Ropinirole and Pramipexole Promote Structural Plasticity in Human iPSC-Derived Dopaminergic Neurons via BDNF and mTOR Signaling

Figure 1

Differentiation of F3 human iPSCs in dopaminergic neurons. (a) Diagram of the time and conditions used for the differentiation of dopaminergic neurons from hiPSCs. (b–f) Representative images of dual immunofluorescence indicating coexpression of (b) LMX1-A (green) and FOXA2 (red) in dopaminergic progenitors at day 11; (c) TH (green) and MAP2 (red) in neurons at day 30; (d) TH (green) and DAT (red); (e) TH (red) and GLUR2 (green); and (f) TH (green) and VMAT2 (red) in DA neurons at day 70. (g, h) Dual immunofluorescence of (g) TH (red) and GAD67 (green) and (h) TH (red) and VGLUT2 (green) in neuronal cultures at day 70. Cell nuclei were stained with DAPI (blue). Scale bar: (b, c) = 40 μm; (d, e, g, and h) = 20 μm; and (f) = 10 μm. (i, j) Semiquantitative RT-PCR analysis of (i) gene expression at the iPSC stage and at day 11, 19, 50, and 70 of dopaminergic differentiation and (j) expression of D2 and D3 receptors at the iPSC stage and at day 11, 19, 70, 80, and 90 of dopaminergic differentiation (negative controls contain PCR Master Mix and primers, but no cDNA). (k) Dopamine level measured by HPLC in the supernatant of F3 DA cultures (day 70) following incubation with the DAT inhibitor GBR12935 (GBR) or vehicle (V) over a time course (0, 3, 6, 9, and 24 hrs after administration). Data are expressed as mean ± SEM ( versus vehicle at 24 hrs, versus vehicle at 9 hrs, and and versus the corresponding treatment (GBR or V) at time 0 h; post hoc Bonferroni’s test). KSR, knockout serum replacement; LDN, LDN193189; SB, SB431542; Shh, Shh C25II; FGF8, fibroblast growth factor 8; Purm, purmorphamine; CHIR, CHIR99021; BDNF, brain-derived neurotrophic factor; AA, ascorbic acid; cAMP, dibutyryl cAMP; TGFβ, transforming growth factor type β3; GDNF, glial cell line-derived neurotrophic factor.

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