Chemical induction of hESC into goblet cells

Protocols for chemical induction of GCs

1. hESC were obtained from the Otwo Biotech Inc., the hESC were isolated from the newborn foreskin. hESC were grown on plates coated with μg/cm² collagen IV (ColIV, Sigma-Aldrich) in EpiLife medium (Invitrogen, #MEPI500CA) supplemented with 0.06 mM Ca2⁺, 1% Human Keratinocyte Growth Supplement (Invitrogen, #S0015), and 1% P/S (Invitrogen). Cultures were routinely maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

2. 6-well plates, 24-well plates, or glass coverslips were first coated with collagen I (Corning, 354236) at 10 μg/cm² at room temperature for one hour, and then carefully aspirate remaining solution. Use PBS to rinse the well to remove acid, and plates are air dried. 

3. hESC were digested and passaged with TrypLE™ (Invitrogen) and then plated onto collagen I-coated wells in the EpiLife medium. When cells reached 70 to 80% confluence, the culture medium was replaced by the chemical induction medium consisting of DMEM/F12 (Invitrogen, #10565018), 0.5% N2 (Gibco, #17502048), 0.5% B27 (Gibco, #17504044), and 1% P/S, supplemented with 5 mM Repsox (MedChem Express, #HY-13012), 3 mM CHIR99021 (MedChem Express, #HY-10182), bFGF (10 ng/ml; PeproTech, #100-18B-50), and BMP4 (10 ng/ml; PeproTech, #120-05ET-10). The chemical induction medium was refreshed every 2 days. After 6 to 8 days of induction, hESC were induced into GCs, and the induced GCs were maintained in the chemical induction medium for the following use. 

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