Morphological And Functional Properties Of Hair-cell-like Cells In Mammalian Cochlea Induced By Math1

Objects Mathl is a basic helix-loop-helix transcription factor and play a key role in hair cell development. In this research, we described the process of the supporting cells trans-differentiated into hair cells and compare the expression of inner ear hair cells specific markers in newly differentiated and native hair cells to identify the phenotype morphologically. We also tried to explain the mechanism of trans-differentiation from supporting cells into hair cells. Most importantly, we tried to explore if the newly differentiated hair cells also possess the functional properties as the normal hair cells.Methods Newborn 3-day-old postnatal SD rats were used for experiments. We cultured the cochleae and infected them with Ad5-EGFP (control) or Ad-EGFP-Mathl (experimental).First, we fixed them at different time points with paraformaldehyde and performed immune-fluorescence to detect if Mathl could induce the hair cells generation and express the relative specific protein; Second, Brdu incorporate assay was used to detect the mechanism of supporting cells trans-differentiated into hair cells; Third, lmM neomycin trisulfate salt hydrate and 3?M FM1-43 were used to detected the presence of mechano-electical transduction (MET); Finally, Patch clamp technique was used to detected the function of newly differentiated hair cells, which inculed the ionic currents, action potential and mechano-electical transduction currents.Results?Three days after Ad5-EGFP-Mathl infection, newly ectopic hair cells could be examined at LER, which was myosinVIIA positive; However, we could only detect newly ectopic hair cells at GER from five days after Mathl infection; Calcium buffering protein, such as calbindin, calmodulin, calretinin and PMCA2 were also the specific marker of inner ear hair cells, could be detected at the newly differentiated hair-cell-like cells; Phlloidin staining showed the stereocilia-like bundles in the apical aspect of the newly differentiated hair cells.?Brdu incorporation assay results suggested that supporting cells directly trans-differentiated into hair cells. Thy process did not depend on the cell division of supporting cells. Mathl expression level was one of factors to induce hair cells generation;?1mM neomycin could induce the hair cells loss of newly differentiated hair cells;5 days after Mathl infection, newly differentiated hair cells could uptake FM1-43. These two phenomenons indicated the presence of MET in the newly differentiated hair cells.?Supporting cells infected with EGFP alone did not express inward sodium current; However, newly differentiated hair cells could express sodium current from 2 days after Mathl infection; the amplitude of current density increased at day 5 and then decrease.?Supporting cells infected with EGFP alone did not express calcium current; However, newly differentiated hair cells could expressed calcium current from 3 days after Mathl infection, the amplitude of current density increased with the time;?Supporting cells infected with EGFP alone only expressed a small sustained potassium current; However, newly differentiated hair cells could expressed a transient potassium current at 5 days after Mathl infection; then, a sustained potassium current substituted the transient current 10 days after infection;?Mathl induced newly differentiated hair cells could be detected HCN currents. The amplitude of the current density decreased and disappeared with time.?We could record the evoked action potential (EvAP) in most of the newly trans-differentiated hair cells; We also could get the spontaneous action potential (SAP) in 3 of the hundreds of cells;?7 days after Mathl infection, we also could get the MET current from the newly differentiated hair cells.Conclusions?We successfully constructed the model to generate the newly differentiated hair cells by using Ad5-EGFP-Mathl in newborn rats cochleae;?These newly differentiated hair cells have been confirmed to express the specific marker of normal hair cells.?Supporting cells in LER directly trans-differentiated into hair cells, without cells division.?Neomycin sensitive and FM1-43 loading experiments indicated that MET was in the presence of newly trans-differentiated hair cells from 5 days after Mathl infection;?Ionic currents, action potential changes in different stage combined with MET recorded from newly differentiated hair cells, mimicked a developing process of normal hair cells, strongly indicated that the hair cells were already a functional hair cells. Taken together, the newly differentiated hair cells induced by Mathl in LER not only possessed the morphological properties like normal hair cells, but also they were already developing and functional hair cells.