Imr90

Human pluripotent stem cells (IMR90, H9, and HES2) were provided by WiCell Research Institute (Madison, WI), and passaged 33–49. Cells were cultured on Matrigel gel (Corning)-coated plates in mTeSR (Stem Cell Technologies) or E8 medium (Thermo Fisher Scientific, Waltham, MA). Cells were passaged with ResLR (Stem Cell Technologies), washed, and replated at a dilution of 1:5 or 1:10.

Isogenic iPSC lines were obtained from Colman A et al. [18 (link)]. In brief, the female patient carried a heterozygous mutation with a 32 bp deletion within the 3′ coding region of the MeCP2 gene (MeCP2-1155del32) [18 (link)]. In this report, the isogenic iPSC lines that expressed only the specific MeCP2 allele (either wildtype or mutant) were denoted as MeCP2^wildtype or MeCP2^mutant, respectively. For normal controls, a non-disease iPSC line, IMR90 (female, WiCell, Madison, WI, USA) was used.

All the three hPSC lines, which includes human embryonic cell lines H9 and H1, and human induced pluripotent stem cell IMR90 were obtained from WiCell Research Institute, Inc. (Madison, WI, USA) and followed the same maintenance and differentiation protocols. Cells were cultured in hPSC-qualified Matrigel^TM (354277, BD Biosciences) coated cell culture plates using mTeSR^TM1 medium (05850, StemCell^TM Technologies). Mechanical scraping was applied during normal passaging in order to only get undifferentiated hPSC colonies after Dispase (07923, StemCell^TM Technologies) treatment. To induce mesoendodermal differentiation, cells were cultured in basal STEMdiff^TM APEL^TM medium (05210, StemCell^TM Technologies) supplemented with 100 ng/ml Activin A (338-AC-025, R&D Systems), 25 ng/ml BMP4 (314–BP–010, R&D Systems) and 10 ng/ml FGF2 (233–FB–025, R&D Systems).
The adult human dermal fibroblast (aHDF) was obtained from Lonza (Singapore) and cultured in DMEM high glucose medium (10569–010, Gibco) supplemented with 10% FBS (SV30160.03, Thermo Scientific Hyclone) and 1% Pen-Strep (09367-34, Nacalai Tesque). To get single cell suspension for cell seeding, the cells were washed with 1X PBS three times and treated with 0.25% Trypsion-EDTA (25200-114, Gibco) at 37 °C for 3–4 min.

hESC line H9 and human iPSC line IMR90 acquired from the WiCell Research Institute (Madison, WI, USA) were routinely maintained in mTeSR1 medium (StemCell Technologies, Vancouver, BC, Canada) on growth factor reduced Matrigel-coated tissue culture dishes at 37 °C and 5% CO₂ as described previously [4 (link),5 (link),39 (link)]. Cells were passaged by 1 mg/mL of dispase treatment and scraping every 3–4 days, with a split ratio of 1:3–1:6. The culture medium was exchanged daily. The morphology of cell colonies was examined daily, and spontaneously differentiated colonies were removed to ensure the maintenance of undifferentiated state of hPSCs. To characterize hESC and iPSC behavior under various conditions, ~5 × 10⁴ cells/cm² were seeded to the PET porous membrane surface with 2 × 10⁶ pores/cm² (EMD Millipore, Billerica, MA, USA) after Matrigel coating (Corning Inc., New York, NY, USA). Cells seeded onto Matrigel coated tissue culture polystyrene dishes, referred to as TCP, served as a control. Cell doubling time (t_d) was calculated as follows: $\frac{dx}{dt}=\mu x$
$t_d=\frac{\ln 2}{\mu }$
where x is the cell concentration and µ is the specific growth rate. hPSC numbers were counted at 24-h intervals during the culture using a hemocytometer after trypan blue staining.

Patient-derived iPSC lines with C9ORF72 mutations were obtained from the Target ALS stem cell core at RUCDR Infinite Biologics (Target ALS IDs TALS9-9.3 and TALS9-9.5; they are referred as ‘C9-ALS 1’ and ‘C9-ALS 2’ in this study, respectively) (Piscataway Township, NJ, USA) and Cedars-Sinai iPSC stem cell core (four iPSC lines named 28i, 29i, 30i and 52i; referred to here as ‘C9-ALS 3, 4, 5 and 6’) (Los Angeles, CA, USA). A human embryonic stem cell line WA09 (‘ESC 1’) and human iPSC line IMR90 (‘iPSC 1’) were obtained from WiCell (Madison, WI, USA), and iPSC line TD-A-47 (‘iPSC 2’) was obtained from Cellular Dynamics International (Madison, WI, USA). Stem cell lines from Cedars-Sinai were cultured on a mouse embryonic fibroblast feeder layer (Thomson et al., 1998 (link)), and lines from WiCell and Target ALS were cultured using a feeder-free protocol (Ludwig et al., 2006a (link),b (link)).