Differentiation of human induced pluripotent stem cells in William's E initiation medium supplemented with 3-bromopyruvate and 2-deoxy-D-glucose

Abstract

Hepatocyte selection medium (HSM) is deprived of glucose and supplemented with galactose, and is based on Leibovitz's-15 (L15) medium. HSM may promote the differentiation of human induced pluripotent stem (iPS) cells towards hepatocyte lineage. These culture conditions result in increased expression of galactokinase (GALK)-1 and GALK2. However, iPS cells do not survive in HSM. Two potential alternatives to glucose deprivation are treatment with 3-bromopyruvate (3BP), an analogue of pyruvate, and 2-deoxy-D-glucose (2DG), an analogue of glucose. The promoters of GALK1 and GALK2 were subcloned using the pMetLuc2 reporter plasmid to make pMetLuc2/GALK1 and pMetLuc2/GALK2, respectively. 201B7 human iPS cells were transfected with the reporter plasmids, cultured in HSM and analyzed by luciferase assay. Furthermore, 201B7 cells were cultured in L15, William's E (WE) or Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DF12) supplemented with 3BP, 2DG or a combination of the two, for 15 days, and subjected to reverse transcription-quantitative polymerase chain reaction to measure the levels of α-fetoprotein (AFP) mRNA expression. Metridia luciferase activity was significantly higher in cells cultured in HSM compared with those in ReproFF medium (P<0.05). 3BP and 2DG treatment, alone or in combination, decreased AFP expression levels in cells cultured in L15 and DF12. The combination of 3BP+2DG increased the expression levels of AFP in WE. Without 3BP or 2DG, AFP expression was higher in L15 compared with WE or DF12. The promoters of GALK1 and GALK2 were activated in 201B7 cells cultured in HSM, enabling survival using galactose as an energy source. 3BP and 2DG supplementation in WE medium may promote the differentiation of iPS cells to the hepatocyte lineage.