Dopaminergic differentiation of the Nurr1-expressing immortalized mesencephalic cell line CSM14.1 in vitro.
The use of neural stem cells as grafts is a potential treatment for Parkinson's disease, but the potential of stem cells to differentiate into dopaminergic neurones requires investigation. The present study examined the in vitro differentiation of the temperature-sensitive immortalized mesencephalic progenitor cell line CSM14.1 under defined conditions. Cells were derived from the mesencephalic region of a 14-day-old rat embryo, retrovirally immortalized with the Large T antigen and cultured at 33 degrees C in DMEM containing 10% fetal calf serum (FCS). For differentiation, the temperature was elevated at 39 degrees C and FCS was reduced (1%). Using histology, immunocytochemical detection of the stem cell marker Nestin and the neuronal marker MAP5 and, in addition, Western blotting to determine the presence of neurone-specific enolase and the neurone nuclei antigen we demonstrated a differentiation of these cells into neuronal cells accompanied by a decrease in Nestin production. In Western blots, we detected the orphan nuclear receptor Nurr1 in these cells. This was followed by a time-dependent up-regulation of the enzymes tyrosine hydroxylase and aldehyde dehydrogenase 2 characteristic of mature dopaminergic neurones. Our in vitro model of dopaminergic cell differentiation corroborates recent in vivo observations in the developing rodent brain.
Haas
Stefan Jean-Pierre
SJ
Institute of Anatomy, University of Rostock, Germany.
Wree
Andreas
A
D000444:Aldehyde Dehydrogenase / Q000032:analysis; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000818:Animals; D000957:Antigens, Viral, Tumor; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western / Q000379:methods; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins; D004298:Dopamine / Q000502:physiology*; D007150:Immunohistochemistry / Q000379:methods; D007381:Intermediate Filament Proteins / Q000032:analysis; D008636:Mesencephalon / Q000196:embryology*; D008869:Microtubule-Associated Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins; D064231:Nestin; D009474:Neurons / Q000737:chemistry*; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D051381:Rats; D013234:Stem Cells / Q000502:physiology*; D014157:Transcription Factors / Q000032:analysis*; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis
D000957:Antigens, Viral, Tumor; D015415:Biomarkers; D004268:DNA-Binding Proteins; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C577484:NES protein, human; C539882:NR4A2 protein, human; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; C539884:Nr4a2 protein, rat; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D014157:Transcription Factors; C085284:microtubule-associated protein 1B; D014446:Tyrosine 3-Monooxygenase; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D010751:Phosphopyruvate Hydratase; D004298:Dopamine