Yohichi Mochizuki

Alteration of expression of liver-enriched transcription factors in the transition between growth and differentiation of primary cultured rat hepatocytes

In the present study, we showed the role of the liver‐enriched transcription factors in the transition during which proliferating hepatocytes become quiescent. We used primary rat hepatocytes cultured in modified L‐15 medium. The cells proliferated and, after the addition of 2% dimethyl sulfoxide (DMSO) from day 4, they stopped growing and gradually differentiated. During hepatic proliferation, expression of hepatocyte nuclear factors (HNF)1α, HNF4, C/EBPα, and C/EBPβ mRNAs was depressed, whereas that of HNF3α and HNF3β transcripts was enhanced. After the addition of DMSO, the expression of HNF1α, HNF3γ, and HNF4 returned to the level in isolated cells and HNF1β mRNA expression gradually increased. However, expression of C/EBPα and C/EBPβ mRNAs was partially recovered. The mitoinhibitory agents, IL‐1β, IL‐6, TGF‐β, and activin A, were examined to determine whether they could induce differentiation of proliferating hepatocytes as shown in cells treated with DMSO. Although these factors inhibited cell growth, the cells did not differentiate. The expression pattern of HNF3γ mRNA was quite different in the cells cultured with DMSO and those cultured with cytokines. Therefore, hepatic differentiation requires not only inhibition of DNA synthesis but also induction of appropriate transcription factors. Thus, expression of HNF3γ, C/EBPα, and C/EBPβ may be necessary for hepatocytes to acquire highly differentiated functions in addition to coexpression of certain amounts of transcripts of HNF1α, HNF1β, HNF3α, HNF3β, and HNF4 as well as suppression of C/EBPδ. J. Cell. Physiol. 174:273–284, 1998.

Morphological changes induced by extracellular matrix are correlated with maturation of rat small hepatocytes

Small hepatocytes (SHs), which are known to be hepatic progenitor cells, were isolated from an adult rat liver. SHs in a colony sometimes change their shape from small to large and from flat to rising/piled‐up. The aim of the present study is to clarify whether the alteration of cell shape is correlated with the maturation of SHs and whether extracellular matrix (ECM) can induce the morphological changes of SHs. We used liver‐enriched transcription factors (LETFs) such as hepatocyte nuclear factor (HNF) 4α, HNF6, CCAAT/enhancer binding proteins (C/EBP) α, and C/EBPβ, tryptophan 2,3‐dioxygenase (TO), and serine dehydratase (SDH) as markers of hepatic maturation. To enrich the number of SH colonies, the colonies were isolated from dishes and replated. Replated colonies proliferated and the average number of cells per colony was about five times larger at day 9 than at day 1. When the cells were treated with laminin, type IV collagen, a mixture of laminin and type IV collagen, Matrigel™ or collagen gel (CG), only the cells treated with Matrigel dramatically changed their shape within several days and had reduced growth activity, whereas the cells treated with other ECM did not. HNF4α, HNF6, C/EBPα, C/EBPβ, and TO were well expressed in the cells treated with Matrigel. Furthermore, addition of both glucagon and dexamethasone dramatically induced the expression of SDH mRNA and protein in the cells treated with Matrigel. In conclusion, morphological changes of SHs may be correlated with hepatic maturation and basement membrane (BM)‐like structure may induce the morphological changes of SHs. J. Cell. Biochem. 87: 16–28, 2002.

Growth and maturation of small hepatocytes.

Proliferation of adult rat hepatocytes is observed in serum‐free Dulbeccos modified Eagles medium (DMEM) supplemented with 10 mmol/L nicotinamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a less‐differentiated appearance, immunocytochemically and ultrastructurally they possess hepatic characteristics. The size of small hepatocytes is one‐third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes can be subcultured for several passages. Furthermore, the cells are rich in the supernatant following 50 g centrifugation for 1 min after collagenase liver perfusion. When the cells are cultured in DMEM supplemented with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2‐phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A small hepatocyte divides to form a colony and the number of cells reaches more than 100 within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature hepatocytes. Immunoreactivity to connexin 32 and well‐developed bile canaliculus structures are often observed in the cell‐cell borders. Thus, we suggest that small hepatocytes may be considered to be ‘committed progenitor cells’ that can further differentiate into mature hepatocytes.

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes

SummaryPhenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles’ medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, α-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was α-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. γ-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.

REDIFFERENTIATION OF PROLIFERATED RAT HEPATOCYTES CULTURED IN L15 MEDIUM SUPPLEMENTED WITH EGF AND DMSO

SummaryPrimary adult rat hepatocytes were cultured in serum-free L15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml epidermal growth factor in a 5% CO2:95% air incubator. The number of cells increased and reached about 180% of the initial value by Day 4, and after 2% dimethyl sulfoxide (DMSO) was added to the culture medium at Day 4, the cells continued to proliferate until Day 6. The number of cells reached about 210% at Day 6 and they were well maintained until Day 18. The cell number gradually decreased with time in culture, but many cells remained for more than 2 mo. On the other hand, without 2% DMSO, the cells proliferated until Day 5, but thereafter they rapidly decreased. After DMSO addition, albumin and transferrin were secreted into the medium and the production of both proteins continued for more than 2 mo. Immunocytochemically both proteins were strongly stained in the cells treated with 2% DMSO. Although the expression of G6Pase in the cells disappeared at Day 6 without DMSO, the cells treated with 2% DMSO recovered G6Pase activity at Day 16. In addition, induction of peroxisomes by 2 mM sodium clofibric acid was clearly shown in the hepatocytes at Day 14 and Day 25 using enzyme-cytochemistry. Ultrastructurally, DMSO-treated hepatocytes had many mitochondria and large peroxisomes with a crystalline nucleoid, and both gap junctions and desmosomes were well developed between the cells even at Day 40. Thus, the number of cells doubled, some differentiated functions of the primary hepatocytes were well restored by the use of 2% DMSO, and these functions were maintained for more than 2 mo.

Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells

BACKGROUND/AIMSnHybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs).nnnMETHODSnColonies of SHs were collected and replated on a collagen sponge. Hepatic functions were examined by ELISA, immunoblotting, and Northern blotting. Cells in the sponge were characterized by immunocytochemistry and transmission electron microscopy. Urea synthesis was measured and metabolization of fluorescein diacetate was examined.nnnRESULTSnSHs could proliferate and expand to form a hepatic organoid in the sponge. Albumin secretion and other hepatic protein production of the cells in the sponge increased with time in culture and the amounts were much larger than for those obtained from cells grown on dishes. Morphologically and functionally differentiated hepatocytes were observed and some CK19-positive cells formed duct-like structures within the sponge. Excretion of fluorescein was observed in bile canaliculi.nnnCONCLUSIONSnHepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs.

Immortal Epithelial Cells of Normal C3H Mouse Liver in Culture: Possible Precursor Populations for Spontaneous Hepatocellular Carcinoma

Long-term culture of primary hepatocytes derived from normal young male C3H/HeNJclMTV+ (C3H) and C57BL/6NJcl (C57) mice, respectively known for very high and low incidences of spontaneous hepatoma, resulted in development of multiple slowly growing epithelial colonies in the C3H case, the number of colonies being increased five-fold when 1.5 mM phenobarbital was added to the culture medium. On the other hand, the primary culture cells from C57 mouse liver gave rise to such epithelial colonies only very rarely, even with phenobarbital. Immunohistochemical investigation revealed alpha-fetoprotein and/or albumin production by the colony cells and ultrastructural analysis also revealed some hepatocytic features in them. Subculturing of individual colonies gave rise to cell lines which could be repeatedly passaged. Two of five lines implanted into athymic nude mice manifested tumorigenicity, the resultant neoplasms being diagnosed as a trabecular hepatocellular carcinoma and an adenocarcinoma. The experimental data suggest that the colony-forming immortal epithelial cells possibly represent early phase precursors of spontaneous mouse hepatocellular carcinomas. This culture system is expected to be useful for future elucidation of the mechanisms underlying spontaneous mouse hepatocarcinogenesis.

Proliferation of rat small hepatocytes after long-term cryopreservation

BACKGROUND/AIMSnThe demand for clinical use of hepatocytes is escalating because cell transplantation will be an alternative to orthotopic liver transplantation and the shortage of liver donors is a serious problem throughout the world. However, the supply of fresh differentiated hepatocytes is limited and methods for cryopreservation of hepatocytes that can proliferate with hepatic functions are not satisfactorily established.nnnMETHODSnColonies of small hepatocytes were collected and then maintained at -80 degrees C for more than 6 months. Albumin secretion and mRNA expression of thawed cells were measured by enzyme linked immunosorbent assay and Northern blotting, respectively, and the expression of hepatic functions was examined by immunoblotting. The ultrastructure of cryopreserved cells was also examined.nnnRESULTSnAbout 60% of the cryopreserved colonies attached on dishes and then proliferated. The average area of small hepatocyte colonies was about 7.5 times larger at day 15 than at day 1. Albumin production increased with time in culture. In addition, the cells produced other serum proteins such as transferrin and fibrinogen, and expressed carbamoyl phosphate synthetase I and tryptophan 2,3-dioxygenase.nnnCONCLUSIONSnSmall hepatocytes maintain growth ability and hepatic differentiated functions even after long-term cryopreservation.

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

SummaryA highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.

Interaction of Interleukin‐1 and Interferon‐γ on Fibroblast Growth Factor‐induced Angiogenesis

The interaction of interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) actions on several aspects of angiogenesis in vitro and in vivo was studied. The proliferation and migration of human umbilical vein endothelial cells cultured with basic fibroblast growth factor (bFGF) were synergistically inhibited by cotreatment with IL‐1 and IFN‐γ. Endothelial cell adhesion to collagen was suppressed by IL‐1 and the effect was slightly enhanced by the combination of IL‐1 and IFN‐γ. Local administration of IL‐1 (10,000 U) and IFN‐γ (1,000 U) inhibited bFGF‐induced angiogenesis in the skin of mice, and synergistic inhibitory activity of the combination was demonstrated. Expression of FGF receptors was strongly downregulated by the combination, whereas expressions of epidermal growth factor (EGF) receptors, integrin β1 and integrin β3 were not. EGF partially abrogated the growth‐inhibitory effects of IL‐1 and IFN‐γ. These findings indicate that IL‐1 and IFN‐γ are each able to act an angiogenesis inhibitor in a situation where FGF plays a major role in angiogenesis, and the activity is synergistically enhanced when they are used in combination.