Endogenous Cardiac Repair in Humans

描述（由申请人提供）： 直到最近，心脏一直被认为是一个终末分化的器官，没有新的心肌细胞（CM）形成的能力。这种观点似乎是不正确的，因为我们和其他人已经能够从人类心肌中分离出心脏来源的祖细胞（CDPC）。扩展这些结果，我们最近的研究表明，表达干细胞表面标志物c-kit的细胞可以从人心脏分离后立即复苏，随后诱导分化成CM通过短期共培养与新生大鼠心室肌细胞（RVMs）。虽然我们通常在衰竭的心脏比非衰竭的心脏中发现更多的c-kit+细胞，但通过移植替换这些衰竭的心脏的需要突出了天然心脏修复机制的不足。基于这些发现，我们广泛的工作假设是，在衰竭的人类心脏中增加的c-kit+ CDPC包括谱系阴性的c-kit+和c-kit+/CD 45（dim-moderate）细胞，它们各自能够在体外形成新的肌细胞。在这种情况下，本提案的目的是量化和表征这些不同的干/祖细胞亚群在人类心脏内，重点是阐明其复制和CM分化的功能能力。我们的第一个目标是确定什么类型的干/祖细胞存在于正常和失败的人类心脏。我们将根据荧光显微镜和流式细胞术对分解的心肌细胞进行免疫分型，确定不同的干细胞/祖细胞亚群，并在同一心脏的组织切片中进行补充研究，以确定其分布。我们的第二个目的是根据静态分析（端粒长度，端粒酶活性和p16 INK 4a表达）和增殖率的功能评估的组合来表征所选CDPC亚群的复制能力。我们的第三个目标是表征来自人类心脏的选定CDPC亚群的心肌生成潜力。这些研究将确定标准化共培养条件下分选亚群CM分化的速率和频率，确定新生大鼠肌细胞诱导CM分化是否需要细胞接触，并鉴定促进或增加选定CDPC亚群体外CM分化速率的分泌因子（趋化因子或生长因子）。这一建议的临床/治疗意义是基于这样的前提，即对内源性心脏干/祖细胞亚群的增殖和心肌发生潜力的认识将促进治疗性心脏再生的进展，有或没有细胞治疗本身。公共卫生相关性：在最近成功的实验中，我们已经从人类心脏中分离出常驻干/祖细胞，并在体外诱导其分化为收缩心肌细胞。基于这些发现，本研究的重点是表征这些干/祖细胞的选定亚群，重点是阐明它们复制和分化为功能心肌细胞的能力。我们认为，对内源性心脏干细胞/祖细胞的心肌发生潜力的认识将促进治疗性心脏再生的进展，无论是否有细胞治疗本身。

DESCRIPTION (provided by applicant): Until recently, the heart has been viewed as a terminally differentiated organ with no capacity for new cardiac myocyte (CM) formation. This view appears to be incorrect, in that we and others have been able to isolate cardiac-derived progenitor cells (CDPCs) from human myocardium. Extending these results, our recent studies indicate that cells expressing the stem cell surface marker c-kit can be isolated from human hearts immediately after explantation and subsequently induced to differentiate into CM via short-term co-culture with neonatal rat ventricular myocytes (RVMs). Though we typically find more c-kit+ cells usually in failing vs. nonfailing hearts, the need to replace these failing hearts via transplantation highlights the inadequacy of native cardiac repair mechanisms. Based on these findings, our broad working hypothesis is that increased c-kit+ CDPCs in failing human hearts include both lineage-negative c-kit+ and c-kit+/CD45(dim-moderate) cells that are each capable of new myocyte formation in vitro. In this context, the objective of this proposal is to quantify and characterize these distinct subpopulations of stem/progenitor cells within human hearts with an emphasis on elucidating their functional capacity for replication and CM differentiation. Our first aim is to identify what types of stem/progenitor cells are present in normal and failing human hearts. We will define distinct stem/progenitor subpopulations based on immunotyping of disaggregated myocardial cells with fluorescence microscopy and flow cytometry and perform complementary studies in tissue sections from the same hearts to define their distribution. Our second aim is to characterize replicative capacity of the selected CDPC subpopulations based on a combination of static assays (telomere length, telomerase activity and and p16INK4a expression) and functional assessment of proliferation rates. Our third aim is to characterize the cardiac myogenic potential of selected CDPC subpopulations derived from human hearts. These studies will define the rates and frequency of CM differentiation for sorted subpopulations under stardardized co-culture conditions, define whether cell contact is required for induction of CM differentiation by neonatal rat myocytes and identify secreted factors (chemokines or growth factors) that promote or augment rates of in vitro CM diffentiation in selected CDPC subpopulations. The clinical/therapeutic significance of this proposal is based on the premise that insights into the proliferative and cardiomyogenic potential of endogenous cardiac stem/progenitor cell subpopulations will promote progress towards therapeutic cardiac regeneration with or without cell therapy per se. PUBLIC HEALTH RELEVANCE: In recent successful experiments, we have isolated resident stem/progenitor cells from human hearts and induced their in vitro differentiation into contracting cardiac myocytes. Building on these findings, this research is focused on characterizing selected subpopulations of these stem/progenitor cells with an emphasis on elucidating their capacity for replication and differentiation into functioning cardiac myocytes. We contend that insights into the cardiomyogenic potential of endogenous cardiac stem/progenitor cells will promote progress towards therapeutic cardiac regeneration with or without cell therapy per se.