Dissecting embryonic axis formation using micropatterned hESC colony architecture

描述(由申请人提供)：在体内，细胞分配到三个早期胚胎胚层中，遵循特定的时空顺序。作为对生长因子的响应，人类胚胎干细胞(HESCs)可以在培养中产生三层胚层，但分化是随机的和空间无序的。我们已经证明，当人类胚胎干细胞生长在特定大小和形状的微图案表面上时，它们与哺乳动物胚胎中的基因表达空间模式具有定量可重复性的差异。这是第一次在体外展示有序的生殖层模式，并为研究目前不可能在完整的哺乳动物胚胎，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介质中自发发展，该系统提供了一个用于分析细胞-细胞信号的定量表型。这笔赠款的目标是建立微模式细胞培养，作为研究人类胚胎干细胞信号动力学和空间模式的系统。我们建议通过在我们的底物上分化小鼠上皮细胞干细胞(与hESC最接近的类似物)并与从商业来源购买的小鼠胚胎进行比较来进一步验证我们的方法。我们将刺激形成早期哺乳动物胚胎图案的转化生长因子、骨形态发生蛋白和Wnt通路，并遵循这些通路的转录效应器以及所产生的生殖层标记的模式。我们将利用荧光记者的组合来设计细胞系，用于信号和命运，并将这些用于时间推移成像。结果将符合表型计算模型，这将允许我们合理地设计具有定义的剂量和时间的配体组合，从而产生定义的结果。 我们还将操纵分泌配体(激活剂和抑制物)的水平，这些配体负责在我们的微模式集落上的胚胎中发出旁分泌信号，并进一步量化细胞与细胞的相互作用。使用微流控技术对分泌配体进行物理分析将被用来量化配体运输的机制。

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.