Fig. 1.

Generation and characterization of iPSCs and MNs from ALS patients and controls. a Semi-quantitative PCR of Oct4, Nanog, Sox2, and Rex1 in patient iPSCs, control iPSCs, human ESC (ES), and human fibroblasts (HF). b Immunocytochemical characterization of iPSCs for the pluripotency markers Tra1-60, SSEA4, Oct-4, and Nanog from 2/2, 3/1-2, and 3/2 lines. Scale bar = 50 µm. c Sequencing confirming the heterozygeous R521H and P525L FUS mutations in patient-derived iPSCs and the absence of FUS mutations in the control iPSC lines. d Hematoxylin–eosin staining identifying three germ layers in teratomas formed after iPSC injections in immunodeficient mice. Neural-like tissues represent ectoderm, cartilage represents mesoderm, and gut epithelium represents endoderm. e Schematic protocol of motor neuron differentiation. Y Y-27632, SB SB 431542, LDN LDN-193189, CHIR CHIR99021, RA retinoic acid, SAG smoothened agonist, DAPT a γ-secretase inhibitor, NEPs neuroepithelial stem cells, MNPs motor neuron progenitors, sMNs spinal motor neurons, BDNF brain-derived neurotrophic factor, GDNF glial cell line-derived neurotrophic factor, CNTF, ciliary neurotrophic factor. f Staining of different motor neuron markers (Hb9, Isl1, ChAT, SMI-32, Synapsin1), and DAPI in mutant FUS expressing and control cells at day 38. Scale bar = 50 µm. g Quantification of Isl1-positive (upper panel), ChAT-positive (middle panel), and SMI-32-positive (lower panel) cells expressed relative to the total number of DAPI-labeled cells. N = 10 images per line. Data are represented as mean ± SEM