Dedifferentiation of cardiomyocytes into cardiac progenitor cells

描述（由申请人提供）：成年哺乳动物心脏中的心脏祖细胞（CPCs）通过干细胞相关抗原、多能性以及内源性和医源性心肌再生能力来鉴定。缺血损伤后，将CPCs注入心脏，可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。有些（如果不是大多数的话）cpc可能来自心脏内的先天胚胎起源；外源性血源性前体细胞也能在心脏损伤后形成。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。心肌细胞发育的既定模式假设了从前体细胞通过新生儿心肌细胞到成熟心脏细胞的单向转运，成熟心脏细胞不再能够分裂和增殖。相比之下，成年斑马鱼的心肌细胞可以快速地重新进入细胞周期，但即使是它们也没有被证明能够去分化到多能状态。该R01最初拨款期的主要发现是对出生后哺乳动物心肌细胞去分化和获得心肌细胞特征的能力的认识。进度报告中提出了支持这一主张的初步数据。去分化心肌细胞成为心肌细胞的能力，如果被本文提出的工作证实，将从根本上改变我们对成人心脏及其潜在可塑性的看法。出于这个原因，我们试图通过多种互补的实验方法来证明心肌细胞向cpc的去分化；描述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。基因细胞命运定位将被用来确定确认的心肌细胞去分化，表达c-kit，并能够分化成至少两个心脏谱系。需要解决的具体问题包括：去分化心肌细胞重新进入细胞周期的频率和条件？基因表达、microrna和细胞周期调节蛋白的哪些变化是去分化过程的基础？条件介质中的自分泌/旁分泌因子在心肌细胞去分化中重要吗？去分化心肌细胞表达干细胞抗原吗？它们是多能的吗？它们能克隆繁殖吗？去分化心肌细胞能否在体外再分化和转分化？当注射到受伤的心脏时，去分化的心肌细胞是否会移植、分化和改善功能？基因定义的心肌细胞在损伤后会原位去分化吗？梗死后内源性心肌再生是否反映心肌细胞的去分化和随后的多系分化？这项工作验证了CPCs可能产生于去分化以及胚胎发生或血源性播种的观点。

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.