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Figure 2 Neuron differentiation and validation

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1 Figure 2 Neuron differentiation and validation
Neuron differentiation and validation (A) Work-flow of fibroblast-derived induced pluripotent stem cell (iPSC) differentiation into motor neurons; Wnt signaling pathway activator (WNT act), retinoic acid (RA), Sonic hedgehog (SHH), growth factors (GF; BDNF, IGF-1 and CNTF), Poly-d-lysine (PDL). (B) Validation of the expression of neural transcripts MAP2 and TUBB3 against GAPDH by quantitative PCR (qPCR) in total culture RNA of patient 1 clones 1 (Pt C1) and 2 (Pt C2) and controls 1-3 (ctr 1-3) after motor neuron differentiation. (C) Immunocytochemical analysis of MAP2 (green) and TUBB3 (red) proteins in patient 1 and control neuronal cultures. (D) Validation of the expression of motor neural transcripts ISL1, MNX1, and CHAT by qPCR as in B. (E) Immunocytochemical analysis of ISL1 (red) and NEFM (green) protein in patient 1 and control neuronal cultures. ISL1-positive neurons are shown in larger cell clusters in the final differentiation stage (day 14 of in PDL + laminin-coated plates). (F) Expression of intermediate filament subunits neurofilament medium (NEFM), neurofilament heavy (NEFH), and neurofilament light (NEFL) by qPCR as in B. The bars in each graph represent mean levels ± SD, n = 3 for each cell line. All scale bars 50 μm. 4′,6-diamidino-2-phenylindole (DAPI) indicates nuclear staining. Markus T. Sainio et al. Neurol Genet 2018;4:e244 Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

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