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

项目概述：美国每年约有79万例心肌梗塞(MI)病例。 通常，心肌梗死进展为心力衰竭，患者在5年内死亡的风险很高。 诊断。虽然动物模型提供了一个有价值的MI模型系统，但物种间的差异导致 对人体心肌的不准确概括。为了解决这个问题，我们的实验室最初开发了3D人类 HPSC-CMS，人原代成人心脏成纤维细胞(成人CFbs)自组装的心脏有机体， 内皮细胞和间质细胞。此外，我们利用了3D人体心脏的氧气扩散限制。 有机化合物与慢性肾上腺素能刺激一起产生心肌梗死后心脏的器官模型。这个 人心肌梗死后器质性重现的转录、结构和功能特征 心肌。然而，在我们目前的器官中，使用原代非心肌细胞群体限制了它们的 有可能模拟患者特定的心肌。为了开发人类等基因心脏器官，我们正在 与威斯康星大学麦迪逊分校的肖恩·帕莱切克博士合作从 在我们的心脏器官模型中，人多能干细胞(HPSC)将取代成体cFbs。帕莱切克博士的实验室已经 在两种不同的谱系中指导hPSC分化为心脏成纤维细胞(hPSC-cFbs)的专业知识： 心外膜来源的成纤维细胞(hPSC-CFB(EPIC)S)和第二心脏场祖细胞来源的成纤维细胞(hPSC-CFB) 首席财务官S)。虽然这两个谱系都会导致心脏纤维化，而且在功能上相似，但在小鼠心脏中， 心外膜是心脏成纤维细胞的主要来源，但也有一小部分。 来自心内膜。此外，hPSC-CFB(EPIC)S可能需要增强成熟度 取代人类成人cFbs，因为我们的初步数据表明，长期培养改善了细胞组织 心脏组织中hPSC-CFB(SHFP)S与成人CFB组织中hPSC-CFB的比较中环 这项提议的假设是，高传代hPSC-CFB(EPIC)S将最好地复制成人-CFB转录本 和功能性。这项建议的创新之处在于，我们将首次确定合适的hPSC-CFB 取代成人cFbs的人群，以开发人体心肌的等基因3D器官型模型。我们的 长期目标是开发患者特有的心脏器官，用于体外疾病建模和药物测试。 因此，我们将追求以下两个目标：1)确定高通道hPSC的有效性- Cfb(史诗)S复制成人cFb的转录和功能，2)确定有效性 高传代hPSC-CFB(EPIC)-S构建的人心肌梗死后模型的研究 心肌与抗心肌梗死治疗的反应性。我们还将进行单细胞rna-seq检测。 我们的梗塞方案对hPSC-CFB(EPIC)的异质性S的反应。这项研究的完成将 为建立等基因的人类心肌模型提供了第一步。单细胞rna-seq研究将揭示 心肌梗死后人心肌成纤维细胞的不同作用/亚群。

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.