**4. Induced pluripotent stem cells from patients: how to model muscle disease in the Petri dish**

The possibility to direct cell differentiation from human PSCs opens the door for the devel‐ opment of massive platforms for the study of muscle differentiation and disease progression. Moreover, the possibility to combine gene-editing strategies allowing for the correction of the genetic disorder leading to muscle disease, together with the generation of myogenic cells from patients' cells, represents an unprecedented opportunity for the establishment of *in vitro* systems for the study of MDs.

So far, different groups have demonstrated the suitability of patient iPSCs approaches in order to model MDs. Abujarour and colleagues [41] have derived myotubes from Duchenne Muscular Distrophy (DMD) and Becker Muscular Distrophy (BMD) hiPSCs. In particular, authors showed that myotubes derived from MDM and BMD iPSCs could respond to insulinlike growth factor 1(IGF-1) and wingless-type MMTV integration site family member 7A (Wnt7a) in a similar manner to primary myotubes. These results point out that iPSC derived from MDM and BMD patients have no intrinsic barriers preventing from myogenesis, and thus represent a scalable source of normal and dystrophic myoblast for further use in disease modelling and drug discovery.

Recently, Tedesco and colleagues [71] generated iPSCs from fibroblasts and myoblasts from limb-girdle muscular dystrophy 2D (LGMD2D) patients, developing the first protocol for the derivation of mesoangioblast-like cells from these iPSCs. Moreover, authors expanded and genetically corrected patient iPSC-derived mesoangioblasts *in vitro* by means of a lentiviral vector for the expression of human α-sarcoglycan in striated muscle cells. When LGMD2D disease free iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immu‐ nodeficient mice authors showed that they were capable to generate muscle fibers expressing α-sarcoglycan. Interestingly, when the same experiments were conducted using mousederived mesoangioblasts authors showed a functional amelioration of the dystrophic pheno‐ type and restoration of the depleted progenitors in α-sarcoglycan-null immunodeficient mice. Overall, Tedesco and colleagues showed that transplantation of genetically corrected meso‐ angioblast-like cells derived from iPSCs from LGMD2D patients could represent a novel therapeutic approach for these patients.

Notably, other authors have shown the possibility to generate PDGFR-α+ from hESCs [83]. However, those same authors showed few engraftments of transplanted hESCs-derived myogenic cells into injured skeletal muscle. Interestingly, the same authors have recently demonstrated that, by incorporating Wnt3a in culture medium, myogenic commitment is rapidly achieved from hESCs, and more significantly, that those cells can contribute to finally regenerate cardiotoxin-injured skeletal muscle of NOD/SCID mice [84]. In the same line, other authors have demonstrated that the inhibition of GSK3B and treatment with FGF2 could specifically promote skeletal muscle differentiation. In particular, Xu and colleagues [85] have demonstrated that simultaneous inhibition of GSK3B, activation of adenyl cyclase and stimulation with FGF2 during EBs formation could promote the generation of myogenic precursors that terminally differentiate *in vitro* and act as satellite cells upon transplantation. Also, Borchin and colleagues [86] have shown that human PSCs can be differentiated towards

**4. Induced pluripotent stem cells from patients: how to model muscle**

The possibility to direct cell differentiation from human PSCs opens the door for the devel‐ opment of massive platforms for the study of muscle differentiation and disease progression. Moreover, the possibility to combine gene-editing strategies allowing for the correction of the genetic disorder leading to muscle disease, together with the generation of myogenic cells from patients' cells, represents an unprecedented opportunity for the establishment of *in vitro*

So far, different groups have demonstrated the suitability of patient iPSCs approaches in order to model MDs. Abujarour and colleagues [41] have derived myotubes from Duchenne Muscular Distrophy (DMD) and Becker Muscular Distrophy (BMD) hiPSCs. In particular, authors showed that myotubes derived from MDM and BMD iPSCs could respond to insulinlike growth factor 1(IGF-1) and wingless-type MMTV integration site family member 7A (Wnt7a) in a similar manner to primary myotubes. These results point out that iPSC derived from MDM and BMD patients have no intrinsic barriers preventing from myogenesis, and thus represent a scalable source of normal and dystrophic myoblast for further use in disease

Recently, Tedesco and colleagues [71] generated iPSCs from fibroblasts and myoblasts from limb-girdle muscular dystrophy 2D (LGMD2D) patients, developing the first protocol for the derivation of mesoangioblast-like cells from these iPSCs. Moreover, authors expanded and genetically corrected patient iPSC-derived mesoangioblasts *in vitro* by means of a lentiviral vector for the expression of human α-sarcoglycan in striated muscle cells. When LGMD2D disease free iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immu‐ nodeficient mice authors showed that they were capable to generate muscle fibers expressing α-sarcoglycan. Interestingly, when the same experiments were conducted using mousederived mesoangioblasts authors showed a functional amelioration of the dystrophic pheno‐

Pax3/Pax7 double positive cells after GSK3B and FGF2 exposure.

**disease in the Petri dish**

344 Muscle Cell and Tissue

systems for the study of MDs.

modelling and drug discovery.

In the same line, other authors [36] have generated iPSCs from patients affected by Miyoshi myopathy (MM), a congenital distal myopathy caused by mutations in dysferlin. Specifically, authors demonstrated that the expression of full-length dysferlin could restore the MM associated phenotype in myotubes differentiated from MM-iPSCs. In the same line, Yasuno and colleagues [87] have shown the possibility to generate iPSCs from patients affected by Carnitine palmitoyltransferase II (CPT II) deficiency, an inherited disorder involving Boxidation of long-chain fatty acids (FAO).

Very recently, Li and colleagues [88] have demonstrated the possibility to correct iPSCs derived from DMD patients by means of three different strategies: exon skipping, frameshifting, and exon knock-in. In their hands, exon knock-in was the most effective approach. The work of Li reveals the suitability of iPSC technology for the generation of iPSC-based approaches for MDs modelling and therapy.

Overall, these recent advances set the bases for the generation of a previously nonexisting tool for the study of MDs. The possibility to generate human models for the study of MDs by means of iPSC technology opens the door for the development of novel therapeutic compounds for MD treatment, and more importantly, to increase our understanding of MDs and muscle development.
