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

Human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation in a medium without glucose and supplemented with galactose, oncostatin M and small molecules [hepatocyte differentiation inducer (HDI)]. To clarify the metabolic differences between iPS cells in HDI and ReproFF (undifferentiated state), a metabolome analysis was performed. iPS cells were cultured in a medium without glucose and supplemented with galactose, as well as 1 mM of calcium lactate, sodium lactate or lactic acid. After 7 days of culture, the cells were subjected to reverse transcription-quantitative PCR analysis. The galactose-1-phosphate concentration was significantly higher in cells cultured in HDI than in those cultured with ReproFF. The lactate concentration in the HDI group was significantly lower than that in the ReproFF group. The expression levels of α-feto protein and albumin were significantly higher in the groups cultured with calcium lactate, sodium lactate and lactic acid as compared with ReproFF. It was suggested that lactate promoted the survival of iPS cells cultured in a medium without glucose and supplemented with galactose. Under these conditions, iPS cells begin to differentiate into a hepatocyte lineage. Lactate may be applied to produce hepatocytes from iPS cells more efficiently.

Oncostatin M in William's E medium is suitable for initiation of hepatocyte differentiation in human induced pluripotent stem cells.

William's E (WE) is a suitable medium for the differentiation of human induced pluripotent stem (iPS) cells to the hepatocyte lineage. The aim of the present study was to investigate various growth factors in their ability to promote hepatocyte differentiation of iPS cells in WE medium. Human iPS 201B7 cells were cultured in WE medium supplemented with growth factors, and mRNA expression levels and promoter activities of α­fetoprotein (AFP) and albumin were examined by reverse transcription­quantitative polymerase chain reaction and luciferase assay, respectively. In addition, time course analysis of AFP mRNA expression was performed in 201B7 cells cultured in WE medium supplemented with oncostatin M. The results demonstrated that mRNA expression levels of AFP were significantly elevated by most growth factors tested as supplements in WE medium, except all­trans retinoic acid, compared with cells cultured in ReproFF (a medium that maintains pluripotency). The highest increase in AFP mRNA expression levels was observed by oncostatin M stimulation. Albumin mRNA expression levels were increased by all­trans retinoic acid and insulin­transferrin­selenium supplementation in WE medium compared with cells cultured in ReproFF. Oncostatin M supplementation significantly stimulated the promoter activity of the AFP gene, but no growth factor tested significantly stimulated the promoter activity of the albumin gene. By time course analysis, significant increase of AFP mRNA expression was observed on the sixth day post­stimulation, compared with cells cultured in WE medium alone. In conclusion, the present study demonstrated that oncostatin M supplementation in WE medium was sufficient to initiate hepatocyte differentiation in iPS cells.

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

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.

Single-step protocol for the differentiation of human-induced pluripotent stem cells into hepatic progenitor-like cells.

Induced pluripotent stem (iPS) cells are ideal sources of hepatocyte for transplantation into patients experiencing hepatic failure. Growth and transcription factors were analyzed to design a single-step protocol for the differentiation of iPS cells into hepatocytes. The expression of transcription factors was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and compared among iPS cells, as well as fetal and adult liver cells. iPS cells were cultured with growth factors and RT-PCR was performed to analyze the expression of transcription factors. iPS cells were introduced with transcription factors, cultured with growth factors and subjected to real-time quantitative PCR. Indocyanine green (ICG) was added to the medium as a hepatocyte marker. Sox17, GATA4, GATA6, FoxA2, HEX, HNF4α and C/EBPα were expressed in fetal and adult liver cells, but not in iPS cells. Sox17, GATA6 and HNF4α were expressed after exposure a combination of oncostatin M, epidermal growth factor, retinoic acid, dexamethasone and ITS (OERDITS). When iPS cells were introduced with FoxA2, GATA4, HEX and C/EBPα and cultured with OERDITS for 8 days, the cells expressed α-fetoprotein, δ-like (Dlk)-1 and γ-glutamyl transpeptidase (GTP), and ICG uptake was observed. Exposure to FoxA2, GATA4, HEX and C/EBPα and culturing with OERDITS supplementation potentially serves as a single-step inducer for the differentiation of iPS cells into hepatic progenitor-like cells within 8 days.

Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine.

In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.