Figure 4.

The expression of miR-218 in iDAn improves dopaminergic differentiation. a, Procedural scheme for trans-differentiation of MEFs into iDAn after miR-218-1 or miR-218-2 overexpression. MEFs from E14.5 tgTH^GFP/+ mouse embryos were infected with the transcription factors Ascl1, Nurr1, and Lmx1a (ANL) alone or in combination with the miR-218-1 (or miR-218-2) cloned upstream Ires-mCherry sequence and differentiated for 2 weeks. b, qPCR for pre-miR-218-1 and pre-miR-218-2 on iDAn overexpressing the two different isoforms. U6 snRNA and sno-202 RNA were used for normalization. Data are mean ± SEM of 2^−ΔCt values. *p < 0.05, n = 4; Kruskal–Wallis test plus Dunn's multiple comparison correction. c, The expression of Th, Pitx3, Vmat2, and Dat was analyzed by qPCR in iDAn overexpressing miR-218-1. Data are mean ± SEM from three independent experiments. *p < 0.05 (Student's t test). d, iDAn trans-differentiation efficiency is shown in the representative pictures obtained through the overexpression of Ascl1, Nurr1, and Lmx1a (herein ANL) in combination with the miR-218-1-Ires-Cherry into MEFs isolated from tgTH^GFP/+ mice. Images were acquired with DMI6000 at 20× magnification. Scale bar, 100 µm. e, The number of iDAn was evaluated by counting Th-GFP⁺ or Th-GFP⁺/mCherry⁺ cells. Manual counting was performed at least on five different fields for each well in four independent trans-differentiation experiments (n > 20) and normalizing for the analyzed surface. Data are mean ± SEM. ANL: 27.09 ± 3.82/mm² versus ANL218-1: 46.63 ± 3.7/mm² versus ANL218-2: 47.47 ± 6.05/mm²; respectively, p = 0.0055 and p = 0.0298 with one-way ANOVA nonparametric Kruskal–Wallis test and Dunn's multiple comparison correction. n ≥ 3 biological x2 technical. f, g, Ctrl (ANL) and miR-218-1-Ires-mCherry (ANL+miR218-1) overexpressing iDAn analyzed by FACS. h, The number of total TH-GFP⁺ cells was normalized with Cherry⁺ miR-218-1 (miR-218-2) cells. Data are mean ± SEM. ANL: 9.11 ± 1.3 versus ANL218-1: 32.99 ± 3.51, p = 0.0049; ANL versus ANL218-2: 31.02 ± 2.57, p = 0.0099; one-way ANOVA nonparametric test Kruskal–Wallis test and Dunn's multiple comparison correction. n ≥ 3 biological x2 technical. j-l, Functional characterization of iDAn obtained by trans-differentiation of MEFs with and without miR-218-1. iDAn were identified as TH-GFP⁺ or as double positive: TH-GFP⁺, miR-218-mCherry⁺, revealed that voltage-dependent Na⁺(i) and K⁺(j) currents displayed nearly overlapping current to voltage relationships in the two groups Data are mean ± SEM. Two-way repeated-measures ANOVA, Bonferroni test (not significant). Nevertheless, iDAn overexpressing miR-218-1 in combination with ANL produce a higher number of APs (k) in response to depolarizing current injections (15-105 pA, 10 pA increment, 300 ms) compared with iDAn generated only with ANL (TH-GFP⁺) Data are mean ± SEM. 65 pA and 75 pA, p < 0.05; 85-105 pA, p < 0.01; two-way repeated-measures ANOVA followed by Tukey test. Representative traces were reported in the quadrant.