Optimization of the Chemical-Physical Environments for Stem Cell Differentiation

描述（由申请人提供）：多能性人胚胎干细胞（hESCs）可以在体外分化为多种细胞类型，包括心血管细胞（心肌细胞、血管内皮细胞和平滑肌细胞）。体外控制干细胞生长和分化的能力对于成功应用分化细胞进行细胞基础治疗至关重要。适当的细胞类型致力于一个理想的谱系，连同心血管系统的相关元素，可用于治疗心血管疾病。本研究采用一种由化学-物理综合微环境组成的新型中-高通量微阵列平台来确定控制hESC命运的最佳条件。这些包括(1)固定化生长因子（GFs）和细胞外基质蛋白（ECMPs）， (2) ECM的机械特性，以及(3)作用于细胞的外部机械力。目前的知识表明，这些参数（即GFs、ECMPs、底物刚度以及机械载荷）中的每一个都在调节干细胞命运中发挥作用，但单独作用时效率很低。由于干细胞在发育过程中受到多种微环境因素的影响，因此有必要研究当前研究中提出的生态位多因子复杂性的组合效应。在本研究中，我们将研究ECM蛋白（ECMPs）和GFs对hESCs（联邦批准的WA01和WA09细胞系）分化的组合作用。我们设计了一种水凝胶系统来控制基质的硬度，覆盖了不同组织中遇到的范围，用于干细胞培养中的ECMP阵列。我们还将把我们实验室开发的流动和拉伸装置整合到微阵列系统中，以评估外部机械力在调节ECMP/GF/刚度平台上hESCs细胞命运中的作用。这种新型的组合微阵列系统允许对化学-物理微环境进行全面测试，以选择hESC生长和分化的最佳条件。将这些经过优化选择的hESCs转化为心血管组织工程，将为人工血管、血管生成贴片和心力衰竭细胞替代的发展提供机会，这将反过来改善心血管疾病患者的医疗保健和我们公民的福祉。

DESCRIPTION (provided by applicant): Pluripotent human embryonic stem cells (hESCs) can be differentiated in vitro into multiple cell types, including cardiovascular cells (cardiomyocytes, vascular endothelial and smooth muscle cells). The ability to control the growth and differentiation of stem cells in vitro is essential for the successful application of differentiated cells for cell-based therapies. The appropriate cell types committed to a desired lineage, together with the relevant elements of the cardiovascular system, can be used to treat cardiovascular diseases. The proposed research on the identification of the optimal condition for controlling hESC fate uses a novel medium-high throughput microarray platform composed of comprehensive chemical-physical microenvironments. These include (1) immobilized growth factors (GFs) and extracellular matrix proteins (ECMPs), (2) mechanical properties of the ECM, and (3) external mechanical forces acting on the cells. Current knowledge indicates that each of these parameters (i.e., GFs, ECMPs, substrate rigidities, as well as mechanical loadings) plays roles in regulating stem cell fate, but the efficiency is low when acting alone. Since stem cells experience the influence of multiple microenvironmental factors which change during the developmental stages, it is essential to examine the combinatory effects of multi-factorial complexities of the niches, as proposed in the current research. In the proposed research, we will investigate the combinatorial effects of ECM proteins (ECMPs) and GFs on the differentiation of hESCs (Federally approved WA01 and WA09 cell lines). We have designed a hydrogel system to control the rigidities of matrices, covering a range encountered in different tissues, for our ECMP array in stem cell culture. We will also incorporate the flow and stretch devices developed in our lab into the microarray system to assess the roles of external mechanical forces in regulating the cell fate of hESCs on the ECMP/GF/Rigidity platform. This novel combinatory microarray system allows the comprehensive testing of the chemical-physical microenvironment for the choice of optimal conditions for hESC growth and differentiation. The use of such optimally chosen hESCs for translation to cardiovascular tissue engineering will provide the opportunity to significantly advance the development of artificial vessels, angiogenesis patches, as well as cell replacement for heart failure, which will in turn improve the healthcare of patients with cardiovascular diseases and the wellbeing of our citizens. PUBLIC HEALTH RELEVANCE: Pluripotent human embryonic stem cells can be differentiated into multiple cell types, including cells in the cardiovascular systems. The appropriate cell types with the associated networks of vascular cells can be used to treat heart, lung, and blood diseases. This project is aiming at developing a novel systematic approach to understand, define, and ultimately control the process of stem cell differentiation, with the ultimate goal of developing tools of regenerative medicine to treat cardiovascular diseases.