Endogenous Cardiac Repair in Humans

人类内源性心脏修复

基本信息

批准号：
8034259
负责人：
Kenneth Ber Margulies
金额：
$ 45.4万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-15 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8034259
关键词：
Activities of Daily Living Affect Age Apoptosis Biological Assay Biological Markers Biopsy CDKN2A gene Cardiac Cardiac Myocytes Cell Therapy Cell surface Cells Clinical Coculture Techniques Conditioned Culture Media Contracts Data Development Endothelial Cells Flow Cytometry Fluorescence Microscopy Frequencies Gene Expression Growth Factor Health Heart Heart failure Human Immunophenotyping In Vitro Integration Host Factors Intrinsic factor Length Muscle Cells Myocardial Myocardial Infarction Myocardium Natural regeneration Neonatal Organ PTPRC gene Patients Phylogeny Population Preparation Principal Investigator Process Proto-Oncogene Protein c-kit Rattus Research Reverse Transcriptase Polymerase Chain Reaction Rodent Smooth Muscle Myocytes Sorting - Cell Movement Stem cells Techniques Telomerase Therapeutic Time Tissues Transplantation Ventricular Work activity marker base chemokine empowered improved insight progenitor regenerative repaired research study senescence stem telomere

项目摘要

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.

描述（由申请人提供）：直到最近，心脏一直被视为一种终极分化的器官，没有新的心肌细胞（CM）形成。这种观点似乎是不正确的，因为我们和其他人已经能够将心脏衍生的祖细胞（CDPC）与人心肌分离。扩展了这些结果，我们最近的研究表明，表达干细胞表面标记C-KIT的细胞可以在膨胀后立即从人的心脏中分离出来，随后通过与新生儿大鼠心室心肌细胞（RVMS）通过短期共培养而诱导将CM分化为CM。。尽管我们通常在失败的心脏与非福利心脏中发现更多的C-KIT+细胞，但通过移植替换这些失败的心脏的需求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，在失败的人心脏中增加的C-KIT+ CDPC包括谱系阴性C-KKIT+和C-KWIT+/C-KIT+/CD45（Dim-Mimenterate）细胞，这些细胞每个人都能在体外形成新的心肌细胞。。在这种情况下，该提案的目的是量化和表征人类心中茎/祖细胞的这些独特的亚群，重点是阐明其复制和CM分化的功能能力。我们的第一个目的是确定在正常和失败的人心中存在哪些类型的茎/祖细胞。我们将基于用荧光显微镜和流式细胞术对分解的心肌细胞的免疫分型来定义不同的茎/祖细胞亚群，并在同一心脏的组织切片中进行互补研究以定义其分布。我们的第二个目的是根据静态测定（端粒长度，端粒酶活性和P16INK4A表达）和增殖率的功能评估，表征所选CDPC亚群的复制能力。我们的第三个目的是表征从人类心脏中衍生的选定CDPC亚群的心脏肌源潜力。这些研究将定义在明星化的共培养条件下分类亚群的CM分化的速率和频率，并确定是否需要细胞接触来通过新生大鼠肌细胞诱导CM分化并识别促进或增强的分泌因子（趋化因子或生长因子）选定CDPC亚群中体外CM扩散的速率。该提案的临床/治疗意义基于以下前提：内源性心脏干/祖细胞亚群的增殖和心肌生成潜力的见解将促进对或不接受细胞治疗的治疗性心脏再生的进展。公共卫生相关性：在最近的成功实验中，我们从人心脏中分离了居民的茎/祖细胞，并将其体外分化诱导为收缩的心肌细胞。在这些发现的基础上，这项研究的重点是表征这些茎/祖细胞所选亚群，重点是阐明其复制能力和分化为功能性心肌细胞的能力。我们争辩说，对内源性心脏干/祖细胞的心肌生成潜力的见解将促进在有或没有细胞治疗本身的情况下，朝着治疗性心脏再生的进展。