Understanding Cardiac Progenitors to deliver Regenerative Medicine and Disease Modelling

心脏病，包括心肌损伤，在世界范围内导致的死亡人数比任何其他疾病都多。这意味着心脏再生的策略是迫切需要的，例如心肌梗死后的细胞替代疗法和体外培养皿中的疾病模型。从人类多能干细胞(HPSCs)获得包括心肌细胞在内的心血管细胞提供了能够彻底改变心脏病学的治疗潜力，但到目前为止，这一承诺尚未实现。目前，由于我们对心肌细胞发育的理解和控制能力较差，主要是心脏祖细胞(CPC)规范、自我更新和分化等事件，阻碍了进展。另一个主要障碍是缺乏与人类胚胎发生相比较的分子基准，这是在体外验证细胞所必需的。因此，目前，不受控制的、异质分化的心肌细胞特性的混合体出现。由此产生的细胞不足以进行准确的疾病建模(疾病表型高度可变)，如果移植可能会危及生命(导致心律失常)。我的研究包括三个主要部分，它们的交付将释放学术研究、临床和工业应用的潜力：1)发现CPC的身份和如何控制它们，2)鉴定心室肌细胞分化和3)在活体环境中测试该系统。揭开心肌细胞发育的复杂性的挑战是分化是异质性的，CPC是多样化的，细胞沿着分化轨迹以不同的速度前进。为了克服这一点，我创建了一个与克隆谱系追踪和命运评估相联系的单细胞基因组学体外模型系统。这种综合和公正的方法将使我能够识别与CPC自我更新和心肌细胞分化潜力相关的基因表达和染色质特征。体外扰动实验，结合对人类胚胎发生过程中天然细胞的比较测量，将帮助我确定最具功能影响力和真实性的候选细胞。总之，这些实验将提供知识和工具，以分离、扩展和区分最理想的CPC身份-那些有可能被准确编程为特定靶细胞类型的身份，例如室性心肌细胞(疾病建模和细胞治疗的关键靶细胞)。为了了解如何准确地控制CPC向心肌细胞的下游分化，我将利用人心肌发育过程中的基因表达和染色质结构作为体外分化的基准。信号通路与关键转录因子的调控和表达之间的关系将被系统地解决，以实现准确的、谱系特异性的CPC分化。最后，为了验证这些在CPC维持、跟踪和分化方面的进展与翻译的相关性，我将与国际合作者合作，评估移植到动物模型后的细胞行为。总而言之，通过提供具有直接临床和工业重要性的研究创新，该项目与英国政府的产业战略密切一致。预测的进展将有利于在再生医学、心脏病模型、生物标记物识别和药物筛选方面的应用。此外，它还将使我成为该领域的全球领导者，并通过与行业合作，成为实现这些翻译目标的持久力量。

Heart disease, including damage to heart muscle, kills more people worldwide than any other illness. This means strategies for cardiac regeneration, such as cell replacement therapy after myocardial infarction and 'disease in a dish' modelling in vitro, are urgently needed. Access to cardiovascular cells including cardiomyocytes from human pluripotent stem cells (hPSCs) offers therapeutic potential capable of revolutionising cardiology but, as yet, the promise is unmet. Progress is currently hindered by our poor understanding of, and thus ability to control, cardiac cell development - principally the events of cardiac progenitor cell (CPC) specification, self-renewal and differentiation. The lack of comparative molecular benchmarking with human embryogenesis, required to authenticate cells in vitro, is another major barrier. By consequence, at present, mixtures of poorly specified cardiomyocyte identities emerge from uncontrolled, heterogeneous differentiation. The resultant cells are inadequate for accurate disease modelling (disease phenotypes are highly variable) and would be potentially life-threatening (arrhythmogenic) if transplanted. There are three major components to my study, delivery of which will unlock potential for academic research, clinical and industrial applications: 1) discovering the identity of CPCs and how to control them, 2) authenticating ventricular cardiomyocyte differentiation and 3) testing the system in an in vivo context.The challenge to unravelling the complexities of cardiac cell development is that differentiation is heterogeneous, CPCs are diverse, and cells progress along the differentiation trajectory at different rates. To overcome this, I have created an in vitro model system of single cell genomics linked to clonal lineage tracing and fate assessment. This integrated and unbiased approach will enable me to identify the gene expression and chromatin signatures pertinent to CPC self-renewal and cardiomyocyte differentiation potential. Perturbation experiments in vitro, combined with comparative measurements of the native cells in human embryogenesis, will help me identify the most functionally influential and authentic candidates. Together, these experiments will deliver the knowledge and tools to isolate, expand and differentiate the most desirable CPC identities - those with potential to be accurately programmed to specific target cell types e.g. ventricular cardiomyocytes (a key target for disease modelling and cell therapy). To learn how to accurately control downstream differentiation of CPCs to ventricular cardiomyocytes, I will use the gene expression and chromatin structure of human ventricular myocardium through development to benchmark differentiation in vitro. The relationship between signalling pathways and the regulation and expression of key transcription factors will be systematically resolved, to achieve the accurate, lineage-specific differentiation of CPCs. Finally, to validate these advances in CPC maintenance, tracking and differentiation to translational relevance, I will work in partnership with international collaborators to assess cell behaviour following transplantation into an animal model. In summary, by delivering research innovation of immediate clinical and industrial importance, this project is closely in tune with the UK Government's Industrial Strategy. The predicted advances will benefit applications in regenerative medicine, cardiac disease modelling, biomarker identification and drug screening. Moreover, it will also allow me to establish myself as a global leader in the field and, by partnering with industry, a persistent force towards achieving these translational goals.