Therapeutic Mechanisms of Human CD34 Exosomes

描述（由申请人提供）：干细胞治疗，包括最近的CD34+细胞治疗，是一种有希望改善心脏再生和功能的治疗方法。CD34+细胞移植的益处似乎主要是通过缺血心肌中CD34+细胞分泌的旁分泌因子增加血管新生而发生的。然而，导致CD34+细胞诱导的血管生长和治疗恢复的确切机制尚不清楚。这种缺乏机制的认识是心脏干细胞治疗成功的关键障碍，因为移植干细胞的再生功效受到其在缺血心肌中较差的生存能力和保留能力的限制。因此，为了解决这些局限性并开发新的替代方法，我们必须设法了解导致治疗恢复的机制。我们之前发表的数据和我们的初步数据显示，人CD34+细胞的旁分泌含有称为外泌体（即CD34+外泌体）的膜结合纳米囊泡，它们与细胞相似，具有血管生成和治疗作用。此外，CD34+外泌体携带并将促血管生成mirna（如miR-126）转移到心肌缺血内皮细胞并诱导其血管生成活性。我们建议的基本基础是利用CD34+外泌体的再生潜力和通信能力来增强治疗方法。我们的中心假设是，通过人类CD34+细胞旁分泌释放的外泌体通过将mirna直接转移到心脏细胞中介导心肌修复。我们的目标是建立CD34+干细胞衍生的外泌体作为缺血性心肌修复的新型无细胞治疗实体，并全面了解外泌体诱导心肌血管生成和治疗恢复的分子机制。这项创新研究对于理解CD34+细胞治疗的机制和解锁祖细胞衍生外泌体的转化潜力具有重要意义。我们将从三个方面验证我们的假设：1)确定无细胞CD34+外泌体在小鼠心肌缺血模型中的治疗效果，并确定CD34+外泌体分泌是否是CD34+细胞治疗的关键机制之一；2)通过研究缺血心脏中CD34+外泌体的转运和信号机制，表征CD34+外泌体诱导血管生成的分子机制；3)确定miR-126在小鼠心肌梗死模型中介导CD34+外泌体的有益作用的程度。本提案中描述的实验将通过表征CD34+干细胞治疗的主要但尚未定义的机制来探索关键的科学问题。我们的研究将为利用人类干细胞外泌体作为一种合适的无细胞替代品的新型治疗方法奠定基础。利用外泌体相对于细胞的许多实际和技术优势，将外泌体应用于心血管再生医学，具有推进细胞治疗的潜力。

DESCRIPTION (provided by applicant): Stem cell-based therapies, including the recent CD34+ cell therapy, are a promising therapeutic approach for improving cardiac regeneration and function. The benefits of CD34+ cell transplantation appear to occur primarily via increase in vascular angiogenesis by the CD34+ cell-secreted paracrine factors in the ischemic myocardium. However, the precise mechanisms that lead to CD34+ cell-induced vessel growth and therapeutic recovery are poorly understood. This lack of mechanistic insight is a critical barrier to the success of cardiac stem cell therapy, as the regenerative efficacy of the transplanted stem cells is limited by their poor viability and retention in the ischemic myocardium. Therefore, to address these limitations and to develop novel alternate approaches, we must seek to understand the mechanisms that lead to therapeutic recovery. Our previously published data and our preliminary data have revealed that the paracrine secretion from human CD34+ cells contain membrane-bound nano-vesicles called exosomes (i.e. CD34+ exosomes), which are angiogenic and therapeutic similar to the cells. Further, CD34+ exosomes carry and transfer proangiogenic miRNAs, such as miR-126, to ischemic endothelial cells in the myocardium and induce their angiogenic activity. The fundamental basis of our proposal is to harness the regenerative potential and communication power of CD34+ exosomes to augment therapeutic approaches. Our central hypothesis is that exosomes released via paracrine secretion of human CD34+ cells mediate myocardial repair by direct transfer of miRNAs to cells in the heart. Our goal is to establish CD34+ stem cell-derived exosomes as a novel cell-free therapeutic entity for ischemic myocardial repair and to develop a comprehensive understanding of the molecular mechanisms of exosomes-induced cardiac angiogenesis and therapeutic recovery. This innovative study is important to understand the mechanisms of CD34+ cell therapy and to unlock the transformative potential of progenitor cell-derived exosomes. We will test our hypothesis in three specific aims: 1) Determine the therapeutic efficacy of cell-free CD34+ exosomes in a murine model of myocardial ischemia and determine whether CD34+ exosomes secretion is one of the key mechanisms of CD34+ cell therapy; 2) Characterize the molecular mechanisms of CD34+ exosomes-induced angiogenesis by studying the CD34+ exosomes trafficking and signaling mechanisms in the ischemic heart; 3) Establish the extent to which the beneficial effects of CD34+ exosomes are mediated by miR-126 in a murine model of MI. The experiments described in this proposal will explore key scientific questions by characterizing the predominant, but as yet undefined, mechanism of CD34+ stem cell therapy. Our studies will lay foundation to a novel therapeutic approach by using exosomes from human stem cells as a suitable cell-free alternative. It has the potential to advance cell- based therapis by exploiting many practical and technical advantages of exosomes relative to cells for application in cardiovascular regenerative medicine.