Dedifferentiation of cardiomyocytes into cardiac progenitor cells

描述(申请人提供)：成年哺乳动物心脏中的心脏前体细胞(CPC)通过干细胞相关抗原、多能性以及它们在内源性和医源性再生心肌的能力来鉴定。当CPC在缺血损伤后被注射到心脏中时，产生有限的多系分化(心肌细胞、内皮细胞、血管平滑肌细胞)和功能益处。CPC的起源至今尚不清楚。一些，如果不是大多数，可能是来自心脏内的先天胚胎起源；外源性的血源性前体细胞也可以在损伤后种植心脏。另一个被忽视的潜在起源机制是去分化，这是一个成熟的、特化的细胞重新获得增殖能力和增强可塑性的过程。已建立的心肌细胞发育范式假定，从前体细胞到新生心肌细胞再到不能再分裂和增殖的成熟心肌细胞是单向的。相比之下，成年斑马鱼心肌细胞迅速重新进入细胞周期，但即使是它们也没有被证明去分化为多潜能状态。在R01的初始授权期内的主要发现是认识到出生后哺乳动物心肌细胞去分化和获得CPC特征的能力。支持这一说法的初步数据载于进度报告。所提出的去分化心肌细胞成为CPC的能力，如果被这里提出的工作所证实，将从根本上改变我们对成人心脏及其潜在可塑性的看法。为此，我们试图通过多种互补的实验方法来证明心肌细胞去分化为CPC；表征潜在的分子和细胞机制；并建立去分化的病理生理学相关性。我们将测试细胞培养促进去分化、细胞周期重新进入和获得“干性”的想法。遗传细胞命运图谱将被用来确定确认的心肌细胞去分化，表达c-kit，并能够分化为至少两个心脏谱系。需要解决的具体问题包括：去分化的心肌细胞重新进入细胞周期的频率和条件？基因表达、microRNAs和细胞周期调节蛋白的哪些变化是去分化过程的基础？条件培养液中的自分泌/旁分泌因子在心肌细胞去分化中是否重要？去分化的心肌细胞表达干细胞抗原吗？它们是全能的吗？它们能被克隆繁殖吗？去分化的心肌细胞能在体外重新分化和转分化吗？当被注射到受损的心脏中时，去分化的心肌细胞是否会植入、分化和改善功能？基因定义的心肌细胞对损伤的反应是否原位去分化？心肌梗死后内源性心肌再生是否反映了心肌细胞的去分化和随后的多向分化？这项工作测试了CPC可能来自去分化以及胚胎发生或血源性播种的想法。 与公共健康相关：拟议的工作重点是心脏与生俱来的再生能力，以及如何利用这种能力来改进心脏病的治疗。我们试图了解心脏中先天祖细胞的起源机制，这些先天祖细胞通常具有修复日常生活中的磨损的功能。我们将测试成熟的心脏细胞可以倒退并恢复先天祖细胞特征的想法，这一想法具有重要的生物学、病理生理学和治疗意义。

DESCRIPTION (provided by applicant): Cardiac progenitor cells (CPCs) in the adult mammalian heart are identified by stem cell-related antigens, multipotency, and by their ability to regenerate myocardium endogenously and iatrogenically. CPCs, when injected into the heart after ischemic injury, produce limited multilineage differentiation (cardiomyocytes, endothelial cells, vascular smooth muscle cells) and functional benefits. The origin of CPCs is as-yet unclear. Some if not most CPCs are likely of innate embryonic origin within the heart; exogenous bloodborne precursor cells can also seed the heart after injury. Another potential mechanism of origin that has been overlooked is that of dedifferentiation, a process whereby mature, specialized cells regain proliferative ability and augmented plasticity. The established paradigm for cardiomyocyte development posits a one-way transit from precursor cells through neonatal cardiomyocytes on to mature heart cells, which can no longer divide and proliferate. Adult zebrafish cardiomyocytes, in contrast, re-enter the cell cycle with alacrity, but even they have not been demonstrated to dedifferentiate to a state of multipotency. The major discovery in the initial grant period of this R01 was the recognition of the ability of postnatal mammalian cardiomyocytes to dedifferentiate and acquire features of CPCs. Preliminary data supporting this claim are presented in the progress report. The proposed ability of dedifferentiated cardiomyocytes to become CPCs, if verified by the work proposed here, would change fundamentally our view of the adult heart and its potential plasticity. For this reason, we seek to demonstrate cardiomyocyte dedifferentiation to CPCs by multiple complementary experimental approaches; to characterize the underlying molecular and cellular mechanisms; and to establish the pathophysiological relevance of dedifferentiation. We will test the idea that cell culture promotes dedifferentiation, cell cycle re- entry, and acquisition of "stemness". Genetic cell fate mapping will be utilized to establish that confirmed cardiomyocytes dedifferentiate, express c-kit, and are capable of differentiating into at least two cardiac lineages. Specific questions to be addressed include: How often and under what conditions do dedifferentiating cardiomyocytes re-enter the cell cycle? What changes in gene expression, microRNAs, and cell cycle regulatory proteins underlie the dedifferentiation process? Are autocrine/paracrine factors in conditioned media important in cardiomyocyte dedifferentiation? Do dedifferentiated cardiomyocytes express stem cell antigens? Are they pluripotent? Can they be clonally proliferated? Can dedifferentiated cardiomyocytes re-differentiate and transdifferentiate in vitro? When injected into injured hearts, do dedifferentiated cardiomyocytes engraft, differentiate and improve function? Do genetically-defined cardiomyocytes dedifferentiate in situ in response to injury? Does endogenous myocardial regeneration after infarction reflect dedifferentiation and subsequent multilineage differentiation of cardiomyocytes? The work tests the idea that CPCs may arise from dedifferentiation as well as embryogenesis or bloodborne seeding. PUBLIC HEALTH RELEVANCE: The proposed work focuses on the heart's innate ability to regenerate, and how that ability might be tapped to improve therapies for heart disease. We seek to understand the mechanism of origin of innate progenitor cells in the heart that normally function to repair the wear and tear of daily life. We will test the idea that mature heart cells can go backwards and regain the features of innate progenitor cells, an idea which has important biological, pathophysiological and therapeutic implications.