Geltrex ldev free hesc qualified reduced growth factor basement membrane matrix

BJ and PCS201 fibroblast cells, grown in DMEM + 10% FBS media, were dissociated by trypsin and collected by centrifugation. Human iPSCs (originated from BJ and PCS201 fibroblast cells) were grown in mTeSR media (STEMCELL Technologies) under Geltrex^TM LDEV-Free hESC-qualified reduced growth factor basement membrane matrix (Thermo Fisher Scientific). Cells were dissociated by accutase (Thermo Fisher Scientific) and collected by centrifugation. Cells were lysed and their protein content digested into peptides by trypsin. After desalting, the peptides were labeled with TMT reagents. The labeled samples were equally mixed and further fractionated by neutral pH reverse phase liquid chromatography. In general, 10 fractions were collected and further analyzed by low pH reverse phase LC-MS/MS. During ion fragmentation, the TMT regents are cleaved to produce reporter ions for quantification. The collected data were searched against a database to identify peptides. While the peptides were identified by MS/MS, the quantification was achieved by the fragmented reporter ions in the same MS/MS scans. The peptide quantification data were then corrected for mixing errors and summarized to derive protein quantification results. Statistical analysis was performed to determine cutoff for altered proteins and to evaluate associated false discovery rate.

The human male iPSC line denoted as SV20 was obtained from the University of Pennsylvania, where it was generated, and validated by the expression of pluripotency markers and differentiation into various cell types in multiple studies (Pagliaroli et al., 2021 (link); Pashos et al., 2017 (link); Yang et al., 2015 (link); Zhang et al., 2015 (link)). The human female iPSC line GM 23716 was obtained from the Coriell Institute for Medical Research (Camden, NJ, USA) and validated by the expression of pluripotency markers and differentiation into cranial neural crest cells in a previous study (Pagliaroli et al., 2021 (link)). The human female iPSC line 21792 and all three chimpanzee iPSC lines were obtained from the laboratory of Yoav Gilad at the University of Chicago and validated in previous studies (Gallego Romero et al., 2015 (link); Ward et al., 2018 (link)).
The iPSC lines were expanded in feeder-free, serum-free mTeSR1 medium (STEMCELL Technologies). Cells were passaged ∼1:10 at 80% confluency using ReLeSR (STEMCELL Technologies) and small cell clusters (50-200 cells) were subsequently plated on tissue culture dishes coated overnight with Geltrex LDEV-Free hESC-qualified reduced growth factor basement membrane matrix (Thermo Fisher Scientific).

iPSC cell lines WTSIi002-A (https://cells.ebisc.org/WTSIi002-A/, iPSC line1) and WTSIi008-A (https://cells.ebisc.org/WTSIi008-A/, iPSC line2) were purchased from EBISC (European Bank for Induced pluripotent Stem Cells) and maintained on Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific) in Essential 8 Flex Media Kit (Thermo Fisher Scientific) with 0.1% penicillin/streptomycin. Cultures were fed every other day and passaged every 5 to 7 days by ReLeSR (STEMCELL Technologies).

iPSC cell line WTSIi002 purchased from EBISC (European bank for induced pluripotent cells) was maintained on feeder-free conditions on Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (A1413302; Thermo Fisher Scientific) in Essential 8 Flex Media Kit (A2858501; Thermo Fisher Scientific) with 0,1% penicillin–streptomycin (15140122; Thermo Fisher Scientific).

Human iPSCs were seeded at 2.0 × 10⁵ cells/well on Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific)-coated six-well plates in the presence of 10-μM Y-27632. Approximately 24 h after seeding, culture medium was switched to PSC Neural Induction Medium (Thermo Fisher Scientific) containing Neurobasal Medium and Neural Induction Supplement. Neural Induction Medium was changed every other day from day 0 to day 6 of neural induction. From day 4 of neural induction, media volume was increased 2-fold. At day 7 of neural induction, primitive NSCs were dissociated using StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific) and then seeded at 1.0 × 10⁶ cells/well on Geltrex-coated six-well plates in Neural Expansion Medium containing 50% Neurobasal Medium, 50% Advanced DMEM/F12, Neural Induction Supplement, 0.25% AlbuMAX I Lipid-Rich bovine serum albumin (BSA), 1× penicillin−streptomycin solution, and 10-μM Y-27632. Culture medium without Y-27632 was changed every other day until NSCs reached confluence.