Multipotent Neural Stem Cells from the Adult Tegmentum with Dopaminergic Potential Develop Essential Properties of Functional Neurons

Abstract

Neurogenesis in the adult brain occurs within the two principal neurogenic regions: the hippocampus and the subventricular zone of the lateral ventricles. The occurrence of adult neurogenesis in non-neurogenic regions, including the midbrain, remains controversial, but isolation of neural stem cells (NSCs) from several parts of the adult brain, including the substantia nigra, has been reported. Nevertheless, it is unclear whether adult NSCs do have the capacity to produce functional dopaminergic neurons, the cell type lost in Parkinson's disease. Here, we describe the isolation, expansion, and in vitro characterization of adult mouse tegmental NSCs (tNSCs) and their differentiation into functional nerve cells, including dopaminergic neurons. These tNSCs showed neurosphere formation and expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1, Neurog2, and Olig2, the NSC markers Nestin and Musashi1, and the proliferation markers Ki67 and BrdU (5-bromo-2-deoxyuridine). The cells showed typical propidium iodide–fluorescence-activated cell sorting analysis of slowly dividing cells. In the presence of selected growth factors, tNSCs differentiated into astroglia, oligodendroglia, and neurons expressing markers for cholinergic, GABAergic, and glutamatergic cells. Electrophysiological analyses revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels, action potentials, as well as currents induced by GABA (γ-aminobutyric acid), glutamate, and NMDA (N-methyl-d-aspartate). Clonal analysis demonstrated that individual NSCs retain the capacity to generate both glia and neurons. After a multistep differentiation protocol using co-culture conditions with PA6 stromal cells, a small number of cells acquired morphological and functional properties of dopaminergic neurons in culture. Here, we demonstrate the existence of adult tNSCs with functional neurogenic and dopaminergic potential, a prerequisite for future endogenous cell replacement strategies in Parkinson's disease.