Improving cardiovascular disease modeling using human pluripotent stem cell-derived cardiac fibroblasts

使用人类多能干细胞来源的心脏成纤维细胞改善心血管疾病模型

• 批准号: 10462472
• 负责人: Charles Matthew Kerr
• 金额: $ 4.6万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462472
• 关键词: 3-Dimensional; Address; Adipose tissue; Adrenergic Agents; Adult; Animal Model; Biological Models; Cardiac; Cardiac Myocytes; Cardiovascular Diseases; Cause of Death; Cells; Chronic; Data; Deposition; Development; Diagnosis; Diffusion; Disease model; Effectiveness; Endocardium; Endothelial Cells; Epicardium; Extracellular Matrix; Fibroblasts; Genes; Genetic; Genetic Transcription; Goals; Heart; Heart failure; Heterogeneity; Human; In Vitro; Infarction; Inflammatory; Injury; Lead; Metabolic; Metabolism; Modeling; Morphology; Mus; Myocardial Infarction; Myocardium; Organoids; Oxygen; Patients; Population; Protein Secretion; Protocols documentation; RNA analysis; Role; Source; Stromal Cells; Structure; System; Therapeutic; Time; Umbilical vein; United States; Universities; Ventricular; Wisconsin; coronary fibrosis; cytokine; drug testing; effectiveness evaluation; fetal; high risk; human RNA sequencing; human model; human pluripotent stem cell; improved; in vivo; innovation; mortality risk; mouse model; progenitor; regenerative therapy; response; self assembly; single-cell RNA sequencing; stem cell differentiation; stem cells; transcriptome sequencing; transcriptomics

Project Summary: Annually, there are ~790,000 cases of myocardial infarction (MI) in the United States. Typically, MI progresses into heart failure where patients have a high risk of mortality within 5 years after diagnosis. While animal models provide a valuable model system of MI, interspecies differences lead to inaccurate recapitulation of human myocardium. To address this, our lab originally developed 3D human cardiac organoids through self-assembly of hPSC-CMs, human primary adult cardiac fibroblasts (adult-cFbs), endothelial cells, and stromal cells. Further, we leveraged the oxygen diffusion limitation in 3D human cardiac organoids along with chronic adrenergic stimulation to generate an organotypic model of post-MI hearts. The human cardiac infarct organoids recapitulated transcriptional, structural and functional hallmarks of post-MI myocardium. However, the use of primary, non-myocyte cell populations in our current organoids limit their potential to mimic patient-specific myocardium. To develop human isogenic cardiac organoids, we are collaborating with Dr. Sean Palecek at the University of Wisconsin-Madison to derive cardiac fibroblasts from human pluripotent stem cells (hPSC) to replace adult-cFbs in our cardiac organoid model. Dr. Palecek’s lab has developed expertise to direct hPSC differentiation into cardiac fibroblasts (hPSC-cFbs) in 2 different lineages: epicardial-derived fibroblasts (hPSC-cFb(EpiC)s) and second heart field progenitor-derived fibroblasts (hPSC- cFb(SHFP)s). While both lineages contribute to cardiac fibrosis and are functionally similar, in murine hearts, the epicardium is the predominate source of ventricular cardiac fibroblasts while a small population arise from the endocardium. In addition, the enhanced maturation may be needed for the hPSC-cFb(EpiC)s to replace human adult-cFbs, as our preliminary data that showed that prolonged culture improved cell organization of hPSC-cFb(SHFP)s in cardiac organoids when compared to that of adult-cFb organoids. The central hypothesize of this proposal is that high passage hPSC-cFb(EpiC)s will best replicate adult-cFb transcriptomics and functionality. The proposal is innovative in that, for the first time, we will identify a suitable hPSC-cFb population to replace adult-cFbs to develop an isogenic 3D organotypic model of human myocardium. Our long-term goal is to develop patient-specific cardiac organoids for in vitro disease modeling and drug testing. Accordingly, we will pursue the following two Aims: 1) Determine the effectiveness of high passage hPSC- cFb(EpiC)s to replicate the transcriptomics and functionality of adult cFbs, and 2) Determine the effectiveness of human cardiac organoids composed of high passage hPSC-cFb(EpiC)s in modeling post-MI human myocardium and responsiveness to anti-MI therapeutics. We also will perform single cell RNA-seq to examine the heterogeneity of hPSC-cFb(EpiC)s in response to our infarction protocol. Completion of this study would provide the first step towards an isogenic human myocardium model. The single cell RNA-seq studies will reveal the various roles/subpopulations of cardiac fibroblasts in post-MI human myocardium.

项目概述：美国每年约有790，000例心肌梗死（MI）病例。 通常，MI进展为心力衰竭，其中患者在MI后5年内具有高死亡风险。 诊断.虽然动物模型提供了MI的有价值的模型系统，但种间差异导致 对人体心肌的不准确再现。为了解决这个问题，我们的实验室最初开发了3D人体 通过hPSC-CM，人原代成体心脏成纤维细胞（adult-cFbs）， 内皮细胞和基质细胞。此外，我们利用3D人体心脏中的氧扩散限制， 类器官沿着慢性肾上腺素能刺激以产生MI后心脏的器官型模型。的 人类心肌梗死类器官再现了MI后转录、结构和功能标志 心肌然而，在我们目前的类器官中使用原代非肌细胞细胞群限制了它们的功能。 模拟患者特异性心肌的潜力。为了开发人类同基因心脏类器官，我们 与威斯康星大学麦迪逊分校的Sean Palecek博士合作，从 人多能干细胞（hPSC）替代我们的心脏类器官模型中的成体cFbs。帕莱切克博士的实验室已经 开发了指导hPSC分化为2种不同谱系的心脏成纤维细胞（hPSC-cFbs）的专业知识： 心外膜来源的成纤维细胞（hPSC-cFb（EpiC）s）和第二心脏区域祖细胞来源的成纤维细胞（hPSC-cFb（EpiC）s）。 cFb（SHFP）s）。虽然这两种谱系都有助于心脏纤维化并且功能相似，但在小鼠心脏中， 心外膜是心室心肌成纤维细胞的主要来源， 从子宫内膜。此外，hPSC-cFb（EpiC）可能需要增强的成熟， 我们的初步数据显示，长期培养改善了细胞组织， hPSC-cFb（SHFP）在心脏类器官中的表达与成人-cFb类器官相比。中央 这一建议假设是高传代hPSC-cFb（EpiC）将最好地复制成人cFb转录组 和功能性。该提案具有创新性，因为我们将首次确定合适的hPSC-cFb 群体来替代成人cFbs以开发人心肌的同基因3D器官型模型。我们 长期目标是开发用于体外疾病建模和药物测试的患者特异性心脏类器官。 因此，我们将追求以下两个目标：1）确定高传代hPSC的有效性- c Fb（EpiC）s复制成人cFb的转录组学和功能性，以及2）确定其有效性 由高传代hPSC-cFb（EpiC）组成的人心脏类器官在MI后人模型中的表达 心肌和对抗MI治疗剂的反应性。我们还将进行单细胞RNA-seq， hPSC-cFb（EpiC）对我们的梗塞方案的响应的异质性。完成这项研究将 提供了迈向同基因人类心肌模型的第一步。单细胞RNA-seq研究将揭示 心肌梗死后人心肌中心脏成纤维细胞的各种作用/亚群。