Expression of MRP2 and MDR1 transporters and other hepatic markers in rat and human liver and in WRL 68 cell line

Here we describe a comparative study of phenotypic properties of hepatic cells in situ and in vitro. We analyzed the expression levels and distribution patterns of ABC transporters MRP2 and MDR1, pan-cytokeratin, cytokeratin 18, albumin, alpha-fetoprotein and the specific hepatocyte marker OCH1E5 in the fetal and adult rat as well as human liver tissue and in human fetal hepatocytes of WRL 68 cell line using peroxidase immunohistochemistry or immunofluorescence. Transporters MRP2 and MDR1 were expressed in all examined liver tissues, except rat ED13 embryo. The immunopositivity of these proteins was localized to the canalicular membrane of differentiating and mature hepatocytes but in the later developmental stages and in the adult liver tissues it was also found in the apical membrane of cholangiocytes. In WRL 68 cells, MRP2 and MDR1 immunoreactivity appeared after 5-6 days of cultivation and both transporters were fully expressed in the plasmalemma and in the cytoplasm 9 days after the passage. In conclusion, we observed only moderate variances reflecting diverse ontogenetic phases between the fetal and adult liver tissue. To study functions of hepatocytes in vitro, WRL 68 cells have to differentiate prior to the examination. Our findings indicate that WRL 68 cells can undergo differentiation in vitro and their antigenic profile closely resembles hepatocytes in the human liver.     

Occurrence of alpha-fetoprotein-containing hepatocytes in human embryos and fetuses: an immunohistochemical study using the light and ultrastructural immunoperoxidase methods

The occurrence of alpha-fetoprotein (AFP)-containing hepatocytes in 12 human embryos and fetuses ranging from 30-32 days of estimated ovulation age (Streeter's horizon XIV) to 16-17 weeks of estimated menstrual age was investigated using the direct (horseradish peroxidase (HRP)-labeled anti-human AFP horse IgG(Fab')2) and/or indirect immunoperoxidase methods. Under light microscopy, the AFP-containing hepatocytes were successfully demonstrated in paraffin-embedded liver tissues fixed with 4% paraformaldehyde(PFA)-picric acid solution containing 0.5% glutaraldehyde (GA). As a result of the studies of the human fetal livers at different developmental stages, only a small number of AFP-containing hepatocytes were initially identified immunohistochemically at the stage of Streeter's horizon XIX. Ultrastructural immunohistochemistry (the block-staining method using anti-human AFP horse IgG(Fab')2) revealed that the immunoreactive products against AFP were demonstrated on the ribosomal granules of the rough endoplasmic reticulum (rER), outer nuclear envelopes, free ribosomes, and Golgi's apparatus, and also in the cisternal lumina of the ER. No reactive products were noted in the nuclei or mitochondria. Our observations confirmed the presence of AFP-producing ability of the hepatocytes and the ultrastructural localization of the sites of protein synthesis in the early stage of development of human livers. Furthermore, we describe the extreme usefulness of the block-staining method for demonstrating the subcellular localization of AFP using HRP-labeled reduced anti-AFP antisera on liver tissues fixed with 4% PFA-picric acid solution containing 0.5% GA.     

alpha-Fetoprotein production and erythropoiesis in cell cultures of human fetal liver.

alpha-Fetoprotein and the synthesis of heme associated with hemoglobin were measured simultaneously in short-term cultures of human fetal liver cells to correlate the relationship of alpha-fetoprotein to erythroid cell function. Both synthetic processes decreased exponentially during the first 5 days of culture. The use of media supplemented with different batches of fetal calf serum and porcine portal vein serum indicated that the optimal conditions for the production of alpha-fetoprotein were different from those required for the synthesis of heme associated with hemoglobin. Moreover, the alpha-fetoprotein-producing cells could be separated from erythroid cells after velocity sedimentation in Ficoll gradients. Although it is well known that erythropoiesis and alpha-fetoprotein production occur simultaneously during ontogenesis, alpha-fetoprotein itself (0.01-100 micron g/ml) did not stimulate heme synthesis in liver erythroid cells. Erythropoietin did not stimulate alpha-fetoprotein production. It is concluded that there is no cause-effect relationship between alpha-fetoprotein production and erythroid cell fuction in human fetal liver cells and that the two processes occur independently in different cell types.     

