Association of a single nucleotide polymorphism in TNIP1 with type-1 autoimmune hepatitis in the Japanese population.

Several studies reported that autoimmune diseases share a number of susceptibility genes. Of these genes, a SNP rs7708392 in TNIP1 was reported to be associated with systemic lupus erythematosus (SLE). Autoimmune hepatitis (AIH), a rare chronic progressive liver disease, shares some clinical features with SLE. Therefore, we investigated whether the SNP is associated with Japanese AIH. An association study of rs7708392 was conducted in 343 Japanese AIH patients and 828 controls. We found that rs7708392 is associated with AIH (P = 0.0236, odds ratio (OR) 1.26, 95% confidence interval (CI): 1.03-1.54), under the allele model for C allele. Significant differences of clinical characteristics of the AIH patients with or without G allele of rs7708392 were not detected. Of interest, the association was stronger in AIH without HLA-DRB1*04:05 allele (P = 0.0063, Q = 0.0127, OR 1.48, 95% CI: 1.12-1.96), though the association was not detected in AIH with DRB1*04:05. The C allele of rs7708392 was associated with AIH, especially AIH without DRB1*04:05, an already established risk factor.

Association of a single nucleotide polymorphism upstream of ICOS with Japanese autoimmune hepatitis type 1.

Autoimmune hepatitis (AIH) is an uncommon chronic autoimmune liver disease. Several studies reported the association of polymorphisms between CD28, CTLA4 and ICOS gene cluster in 2q33.2 with autoimmune or inflammatory diseases. The previous genome-wide association study on type 1 AIH in a European population has reported a risk G allele of a single nucleotide polymorphism (SNP), rs4325730, in this region. Here, we conducted an association study of this SNP with type 1 AIH in a Japanese population, as a replication study.An association study of rs4325730 was conducted in 343 Japanese AIH patients and 315 controls.We found that rs4325730 is associated with AIH (P=0.0173, odds ratio (OR) 1.30, 95% confidence interval (CI) 1.05-1.62, under the allele model for G allele, P=0.0070, OR 1.62, 95% CI 1.14-2.31, under the dominant model for G allele). This SNP was strongly associated with definite AIH (P=0.0134, OR 1.36, 95% CI 1.07-1.74; under allele model for G, P=0.0035, OR 1.85, 95% CI 1.22-2.81, under dominant model for G).This is the first replication association study of rs4325730 upstream of ICOS with AIH in the Japanese population and rs4325730G is a risk allele.

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

Transcription factors and culture media were investigated to determine the condition to initiate the differentiation of human-induced pluripotent stem (iPS) cells most efficiently. The expression of genes in human adult liver was compared with that in 201B7 cells (iPS cells) using cDNA microarray analysis. Episomal plasmids expressing transcription factors were constructed. 201B7 cells were transfected with the episomal plasmids and cultured in ReproFF (feeder-free media maintaining pluripotency), Leibovitz-15 (L15), William's E (WE), or Dulbecco's modified Eagle medium/Nutrient F-12 Ham (DF12) for 7 days. RNA was isolated and subjected to real-time quantitative PCR to analyze the expression of alpha-feto protein (AFP) and albumin. cDNA microarray analysis revealed 16 transcription factors that were upregulated in human adult liver relative to that in 201B7 cells. Episomal plasmids expressing these 16 genes were transfected into 201B7 cells. CCAAT/enhancer-binding protein alpha (CEBPA), CCAAT/enhancer-binding protein beta (CEBPB), forkhead box A1 (FOXA1), and forkhead box A3 (FOXA3) up-regulated AFP and down-regulated Nanog. These four genes were further analyzed. The expression of AFP and albumin was the highest in 201B7 cells transfected with the combination of CEBPA, CEBPB, FOXA1, and FOXA3 and cultured in WE. The combination of CEBPA, CEBPB, FOXA1, and FOXA3 was suitable for 201B7 cells to initiate differentiation to the hepatocyte lineage and WE was the most suitable medium for culture after transfection. J. Cell. Biochem. 117: 2001-2009, 2016. © 2016 Wiley Periodicals, Inc.

