hPSC lines, H1, Hues8, and FUCCI-H9, were cultured under feeder-free on hESC-qualified Matrigel (BD Biosciences) in E8 media (Stemcell Technologies) with 30% of irradiated mouse embryonic fibroblasts (iMEFs) conditional media. FUCCI-H9 hPSCs were kindly provided by S. Dalton29 (link) and were maintained under G418 sulfate (Sigma, 200 ug/ml) and Puromycin (0.1 ug/ml) selections. Cells were passaged every 3–5 days at 80% confluent with TrypLE Express (Invitrogen). After dissociation, cells were plated in E8 media with 10 uM Y-27632, (StemGent) for 24 hrs. After 24 hrs media, without Y-27632, was replenished daily. iMEF conditional media was prepared by incubating iMEFs with hPSC media without bFGF for 24 hrs for 7 days. Collected media was filtered, flash frozen and stored at −80 oC.
To maintain neural progenitors, cells were cultured in NPM media: DMEM/F12: Neurobasal media at a 1:1 ratio +0.5xB27 +0.5xN2 + 2 mM Glutamax +20 ng/mL bFGF (R&D Systems) +20 ng/mL EGF (R&D Systems) and media was replenished every four days. To split cells, cells were washed with PBS and incubated for 5 min at 37 oC in Accutase (Innovative Cell Technologies). Cells were washed, spun down at 1000 g for 5 min, and replated in NPM media on Geltrex-coated plates.
To maintain neural progenitors, cells were cultured in NPM media: DMEM/F12: Neurobasal media at a 1:1 ratio +0.5xB27 +0.5xN2 + 2 mM Glutamax +20 ng/mL bFGF (R&D Systems) +20 ng/mL EGF (R&D Systems) and media was replenished every four days. To split cells, cells were washed with PBS and incubated for 5 min at 37 oC in Accutase (Innovative Cell Technologies). Cells were washed, spun down at 1000 g for 5 min, and replated in NPM media on Geltrex-coated plates.
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