Collaborative Research: Engineering Human 3D Cardiac Tissue Model of Hypertrophic Cardiomyopathy

目前，人类诱导多能干细胞（hiPSC）技术（一项获得诺贝尔奖的技术，可以将丰富的人类细胞，如皮肤细胞和脂肪细胞转化为干细胞，这些干细胞可以产生身体中的所有其他细胞类型）已经使得在细胞培养中将人类心脏病建模为“盘中疾病”成为可能。“然而，目前hiPSC疾病建模的巨大挑战是，这些模型倾向于将疾病简化为单个缺陷基因的结果，而没有考虑到遗传-环境相互作用的许多其他影响。具体来说，肥厚型心肌病（HCM），一种心脏细胞扩大导致心脏壁膨胀的疾病，是年轻人和运动员心脏性猝死的主要原因，这表明身体压力增加了由于遗传因素而已经处于风险中的患者发生心力衰竭的风险。因此，为了开发有用的“HCM-in-a dish”模型系统，有必要精确地控制施加在hiPS衍生的心脏组织上的环境应力。 该项目的重点是MYBPC 3基因，这是最常见的突变HCM基因之一。 虽然已经建立了这种关系，但MYBPC 3突变导致HCM的机制尚不清楚。因此，本项目的主要目标是研究HCM特征与MYBPC 3表达减少之间的相关性，以及这如何受到心脏组织环境应激增加的影响。 这项工作成功的关键是创建HCM的功能/跳动3D心脏组织模型，这可以更好地了解遗传缺陷如何与细胞和组织环境联合收割机结合以启动和推进疾病。更广泛地说，该项目中开发的策略可以应用于研究其他心脏疾病，并可能导致疾病管理和治疗的新疗法。这种新方法（需要hiPSC技术，心脏组织工程，先进的3D生物打印以及材料加工和表征）将为高中生，本科生和研究生提供重要且目前无法获得的机会，以获得令人兴奋的研究经验和最先进的生物医学工程和纳米技术培训。这将通过课程、研讨会和讲习班的形式进行协调、结构化的教学和评估，以及参与研究实验室环境来完成。本项目的主要目标是建立一个基于等基因的人类诱导多能干细胞（hiPSs）的肥厚型心肌病（HCM）组织模型，用于研究遗传缺陷如何与细胞和组织环境相互作用以引发和发展疾病。 HCM是年轻人和运动员心脏性猝死的主要原因，这表明身体压力增加了HCM相关遗传易感性患者发生心力衰竭的风险。该项目的重点是MYBPC 3基因，最常见的突变HCM基因之一，虽然MYBPC 3突变导致HCM的分子机制仍然难以捉摸。该项目的中心假设是HCM表型的严重程度将依赖于MYBPC 3基因表达和蛋白质含量的降低（单倍不足），这可能会因来自hiPSC的心脏微组织（hiPS-microCT）的环境压力增加而加剧。 将通过整合以下内容建立微组织模型：1）用于理解与MYBPC 3突变相关的人类特异性HCM疾病机制的hiPSC技术，2）用于在具有可控生物力学应力的丝状基质上创建三维（3D）hiPS-microCT的基于激光的生物打印方法，和3）用于产生具有相同遗传背景的MYBPC 3功能丧失突变的基因编辑方法（同基因型）与野生型（WT）相比，MYBPC 3基因表达呈剂量依赖性降低。本研究计划有三个目标：1）基于主要表型指标，将MYBPC 3缺陷型同基因hiPS-microCT的生物力学应力与HCM疾病严重程度相关联; 2）将MYBPC 3缺陷型同基因hiPS-microCT中的单倍不足水平与不同生物力学应力下的HCM疾病严重程度相关联;阐明MYBPC 3相关HCM在应激诱导疾病进展中的分子机制。hiPSC技术、3D生物打印、基因编辑方法和组织工程方法的组合为开发下一代基于hiPSC的疾病特异性体外临床前组织模型提供了巨大潜力。该模型将是研究临床异质性相关的基因型-表型相关性，阐明人类心肌病的疾病进展，以及开发疾病管理和治疗的新治疗策略的重大进步。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

Currently, human induced pluripotent stem cell (hiPSC) technology (a Nobel Prize winning technology that can turn abundant human cells, such as skin cells and fat cells, into stem cells that can give rise to every other cell type in the body) has made it possible to model human heart diseases in cell culture as a "disease-in-a-dish." However, the grand challenge in current hiPSC disease modeling is that these models have tended to simplify the diseases as being a result of a single defective gene without taking into account the many other influences of genetic-environmental interactions. Specifically, hypertrophic cardiomyopathy (HCM), a condition in which the heart cells enlarge causing the heart wall to thicken, is the leading cause of sudden cardiac death among young adults and athletes, which indicates that physical stress increases the risk of developing heart failure in the patients already at risk due to genetic factors. Therefore, to develop useful "HCM-in-a dish" model systems, it is necessary to precisely control the environmental stress exerted on hiPS-derived cardiac tissues. This project focuses on the MYBPC3 gene, which is one of the most frequently mutated HCM genes. Though the relationship has been established, the mechanisms by which MYBPC3 mutations lead to HCM are not known. Thus, the primary goal of this project is to investigate the correlation between HCM characteristics and reduced MYBPC3 expression, and how this could be influenced by the increase of environmental stress to the cardiac tissues. Key to the success of this effort is creating a functional/beating 3D cardiac tissue model of HCM, which offers better understanding of how the genetic defects combine with the cellular and tissue environment to