Regulating Embryonic Stem Cell Growth & Differentiation by Colony Confinement

描述(申请人提供)：人类胚胎干细胞(HESCs)具有组织工程和再生医学应用的巨大潜力，因为它具有多能性和无限自我更新两种特性的独特组合。因此，hESCs可作为细胞治疗所需的安全、无限的细胞来源。为了实现这一潜力，人类胚胎干细胞研究人员要求培养系统允许未分化的人类胚胎干细胞的强劲扩张，提供人类胚胎干细胞的有效存储和恢复，并沿所需的发育谱系直接分化人类胚胎干细胞。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时空呈现、固定化的细胞外基质成分、机械刺激和细胞与细胞的接触。在最近的工作中，我们和其他人已经证明，构建将包括hESCs在内的各种细胞类型限制在二维模式或三维孔洞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(包括生存、增殖和分化)产生显著影响。我们开发了一种方法，通过在微孔之间涂上细胞和蛋白质排斥涂层并将细胞外基质蛋白吸附到孔内的表面，将HESCs限制在通过软光刻构建的聚合物微孔中。当菌落填满油井时，培养物中的每个菌落都具有相同的大小和形状，因为生长在油井外的部分菌落被流体剪切去除。因此，这些微孔是评估集落形态对hESC生长和分化的影响的有价值的工具。我们组建了一支拥有材料科学、hESC细胞生物学、细胞生理学和细胞工程专业知识的研究团队，研究如何将微孔限制与其他微环境刺激结合起来设计hESC培养系统。我们的实验将利用来自NIH hESC干细胞注册处的hESC细胞系WA01和WA09。在这项研究中，我们提出了以下目标：(1)促进未分化的hESCs的高密度培养；(2)提供冷冻后可存活的未分化的hESCs的有效恢复；(3)引导hESCs沿着所需的发育谱系分化： 1.评价hESC集落限制对细胞生长和分化的影响 2.确定集落限制对以下未分化hESCs恢复的影响 超低温保存 3.定量培养人胚胎干细胞来源的心肌细胞的最佳拟胚体大小 4.研制机械应变应用于约束高强混凝土的力学柔顺微孔 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