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.

Diffusion-weighted whole-body imaging with background body signal suppression/T2 image fusion and positron emission tomography/computed tomography of upper gastrointestinal cancers.

PURPOSE: Diffusion-weighted whole-body imaging with background body signal suppression/T2 image fusion (DWIBS/T2) strongly contrasts cancerous tissue against background healthy tissues. Positron emission tomography/computed tomography (PET/CT) applies the uptake of 18-fluorodeoxyglucose in the diagnosis of cancer. Our aim was to compare DWIBS/T2 and PET/CT in patients with upper gastrointestinal cancers. METHODS: Patient records, including imaging results from July 2012 to March 2015, were analyzed retrospectively. Four men (age, 72.5 ± 5.3 years) and ten women (age, 71.6 ± 4.0 years) were enrolled in this study. The numbers of patients with esophageal cancer, gastric cancer, gastrointestinal stromal tumor, and duodenal cancer were one, eight, three, and two, respectively. RESULTS: Six out of eight patients with gastric cancer had positive results on both DWIBS/T2 and PET/CT. The diameter and depth of invasion of gastric cancer was larger in patients with positive DWIBS/T2 and PET/CT findings than those with negative findings. These results suggested that patients with gastric cancer with larger pixel numbers might tend to show positive results with DWIBS/T2. CONCLUSIONS: DWIBS/T2 and PET/CT have similar sensitivity for the diagnosis of upper gastrointestinal cancer. The diameter and depth of invasion affected the detectability of gastric cancer.

Diffusion-weighted whole body imaging with background body signal suppression/T2 image fusion is negative for patients with intraductal papillary mucinous neoplasm.

BACKGROUND/AIMS: One major problem with Intraductal papillary mucinous neoplasm (IPMN) is the appearance of pancreatic duct adenocarcinoma. Diffusion-weighted whole body imaging with background body signal suppression (DWIBS) provides hyperintense signals in cases of cancer. DWIBS and T2 image fusion (DWIBS/T2) provides functional information in anatomical settings, and is useful for the detection of cancer with strong contrast against surrounding tissues. DWIBS/T2 signals were analyzed in patients with IPMN to investigate positive or negative results. METHODOLOGY: Patient records were analyzed retrospectively regarding IPMN. None showed high-risk stigmata or worrisome features. To rule out T2 shine-through or differentiate malignant lesions from non-malignant causes of restricted diffusion, positive ADC maps were produced from the recorded ADC values. RESULTS: None of the patients with IPMN had features of malignant progression. No mural nodules were detected by endoscopic ultrasonography. IPMN was hyperintense with DWIBS/T2 and the ADC map. This finding suggested that the hyperintense values of IPMN were T2 shine-through. These results showed that none of the IPMNs were positive with DWIBS/T2. CONCLUSION: DWIBS/T2 was negative for patients with IPMN. DWIBS/T2 might be useful for the evaluation of malignant progression, in addition to observation.

Factors affecting the detection of colorectal cancer and colon polyps on screening abdominal ultrasonography.

BACKGROUND/AIMS: The aim of this study was to identify factors affecting the detection of colorectal cancer (CRC) and colon polyps (CPs) using abdominal ultrasonography (US). METHODOLOGY: Patient records were analyzed retrospectively. Those diagnosed as having either CRC or CPs by colonoscopy performed after screening abdominal US were enrolled. The diagnostic criterion for CRC was an irregularly thickened wall or mass. CPs were diagnosed as spherical or ovoid hypoechoic lesions arising within the colonic lumen as seen on abdominal US. RESULTS: Sixteen patients had a total of 16 CRC lesions and 11 patients had a total of 17 CPs. All CRC lesions invaded deeper than the subserosa. Cancer cell invasion limited to the submucosa was noted in the two 1.5-cm CPs. Detection of these lesions was not associated with invasion to lymph or blood vessels. These results suggest that wall thickening might be the consequence of cancer cells invading below the subserosa, thereby resulting in the lesions becoming detectable on abdominal US. CONCLUSIONS: Detection of CRC and CPs on abdominal US was associated with lesion size and depth of invasion.

