Thomas Matthew A, Fahey Megan J, Pugliese Brenna R, Irwin Rebecca M, Antonyak Marc A, Delco Michelle L
Cornell University College of Veterinary Medicine, Department of Clinical Sciences, Ithaca, NY, United States.
Cornell University College of Veterinary Medicine, Department of Molecular Medicine, Ithaca, NY, United States.
Front Bioeng Biotechnol. 2022 Aug 19;10:870193. doi: 10.3389/fbioe.2022.870193. eCollection 2022.
Cartilage and other skeletal soft tissues heal poorly after injury, in part due to their lack of vascularity and low metabolic rate. No pharmacologic approaches have proven effective in preventing chronic degenerative disease after joint injury. Mesenchymal stromal cells (MSCs) have been investigated for their ability to treat pain associated with osteoarthritis (OA) and preserve articular cartilage. Limitations of MSCs include variability in cell phenotype, low engraftment and retention rates, and inconsistent clinical outcomes. Therefore, acellular biologic therapies such as extracellular vesicles (EVs) are currently being investigated. MSC-derived EVs have been found to replicate many of the therapeutic effects of their cells of origin, but the mechanisms driving this remain unclear. Recent evidence in non-orthopedic tissues suggests MSCs can rescue injured cells by donating mitochondria, restoring mitochondrial function in recipient cells, preserving cell viability, and promoting tissue repair. Our group hypothesized that MSCs package mitochondria for export into EVs, and that these so-called "mitoEVs" could provide a delivery strategy for cell-free mitochondria-targeted therapy. Therefore, the goals of this study were to: 1) characterize the vesicle fractions of the MSCs secretome with respect to mitochondrial cargoes, 2) determine if MSC-EVs contain functional mitochondria, and 3) determine if chondrocytes can take up MSC-derived mitoEVs. We isolated exosome, microvesicle, and vesicle-free fractions from MSC-conditioned media. Using a combination of dynamic light scattering and nanoparticle tracking, we determined that MSC-EV populations fall within the three size categories typically used to classify EVs (exosomes, microvesicles, apoptotic bodies). Fluorescent nanoparticle tracking, immunoblotting, and flow cytometry revealed that mitochondrial cargoes are abundant across all EV size populations, and mitoEVs are nearly ubiquitous among the largest EVs. Polarization staining indicated a subset of mitoEVs contain functional mitochondria. Finally, flow cytometry and fluorescent imaging confirmed uptake of mitoEVs by chondrocytes undergoing rotenone/antimycin-induced mitochondrial dysfunction. These data indicate that MSCs package intact, functional mitochondria into EVs, which can be transferred to chondrocytes in the absence of direct cell-cell interactions. This work suggests intercellular transfer of healthy MT to chondrocytes could represent a new, acellular approach to augment mitochondrial content and function in poorly-healing avascular skeletal soft tissues.
软骨和其他骨骼软组织损伤后愈合较差，部分原因是它们缺乏血管且代谢率低。尚无药物疗法被证明对预防关节损伤后的慢性退行性疾病有效。间充质基质细胞（MSCs）因其治疗骨关节炎（OA）相关疼痛和保护关节软骨的能力而受到研究。MSCs的局限性包括细胞表型的变异性、低植入率和保留率以及不一致的临床结果。因此，目前正在研究细胞外囊泡（EVs）等无细胞生物疗法。已发现源自MSCs的EVs可复制其来源细胞的许多治疗效果，但其驱动机制仍不清楚。非骨科组织的最新证据表明，MSCs可通过捐赠线粒体来拯救受损细胞，恢复受体细胞中的线粒体功能，维持细胞活力并促进组织修复。我们的研究小组推测，MSCs将线粒体包装进EVs中，而这些所谓的“线粒体EVs”可为无细胞线粒体靶向治疗提供一种递送策略。因此，本研究的目标是：1）根据线粒体货物对MSCs分泌组的囊泡部分进行表征，2）确定MSC-EVs是否含有功能性线粒体，3）确定软骨细胞是否能摄取源自MSCs的线粒体EVs。我们从MSC条件培养基中分离出外泌体、微囊泡和无囊泡部分。通过结合动态光散射和纳米颗粒跟踪，我们确定MSC-EV群体属于通常用于分类EVs的三种大小类别（外泌体、微囊泡、凋亡小体）。荧光纳米颗粒跟踪、免疫印迹和流式细胞术显示，线粒体货物在所有EV大小群体中都很丰富，并且线粒体EVs在最大的EVs中几乎普遍存在。极化染色表明一部分线粒体EVs含有功能性线粒体。最后，流式细胞术和荧光成像证实了遭受鱼藤酮/抗霉素诱导的线粒体功能障碍的软骨细胞摄取了线粒体EVs。这些数据表明，MSCs将完整的功能性线粒体包装进EVs中，在没有直接细胞间相互作用的情况下，这些EVs可转移至软骨细胞。这项工作表明，将健康线粒体细胞间转移至软骨细胞可能代表一种新的无细胞方法，可增加愈合不良的无血管骨骼软组织中的线粒体含量和功能。
