Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

Primary hepatocytes lose their differentiated functions rapidly when in culture. Our aim was to maintain the differentiated status of hepatocytes in vitro by means of vital hepatic stellate cells (HSCs), their soluble and particulate factors and lipid extracts. Hepatocytes were placed into collagen-coated culture dishes in the presence of HSCs at different ages of pre-culture, with or without direct cell to cell contacts, at different cell ratios and in monoculture with cellular HSC components in place of vital cells. Changes in morphology and enhancement of phosphoenolpyruvate carboxykinase (PCK) activity by glucagon were used to determine the differentiated status of hepatocytes in 2d-short-term culture. HSCs proved able to maintain the differentiated function of hepatocytes in co-culture either by direct cell contacts or via factors derived from HSC-conditioned medium. In comparison, however, without cellular contact to hepatocytes five to ten times as many HSCs were necessary to increase the PCK activity to the same degree as in the presence of intercellular contacts. Whereas stimulation in the presence of HSC/hepatocyte contacts was independent of HSC culture age only quiescent, resting HSCs (precultured for 1–2 d) were able to stimulate hepatocytes significantly via soluble factors. Culturing of hepatocytes with a lipid extract or a particulate fraction from HSCs clearly displayed a very strong beneficial effect on enzyme activity and morphology. HSCs maintain hepatocyte function and structure through preferentially cell-bound signalling and transfer of lipids.     

Long-term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone

Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co-culture system, the effect of sinusoidal liver cells on hepatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co-cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of alpha 1-fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co-cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte-stabilizing activity was also produced by a pulmonary endothelial cell line.     

Radiation inactivation studies of hepatic sinusoidal reduced glutathione transport system

Sinusoidal transport of reduced glutathione (GSH) is a carrier-mediated process. Perfused liver and isolated hepatocyte models revealed a low-affinity transporter with sigmoidal kinetics (K(m) approximately 3.2-12 mM), while studies with sinusoidal membrane vesicles (SMV) revealed a high-affinity unit (K(m) approximately 0.34 mM) besides a low-affinity one (K(m) approximately 3.5-7 mM). However, in SMV, both the high- and low-affinity units manifested Michaelis-Menten kinetics of GSH transport. We have now established the sigmoidicity of the low-affinity unit (K(m) approximately 9) in SMV, consistent with other models, while the high-affinity unit has been retained intact with Michaelis-Menten kinetics (K(m) approximately 0.13 mM). We capitalized on the negligible cross-contributions of the two units to total transport at the low and high ends of GSH concentrations and investigated their characteristics separately, using radiation inactivation, as we did in canalicular GSH transport (Am. J. Physiol. 274 (1998) G923-G930). We studied the functional sizes of the proteins that mediate high- and low-affinity GSH transport in SMV by inactivation of transport at low (trace and 0.02 mM) and high (25 and 50 mM) concentrations of GSH. The low-affinity unit in SMV was much less affected by radiation than in canalicular membrane vesicles (CMV). The target size of the low-affinity sinusoidal GSH transporter appeared to be considerably smaller than both the canalicular low- and high-affinity transporters. The high-affinity unit in SMV was markedly inactivated upon irradiation, revealing a single protein structure with a functional size of approximately 70 kDa. This size is indistinguishable from that of the high-affinity GSH transporter in CMV reported earlier.     

Multihormonal regulation of hepatic sinusoidal Ntcp gene expression

Bile acids are efficiently removed from sinusoidal blood by a number of transporters including the Na+-taurocholate-cotransporting polypeptide (Ntcp). Na+-dependent bile salt uptake, as well as Ntcp, are expressed twofold higher in male compared with female rat livers. Also, estrogen administration to male rats decreases Ntcp expression. The aims of this study were to determine the hormonal mechanism(s) responsible for this sexually dimorphic expression of Ntcp. We examined castrated and hypophysectomized rats of both sexes. Sex steroid hormones, growth hormone, thyroid, and glucocorticoids were administered, and livers were examined for changes in Ntcp messenger RNA (mRNA). Ntcp mRNA and protein content were selectively increased in males. Estradiol selectively decreased Ntcp expression in males, whereas ovariectomy increased Ntcp in females, confirming the importance of estrogens in regulating Ntcp. Hypophysectomy decreased Ntcp mRNA levels in males and prevented estrogen administration from decreasing Ntcp, indicating the importance of pituitary hormones. Although constant infusion of growth hormone to intact males reduced Ntcp, its replacement alone after hypophysectomy did not restore the sex differences. In contrast, thyroid hormone and corticosterone increased Ntcp mRNA in hypophysectomized rats. Sex differences in Ntcp mRNA levels were produced only when the female pattern of growth hormone was administered to animals also receiving thyroid and corticosterone. Thyroid and dexamethasone also increased Ntcp mRNA in isolated rat hepatocytes, whereas growth hormone decreased Ntcp. These findings demonstrate the essential role that pituitary hormones play in the sexually dimorphic control of Ntcp expression in adult rat liver and in the mediation of estrogen effects.     

