Dissecting embryonic axis formation using micropatterned hESC colony architecture

描述（由申请方提供）：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。人类胚胎干细胞（human embryonic stem cells，hESC）在生长因子的作用下可以分化为三个胚层，但分化是随机的，空间上是无序的。我们已经证明，当人胚胎干细胞生长在微图案化的表面上，在确定的大小和形状的集落，他们分化与定量再现的空间模式的基因表达暗示那些在哺乳动物胚胎。这是第一次在体外展示有序的胚层图案，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。由于空间模式在充分混合的培养基中自发地发展，因此该系统提供了用于分析细胞-细胞信号传导的定量表型。该基金的目标是建立微模式化细胞培养系统，用于研究hESC的信号动力学和空间模式。我们建议通过在我们的基质上分化小鼠上胚层干细胞（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测定。我们将刺激形成早期哺乳动物胚胎的TGF-β、BMP和Wnt通路，并及时跟踪这些通路的转录效应子以及由此产生的胚层标记物模式。我们将用荧光报告分子的组合来设计细胞系，用于信号传导和命运，并将其用于延时成像。结果将适合于表型计算模型，该模型将允许我们合理地设计配体的组合，其具有产生确定结果的确定剂量和时间。 我们还将操纵分泌的配体（激活剂和抑制剂）的水平，这些配体负责我们微图案集落上胚胎中的旁分泌信号传导，并进一步量化细胞-细胞相互作用。使用微流体的分泌配体的物理测定将用于定量配体转运的机制。

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.