ConProject-001

基本信息

批准号：
10649483
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
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/10649483
关键词：
3-Dimensional AMOT gene Acceleration Amniotic Sac BMP4 Beds Biological Models Cell Differentiation process Cell Nucleus Cells Couples Cyst Data Development Early Diagnosis Embryo Embryonic Development Endoderm Epiblast Epithelium 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 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

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.

羊膜膜形成一个充满坚硬液体的囊，可保护发育的胚胎，对于成功的怀孕。羊膜发生在人类发展的早期开始，因为胚胎会进入子宫壁：内部细胞质量最初偏振以形成带有中央管腔的多能囊肿，然后这种两极分化的囊肿的一半失去多能标记并成为平方（前瞻性羊膜ecotderm）而另一侧（层状）仍然多能。胃分布在层细胞上此后不久。这些早期的植入后发展步骤无法在人类中学习，在我们对羊膜命运的确定和羊膜囊的形成方面的了解中留下了巨大的差距，尽管这些事件对发展中的胚胎的生存至关重要。新的体外模型可以帮助缩小差距：在特定3D条件下培养的人类多能干细胞（HPSC）形式偏光多能囊肿，从而自信地将自组织化为完全由羊膜组成的对称囊肿细胞（90-95％）以及类似于羊膜囊样结构（5-10％）的不对称囊肿。不对称图案囊肿镜面卡内基阶段5C人类胚胎，称为“植入后羊膜囊胚胎或“ PASE”。囊肿形成在培养物中逐渐发生。实时成像表明不对称囊肿是由囊肿的一个极点在囊肿的一个极点扁平而产生的。当以多焦点模式发生变平时，会产生对称囊肿。从机械上讲，最初的触发器羊膜分化是机械的，这会导致推定羊膜细胞激活BMP信号既需要且足够的羊膜生成的程序。在培养的5天时，PASE包含不同的羊膜，层细胞和边界细胞的种群； epiblast细胞启动EMT运动类似过度。我们将利用这种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。新型基于Piggybac的基于hESC的基因修饰工具将协助这些研究。目标2）建立基因激活的层次结构，从而导致羊膜发生和成熟PASE的开发。单细胞RNASEQ将用于剖析转录级联涉及对称性破裂，扩散羊Amniogenogeny，边界形成和EMT EMT的主动性动作。目标3）在功能上测试控制羊膜命运的转录因子。遗传缺失和过表达研究将用于探索羊膜命运的几个潜在主调节剂的作用。总体而言，这里提出的工作将极大地加速有关关键但以前的发现空间无法访问的后植入事件，因此将对早期理解具有巨大的影响影响胚胎发育和人类生育的过程。