Short term regulation by lysine of ornithine decarboxylase activity in Dictyostelium discoideum

A transitory increase in ornithine decarboxylase activity has been observed soon after food removal from Dictyostelium discoideum amoeba. This increase can be prevented by supplementation of the differentiation buffer with the 11 amino acids known for their ability to retard the development of this slime mold. Lysine can replace the amino acid mixture with an apparent inhibition constant of 50 micromolar. This inhibition by lysine, which was only observed in vivo, took place within 5 min and was readily reversed upon lysine removal.     

The importance and regulation of hepatic glutathione.

Glutathione plays a key role in the liver in detoxification reactions and in regulating the thiol-disulfide status of the cell. Glutathione synthesis is regulated mainly by the availability of precursor cysteine and the concentration of glutathione itself which feeds back to regulate its own synthesis. Degradation of hepatic glutathione is principally regulated by the efflux of reduced and oxidized glutathione into both sinusoidal plasma and bile. In addition, glutathione may be consumed in conjugation reactions. Under conditions of oxidative stress, the liver exports oxidized glutathione into bile in a concentrative fashion, whereas under basal conditions, mainly reduced glutathione is exported into bile and blood. The mechanism of export of reduced glutathione into bile and sinusoidal blood is poorly understood.     

3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.     

Short term regulation of cell-cell communication in TM3 Leydig cells. A perforated patch study

Determination of the junctional conductance (g(j)) in TM3 Leydig cells by the dual whole cell patch clamp technique (DWCPC) shows that coupling undergoes a rapid and irreversible run down. Addition of ATP or cAMP derivatives to the pipette solution has been shown to prevent this phenomenon in several tissues, but this same treatment is unable to inhibit run down in Leydig cells. Because the run down in junctional conductance may pose serious problems to the interpretation of results, we also measured g(j) by using the double perforated patch clamp technique (DPPT). Access to the cell interior was achieved by adding 200 microgram/ml of nystatin to the pipette solution. With this method, run down in g(j) was greatly reduced, amounting to no more than 5% of the initial value. Exposure of the cells, under DWCPC or DPPT, to dibutyryl cAMP or to tumor promoting agent (TPA) led to a decrease in cell to cell communication. Staurosporine, a PKC inhibitor, increased g(j) and was able to prevent and reverse the uncoupling action of cAMP or TPA. Our results indicate that cell-cell communication in Leydig cells is down regulated by both protein kinases A and C, interacting in a complex manner.     

Low content of hepatic reduced glutathione in patients with Wilson's disease

In five of six patients with symptomatic Wilson's disease (WD) with increased hepatic copper content, increased renal copper excretion, and decreased serum concentrations of ceruloplasmin, significantly low levels of hepatic reduced glutathione (GSH) were found. Three of these patients showed increased levels of oxidized glutathione which in part could account for the missing GSH. These changes may result from increased lipid peroxidation due to the rise of intracellular copper concentration. Furthermore, WD patients showed a 50% decrease in the activity of hepatic GSH S-transferases. From these results we conclude that the disturbance in the hepatic glutathione system of patients with symptomatic WD may contribute to the perpetuation of liver damage. These patients, additionally, may be predisposed to an increased sensitivity to drugs interacting with glutathione.     

Distribution of hepatic phosphofructokinase isozymes in parenchymal and sinusoidal cells.

