Stem cell oxygenation and ischemic tissue regeneration

项目负责人/主要研究者（末、首、中）：关健军 项目摘要 严重肢体缺血（CLI）是一种严重的外周动脉疾病，肢体丧失率和死亡率很高。是 其特征在于低血液灌注、广泛的组织缺血和退化的骨骼肌。快速血管化 恢复血液灌注和快速肌肉再生以恢复正常功能，代表了 CLI治疗。目前还没有有效的治疗方法，尽管干细胞治疗是最有效的治疗方法之一。 有前途的战略。大多数类型的干细胞主要通过旁分泌促进血管化和肌肉再生 一些细胞还可以分化为内皮细胞和骨骼肌细胞。目前，干细胞 治疗经历低的功效，主要是由于在极低的细胞存活率和旁分泌效应下， 缺血肢体氧合情况<1%。在这个项目中，我们提出了一种新的细胞输送系统， 释放适当浓度的O2以同时提高干细胞存活和旁分泌效应， 导致快速血管化和肌肉再生。旁分泌效应同时提供多重生长 血管形成和肌肉再生的关键因素，这不能通过生长因子容易地实现 疗法 在我们的初步研究中，我们已经创建了一个基于水凝胶的细胞输送系统，释放O2。测试时 使用骨髓来源的间充质干细胞（MSC），释放的O2增加了细胞存活， 在体外缺血条件下，不增加活性氧（ROS）含量。它还上调了MSC 旁分泌作用，特别是在分泌促血管生成/促肌生成生长因子如PDGF和IGF-1方面。 植入缺血肢体后，O2释放细胞递送系统不仅增加了MSC的存活，而且 在4周内也完全恢复了血液灌注和肌肉收缩力。骨髓间充质干细胞对血管化的作用 主要来自旁分泌效应，因为只有低百分比的细胞分化为内皮细胞。 同时，MSC的旁分泌效应和肌源性分化有助于肌肉再生。这些 初步数据表明，增加MSC存活率和旁分泌效应可以显著增强 血管化和肌肉再生。然而，细胞存活和旁分泌效应并不总是 同时增加。 基于我们的初步研究和上述讨论，我们假设， 同时增加MSC存活和旁分泌效应的最佳O2释放曲线，将显著 促进缺血肢体血管化和肌肉再生。 目的#1将检验最佳O2释放曲线将促进MSC存活和旁分泌效应的假设 在局部缺血的情况下。 目标#2将使用后肢缺血模型测试所创建的细胞递送系统的功效。 这个项目是创新的，因为它开发了一个安全和长期的O2释放系统，以建立作用， 机制和控制O2释放在增加干细胞存活和旁分泌效应中的功效。 缺血组织加速再生。所提出的干细胞递送系统也是翻译的。 OMB编号0925-0001/0002（2016年3月修订版，批准至2018年10月31日）

Program Director/Principal Investigator (Last, First, Middle): GUAN, JIANJUN Project Summary Critical limb ischemia (CLI) is a severe peripheral artery disease with high rates of limb loss and mortality. It is featured by low blood perfusion, extensive tissue ischemia, and degenerated skeletal muscle. Quick vascularization to restore blood perfusion, and fast muscle regeneration to restore normal function, represent the optimal goals for CLI treatment. Currently there is no efficient treatment available, although stem cell therapy is one of the most promising strategies. Most of stem cell types promote vascularization and muscle regeneration mainly by paracrine effects while some may also differentiate into endothelial and skeletal muscle cells. However, current stem cell therapy experiences low efficacy largely due to inferior cell survival and paracrine effects under the extremely low oxygen condition (<1%) of ischemic limbs. In this project, we propose a new cell delivery system that continuously releases appropriate concentration of O2 to simultaneously improve stem cell survival and paracrine effects, resulting in quick vascularization and muscle regeneration. Paracrine effects concurrently provide multiple growth factors critical for vascularization and muscle regeneration, which cannot be readily achieved by growth factor therapy. In our preliminary studies, we have created a hydrogel-based cell delivery system that releases O2. When tested using bone marrow-derived mesenchymal stem cells (MSCs), the released O2 increased cell survival under ischemic conditions in vitro without increasing reactive oxygen species (ROS) content. It also upregulated MSC paracrine effects especially in terms of secreting proangiogenic/promyogenic growth factors like PDGF and IGF-1. After implanting into ischemic limbs, the O2 releasing cell delivery system not only augmented MSC survival, but also fully restored blood perfusion and muscle contractility in 4 weeks. The contribution of MSCs to vascularization is mainly from paracrine effects as only a low percentage of cells were differentiated into endothelial cells. Meanwhile, both MSC paracrine effects and myogenic differentiation contributed to muscle regeneration. These preliminary data suggest that increasing both MSC survival and paracrine effects can significantly enhance vascularization and muscle regeneration in ischemic limbs. Yet, cell survival and paracrine effects do not always increase concurrently. Based on our preliminary studies and above discussion, we hypothesize that stem cell delivery systems with optimal O2 release profiles that simultaneously increase MSC survival and paracrine effects, will significantly accelerate vascularization and muscle regeneration in ischemic limbs. Aim #1 will test the hypothesis that optimal O2 release profiles will promote MSC survival and paracrine effects under ischemic conditions. Aim #2 will test efficacy of the created cell delivery systems using a model of hindlimb ischemia. This project is innovative because it develops a safe and long-term O2 release system to establish the role, mechanism, and efficacy of controlled O2 release in augmenting both stem cell survival and paracrine effects in ischemic tissues for accelerated regeneration. The proposed stem cell delivery system is also translational. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page