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Engineering Human Heart Tissues with Polyploid Cardiomyocytes

用多倍体心肌细胞改造人类心脏组织

基本信息

批准号：
10616611
负责人：
Nenad Bursac
金额：
$ 54.46万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10616611
关键词：
3-Dimensional ATAC-seq Acceleration Adult Affect Biological Assay Biomechanics Biomedical Engineering Bioreactors CRISPR interference CRISPR screen CRISPR-mediated transcriptional activation Cardiac Cardiac Myocytes Cell Cycle Cell Line Cell Size Cell Therapy Characteristics Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Cytokinesis DNA Development Diploidy Disease model Dominant-Negative Mutation Dorsal EP300 gene Electrophysiology (science)Endowment Engineering Environment Epigenetic Process Exhibits Failure Fatty Acids Foundations Future Gene Expression Generations Genes Genetic Genotype Goals Heart Heart Diseases Hormonal Human Human Engineering In Vitro Insulin-Like Growth Factor I Ischemia Lentivirus Libraries Mechanics Metabolic Metabolism Methodology Microtubules Mitochondria Mitogens Mitosis Molecular Mononuclear Mus Myocardium Names Outcome Study Output Oxidative Stress Pharmacologic Substance Phenotype Physiological Ploidies Polyploidy Proliferating Property Proteins Regenerative Medicine Reperfusion Injury Reporting Repression Research Rodent Role Signal Transduction Source Stimulus Stress Structure System Testing Thyroid Hormones Tissue Engineering Tissues Workload aurora B kinase candidate identification cardiac tissue engineering cardiogenesis catalyst comparison control complex IV density drug development drug testing experience genetic inducer genome editing genome-wide human disease improved in vivo indexing induced pluripotent stem cell mechanical load mimetics novel organ on a chip overexpression pharmacologic postnatal postnatal development regenerative therapy response segregation stable cell line three dimensional cell culture tissue culture trait transcriptome sequencing transcriptomics

项目摘要

Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of functional cardiomyocytes (hiPSC-CMs) for use in disease modeling, drug development, and regenerative therapies. In particular, use of hiPSC-CM-derived microtissues in microphysiological (“organ-on-chip”) systems holds promise as the future mainstay of pharmaceutical research and a platform to improve our understanding of genotype-phenotype relationships in mono- and polygenic diseases of the human heart. However, a major obstacle to wide-spread use of hiPSC-CMs in these applications are their immature properties including small cell size, lack of T-tubules, predominantly glycolytic metabolism, and reduced functional output, to name a few. One important aspect of postnatal CM maturation - increased ploidy - has been largely understudied. Namely, in vitro cultured hiPSC-CMs are predominantly mononuclear and diploid, while the adult human myocardium consists of ~90% polyploid CMs. We thus propose to investigate potential roles of polyploidy in hiPSC-CM maturation, and specifically, to explore if engineered polyploidy of hiPSC-CMs can endow human engineered cardiac tissues (hECTs) with increased functionality and maturity compared to control tissues made from primarily diploid CMs. Our preliminary results show that stable hiPSC-CM polyploidy induced genetically or pharmacologically results in increased size and mitochondrial density of hiPSC-CMs, as well as contractile strength and conduction velocity of hECTs. In the proposed studies, we will further examine roles of CM polyploidization in structural, functional, and metabolic maturation of hECTs and investigate polyploidy-induced transcriptomic and epigenetic changes in hiPSC-CMs. Furthermore, using a novel bioreactor with capacity to dynamically control applied mechanical preload and afterload to hECTs, we will investigate the relationships between developmentally-mimetic regimes of mechanical loading and CM polyploidy. We will also determine if polyploidy sensitizes hiPSC-CMs to hypertrophic stimuli in vitro and protects hECTs from oxidative stress in vitro and ischemic damage in vivo. Finally, we will apply CRISPR/Cas9 screening methodologies to identify genetic inducers of terminal maturation in already polyploidy hiPSC-CMs and will perform additional screens in both diploid and polyploid hiPSC-CMs to identify candidate mitogens that can promote CM cell cycle activity. By successfully completing these studies, we expect to improve our understanding of physiological roles of polyploidy in cardiac development and to establish the foundation for the future translational uses of engineered cardiac tissues in disease modeling, drug development, and cardiac therapies.

人诱导多能干细胞（hiPSC）代表了一种潜在的无限的功能性干细胞来源。本发明提供了用于疾病建模、药物开发和再生疗法的心肌细胞（hiPSC-CM）。在特别地，hiPSC-CM衍生的微组织在微生理学（“器官芯片”）系统中的使用保持了作为未来药物研究的支柱和平台，以提高我们的理解，人类心脏单基因和多基因疾病的基因型-表型关系。然而一个主要在这些应用中广泛使用hiPSC-CM的障碍是它们不成熟的性质，细胞大小、缺乏T-小管、主要是糖酵解代谢和减少的功能输出，仅举几例。出生后CM成熟的一个重要方面-倍性增加-在很大程度上研究不足。也就是说，体外培养的hiPSC-CM主要是单核细胞和二倍体，而成人心肌细胞主要是单核细胞和二倍体。由约90%的多倍体CM组成。因此，我们建议研究hiPSC-CM中多倍体的潜在作用成熟，并且具体地，探索hiPSC-CM的工程化多倍性是否可以赋予人类工程化的心脏组织（hECTs），与对照组织相比，其功能和成熟度增加，主要是二倍体CM。我们的初步结果表明，稳定的hiPSC-CM多倍体诱导遗传或 HiPSC-CM的大小和线粒体密度增加， hECT的强度和传导速度。在建议的研究中，我们会进一步探讨中医的角色， hECTs的结构、功能和代谢成熟中的多倍化，并研究多倍化诱导的hECTs hiPSC-CM中的转录组学和表观遗传学变化。此外，使用具有以下能力的新型生物反应器，动态控制对hECT施加的机械预载荷和后载荷，我们将研究它们之间的关系。机械负荷和CM多倍性的发育模拟机制之间的关系。我们还将确定如果多倍体在体外使hiPSC-CM对肥大刺激物敏感，并在体外保护hECTs免受氧化应激，体外和体内缺血性损伤。最后，我们将应用CRISPR/Cas9筛选方法来识别在已经多倍化的hiPSC-CM中终末成熟的遗传诱导剂，并将进行额外的筛选在二倍体和多倍体hiPSC-CM中鉴定可促进CM细胞周期活性的候选有丝分裂原。通过成功地完成这些研究，我们希望提高我们的生理作用的理解，心脏发育中的多倍性，并为工程化的心脏组织在疾病建模、药物开发和心脏治疗中的应用。