Study of liver differentiation in vitro

A clonal rat fetal liver cell line that expresses the functions of differentiated liver cells under controllable conditions has been established. Normal fetal liver cells were transformed by a temperature-sensitive A (tsA) mutant (tsA209) of simian virus 40. At the permissive temperature (33 degrees C), the tsA209-transformed liver cell line (RLA209-15) can be cultured indefinitely and cloned readily. The RLA209-15 cells were temperature sensitive for maintenance of the transformed phenotype. These transformed liver cells selectively lost four characteristics of the transformed phenotype at the restrictive temperature (40 degrees C): generation time of the cells increased, the saturation density decreased, the efficiency of growth on nontransformed cell layers decreased, and the ability to clone in soft agar was lost. The transformation can be reversed simply by a shift in temperature. RLA209-15 fetal liver cells synthesized alpha-fetoprotein albumin, and transferrin. At 33 degrees C, the levels of these liver proteins were relatively low. At 40 degrees C the transformed phenotype was lost and the levels of alpha-fetoprotein, albumin, and transferrin were greatly increased. At the restrictive temperature, maximal induction of the synthesis of alpha-fetoprotein, albumin, and transferrin was achieved 3-4 d after the upward shift in temperature. The synthesis of alpha-fetoprotein then decreased; the synthesis of albumin and transferrin, however, was maintained. A second phase of albumin and transferrin synthesis was observed in all cultures after 6 d or more at 40 degrees C. Alpha-Fetoprotein, albumin, and transferrin secreted by RLA209-15 cells were immunologically indistinguishable from authentic alpha-fetoprotein, albumin, and transferrin, respectively. RLA209-15 cells, like primary cultures of hepatocytes and a simian virus 40 tsA255-transformed fetal liver cell line (RLA255-4) reported earlier from this laboratory, responded to glucagon with markedly elevated levels of cyclic AMP. Thus, it appears that glucagon receptors characteristic of hepatocytes are retained in the simian virus 40 tsA-transformed fetal liver cells.     

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.     

Rat corticosteroid-binding globulin (CBG) biosynthesis by fetal hepatocytes in culture

The ability of fetal liver to synthesize and secrete corticosteroid-binding globulin (CBG) was investigated using primary cultures of hepatocytes transplanted from 15- and 18-day-old rat fetuses. Analysis of culture medium, after labelling with [14C]leucine, by crossed immunoelectrophoresis, clearly demonstrated CBG, albumin and alpha-fetoprotein (AFP) secretion. The relative rate of CBG secretion was more important at the younger stage. Indirect immunofluorescence permitted localization of CBG in fetal hepatocytes. The results suggest that the high level of CBG found in fetal serum is mainly produced by the liver of the fetus itself.     

Alpha-fetoprotein in the liver of embryonic and newborn rats]

The localization of alpha-fetoprotein in the liver of embryos and newborn Wistar rats was determined by the method of fluorescent antibodies. This protein was found in the cytoplasm of some hepatocytes, endothelium of blood vessels and erythroid blood cells of embryos and new born rats. It was never found in the blood-forming and Kupffer cells of the liver, as well as in the epithelium of bile ducts. The hepatocytes containing this protein were located in the liver lobes near the central veins. Their number and the intensity of fluorescence decreased with the age of animals.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Hepatic progenitor cells in human fetal liver express the oval cell marker Thy-1

Hepatic progenitor cells play a major role in regenerating diseased liver. In rodents, progenitors forming hepatocytes or cholangiocytes are identified by the stem cell marker Thy-1. The aim of this study was to ascertain whether progenitor cells expressing Thy-1 could be identified in human fetal liver. Midtrimester human fetal liver was immunostained for Thy-1, cytokeratins 18 and 19, vimentin, CD34, CD45, and fibrinogen. Thy-1+ and Thy-1+CD34+ populations were purified using fluorescence-activated cell sorting (FACS). Immunofluorescence and mRNA expression were used to examine the bipotential nature of purified stem cells. We found that Thy-1+ cells were concentrated in portal tracts but were also scattered in parenchyma. In FACS-prepared cells, 0.18-3.08% (median 0.65%, n = 14) of cells were Thy-1+. Immunophenotyping revealed that some Thy-1+ cells coexpressed cytokeratins 18 and 19, others, fibrinogen and cytokeratin 19. RT-PCR demonstrated that Thy-1+ cells expressed mRNA for Thy-1, cytokeratin 18, and cytokeratin 19, and Thy-1+CD34+ cells expressed mRNA for alpha-fetoprotein, transferrin, and hepatocyte nuclear factor-4alpha. Thy-1+ cells were identified in fetal liver. These cells expressed several lineage markers, including coexpression of biliary and hepatocellular proteins and mRNA. These data suggest that Thy-1 is a marker of liver stem cells in human fetal liver.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

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.     

