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.

Lactate promotes survival and hepatocyte differentiation of human induced pluripotent stem cells in a medium without glucose and supplemented with galactose

“…Previous studies by our group showed that iPS differentiation toward the hepatocyte lineage was promoted in medium without glucose and supplemented with galactose (18,19). Hepatocyte differentiation is promoted by inhibiting glycolysis with 3-bromopyruvate and 2-deoxy-d-glucose, pyruvate and glucose analogs, respectively (29). The current data are consistent with those of the previous studies by our group.…”

“…The present RT-qPCR results indicated that AFP and albumin levels were upregulated in cells in HSM with calcium lactate. It was suggested that hepatocyte differentiation was promoted by HSM with lactate, as these two genes are hepatocyte markers (28,29). Previous studies by our group showed that iPS differentiation toward the hepatocyte lineage was promoted in medium without glucose and supplemented with galactose (18,19).…”

Interaction of stem cells derived from maxillary and mandibular bone with salts from culture medium

“…Importantly, the medium played an integral role in establishing a good manufacturing practice (cGMP)compliant process for the manufacturing of hiPSCs that can be used for generating clinically relevant cell types for cell replacement therapy applications [49]. Multiple various modified mediums were proposed, in order to provide a reliable, reproducible and controllable cell culture systems and promote the proliferation and growth of the seeded cells [50][51][52].…”

Innovation for hepatotoxicity in vitro research models: A review

“…For example, Kondo et al found that iPSCs could be converted to high‐purity, differentiated hepatocytes in highly enriched modified medium that contained galactose, phenylalanine, ornithine and oncostatin M, but was deprived of glucose, tyrosine, arginine and pyruvic acid. We have made a list to illustrate the supplements that increase maturity in Table (Kondo et al, ; Tomizawa et al, ; Tomizawa et al, , Tomizawa et al, , Tomizawa et al, , Tomizawa et al, , Tomizawa et al, ) (Table ).…”