In a recent study Scheffer

In a recent study (Scheffer et al., 2015), the expression of Lmod3 during natural mouse development was found to be restricted to cochlear HCs and that of Dnah5 to vestibular HCs, while double-positive cells were not produced. In the present study, immunocytochemical analyses showed that the cochlear HC marker Lmod3 and vestibular HC marker Dnah5 were expressed in 79.8±2.7% and 51.3±6.3%, respectively, of myosin6-immunopositive cells (Fig. 3D). These results suggested that some of the HC-like cells had co-expression of Lmod3 and Dnah5, though we unfortunately could not confirm their simultaneous expression because an appropriate set of JNJ26481585 to recognize them was not available. Restricted expression of Lmod3 and Dnah5, as observed in naturally developed HCs (Scheffer et al., 2015), was not recapitulated in the present combined method. Continuous overexpression of Math1 during the culture period explains the dysregulated expression of Lmod3 and Dnah5. Math1 expression in the cochlea begins on embryonic day (ED) 13.5 and continues until postnatal day 3–7, or possibly even into adulthood (Lumpkin et al., 2003; Lanford et al., 2000; Scheffer et al., 2007). On the other hand, Math1 expression in vestibular cells begins on ED12.5, then spreads throughout sensory epithelium by E14.5 and is down-regulated during the first postnatal week (Shailam et al., 1999; Bermingham et al., 1999). To recapitulate reproduce the expression pattern of the Math1 gene that occurs during natural development, precise regulation of its expression may be required. Recently, use of small molecular weight compounds such as DAPT has been reported to increase endogenous Math1 gene expression (Ren et al., 2016). Therefore, instead of direct gene induction by gene transfer, chemicals known to be Math1 inducers may function well with gene-engineered ES cells and simplify the present method.
Conclusion In summary, we investigated the efficacy of a combination of Math1-ES cells and use of ST2-CM, termed the HIST2 method, for inducing HC-like cells. Our results showed that Math1 expression increased the production of both cochlear- and vestibular-HC-like cells. The following are the supplementary data related to this article.
Authors\' contributions
Conflicts of interest
Acknowledgments We thank Dr. H. Niwa (Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan) for the ES cell lines and S. Shimada for the technical assistance. This research was supported by the Osaka Medical Research Foundation for Intractable Diseases and Grant-in-Aid for Scientific Research (B) (to Y.O. and M.Y.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (KAKENHI) (16H05482).
Introduction Over the past few years, several studies using hPS cells focused on the use of transgene-free strategies to recapitulate embryonic skeletal myogenesis in vitro (Barberi et al., 2007; Chal et al., 2016; Hwang et al., 2013; Shelton et al., 2014; Xi et al., 2017; Zheng et al., 2006). However, most of the current transgene-free myogenic differentiation protocols have limitations for potential clinical translation. The primary problems are the heterogeneity of cell preparations and the lack of evidence for the in vivo regenerative potential of generated myogenic cells (Kim et al., 2017). To date, the most homogeneous approaches for the generation of a myogenic-restricted population from PS cells relies on the introduction of exogenous DNA for the myogenic regulatory factor MYOD (Albini et al., 2013; Goudenege et al., 2012; Tedesco et al., 2012; Young et al., 2016) or PAX7 (Darabi et al., 2012; Kim et al., 2017), a paired box transcription factor essential for the commitment and maintenance of muscle stem cells (Gros et al., 2005; Lagha et al., 2008; Seale et al., 2000). Few of these studies have documented transplantation of PS-cell derived progenitors, but of those that have, most have shown minimal engraftment (Barberi et al., 2007; Hwang et al., 2013; Kim et al., 2017; Zheng et al., 2006). In contrast to these studies, the approach of temporally overexpressing PAX7 enables the generation of large numbers of proliferating PAX7+ myogenic progenitors and when these are transplanted, they contribute to significant skeletal muscle regeneration (Darabi et al., 2012; Kim et al., 2017; Magli et al., 2017). To date, the PAX7 approach has used lentiviral (LV) delivery of PAX7, which introduces the possibility of insertional mutagenesis.