Sertoli cell-secreted factors have promyogenic and antifibrotic properties on human DMD myoblasts with different mutations.

Duchenne muscular dystrophy (DMD) is a recessive X-linked lethal disease affecting one over 5,000 live male births in which mutations in the dystrophin gene (DMD) lead to lack of a functional protein resulting in susceptibility of myofibers to rupture during contraction. Muscles of DMD patients or experimental models of DMD show progressive necrosis of the myofibers, chronic inflammation and reactive regeneration, which lead to exhaustion of muscle precursor cell pool and replacement of myofibers with fibrous and fatty tissues.1 Although multiple therapeutic approaches have been explored in the last decades and are still under investigation, the standard therapy to DMD remains the use of glucocorticoids despite their limited efficacy and undesired side effects.2 We set up a preclinical approach based on the peculiar properties of Sertoli cells (SeC)3,4 a cell type of the seminiferous tubules of the testis in which they favor the maturation of developing germ cells and protect them against the host immune system. Besides creating a physical barrier (the blood-testis barrier), SeC secrete a plethora of trophic and immunomodulatory factors that confer to these cells the ability to survive long time after engraftment, and protect allo- and xenogenic engraftments of tissues and organs.5 SeC isolated from specific pathogens free (SPF) pre-pubertal pigs were encapsulated in highly biocompatible alginate-based microcapsules (MC-SeC) and injected into the peritoneal cavity of mdx mice, an experimental model of DMD, in the absence of pharmacological immunosuppression.3,4A single i.p. injection of MC-SeC resulted in amelioration of muscle morphology and performance as a consequence of the secretion by SeC of anti-inflammatory factors and heregulin β1, an inducer of the dystrophin paralogue, utrophin, opening new routes in the treatment of DMD. However, information is lacking about possible direct effects of SeC on myoblasts/myotubes. Here, we show that SeC secrete factors able to stimulate cell proliferation in the early phase of the myogenic differentiation process in murine C2C12 and human (healthy and DMD) myoblasts. In DMD myoblasts, SeC-derived factors delayed the expression of the muscle-specific terminal differentiation markers, myogenin and myosin heavy chain (MyHC)-II in the early phase (24h) of the differentiation process; nevertheless, SeC stimulated terminal differentiation (6 days) in these cells, suggesting that the promitogenic activity of SeC does not affect the myogenic potential. Moreover, SeC inhibit the expression of the myofibroblast transdifferentiation markers, COL1A1, FN1 and CTGF/CCN2 in DMD myoblasts, suggesting an antifibrotic effect of the SeC-derived factors. Finally, SeC induced utrophin expression in preformed DMD myotubes regardless of the mutation, with the same mechanism reported in dystrophic mice. Altogether, these results further support the use of MC-SeC or SeC-derived factors in the treatment of DMD patients, and suggest that SeC-based approaches might be useful also in improving the early phase of muscle regeneration, during which myoblasts have to proliferate in order to reach the critical amount required to replace the damaged muscle mass. Keywords: Duchenne muscular dystrophy; Sertoli cell; muscle differentiation; fibrosis. References 1. Davies KE, Nowak KJ. Molecular mechanisms of muscular dystrophies: old and new players. Nat Rev Mol Cell Biol. 2006;10:762-73. doi: 10.1038/nrm 2024 2. Muntoni F, Fisher I, Morgan, JE, Abraham D. Steroids in Duchenne muscular dystrophy: from clinical trials to genomic research. Neuromuscul. Disord. 2002;1:S162-5. doi: 10.1016/s0960-8966 (02)00101-3 3. Chiappalupi S, Luca G, Mancuso F, Madaro L, Fallarino F, Nicoletti C, Calvitti M, Arato I, Falabella F, Salvadori L, Di Meo A, Bufalari A, Giovagnoli S, Calafiore R, Donato R, Sorci G. Intraperitoneal injection of microencapsulated Sertoli cells restores muscle morphology and performance in dystrophic mice. Biomaterials. 2016;75:313-26. doi:10.1016/j.biomaterials. 2015. 10.029 4. Chiappalupi S, Luca G, Mancuso F, Madaro L, Fallarino F, Nicoletti C, Calvitti M, Arato I, Falabella F, Salvadori L, Di Meo A, Bufalari A, Giovagnoli S, Calafiore R, Donato R, Sorci G. Effects of intraperitoneal injection of microencapsulated Sertoli cells on chronic and presymptomatic dystrophic mice. Data in Brief. 2015;5:1015-21. doi:10.1016/j.dib.2015.11.016 5. Chiappalupi S, Salvadori L, Luca G, Riuzzi F, Calafiore R, Donato R, Sorci G. Do porcine Sertoli cells represent an opportunity for Duchenne muscular dystrophy? Cell Proliferation. 2019;26:e12599; doi: 10.1111/cpr.12599.