Differentiation of human dopaminergic neurons.

<p>A) hiPS cells were directed to differentiate into dopaminergic neurons. Following midbrain patterning, neural progenitor cells were manually passaged (day 16+). Dopaminergic neurons were matured for a total of 6–10 weeks before experimentation. B) RT-PCR analysis of gene expression in cells at different stages of differentiation (EB: day 0, Rosette: day 16, Neuron: day 35). Pluripotency genes (OCT4 and SOX2) were down-regulated as cells differentiate and concurrently midbrain dopaminergic gene expression was upregulated. Midbrain dopaminergic neuronal markers (PITX3, NURR1, EN1, TH and GIRK2) were strongly expressed in differentiated neuronal populations. C) Immunocytochemical staining for TH (bottom panels) revealed an abundant population of neurons. Large clusters of neurons expressed the floorplate marker FOXA2 (top panels). Scale bar: 70 µm. D) The midbrain transcription factors, PITX3 and NURR1 were expressed in differentiated neurons. Scale bar: 70 µm. E) Efficiency of dopaminergic differentiation was quantified from at least 3 independent differentiations. iPS- NHDF1 and 2 lines differentiated with similar efficiencies into neurons (39.59% ±3.56 and 41.73% ±2.02, expressed as Tuj1/DAPI). The proportion of neurons that were dopaminergic was also similar (NHDF1: 50.35% ±3.4 and NHDF2: 52.54% ±2.76). The total number of cells that were TH⁺ were 19.21% ±2.05 and 21.5% ±1.53 for NHDF1 and NHDF2, respectively. Data are expressed as the mean ± SEM. F) Co-labelling of differentiated neurons revealed expression of dopaminergic neuronal proteins such as DAT and VMAT2 together with TH. The A9 dopaminergic neuron marker, GIRK2 was also expressed in some TH-positive cells. Scale bar: 70 µm. G) Analysis of MAPT exon splicing indicated that differentiated neurons have a splicing pattern similar to that of the human adult cortex. Exon skipping of exons 2, 3 and 10 was observed in undifferentiated hiPSCs, which is typical of embryonic neural tissues.