Math1Induced Auditory Sensory Epithelium Regeneration In Vitro

Objective Mathl gene overexpression can induce new hair cell production in mammalian inner ear, but hearing function restore is far more away from single cell replacement. In this study, we explored two regions of non-sensory epithelial cells which are competent to become new hair cells by mis-expression of transcript factor math1, and described concepts of hair-supporting cell unit regeneration and overall regeneration, rather than replacement of single cells.Methods The experimental animals were neonatal rats. Ad-math1-EGFP vectors and Ad-EGFP, which is adenovirus encoding EGFP and mathl gene or single EGFP gene, were constructed, and then the efficiency of the vectors were examined. Application of in vitro culture of cochlea were overexpressed mathl gene, thus produced ectopic new hair cells in non-sensory epithelium, including greater epithelial region(GER) in the medial side of the organ of Corti, and lateral non-sensory region(LNSR). Immunofluorescence labeling was used to identify fate of infected cells. Scanning electron microscopy and confocal laser microscope were used to observe the morphological change of new sensory cells and interaction between infected cells. Tissue co-culture was used to determine the movement of the individual cell and the whole epithelial region, and the difference between culture sections near the basal end and the one nearer the apex end of cochlea. The movement as a whole epithelial region was recording by live cell scan array for24h. The cue for the cell movement was explored by co-culture in different conditions.Results1) Greater epithelial region (GER) inside the organ of Corti and the lateral non-sensory region outside the organ of Corti (LNSR) were both able to produce new ectopic hair cells induced by mathl overexpression in vitro.2) New stereocilum were produced, accompanied by lateral links assembling and then reduction. Tip link production was occasionally.3) Hair bundles of new hair cells from GER was more mature than that from LNSR in general.4) New hair cell-supporting cell unit was established in this process. The fate of supporting cell from the non-sensory region infection was probably affected by adjacent new hair cells. And the new supporting cell moved to a lower cell layer as in the original hair-supporting cell unit.5) Individual new hair cell from the GER joined in the organ of Corti, while new hair cells from the LNSR integrated and mixed with original hair cells in organ of Corti as a whole epithelial movement, similar to convergent extension in the development of organ of Corti.6) The integration of LNSR new sensory epithelial presented basal-apex gradient, another character in development process of organ of Corti.7) The cue of this integration movement is from the organ of Corti, and relied on the existence of cell connection or junction.Conclusion1) There are some high proliferating cells in the postnatal cochlear to produce flat epithelial cells of LNSR.2) GER and LNSR are both potential sources for new hair cells in the inner ear.3) Mathl overexpression cells can trans-differentiated to both new hair cells and new supporting cells, and the cell fate was affected by adjacent cell interactions, and probably the expression amount and time difference between adjacent cells.4) New hair cells from LNSR moved as a whole pre-sensory region towards and finally integrated into organ of Corti, in which the cue was from organ of Corti, and the spread was through the cell junction, from cell to cell.