Mtesr plus 5x supplement

Human patient derived CHOP10.2P wildtype iPSCs on mouse embryonic fibroblasts (MEFs) were obtained from the Human Pluripotent Stem Cell Core at the Children’s Hospital of Philadelphia. The iPSCs were transitioned into feeder-free conditions inhouse and maintained as feeder-free cultures in mTeSR Plus basal medium supplemented with mTeSR Plus 5X supplement (StemCell Technologies). The cultures were grown in multiwell plates coated with Matrigel^® hESC-Qualified Matrix (Corning) and maintained in an antibiotic-free environment at 37°C with 5% CO₂. Medium was changed every other day and the cells were passaged as colonies with Versene when >70% confluent.
For luciferase assays, the iPSCs were passaged as single cells using Accutase and plated into Matrigel-coated 24-well plates at a density of 50,000 cells/well with 10 μM ROCK Inhibitor. 24 hours post-seeding, the medium containing ROCK Inhibitor was replaced with fresh mTeSR Plus complete medium.

HeLa cells [American Type Culture Collection (ATCC)] were cultured in Dulbecco’s modified Eagle’s medium (Gibco; 11995-065) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Human iPSCs derived from a PD patient harboring a parkin loss-of-function mutation (B125: homozygous c.1072del, male, age of onset: 43) as well as a CRISPR-generated isogenic control were maintained on Cultrex-coated (R&D Systems; 3434-010-02) six-well plates, cultured in mTeSR Plus Basal Medium (STEMCELL Technologies; 100-0274) with mTeSR Plus 5X Supplement (STEMCELL Technologies; 100-0275) and were passaged every 5 to 6 days. All cells were maintained at 37°C in a 5% CO₂ incubator and were previously verified by cytochrome c oxidase subunit I and short tandem repeat testing and tested for mycoplasma contamination. HeLa cells were transfected using X-tremeGENE HP (Roche; 6366546001). iPSC-derived dopaminergic neurons were transduced with the lentiviral constructs described below. For live-cell imaging, cells were grown on four-chamber glass bottom dishes (Cellvis; D35C4-20-1.5-N).