Induced pluripotent stem cells (iPSCs) were differentiated into hepatocytes according to the previously published protocol35 (link). In brief, cells were seeded into growth factor-reduced Matrigel-coated 6-well plates (2 million cells per well) in STEMdiff definitive endoderm basal medium (StemCell Technologies) containing Supplements A and B and cultivated for one day. From day 2 to 4, medium was exchanged daily and only contained Supplement B. At day 5, cells were detached and re-seeded into 96-well plates with a density of 12,000 cells per well. Medium was changed to Differentiation medium for hepatic specification: High-glucose DMEM/F12 (Gibco); 10% KOSR (Gibco); 1% Glutamin (Gibco); 1% Non-essential amino acids (Gibco); 1% Pen/Strep (Gibco); 100 ng/ml Human Growth Factor (peprotech); 1% DMSO; 10 µM Rock inhibitor Y27632 (Millipore).
From day 7 to 13, medium was changed to Differentiation medium without Rock inhibitor and refreshed daily. At day 14, medium was changed to Cultivation medium, which contained 10−7 M Dexamethasone (Sigma Aldrich) instead of Human Growth Factor and DMSO. This medium was also changed daily and used during treatments and further analysis.
To assess the level of iPSC differentiation into hepatocytes, expression levels of markers were analyzed on mRNA level by qRT-PCR: ALB, ABCC2, CYP1A1, HNF4A and RXRA.
From day 7 to 13, medium was changed to Differentiation medium without Rock inhibitor and refreshed daily. At day 14, medium was changed to Cultivation medium, which contained 10−7 M Dexamethasone (Sigma Aldrich) instead of Human Growth Factor and DMSO. This medium was also changed daily and used during treatments and further analysis.
To assess the level of iPSC differentiation into hepatocytes, expression levels of markers were analyzed on mRNA level by qRT-PCR: ALB, ABCC2, CYP1A1, HNF4A and RXRA.
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