UNS:Role of Cell-Mediated ECM Remodeling in Pluripotent Stem Cell Differentiation

1508950 Palecek，Sean P.阻碍人类多能干细胞(HPSCs)前景实现的一个重要障碍是无法从hPSCs高效地生物制造正常细胞，这在很大程度上是由于对hPSCs如何决定如何分化为更专业的细胞类型的不完全理解。这些细胞命运的决定受到细胞环境中存在的化学和机械信号的影响。该项目将研究细胞外基质(ECM)环境的细胞重塑如何影响向各种实验谱系的分化，包括心脏细胞、血管细胞、脑细胞和皮肤细胞。这种对干细胞分化机制的深入理解将有助于设计出更有效、更明确的方案，将干细胞引导到所需谱系的细胞。因此，该项目的结果将直接影响到在实验室规模或在生物制造应用中生产干细胞来源的细胞和组织。该项目将检验hPSC在分化过程中细胞外基质重塑是干细胞分化命运的关键调节因素的假设。该项目将确定通过ECM重塑调节hPSC命运的新的自分泌/旁分泌机制，并展示在hPSC分化过程中设计增强或抑制ECM重塑的材料和支架的重要性。为了验证这一假设，蛋白质组学分析将用于鉴定hPSC定向分化为心肌细胞、内皮细胞、神经元和角质形成细胞过程中产生的ECM成分。这些实验将确定候选细胞合成的ECM蛋白，这些蛋白调控特定的hPSC分化命运。为了验证ECM合成是有效定向分化所必需的预测，将采用CRISPR/Cas9介导的ECM基因敲除实验来评估抑制基质产生如何影响hPSC向心肌细胞、血管内皮细胞、神经元和角质形成细胞的分化。最后，为了验证通过ECM的蛋白降解而导致的基质重塑影响hPSC分化效率的假设，将在定向分化过程中监测基质金属蛋白酶的表达和活性，然后使用化学抑制剂和基因敲除来量化这些蛋白水解酶在定向分化过程中的作用。该计划中描述的教育和推广活动还将培训研究生和本科水平的干细胞工程师，并将向K-12学生、K-12教师、本科生和普通公众提供干细胞科学和工程的技术、伦理和政治方面的推广。该奖项由CBET生物技术和生化工程计划获得，由材料研究部生物材料计划共同资助。

1508950Palecek, Sean P. A significant roadblock impeding realization of the promise of human pluripotent stem cells (hPSCs) is the inability to efficiently biomanufacture normal cells from hPSCs, in a large part resulting from an incomplete understanding of how hPSCs decide how to differentiate to more specialized cell types. These cell fate decisions are influenced by chemical and mechanical signals present in the cell environment. This project will investigate how cell remodeling of their extracellular matrix (ECM) environment affects differentiation to various testbed lineages including heart cells, blood vessel cells, brain cells, and skin cells. This deeper understanding of mechanisms of stem cell differentiation will then inform design of more efficient, defined protocols for guiding stem cells to cells in desired lineages. Thus, the project outcomes will have direct implications on producing stem cell-derived cells and tissues at lab scale or in biomanufacturing applications.This project will test the hypothesis that extracellular matrix remodeling by hPSCs during differentiation is a key regulator of stem cell differentiation fate. The project will identify novel autocrine/paracrine mechanisms of regulation of hPSC fate via ECM remodeling, and demonstrate the importance of designing materials and scaffolds that enhance or inhibit ECM remodeling during hPSC differentiation. To test this hypothesis, proteomic analysis will be used to identify ECM components produced during hPSC directed differentiation to cardiomyocytes, endothelial cells, neurons, and keratinocytes. These experiments will identify candidate cell-synthesized ECM proteins that regulate specific hPSC differentiation fates. To test the prediction that ECM synthesis is necessary for efficient directed differentiation, CRISPR/Cas9-mediated ECM gene knockout experiments will be employed to assess how inhibiting matrix production affects hPSC differentiation to cardiomyocytes, vascular endothelial cells, neurons, and keratinocytes. Finally, to test the hypothesis that matrix remodeling via proteolytic degradation of ECM affects hPSC differentiation efficiency, matrix metalloprotease expression and activity will be monitored during directed differentiation, then chemical inhibitors and genetic knockouts will be used to quantify the contributions of these proteases during directed differentiation. Education and outreach activities described in this proposal will also train stem cell engineers at the graduate and undergraduate levels, and will provide outreach to K-12 students, K-12 teachers, undergraduate students, and the general public on technical, ethical, and political aspects of stem cell science and engineering.This award by the Biotechnology and Biochemical Engineering Program of CBET is co-funded by the Biomaterials Program of the Division of Materials Research.