RESUMO
Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.
