Supplementary Figure 9: Generation and characterization of induced pluripotent cells and motor neurons.

(a) Cytogenetic analysis of selected clone confirmed normal karyotype by G-banding staining. Representative image of 20 examined cells is shown. (b–d) Characterization of selected iPSC clone by immunofluorescence. Pluripotency was confirmed by staining for the following stem cells markers: (b) Nanog and Sox2 (c) Oct4 and SSEA4 (d) TRA-1-81 and SSEA1. Experiment was repeated 3 times independently with similar results. (e) Schematic timeline of iPS cells differentiation into motor neurons. (f) Immunofluorescence staining of motor neurons differentiated from human iPS cells demonstrates expression of the motor neurons precursor marker homeobox gene HB9, at day 21 of differentiation. (g) Bright field image of iPSC-derived motor neuron cultures. (h) Immunofluorescence staining demonstrates motor neurons maturation by expression of the axonal neurofilament heavy subunit (NF-H; green) and the microtubule-associated protein 2 (MAP2; red), at day 28 of differentiation. Experiments in f-h were reproduced 3 times independently with similar results. (i) Cell viability assay was performed in iPSC-derived motor neurons treated with control ASOs (mean = 78%) or with ASOs targeting TDP-43 (mean = 71.5%) or stathmin-2 (mean = 73.5%) for 20 days. Percentages of viable cells are plotted, 4 biologically independent motor neuron cultures are shown, error bars represent SEM.