Department of Toxicogenomics, Maastricht University Medical Centre+, 6229 HX Maastricht, The Netherlands.
School for Developmental Biology and Oncology (GROW), Maastricht University Medical Centre+, 6229 HX Maastricht, The Netherlands.
Int J Mol Sci. 2023 Jan 31;24(3):2679. doi: 10.3390/ijms24032679.
In 25% of patients with mitochondrial myopathies, pathogenic mitochondrial DNA (mtDNA) mutation are the cause. For heteroplasmic mtDNA mutations, symptoms manifest when the mutation load exceeds a tissue-specific threshold. Therefore, lowering the mutation load is expected to ameliorate disease manifestations. This can be achieved by fusing wild-type mesoangioblasts with mtDNA mutant myotubes. We have tested this in vitro for female carriers of the m.3271T>C or m.3291T>C mutation (mutation load >90%) using wild-type male mesoangioblasts. Individual fused myotubes were collected by a newly-developed laser capture microdissection (LCM) protocol, visualized by immunostaining using an anti-myosin antibody. Fusion rates were determined based on male-female nuclei ratios by fluorescently labelling the Y-chromosome. Using combined 'wet' and 'air dried' LCM imaging improved fluorescence imaging quality and cell yield. Wild-type mesoangioblasts fused in different ratios with myotubes containing either the m.3271T>C or the m.3291T>C mutation. This resulted in the reduction of the mtDNA mutation load proportional to the number of fused wild-type mesoangioblasts for both mtDNA mutations. The proportional reduction in mtDNA mutation load in vitro after fusion is promising in the context of muscle stem cell therapy for mtDNA mutation carriers in vivo, in which we propose the same strategy using autologous wild-type mesoangioblasts.
在 25%的线粒体肌病患者中，致病性线粒体 DNA（mtDNA）突变是病因。对于异质性 mtDNA 突变，当突变负荷超过组织特异性阈值时，症状就会显现。因此，降低突变负荷有望改善疾病表现。这可以通过将野生型间充质成肌细胞与 mtDNA 突变肌管融合来实现。我们使用野生型雄性间充质成肌细胞对携带 m.3271T>C 或 m.3291T>C 突变（突变负荷>90%）的女性携带者进行了体外测试。通过新开发的激光捕获显微切割（LCM）方案收集单个融合肌管，使用抗肌球蛋白抗体进行免疫染色进行可视化。融合率基于雄性-雌性核比值通过荧光标记 Y 染色体来确定。使用联合的“湿”和“空气干燥”LCM 成像提高了荧光成像质量和细胞产量。野生型间充质成肌细胞以不同的比例与含有 m.3271T>C 或 m.3291T>C 突变的肌管融合。对于这两种 mtDNA 突变，融合后 mtDNA 突变负荷的比例降低与融合的野生型间充质成肌细胞的数量成正比。体外融合后 mtDNA 突变负荷的比例降低，为体内携带 mtDNA 突变的肌肉干细胞治疗提供了希望，我们建议使用自体野生型间充质成肌细胞采用相同的策略。
