A time course analysis of the electrophysiological and gene expression properties during differentiation of hair cell-like cells in culture.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are multipotent cells that have attracted considerable attention in regenerative medicine. Current in vitro test focus on biochemical assays of hair cell-like cells (HCLCs) derived from BMSCs associated with changes in electrophysiological properties. HCLCs were produced from BMSCs by culturing BMSCs with B27, EGF, FGF, and IGF-1. RNA Sequencing studies, immunocytochemistry (ICC) and double immunofluorescence staining were used to test hair cell-associated markers on day 17 and 21-26. Next, we performed whole-cell patch-clamp recording by utilizing current- and voltage-clamp techniques to assess changes in membrane potential and ionic currents during differentiation. Immunostaining assay reveals significant expression of myosin VIIA and SOX2 in cultured hair cells on day 21-26. We have also found 8 enhanced transcripts in differentiated cell genes (Wnt7a, Mgat5b, Myo7a, Pou4f3, SOX2, Atoh1, Map2k3, Actin) using RNA Sequencing. Electrophysiological results indicate that cells undergoing differentiation had an average resting membrane potential (RMP) of -11.93 ± 0.89 mV on day 17 and -58.96 ± 1.10 mV on days 21-26. Differentiated HCLCs displayed a mean resting membrane resistance of 171.66 ± 29.12 MΩ, membrane time constant of 10.73 ± 0.45 ms and membrane capacitance of 0.0625 ± 0.0087 pF, following 21-26 days in culture. Our results also showed cultured HCLCs express transcriptomic profile of this cell type. These findings indicate that alterations in RMP may serve as a valuable indicator for distinguishing HCLCs differentiation potential from BMSCs.