initiate and advance the disease. More broadly, the strategies developed in this project could be applied to studying other cardiac diseases and potentially lead to new therapies for disease management and treatment. This new approach (which requires hiPSC technology, cardiac tissue engineering, advanced 3D bioprinting, and materials processing and characterization) will provide significant and presently unavailable opportunities for high school, undergraduate and graduate students to have exciting research experiences and state-of-the-art training in biomedical engineering and nanotechnology. This will be accomplished with coordinated, structured instruction and assessments in the form of coursework, seminars, and workshops, as well as with participation in the research laboratory environment.The primary goal of this project is to establish an isogenic, human induced pluripotent stem cell (hiPSs) based tissue model of hypertrophic cardiomyopathy (HCM), for studying how genetic defects interplay with the cellular and tissue environment to initiate and progress the disease. HCM is the leading cause of sudden cardiac death among young adults and athletes, which indicates that physical stress increases the risk of developing heart failure in patients with HCM-related genetic predispositions. This project focuses on the MYBPC3 gene, one of the most frequent mutated HCM genes, though molecular mechanisms by which MYBPC3 mutations lead to HCM remain elusive. The central hypothesis of this project is that the severity of HCM phenotype would be dose-dependent on the reduction of MYBPC3 gene expression and protein content (haploinsufficiency), which could be exacerbated by the increase of environmental stress to cardiac microtissues derived from hiPSCs (hiPS-microCTs). The microtissue model will be established by integrating: 1) hiPSC technology for understanding human-specific HCM disease mechanisms associated with MYBPC3 mutations, 2) laser-based bioprinting method for the creation of three-dimensional (3D) hiPS-microCTs on the filamentous matrices with controllable biomechanical stress, and 3) gene-editing approach for the generation of MYBPC3 loss-of-function mutations with identical genetic background (isogenic) as wild type (WT) and dose-dependent reduction of MYBPC3 gene expression. The research plan is organized under three objectives: 1) To correlate the biomechanical stress presented to the MYBPC3 deficient isogenic hiPS-microCTs with the HCM disease severity based on the primary phenotypic metrics; 2) To correlate the haploinsufficiency level in the MYBPC3 deficient isogenic hiPS-microCTs with the HCM disease severity under different biomechanical stress; and 3) To elucidate the molecular mechanisms involved in the stress-induced disease progression of MYBPC3-associated HCM. The combination of hiPSC technology, 3D bioprinting, gene editing method and tissue engineering approaches provides great potential in the development of next generation hiPSC-based disease-specific in vitro preclinical tissue models. This model will be a significant advancement for investigating genotype-phenotype correlation associated with the clinical heterogeneity, elucidating the disease progression in human cardiomyopathies, and developing new therapeutic strategies for disease management and treatment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.