Matrigel coated culture dishes

The hiPS cell line was previously generated by our group. This cell line derives from an unaffected male donor and has an APOE 3/3 genotype. The line was analyzed for pluripotency markers expression by quantitative PCR and for chromosomal abnormalities by karyotyping, and was able to differentiate into the three germ layers. Whole exome sequencing of the parental hiPSC line confirmed the absence of rare coding variants in the SORL1 gene, as well as in APP, PSEN1, PSEN2, TREM2, ABCA7 and other genes causing Mendelian forms of dementia [30 (link)]. hiPSCs were cultured on feeder-free conditions in mTeSR Plus medium (STEMCELL Technologies, Vancouver, Canada) on Matrigel-coated culture dishes (Corning, Corning, NY, USA) diluted in DMEM-F12 according to manufacturer’s instructions in a 37 °C/5% CO₂ incubator. Cells were split when they reached 80% confluency using StemPro Accutase (Thermo Fisher Scientific) and plated in 10 µM ROCK inhibitor (StemGent, Cambridge, MA, USA) supplemented medium. Medium was refreshed the next day to remove ROCK inhibitor. Differentiating colonies were removed from the plate before splitting. Cell lines were confirmed to be free of mycoplasma.

Experiments on hESCs have been approved by the Agence de la Biomedecine and informed consent was obtained from all subjects. The hESC lines (H1 and H9, obtained from the WiCell Research Institute, and WIBR2, obtained from the Whitehead Institute for Biomedical Research) were maintained on Matrigel-coated culture dishes (Corning) in mTeSR1 medium (Stem Cell Technologies) and were cultured in a 37 °C incubator with 20% O₂ and 5% CO₂. Cells were routinely passaged as clumps using gentle Cell Dissociation Reagent (Stemcell Technologies) approximately every 3–4 days. For experiments requiring single-cell suspension, cells were detached using Accutase (Stemcell Technologies) and plated on mTeSR1 supplemented with 10 µM of ROCK inhibitor (Y-27632, Merck Millipore). Differentiation experiments were carried out for 10 days on gelatin-coated 6-well plates by transitioning cells in Dulbecco’s modified Eagle medium (ThermoFisher Scientific) with 10% of fetal bovine serum, 2 mM l-glutamine, and 0.1 mM non-essential amino acids (ThermoFisher Scientific).