Resolving myocardial regeneration by tracking commitment of c-Kit+ cells

项目摘要 干细胞介导的心脏修复是心血管研究的一个令人兴奋和有争议的领域， 有可能产生新的，革命性的治疗心脏病的疗法。广泛 对确定有助于心脏形成、体内平衡和再生的细胞类型的研究已经产生了 几种候选细胞，包括表达c-Kit+的成体心脏干细胞和祖细胞， 在I期临床试验中使用，证明了许多患者的有益结果以及安全性 以及这种治疗方法的可行性尽管包括SCIPIO临床试验在内的许多有希望的研究， c-kit+细胞群在心脏再生中的重要性在许多人中一直存在争议 心血管研究人员和临床医生。因此，进一步的调查进行了严格的 实验设计、方法学和解释对于解决当前的模糊性和推进 心脏再生治疗领域本着这种精神，该提案确定了c-Kit+细胞的作用， 使用一种新的、可诱导的转基因小鼠模型对c-Kit进行基因标记， 在发育过程中、损伤后和衰老的心脏中表达细胞。与“敲入”相反， 在许多最近的c-Kit谱系追踪模型中采用的方法， 内源性c-Kit，本文提出的策略利用了有效的外源性c-Kit启动子的过表达 构建体以驱动rtTA反式激活因子的表达。该构建体与受调控的启动子组合， 四环素反应元件（TRE）诱导TRE控制的报告基因转录， 给予强力霉素。c-Kit+细胞以时间依赖性方式被短暂或永久标记， 这取决于TRE控制的合作伙伴。初步结果表明，c-Kit细胞包含更多样化的 人口比以前假设的，需要更细致的了解基本的c-Kit生物学 和表达模式。具体目的是：1）c-Kit+干细胞在组织培养期间有助于成人心脏组织 使用体内谱系追踪的遗传模型证明的稳态和修复，2）c-Kit+细胞 3）c-Kit+细胞有助于衰老过程中的稳态 心该提议的创新在于用于标记和跟踪c-Kit细胞的方法，使得内源性 c-Kit生物学保持完整，而过表达赋予更高的敏感性，因此更广泛 c-Kit细胞群的标记。这些研究的意义在于澄清现有的未解决的 通过建立c-Kit细胞表达和参与心脏形成和修复来解决该领域的争议。 总的来说，这些研究将提供定义c-Kit+心脏的重要性的基本信息。 祖细胞群体作为用于治疗心脏病的基于细胞的疗法的试剂。

Project Summary Stem cell mediated cardiac repair is an exciting and controversial area of cardiovascular research that holds the potential to produce novel, revolutionary therapies for the treatment of heart disease. Extensive investigation to define cell types contributing to cardiac formation, homeostasis and regeneration has produced several candidates, including adult cardiac c-Kit+ expressing stem and progenitor cells that have even been employed in a Phase I clinical trial demonstrating beneficial outcome for many patients together with safety and feasibility of this therapeutic approach. Despite many promising studies including the SCIPIO clinical trial, the significance of the c-kit+ cell population in cardiac regeneration is persistently debated among many cardiovascular researchers and clinicians. Therefore, further investigation conducted with rigorous experimental design, methodology, and interpretation is essential to resolve current ambiguities and advance the field of cardiac regenerative therapy. In that spirit, this proposal determines the role of c-Kit+ cell participation in cardiac repair using a novel, inducible transgenic mouse model to genetically tag c-Kit expressing cells during development, following injury and in the aging heart. In contrast to “knock-in” approaches employed in many recent c-Kit lineage-tracing models that generate a hemizygous genotype for endogenous c-Kit, the strategy proposed here exploits overexpression of a validated exogenous c-Kit promoter construct to drive expression of the rtTA transactivator. This construct in combination with promoters regulated by the tetracycline responsive element (TRE) induces transcription of the TRE controlled reporter upon administration of doxycycline. c-Kit+ cells are tagged in a time-dependent manner transiently or permanently, depending upon the TRE controlled partner. Preliminary results show that c-Kit cells comprise a more diverse population than previously assumed, necessitating a more nuanced understanding of fundamental c-Kit biology and expression patterns. Specific Aims are: 1) c-Kit+ stem cells contribute to adult cardiac tissue during tissue homeostasis and repair as demonstrated using genetic models for lineage tracing in vivo, 2) c-Kit+ cells contribute to cardiac formation during development and 3) c-Kit+ cells contribute to homeostasis in the aging heart. The innovation of this proposal is in the approach used to tag and track c-Kit cells such that endogenous c-Kit biology remains intact while overexpression confers greater sensitivity and therefore more extensive labeling of the c-Kit cell population. The significance of these studies is to clarify existing unresolved controversies in the field by establishing c-Kit cell expression and participation in cardiac formation and repair. Collectively, these studies will contribute essential information defining the importance of c-Kit+ cardiac progenitor populations as reagents for cell based therapy in the treatment of heart disease.