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

• 批准号: 9269241
• 负责人: ALI H BRIVANLOU
• 金额: $ 35.17万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 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/9269241
• 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; Genetic Transcription; Germ Layers; Goals; Grant; Growth Factor; Heterogeneity; Human; Human Development; Image; In Vitro; Investigation; Ligands; Light; Maps; Mathematics; Methods; Microfluidics; Microscopy; Modeling; Mus; Outcome; Paracrine Communication; Pathway interactions; Pattern; Phenotype; Protocols documentation; Recording of previous events; Regenerative Medicine; Reporter; Reproducibility; Shapes; Signal Transduction; Sodium Chloride; Source; Stem cells; Suggestion; Surface; System; Techniques; Testing; Time; Transforming Growth Factor beta; Work; analog; design; embryonic stem cell; experimental study; high dimensionality; human embryonic stem cell; in vivo; induced pluripotent stem cell; inhibitor/antagonist; intercellular communication; predictive modeling; progenitor; public health relevance; regenerative therapy; response; success; time use; tool

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（由适用提供）：在体内分配细胞为三个早期胚胎细菌层的分配遵循特定的露台 - 周期序列。为了响应生长因子，人类胚胎干细胞（HESC）可以在培养中产生三个细菌层，但分化是随机的，并且在空间上无序。我们已经证明，当在定义大小和形状的菌落中生长在微图案的表面上时，它们会与基因表达的定量可重复可重复的空间模式进行区分，这些模式暗示了哺乳动物胚胎中的基因表达。这是在体外模式的有序细菌层的首次演示，并为对早期发育的研究打开了目前无法在完整的哺乳动物胚胎（尤其是人类胚胎）中进行的研究之门。由于在混合良好的培养基中赞助的空间模式发展，该系统提供了一种定量表型，可以通过该表型来分析细胞细胞信号传导。该赠款的目的是建立微型图案细胞培养物，一种用于研究hESC中信号传导动力学和空间图案的系统。我们建议通过区分底物上的小鼠培养基干细胞（最接近hESC的类似物）来进一步验证我们的评估，并与从商业来源购买的小鼠胚胎进行比较。我们将刺激对早期哺乳动物胚胎进行模拟的TGF-ß，BMP和WNT途径，并遵循这些途径的转录效应以及随时间的胚芽层标记模式。我们将使用荧光报告和命运的荧光记者组合来设计细胞系，并将其用于延时成像。结果将适合表型计算模型，该模型将使我们能够合理地设计具有定义剂量和时机的配体的组合，从而产生定义的结果。 我们还将操纵导致微型图案菌落胚胎中旁分泌信号的分泌配体（激活剂和抑制剂）的水平，并进一步量化细胞细胞相互作用。使用微流体物质的分泌配体的物理测定将被聘请以量化配体转运的机制。