The distribution profile of the isozymes of phosphofructokinase (PFK) in different cell types of rat liver is established using the techniques of electrophoresis and immunodiffusion. Agarose gel electrophoresis of the extracts of parenchymal cells, Kupffer or sinusoidal cells, and whole liver indicated that two PFK isozymes are present in whole liver and that the faster moving hepatic PFK isozyme is present only in parenchymal cells; whereas, the slower moving hepatic PFK isozyme is only in sinusoidal cells. Immunodiffusion studies using antiserum specific for the major hepatic PFK isozyme (PFK-L₂) revealed that PFK-L₂ is present only in whole liver or parenchymal cell extracts and is absent from sinusoidal cells. It is apparent that the other hepatic PFK isozyme (PFK-L₁) is normally found only in sinusoidal cells.     

Leucine stimulates the secretion of hepatocyte growth factor by hepatic stellate cells

Branched-chain amino acids (BCAAs) modulate various cellular functions, in addition to providing substrates for the production of proteins. In this study, we examined the effect of BCAAs on the secretion of hepatocyte growth factor (HGF) by hepatic stellate cells. A hepatic stellate cell clone was cultured in medium supplemented with various concentrations of valine, leucine, or isoleucine. Of these BCAAs, leucine markedly induced an increase in the levels of HGF in the medium in a dose-dependent manner. The addition of valine or isoleucine had no significant effect on HGF levels in the medium. The difference in levels of HGF in the medium between leucine-treated and non-treated cells was enhanced by the incubation period. These results demonstrate that, among BCAAs, leucine stimulates the secretion of HGF by cultured hepatic stellate cells.     

Dissociation between rate of hepatic lipoprotein secretion and hepatocyte microtubule content

The fact that colchicines inhibits hepatic secretion of very low density lipoprotein (VLDL) particles has been interpreted to mean that microtubules are involved in hepatic VLDL secretion. To further define this relationship, we have attempted to see if changes in hepatic VLDL secretion are associated with changes in hepatocyte microtubule or tubulin content. Accordingly, hepatic secretion of VLDL was increased in rats, and the hepatocyte content of both microtubules (using quantitative morphometric methods) and tubulin (using a time-decay colchicine binding assay) was determined. In acute experiments, VLDL secretion was increased by perfusion of isolated rat livers for 2 h with varying concentrations of free fatty acids (FFA). Results indicate that hepatic VLDL triglyceride (TG) secretion at perfusate FFA levels of 0.7 μEq/ml is threefold greater (P < 0.01) than when livers are perfused without added FFA. However, no differences are observed in the content of microtubules in these livers: specifically, microtubules occupy 0.029 percent of hepatocyte cytoplasm in livers perfused without FFA and 0.030 percent of cytoplasm in livers perfused with FFA. In chronic experiments, rats were fed for 1 wk with either standard rat chow or a hyperlipidemic (sucrose/lard) diet. With the experimental diet, plasma triglyceride levels increase threefold over controls, and liver VLDL-TG production, as determined by [(3)H]glycerol turnover studies, is 55 percent greater (P < 0.01) than controls. However, microtubules occupy 0.027 percent of the cytoplasm of hepatocyte cytoplasm whether rats are on standard or hyperlipidemic diets. Furthermore, the tubulin content of isolated hepatocytes does change, and represents 1 percent of hepatocyte soluble protein, irrespective of diet. These results suggest that increases in hepatic VLDL secretion can occur without any demonstrable change in hepatocyte assembled microtubule or tubulin content, and raise questions as to the role played by microtubules in hepatic VLDL secretion.     

Modulation of fetal and neonatal rat hepatocyte functional activity by glucocorticoids in co-culture

Fetal and neonatal rat hepatocytes were cultured alone or in association with another liver epithelial cell type, in a medium with or without hydrocortisone. Secretion of albumin and alpha-fetoprotein decreased in pure hepatocyte culture, whereas in co-culture it remained stable for several days. Furthermore, addition of hydrocortisone to the co-culture medium induced a rapid increase in albumin production which was maintained at a high level. In contrast, alpha-fetoprotein production was inhibited. At the same time, an abundant extracellular material was secreted between and around hepatocyte colonies. The results demonstrate that the reciprocal relation between albumin and alpha-fetoprotein production which occurs during in vivo perinatal hepatocyte maturation is also observed in vitro. Both cell-cell contacts and glucocorticoids play a key role in this process. It appears that fetal and neonatal hepatocytes can maturate when maintained in a co-culture system.     

Maintenance of hepatocyte functions in coculture with hepatic stellate cells

Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.