ConProject-001

ConProject-001

• 批准号: 10427299
• 负责人: Kenichiro Taniguchi
• 金额: $ 15.29万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 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/10427299
• 关键词: 3-Dimensional; AMOT gene; Amniotic Sac; BMP4; Beds; Biological Models; Cell Differentiation process; Cell Nucleus; Cells; Couples; 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; mechanical signal; mechanotransduction; mouse model; novel; overexpression; pluripotency; programs; prospective; self organization; single-cell RNA sequencing; species difference; three dimensional cell culture; tool; transcription factor

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.

羊膜形成一个坚韧的充满液体的囊，保护发育中的胚胎，对 一个成功的怀孕。羊膜形成在人类发育早期就开始了，因为胚胎被植入到 子宫壁：内细胞团首先极化，形成中央管腔的多能囊，然后， 这个极化的囊肿中有一半失去了多能标记，变成了鳞状(预期的 羊膜生态胚层)，而另一侧(外胚层)保持多能性。原肠形成始于外胚层 在那之后不久就到了一边。这些植入后的早期发育步骤无法在人类身上进行研究， 在我们对羊膜命运的决定和羊膜囊形成的认识上留下了巨大的空白， 尽管这些事件对发育中胚胎的存活至关重要。一种新的体外模型 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)形成 极化多能囊自发自组织成完全由羊膜组成的对称囊 细胞(90%-95%)以及类似羊膜囊样结构的不对称囊(5%-10%)。不对称地 花纹囊与卡内基5c期人类胚胎相似，被称为“植入后羊膜囊”。 胚状体“或”PASE“。在培养的五天内逐渐形成包囊。现场成像显示， 非对称性包囊是由于包囊一端的灶性扁平和羊膜命运的侧向展开所致； 当以多焦点模式发生扁平化时，就会出现对称性的囊肿。从机制上讲， 羊膜分化是机械性的，这可能导致羊膜细胞激活BMP信号。 对羊膜发生既必要又充分的计划。在培养5天时，PASE含有不同的 羊膜、外胚层和边界细胞群；外胚层细胞启动EMT运动类似于 原肠形成。我们将利用这一强大的体外模型来实现以下目标：目标1)探索如何 机械信号激活BMP信号。基于PiggyBac的人类胚胎干细胞基因修饰新工具 为这些研究提供帮助。目的2)建立导致羊水生成和 成熟Pase的发育。单细胞RNAseq将被用来剖析 伴随着对称性破坏、扩散性羊膜形成、边界形成和外胚层EMT的启动 动静。目的3)从功能上检测控制羊膜命运的转录因子。基因缺失和 过度表达研究将被用来探索几个潜在的羊膜命运主要调节者的作用。 总体而言，这里提出的工作将极大地加快发现关键但以前的 无法接触到的植入后事件，因此将对早期理解有巨大的影响 影响胚胎发育和人类生育能力的过程。