Skeletal muscles are involved in posture, strength, and movement. Their function is impaired by pathologies such as facioscapulohumeral dystrophy (FSHD), an autosomal dominant inherited disease. FSHD is characterized by progressive and asymmetric weakening of the facial, shoulder, and humeral muscles. In the 1990s, research linked the onset of FSHD to the 4q35 locus and then to the D4Z4 macrosatellite repeat. However, there is no relevant animal model and no reference treatment for the 3rd most common inherited neuromuscular disorder.

We model FSHD using human induced Pluripotent Stem Cells (hiPSCs), generating muscle fibers innervated by motor neurons (MN). Then, we explore the contribution of MN and fibroadipogenic progenitors (FAP) cells to the pathophysiology and to muscle regeneration mechanisms. MN contribute to muscle maturation and contractile function. FAP, resident in muscle, play a key role in regulating the extracellular matrix and tissue regeneration; their deregulation promotes fibrosis and fatty infiltration, observed in FSHD muscles.

For this study, a 30-day differentiation into muscle fibers innervated by MN, implemented by the team, was characterized by flow cytometry and bulk RNASeq. Two conditions were tested pro-fibrogenic (addition of TGFβ1, D8–17) and pro-adipogenic (adipogenic cocktail, D8–33). In addition, the electrophysiological activity of MN was assessed on multielectrode arrays.

Flow cytometry revealed a transient stromal population (CD73+/CD90+/CD201+) observed between D8 and D17, suggesting an intermediate phenotype close to FAP, possibly involved in stromal dynamics or supporting muscle differentiation. The addition of TGFβ1 activated pro-fibrotic pathways, with an increase in the expression of TGFB1 and FN1, a decrease in ACTA1 and the induction of fibroblast/myofibroblast markers (COL1, α-SMA), reflecting a fibro-mesenchymal phenotype. Adipogenic induction led to an increased expression of adipocyte markers (ADIPOQ, FABP4, PPARG) and some lipid accumulation (Oil RedO staining). MN derived from patient hiPSCs exhibit increased spontaneous electrical activity (number of spikes) and altered maturity (burst frequencies), suggesting increased excitability and/or impaired functional maturation.

The identification of a transient stromal population, the demonstration of pro-fibrotic and adipogenic differentiation, as well as altered MN excitability, provide new insights into the contribution of MN and FAP to the pathophysiology of FSHD.