A New Extensively Characterised Conditionally Immortal Muscle Cell-Line for Investigating Therapeutic Strategies in Muscular Dystrophies

A new conditionally immortal satellite cell-derived cell-line, H2K 2B4, was generated from the H2Kb-tsA58 immortomouse. Under permissive conditions H2K 2B4 cells terminally differentiate in vitro to form uniform myotubes with a myogenic protein profile comparable with freshly isolated satellite cells. Following engraftment into immunodeficient dystrophin-deficient mice, H2K 2B4 cells regenerated host muscle with donor derived myofibres that persisted for at least 24 weeks, without forming tumours. These cells were readily transfectable using both retrovirus and the non-viral transfection methods and importantly upon transplantation, were able to reconstitute the satellite cell niche with functional donor derived satellite cells. Finally using the Class II DNA transposon, Sleeping Beauty, we successfully integrated a reporter plasmid into the genome of H2K 2B4 cells without hindering the myogenic differentiation. Overall, these data suggest that H2K 2B4 cells represent a readily transfectable stable cell-line in which to investigate future stem cell based therapies for muscle disease.本研究从H2Kb-tsA58永生化小鼠（immortomouse）中构建得到一株全新的条件永生化肌卫星细胞来源细胞系H2K 2B4。在允许性培养条件下，H2K 2B4细胞可在体外发生终末分化，形成均一的肌管（myotube），其肌源性蛋白表达谱（myogenic protein profile）与新鲜分离的肌卫星细胞（satellite cell）相当。将该细胞系移植至免疫缺陷型肌营养不良蛋白缺陷（dystrophin-deficient）小鼠体内后，H2K 2B4细胞可再生宿主肌肉，生成供体来源的肌纤维（myofibre），该肌纤维可稳定存在至少24周且未形成肿瘤。该细胞可通过逆转录病毒（retrovirus）及非病毒转染方法实现高效转染；尤为关键的是，移植后其可重建具备功能的供体来源肌卫星细胞龛（satellite cell niche）。最后，本研究借助Ⅱ类DNA转座子（Class II DNA transposon）睡美人（Sleeping Beauty）系统，成功将报告质粒（reporter plasmid）整合至H2K 2B4细胞的基因组中，且未对其肌源性分化能力造成影响。综上，上述实验结果表明，H2K 2B4细胞是一种可高效转染的稳定细胞系，可用于未来肌肉疾病干细胞疗法的相关研究。