Modeling Patient-Specific Muscular Dystrophy Phenotypes and Therapeutic Responses in Reprogrammed Myotubes Engineered on Micromolded Gelatin Hydrogels

Abstract

In vitro models of patient-derived muscle allow for more efficient development of genetic medicines for the muscular dystrophies, which often present mutation-specific pathologies. One popular strategy to generate patient-specific myotubes involves reprogramming dermal fibroblasts to a muscle lineage through MyoD induction. However, creating physiologically relevant, reproducible tissues exhibiting multinucleated, aligned myotubes with organized striations is dependent on the introduction of physicochemical cues that mimic the native muscle microenvironment. Here, we engineered patient-specific control and dystrophic muscle tissues in vitro by culturing and differentiating MyoD–directly reprogrammed fibroblasts isolated from one healthy control subject, three patients with Duchenne muscular dystrophy (DMD), and two Limb Girdle 2A/R1 (LGMD2A/R1) patients on micromolded gelatin hydrogels. Engineered DMD and LGMD2A/R1 tissues demonstrated varying levels of defects in α-actinin expression and organization relative to control, depending on the mutation. In genetically relevant DMD tissues amenable to mRNA reframing by targeting exon 44 or 45 exclusion, exposure to exon skipping antisense oligonucleotides modestly increased myotube coverage and alignment and rescued dystrophin protein expression. These findings highlight the value of engineered culture substrates in guiding the organization of reprogrammed patient fibroblasts into aligned muscle tissues, thereby extending their value as tools for exploration and dissection of the cellular and molecular basis of genetic muscle defects, rescue, and repair.

Satellite cell contribution to disease pathology in Duchenne muscular dystrophy

“…Myotubes derived from DMD myoblasts have also been observed to have reduced expression of sarcomeric genes, likely contributing to the characteristic sarcomeric instability seen in DMD skeletal muscle ( Barthélémy et al, 2022 ). Moreover, DMD patient derived human induced pluripotent stem cells differentiated into myotubes have also been shown to undergo pronounced calcium ion (Ca 2+ ) influx upon in vitro contraction ( Shoji et al, 2015 ; Yoshioka et al, 2021 ).…”

Regulation of Satellite Cells Functions during Skeletal Muscle Regeneration: A Critical Step in Physiological and Pathological Conditions

“…Functionally, increased myoblast proliferation, altered chemotaxis, and accelerated differentiation have been described in myoblasts isolated from dystrophic mice and patients [ 134 ]. In vitro differentiation of human pluripotent stem cells lacking dystrophin and DMD myoblasts displayed mis-organization of sarcomeres, contributing to myofibers instability, mis-localization of proteins belonging to the dystrophin-associated complex, myotubes branching, contraction defects and hyperactivation of calcium signaling [ 135 , 136 , 137 ].…”