THE INNER EAR OF SEVEN DAYS OLD MICE CONTAINES PLURIPOTENT STEM CELLS

Abstract

The purpose of the ongoing research is to improve our current skills and knowledge in stem cell isolation, cultivation and differentiation from the utricular and saccular epithelia of young mice. We harvested utricles and sacculi from 7 days old NMRI mice. Utricles were trypsinized in order to isolate single cells. Obtained cells were cultivated in DMEM with F12 Nutrient mixture, B27, N2 supplement, IGF-1 and EGF. Sphere pluripotency was established with stem cell markers Nanog and Oct-4. We mechanically dissociated primary spheres and cultivated. Secondary spheres were placed on fibronectin coated tissue culture slide chambers in the absence of IGF-1 and EGF. Cells were characterized by immunofluorescence and immunohistochemistry for myosinVIIA (hair cell marker) and nestin (intermediate filament VI marker). Vestibular epithelia contain pluripotent stem cells able to form cell clusters (spheres). Pluripotency was demonstrated by the expression of nanog, oct 4 and nestin markers (cell progenitors). Also sphere dissociation and separate cultivation of the isolated cells lead to the formation of larger spheres and in higher amounts compared to the original spheres, proving that these cells are not only pluripotent but also capable of self-renewal. Single spheres harvested and cultivated on fibronectin produced through differentiation different cell types including neuron like-cells, positive for myosin VIIA and nestin.

Keywords

• stem cell isolation
• cultivation
• differentiation
• inner ear