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.

人诱导的多能干细胞（HIPSC）代表了潜在的无限功能来源用于疾病建模，药物开发和再生疗法的心肌细胞（HIPSC-CMS）。特别是，在微生物生理（“芯片”）系统中使用HIPSC-CM衍生的微作用承诺是药物研究的未来支柱，也是提高我们对理解的平台人心的单型和多基因疾病中的基因型 - 表型关系。但是，一个专业在这些应用中广泛使用HIPSC-CM的障碍是其不成熟的特性，包括小细胞大小，缺乏T管，主要是糖酵解代谢，并降低功能输出，仅举几例。产后CM成熟的一个重要方面 - 增加了倍性 - 已在很大程度上被理解。即，体外培养的HIPSC-CM主要是单核和二倍体，而成年人类心肌由〜90％的多倍体CM组成。因此，我们建议研究多倍体在HIPSC-CM中的潜在作用成熟，特别是要探索HIPSC-CM的设计多倍体是否可以赋予人类工程与由对照组织相比主要二倍体CM。我们的初步结果表明，稳定的HIPSC-CM多倍体诱导的遗传或从药理上导致HIPSC-CMS的大小和线粒体密度增加以及收缩高强度和传导速度。在拟议的研究中，我们将进一步研究广告的作用参亚的结构，功能和代谢成熟中的多倍体化，并研究多倍体诱导的 HIPSC-CMS的转录组和表观遗传变化。此外，使用具有能力的新型生物反应器动态控制的应用机械预加载和后负载到hects，我们将研究关系在机械载荷的发育模拟状态和商业多倍体之间。我们还将确定如果多倍体在体外感觉到HIPSC-CMS对肥厚的刺激，并保护Hect免受氧化应激体内体外和缺血损伤。最后，我们将应用CRISPR/CAS9筛选方法来识别末端成熟的遗传诱导剂在已经多倍的HIPSC-CMS中，将执行其他筛选在二倍体和多倍体HIPSC-CM中，都可以鉴定可以促进CM细胞周期活性的候选有丝分裂剂。通过成功完成这些研究，我们希望提高我们对身体角色的理解心脏发展中的多倍体，并为未来的翻译用途奠定了基础疾病建模，药物发育和心脏疗法中的心脏组织。