Mtesr plus

Female H9 (WA09; RRID: CVCL_9773) hESCs were obtained from WiCell and cultured in either mTeSR1 (Stem Cell Technologies 85850) for at least one passage before differentiation into CNCCs or mTeSR Plus (Stem Cell Technologies 100–0276) for gene editing, single-cell cloning, expansion and maintenance. hESCs were grown on Matrigel growth factor reduced basement membrane matrix (Corning 354230) at 37 °C. hESCs were fed every day for mTeSR1 or every 2 days for mTeSR Plus and passaged every 5–6 days using ReLeSR (Stem Cell Technologies 05872).
HEK293FT cells were obtained from Invitrogen (R70007) and cultured in complete medium (DMEM-HG (GE Healthcare Life Science SH30243.01), 10% FBS, 1× Non-essential amino acids (Gibco 1114-0050), 1× GlutaMAX (Gibco 4109-0036), 1× antibiotic–antimycotic (Gibco 1524-0062)). Cells were fed every other day and passaged every 2–3 days using trypsin–EDTA (Gibco 25200072).

For human induced pluripotent stem cell (hiPSCs; SCVI-113; Stanford, CA, USA) cultures, cells were maintained in 6-well tissue culture plates coated in hESC-qualified Matrigel Matrix (Corning, Corning, NY, USA) or LDEV-free reduced growth factor Geltrex Matrix (Thermo Fisher, Waltham, MA, USA) at 1:100 dilution with daily feeding with mTESR Plus (StemCell Technologies, Vancouver, BC, Canada) maintenance media. Upon reaching 70% confluency, cells were dissociated in PBS with 0.5 mM EDTA (Thermo Fisher) for 11 min at 37 °C and passaged at a 1:18 splitting ratio. First 24 h after replating included 10 µM of Y-27632 (StemCell Technologies, Vancouver, BC, Canada) in mTESR Plus media.

The use of CW20107 (from California’s Stem Cell Agency - CIRM) and KOLF2.2J (from The Jackson Laboratory) was part of the SSPsyGene consortium agreement on the common cell lines. The other 4 hiPSC lines (CD14, CD19, 8565612726, 8129019249) were specific to the MiNND project and were from Duan lab (Shi et al., 2009 (link); Zhang et al., 2023 (link); Zhang et al., 2020 (link)). Only 3 (CW20107, KOLF2.2J and CD14) of the 6 cell lines were used in the current report. Detailed cell line information is described in Table S2. The hiPSCs were maintained in mTeSRPlus (StemCell #100–0276) with primocin (Invitrogen #ant-pm-1) on tissue culture plates coated with matrigel (Fisher Scientific #08–774-552) or geltrex (Fisher Scientific #A1413202) throughout the mutagenesis process. The Institutional Review Board (IRB) of NorthShore University HealthSystem approved study.

SH-SY5Y human neuroblastoma cells were obtained from ATCC (CRL-2266) and maintained 1:1 EMEM/F12 media (ATCC #30–2003/Thermo #11765062) supplemented with 10% FBS and 1% Penicillin-Streptomycin (Thermo Fisher #15140122). SH-SY5Y cells were differentiated by treating with 10 μM all-trans retinoic acid (Sigma #R2625) for 6 days in EMEM/F12 media supplemented with 10% FBS and 1% Penicillin-Streptomycin, followed by 4 days of treatment with 50 ng/mL of BDNF (Peprotech #450-02B) in EMEM/F12 media supplemented with only 1% Penicillin-Streptomycin. HEK293FT were maintained in high glucose DMEM media with 10% FBS and 1% Penicillin-Streptomycin. The WTC11 and isogenic progranulin KO iPSC cell lines [22 (link), 23 ] were gifted by Bruce Conklin’s lab (Gladstone Institute, UCSF). For routine culture, iPSCs were plated in hESC matrigel (Corning #354277) coated plate with mTeSR plus (Stemcell Technologies #05825) and ROCKi (Stemcell Technologies #72304). iPSCs were maintained in mTeSR plus until confluent for lysate collection. Cells were washed with PBS and dissociated with Accutase (Thermo Fisher Scientific #A1110501).