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

项目摘要/摘要 周围神经损伤(PNI)是临床常见而复杂的挑战。自体神经移植仍是“黄金” 标准“重建PNI有很大的间隙，但供体神经的可用性有限，几个严重的 供体部位的发病率严重阻碍了其临床应用。组织工程的组合应用 含有支持细胞和生物活性因子的神经引导导管(NGCs)有可能成为替代材料 自体神经移植。基于间充质干细胞(MSC)的治疗在再生方面显示出希望 由于它们具有多能性、免疫调节/消炎和再生潜力。这个 骨髓间充质干细胞的治疗效果很可能依赖于其分泌体，尤其是分泌细胞外。 囊泡通过转运多种生物活性物质，在细胞间的通讯中发挥重要作用。 分子，如脂类、蛋白质和非编码RNA。由于它们的治疗效果与父母相当 细胞，MSC-EVS已被探索为治疗一系列疾病的新型无细胞疗法。然而， MSC-EVS在周围神经再生中的潜在应用在很大程度上仍未被探索。我们最近做了 研究表明，人牙龈来源的间充质干细胞(GMSCs)及其释放的EV具有类似的治疗效果 大鼠味蕾/味觉神经再生及挤压伤修复/再生的影响 小鼠坐骨神经可能通过促进有髓雪旺细胞的重编程而修复 表型。我们的初步研究表明，GMSC-EVS注入定制的AxoGuard神经 连接器显著提高了大鼠坐骨神经横断后的再生效率。穿过 蛋白质组学方面，我们鉴定了一组显著富含GMSC-EVS的蛋白质因子，其中 乳脂球-表皮生长因子(MFG-E8)因其多效性而备受关注 生物功能。我们的初步数据还表明，MFG-E8激活了pSTAT3并上调了基因 参与调节雪旺细胞修复音型转换的表达。我们假设 富含MFG-E8在GMSC-EVS介导的神经再生治疗中的重要作用 通过促进雪旺细胞的修复表型转换。我们提出了两个具体目标来测试 假设：1)确定富含GMSC-EVS的MFG-E8对修复表型的多效性作用 2)研究MFG-E8在GMSC EV介导的治疗中的关键作用 对大鼠坐骨神经横断后再生的影响。我们的长期目标是开发一种无细胞/干细胞- 作为一种潜在的替代自体神经移植用于PNI再生的产品，具有较大的缺口。结果 这项研究将引导我们提交R01拨款申请，使我们能够更好地了解 MSC-EV介导的神经再生治疗作用机制及进一步探讨 MSC-EVS在大动物神经再生和早期人类临床试验中的安全性和有效性。

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.