P21. Induction of inner ear hair cells from mouse ES cells using stromal cells

Abstract

Introduction Earing loss is mainly due to an inability of cochlear sensory epithelia to replace lost hair cells of the inner ear. Embryonic stem (ES) cells have shown promise for cell therapy in a range of organs, because of their potential for self-renewal and pluripotency. In the present study, we investigated the differentiation of ES cells into inner ear hair cells (IEHCs) using differentiation-inducing activity (DIA) in culture supernatants of PA6 and ST-2 stromal cells. Methods Various media, including DMEM with 10% FBS, DMEM+KOS, DMEM+N2M, DMEM+N2M+FGF2, PA6-conditioned medium (PA-CM), and ST-2-CM, were investigated in regard to their effects on the differentiation of 4-day embryoid bodies (EBs) into IEHC-like cells. EBs were cultured in media for 14 days, after which the expressions of hair cell markers, including myosin 7a, myosin 6, Brn3c, and a9AchR, were examined by RT-PCR and immunocytochemistry. Results Prominent neuritic outgrowths were observed emerging from the EB aggregates in cultures with ST2-CM, while such distinguished formation of neurites was not observed in cultures with the other media. PA6-CM-induced expressions of MAP2 and GFAP, but not those of inner ear hair cell markers, while it promoted the induction of hair cell markers, as shown by both RT-PCR and immunocytochemistry findings. Conclusion Inner ear hair cell marker-positive cells were generated from ES cells after culturing in ST2-CM, suggesting that secreted DIA may be sufficient to induce IEHCs from ES cells.