Molecular Engineering of Bioactive Hydrogels

描述(申请人提供)：人类胚胎干细胞(HESCs)作为治疗疾病和损伤(如组织工程和重建、糖尿病、帕金森病、白血病、充血性心力衰竭等)的细胞来源具有很强的潜力。成功地将hESC整合到这类疗法将取决于三个关键步骤：无分化的扩增(即自我更新)、分化为特定的细胞类型或细胞类型的集合，以及促进其存活和与现有组织的功能整合。然而，在这些步骤中的每个步骤中控制细胞行为将需要对细胞微环境进行精确控制。这在目前的hESC培养系统中构成了一个主要的挑战，从与饲养层细胞的共培养到细胞在复杂的细胞外基质蛋白上培养的无血清系统。所有这些系统都涉及动物或人类蛋白质，这会给病原体传播、免疫排斥、重复性有限以及扩大到临床过程带来问题。为了达到再生医学的预期目标，在体外和体内精确控制干细胞群体的存活、增殖和分化的方法是必要的。在这里，我们建议建立一个完全合成的环境来精确控制hESC在培养中的自我更新。具体地说，我们将设计一个可调节的、定义明确的环境，提供一个完全“合成细胞外基质”(ECM)和化学定义的介质来控制hESCs的自我更新/扩张。此外，我们还将 开发高通量方法来鉴定合成多肽配体，用于合成ECM的功能化和促进hESC的自我更新。如果hESCs能够在这种完全合成的微环境中获得和维持，那么就有可能消除与鼠或人饲养层相关的病原体传播，为hESCs的大规模生产提供可扩展的基础，并为进一步开发控制HES细胞分化的研究提供精确的基础。此外，其结果将是一个技术平台，可以普遍应用于众多干细胞群体，并用于研究自我更新的基本生物学/发育机制。公共卫生相关性：新型生物活性材料的开发在对细胞功能进行精确控制方面具有巨大潜力，无论是在基础生物学研究方面，还是在组织工程和再生医学中的应用方面。例如，开发合成的生物活性材料系统来促进人类胚胎干细胞的自我更新和扩张将有许多生物医学应用，包括设计治疗肌肉、骨骼、脑、心脏、肝脏、胰腺和其他组织的疾病或损伤的方法。这项提案中描述的干细胞生物学、材料科学、分子生物学和生物工程的新组合将非常适合于解决一个重要的问题，即在生物学、工程学和医学的交界处的干细胞控制。

Description (provided by applicant): Human embryonic stem cells (hESCs) have strong potential as sources of cells for the treatment for disease and injury (e.g. tissue engineering and reconstruction, diabetes, Parkinson's Disease, leukemia, congestive heart failure, etc.). The successful integration of hESC into such therapies will hinge upon three critical steps: their expansion without differentiation (i.e., self-renewal), their differentiation into a specific cell type or collection of cell types, and the promotion of their survival and functional integration into existing tissue. However, controlling cell behavior during each of these steps will require precise control over the cellular microenvironment. This poses a major challenge ex vivo in current hESC culture systems, which range from co-culture with feeder cells to serum-free systems where cells are cultured on complex extracellular matrix proteins. All such systems involve animal or human proteins, which pose problems for pathogen transmission, immune rejection, limited reproducibility, and scale up to a clinical process. To achieve the intended goals of regenerative medicine, methods for the precise control of the survival, proliferation, and differentiation of stem cell populations in vitro and in vivo are necessary. Here, we propose to develop a completely synthetic environment to precisely control hESC self-renewal in culture. Specifically, we will engineer a tunable and well-defined environment presenting a completely "synthetic extracellular matrix" (ECM) and chemically-defined media to control the self-renewal/expansion of hESCs. Furthermore, we will develop high throughput approaches to identify synthetic peptide ligands for functionalization to the synthetic ECM and promotion of hESC self-renewal. If hESCs can be derived and maintained within this fully synthetic microenvironment, then it will be possible to eliminate pathogen transmission associated with mouse or human feeder layers, provide a scalable basis for large-scale production of hESCs, and provide a precise base for further development to control hES cell differentiation. Furthermore, the result will be a technology platform that can be generally applied to numerous stem cell populations and used to investigate the basic biological/developmental mechanisms underlying self-renewal. Public Health Relevance: The development of novel, bioactive materials has significant potential for exerting precise control over cell function, both for fundamental biological studies and applications in tissue engineering and regenerative medicine. For example, developing synthetic, bioactive material systems to promote the self-renewal and expansion of human embryonic stem cells will have numerous biomedical applications including the design of therapies for disease or injury in the muscle, bone, brain, heart, liver, pancreas, and other tissues. The novel blend of stem cell biology, materials science, molecular biology, and bioengineering described in this proposal will be well suited to addressing an important problem, i.e. stem cell control, at the interface of biology, engineering, and medicine