Adult human olfactory neural progenitors cultured in defined medium

Neurosphere-forming cells (NSFCs) derived from primary cultures of adult human olfactory epithelium were established in minimum essential medium (MEM) with Hanks balanced salts and 10% heat-inactivated fetal bovine serum (FBS). A totally defined medium (DM) was employed to examine their proliferation, lineage restriction and differentiation. DMEM/F12 (DF) was found to support NSFCs and served as the base medium for this study. NSFCs were adapted to the DM through serial serum reductions at successive feedings. NSFCs in DF supplemented with N2, B27 or insulin attained saturation density and formed extensive processes. Immunolocalization of lineage specific markers [i.e., nestin, beta-tubulin III, peripherin, neural cell adhesion molecule, A2B5, O4, microtubule-associated-protein-2 (MAP2) and glial fibrillary acidic protein], as well as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and ornithine decarboxylase assays were employed to characterize the NSFCs. The effects of trophic factors including epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), neurotrophic factors (NT-3), and basic fibroblast growth factor (bFGF) were evaluated. With the reduction of serum and the addition N2, B27, and other nutrients, there was a change in lineage restriction including an increase the expression of A2B5 and other glial markers as well as the expression of mature neuronal markers with a simultaneous reduction of nestin reactivity. NSFCs proliferated and maintained their pluripotency for over a year in the DM. Further studies will determine the utility of NSFCs for cell replacement therapy.