Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, 187-8502, Japan.
Department of NCNP Brain Physiology and Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Sci Rep. 2024 Jan 28;14(1):2329. doi: 10.1038/s41598-024-52530-5.
Human urine-derived cells (UDCs) are primary cultured cells originating from the upper urinary tract and are known to be multipotent. We previously developed MYOD1-transduced UDCs (MYOD1-UDCs) as a model recapitulating the pathogenesis of Duchenne muscular dystrophy (DMD) caused by a lack of dystrophin. MYOD1-UDCs also allow evaluation of the efficacy of exon skipping with antisense oligonucleotides. However, despite the introduction of MYOD1, some MYOD1-UDCs failed to form myotubes, possibly because of heterogeneity among UDCs. Here, we carried out single-cell RNA-sequencing analyses and revealed that CD90/Thy-1 was highly expressed in a limited subpopulation of UDCs with high myogenic potency. Furthermore, CD90-positive MYOD1-UDCs, but not CD90-negative cells, could form myotubes expressing high levels of myosin heavy chain and dystrophin. Notably, overexpression of CD90 in CD90-negative MYOD1-UDCs did not enhance myogenic differentiation, whereas CD90 suppression in CD90-positive UDCs led to decreased myotube formation and decreased myosin heavy chain expression. CD90 may thus contribute to the fusion of single-nucleated MYOD1-UDCs into myotubes but is not crucial for promoting the expression of late muscle regulatory factors. Finally, we confirmed that CD90-positive MYOD1-UDCs derived from patients with DMD were a valuable tool for obtaining a highly reproducible and stable evaluation of exon skipping using antisense oligonucleotide.
人尿源性细胞（UDC）是源自上尿路的原代培养细胞，已知具有多能性。我们之前开发了 MYOD1 转导的 UDC（MYOD1-UDC）作为一种模型，可重现由于缺乏肌营养不良蛋白而导致的杜氏肌营养不良症（DMD）的发病机制。MYOD1-UDC 还可以评估反义寡核苷酸的外显子跳跃的疗效。然而，尽管引入了 MYOD1，一些 MYOD1-UDC 未能形成肌管，可能是因为 UDC 之间存在异质性。在这里，我们进行了单细胞 RNA-seq 分析，结果表明 CD90/Thy-1 在具有高成肌能力的 UDC 的有限亚群中高度表达。此外，CD90 阳性的 MYOD1-UDC 可以形成肌管，表达高水平的肌球蛋白重链和肌营养不良蛋白，而 CD90 阴性细胞则不能。值得注意的是，CD90 过表达在 CD90 阴性的 MYOD1-UDC 中并没有增强成肌分化，而 CD90 在 CD90 阳性的 UDC 中的抑制导致肌管形成减少和肌球蛋白重链表达减少。因此，CD90 可能有助于将单核的 MYOD1-UDC 融合成肌管，但对于促进晚期肌肉调节因子的表达并不是至关重要的。最后，我们证实了源自 DMD 患者的 CD90 阳性 MYOD1-UDC 是一种非常有价值的工具，可用于使用反义寡核苷酸获得高度重现性和稳定的外显子跳跃评估。
