Primary Culture of Chicken Hepatocytes as an in Vitro Model for Determining the Influence of Dioxin

An easy method for primary culture of chicken hepatocytes was developed to study the influence of dioxin on birds. Chicken hepatocytes could maintain gene expression and protein secretion of albumin for a long period in serum-free medium with free atmosphere exchange at 37°C. Moreover, the cells showed a sensitive response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by monitoring the expression of P450 1A, theta GST (θ-GST) and albumin genes.     

The role of fetal and adult hepatocyte extracellular matrix in the regulation of tissue-specific gene expression in fetal and adult hepatocytes

We explored the effect of extracellular matrix (ECM) produced by fetal and adult hepatocytes on tissue-specific gene expression and proliferation of fetal and adult hepatocytes. Adult hepatocytes ECM strongly induced expression of both albumin and HNF-4 in adult hepatocytes. In contrast, fibroblast ECM reduced the expression of mRNAs for albumin and alpha-fetoprotein in fetal hepatocytes. Adult hepatocytes ECM also increased the activity of liver-specific enzymes of adult hepatocytes (DPP IV and glucose-6-phosphatase) in both fetal and adult hepatocytes, while fetal hepatocyte-derived ECM increased activity of the fetal hepatocyte enzyme GGT in fetal hepatocytes. Fibroblast ECM was inhibitory for the activity of all enzymes assayed. Removal of heparin chains from the various matrices by pretreatment of the ECM with heparinase resulted in reduction of glucose-6-phosphatase and DPP IV in adult hepatocytes. Removal of chondroitin sulfate chains from fetal hepatocyte-derived ECM resulted in loss of induction of GGT in the fetal cells. Fetal hepatocytes proliferated best on adult hepatocyte-derived ECM. Adult hepatocytes showed only modest proliferation on both fetal and adult hepatocytes ECM and their growth was inhibited by fibroblast ECM. In conclusion, adult hepatocyte ECM better supports the expression of adult genes, whereas fetal hepatocyte ECM induced expression of fetal genes. Fibroblast derived-ECM was inhibitory for both proliferation and tissue-specific gene expression in fetal and adult hepatocytes. The data support a role for heparan sulfate being the active element in adult ECM, and chondroitin sulfate being the active element in fetal ECM.     

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.     

Chimeric molecule IL-6/soluble IL-6 receptor is a potent mitogen for fetal hepatocytes

A novel recombinant molecule, termed IL-6c and consisting of a chimera of interleukin 6 (IL-6) and its soluble receptor is extremely potent in stimulating proliferation of hematopoietic progenitors. We investigated the effect of the IL-6c on the proliferation and differentiation of E14 fetal hepatocytes. IL-6c, in a dose-dependent manner, stimulated proliferation of E14 fetal rat hepatocytes. Adult hepatocyte mitogens together with IL-6c showed no further effect on proliferation. Hematopoietic stem cells mitogens SCF and flt3 ligand (FL) were also mitogenic for fetal hepatocytes, but did not further enhance the effect of IL-6c on cell proliferation. IL-6c decreased expression of fetal markers alpha-fetoprotein (AFP) and gamma-glutamyltranspeptidase, and induced expression of adult enzyme glucose-6-phosphatase (Gluc-6-P) in E14 hepatocytes. On the other hand, IL-6c strongly reduced, in a dose-dependant manner, expression of albumin and tyrosine aminotransferase (TAT). However, when the cells were grown for 3 days with IL-6c, and IL-6c was removed for the next 5 days, expression of albumin and TAT returned to levels found in control cultures. In conclusion, IL-6c stimulated proliferation and affected gene expression in fetal hepatocytes in culture.     

Methylation of the serum albumin gene as compared to the Kirsten-ras oncogene in hepatocytes and non-parenchymal cells of rat liver

The extent of methylation of a gene, i.e. percent of cytosine present as 5-methylcytosine, is correlated with its activity. Hypermethylation is associated with non-expression, whereas hypomethylation is a necessary but not sufficient condition for expression. In this study, the methylation state of the serum albumin gene as compared to the Kirsten-ras (Ki-ras) oncogene was assessed in hepatocytes and non-parenchymal cells (NPC) isolated from rat liver. The results of this investigation indicate that the serum albumin gene is hypomethylated in hepatocytes and hypermethylated in NPC. This is consistent with expression of the gene in the former cell type, and non-expression in the latter. In contrast, the Ki-ras oncogene is hypermethylated in both hepatocytes and NPC, suggesting that it is, at most, minimally expressed in normal rat liver.     

