Development of neurospheres from olfactory receptor neuron precursors using a recombinant protein

Abstract

Neurospheres are spherical free-floating cell clusters and composed of neural progenitor cells and stem cell population so that can be differentiated into neurons by environmental modulations. However, the neurosphere culture system is sensitive to experimental conditions such as cell density and medium composition. In this research, we have introduced a new method for stable and reproducible generation of neurospheres, and characterized the neurospheres using primary cultures of rat olfactory receptor neurons (ORNs). In order to establish stable and reproducible neurospheres, a recombinant protein (REP) was employed. The REP was designed for inducing fibronectin-integrin signaling pathway. Neurospheres were produced by incubating freshly dissociated rat ORN precursors on REP coated plates. ORN neurospheres formed on the plates were firstly characterized if stemness is maintained by immunocytochemistry (ICC) using stemness and proliferation markers. We next examined a physiological function of ORNs by performing calcium imaging in differentiated ORNs after neurospheres were moved to laminin coated plates. Also, electrical signals of ORNs activated by odorant stimulation were measured using micro electrode array (MEA) to verify a physiological function of ORNs. Expressions of nestin and Ki67 indicate that ORN neurospheres have innate characteristics of stemness and proliferation. And elevation of intracellular calcium levels upon odorant stimulation in ORNs after differentiation from neurospheres suggests that differentiated ORNs have innate function in odor detection. Moreover, we found that different activating patterns in distinct odors and similar activating patterns in same odors by analyzing electrical signals measured by MEA. Due to the introduction of REP, stable and reproducible neurosphere generation was realized. ORNs derived from neurospheres were steadily activated by odors, and which neurospheres maintain functions of ORNs during cultivation. Our results may facilitate ORN-based bioelectronic nose development for chemical detection and advanced researches for understanding odor encoding process in the mammalian olfactory system.