Isolation, culture optimization and functional characterization of stem cell neurospheres from mouse neonatal olfactory bulb and epithelium.

The olfactory epithelium contains basal cells with stem cell characteristics, which have the capacity to differentiate throughout life into olfactory receptor neurons (ORNs). Here we investigate the in vitro characteristics of stem cells taken from the olfactory bulb (OB) and the olfactory epithelium (OE) of neonatal TIS21 knock-in mice. The major aim of the study was the generation of olfactory neurospheres (ONS) derived from OB and OE of neonatal mice as a tool to further analyze the elementary processes of ORN development. Our data showed that the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) leads to a significant increase in number of ONS derived from OB but not from OE. The differentiation of ONSs led to the formation of different neuronal cell types, in particular to bipolar-shaped cells as well as putative pyramidal-neurons, astrocytes and oligodendrocytes. Immunohistochemical staining confirmed the presence of astrocytes and neurons in both types of ONSs. In order to investigate the functionality of the neurons we performed calcium imaging and patch-clamp experiments. Calcium imaging experiments revealed that the application of high potassium concentration provokes calcium transients. No excitable properties, neither sodium currents nor action potentials, were observed for the bipolar-shaped cells derived from OB and OE neurospheres, which means that these types of cells morphologically defined as putative neuronal cells, were not physiologically active. Interestingly, patch-clamp recordings performed in the pyramidal-shaped cells of OB neurospheres showed sodium and potassium currents as well as action potentials. Our study will help to establish further models in the field of olfactology.