Differentiation of putative hepatic stem cells derived from adult rats into mature hepatocytes in the presence of epidermal growth factor and hepatocyte growth factor

Oval cells, putative hepatic stem cells, can differentiate into a wide range of cell types including hepatocytes, bile epithelial cells, pancreatic cells and intestinal epithelial cells. In this study, we used different growth factor combinations to induce oval cells to differentiate into mature hepatocytes. We isolated and purified oval cells utilizing selective enzymatic digestion and density gradient centrifugation. Oval cells were identified by their morphological characteristics and the strong expressions of OV-6, albumin, cytokeratin (CK)-19 and CK-7. Using a 2-step induction protocol, we demonstrated that oval cells first changed into small hepatocytes, then differentiated into mature hepatocytes. Small hepatocytes were distinguished from oval cells by their morphological features (e.g. round shape and nuclei) and the lack of CK-19 mRNA expression. Mature hepatocytes were identified by their ultrastructural traits and their expressions of albumin, CK-18, tyrosine aminotransferase (TAT), and alpha-1-antitrypsin (alpha-1-AT). Differentiated cells acquired the functional attributes of hepatocytes in that they secreted albumin and synthesized urea at a high level throughout differentiation. Oval cells can thus differentiate into cells with the morphological, phenotypic and functional characteristics of hepatocytes. This 2-step induction procedure could provide an abundant source of hepatocytes for cell transplantation and tissue engineering.     

Optimization of the technique to isolate fetal hepatocytes, and assessment of their functionality by transplantation

In contrast to adult hepatocytes, fetal hepatocytes (FH) are thought to be highly proliferative less immunogenic and more resistant to both cryopreservation and ischemic injury. In the present study, we describe the method for isolation of FH and the relationship between the transplantability of FH into the spleen of analbuminemic rats and expression of albumin mRNA. Rat FH were obtained using the nonperfusion collagenase/DNase digestion method. Nagase analbuminemic rats (NAR), a strain which bears a mutation that determines the impossibility of the normal splicing of the albumin mRNA were used as recipients. The transplanted FH immediately migrated to the liver via portal vein, and anchored there. To assess the functional state of the transplanted cells, one month after transplantation, the expression of the albumin gene was studied in the liver of the recipients.     

Fatty acids alter time dependent loss of apolipoprotein E expression by primary cultures of rat hepatocytes

Although the phenomenon of intracellular apolipoprotein E (apoE) degradation has been reported in other cell types, the fate of newly synthesized apoE in the liver is not well understood. In the present study, we examined the expression (the balance of synthesis, secretion, and degradation) of apoE in primary cultures of rat hepatocytes and compared it with albumin, a typical secretory protein. Synthesis and secretion of [(35)S]apoE was diminished in primary hepatocytes cultured for more than 2 days, in agreement with an observed decrease in apoE mRNA. Cells cultured for 1 day and labeled for up to 4 hours secreted total protein, apoE, and albumin, linearly. The apparent rates of synthesis for apoE and albumin were similar (1,158 vs. 1,334 dpm/mg/min) but rates of their secretion differed significantly (225 vs. 1,159 dpm/mg/min). Pulse-chase experiments indicated that cell-associated [(35)S]albumin was secreted without degradation, whereas significant quantities of newly synthesized apoE were degraded. The overall synthesis and secretion of total proteins, including secretion of apoE, was enhanced by oleic acid (1 mmol/L). However, this effect may not be limited to oleic acid because other fatty acids showed a similar effect on apoE mRNA abundance. In control cells, apoE was found to associate with high density lipoproteins predominantly, although the fraction associated with very low density lipoprotein was increased in hepatocytes incubated with oleic acid. Overall, the findings from this study suggest that the level of apoE expression by primary hepatocytes is dependent on the age of the culture. The study also indicates that the phenomenon of apoE degradation occurs in primary hepatocytes.     

Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte

The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml) showed significantly improved survival and functions (albumin and urea) compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.     

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Sustained production of plasma proteins, notably albumin, is a reliable indicator of the differentiated state of hepatocytes. In this work, we have developed a fetal hepatocyte culture system where studying the regulation of albumin expression in proliferating liver cells. Our results show that under proliferative conditions (i.e., in the presence of EGF) fetal hepatocytes maintain albumin production above control quiescent non-treated cells. Glucagon and noradrenaline have no effect on the proliferation induced by EGF in cultured fetal hepatocytes; however, they act synergistically with the growth factor, increasing intracellular albumin levels. The maximum response is obtained by treatment of cells with EGF and noradrenaline. The stimulatory noradrenergic effect is mimicked by agents that increase cyclic AMP levels (forskolin plus IBMX). However, vasopressin or phorbol esters have no effect on albumin production, neither alone nor in combination with EGF. Dexamethasone, which does not alter the proliferative induction of EGF, increases albumin content. This effect is independent of the proliferative status of the cells and is not enhanced by glucagon, noradrenaline, or cyclic AMP increasing agents. The hormonal changes observed in albumin production partially correlate with changes in mRNA levels. This is the first time that cyclic AMP increasing agents are shown to act synergistically with EGF, increasing the expression of this liver specific gene.     

Regulation of albumin gene expression in a series of rat hepatocyte cell lines immortalized by simian virus 40 and maintained in chemically defined medium.

