侵袭性的因素。同样，通过从患有先兆子痫的母亲所生婴儿的脐带中产生这种多能细胞，我们希望首先重建引起这种疾病的细胞类型。