A Multi-Omics Approach to Discover Metabolic Critical Quality Attributes for Cardiomyocyte Biomanufacturing

项目摘要 人诱导多能干细胞（iPSC）提供了一个有前途的来源，患者特异性心脏细胞。我们 研究小组率先开发了将iPSC分化为心肌细胞（iPSC-CM）的方案，但这些方案 细胞缺乏成熟的成人样表型。CM成熟的一个标志是从糖酵解转变为糖酵解， 葡萄糖氧化为脂肪酸氧化作为主要代谢途径，以及其他代谢变化。 我们的前提是，确定成熟度的代谢关键质量属性（CQA）将提供基本的 深入了解iPSC-CM的表型成熟，促进发现有效成熟策略的新工具 iPSC-CM，以及在iPSC-CM生物制造过程中监测成熟状态的新技术。实现 在此前提下，我们将采用综合定量代谢组学和蛋白质组学方法来分析 代谢物和代谢酶浓度以及代谢途径利用，在iPSC-CM中进行 通过延长培养时间或生物化学/生物力学刺激使其成熟。比较代谢 在体外iPSC-CM成熟过程中向体内发育过程中的代谢转变以及 成熟表型的获得将允许我们绘制发育过程的代谢转变。我们 将进行多变量数据分析，以预测成熟表型的代谢CQA，并建立新的工具 在iPSC-CM生物制造过程中监测这些CQA。因此，拟议的研究将提供基本的 对iPSC-CM成熟和心脏发育过程中代谢途径利用的新见解， 预测代谢CQA，这将有助于监测iPSC-CM的成熟进程， 生物制造我们的具体目标是： 1.描述iPSC-CM分化和成熟期间的代谢转变。在不同的iPSC-CM 由扩展培养、微图案化基质、碳源可用性诱导的成熟阶段， 机电刺激，我们将通过代谢组学量化代谢物和蛋白质浓度， 蛋白质组学和靶向代谢分析。我们将通过分子和功能测定评估成熟 将代谢物和代谢途径利用的变化与成熟表型的获得相关联。 2.评估发育中小鼠心肌细胞的代谢途径富集。我们将使用 代谢组学和蛋白质组学，以分析不同浓度下小鼠CM中的代谢物和蛋白质浓度 在一些实施例中，iPSC-CM在发育阶段中的代谢转变与iPSC-CM成熟期间发生的代谢转变进行比较。 3.确定代谢CQA并开发评估iPSC-CM成熟度的工具， 生物制造我们将使用多变量分析来预测识别成熟状态的代谢CQA iPSC-CM然后，我们将开发检测方法来监测这些CQA（代谢产物和代谢产物的组合）， 通过靶向代谢组学/蛋白质组学以及通过使用CRISPR-Cas9 基因编辑以工程化标记iPSC-CM成熟状态的代谢途径转变的报告物。

PROJECT SUMMARY Human induced pluripotent stem cells (iPSCs) provide a promising source of patient-specific cardiac cells. Our team has pioneered development of protocols to differentiate iPSCs to cardiomyocytes (iPSC-CMs) but these cells lack mature, adult-like phenotypes. One hallmark of CM maturation is a transition from glycolysis and glucose oxidation to fatty acid oxidation as the dominant metabolic pathway, among other metabolic changes. Our premise is that identifying metabolic critical quality attributes (CQAs) of maturity will provide fundamental insight into phenotypic maturation of iPSC-CMs, new tools to facilitate discovery of effective strategies to mature iPSC-CMs, and novel technologies to monitor maturation state during iPSC-CM biomanufacturing. To achieve this premise, we will employ an integrative quantitative metabolomics and proteomics approach to profile metabolite and metabolic enzyme concentrations, and metabolic pathway utilization, in iPSC-CMs undergoing maturation by extended time in culture or biochemical/biomechanical stimulation. Comparing metabolic transitions during in vitro iPSC-CM maturation to metabolic transitions during development in vivo and to acquisition of maturation phenotypes will allow us to map metabolic transitions to developmental processes. We will perform multivariate data analyses to predict metabolic CQAs of maturation phenotypes and build novel tools to monitor these CQAs during iPSC-CM biomanufacturing. Thus, the proposed study will provide fundamental new insight into metabolic pathway utilization during iPSC-CM maturation and cardiac development, and will predict metabolic CQAs that will facilitate monitoring progression of maturation in iPSC-CMs during biomanufacturing. Our specific aims are: 1. Profile metabolic transitions during iPSC-CM differentiation and maturation. At different iPSC-CM maturation stages induced by extended culture, micropatterned substrates, carbon source availability, and electromechanical stimulation, we will quantify metabolite and protein concentrations via metabolomics and proteomics and targeted metabolic assays. We will assess maturation via molecular and functional assays to relate changes in metabolites and metabolic pathway utilization to acquisition of maturation phenotypes. 2. Assess metabolic pathway enrichment in developing murine cardiomyocytes. We will use metabolomics and proteomics to profile metabolite and protein concentrations in murine CMs at different developmental stages and compare to metabolic transitions that occur during iPSC-CM maturation. 3. Identify metabolic CQAs and develop tools for assessment of iPSC-CM maturity during biomanufacturing. We will use multivariate analysis to predict metabolic CQAs that identify maturation state of iPSC-CMs. We will then develop assays to monitor these CQAs (combinations of metabolite and metabolic enzymes) during biomanufacturing via targeted metabolomics/proteomics, and by use of CRISPR-Cas9 gene editing to engineer reporters of metabolic pathway transitions that mark iPSC-CM maturation states.