Human Pluripotent Stem Cell Differentiation with Defined O2 & Protein Engagement

描述(由申请人提供)：本申请涉及广泛的挑战领域(14)干细胞和具体的挑战主题，14-DE-103：增强人类胚胎干细胞(ES)细胞培养系统。这项建议的总体目标是开发和优化一种无饲养层的体外培养系统，该系统具有可控的培养微环境，用于人类多潜能干细胞的生长和定向分化。特别是，高通量方法将用于开发具有优化表面特性的培养材料，其中包括定义的细胞外基质(ECM)成分，这些成分允许精确控制细胞表面(pO2细胞)的氧分压，以提供更多的生理条件，并允许更好地模拟正常发育过程。发育中的胚胎在体内暴露在非常低的氧气水平下，越来越多的证据表明，氧气浓度以及细胞与蛋白质的相互作用是分化和增殖的重要因素。在典型的培养体系中，由于培养液中的梯度，pO2细胞是未知的。目前，还没有商业上可用的方法或设备来在已知的低氧条件下培养细胞。硅橡胶薄膜具有非常高的透氧性，将被用作培养基质，使细胞暴露在与气相相同的氧气水平中。硅橡胶表面将以各种形式进行研究：(1)未经改性，(2)用合成聚合物进行化学改性和功能化，(3)用合成聚合物进行化学改性，其中ECM成分通过物理吸附或共价键连接到合成聚合物上，包括定义的蛋白质及其混合物。为了筛选大量的细胞-细胞外基质-生物材料相互作用，我们将利用我们之前开发的用于细胞培养的高通量微阵列平台，在功能化硅橡胶上创建细胞兼容的ECM蛋白质微阵列。不同的细胞底物将使用组合技术在pO2细胞的不同水平上检测细胞附着、增殖和基因表达模式。使用培养器皿进行大规模培养的验证性实验将与最有希望的组合进行。高通量微阵列平台和培养容器将进行类似的实验，将人类多能干细胞分化为心肌细胞，这已被证明受益于低氧培养，作为模型系统。这项研究的目标的完成将导致消除传统的饲养层和未知的异种蛋白，并开发和验证一个更明确的、更具生理学的培养平台，用于同时变异和研究可溶性因子、ECM-细胞相互作用和pO2细胞，从而使我们能够更好地理解这些因素在人类多能干细胞维持和分化中的作用。这一新颖的、普遍适用的平台将提升该领域的研究能力和质量。多能干细胞在药物筛选、遗传疾病的体外建模方面有着巨大的前景，细胞疗法和拟议的研究将对人类健康产生广泛影响。在最基本的层面上，这项研究将提供生物材料、细胞外基质成分和未分化或分化的人类多能干细胞在模拟生理条件的pO2细胞的不同已知值下的相互作用信息，此前除了常氧培养外，还没有关于这些细胞的数据。这些信息将促进我们在发育和干细胞生物学以及人类多能干细胞技术领域的知识。特别是，我们希望了解ECM蛋白的选择和pO2细胞水平如何相互作用，以帮助定向分化。从更广泛的角度来看，拟议的高通量平台和由此衍生的大规模培养容器是构成在特定生理条件下培养人类多潜能干细胞的使能技术的工具。他们将在培养人类多能干细胞的能力方面改善研究基础设施。所获得的知识和开发的工具可能会对人类健康许多领域的广泛应用产生深远的影响。这将加快药物筛选、遗传疾病体外模型、心脏病、糖尿病和帕金森等神经疾病等重大疾病的病细胞治疗性替换等应用领域的发展，这些疾病每年影响美国数百万人。事实上，将人类多能干细胞分化为心肌细胞，这一拟议的模型系统，本身对于生成用于缺血性心脏病后心脏组织修复的细胞和组织具有很高的兴趣。此外，待检测的培养血管将用于培养心脏组织。这项合作研究将整合生物工程、生物材料和发育生物学领域的最新进展，以设计一个新的培养平台和方案，促进基于细胞的疗法治疗多种人类疾病和其他ES细胞应用。 与公共卫生相关：该项目涉及使用人类干细胞的改进方法，以便它们能够发育成医学上有用的细胞和组织。例如，这可能导致培养出可用于治疗心脏病的功能性心肌细胞。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-DE-103: Enhancing Human Embryonic Stem (ES) Cell Culture Systems. The overall objective of this proposal is to develop and optimize a feeder-free in vitro culture system with a controlled culture microenvironment for human pluripotent stem cell growth and directed differentiation. In particular, high-throughput approaches will be used to develop culture materials with optimized surface properties incorporating defined extracellular matrix (ECM) components that allow for accurate control of oxygen partial pressure of the cell surface (pO2cell) to provide more physiological conditions and allow strategies to better mimic normal development processes. The developing embryo is exposed in vivo to very low oxygen levels, and there is growing evidence that oxygen concentration, together with cell-protein interactions, are important factors in differentiation and proliferation. In typical culture systems, pO2cell is unknown because of gradients in the culture medium. Currently, there are no commercially available methods or equipment for culturing cells under known, hypoxic conditions. Membranes of silicone rubber, which have very high oxygen permeability, will be used as a culture substrate so that cells are exposed to the same oxygen level as in the gas phase. Silicone rubber surfaces will be investigated in various forms: (1) unmodified, (2) chemically modified and functionalized with synthetic polymers, and (3) chemically modified with synthetic polymers to which ECM components are attached by physical adsorption or covalent linkage, including defined proteins and mixtures thereof. Cell compatible, ECM protein microarrays on functionalized silicone rubber will be created with a high-throughput microarray platform that we previously developed for culture of cells in order to screen a large number of cell-ECM-biomaterial interactions. The various cell substrates will be examined for cellular