Hair cell formation in cultures of dissociated cells from the vestibular sensory epithelium of the bullfrog

Hypothesis: Bullfrog vestibular hair cells are capable of regenerating in v itro. Background: Recent studies have established that sensory organs in the inne r ear of vertebrates continue to produce hair cells after birth. However, t he mechanisms responsible for the regulation of this process are not well u nderstood. The current study reports the development of a novel method for the culture of dispersed cells from the bullfrog inner ear. Methods: New hair cell formation in this in vitro preparation was shown by sequential photomicroscopy. Studies with the selective marker for mitotic a ctivity 5-bromo-2-deoxyuridine (BrdU) were done to estimate the level of ce ll proliferation and to quantify postmitotic hair cell formation. Finally, confirmation of cell type was obtained by scanning electron microscopy and by the use of specific markers for hair cells. Results: Once the optimal culture conditions were established in the initia l experiments, the formation of new hair cells was directly visualized in a ll unstained live cultures and fixed preparations without exception. Asymme tric division of progenitor cells, with subsequent differentiation of one o f the daughter cells into new hair cells, also was documented by photomicro scopy. Approximately 12% of the cells were labeled with BrdU, of which 6% w ere hair cells, showing that new hair cell formation was subsequent to mito tic division in vitro. The identity of newly formed hair cells was verified as follows: 1) morphologically by scanning electron microscopy; 2) by posi tive labeling with phalloidin-rhodamine, a marker for actin; and 3) by posi tive calmodulin immunocytochemistry. Conclusions: This study reports the development of an in vitro culture prep aration in which undifferentiated epithelial cells proliferate to become ne w hair cells. Evidence is provided of division of hair cell progenitors and subsequent differentiation of the daughter cells as one of the mechanisms involved in new hair cell formation in the culture preparation. This newly developed cell culture technique provides a powerful tool for further study of the process of hair cell formation in the vestibular end organ.