Figure 3
Neuronal maturation and functionality of midbrain-type DA neurons generated by combinatory exogenous Nurr1 and Foxa2 expression. TH+ DA cells were generated from E14 cortical NPCs by combinatory expression of Nurr1+ Foxa2 using diverse retro-and lentiviral transductions. (a–c) Neuronal maturation of TH+ cells. Representative immunostaining images for TH+ (upper) and TH+/TuJ1+ cells (lower) (a). Neuronal maturity of TH+ DA cells was evaluated by TH+ fiber length (b) and % TuJ1+ cells out of total TH+ cells (c) at D7. *Significantly different from cultures transduced with Retro-pLTR-Nurr1+Retro-pLTR-Foxa2 at P<0.05, n=50 TH+ cells (b), 20 microscopic fields (c). (d–f) Neuronal maturation of TH+ cells during the late differentiation period (D7–D14). Representative images for TH+ cells (left), TH+/Tuj1+ (right upper) and TH+/MAP2+ cells (right lower) at differentiation days 7 and 14 (D7 and D14) (d). TH+ fiber length (e) and co-expression of the TH/Tuj1 (f) at D7 and D14. Significantly different from cultures transduced with Retro-pLTR-Nurr1+Retro-pLTR-Foxa2* and from D7# at P<0.05, n=50 TH+ cells (e), 20 microscopic fields (f). (g–i) Phenotypic and functional analysis of TH+ mDA neuronal cells differentiated from NPCs transduced with Lenti-pUb-Nurr1+Retro-pLTR-Foxa2. Mature midbrain DA neuron phenotypes were analyzed by co-expression of neuronal (MAP2 and NeuN), general DA neuronal (DAT, VMAT2) and midbrain-specific DA neuronal marker (PITX3) (g, h). Measurement of released DA in media (24 h; upper) and evoked by KCl (56 mM; 30 min; lower) from differentiated neurons (i). *Significantly different from the cultures transduced with Retro-pLTR-Nurr1+Retro-pLTR-Foxa2 at P<0.05, n=20 microscopic fields (h) and three independent cultures (i). #Significantly different from the cultures transduced with control vector at P<0.05. Scale bar, 20 μm. DA, dopamine; TH, tyrosine hydroxylase.