attachment, proliferation, and gene expression patterns at various levels of pO2cell using combinatorial techniques. Confirmatory experiments in macro-scale culture using culture vessels will be carried out with the most promising combinations. Similar experiments with the high-throughput microarray platform and culture vessels will be carried out for differentiation of human pluripotent stem cells to cardiomyocytes, which has been shown to benefit from hypoxic culture, as a model system. Accomplishment of the goal of the proposed studies will lead to elimination of conventional feeder layers and undefined xenogenic proteins and to the development and validation of a more well-defined and physiological culture platform for simultaneous variation and study of soluble factors, ECM-cell interactions, and pO2cell, thereby enabling enhancement of our understanding of the role of these factors in maintenance and differentiation of human pluripotent stem cells. This novel, generally applicable platform, will upgrade the capability and quantity of research in this field. Pluripotent stem cells hold enormous promise for drug screening, in vitro modeling of genetic disorders, and cell therapies and the proposed research will have wide impact on human health. At its most basic level, this research will provide information about the interaction of biomaterials, ECM components, and undifferentiated or differentiating human pluripotent stem cells at different known values of pO2cell that mimic physiological conditions, data for which has not previously been available except under normoxic culture. This information will advance our knowledge in the fields of developmental and stem cell biology as well as human pluripotent stem cell technology. In particular, we expect to learn how the choice of ECM proteins and pO2cell levels interact to aid directed differentiation. From a broader standpoint, the proposed high-throughput platform and macro-scale culture vessels that derive therefrom are tools that constitute enabling technology to culture human pluripotent stem cells under defined physiological conditions. They will improve the research infrastructure in terms of the capability for culturing human pluripotent stem cells. The knowledge acquired and tools developed may have profound effects on a wide variety of applications in many fields of human health. This will accelerate developments in applications such as drug screening, in vitro models of genetic disease, therapeutic replacement of diseased cells in major diseases such as heart disease, diabetes, and neural diseases such as Parkinson's, which annually affect millions of people in the United States. Indeed, differentiation of human pluripotent stem cells to cardiomyocytes, the proposed model system, is itself of high interest for generating cells and tissues for repair of cardiac tissues following ischemic heart disease. Furthermore, the culture vessels to be tested will be useful in culturing cardiac tissue. This collaborative study will integrate recent progress in the fields of bioengineering, biomaterials, and developmental biology to design a new culture platform and protocols that will facilitate cell-based therapies for treatment of a multitude of human diseases and other ES cell applications. PUBLIC HEALTH RELEVANCE: This project concerns improved methods to work with human stem cells so that they can develop into medically useful cells and tissues. For example, this can lead to ways to grow functioning heart muscle cells that can be used to treat heart disease.