An association of elevated levels of alkaline phosphatase and gamma-glutamyl transpeptidase with acute cholangitis.

BACKGROUND/AIMS: The early diagnosis of acute cholangitis (AC) is critical for appropriate treatment. METHODOLOGY: Patient records from April 2008 to December 2012 were retrospectively analyzed. Data on white blood cell count and levels of C-reactive protein, total-bilirubin, alkaline phosphatase (ALP), aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase (gamma-GTP) were collected from AC patients on the day they underwent endoscopic retrograde cholangiopancreatography (ERCP) for diagnosis and treatment. Data were collected 3 months before ERCP to analyze the rate of change of these variables. Receiver operating characteristics curve analysis was performed. RESULTS: We enrolled 63 patients with AC and 65 patients with non-AC. The threshold values of ALP and gamma-GTP were 1.09 and 1.30, respectively. CONCLUSIONS: 450 (IU/L) and 100 (IU/L), respectively, were thresholds of ALP and gamma-GTP on the day of ERCP. 1.09 and 1.30, respectively, were thresholds of ALP and gamma-GTP rates of change for the diagnosis of AC.

Activin A is essential for Feeder-free culture of human induced pluripotent stem cells.

Feeder-free culture of human induced pluripotent stem (hiPS) cells is necessary for their clinical application to avoid adverse effects of foreign proteins. hiPS cells were cultured with combinations of activin (A), CHIR99021 (C), basic fibroblast growth factor (F), and leukemia inhibitory factor (L) under feeder-free conditions. Culture was terminated after 12 passages or when the cell morphology changed from pluripotency. Pluripotency was analyzed by alkaline phosphatase (ALP) staining and immunostaining with antibodies to Oct3/4, Nanog, SSEA4, and TRA-1-60. SB431542 (SB), an activin inhibitor, was added to the culture, and the morphology of the cells was observed. hiPS cells cultured with A, AC, and ACL after 12 passages were positive for ALP staining. Oct3/4 was positive in hiPS cells cultured with A, AC, and ACL. hiPS cells were positive for Nanog when cultured with A and AC; however, Nanog signal was weaker in cells cultured with ACL. SSEA4 was positive in hiPS cells cultured with A and AC but almost negative in those cultured with ACL. hiPS cells were positive for TRA-1-60 when cultured with A, AC, and ACL. hiPS cells lose their undifferentiated morphology at six passages when cultured with A + SB, five passages with AC + SB, and nine passages with ACL. We conclude that feeder-free culture of hiPS cells requires A or AC to maintain pluripotency.

Screening ultrasonography is useful for the diagnosis of gastric and colorectal cancer.

BACKGROUND/AIMS: To clarify the usefulness of screening ultrasonography (US) to diagnose gastric and colorectal cancer, patient records were analyzed retrospectively. METHODOLOGY: Ultrasonography was performed for patients with abdominal symptoms. They were then subjected to computed tomography (CT) when diagnosed with gastric cancer, colorectal cancer, or bowel obstruction. Patient records were analyzed retrospectively after final diagnosis of gastric cancer or colorectal cancer by endoscopy, surgery or necropsy. RESULTS: Twelve patients were diagnosed with colorectal cancer and six with gastric cancer. The detailed structure of colorectal cancer was visible as wall thickening with US, while cancer was often illustrated as a mass by CT. Loss of stratification was clear with US in 11 patients. US demonstrated wall thickening in 10 patients and a mass in 1 patient, while CT demonstrated wall thickening in 3 patients and a mass in 8 patients. The structure of colorectal cancer was more obvious when using US than when using CT. One patient demonstrated focal wall thickening with US, but this was not detected by CT. CONCLUSIONS: US is useful for diagnosis of gastric cancer and colorectal cancer. US produces more detailed findings in colorectal cancer than CT.

