For differentiation to AME-E-like cells, partially primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x105/cm2 in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901 and 1μM A8301 (Cat. 2939, Tocris Bio-Techne). 100nM LDN193189 (alternative name DM3189, Cat. 1509, Axon Medchem) or 20ng/ml BMP4 (Miltenyi Biotec) were optionally added to the medium. The medium was changed daily. When the spheres appeared, the medium was refreshed by careful exchange of half volume of the medium, and the volume of the medium per well was increased.
Differentiation to TE-like cells was performed according to Guo et al. (2021) (link) and Io et al. (2021) (link). For differentiation to AME-L-like cells, primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x105/cm2 in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901, 1μM A8301 (Cat. 2939, Tocris Bio-Techne) and 20ng/ml BMP4 (Miltenyi Biotec). The medium was changed daily.
Differentiation to TE-like cells was performed according to Guo et al. (2021) (link) and Io et al. (2021) (link). For differentiation to AME-L-like cells, primed hPSC were dissociated to single cells using TrypLE Express and counted. The cells were plated to Geltrex-coated tissue culture plates at a seeding density of 1x105/cm2 in differentiation medium with 10μM ROCK inhibitor, and further cultured for 5 days. Differentiation medium was prepared as following: N2B27 basal medium, 1μM PD0325901, 1μM A8301 (Cat. 2939, Tocris Bio-Techne) and 20ng/ml BMP4 (Miltenyi Biotec). The medium was changed daily.
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