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.

人类诱导多能干细胞 (hiPSC) 代表了潜在的无限功能来源心肌细胞 (hiPSC-CM) 用于疾病建模、药物开发和再生疗法。特别是，在微生理学（“芯片上器官”）系统中使用 hiPSC-CM 衍生的微组织是有效的有望成为药物研究的未来支柱和提高我们对药物的理解的平台然而，人类心脏单基因和多基因疾病中的基因型-表型关系。 hiPSC-CM 在这些应用中广泛使用的障碍是其不成熟的特性，包括小细胞大小、T 管缺乏、主要是糖酵解代谢以及功能输出减少等等。出生后 CM 成熟的一个重要方面——倍性增加——尚未得到充分研究。体外培养的 hiPSC-CM 主要是单核和二倍体，而成人心肌由约 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，特别是探索 hiPSC-CM 的工程化多倍体是否可以赋予人类工程化与由以下材料制成的对照组织相比，心脏组织（hECT）具有更高的功能和成熟度我们的初步结果表明，稳定的 hiPSC-CM 多倍体是通过遗传或诱导产生的。药理学结果增加 hiPSC-CM 的大小和线粒体密度，以及收缩性 hECT 的强度和传导速度在拟议的研究中，我们将进一步研究 CM 的作用。 hECT 的结构、功能和代谢成熟中的多倍化，并研究多倍体诱导的 hiPSC-CM 中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。动态控制对 hECT 施加的机械预载和后载，我们将研究这些关系我们还将确定机械负荷的发育模拟机制和 CM 多倍体之间的关系。如果多倍体使 hiPSC-CM 在体外对肥大刺激敏感并保护 hECT 免受氧化应激最后，我们将应用 CRISPR/Cas9 筛选方法来识别。已是多倍体 hiPSC-CM 中最终成熟的遗传诱导剂，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。通过成功完成这些研究，我们期望提高我们对生理作用的理解心脏发育中的多倍体，并为未来工程化应用奠定基础疾病建模、药物开发和心脏治疗中的心脏组织。