Therapeutic Potential of Gingival mesenchymal Stem Cell-derived Extracellular Vesicles Enriched with MFG-E8 in Peripheral Nerve Regeneration

项目概要/摘要 周围神经损伤（PNI）是一种常见而复杂的临床挑战。自体神经移植仍然是“黄金 对于具有较大间隙的PNI重建，“标准”，但供体神经的可用性有限， 供体部位的发病率显著阻碍了它们的临床应用。组织工程的组合应用 具有支持细胞和生物活性因子的神经引导导管（NGC）具有成为替代物的潜力 神经自体移植间充质干细胞（MSC）为基础的治疗已显示出再生的承诺， 由于其多能性、免疫调节/抗炎和再生潜力，的 MSC的治疗作用最可能依赖于它们的分泌组，特别是分泌性细胞外基质。 囊泡（EV）通过传递各种生物活性物质在细胞间通讯中发挥重要作用， 分子，例如脂质、蛋白质和非编码RNA。由于其治疗效果与母体药物相当， 细胞，MSC-EV已被探索为一系列疾病的新型无细胞治疗剂。但 MSC-EV在周围神经再生中的潜在用途仍然在很大程度上未被探索。我们最近 显示人牙龈来源的MSC（GMSC）及其释放的EV显示出相当的治疗效果， 对大鼠味蕾/味觉神经再生和挤压损伤修复/再生的影响 可能通过促进有髓鞘雪旺细胞的重编程来修复小鼠坐骨神经 表型。我们的初步研究表明，将GMSC-EV注入定制的AxoGuard Nerve 连接器显着提高其对大鼠坐骨神经横断再生的功效。通过 通过蛋白质组分析，我们鉴定了一组在GMSC-EV中显著富集的蛋白质因子，其中 乳脂球-表皮生长因子-因子VIII（MFG-E8）由于其多效性而特别令人感兴趣 生物功能。我们的初步数据还表明MFG-E8激活pSTAT 3，上调基因表达。 参与调节雪旺氏细胞的修复声型转换的表达。我们假设 富集的MFG-E8在GMSC-EV介导的对神经再生的治疗作用中起主要作用 通过促进雪旺细胞的修复表型转化。我们提出了两个具体目标，以测试 假设：1）确定富含GMSC-EV的MFG-E8对修复表型的多效性影响 2）研究MFG-E8在GMSC EV介导的治疗性转化中的关键作用; 对切断的大鼠坐骨神经再生的影响。我们的长期目标是开发无细胞/干细胞- 的产品作为自体神经移植物的潜在替代品，用于具有大间隙的PNI的再生。结果 从这项研究将导致我们提交R 01赠款申请，这将使我们能够更好地了解 MSC-EV介导的神经再生治疗作用的机制，并进一步探讨 MSC-EV在大型动物和早期人类临床试验中神经再生的安全性和有效性。

PROJECT SUMMARY/ABSTRACT Peripheral nerve injury (PNI) is a common and complex clinical challenge. Nerve autografts remain the “gold standard” for reconstruction of PNI with a large gap, but limited availability of donor nerves and several severe donor site morbidities significantly impede their clinic application. The combinatorial use of tissue engineered nerve guidance conduits (NGCs) with supportive cells and bioactive factors has the potential to be alternatives to nerve autografts. Mesenchymal stem cell (MSC)-based therapy has shown promises in regenerative medicine due to their multipotent, immunomodulatory/anti-inflammatory, and regenerative potentials. The therapeutic effects of MSCs most probably rely on their secretome, particularly the secretory extracellular vesicles (EVs) which play important roles in intercellular communication through transfer of various bioactive molecules e.g. lipids, proteins, and noncoding RNAs. Due to their comparable therapeutic efficacy to parental cells, MSC-EVs have been explored as novel cell-free therapeutics for a spectrum of diseases. However, the potential use of MSC-EVs in peripheral nerve regeneration remains largely unexplored. We have recently shown that human gingiva-derived MSCs (GMSCs) and their released EVs displayed comparable therapeutic effects on regeneration of taste bud/taste sensory nerves of rats and repair/regeneration of crush-injured sciatic nerves of mice possibly by promoting reprogramming of myelinated Schwann cells toward a repair phenotype. Our preliminary study showed that infusion of GMSC-EVs into the customized AxoGuard Nerve Connectors significantly improved their efficacy on regeneration of transected rat sciatic nerves. Through proteomic profiling, we identified a group of protein factors significantly enriched in GMSC-EVs, among which milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is particularly interesting because of its pleiotropic biological functions. Our preliminary data also showed that MFG-E8 activated pSTAT3 and upregulated gene expressions involved in regulating repair phonotypic conversion of Schwann cells. We hypothesize that enriched MFG-E8 contributes a major role in GMSC-EVs mediated therapeutic effects on nerve regeneration by promoting the repair phenotypic conversion of Schwann cells. We proposed two specific aims to test the hypothesis: 1) determine the pleiotropic effects of MFG-E8 enriched in GMSC-EVs on the repair phenotypic conversion of Schwann cells; 2) investigate the critical role of MFG-E8 in GMSC EV-mediated therapeutic effects on regeneration of transected rat sciatic nerves. Our long-term goal is to develop a cell-free/stem cell- based product as a potential alternative to nerve autografts for regeneration of PNI with a large gap. Results from this study will lead us to submit an R01 grant application that will allow us to better understand the mechanisms underlying MSC-EV mediated therapeutic effects on nerve regeneration and to further explore the safety and efficacy of MSC-EVs in nerve regeneration in large animals and early human clinical trials.