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

发现心肌细胞生物制造代谢关键质量属性的多组学方法

基本信息

  • 批准号:
    10435467
  • 负责人:
  • 金额:
    $ 37.54万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2019
  • 资助国家:
    美国
  • 起止时间:
    2019-07-01 至 2024-06-30
  • 项目状态:
    已结题

项目摘要

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.
项目总结 人诱导多能干细胞(IPSCs)提供了一种有希望的患者特异性心肌细胞来源。我们的 该团队率先开发了区分IPSC和心肌细胞的方案(IPSC-CMS),但这些 细胞缺乏成熟的成体样表型。CM成熟的一个标志是从糖酵解和 在其他代谢变化中,葡萄糖氧化为脂肪酸氧化是主要的代谢途径。 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 洞察IPSC-CMS的表型成熟,这是促进发现有效成熟策略的新工具 IPSC-CMS,以及在IPSC-CM生物制造过程中监控成熟状态的新技术。要实现 在这一前提下,我们将采用定量代谢组学和蛋白质组学相结合的方法来描述 IPSC-CMS代谢产物和代谢酶浓度及代谢途径利用 通过延长培养时间或生物化学/生物力学刺激成熟。比较新陈代谢 IPSC-CM体外成熟到体内发育过程中的代谢转变 成熟表型的获得将使我们能够将代谢过渡映射到发育过程。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生物制造过程中监控这些CQA。因此,拟议的研究将为 对IPSC-CM成熟和心脏发育过程中代谢途径利用的新见解,并将 预测有助于监测IPSC-CMS成熟进程的代谢CQA 生物制造。我们的具体目标是: 1.研究IPSC-CM分化成熟过程中的代谢变化。在不同的IPSC-CM 延长培养、微图案化底物、碳源可利用性以及 机电刺激,我们将量化代谢物和蛋白质浓度通过代谢组学和 蛋白质组学和靶向代谢分析。我们将通过分子和功能分析来评估成熟度。 将代谢产物和代谢途径利用的变化与成熟表型的获得联系起来。 2.评价发育过程中小鼠心肌细胞代谢途径的丰富。我们将使用 代谢组学和蛋白质组学分析小鼠CMS在不同时间的代谢物和蛋白质浓度 并与IPSC-CM成熟期发生的代谢转变进行比较。 3.确定新陈代谢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确定成熟状态的代谢CQA IPSC-CMS。然后,我们将开发检测方法来监测这些CQA(代谢物和代谢物的组合 酶)通过靶向代谢组学/蛋白质组学和使用CRISPR-Cas9进行生物制造 基因编辑以工程记者的代谢途径转变,标志着IPSC-CM成熟状态。

项目成果

期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Advances in Manufacturing Cardiomyocytes from Human Pluripotent Stem Cells.
  • DOI:
    10.1146/annurev-chembioeng-092120-033922
  • 发表时间:
    2022-06-10
  • 期刊:
  • 影响因子:
    8.4
  • 作者:
    Floy, Martha E.;Shabnam, Fathima;Simmons, Aaron D.;Bhute, Vijesh J.;Jin, Gyuhyung;Friedrich, Will A.;Steinberg, Alexandra B.;Palecek, Sean P.
  • 通讯作者:
    Palecek, Sean P.
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Sean P Palecek其他文献

Sean P Palecek的其他文献

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{{ truncateString('Sean P Palecek', 18)}}的其他基金

Label-free single-cell imaging for quality control of cardiomyocyte biomanufacturing
用于心肌细胞生物制造质量控制的无标记单细胞成像
  • 批准号:
    10675976
  • 财政年份:
    2023
  • 资助金额:
    $ 37.54万
  • 项目类别:
Mechanisms of Shear Induction of Blood-Brain Barrier Phenotypes in Human iPSC-derived Brain Endothelial Progenitors
人 iPSC 来源的脑内皮祖细胞血脑屏障表型的剪切诱导机制
  • 批准号:
    10328223
  • 财政年份:
    2019
  • 资助金额:
    $ 37.54万
  • 项目类别:
Mechanisms of Shear Induction of Blood-Brain Barrier Phenotypes in Human iPSC-derived Brain Endothelial Progenitors
人 iPSC 来源的脑内皮祖细胞血脑屏障表型的剪切诱导机制
  • 批准号:
    10557176
  • 财政年份:
    2019
  • 资助金额:
    $ 37.54万
  • 项目类别:
A Multi-Omics Approach to Discover Metabolic Critical Quality Attributes for Cardiomyocyte Biomanufacturing
发现心肌细胞生物制造代谢关键质量属性的多组学方法
  • 批准号:
    10218267
  • 财政年份:
    2019
  • 资助金额:
    $ 37.54万
  • 项目类别:
Prevention of Candida biofilms by localized delivery of aurein analogues
通过局部递送金黄色素类似物预防念珠菌生物膜
  • 批准号:
    9221080
  • 财政年份:
    2016
  • 资助金额:
    $ 37.54万
  • 项目类别:
Prevention of Candida biofilms by localized delivery of aurein analogues
通过局部递送金黄色素类似物预防念珠菌生物膜
  • 批准号:
    9813824
  • 财政年份:
    2016
  • 资助金额:
    $ 37.54万
  • 项目类别:
Shear regulated differentiation of hPSCs to brain endothelial cells
hPSC 向脑内皮细胞的剪切调节分化
  • 批准号:
    8619338
  • 财政年份:
    2013
  • 资助金额:
    $ 37.54万
  • 项目类别:
Shear regulated differentiation of hPSCs to brain endothelial cells
hPSC 向脑内皮细胞的剪切调节分化
  • 批准号:
    8723321
  • 财政年份:
    2013
  • 资助金额:
    $ 37.54万
  • 项目类别:
Prevention of C. Albicans Biofilms by Beta-Peptide Release From Thin Films
通过薄膜释放 β 肽来预防白色念珠菌生物膜
  • 批准号:
    8681304
  • 财政年份:
    2011
  • 资助金额:
    $ 37.54万
  • 项目类别:
Prevention of C. Albicans Biofilms by Beta-Peptide Release From Thin Films
通过薄膜释放 β 肽来预防白色念珠菌生物膜
  • 批准号:
    8484784
  • 财政年份:
    2011
  • 资助金额:
    $ 37.54万
  • 项目类别:

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