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，没有形成新的心肌细胞(CM)的能力。这一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c-kit的细胞可以在移植后立即从人心脏中分离出来，然后通过与新生大鼠心室肌细胞(RVMs)的短期共培养诱导分化为CM。虽然我们通常会在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必要性突显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，衰竭心脏中增加的c-kit+CDPC包括谱系阴性的c-kit+和c-kit+/CD45(暗-中等)细胞，每个细胞都能够在体外形成新的心肌细胞。在此背景下，这项建议的目标是量化和描述人类心脏中这些不同的干细胞/祖细胞亚群，重点是阐明它们复制和分化CM的功能能力。我们的第一个目标是确定在正常和衰竭的人类心脏中存在哪些类型的干细胞/祖细胞。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，定义不同的干/祖细胞亚群，并在相同心脏的组织切片中进行补充研究，以确定它们的分布。我们的第二个目标是基于静态分析(端粒长度、端粒酶活性和p16INK4a表达)和增殖率的功能评估相结合的方法来表征选定的CDPC亚群的复制能力。我们的第三个目标是研究人类心脏来源的CDPC亚群的心肌生肌潜能。这些研究将确定在标准化的共培养条件下分选的亚群的CM分化率和频率，确定新生大鼠心肌细胞诱导CM分化是否需要细胞接触，并确定在选定的CDPC亚群中促进或增强体外CM分化率的分泌因子(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一个前提，即对内源性心脏干/祖细胞亚群的增殖和心肌生成潜力的洞察将促进无论是否使用细胞治疗本身，都将在治疗性心脏再生方面取得进展。公共卫生相关性：在最近成功的实验中，我们从人的心脏中分离出驻留的干细胞/祖细胞，并在体外诱导其分化为收缩的心肌细胞。在这些发现的基础上，这项研究的重点是确定这些干细胞/祖细胞的特定亚群的特征，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，深入了解内源性心脏干细胞/祖细胞的心肌再生潜能，将促进治疗心脏再生的进展，无论是否使用细胞治疗本身。

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.