Human embryonic stem cells were maintained and expanded using conventional techniques as previously reported. A HES3-Mixl1 reporter line was cultured using standard techniques. Briefly, cells were expanded and maintained on irradiated mouse embryonic feeder cells (MEFs) in KSR (knockout serum replacement) media supplemented with basic FGF at 10 ng/ml. Cells were passaged enzymatically using TrypLE (Thermo Fisher Scientific, USA) for 3 min, followed by being rinsed in Dulbecco’s minimum essential medium/F12. Before differentiation, cells were adapted to Matrigel-coated (Corning Life Sciences, USA) culturing techniques with MEF-conditioned KSR for one passage. Subsequently, cells were seed on Matrigel-coated six-well plates at a density of 12,000 cells/cm2 in conditioned KSR. The following day, KSR media was replaced with APEL (albumin polyvinylalcohol essential lipids) media with 8 μM CHIR.

For “day 6” cells, CHIR treatment was carried out for 2 days. After 2 days, the media was aspirated and replaced with APEL with FGF9 (200 ng/ml) and heparin (50 μg/ml). Media was replaced after 2 days, and on the sixth day, cells were dissociated with TripLE, rinsed in media and resuspended as described above. These cells were seeded immediately in bioreactors as described below.

For “day 3/4” cells, CHIR treatment was carried out for 4 days in total, with fresh media replaced after 3 days. These cells were then dissociated with TripLE and rinsed and resuspended in APEL with 8 μM in each bioreactor as described below.

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