More than 25 years ago, MyoD was first isolated as a master gene of myogenesis because ectopically expressed MyoD induced differentiation of non-myogenic cells to skeletal muscle [25]. This was the first report of direct reprogramming by transcription factors. Rao et al. transduced human ES cells with a lentiviral vector encoding a doxycycline (DOX)-inducible MyoD. The protocol takes approximately 10 days to induce multinucleated myotubes in vitro with an induction efficiency of over 90 % [26]. Tanaka et al. constructed a PiggyBac transposon vector to overexpress MYOD in the presence of DOX [27]. After the cells are switched to muscle differentiation medium, MYOD starts to activate the genes involved in terminal differentiation such as contractile proteins and muscle-specific enzymes. This protocol is able to differentiate human iPS cells into multinucleated myotubes in a short period. In some iPS lines, however, the efficiency of muscle differentiation is not 100 %. It remains to be determined whether MyoD-induced cells can be safely applied to cell therapy of DMD.

PAX7 drives myogenesis together with PAX3 during development and plays unique roles in postnatal satellite cells (reviewed in [28]). Forced expression of PAX7 during embryoid body (EB) formation successfully induces transplantable myogenic cells from human ES cells [29]. Transplanted PAX7-induced myogenic cells efficiently fuse with host myofibers and restore dystrophin expression and muscle function in immunodeficient mdx mice. In contrast to the MRF family (MYOD, MYF5, MRF4, and myogenin), PAX7 alone does not induce fibroblasts to differentiate into skeletal muscle. Therefore, the differentiation stage of the cells and timing of the expression of PAX7 in human ES/iPS cells seem critical.

Recently, Hosoyama et al. reported a new protocol named “EZ-sphere culture” for derivation of myogenic stem/progenitor cells from human ES/iPS cells [30]. iPS cells are cultured in low-attachment poly(2-hydroxyethyl methacrylate)-coated flasks and a medium generally used for formation of neurospheres but supplemented with high concentrations of human fibroblast growth factor 2 (FGF2) and human epidermal growth factor (EGF) (100 ng/ml each). In 1 week, human ES/iPS cells form free-floating spheres and robustly expand (Fig. 8.3). It takes roughly 6 weeks for a sphere to become myogenic. When plated onto Matrigel^TM-coated dishes, cells forming myospheres start to fuse to form multinucleated myotubes and finally start to twitch. The method is applicable to most human ES and iPS clones including patient-derived iPS cells. We confirmed the myogenic potentials of a variety of disease-specific iPS cells, such as Ullrich congenital muscular dystrophy, Schwartz-Jampel syndrome, congenital myasthenic syndrome, and Duchenne muscular dystrophy (Miyagoe-Suzuki et al. unpublished data). Importantly, some iPS clones fail to form spheres, and some fail to differentiate into the skeletal muscle lineage. This might be correlated to incomplete reprogramming of the iPS cells, but further investigation is needed to determine the cause.

The percentage of myogenic cells produced in cultures using the original protocol described by Hosoyama et al. [30] is not 100 %, and they need purification before transplantation. To increase the efficiency of myogenic differentiation, clarification of the molecular mechanisms by which myogenic cells are induced through sphere formation is required. Myogenic cells induced by the EZ-sphere method fuse with cardiotoxin-injured muscle fibers in host NOD/Scid mice (Miyagoe-Suzuki et al. unpublished data).

So far a limited number of research groups have reported successful engraftment of human iPS cell-derived myogenic cells in animal models. All studies used immunodeficient dystrophin-deficient mdx mice. Recently NSG–mdx ^4Cv mice, which have triple mutations, i.e., NOD/Scid, IL2 receptor gamma chain deficiency, and a mutation in the DMD gene, have been successfully used for xenogeneic transplantation [33]. However, xenogeneic transplantation might underestimate the myogenic potency of human muscle stem/progenitor cells. On the other hand, in many studies, the leg muscles of recipient mice are X-irradiated to suppress endogenous satellite cells and injected with a high dose of cardiotoxin to damage the whole TA muscle and evoke synchronized muscle regeneration. X-irradiation and cardiotoxin injection are not physiological and cannot be applied in the clinical setting. Thus, currently, the efficiency of cell transplantation is being tested in conditions quite different from clinical settings. The results need to be interpreted with caution.

Human iPS cells are heterogeneous in myogenic differentiation potential. Differences are found even among iPS clones from the same donor. One possible reason for the heterogeneity is that some clones are incompletely reprogrammed and retain epigenetic memory of their original cell, which interferes with the differentiation into the intended lineages. Practically, it would be useful to find characteristic gene expression patterns or specific markers of highly myogenic iPS clones instead of empirically testing each iPS clone for myogenic potential.

Human iPS cells vigorously self-renew. Although this property makes them an attractive source for cell transplantation for devastating diseases, the same property seems to be related to tumorigenesis by transplanted undifferentiated cells. Importantly it is suggested that the risk of tumorigenesis differ greatly among iPS clones. Therefore, primarily, “good” iPS clones should be selected for clinical application. How can we know the quality of iPS cells before transplantation? Koyanagi-Aoi et al. reported the gene expression and methylation patterns of “bad” human iPS clones, which retain a significant number of undifferentiated cells after neural differentiation culture and form teratomas when transplanted into mouse brains [34]. Such a molecular signature would facilitate the selection of therapeutic cells.