Dissecting embryonic axis formation using micropatterned hESC colony architecture

描述（由申请人提供）：细胞在体内分配到三个早期胚胎胚层，遵循特定的天时顺序。在生长因子的作用下，人胚胎干细胞（hESCs）在培养过程中可产生三种胚层，但其分化具有随机性和空间无序性。我们已经证明，当hESCs在确定大小和形状的微图案表面生长时，它们与哺乳动物胚胎中可定量再现的基因表达空间模式相分化。这是体外有序胚层模式的首次展示，并为研究目前不可能在完整哺乳动物胚胎，特别是人类胚胎中进行的早期发育打开了大门。由于空间模式在混合良好的培养基中自发发展，该系统提供了定量表型，用于分析细胞-细胞信号传导。该基金的目标是建立微模式细胞培养系统，用于研究hESCs的信号动力学和空间模式。我们打算通过在我们的基质上分化小鼠外胚层干细胞（最接近hESC的类似物），并与从商业来源购买的小鼠胚胎进行比较，进一步验证我们的实验。我们将刺激早期哺乳动物胚胎模式的TGF-ß、BMP和Wnt通路，并及时跟踪这些通路的转录效应物以及由此产生的胚层标记物模式。我们将设计带有荧光报告基因组合的细胞系，用于信号和命运，并将其用于延时成像。结果将适合表型计算模型，这将使我们能够合理地设计配体的组合，确定剂量和时间，产生确定的结果。

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.