Department of Cell Biology and Genetics, Institute for Regenerative Medicine, Texas A&M University School of Medicine, College Station, Texas, USA.
Department of Ophthalmology, Seoul National University College of Medicine, Seoul, South Korea.
J Extracell Vesicles. 2024 Aug;13(8):e12497. doi: 10.1002/jev2.12497.
Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been recognized as promising cytotherapeutics due to their demonstrated immunomodulatory effects in various preclinical models. The immunomodulatory capabilities of EVs stem from the proteins and genetic materials they carry from parent cells, but the cargo contents of EVs are significantly influenced by MSC tissues and donors, cellular age and culture conditions, resulting in functional variations. However, there are no surrogate assays available to validate the immunomodulatory potency of MSC-EVs before in vivo administration. In previous work, we discovered that microcarrier culture conditions enhance the immunomodulatory function of MSC-EVs, as well as the levels of immunosuppressive molecules such as TGF-β1 and let-7b in MSC-EVs. Building on these findings, we investigated whether TGF-β1 levels in MSC-EVs could serve as a surrogate biomarker for predicting their potency in vivo. Our studies revealed a strong correlation between TGF-β1 and let-7b levels in MSC-EVs, as well as their capacity to suppress IFN-γ secretion in stimulated splenocytes, establishing biopotency and surrogate assays for MSC-EVs. Subsequently, we validated MSC-EVs generated from monolayer cultures (ML-EVs) or microcarrier cultures (MC-EVs) using murine models of experimental autoimmune uveoretinitis (EAU) and additional in vitro assays reflecting the Mode of Action of MSC-EVs in vivo. Our findings demonstrated that MC-EVs carrying high levels of TGF-β1 exhibited greater efficacy than ML-EVs in halting disease progression in mice with EAU as well as inducing apoptosis and inhibiting the chemotaxis of retina-reactive T cells. Additionally, MSC-EVs suppressed the MAPK/ERK pathway in activated T cells, with treatment using TGF-β1 or let-7b showing similar effects on the MAPK/ERK pathway. Collectively, our data suggest that MSC-EVs directly inhibit the infiltration of retina-reactive T cells toward the eyes, thereby halting the disease progression in EAU mice, and their immunomodulatory potency in vivo can be predicted by their TGF-β1 levels.
细胞外囊泡 (EVs) 来源于间充质干细胞 (MSCs)，由于其在各种临床前模型中表现出的免疫调节作用，被认为是有前途的细胞治疗药物。EVs 的免疫调节能力源于它们从亲本细胞携带的蛋白质和遗传物质，但 EVs 的货物含量受到 MSC 组织和供体、细胞年龄和培养条件的显著影响，导致功能变化。然而，在体内给药之前，没有替代检测方法来验证 MSC-EVs 的免疫调节效力。在之前的工作中，我们发现微载体培养条件增强了 MSC-EVs 的免疫调节功能，以及 MSC-EVs 中免疫抑制分子如 TGF-β1 和 let-7b 的水平。基于这些发现，我们研究了 MSC-EVs 中的 TGF-β1 水平是否可以作为预测其体内效力的替代生物标志物。我们的研究表明，MSC-EVs 中 TGF-β1 和 let-7b 的水平之间存在很强的相关性，以及它们抑制刺激的脾细胞中 IFN-γ 分泌的能力，从而建立了 MSC-EVs 的生物效力和替代检测方法。随后，我们使用实验性自身免疫性葡萄膜炎 (EAU) 的小鼠模型和反映 MSC-EVs 在体内作用模式的其他体外检测方法，验证了来自单层培养 (ML-EVs) 或微载体培养 (MC-EVs) 的 MSC-EVs。我们的研究结果表明，携带高 TGF-β1 水平的 MC-EVs 在阻止 EAU 小鼠疾病进展以及诱导凋亡和抑制视网膜反应性 T 细胞趋化方面比 ML-EVs 更有效。此外，MSC-EVs 抑制了激活的 T 细胞中的 MAPK/ERK 途径，用 TGF-β1 或 let-7b 处理对 MAPK/ERK 途径显示出相似的作用。总之，我们的数据表明，MSC-EVs 直接抑制视网膜反应性 T 细胞向眼睛的浸润，从而阻止 EAU 小鼠的疾病进展，其体内免疫调节效力可以通过 TGF-β1 水平来预测。
