Stem/Progenitor Cells Derived from the Cochlear Sensory Epithelium Give Rise to Spheres with Distinct Morphologies and Features

Abstract

Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair celllike cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.

Cochlear epithelial of dog fetuses: a new source of multipotent stem cells

“…Similar results were previously obtained by Diensthuber et al (2009) for cultures of cochlear epithelial cells of different mice lineages (BALB/c and Math-1/nuclear green fluorescence protein (nGFP) transgenic mice) using similar culture medium. Media containing DMEM-High glucose and DMEM-F12 (1:1) have been proven to be successful for culturing rodent cochlea cells and were suitable for the applied purposes (Chao et al 2013;White et al 2012;Diensthuber et al 2009;Yerukhimovich et al 2007;Savary et al 2007;Martinez-Monedero and Edge 2007;Doetzlhofer et al 2004;Li et al 2003). In our adherent cultures, cochlear epithelial cells have typical fibroblast-like morphology, as observed for other mesenchymal stem cell cultures, including multipotentiality (Favaron et al 2014;Chao et al 2013;Wenceslau et al 2012).…”

“…In contrast, rodent cochlea tissues so far were cultured in suspension with induction factors N2 and/or B27 (Yerukhimovich et al 2007). Then, cells modified their fibroblast-like appearance into spherical shape with a differentiation potential especially towards neural cell lineages (Diensthuber et al 2009;Yerukhimovich et al 2007). Thus, here the adherent culture condition method was chosen since it seems to be more suitable to produce multipotent stem cell lineages and may be favourable compared to the other approaches.…”

Potential of Pluripotent Stem Cells for the Replacement of Inner Ears

“…The belief that no tissue stem cells might exist in the inner ear was overturned by the finding that stem cells were still present in the vestibular organs of adult mice [19]. Several laboratories adopted a sphere-forming assay to isolate stem/progenitor cells from complex cell mixtures [6,19,20,21,30,33,39,37,48,51,53,54] derived from inner ear tissues. Sphere-forming cells from the utricle of adult mice are pluripotent and can give rise to a variety of cell types, including cells representative of ectodermal, mesodermal and endodermal lineages [19].…”

Serial Analysis of Gene Expression in the Chicken Otocyst

“…This was demonstrated by showing the potential of otic progenitors to differentiate into different cell types that express makers indicative of neurons, hair cells, and supporting cells. Based on these findings, it has been hypothesized that stem cellderived otic progenitors are similar to otocyst cells (Brigande and Heller 2009;Diensthuber et al 2009). …”