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.

Insulin-like growth factor I receptor involvement in proliferation of NOR-P1 cells in serum-free media.

Insulin-like growth factor (IGF)-I is up-regulated in pancreatic cancer tissues. Pancreatic cancer cell lines were analyzed in serum-free media as a model of the fibrous tissues that these cells often invade. Pancreatic cancer surgical specimens were immunostained with anti-IGF-I receptor (IGF-IR)ß antibody. The growth of pancreatic cancer cells in serum-free media was also analyzed. Cell lysates were analyzed for protein by western blot analysis. Cells cultured in the presence of picropodophyllin (PPP), LY294002, or PD98059, were subjected to cell proliferation and scratch assays. In addition, BrdU uptake and apoptosis were analyzed in these cells. IGF-IRß was detected in pancreatic cancer cells invading fibrous tissues. NOR-P1 grew most rapidly in serum-free media. The concentrations of IGF-I and IGF-II in the media were higher in NOR-P1 than the other cell lines. Cell proliferation in NOR-P1 cells was enhanced by IGF-I or IGF-II treatment more than in MIA-Paca2 or PK-1 cells. PPP, LY294002, and PD98059 suppressed proliferation and motility of NOR-P1 cells and inhibited BrdU uptake, while PPP induced apoptosis. IGF-IRß may be a potential therapeutic target to inhibit invasion of pancreatic cancer.

Ultrasonography for leukocytosis or elevated C-reactive protein.

BACKGROUND/AIMS: Patients with abdominal symptoms and leukocytosis or elevated C-reactive protein were subjected to abdominal ultrasonography (US) for correct diagnosis. METHODOLOGY: Patients with abdominal symptoms, leukocytosis and elevated C-reactive protein were enrolled. Those with abnormal liver enzymes or radiographs were excluded since either of them might be a clue to proper diagnosis, such as hepatobiliary diseases or bowel obstruction. RESULTS: Total number of patients was 38. Number of acute diverticulitis, colitis, acute appendicitis and enteritis were 8, 7, 7 and 6, respectively. One patient with pelvis tumor and 2 with colon cancer were successfully diagnosed with abdominal US. Colon cancers were confirmed and pelvis tumor was diagnosed as ovarian squamous cell carcinoma with surgical specimens. Sensitivity and specificity were 100% (95% CI: 44-100%) and 97.1% (95% CI: 85-99%), respectively. CONCLUSIONS: Our data clearly recommended that abdominal US be performed carefully for patients with abdominal symptoms and leukocytosis or elevated CRP since potentially they had malignancies.

[Case of intrahepatic cholangiocarcinoma drained through a fistula of the duodenal bulb].

A 72-year-old woman presented with epigastric discomfort. A low density tumor was found in the hilum and left liver by CT. Since she complained epigastralgia, upper gastrointestinal endoscopy was performed, showing an ulcer in the duodenal bulb, with poorly-differentiated adenocarcinoma seen on a biopsy specimen from the edge of the ulcer. After admission, poorly-differentiated adenocarcinoma cells were also obtained with ultrasound guided aspiration cytology of the liver tumor. We diagnosed intrahepatic cholangiocarcinoma (IHC), and treated with gemcitabine. During chemotherapy, the duodenal ulcer became a fistula, and the liver tumor diminished with bubbles inside it. It was suggested that liquid material of IHC, such as necrotic tissue and mucin, drained to the duodenal bulb during chemotherapy.

