Effective hepatocyte transplantation using rat hepatocytes with low asialoglycoprotein receptor expression.

Development of a reliable method of isolating highly proliferative potential hepatocytes provides information crucial to progress in the field of hepatocyte transplantation. The aim of this study was to develop reliable hepatocyte transplantation using highly proliferative, eg, progenitor-like hepatocytes, based on asialoglycoprotein receptor (ASGPR) expression levels for hepatocyte transplantation. We have previously reported that mouse hepatocytes with low ASGPR expression levels have highly proliferative potential and can be used as progenitor-like hepatocytes. We therefore fractionated F344 male rat hepatocytes expressing low and high levels of ASGPR and determined the liver repopulation capacity of hepatocytes according to low and high ASGPR expression in the liver. Next, 2 x 10(5) cells of each type were transplanted into female liver regenerative model dipeptidyl peptidase-deficient rats, and we estimated the rate of liver repopulation by the transplanted hepatocytes in the host liver, as determined by recognition of the Sry gene on the Y-chromosome. At 60 days after hepatocyte transplantation, the transplanted hepatocytes occupied approximately 76% of the total hepatocyte mass in the case of the transplantation of hepatocytes with low ASGPR expression, but accounted for approximately 12% and 17% of the mass in the case of the transplantation of hepatocytes with high ASGPR expression and unfractionated hepatocytes, respectively. In conclusion, these findings suggest that hepatocytes with low ASGPR expression can result in normal liver function and a high repopulation capacity in vivo. These results provide insight into development of a strategy for effective liver repopulation using transplanted hepatocytes.

Expression and induction of CYP3As in human fetal hepatocytes.

CYP3A4 and CYP3A7 mRNA expression levels were markedly up-regulated by dexamethasone (DEX), but not by rifampicin (RIF). CYP3A5 mRNA level was not increased significantly by DEX, RIF, or phenobarbital. Testosterone 6beta-hydroxylase activity was induced to about 2-fold of control by DEX. However, concomitant treatment with RIF did not alter DEX-mediated induction of CYP3A mRNA expression and testosterone 6beta-hydroxylase activity. DEX-mediated induction of CYP3A mRNA was suppressed in a dose-dependent manner by RU486, a glucocorticoid receptor (GR) antagonist. At 5microM RU486, DEX-mediated induction of CYP3A4, CYP3A5, and CYP3A7 mRNA expression was inhibited almost completely. These results suggest that, in human fetal hepatocytes, PXR is not involved in DEX-mediated induction of CYP3A4 and CYP3A7, and that the induction is mediated directly by GR.

Development of highly functional long-term culture method of liver slice embedded in agarose gel for bioartificial liver.

It is difficult to a produce highly functional bioartificial liver (BAL) using only hepatocytes, because it is believed that liver-specific three-dimensional structure is necessary to maintain high function for BAL. But it is difficult to construct a culture system with liver-specific three-dimensional structure in vitro. To realize a highly functional culture system with liver-specific three-dimensional structure, we developed a culture system using liver slices that keep liver-specific architecture, such as liver lobule and hepatic microvascular system. Liver slices were embedded in agarose gel to maintain them under a moist and three-dimensional environment. We examined the viability and function of liver slices by using various shapes of agarose gel. Liver slices were cultured 1) under stationary condition (control), 2) directly embedded in gel, and 3) embedded in cylindrical gel for good drainage of medium and ventilation of air. The viability and function of the incubated liver slices were evaluated by LDH leakage, histomorphology, and immunohistochemistry. At 10 days, the morphological condition and function of liver slices embedded in cylindrical gel were maintained better than liver slices directly embedded in gel or in the stationary condition. We suggest that high functionality and morphological condition of liver slices could be maintained by embedding in cylindrical gel. In the future, it is possible that this method could be used to develop a highly functional bioartificial liver.

Soluble asialoglycoprotein receptors reflect the apoptosis of hepatocytes.

Cell death is thought to take place through at least two distinct processes: apoptosis and necrosis. There is increasing evidence that dysregulation of the apoptotic program is involved in liver diseases. However, there is no method to simply evaluate apoptosis in the liver tissue at present. It has been reported that the expression of asialoglycoprotein receptors (AGPRs) increases with apoptosis, but there is no report until now that investigates the influence of soluble AGPRs on apoptosis of hepatocytes. Soluble AGPRs have been reported to be present in human serum under physiological conditions. In the present study, in order to investigate the correlation between apoptosis of hepatocytes and soluble AGPR, mouse soluble AGPRs were detected using SDS-PAGE and Western blot analysis was conducted using anti-extracellular mouse hepatic lectin-1 (Ex-MHL-1) antiserum (polyclonal rabbit serum). The mouse soluble AGPRs were present in culture medium and mouse serum when hepatocytes were damaged. The soluble AGPRs increased proportionately, as the number of dead hepatocytes increased. In addition, soluble AGPRs existed more when apoptotic cell death was observed in in vitro and in vivo than when necrotic cell death was observed. The extracellular moiety of MHL-1 exists in the culture medium and mouse serum as a soluble AGPR, but the detailed mechanism of releasing soluble AGPR from hepatocytes has not been revealed yet. We described the first evidence for the relation between quantity of soluble AGPRs with two kinds of cell death: necrosis and apoptosis. Based on the results of our study, soluble AGPRs might become a new marker of apoptosis in the liver tissue and be useful for clinical diagnosis and treatment for liver diseases.

Drug metabolic activity of cultured hepatocytes can synchronize with bile acid concentration in the medium.

The regulation of drug metabolic activity of cultured hepatocytes can be applied to the evaluation of pharmacokinetics, analysis of drug delivery and the bioartificial liver system. It is very difficult to maintain the drug metabolic activity mediated by cytochrome P-450 (CYP) 3A. Recently we found that the CYP3A aminopyrine N-demethylase (AMND) activity of hepatocytes cultured on collagen surface oscillated with culture time. This phenomenon was related to the concentration of bile acid in the culture medium. CYP3A, multidrug resistant gene 2 (MDR2) and heat shock protein 84 (HSP84) mRNA appeared in a manner corresponding to this oscillation. When a large quantity of bile acid was taken up into hepatocytes from the medium, low AMND activity was observed, and these proteins did not appear. When bile acid was secreted and the bile acid concentration inside the hepatocytes was low, high AMND activity was obtained, and these proteins appeared. In order to clarify the mechanism of oscillation between AMND activity and bile acid, 8 microM glycocholic acid was added to the culture medium 15 h before the measurement. No oscillation in AMND activity was observed in the presence of 8 microM glycocholic acid. Bile acid controls the AMND activity in the transcription of hepatocytes.