br Introduction Hearing loss is the most common congenital s

Introduction Hearing loss is the most common congenital sensory deficit (Chan et al., 2010). Approximately 1 child in 1,000 is affected with severe hearing loss at birth or during early childhood, and this is defined as prelingual deafness (Morton, 1991; Petersen and Willems, 2006), with about half of the cases attributable to genetic causes (Birkenhager et al., 2010). There are more than 100 known forms of non-syndromic deafness associated with identified genetic loci (available at http://hereditaryhearingloss.org). Mutations in the Gap Junction Beta 2 gene (GJB2), encoding connexin 26 (CX26), account for up to 50% of cases of non-syndromic sensorineural hearing loss in some populations (Morton and Nance, 2006). CX26 and CX30, which are encoded by GJB6, co-assemble and participate in the formation of gap junctions between cells, and these connexins are the two most abundantly expressed gap junction proteins in the cochlea (Ahmad et al., 2003) (Figure S1 and Movie S1). Gap junctions facilitate the rapid removal of K+ from the ampk pathway of the cochlear hair cells, resulting in K+ recycling back to the endolymph to maintain cochlear homeostasis (Kikuchi et al., 2000). CX26 is also involved in the developmental organization of mammalian cochlea, for example, tunnel of Corti, Nuel\'s space, or spaces surrounding the outer hair cells (Inoshita et al., 2008). CX26 and CX30 form heteromeric and heterotypic channels in most of the cochlear gap junction plaques (GJPs) (Sun et al., 2005) and in in vitro experiments (Yum et al., 2007). Recently, expression of various transcription factors and other proteins in human developmental fetal cochleae from gestational weeks 9–22 were investigated using immunohistochemistry (Locher et al., 2013, 2014), and it has been found that the expression of CX26 and CX30 is detectable in the outer sulcus cells at 18 weeks of gestation (Locher et al., 2015). In our recent study, it was shown that disruption of the CX26 GJP is associated with the pathogenesis of GJB2-related hearing loss and that the assembly of cochlear GJP is dependent on CX26 (Kamiya et al., 2014). It was also reported that cochlear gene transfer of GJB2 using an adeno-associated virus significantly improved GJP formation and auditory function (Iizuka et al., 2015). In our alternative approach, a novel strategy was developed for inner-ear cell therapy with bone marrow mesenchymal stem cells (Kamiya et al., 2007). Induced pluripotent stem cells (iPSCs) can be produced by the reprogramming of somatic cells, and are capable of self-renewal and differentiation into various types of cells such as embryonic stem cells (ESCs) (Takahashi and Yamanaka, 2006). Human cochlear cells are not readily accessible for biopsy or direct drug administration because of anatomical limitations. Therefore, ESCs/iPSCs are an important tool for studying the molecular mechanisms underlying inner-ear pathology as well as for generating cells for replacement therapies. It was recently reported that ESCs/iPSCs could be differentiated into inner-ear progenitor cells by in vitro differentiation in adherent monolayer culture and/or floating aggregation culture (Chen et al., 2012; Koehler and Hashino, 2014; Koehler et al., 2013; Oshima et al., 2010). For recapitulation of neural tissue formation in a three-dimensional (3D) context, floating aggregation culture is advantageous as it allows more flexible adaptation of cell and tissue shapes compared with 2D culture approaches (Eiraku and Sasai, 2012). Eiraku et al. (2011) reported in vitro differentiation of ESCs into cortical tissues when the cells were cultured as floating aggregates in a serum-free medium, thereby establishing the technique of serum-free floating culture of embryoid body-like aggregates with quick re-aggregation (SFEBq culture). Koehler and colleagues reported differentiation of ESCs into inner-ear hair cell-like cells using modified SFEBq methods (Koehler and Hashino, 2014; Koehler et al., 2013). For the establishment of strategies for inner-ear cell therapy or the development of a disease model for GJB2-related hearing loss, it is necessary to develop efficient differentiation methods for inducing iPSCs to form cells with CX26-containing GJPs. Although several studies have demonstrated the induced differentiation of ESCs/iPSCs into CX37/40/43/45-expressing cells (Mauritz et al., 2008; Oyamada et al., 1996, 2013), no such protocol has been reported for the differentiation of ESCs/iPSCs into CX26-expressing cells.