高纯度快速扩增克隆（RECs）是用于功能线粒体转移的理想选择。
Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer.
机构信息
Department of Pediatrics, Faculty of Medicine, Shimane University, 89-1, Enya, Izumo, Shimane, 693-8501, Japan.
Faculty of Nursing, Inner Mongolia Medical University, Hohhot, Inner Mongolia, China.
出版信息
Stem Cell Res Ther. 2023 Mar 16;14(1):40. doi: 10.1186/s13287-023-03274-y.
BACKGROUND
Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer.
METHODS
We established mitochondria-deficient cell lines (ρ A549 and ρ HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors.
RESULTS
Co-culture of ρ cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore.
CONCLUSIONS
RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.
背景
由线粒体 DNA（mtDNA）或核 DNA 突变引起的线粒体功能障碍，这些 DNA 编码了线粒体的组成部分，与各种遗传和先天性疾病有关。这些线粒体疾病不仅损害了能量的产生，还影响了线粒体的功能，且目前尚无有效的治疗方法。间充质干细胞（MSCs）已知会迁移到受损部位并进行线粒体转移。使用传统培养方法培养的 MSCs 表现出异质的细胞特征。相比之下，通过单细胞分选分离的高度纯化的 MSCs，即快速扩增克隆（RECs），具有均匀的 MSCs 功能。因此，我们研究了 RECs 和 MSCs 之间的差异，以评估线粒体转移的效果。
方法
我们使用溴化乙锭建立了线粒体缺陷细胞系（ρ A549 和 ρ HeLa 细胞系）。通过 PCR 和流式细胞术分析证实了从 RECs/MSCs 到 ρ 细胞的线粒体转移。我们检查了包括 ATP、活性氧、线粒体膜电位和耗氧量（OCR）在内的几种线粒体功能。使用转染实验和分子抑制剂，通过抑制微管/隧道纳米管、间隙连接或微泡的途径来鉴定线粒体转移的途径。
结果
ρ 细胞与 MSCs 或 RECs 共培养后，mtDNA 含量得到恢复。与 MSCs 相比，REC 向 ρ 细胞转移了更多的线粒体。与与 MSCs 共培养的细胞相比，与 REC 共培养的 ρ 细胞中线粒体功能的恢复，包括 ATP、OCR、线粒体膜电位和线粒体肿胀，更为优越。每条途径的抑制试验表明，RECs 对胞吞抑制剂 dynasore 敏感。
结论
通过捐赠健康的线粒体，RECs 可能成为治疗与线粒体功能障碍相关疾病的潜在治疗策略。
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