An Optimal Medium Supplementation Regimen for Initiation of Hepatocyte Differentiation in Human Induced Pluripotent Stem Cells

Abstract

Human induced pluripotent stem (hiPS) cells are an ideal source for hepatocytes. Glucose and arginine are necessary for cells to survive. Hepatocytes have galactokinase (GALK), which metabolizes galactose for gluconeogenesis, and ornithine transcarbamylase (OTC), which converts ornithine to arginine in the urea cycle. Hepatocyte selection medium (HSM) lacks both glucose and arginine, but contains galactose and ornithine. Although human primary hepatocytes survive in HSM, all the hiPS cells die in 3 days. The aim of this study was to modify HSM so as to initiate hepatocyte differentiation in hiPS cells within 2 days. Hepatocyte differentiation initiating medium (HDI) was prepared by adding oncostatin M (10 ng/ml), hepatocyte functional proliferation inducer (10 nM), 2,2'-methylenebis (1,3-cyclohexanedione) (M50054) (100 μg/ml), 1× non-essential amino acid, 1× sodium pyruvate, nicotinamide (1.2 mg/ml), L-proline (30 ng/ml), and L-glutamine (0.3 mg/ml) to HSM. HiPS cells (201B7 cells) were cultured in HDI for 2 days. RNA was isolated, used as template for cDNA, and subjected to real-time quantitative polymerase chain reaction. Alpha-fetoprotein, γ-glutamyl transpeptidase, and delta-like 1 were upregulated. Expression of albumin was not observed. Expression of transcription factors specific to hepatocytes was upregulated. The expression of GALK2, OTC, and CYP3A4 were increased. In conclusion, differentiation of 201B7 cells to hepatoblast-like cells was initiated in HDI. Limitations were small number of cells were obtained, and the cells with HDI were not mature hepatocytes.

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

“…A previous study demonstrated that the expression levels of GALK1 and GALK2 are increased in iPS cells cultured in HDI that lacks glucose and is supplemented with galactose (14). These data suggest that glucose deprivation and galactose supplementation affect the transcription of GALK1 and GALK2.…”

“…hepatocyte lineage. One major problem is that iPS cells do not survive in HSM and HDI beyond 3 and 7 days, respectively (14). To overcome this limitation, the present study used 3BP and/or 2DG treatment to investigate an alternative to glucose deprivation and galactose supplementation.…”

“…These data suggest that galactose may be used as an energy source instead of glucose by iPS cells cultured in HSM. Unexpectedly, the expression levels of α-fetoprotein (AFP), a marker of immature hepatocytes, were increased in iPS cells cultured in HSM and HDI (11,14). These data suggest that hepatocyte differentiation may be initiated in glucose-free media that is supplemented with galactose.…”

“…In humans, there are two forms: GALK1 and GALK2 (13). The expression levels of GALK1 and GALK2 increase in iPS cells grown in a modified HSM: hepatocyte differentiation inducer (HDI) (14). These data suggest that galactose may be used as an energy source instead of glucose by iPS cells cultured in HSM.…”

Transcription Factors and Medium Suitable for Initiating the Differentiation of Human‐Induced Pluripotent Stem Cells to the Hepatocyte Lineage

“…This could be attributed to the incubation time of 7 days, which was relatively short for hepatocyte differentiation. Another reason might be that additional growth factors or small molecules were necessary [Tomizawa et al, ]. In future analyses, we plan to conduct investigations with longer incubation after transfection and addition of growth factors and small molecules to WE.…”