Key result
Puromycin-selected bulk MYOD1-hiPSCs rapidly differentiate into contractible skeletal muscle matching clonal cell properties.
Why the study?
Modeling muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles, but existing MYOD1-hiPSC methods require time- and labor-consuming clonal selection and must overcome clonal variations.
Bulk differentiation of MYOD1-hiPSCs using puromycin selection offers a faster, less labor-intensive method to generate functional skeletal muscle for disease modeling compared to clonal selection.
May accelerate scalable hiPSC skeletal muscle modeling; leaves open human translation and therapeutic validation.
Pathophysiological analysis and drug discovery targeting human diseases require disease models that suitably recapitulate patient pathology. Disease-specific human induced pluripotent stem cells (hiPSCs) differentiated into affected cell types can potentially recapitulate disease pathology more accurately than existing disease models. Such successful modeling of muscular diseases requires efficient differentiation of hiPSCs into skeletal muscles. hiPSCs transduced with doxycycline-inducible MYOD1 (MYOD1-hiPSCs) have been widely used; however, they require time- and labor-consuming clonal selection, and clonal variations must be overcome. Moreover, their functionality should be carefully examined. Here, we demonstrated that bulk MYOD1-hiPSCs established with puromycin selection rather than G418 selection showed rapid and highly efficient differentiation. Interestingly, bulk MYOD1-hiPSCs exhibited average differentiation properties of clonally established MYOD1-hiPSCs, suggesting that it is possible to minimize clonal variations. Moreover, disease-specific hiPSCs of spinal bulbar muscular atrophy (SBMA) could be efficiently differentiated via this method into skeletal muscle that showed disease phenotypes, suggesting the applicability of this method for disease analysis. Finally, three-dimensional muscle tissues were fabricated from bulk MYOD1-hiPSCs, which exhibited contractile force upon electrical stimulation, indicating their functionality. Thus, our bulk differentiation requires less time and labor than existing methods, efficiently generates contractible skeletal muscles, and may facilitate the generation of muscular disease models.
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Rashid et al. (2023) studied Muscular disease modeling (Spinal bulbar muscular atrophy). Bulk MYOD1-hiPSCs with puromycin selection vs. Clonal MYOD1-hiPSCs and G418 selection was evaluated on Differentiation efficiency into skeletal muscle (MyoG+ cells, MHC+ nuclei). Bulk MYOD1-hiPSCs established with puromycin selection rapidly and efficiently differentiated into contractible skeletal muscles, exhibiting average differentiation properties of clonally established cells.
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