Human iPS cell culture
All policies of the NIH Intramural research program were followed for the procurement and use of iPS cells. For most studies, the iPS cells used were from the WTC11 line, derived from a healthy 30-year-old male, and obtained from the Coriell cell repository. Infomed consent was obtained from the donor. We confirmed the WTC11 line contained no ALS–FTD mutations in the ALS and FTD risk genes in Supplementary Table 1. For key experiments, an independent line was used, NCRM5. NCRM5 was derived from umbilical cord blood from NIH Center for Regenerative Medicine (CRM), Bethesda, MD, USA. Informed consent was obtained from the donor. All culture procedures were conducted as previously11. In brief, iPS cells were grown on tissue culture dishes coated with human embryonic stem cell-qualified Matrigel (Corning, catalogue no. 354277). They were maintained in Essential 8 Medium (E8; Thermo Fisher Scientific, catalogue (cat.) no. A1517001) supplemented with 10 μM ROCK inhibitor (RI; Y-27632; Selleckchem, cat. no. S1049) in a 37 °C, 5% CO2 incubator. Medium was replaced every 1–2 days as needed. Cells were passaged with accutase (Life Technologies, cat. no. A1110501), 5–10 min treatment at 37 °C. Accutase was removed and cells were washed with PBS before re-plating. Following dissociation, cells were plated in E8 media supplemented with 10 μM RI to promote survival. RI was removed once cells grew into colonies of 5–10 cells.
The following cell line and DNA samples were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research: GM25256.
Data
Publicly available data were obtained from the Gene Expression Omnibus (GEO): iPS cell MNs9, GSE121569; SK-N-DZb, GSE97262; FACS-sorted frontal cortex neuronal nuclei, GSE126543; Riboseq, E-MTAB-10235; targeted RNA-seq, E-MTAB-10237; minigene TDP-43 iCLIP, E-MTAB-10297; SH-SY5Y TDP-43 iCLIP, E-MTAB-11243; and UNC13A-targeted nanopore, E-MTAB-11244.
CRISPRi knockdown in human iPS cells
The human iPS cells…