Figure 1.

Differentiation and Development of hiPSC Line-Induced Human DA Neurons

(A–E) Expression of pluripotency marker (OCT4), precursor cells marker (PAX6), neuron-specific marker (neuron-specific class III β-tubulin, TUJ1), and midbrain dopaminergic neuronal marker (TH) in neuronal populations in differentiation media for 10, 15, and 20 days. (A–D) Densitometry of the combined immunoreactive bands for OCT4 (A), PAX6 (B), TUJ1 (C), and TH (D) normalized to β-actin using NIH ImageJ software. Data are expressed as fold increase relative to DA neurons cultured in differentiation media for 10 days (n = 3 independent experiments; mean ± SEM). (E) Representative immunoblots for OCT4, PAX6, TUJ1, TH, and β-actin. β-Actin was used as a protein loading control.

(F) Real-time qPCR results for gene expression. The expression level of the undifferentiated hiPSC cells was set to 1 (n = 3 independent experiments).

(G and H) Co-expression of TUJ1 and TH in hiPSC-induced neurons cultured in differentiation media for 5, 10, 15, and 20 days. Quantification of neuronal process length of TUJ1-positive neurons and TH-positive DA neurons in differentiation media for different days was performed using NIH ImageJ (G). ^#p < 0.01 compared with day-5 (5d) cells, length of TUJ1 positive dendrites, and ^∗p < 0.01 compared with 5d cells, length of TH positive dendrites (n = 3 independent experiments; mean ± SEM, with 10 cells quantified per experiment). (H) Representative images for immunostaining of TUJ1 (green), TH (red), and co-localization (merge, yellow). Scale bars, 50 μm.

For (A), (D), and (G), statistical analysis was performed using StatView software version 5.0.1 (SAS Institute). One-way ANOVA was used for repeated-measures analysis, followed by Fisher's protected least significant difference for post hoc comparisons. Data are presented as mean ± SEM. Three independent experiments in (A–H); n = 10 cells per group per experiment in (G).