Pluristem

Human iPSCs were maintained on matrigel-coated dishes in mTeSR™1 (STEMCELL Technologies, Temecula, CA, USA) or PluriSTEM™ (EMD Millipore, Vancouver, BC, Canada) medium. Human iPSC colonies were treated with alphazyme (1 ml/well in a six-well plate; PAA, Linz, Austria) for 5 to 10 minutes to obtain single cells. Then 100,000 to 200,000 cells were seeded in each well of a six-well plate having mTeSR™1 or PluriSTEM™ medium with 10 μM Rock inhibitor (Y27632; Sigma-Aldrich) to prevent the cell apoptosis. Twenty-four hours later, the medium was changed to mTeSR™1 or PluriSTEM™ containing TAT–Cre (catalogue number SCR508; EMD Millipore) with different concentrations of 0.5 μM, 1 μM and 2 μM. Cells were incubated with TAT-Cre recombinant protein for 5 hours. Cells were grown for 1 week and colonies were expanded either monoclonally or polyclonally, and then polymerase chain reaction (PCR) was performed to assess transgene deletion. Transgene-deleted clones were expanded and characterized further by immunostaining and differentiation.

We previously described a baseline culture method for baboon iPSCs [31 (link)]. Briefly, mitotically inactivated mouse embryonic feeders (MEFs, EMD Millipore, Billerica, MA) were seeded onto gelatin coated tissue culture plates at a density of 2.6 x 10⁴ cells/cm² in MEF medium (DMEM, 10% Fetal Bovine Serum, 1 mM GlutaMax, 0.1 mM nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin [all from ThermoFisher Scientific, Waltham, MA]). Two days after seeding, the medium was replaced with iPSC media (80% KO-DMEM, 20% knockout serum replacer, 1 mM GlutaMax, 0.1 mM nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 ng/mL FGF2 (ThermoFisher Scientific). biPSCs were then seeded onto the MEFs and resulting colonies were manually passaged once every 5–7 days using a fire-polished glass Pasteur pipette. Media was replaced every 24 hours and cells were maintained at 37°C/5% CO₂. Use of this baseline media formula was compared to four other commercially available media–ReproFF (Stemgent, Lexington, MA), mTeSR1 and TeSR-E8 (both from Stem Cell Technologies, Cambridge MA) and PluriStem (EMD Millipore), following the corresponding manufacturer’s directions.