Liu Wei, Wang Xinyu, Chen Yating, Yuan Jiapei, Zhang Huiyu, Jin Xin, Jiang Yuying, Cao Junjing, Wang Zibin, Yang Shuo, Wang Bingwei, Wu Tinghe, Li Jing
State Key Laboratory of Reproductive Medicine and offspring health, Nanjing Medical University, Nanjing, China.
Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Extracell Vesicles. 2025 Jan;14(1):e70029. doi: 10.1002/jev2.70029.
As functional derivatives of mesenchymal stem cells (MSCs), small extracellular vesicles (sEVs) have garnered significant attention and application in regenerative medicine. However, the technical limitations for large-scale isolation of sEVs and their heterogeneous nature have added complexity to their applications. It remains unclear if the heterogeneous sEVs represent different aspects of MSCs functions. Here, we provide a method for the large-scale production of sEVs subpopulations derived from human umbilical cord mesenchymal stem cells (HucMSCs), utilizing tangential flow filtration combined with size exclusion chromatography. The resulting subpopulations, S1-sEVs and S2-sEVs, exhibited stable variations in size, membrane-marked proteins, and carrying cargos, thereby displaying distinct functions both in vitro and in animal disease models. S1-sEVs, that highly expressed CD9, HRS and GPC1, demonstrated a greater immunomodulatory impact, while S2-sEVs with enriched expression of CD63 and FLOT1/2 possessed enhanced capacities in promoting cell proliferation and angiogenesis. These discrepancies are attributed to the specific proteins and miRNAs they contain. Further investigation revealed that the two distinct sEVs subpopulations corresponded to different biological processes: the ESCRT pathway (S1-sEVs) and the ESCRT-independent pathway represented by lipid rafts (S2-sEVs). Therefore, we propose the potential for large-scale isolation and purification of sEVs subpopulations from HucMSCs with distinct functions. This approach may provide advantages for targeted therapeutic interventions in various MSC indications.
作为间充质干细胞（MSCs）的功能性衍生物，小细胞外囊泡（sEVs）在再生医学中受到了广泛关注并得到应用。然而，sEVs大规模分离的技术限制及其异质性增加了其应用的复杂性。目前尚不清楚异质性的sEVs是否代表了MSCs功能的不同方面。在此，我们提供了一种利用切向流过滤结合尺寸排阻色谱法从人脐带间充质干细胞（HucMSCs）大规模生产sEVs亚群的方法。所得的亚群S1-sEVs和S2-sEVs在大小、膜标记蛋白和携带的货物方面表现出稳定的差异，从而在体外和动物疾病模型中显示出不同的功能。高表达CD9、HRS和GPC1的S1-sEVs表现出更大的免疫调节作用，而富含CD63和FLOT1/2表达的S2-sEVs在促进细胞增殖和血管生成方面具有增强的能力。这些差异归因于它们所含的特定蛋白质和miRNAs。进一步的研究表明，这两个不同的sEVs亚群对应于不同的生物学过程：ESCRT途径（S1-sEVs）和以脂筏为代表的ESCRT非依赖途径（S2-sEVs）。因此，我们提出了从HucMSCs大规模分离和纯化具有不同功能的sEVs亚群的潜力。这种方法可能为各种MSCs适应症的靶向治疗干预提供优势。
