Optimization of the Chemical-Physical Environments for Stem Cell Differentiation

描述(申请人提供)：多能人类胚胎干细胞(HESCs)在体外可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)。在体外控制干细胞的生长和分化的能力是成功将分化细胞应用于基于细胞的治疗的关键。致力于所需谱系的适当细胞类型，连同心血管系统的相关元素，可用于治疗心血管疾病。本研究利用一种新颖的中高通量微阵列平台，由综合的化学-物理微环境组成，用于确定控制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(ECMP)，(2)ECM的机械性能，以及(3)作用于细胞的外部机械力。目前的知识表明，这些参数中的每一个(即GFS、ECMPs、底物刚性以及机械载荷)都在调节干细胞命运方面发挥作用，但单独作用时效率较低。由于干细胞在发育过程中会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多因素复杂的生态位组合效应。在拟议的研究中，我们将研究ECM蛋白(ECMPs)和GFS对hESCs(联邦批准的WA01和WA09细胞株)分化的联合作用。我们设计了一种水凝胶系统来控制基质的刚性，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设备整合到微阵列系统中，以评估外部机械力在调节ECMP/GF/刚性平台上的hESCs细胞命运中的作用。这种新颖的组合微阵列系统允许对化学-物理微环境进行全面测试，以选择适合hESC生长和分化的最佳条件。使用这种最佳选择的人类胚胎干细胞转化为心血管组织工程将提供机会，显著推进人造血管、血管生成贴片以及心力衰竭细胞替代的开发，这将反过来改善心血管疾病患者的医疗保健和我国公民的福祉。 公共卫生相关性：多能人类胚胎干细胞可以分化为多种细胞类型，包括心血管系统中的细胞。合适的细胞类型和相关的血管细胞网络可用于治疗心脏、肺和血液疾病。该项目旨在开发一种新的系统方法来了解、定义并最终控制干细胞分化过程，最终目标是开发用于治疗心血管疾病的再生医学工具。

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.