Department of Orthopaedics, University of Illinois at Chicago, Chicago, Illinois, USA.
Department of Human Epigenetics, Mossakowski Medical Research Center Polish Academy of Science, Warsaw, Poland.
Stem Cells Dev. 2021 Feb;30(4):190-202. doi: 10.1089/scd.2020.0161. Epub 2021 Jan 22.
Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in dystrophin gene. Currently, there is no cure for DMD. Cell therapies are challenged by limited engraftment and rejection. Thus, more effective and safer therapeutic approaches are needed for DMD. We previously reported increased dystrophin expression correlating with improved function after transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin in the mouse models of DMD. This study established new DEC cell line of myoblasts and mesenchymal stem cells (MSC) origin and tested its efficacy and therapeutic potential in / mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MB)] and normal human bone marrow-derived MSC (MSC) from normal donors were performed using polyethylene glycol. Flow cytometry, confocal microscopy, polymerase chain reaction (PCR)-short tandem repeats, polymerase chain reaction-reverse sequence-specific oligonucleotide probe assessed chimeric state of fused MB/MSC DEC cells, whereas Comet assay assessed fusion procedure safety testing genotoxicity. Immunofluorescence and real-time PCR assessed dystrophin expression and myogenic differentiation. Mixed lymphocyte reaction (MLR) evaluated DEC's immunogenicity. To test MB/MSC DEC efficacy in vivo, gastrocnemius muscle of mice were injected with vehicle ( = 12), nonfused MB and MSC ( = 9, 0.25 × 10/each) or MB/MSC DEC ( = 9, 0.5 × 10). Animals were evaluated for 90 days using ex vivo and in vivo muscle strength tests. Histology and immunofluorescence staining assessed dystrophin expression, centrally nucleated fibers and scar tissue formation. Post-fusion, MB/MSC DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls ( < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, < 0.05) correlated with improved muscle strength and function in / mice. The created human MB/MSC DEC cell line introduces novel therapeutic approach combining myogenic and immunomodulatory properties of MB and MSC, and as such may open a universal approach for muscle regeneration in DMD.
杜氏肌营养不良症（DMD）是一种致命的 X 连锁疾病，由肌营养不良蛋白基因突变引起。目前，DMD 尚无治愈方法。细胞疗法受到移植物有限和排斥的挑战。因此，需要更有效和更安全的治疗方法来治疗 DMD。我们之前报道过，在 DMD 的小鼠模型中，移植表达肌营养不良蛋白的嵌合（DEC）细胞后，肌营养不良蛋白表达增加与功能改善相关。本研究建立了新的肌源性和间充质干细胞（MSC）来源的 DEC 细胞系，并在 DMD 的 / 小鼠模型中测试了其疗效和治疗潜力。使用聚乙二醇对同种异体人肌母细胞[正常肌母细胞（MB）]和正常供体来源的正常人类骨髓衍生 MSC（MSC）进行了 15 次体外细胞融合。流式细胞术、共聚焦显微镜、聚合酶链反应（PCR）-短串联重复序列、聚合酶链反应-反向序列特异性寡核苷酸探针评估融合 MB/MSC DEC 细胞的嵌合状态，而彗星试验评估融合程序的安全性和遗传毒性。免疫荧光和实时 PCR 评估肌营养不良蛋白表达和肌生成分化。混合淋巴细胞反应（MLR）评估 DEC 的免疫原性。为了测试 MB/MSC DEC 在体内的疗效，将载体（ = 12）、未融合的 MB 和 MSC（ = 9，0.25 × 10/每个）或 MB/MSC DEC（ = 9，0.5 × 10）注入 / 小鼠的腓肠肌。使用体外和体内肌肉力量测试在 90 天内评估动物。组织学和免疫荧光染色评估肌营养不良蛋白表达、中央核纤维和疤痕组织形成。融合后，确认 MB/MSC DEC 的嵌合状态、肌生成分化和肌营养不良蛋白表达。MLR 显示与对照相比，DEC 的免疫反应降低（ < 0.05）。在 DEC 移植后 90 天，/ 小鼠的肌营养不良蛋白表达增加（20.26%±2.5%， < 0.05）与肌肉力量和功能的改善相关。创建的人 MB/MSC DEC 细胞系引入了一种新的治疗方法，结合了 MB 和 MSC 的成肌和免疫调节特性，因此可能为 DMD 的肌肉再生开辟一种通用方法。