Oct3/4 is potentially useful for the suppression of the proliferation and motility of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) cells are immature compared with healthy mature hepatocytes. Transcription factors serve a role in hepatocyte differentiation. The expression levels of transcription factors in HCC cell lines have been investigated to determine potential therapeutic targets. In the present study, the HLE, HLF, PLC/PRF/5, Huh-7, Hep3B, Huh-6 and HepG2 HCC cell lines were subjected to reverse-transcription polymerase chain reaction (RT-PCR) of transcription factors, including NANOG, Oct3/4, GATA binding protein 4 (GATA4), GATA6 and hematopoietically expressed homeobox (HHEX). In addition, these cell lines were analyzed using RT-quantitative PCR (RT-qPCR) of NANOG and Oct3/4. The 201B7 human induced pluripotent stem cells were evaluated as a model of pluripotent cells. The HLF cells were transfected with Oct3/4 small interfering RNA (siRNA) and used in an MTS colorimetric assay and a scratch assay. NANOG was not expressed in any of the cell lines. However, GATA4, GATA6 and HHEX were expressed in the majority of the HCC cell lines. In addition, NANOG and Oct3/4 were expressed in 201B7 cells. Oct3/4 was expressed in HLE, HLF and Hep3B cells; however, its expression levels were significantly reduced compared with those in 201B7 cells. RT-qPCR demonstrated that the expression of Oct3/4 siRNA suppressed the proliferation and motility of HLF cells. Oct3/4 siRNA may be a potentially effective therapy for the suppression of the proliferation and motility of HCC cells.

Serum cytokine profiles and Mac-2 binding protein glycosylation isomer (M2BPGi) level in patients with autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an autoimmune liver disease that is characterized by a progressive destruction of the liver parenchyma and the development of liver fibrosis. We aimed to examine the relationship between circulating cytokines/chemokines and the Mac-2 binding protein glycosylation isomer (M2BPGi) levels in Japanese patients with autoimmune hepatitis (AIH).We investigated the relationship between circulating cytokines/chemokines and M2BPGi levels in Japanese patients with AIH. Seventy-seven patients with well-documented AIH were enrolled in the National Hospital Organization (NHO)-AIH-liver-network database. We measured the serum levels of 20 cytokines in 31 selected AIH patients before and after steroid treatment using multisuspension cytokine array.Eleven cytokines and soluble adhesion molecules were increased in untreated AIH patients compared with treated AIH patients. Among these cytokines and soluble adhesion molecules, soluble intercellular adhesion molecule-1 (sICAM-1) and interferon-γ-inducible protein 10 (IP-10) were most downregulated by steroid therapy in AIH patients. We measured serum sICAM-1 and IP-10 by ELISA and found the levels were significantly higher in AIH patients (n = 77) compared with chronic viral hepatitis C patients (n = 32). Furthermore, there was a positive correlation between sICAM-1 or IP-10 and alanine aminotransferase, total bilirubin, and circulating M2BPGi levels. M2BPGi levels were increased in AIH patients with high stages of liver fibrosis. Additionally, M2BPGi levels were correlated with the histological grade of inflammation in AIH. Circulating M2BPGi levels were significantly reduced by steroid treatment in AIH patients.sICAM-1 and IP-10 are useful markers to assess immune-mediated hepatitis activity in AIH and they correlate with circulating M2BPGi. Serum M2BPGi levels increased in untreated AIH patients with active hepatitis and were decreased by steroid therapy. M2BPGi reflects autoimmune-mediated hepatic inflammation as well as liver fibrosis.

Reciprocal expression of CCAAT/enhancer binding proteins alpha and beta in hepatoblastomas and its prognostic significance.

