Masanobu Maruyama

Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT.

Background. Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT). Methods. SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS). Results. One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel. Conclusion. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers

Background. Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT). Methods. Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3–TWNT-1 co-culture system. Results. TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis. Conclusions. TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.

Lentiviral transfer of the LacZ gene into human endothelial cells and human bone marrow mesenchymal stem cells.

Because one of the attractive characteristics of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors is that it can infect even nondividing cells, a lentivirus-mediated gene delivery system is currently being paid a great deal of attention as an innovative tool for gene transfer into target cells. The purpose of the work was to investigate the efficacy of lentiviral transfer of the LacZ gene into human umbilical vein endothelial cells (HUVECs) and human bone marrow mesenchymal stem cells (HMSCs) in vitro. For the present study, a vesicular stomatitis virus G-protein (VSV-G)-pseudotyped lentiviral vector encoding the E. coli LacZ gene tagged with nuclear localization signal (NLS) was generated in 293T cells by means of the three-plasmid system. The resulting lentiviral vector, LtV-NLS/LacZ, was allowed to infect HUVECs and HMSCs. Approximately 70% of HUVECs were positive for LacZ expression and 50% of HMSCs showed LacZ activity. There was no significant difference in transduction efficacy between early and late-passage phases in both cells. LtV-NLS/LacZ-transduced HUVECs showed gene expression of endothelial markers including CD34 and flt-1 and KDR/flk-1 of vascular endothelial growth factor (VEGF) receptors and had angiogenic potential as efficiently as primarily cultured HUVECs in a Matrigel assay. These findings provide evidence that lentiviral vectors are efficient tools for gene transfer and expression in human endothelial cells and stem cells that could be useful for tissue engineering.

Cryopreservation of primarily isolated porcine hepatocytes with UW solution.

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbeccos modified Eagles medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

Hepatocyte isolation and transplantation in the pig.

Hepatocyte transplantation (HTX) has received great expectation for the treatment of a wide spectrum of liver diseases. Considering the severe shortage of human livers for hepatocyte isolation, porcine hepatocytes are an attractive alternative to normal human hepatocytes. To develop such therapy, establishment of an efficient hepatocyte isolation and transplantation model that enables accurate assessment of safety and efficacy of HTX is extremely important. Porcine hepatocytes were isolated from a surgically removed liver segment with a four-step retrograde perfusion using dispase and collagenase. The resultant hepatocytes of > 84% viability were used for transplantation experiment in a pig model of acute liver failure induced by intravenous administration of D-galactosamine (D-gal) (0.5 mg/kg). Twenty-four hours after D-gal injection, transplantation of freshly isolated porcine hepatocytes (1 x 10(9)) was safely conducted and prolonged the survival of D-gal-treated pigs. We describe an efficient porcine hepatocyte isolation and subsequent cell transplantation in pigs with D-gal-induced liver failure.

Sevelamer Hydrochloride Exacerbates Metabolic Acidosis in Hemodialysis Patients, Depending on the Dosage

Abstract:  Sevelamer hydrochloride, as a phosphate binder that contains neither aluminum nor calcium, is expected to improve the prognosis of dialysis patients. However, sevelamer hydrochloride has been reported to lower the serum bicarbonate level. In the present study, we performed a retrospective study on the potential influences of sevelamer hydrochloride on metabolic acidosis in hemodialysis patients. The subjects were 72 patients who underwent hemodialysis at our hospital. Thirty‐six patients taking sevelamer hydrochloride and 36 patients matched for sex, diabetes mellitus, age and duration of dialysis who were not taking sevelamer hydrochloride were studied. We assigned the 36 patients who had been taking sevelamer hydrochloride to the ‘sevelamer group’, and the 36 patients not taking sevelamer hydrochloride were the control group. Statistical significance was evaluated by a t‐test and Pearsons correlation coefficient. In the sevelamer group, the mean levels of bicarbonate, base excess and pH decreased significantly after administration, compared with the values before administration, but in the control group, aggravation of acidosis was not seen. The levels of bicarbonate, base excess and pH after the medication of sevelamer hydrochloride were found to be significantly and negatively correlated with the daily dose of sevelamer hydrochloride. The levels were also found to be significantly and negatively correlated with the cumulative dose of sevelamer hydrochloride; however, the value of the mean levels of chlorine and the anion gap did not increase with sevelamer hydrochloride. Sevelamer hydrochloride caused metabolic acidosis in a dose‐dependent manner in hemodialysis patients without hyperchloremia.

Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside.

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 μg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbeccos modified Eagles medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 μg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 μg/ml) would be a useful cold preservation means for the development of cell therapies.

Transduction of immortalized human hepatocytes with p21 to enhance differentiated phenotypes.

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G1 phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immuno-fluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G1 checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.

Lowering of oxidative stress in hemodialysis patients by dialysate cleaning: in relation to arteriosclerosis.

Abstract:  The objective of this study was to investigate changes in oxidative stress associated with the cleaning of the dialysate. Thirty‐six dialysis patients were studied. Changes in soluble CD‐14 (sCD‐14), malondialdehyde‐low‐density lipoprotein (MDA‐LDL), and oxidized‐LDL (Ox‐LDL) were monitored for 1 year before and 1 year after dialysate cleaning. The mean endotoxin (ET) level in the dialysate had previously been confirmed to decrease from 39.0 EU/L to an undetectable level after the cleaning. The mean levels of sCD‐14, MDA‐LDL, and Ox‐LDL decreased significantly after the cleaning (sCD‐14, P < 0.0001; MDA‐LDL, P < 0.001; Ox‐LDL, P < 0.001). One year after the cleaning, six cases still showed high levels of MDA‐LDL and Ox‐LDL. Cardiovascular events occurred in four of those six cases within 2.8 years after the cleaning. These four patients suffered from strong oxidative stress during dialysis, even after the cleaning. We therefore concluded that high levels of MDA‐LDL and Ox‐LDL are improved in dialysis patients by cleaning of the dialysate. These results indicate that even a dialysate containing 50 EU/L or less ET may stimulate monocytes and cause oxidative stress. They also suggest that even low levels of ET may aggravate arteriosclerosis in dialysis patients. Thus, in order to prevent cardiovascular events in dialysis patients, it is necessary to purify the dialysate.

Lentiviral vector: A useful tool for transduction of human liver endothelial cells

Endothelial cells play multiple roles in pathophysiologic processes and are increasingly being recognized as target cells of gene therapy. Lentiviral vectors derived from human immunodeficiency virus type 1 have an ability to infect both dividing and nondividing cells and currently receive a great deal of attention as an innovative tool for transduction of target cells. The purpose of the present work was to evaluate the efficacy of a lentiviral vector for transducing human liver endothelial cells (HLECs) in vitro. For the present study, a pseudotyped lentiviral vector encoding a green fluorescent protein (GFP) gene, LtV-GFP, was generated by means of FuGENE 6 method and allowed to infect HLECs. Approximately 95% of HLECs were positive for GFP expression after LtV-GFP infection at a multiplicity of infection of 10. Notably, LtV-GFP transduced HLECs had stable and long term GFP expression, showed gene expression of endothelial markers including CD 34, factor VIII, flt-1, KDR/flk-1 and HGF, and maintained in vitro angiogenic potential in a Matrigel assay to the same extent as primarily cultured HLECs. These findings provide evidence that lentivirus based gene delivery is an efficient tool for transduction of endothelial cells that could be considered for cell and gene therapies and hybrid artificial organs.