Dissecting embryonic axis formation using micropatterned hESC colony architecture

使用微图案 hESC 集落结构剖析胚胎轴形成

基本信息

批准号：
9065180
负责人：
ALI H BRIVANLOU
金额：
$ 34.82万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-15 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9065180
关键词：
Architecture Biological Assay Capsicum Cell Communication Cell Culture Techniques Cell Line Cell Separation Cells Clinic Communities Computer Simulation Data Development Disease Dose Embryo Embryology Engineering Epiblast Exposure to Eye Gene Expression Profile Germ Layers Goals Grant Growth Health Heterogeneity Human Human Development Image In Vitro Investigation Life Ligands Light Maps Methods Microfluidics Microscopy Modeling Mus Outcome Paracrine Communication Pathway interactions Pattern Phenotype Protocols documentation Recording of previous events Regenerative Medicine Reporter Shapes Signal Transduction Sodium Chloride Source Stem cells Surface System Techniques Testing Time Work analog design embryonic stem cell human embryonic stem cell in vivo induced pluripotent stem cell inhibitor/antagonist intercellular communication predictive modeling progenitor regenerative therapy research study response success time use tool

项目摘要

DESCRIPTION (provided by applicant): The allocation of cells into the three early embryonic germ layers, in vivo, follows a specific patio-temporal sequence. In response to growth factors, human embryonic stem cells (hESCs) can generate the three germ layers in culture, but differentiation is random and spatially disordered. We have demonstrated that when hESCs are grown on micro-patterned surfaces in colonies of defined size and shape, they differentiate with quantitatively reproducible spatial patterns of gene expression suggestive of those in mammalian embryos. This is the first demonstration of ordered germ layer patterning in vitro and opens the door to investigations of early development currently impossible to perform in intact mammalian embryos, particularly human embryos. Since the spatial pattern develops spontaneously in a well-mixed medium, this system affords a quantitative phenotype with which to analyze cell-cell signaling. The goal of this grant is to establish micro patterned cell cultureas a system for studying signaling dynamics and spatial patterning in hESCs. We propose to further validate our assay by differentiating mouse epiblast stem cells (the closest analogue to hESC) on our substrates and comparing with mouse embryos purchased from commercial sources. We will stimulate the TGF-ß, BMP, and Wnt pathways that pattern the early mammalian embryo and follow the transcriptional effectors for these pathways as well as the resulting pattern of germ layer markers in time. We will engineer cell lines with combinations of fluorescent reporters for signaling and fate and use these for time-lapse imaging. The results will be fit to a phenotypic computational model that will permit us to rationally design combinations of ligands with defined doses and timings that produce a defined outcome. We will also manipulate the levels of secreted ligands (both activators and inhibitors) that are responsible for paracrine signaling in the embryo on our micro patterned colonies and further quantify cell-cell interactions. Physical assays for secreted ligands using microfluidics will be employed to quantify mechanisms of ligand transport.

描述（申请人提供）：在体内，形成三个早期胚胎胚层的细胞遵循特定的时空分配序列。人类胚胎干细胞（hESC）可以响应生长因子产生三个胚层。但我们已经证明，当 hESC 在具有确定大小和形状的集落的微图案表面上生长时，它们会以定量可重复的基因表达空间模式进行分化。这是哺乳动物胚胎中有序胚层模式的首次演示，并为目前在完整哺乳动物胚胎（尤其是人类胚胎）中不可能进行的早期发育研究打开了大门，因为空间模式是在充分混合的情况下自发发育的。培养基中，该系统提供了用于分析细胞间信号传导的定量表型，这项资助的目标是建立分析微图案细胞培养物作为研究 hESC 信号动力学和空间图案的系统。差异化我们将小鼠外胚层干细胞（与 hESC 最接近的类似物）置于我们的基质上，并与从商业来源购买的小鼠胚胎进行比较，我们将刺激形成早期哺乳动物胚胎的 TGF-β、BMP 和 Wnt 通路，并跟踪这些细胞的转录效应子。我们将利用荧光产生器的组合来设计细胞系，以进行信号传导和命运，并将其用于延时成像。我们合理地设计具有确定剂量和时间的配体组合，以产生确定的结果。我们还将操纵微图案集落上负责胚胎中旁分泌信号的分泌配体（激活剂和抑制剂）的水平，并进一步量化细胞与细胞之间的相互作用，使用微流体对分泌配体进行物理测定，以量化机制。配体运输。