Regulating Embryonic Stem Cell Growth & Differentiation by Colony Confinement

描述（由申请人提供）：人类胚胎干细胞（hESC）由于其多能性和无限自我更新能力这两种特性的独特组合，在组织工程和再生医学应用方面具有巨大的潜力。因此，hESC 可以作为细胞疗法所需细胞的安全、无限供应。为了实现这一潜力，hESC 研究人员需要能够强劲扩增未分化 hESC 的培养系统，提供 hESC 的高效储存和恢复，以及沿所需发育谱系直接分化 hESC。此类培养系统的设计需要了解各种微环境信号如何影响 hESC 生长和分化。这些信号包括可溶性蛋白质和其他化学因子、固定的细胞外基质成分、机械刺激和细胞与细胞接触的空间和时间呈现。 在最近的工作中，我们和其他人已经证明，构建将包括 hESC 在内的多种细胞类型限制在 2 维模式或 3 维孔中的微环境可以对细胞内信号转导途径、基因表达和细胞表型（包括存活、增殖和分化）产生巨大影响。我们开发了一种将 hESC 限制在由软光刻构建的聚合物微孔中的方法，通过用细胞和蛋白质排斥涂层处理微孔之间的表面并将细胞外基质蛋白吸附到孔内的表面。当菌落充满孔时，培养物中的每个菌落具有相同的大小和形状，因为在孔外生长的菌落部分被流体剪切去除。因此，这些微孔可作为评估集落形态对 hESC 生长和分化影响的有价值的工具。我们组建了一支在材料科学、hESC 细胞生物学、细胞生理学和细胞工程方面具有专业知识的研究团队，以解决如何将微孔限制与其他微环境刺激结合使用来设计 hESC 培养系统。我们的实验将利用来自 NIH hESC 干细胞登记处的 hESC 细胞系 WA01 和 WA09。在本研究中，我们提出以下目标，即开发一种基于微孔的培养系统，该系统（1）促进未分化 hESC 的高密度培养，（2）在冷冻保存后有效恢复活的未分化 hESC，以及（3）指导 hESC 沿所需的发育谱系分化： 1. 评估 hESC 集落限制对特定培养基中生长和分化的影响 2. 确定集落限制对以下未分化 hESC 恢复的影响 冷冻保存 3. 量化 hESC 衍生心肌细胞生成的最佳胚体大小 4. 开发机械顺应性微孔，将机械应变应用于受限 hESC 殖民地

DESCRIPTION (provided by applicant): Human embryonic stem cells (hESCs) hold tremendous potential for tissue engineering and regenerative medicine applications because of their unique combination of two properties, pluripotency and the capacity for infinite self-renewal. Thus, hESCs may serve as a safe, limitless supply of desired cells for cell-based therapies. To realize this potential, hESC researchers require culture systems that permit robust expansion of undifferentiated hESCs, provide efficient storage and recovery of hESCs, and direct differentiation of hESCs along desired developmental lineages. Design of such culture systems requires an understanding of how a variety of microenvironmental signals affect hESC growth and differentiation. These signals include spatial and temporal presentation of soluble proteins and other chemical factors, immobilized extracellular matrix components, mechanical stimuli, and cell-cell contact. In recent work we and others have demonstrated that constructing microenvironments that confine a variety of cell types, including hESCs, to 2-dimensional patterns or to 3-dimensional wells can have dramatic effects on intracellular signal transduction pathways, gene expression, and cell phenotype, including survival, proliferation, and differentiation. We have developed a method to confine hESCs to polymer microwells, constructed by soft lithography, by treating the surfaces between the microwells with a cell and protein repulsive coating and adsorbing extracellular matrix proteins to the surfaces inside the wells. As colonies fill the wells, each colony in the culture has the same size and shape as portions of the colony that grow outside the well are removed by fluid shear. Thus, these microwells serve as a valuable tool to assess the effects of colony morphology on hESC growth and differentiation. We have assembled a research team with expertise in materials science, hESC cell biology, cell physiology, and cell engineering to address how microwell confinement can be used in conjunction with other microenvironmental stimuli to design hESC culture systems. Our experiments will utilize hESC cell lines WA01 and WA09 from the NIH hESC Stem Cell Registry. In this study, we propose the following aims to develop a microwell-based culture system that (1) facilitates high density culture of undifferentiated hESCs, (2) provides efficient recovery of viable, undifferentiated hESCs following cryopreservation, and (3) directs differentiation of hESCs along desired developmental lineages: 1. Assess effects of hESC colony confinement on growth and differentiation in defined medium 2. Determine impact of colony confinement on recovery of undifferentiated hESCs following cryopreservation 3. Quantify optimum embryoid body size for generation of hESC-derived cardiac myocytes 4. Develop mechanically-compliant microwells for application of mechanical strain to confined hESC colonies