Liver gene expression and increase in albumin synthesis by fetal hepatocytes transplanted into analbuminemic rats

This report describes the evolution of hepatocytes isolated from 21-day fetuses and transplanted into spleens of Nagase analbuminemic rats which have negligible serum albumin levels due to a mutation affecting albumin mRNA processing. Albumin and alpha-fetoprotein expression, in addition to other parameters related to cellular proliferation status (thymidine kinase and proliferating cell nuclear antigen expression) were studied as indicative of the behavior and evolution of the cells. In recipient rats, only a few clusters of hepatocytes could be observed in the red pulp of the spleen 24 h after transplantation. The fetal hepatocytes migrated to the liver and could be seen in portal branches immediately after transplantation. Fifteen days later, albumin mRNA was detected in recipient livers and was expressed throughout the entire 3-month study. Alpha-fetoprotein was not detected. Cell proliferation was not relevant, although 3 months after transplantation, the proliferation rates appeared to show a tendency to increase. These data demonstrate that fetal hepatocytes transplanted into spleen migrate to liver, settle there and acquire an adult phenotype free of malignant transformation. Our study is a first step towards the thorough understanding of fetal hepatocyte transplantation. The next steps will involve in-depth studies of the possibilities of genetic manipulation to achieve a high degree of repopulation/expression, employing the least possible number of donor cells, and of how the cells reach the liver parenchyma, overcoming the endothelial barrier.     

Pluripotent stem cell-derived hepatocyte-like cells

Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~ 0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are poorly understood, which has hampered the field in its efforts to induce further maturation of iPS-derived hepatic lineage cells. This review analyzes recent developments in the derivation of hepatocyte-like cells, and proposes important points to consider and assays to perform during their characterization. In the future, we envision that iHLCs will be used as in vitro models of human disease, and in the longer term, provide an alternative cell source for drug testing and clinical therapy.     

Kinetics of albumin- and alpha-fetoprotein-production during rat liver development

Synthesis of most of the plasma proteins is one of the main functions of the hepatocytes. Albumin synthesis is quantitatively the most abundant. In the present study we investigated albumin- and alpha-fetoprotein-gene-expression, and the function of the secretory apparatus during rat liver development. To this purpose we used the method of radioactive biosynthetic labeling of newly synthesized albumin and alpha-fetoprotein (AFP) to monitor the secretory capacity of endodermal cells derived from ventral foregut region (embryonic day 10, E10), and of embryonic and fetal hepatoblasts. Synthesis and secretion of albumin and AFP were already detected in the low numbered ventral foregut endodermal cells; fibrinogen synthesis was detectable in the E12 hepatoblasts, which were in higher number. The whole secretory machinery was functional from the earliest stages of liver development, and the speed of secretion was comparable with that of the adult hepatocytes. There was almost 4-fold increase of hepatoblasts cell volume in fetal stage compared with embryonic stage. The model used suggests that the hepatocyte secretory apparatus is already functional before the emergence of the liver bud. This is the first comparative report to analyze the hepatocyte secretory function, cell proliferation and cell volume during liver development.     

Purification of fetal rat hepatocytes

Fetal rat hepatocytes of greater than 90% purity from 16 and 20 day fetuses were prepared by combining the use of sedimentation at unit gravity and discontinuous Percoll gradients. With slight modification the method could also be used to purify 13 day fetal rat hepatocytes. At each day examined the method produced several subpopulations of hepatocytes. Hepatocytes were identified by morphology and the presence of alpha-fetoprotein. Fetal hepatocytes purified in this way were able to attach to a substratum, and retained the ability to synthesize alpha-fetoprotein in short-term culture.     

Modulation of alpha-fetoprotein, albumin and transferrin gene expression by cellular interactions and dexamethasone in cocultures of fetal rat hepatocytes

Fetal hepatocytes were cultured alone or in association with primitive biliary cells (RLEC) in the presence or absence of dexamethasone. Cell-cell contacts were established 3 h or five days after hepatocyte seeding and their effects on hepatocyte growth and functional activities were evaluated in the presence or absence of dexamethasone. Establishment of cellular interactions with RLEC in coculture decreased hepatocyte growth, while it stimulated production of alpha-fetoprotein, albumin and transferrin. Addition of dexamethasone to coculture inhibited alpha-fetoprotein secretion and maintained the synthesis rate of albumin and transferrin together with an additional inhibition of DNA synthesis. The levels of mRNAs corresponding to the three proteins were also measured. We observed that the levels of alpha-fetoprotein, albumin and transferrin secretion in cocultures maintained in the presence or absence of dexamethasone were well correlated with the relative amounts of their corresponding mRNAs. Consequently, it may be assumed that the primitive mechanism involved in the increased functional activity of fetal hepatocytes in coculture is of pretranslational origin. Furthermore, the present data provide evidence that heterotypic interactions and dexamethasone act as distinct modulators of growth and maturation of fetal rat hepatocytes.