Hepatoblastoma is one of the common pediatric solid tumors with frequent mutation of the beta-catenin gene which might be an early event of its carcinogenesis. However, the detailed molecular mechanism is still unknown. We studied the expression levels of CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta, which regulate differentiation and growth of embryonic hepatocytes, to establish whether or not they were involved in affecting the clinical behavior of hepatoblastoma. The quantitative real-time reverse transcriptase-PCR revealed that expression of C/EBPalpha mRNA was significantly up-regulated in tumors 223% (p=0.013) as compared with that in adjacent normal livers, while expression of C/EBPbeta was down-regulated to 27% (p=0.002). Of interest, the immunohistochemical analysis showed that expression of C/EBPalpha was higher and that of C/EBPbeta lower in the poorly differentiated tumor cells than in the well-differentiated cells within the same tumor. Furthermore, high expression of C/EBPalpha (p=0.047) as well as low expression of C/EBPbeta (p=0.025) was significantly associated with poor prognosis of the patients. Cox hazard model suggested that expression of C/EBPalpha and that of C/EBPbeta were independent indicators to predict the prognosis from age but not from histology. Thus, expression of C/EBP proteins may play an important role in the genesis and clinical behavior of hepatoblastoma probably by inducing different stages of arrest of differentiation.

Transcriptional regulatory regions of the survivin gene activate an exogenous suicide gene in human tumors and enhance the sensitivity to a prodrug.

Selective expression of a therapeutic gene in tumors contributes to the efficacy and the safety of cancer therapy. Regulatory regions of genes that are preferentially expressed in tumors have been examined. The regions of the survivin gene exhibited the greatest activity in human hepatocellular carcinoma cells. Deletion of the survivin regulatory region from the 5'-side demonstrated that the 0.5 kb and the 1.4 kb fragments possessed a strong promoter activity with relative tumor specificity. Human tumors transfected with the herpes simplex virus-thymidine kinase gene, that was powered by the survivin region, became susceptible to a prodrug, ganciclovir. Although survivin gene expression was up-regulated in the G2/M-phase of the cell cycle, taxol or vincristine treatment, which induce cell cycle arrest at the M-phase, did not enhance the transcriptional activity of the survivin promoter. These data collectively suggest that the survivin regulatory region induces the expression of an exogenous gene in tumors, but the transcriptional activity is not directly linked with M-phase induction.

Differentiation of human induced pluripotent stem cells in William's E initiation medium supplemented with 3­bromopyruvate and 2­deoxy­d­glucose.

Hepatocyte selection medium (HSM) is deprived of glucose and supplemented with galactose, and is based on Leibovitz's­15 (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 3­bromopyruvate (3BP), an analogue of pyruvate, and 2­deoxy­d­glucose (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 F­12 Ham (DF12) supplemented with 3BP, 2DG or a combination of the two, for 15 days, and subjected to reverse transcription­quantitative polymerase chain reaction to measure the levels of α­fetoprotein (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.

Proliferation and motility of hepatocellular, pancreatic and gastric cancer cells grown in a medium without glucose and arginine, but with galactose and ornithine.

Human primary hepatocytes are able to survive in a medium without glucose and arginine, but supplemented with galactose and ornithine (hepatocyte selection medium; HSM). To address the possibility of the application of HSM in cancer therapy, hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells were cultured in HSM. Cell proliferation was analyzed using an MTS assay. Morphological changes were analyzed using hematoxylin and eosin staining. Apoptosis was analyzed using a TUNEL assay and cell motility was assessed with a scratch assay. Cell proliferation was significantly suppressed in cell lines grown in HSM (P<0.01 in all the cell lines). Hematoxylin and eosin staining revealed pyknotic nuclei, suggesting that these cells had undergone apoptosis. The number of TUNEL-positive cells was significantly increased in HSM. In the scratch assay, the distance between the growing edge and the scratched edge was significantly lower (P<0.01 in all the cell lines) in cells cultured in HSM, compared with those grown in Dulbecco's modified Eagle's medium or RPMI-1640. Therefore, the proliferation and motility of hepatocellular carcinoma cells, pancreatic cancer cells and gastric cancer cells was suppressed, and these cells subsequently underwent apoptosis in a medium without glucose and arginine, but containing galactose and ornithine.