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 mdx 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 mdx/scid mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MB^N)] and normal human bone marrow-derived MSC (MSC^N) 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^N/MSC^N 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^N/MSC^N DEC efficacy in vivo, gastrocnemius muscle of mdx/scid mice were injected with vehicle (n = 12), nonfused MB^N and MSC^N (n = 9, 0.25 × 10⁶/each) or MB^N/MSC^N DEC (n = 9, 0.5 × 10⁶). 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^N/MSC^N DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls (P < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, P < 0.05) correlated with improved muscle strength and function in mdx/scid mice. The created human MB^N/MSC^N 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.

中文翻译：

---

表达肌营养不良蛋白的成肌细胞/间充质干细胞来源的嵌合人细胞移植可改善杜氏肌营养不良模型的功能


杜氏肌营养不良症 (DMD) 是一种由肌营养不良蛋白基因突变引起的致命性 X 连锁疾病。目前，DMD 尚无治愈方法。细胞疗法面临有限的植入和排斥的挑战。因此，DMD 需要更有效、更安全的治疗方法。我们之前报道过，在 DMD 的mdx小鼠模型中，移植表达肌营养不良蛋白的成肌细胞来源的嵌合 (DEC) 细胞后，肌营养不良蛋白表达增加，与功能改善相关。本研究建立了新的成肌细胞和间充质干细胞（MSC）来源的DEC细胞系，并测试了其在DMD mdx / scid小鼠模型中的功效和治疗潜力。使用聚乙二醇对来自正常供体的同种异体人成肌细胞[正常成肌细胞（MB ^N ）]和正常人骨髓来源的MSC（MSC ^N ）进行15次离体细胞融合。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 ^N /MSC ^N DEC细胞的嵌合状态，而彗星测定评估融合程序安全性测试遗传毒性。免疫荧光和实时 PCR 评估肌营养不良蛋白表达和肌源性分化。混合淋巴细胞反应（MLR）评估DEC的免疫原性。为了测试 MB ^N /MSC ^N DEC 体内功效， mdx/scid小鼠的腓肠肌注射载体 ( n = 12)、未融合的 MB ^N和 MSC ^N ( n = 9, 0.25 × 10 ⁶ /每只) 或 MB ^N /MSC ^N DEC ( n = 9, 0.5 × 10 ⁶ )。使用离体和体内肌肉力量测试对动物进行为期 90 天的评估。组织学和免疫荧光染色评估肌营养不良蛋白表达、中央有核纤维和疤痕组织形成。融合后，MB ^N /MSC ^N DEC 嵌合状态、肌原性分化和肌营养不良蛋白表达得到证实。与对照组相比，MLR 表明 DEC 的免疫反应降低（ P < 0.05）。 DEC 移植后 90 天， mdx / scid小鼠中肌营养不良蛋白表达的增加（20.26% ± 2.5%， P < 0.05）与肌肉力量和功能的改善相关。创建的人类 MB ^N /MSC ^N DEC 细胞系引入​​了结合 MB 和 MSC 的生肌和免疫调节特性的新型治疗方法，因此可能为 DMD 肌肉再生开辟一种通用方法。