ConProject-002

基本信息

批准号：
10192780
负责人：
Kenichiro Taniguchi
金额：
$ 14.87万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-16 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10192780
关键词：
3-Dimensional AMOT gene Amniotic Sac BMP4 Beds Biological Models Cell Differentiation process Cell Nucleus Cells Couples Cues Cyst Data Development Embryo Embryonic Development Endoderm Epiblast Epithelial Equilibrium Ethics Event Fertility Gel Gene Activation Generations Genetic Genetic Engineering Genetic Transcription Goals Human Human Development Image Implant Inner Cell Mass Investigation Knowledge Light Liquid substance Location Manuscripts Mechanics Membrane Mesoderm Modeling Modification Molecular Molecular Probes Monkeys Movement Mus Nature Nuclear Pathway interactions Pattern Population Pregnancy Primates Process Rodent Role Side Signal Transduction Signaling Molecule Site Structure Supporting Cell System Testing Tight Junctions Up-Regulation Uterus Validation Work amnion base blastocyst cell type gastrulation human embryonic stem cell human model human pluripotent stem cell implantation improved in vitro Model inhibitor/antagonist mechanotransduction mouse model novel overexpression pluripotency programs prospective self organization single-cell RNA sequencing species difference three dimensional cell culture tool transcription factor

项目摘要

The amniotic membrane forms a tough fluid-filled sac that protects the developing embryo and is essential for a successful pregnancy. Amniogenesis initiates early in human development as the embryo implants into the uterine wall: the inner cell mass first polarizes to form a pluripotent cyst with central lumen and subsequently, one half of this polarized cyst loses pluripotency markers and becomes squamous in nature (the prospective amniotic ecotderm) while the other side (the epiblast) remains pluripotent. Gastrulation begins on the epiblast side soon thereafter. These early post-implantation developmental steps are inaccessible to study in humans, leaving an enormous gap in our knowledge about amnion fate determination and formation of the amniotic sac, despite the central importance of these events to the survival of the developing embryo. A new in vitro model can help to close that gap: human pluripotent stem cells (hPSC), cultured in specific 3D conditions, form polarized pluripotent cysts that spontaneously self-organize into symmetric cysts composed entirely of amnion cells (90-95%) as well as asymmetric cysts that resemble amniotic sac-like structures (5-10%). Asymmetrically patterned cysts mirror Carnegie stage 5c human embryos and are called “post-implantation amniotic sac embryoids” or “PASE”. Cyst formation occurs progressively over five days in culture. Live imaging shows that asymmetric cysts arise from focal flattening at one pole of the cyst and laterally spreading of amnion fate; symmetric cysts arise when flattening occurs in a multi-focal pattern. Mechanistically, the initial trigger for amnion differentiation is mechanical and that this causes presumptive amnion cells to activate a BMP signaling program that is both necessary and sufficient for amniogenesis. At 5 days of culture, PASE contain distinct populations of amnion, epiblast and boundary cells; epiblast cells initiate EMT movements similar to gastrulation. We will exploit this robust in vitro model to accomplish the following goals: Aim 1) Explore how mechanical signals activate BMP signaling. Novel PiggyBac-based tools for genetic modification of hESC will aid in these studies. Aim 2) Establish the hierarchy of gene activation that results in amniogenesis and development of mature PASE. Single cell RNAseq will be used to dissect the transcriptional cascade that accompanies symmetry breaking, spreading amniogenesis, boundary formation and initiation of epiblast EMT movements. Aim 3) Functionally test transcription factors that control amnion fate. Genetic deletion and overexpression studies will be used to explore the role of several potential master regulators of amnion fate. Overall, the work proposed here will greatly accelerate the pace of discovery regarding critical but previously inaccessible post-implantation events and thus will have enormous implications for understanding early processes that impact embryonic development and human fertility.

羊膜形成坚韧的充满液体的囊，保护发育中的胚胎，对于胚胎发育至关重要当胚胎植入子宫时，羊膜形成就在人类发育的早期开始。子宫壁：内细胞团首先极化形成具有中央腔的多能囊肿，随后，这种极化囊肿的一半失去了多能性标记并变成鳞状（预期的）羊膜生态真皮），而另一侧（外胚层）保持多能性，原肠胚形成从外胚层开始。此后不久，这些早期植入后发育步骤无法在人类身上进行研究，我们对羊膜命运决定和羊膜囊形成的知识存在巨大差距，尽管这些事件对于发育中的胚胎的存活至关重要，但新的体外模型。可以帮助缩小这一差距：在特定 3D 条件下培养的人类多能干细胞 (hPSC) 会形成极化多能囊肿自发自组织成完全由羊膜组成的对称囊肿细胞（90-95%）以及类似羊膜囊样结构的不对称囊肿（5-10%）。图案囊肿反映了卡内基 5c 期人类胚胎，被称为“植入后羊膜囊” 实时成像显示，在培养过程中，囊肿逐渐形成。不对称囊肿是由于囊肿一极的局部扁平化和羊膜命运的横向扩散而引起的；当以多焦点模式发生扁平化时，就会出现对称性囊肿，这是最初的触发因素。羊膜分化是机械性的，这会导致假定的羊膜细胞激活 BMP 信号传导培养 5 天时，PASE 包含独特的羊膜生成所需的程序。羊膜、外胚层和边界细胞群启动 EMT 运动，类似于我们将利用这个强大的体外模型来实现以下目标：目标 1) 探索如何实现机械信号激活 BMP 信号转导，用于 hESC 基因修饰的新型 PiggyBac 工具将目标 2) 建立导致羊膜生成的基因激活层次。成熟 PASE 的开发将用于剖析转录级联反应调节对称性破坏、羊膜生成扩散、边界形成和外胚层 EMT 启动目标 3) 功能测试控制羊膜命运的转录因子。过度表达研究将用于探索羊膜命运的几种潜在主调节因子的作用。总体而言，这里提出的工作将大大加快发现关键但以前的问题的步伐难以接近的植入后事件，因此将对早期理解产生巨大影响影响胚胎发育和人类生育能力的过程。