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Hepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE) medium, followed by a 2-day culture in HDI.Expression levels of α-feto protein (AFP) were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12). 201B7 cells expressed the highest AFP and albumin (ALB) when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation) and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition).201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.

Recent studies have demonstrated that micro (mi)RNA molecules can be detected in the circulation and can serve as potential biomarkers of various diseases. This study used microarray analysis to identify aberrantly expressed circulating miRNAs in patients with type 1 autoimmune hepatitis (AIH) compared with healthy controls. Patients with well-documented and untreated AIH were selected from the National Hospital Organization (NHO)-AIH-liver-network database. They underwent blood sampling and liver biopsy with inflammation grading and fibrosis staging before receiving treatment. To further confirm the microarray data, circulating expression levels of miR-21 and miR-122 were quantified by real-time quantitative polymerase chain reaction in 46 AIH patients, 40 patients with chronic hepatitis C (CHC), and 13 healthy controls. Consistent with the microarray data, serum levels of miR-21 were significantly elevated in AIH patients compared with CHC patients and healthy controls. miR-21 and miR-122 serum levels correlated with alanine aminotransferase levels. Circulating levels of miR-21 and miR-122 were significantly reduced in AIH patients with liver cirrhosis, and were inversely correlated with increased stages of fibrosis. By contrast, levels of circulating miR-21 showed a significant correlation with the histological grades of inflammation in AIH. We postulate that aberrantly expressed serum miRNAs are potential biomarkers of AIH and could be implicated in AIH pathogenesis. Alternations of miR-21 and miR-122 serum levels could reflect their putative roles in the mediation of inflammatory processes in AIH.

BACKGROUND: Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine. MATERIALS AND METHODS: Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM. RESULTS: The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells. CONCLUSION: Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.