Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
CellCOM Research Group, Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde (SERGAS), Universidade da Coruña (UDC), A Coruña, Spain.
Stem Cell Res Ther. 2024 Oct 10;15(1):359. doi: 10.1186/s13287-024-03932-9.
The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin 43 (Cx43, encoded by GJA1) and the truncated, internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes.
Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1 + and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 h in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer.
Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes.
Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.
间质基质细胞（MSCs）之间的细胞内线粒体转移现象已显示出在损伤后改善组织愈合的潜力，并有可能治疗骨关节炎（OA）等退行性疾病。最近已经记录了 MSC 向软骨细胞的线粒体转移，但转移的机制尚不清楚。全长连接蛋白 43（Cx43，由 GJA1 编码）和截断的、内部翻译的同工型 GJA1-20k 已被牵连在高度氧化细胞之间的线粒体转移，但尚未在骨科组织中进行探索。在这里，我们的目标是研究 Cx43 在 MSC 向软骨细胞线粒体转移中的作用。在这项研究中，我们检验了以下假设：（a）当软骨细胞处于氧化应激状态时，MSC 向软骨细胞的线粒体转移增加；（b）MSC Cx43 表达介导线粒体向软骨细胞的转移。
使用叔丁基过氧化氢（t-BHP）诱导永生化人软骨细胞发生氧化应激，并评估线粒体膜去极化和活性氧（ROS）的产生。使用慢病毒载体对人骨髓来源的 MSC 进行线粒体荧光转导。使用 siRNA 敲低 MSC Cx43 表达或使用慢病毒转导过表达（GJA1+和 GJA1-20k+）。将软骨细胞和 MSC 共培养 24 小时，直接接触或使用 Transwell 分离。使用流式细胞术定量线粒体转移。固定共培养物并用肌动蛋白和 Cx43 染色，以在转移过程中可视化细胞-细胞相互作用。
t-BHP 应激的软骨细胞中线粒体转移明显增加。接触共培养的线粒体转移明显高于 Transwell 共培养。共焦图像显示 MSC 和软骨细胞之间存在直接细胞接触，其中 Cx43 染色在 MSC 中含有线粒体的肌动蛋白细胞延伸的末端富集。MSC Cx43 表达与向软骨细胞转移的线粒体数量相关；敲低 Cx43 显著降低转移，而过表达 Cx43 则显著增加转移。有趣的是，GJA1-20k 的表达与 MSC 向软骨细胞转移线粒体的发生率高度相关。
MSC 中 GJA1-20k 的过表达增加了向软骨细胞的线粒体转移，这突出了 GJA1-20k 作为一种潜在的靶点，用于促进 MSC 中线粒体转移作为 OA 软骨组织修复的再生治疗。