A series of simian virus 40 (SV40)-immortalized hepatocyte cell lines were characterized for albumin production, the regulation of albumin production, and the expression of other liver-specific genes. This series of cell lines is particularly useful for studying the regulation of hepatocyte gene expression because the cell lines express liverlike levels of a number of liver-specific functions and do so while growing in a chemically defined medium. SV40-immortalized hepatocyte cell lines were derived from colonies of albumin-producing epithelial cells that arose after primary hepatocytes maintained in chemically defined medium were transfected with SV40 DNA. Some cell lines secreted albumin at levels equal to or greater than those secreted by freshly plated primary hepatocytes, and all but one line continued to produce albumin for more than 20 passages. The variation in albumin secretion among cell lines reflected differences in the amount of albumin produced per cell and not in the percentage of albumin-producing cells in each line. The characterization of selected cell lines showed that albumin production was regulated by cell density during the growth cycle. Albumin production in most cell lines was also regulated by dexamethasone; however, one cell line continued to produce high levels of albumin when the cells were grown in medium lacking dexamethasone, demonstrating that although glucocorticoid can induce albumin production in some cell lines, it is not required for high levels of albumin production by all cells in culture. Regulation of albumin production measured at the level of protein secretion was paralleled by changes in steady-state levels of a 2.3-kilobase albumin RNA. Albumin-producing SV40-immortalized hepatocytes secreted a variety of other plasma proteins, including transferrin, hemopexin, and the third component of complement. These cells also expressed tyrosine aminotransferase activity that was inducible by dexamethasone. Alpha-fetoprotein production was not detected in any of the cell lines examined.     

Regulators of fetal liver differentiation in vitro

Seventeen-day-old fetal rat hepatocytes were employed to examine factors required to promote differentiation in vitro. In the absence of effectors, primary fetal hepatocytes dedifferentiated, as characterized by the rapid decline in synthesis of fetal alpha-fetoprotein (AFP), albumin, and transferrin. On the other hand, cells maintained in the presence of glucocorticoid hormone produced high levels of albumin and transferrin. Glucocorticoid could not prevent the decline in fetal AFP synthesis, but induced synthesis of the 65K variant AFP--the major AFP species produced by adult rat liver. Fetal hepatocytes maintained in the presence of 8-bromo-cAMP (8-BrcAMP), or methyl isobutyl xanthine (MIX), an agent that increases intracellular cAMP levels, synthesized high levels of fetal AFP and albumin but reduced levels of transferrin. Both glucocorticoid and 8-BrcAMP or MIX induced expression of adult liver-specific genes such as tyrosine aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK), suggesting that these fetal hepatocytes have matured. Cells maintained in the presence of glucocorticoid hormone and MIX (or 8-BrcAMP) contained more albumin, TAT, and PEPCK mRNAs and synthesized increased amounts of the 65K variant AFP than those with either agent alone. However, the glucocorticoid/MIX cells produced intermediate levels of the fetal AFP and transferrin. Our data indicate that both glucocorticoid hormone and cAMP are necessary for optimal differentiation of fetal hepatocytes in vitro.     

Variability in the intensity of albumin synthesis in mouse hepatocytes

Using immunoenzyme method followed by the cytophotometrical determination of individual protein content in single cells, a comparative analysis was made of albumin contents in hepatocytes of inbred mouse stocks significantly differing in their albumin contents in the blood. Some reasons of heterogeneity of parenchymal liver cells in respect to albumin synthesis were identified. The content of albumin in hepatocytes was shown to correlate with the cell ploidy. A probable contribution of the tissue level to gene expression control is discussed.     

Differentiation of mesenchymal cells derived from human amniotic membranes into hepatocyte-like cells in vitro

Mesenchymal stem cells are believed to be involved in the formation of mesenchymal tissues, including bone, cartilage, muscle, tendon and adipose tissue. Interestingly, it has previously been reported that mesenchymal stem cells could also differentiate into endoderm-derived cells, such as hepatocytes. The amniotic membrane contains mesenchymal cells and is a readily available human tissue. Therefore, we investigated the potential of mesenchymal cells derived from human amniotic membrane (MC-HAM) to differentiate into hepatocytes. We analyzed the expression of hepatocyte-specific genes in MC-HAM before and after induction of differentiation into hepatocytes. We observed the expression of mRNAs encoding albumin, a-fetoprotein, cytokeratin 18 and alpha1-antitrypsin, but not those encoding glucose-6-phosphatase or ornithine transcarbamylase, prior to the induction of differentiation. However, immunocytochemistry revealed that albumin and alpha-fetoprotein were abundantly produced only after the induction of differentiation into hepatocytes. In addition, we observed the storage of glycogen, a characteristic feature of hepatocytes, using periodic acid-Schiff staining of MC-HAM induced to differentiate into hepatocytes. Overall, MC-HAM appear to be able to differentiate into cells possessing some characteristics of hepatocytes. Although further studies should be carried out to determine whether such in vitro-differentiated cells can function in vivo as hepatocytes. These cells may be useful in various applications that require human hepatocytes.     

Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes

We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.