(A) Schematic of the CM treatment experiments. Media conditioned in Ctx- or VM-Ast (or Ctx-NPC as control) was collected and added to cultured VM-NPCs during the differentiation period. (B) Representative images for TH⁺ DA neurons at D12. Scale bar: 100 μm. Insets, enlarged images of the boxed areas (original magnification, ×400). (C) TH⁺DA neuronal yields at D6. *P < 0.05, significantly different from control. n = 3 cultures for each group. (D and E) Morphometric measurement of neurite outgrowth assessed by a time-lapse imaging system. VM-NPCs at D3 were treated with the CMs. Neurite lengths (D) and branch points of the neurites (E) in 27 randomly selected microscopic fields from 3 independent cultures were automatically analyzed for 42 hours using IncuCyte’s NeuroTrack software. (F–J) Expression of mature neuronal (NeuN) and mDA neuronal (Foxa2, Nurr1) markers in the differentiated DA neurons. I and J depict percentage of TH⁺ cells expressing the markers and the expression levels (MFI) in individual TH⁺ DA neurons, respectively. *P < 0.01, significantly different from control; ^#P < 0.01, significantly different from Ctx-Ast, 1-way ANOVA. n = 9 microscopic fields (I) and n = 32–36 TH⁺ cells (J). (K–M) Resistance of DA neurons against a toxic stimulus. TH⁺ DA neurons differentiated in the presence of Ctx- or VM-Ast-CM (Ctx-NPC-CM as the control) at D12 were exposed to H₂O₂ (1,000 μM) for 8 hours and viable TH⁺ cells were counted on the following day (L). Shown in K are representative TH⁺ cell images after H₂O₂ treatment. Scale bars: 100 μm. Insets, high-powered images of the boxed areas (original magnification, ×400). Fiber lengths of surviving TH⁺ cells were also estimated (M). *P < 0.01; ^#P < 0.01, 1-way ANOVA. n = 3 independent experiments (2–3 wells/experiment) (L) and n = 80–90 TH⁺ cells (M). (N) Expression of neurotrophic genes in the cultured Ctx-Ast, VM-Ast, and Ctx-NPCs, estimated by real-time PCR (qPCR) analyses. *P < 0.01; ^#P < 0.05, 1-way ANOVA. n = 3.