Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen

Abstract

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.

Cooperation of Nuclear Fibroblast Growth Factor Receptor 1 and Nurr1 Offers New Interactive Mechanism in Postmitotic Development of Mesencephalic Dopaminergic Neurons

“…The SV40-immortalized rat ventral mesencephalic neuronal progenitor cells (SV40i-VM-NPCs) were described previously (30). The clone C2 was seeded in N2 medium (DMEM/Ham's F-12, 1% N2 supplement (100ϫ), 0.25% BSA, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mg/ml penicillin/streptomycin) containing 3% FCS and afterward cultivated in serum-free N2 medium.…”

“…The SV40i-VM-NPC cell line was previously established in our laboratory by stable transfection of the simian virus 40 (SV40) large T-antigen into primary neuronal ventral mesencephalic progenitor cells, derived from E12.5 rat embryos (30). In this cell line, Nurr1 and FGFR1 are endogenously expressed, and nuclear co-localization of both proteins was analyzed by immunocytochemistry and confocal microscopy (Fig.…”

“…Interaction of Nuclear FGFR1 and Nurr1-We used a SV40 immortalized ventral mesencephalic neuronal progenitor cell line (SV40i-VM-NPC) that expresses genes associated with mDA development and provides ample material for immunoprecipitation (30). The SV40i-VM-NPC cell line was previously established in our laboratory by stable transfection of the simian virus 40 (SV40) large T-antigen into primary neuronal ventral mesencephalic progenitor cells, derived from E12.5 rat embryos (30).…”

“…3D). We performed a detailed quantitative co-localization analysis using an intensity correlation algorithm (30,31). FGFR1 and Nurr1 showed a mutual dependent localization (Fig.…”

Highly Malignant Behavior of a Murine Oligodendrocyte Precursor Cell Line following Transplantation into the Demyelinated and Nondemyelinated Central Nervous System

“…brain, spinal cord, or the peripheral nerve (32,48,51,64,65). This implies that additional factors are defining Contrary to the rat CG4 cell line, BO-1 cells did neither require the addition of the growth-promoting B104 conthe malignant potential of the transplanted cells.…”