Facioscapulohumeral muscular dystrophy (FSHD) is caused by sporadic misexpression of the transcription factor double homeobox 4 (DUX4) in skeletal muscles. So far, monolayer cultures and animal models have been used to study the FSHD disease mechanism and for FSHD therapy development, but these models do not fully recapitulate the disease and there is a lack of knowledge on how DUX4 misexpression leads to skeletal muscle dysfunction. To overcome these barriers, we have developed a three-dimensional tissue engineered skeletal muscle (3D-TESM) model by generating genetically matched myogenic progenitors (MPs) from human induced pluripotent stem cells of three mosaic FSHD patients. 3D-TESMs derived from genetically affected MPs recapitulate pathological features including DUX4 and DUX4 target gene expression, smaller myofiber diameters, and reduced absolute forces upon electrical stimulation. RNA sequencing data illustrates increased expression of DUX4 target genes in 3D-TESMs compared to two-dimensional (2D) myotubes, and cellular differentiation was improved by 3D culture conditions. Treatment of 3D-TESMs with three different small molecules identified in drug development screens in 2D muscle cultures showed no improvements, and sometimes even declines, in contractile force and sarcomere organization. These results suggest that these compounds either have a detrimental effect on the formation of 3D-TESMs, an effect that might have been overlooked or was challenging to detect in 2D cultures and in vivo models, and/or that further development of the 3D-TESM model is needed. In conclusion, we have developed a 3D skeletal muscle model for FSHD that can be employed for preclinical research focusing on DUX4 expression and downstream pathways of FSHD in relation to contractile properties. In the future, we expect that this model can also be used for preclinical drug screening.

中文翻译：

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三维组织工程骨骼肌建模面肩肱肌营养不良症


面肩肱肌营养不良症 （FSHD） 是由骨骼肌中转录因子双同源框 4 （DUX4） 的散发性错误表达引起的。到目前为止，单层培养物和动物模型已被用于研究 FSHD 疾病机制和 FSHD 疗法的发展，但这些模型并不能完全概括疾病，并且缺乏关于 DUX4 错误表达如何导致骨骼肌功能障碍的知识。为了克服这些障碍，我们通过从三名马赛克 FSHD 患者的人类诱导多能干细胞中生成遗传匹配的肌源性祖细胞 （MP），开发了一种三维组织工程骨骼肌 （3D-TESM） 模型。源自受遗传影响的 MP 的 3D-TESM 概括了病理特征，包括 DUX4 和 DUX4 靶基因表达、更小的肌纤维直径和电刺激时降低的绝对力。RNA 测序数据表明，与二维 （2D） 肌管相比，3D-TESM 中 DUX4 靶基因的表达增加，并且 3D 培养条件改善了细胞分化。在 2D 肌肉培养物的药物开发筛选中鉴定的三种不同小分子对 3D-TESM 进行治疗，收缩力和肌节组织没有改善，有时甚至下降。这些结果表明，这些化合物要么对 3D-TESM 的形成产生不利影响，这种影响可能被忽视或在 2D 培养物和体内模型中难以检测，和/或需要进一步开发 3D-TESM 模型。 总之，我们开发了一种用于 FSHD 的 3D 骨骼肌模型，可用于临床前研究，重点关注 DUX4 表达和 FSHD 与收缩特性相关的下游途径。未来，我们预计该模型也可用于临床前药物筛选。