Pluripotent cells were differentiated as discussed previously
[14 (link),18 ]. Briefly, pluripotent cells cultured on E-cadherin-IgG Fc were harvested using Accutase (Millipore) and plated onto 6-well tissue culture-treated plates pre-coated with 2 mg/ml Matrigel (Geltrex; Invitrogen). Typically, one 100 mm dish of cells cultured on E-cadherin-IgG Fc provided enough cells for 2 wells of a 6-well plate. Approximately 24 hours after seeding the cells onto Matrigel, when the cells were 85-95% confluent, differentiation was initiated by culture for 5 days with 50 ng/ml Activin A (R&D Systems) in RPMI/B27 (without Insulin) supplement (Invitrogen) under ambient oxygen/5%CO2. In addition, we included 10 ng/ml BMP4 (Peprotech) and 20 ng/ml FGF-2 (Invitrogen) for the first 2 days. This resulted in reproducible differentiation into definitive endoderm at efficiencies of greater than 80%. Cells were cultured for 5 days with 20 ng/ml BMP4 (Peprotech)/10 ng/ml FGF-2 (Invitrogen) in RPMI/B27 (containing Insulin) under 4%O2/5%CO2, then 5 days with 20 ng/ml HGF (Peprotech) in RPMI/B27 (containing Insulin) under 4%O2/5%CO2, and finally for 5 days with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO2.
[14 (link),18 ]. Briefly, pluripotent cells cultured on E-cadherin-IgG Fc were harvested using Accutase (Millipore) and plated onto 6-well tissue culture-treated plates pre-coated with 2 mg/ml Matrigel (Geltrex; Invitrogen). Typically, one 100 mm dish of cells cultured on E-cadherin-IgG Fc provided enough cells for 2 wells of a 6-well plate. Approximately 24 hours after seeding the cells onto Matrigel, when the cells were 85-95% confluent, differentiation was initiated by culture for 5 days with 50 ng/ml Activin A (R&D Systems) in RPMI/B27 (without Insulin) supplement (Invitrogen) under ambient oxygen/5%CO2. In addition, we included 10 ng/ml BMP4 (Peprotech) and 20 ng/ml FGF-2 (Invitrogen) for the first 2 days. This resulted in reproducible differentiation into definitive endoderm at efficiencies of greater than 80%. Cells were cultured for 5 days with 20 ng/ml BMP4 (Peprotech)/10 ng/ml FGF-2 (Invitrogen) in RPMI/B27 (containing Insulin) under 4%O2/5%CO2, then 5 days with 20 ng/ml HGF (Peprotech) in RPMI/B27 (containing Insulin) under 4%O2/5%CO2, and finally for 5 days with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO2.
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