ISOLATION AND CHARACTERIZATION OF VESTIBULAR STEM CELLS

Introduction The purpose of the ongoing research is to improve our current skills and knowledge in stem cell isolation, cultivation and differentiation from the vestibular epithelia of young mice. Matherials and Methods We harvested utricles and sacculi from 7 days old mice. Obtained cells were cultivated at 37oC and 5% CO2 in DMEM with F12 Nutrient mixture, B27, N2 supplement, IGF-1 and EGF. Sphere pluripotency was established with Nanog and Oct-4 stem cell markers. We mechanically dissociated primary spheres and cultivated them. Cells were characterized by immunofluorescence and immunohistochemistry for myosinVIIA (hair cell marker), nestin (intermediate filament VI marker) and beta III tubulin. Results We proved that vestibular epithelia contains pluripotent stem cells which formed spheres. Sphere-derived cells’ pluripotency was demonstrated by the expression of nanog, oct 4 and nestin markers. We obtained through differentiation different cell types including neuronal cells which were positive for myosin VIIA, nestin, beta III tubulin. We wanted to know the type of neurons and performed Brn3a staining for immature neurons, and  TrKB staining for mature neurons. We cultivated vestibular stem cells on different substrates to observe their behavior. Conclusions Cells obtained from utricular epithelia are pluripotent because they express nanog, oct 4,  nestin, characteristic for cell progenitors and they generate spheres.