Engineered skeletal muscle recapitulates human muscle development, regeneration and dystrophy

Abstract

Background Human pluripotent stem cell-derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for the derivation of skeletal muscle cells and their utility in skeletal muscle tissue engineering with the aim to model skeletal muscle regeneration and dystrophy in vitro. Methods Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors followed by primary and secondary foetal myogenesis into three-dimensional muscle. To simulate Duchenne muscular dystrophy (DMD), a patient-specific induced pluripotent stem cell line was compared to a CRISPR/Cas9-edited isogenic control line. ResultsThe established skeletal muscle differentiation protocol robustly and faithfully recapitulates critical steps of embryonic myogenesis in two-dimensional and three-dimensional cultures, resulting in functional human skeletal muscle organoids (SMOs) and engineered skeletal muscles (ESMs) with a regeneration-competent satellite-like cell pool. Tissue-engineered muscle exhibits organotypic maturation and function (up to 5.7 ± 0.5 mN tetanic twitch tension at 100 Hz in ESM). Contractile performance could be further enhanced by timed thyroid hormone treatment, increasing the speed of contraction (time to peak contraction) as well as relaxation (time to 50% relaxation) of single twitches from 107 ± 2 to 75 ± 4 ms (P < 0.05) and from 146 ± 6 to 100 ± 6 ms (P < 0.05), respectively. Satellite-like cells could be documented as largely quiescent PAX7 + cells (75 ± 6% Ki67 À ) located adjacent to muscle fibres confined under a laminin-containing basal membrane. Activation of the engineered satellite-like cell niche was documented in a cardiotoxin injury model with marked recovery of contractility to 57 ± 8% of the pre-injury force 21 days post-injury (P < 0.05 compared to Day 2 post-injury), which was completely blocked by preceding irradiation. Absence of dystrophin in DMD ESM caused a marked reduction of contractile force (À35 ± 7%, P < 0.05) and impaired expression of fast myosin isoforms resulting in prolonged contraction (175 ± 14 ms, P < 0.05 vs. gene-edited control) and relaxation (238 ± 22 ms, P < 0.05 vs. gene-edited control) times. Restoration of dystrophin levels by gene editing rescued the DMD phenotype in ESM. Conclusions We introduce human muscle models with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease and repair.

Dystrophin is a mechanical tension modulator

“…For this reason, we investigated an established iPSC line using cells from a Duchenne patient with an exon 48-50 deletion (‘DMD51’) in the dystrophin gene and compared it to the ‘Cor51’ iPSC line that was genetically corrected via exon 51 skipping (Figure 4 a) [30] . The isogenic cell lines were differentiated into skeletal myocytes as previously described [51] and used to bioengineer skeletal muscles in our glass bottom chamber over a period of 4 weeks.…”

“…DMD51 and isogenic Cor51 lines were maintained on Matrigel-coated flasks in StemMACS iPSC Brew medium. Directed differentiation into skeletal myocytes was performed as previously reported by Shahriyari et al 2022 [51] .…”

“…For this reason, numerous in vitro strategies have emerged in recent years for cultivating self-organizing, human multinucleated myotubes within 3D extracellular matrix (ECM) scaffolds [31,2,1,22] . Based on this, the first 3D bioengineered human skeletal muscle disease models were reported only recently, and offer great opportunities to further fundamental research and drug development [32,44,42,17,55,51] . By contrast to 2D myotube culture, these approaches support studies of the force generating capacity of normal and DMD muscle tissue upon stimulating a contraction.…”

Genistein Promotes Skeletal Muscle Regeneration by Regulating miR-221/222

“…Skeletal muscle regeneration is a dynamic regulatory process. Skeletal muscle undergoes a series of complex processes from destruction, repair, to remodeling after injury, and a large number of cytokines are involved in this process [ 6 ]. The activation, proliferation, and differentiation of satellite cells are the most critical stages in the process of skeletal muscle regeneration.…”

“…Macrophages are the main inflammatory cells in injured muscles, mainly by secreting inflammatory mediators such as TNFα and IL6 , phagocytosing necrotic muscle tissue, and providing an attachment environment for new myotubes [ 12 , 13 ]. Overall, in the early stages of muscle injury, inflammatory cells and immune cells are recruited to the site of injury, M1 macrophages and T cells maintain a pro-inflammatory environment and clean up necrotic and damaged areas, and satellite cells are activated to enter the cell cycle [ 6 , 10 ]; in the middle and late stages of muscle injury, the pro-inflammatory environment is converted to an anti-inflammatory environment, dominated by M2 macrophages. M2 macrophages and regulatory T cells (tregs) promote the differentiation of satellite cells and maturation of newly formed muscle fibers until muscle damage repair is completed [ 8 , 11 ].…”