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）具有组织工程和再生医学应用的巨大潜力，因为它们具有两种特性，即多能性和无限自我更新能力。因此，hESC可以作为一种安全的、无限的细胞来源，用于基于细胞的治疗。为了实现这一潜力，hESC研究人员需要培养系统，该培养系统允许未分化hESC的稳健扩增，提供hESC的有效储存和回收，以及hESC沿沿着期望的发育谱系的直接分化。设计这样的培养系统需要了解各种微环境信号如何影响hESC的生长和分化。这些信号包括可溶性蛋白质和其他化学因子的空间和时间呈现、固定的细胞外基质组分、机械刺激和细胞-细胞接触。 在最近的工作中，我们和其他人已经证明，构建微环境，限制各种类型的细胞，包括hESC，二维模式或三维威尔斯孔可以有显着的影响细胞内信号转导途径，基因表达，细胞表型，包括生存，增殖和分化。我们已经开发了一种将hESC限制在通过软光刻构建的聚合物微孔威尔斯中的方法，通过用细胞和蛋白质排斥涂层处理微孔威尔斯之间的表面并将细胞外基质蛋白吸附到威尔斯内的表面。当集落填充威尔斯孔时，培养物中的每个集落具有相同的大小和形状，因为生长在孔外的部分殖民地通过流体剪切去除。因此，这些微孔作为一个有价值的工具，以评估集落形态对人胚胎干细胞的生长和分化的影响。我们已经组建了一个研究团队，具有材料科学，hESC细胞生物学，细胞生理学和细胞工程方面的专业知识，以解决如何将微孔限制与其他微环境刺激结合使用，以设计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WA 01和WA 09。在这项研究中，我们提出了以下目标来开发基于微孔的培养系统，其（1）促进未分化的hESC的高密度培养，（2）在冷冻保存后提供活的未分化的hESC的有效回收，和（3）引导hESC沿着期望的发育谱系分化： 1.评估hESC集落限制对限定培养基中生长和分化的影响 2.确定集落限制对未分化的hESC恢复的影响， 冷冻保存 3.量化用于产生hESC衍生的心肌细胞的最佳胚状体大小 4.开发机械顺应性微孔，用于将机械应变应用于受限的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