Directed Differentiation of hESCs

The hESC lines H1, Hes2-ICN1-ER™ and HES3-RUNX1C^GFP/w -EGFP were maintained on irradiated mouse embryonic fibroblasts in hESC media as described previously^{2 (link)}. For differentiation, hESCs were cultured on Matrigel-coated plasticware (BD Biosciences) for 24 hours, followed by embryoid body generation, as described previously^{2 (link)}. Briefly, the undifferentiated hESCs were dissociated with Tryp-LE (GIBCO) treatment, followed by scraping. Aggregates were resuspended in StemPro-34 (Invitrogen), supplemented with penicillin/streptomycin (10 ng/mL), L-glutamine (2mM), ascorbic acid (1mM), monothioglycerol (MTG, 4×10⁻⁴M; Sigma), transferrin (150 μg/mL) (referred to as “supplemented StemPro-34”) and BMP-4 (10 ng/mL), and cultured in 6-well low-cluster plates (Corning) in a volume of 2 mL per well. Following 24 hours, bFGF was added to a final concentration of 5ng/mL. At day 2, the developing EBs were harvested, washed and resuspended in supplemented StemPro-34 with BMP-4, bFGF and either CHIR99021 (3 μM, Stemgent), IWP2 (3μM, Tocris Bioscience) or Activin-A (1ng/ml) as indicated. After 24 hours, the EBs were again harvested and resuspended in supplemented StemPro-34 containing VEGF (15 ng/mL), bFGF (5 ng/mL), IL-6 (10 ng/mL) and IL-11 (5 ng/mL) and cultured for 48 hours. At this stage, the EBs were fed with 2ml of the same media containing also SCF (50 ng/mL final), IGF-1 (25 ng/mL final) and EPO (2 U/mL final) and cultured until day 8. Cultures were maintained in a 5% CO₂/5% O₂/90% N₂ environment. All recombinant factors are human and were purchased from R&D Systems. Where indicated 4-OHT (1 μM, Sigma), GSI (L-685,458, 10 μM, R&D Systems) PI3Ki (LY294002, 10 μM R&D System), MEKi (PD0325901, 1 μM, Stemgent) were included.