Fig. 2

Characterization of iPSC-derived DA neurons with LRRK2 mutations. a Diagram showing the DA differentiation protocol used for neural induction of human iPSC lines. b Temporal gene expression analyzed by qRT-PCR at three time points: induction (3 weeks), expansion (4–5 weeks), and maturation (>6 weeks). Each point represents the mean ± SEM of at least two independent differentiation experiments. c Representative images of mature neuronal cultures showing expression of neuronal (βIII-tubulin, Tau, and α-synuclein) and dopaminergic (TH, NURR1) markers. Nuclei were counterstained with Hoechst. Scale bars: 50 μm. d Quantification of immunostainings. Data are represented as mean ± SEM of counts from at least two different lines for each genotype. e Representative western blot analyses of TH, Tau, and GFAP with βIII-tubulin as loading control in iPSC-derived mature neurons. f Representative immunoblots and quantification of LRRK2 expression in mature neuronal cultures. α-tubulin was the loading control and data were normalized to control WT neurons. Bars represent the mean ± SEM of at least two different lines per genotype. DIV days in vitro, GEL gelatin, POL poly-ornithine, FBN fibronectin, LMN laminin, N2 N2 supplement, bFGF basic fibroblast growth factor, SAG smoothened agonist, LDN LDN-193189, CHIR CHIR99021, SB SB431542, BDNF brain-derived neurotrophic factor, AA ascorbic acid, B27 B27 supplement, dbcAMP dibutyryl cyclic adenosine monophosphate, TGFβIII transforming growth factor βIII, GDNF glial derived neurotrophic factor. See Additional file 2 for uncropped blots