Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell–derived skeletal muscle in vitro

Abstract

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers

“…Additionally, we found that transitional and mature myofibers frequently show fast motions as consequences of active contractions (Figure S6A, B and Movie S6). Similar active contractions have been observed when severing immature myofibrils in Drosophila, as a consequence of laser-induced calcium release from the sarcoplasmic reticulum [14] and have also been observed spontaneously in mature human myofibers after applying the same differentiation protocol [19]. This demonstrates that transitional myofibers harbour already contractile sarcomeres.…”

“…We generated human iPSC-derived myogenic precursors by differentiating NCRM1 iPS cells for 20-30 days according to [18]. These cultures were then dissociated and filtered through a 70 µm and then a 40 µm cell filter to obtain singlecell suspensions, and replated in SKGM-2 medium for 1 to 2 days to generate cultures enriched in myogenic precursors prior to freezing [18,19]. Frozen myogenic precursors were then thawed, seeded on Matrigel-coated ibidi® glass-bottom dishes and grown in proliferation medium (SKGM-2) for 2-3 days before switching to KCTiP differentiation medium (see methods).…”

“…As a first step to investigate the molecular changes at day 7 of differentiation, we analysed a previously generated transcriptomics dataset comparing similar proliferative cultures (SKGM-2 medium) with day 7 and day 15 of myogenic differentiation (KCTiP medium) [19]. After verifying the high quality of the transcriptomics data set with principal components analysis (Figure S4A, B, Table S1) we performed Mfuzz clustering [22], choosing 10 clusters as optimal for our data set (see methods).…”

“…Here we set out to test the self-organisation capacity of human muscle tissue in vitro when developing on a substrate lacking any localised guidance, attachment or other mechanical cues. We employed a recently developed human induced pluripotent stem cell (iPSC) differentiation protocol that can efficiently generate myogenic precursors which further differentiate robustly into striated skeletal muscle fibers in vitro [17][18][19][20]. This method allowed us to obtain large quantities of isogenic human myogenic precursors of defined developmental history, in contrast to primary myoblast cultures from donors.…”

“…This method allowed us to obtain large quantities of isogenic human myogenic precursors of defined developmental history, in contrast to primary myoblast cultures from donors. Furthermore, CRISPR-based genome engineering enabled us to tag sarcomeric protein components of choice to follow their live dynamics in vitro [19,21].…”