Shinichiro Ikeda

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.

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

BACKGROUND/AIMS Hybrid 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). METHODS Colonies 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. RESULTS SHs 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. CONCLUSIONS Hepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs.

Proliferation of rat small hepatocytes after long-term cryopreservation

BACKGROUND/AIMS The 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. METHODS Colonies 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. RESULTS About 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. CONCLUSIONS Small hepatocytes maintain growth ability and hepatic differentiated functions even after long-term cryopreservation.

Surgical influence on Th1/Th2 balance and monocyte surface antigen expression and its relation to infectious complications

Malignancy and operation for it cause several alterations in immune function that are considered to be concerned with the development of infectious complications. Forty-three patients who underwent curative surgery for gastrointestinal malignancies were entered into this study and were divided into two groups, those with and those without postoperative infection. Changes in the proportion of Th1/Th2 subsets in CD4+ T cells and the expression of human leukocyte antigen (HLA)-DR and CD16a molecules on monocytes were measured by flow cytometry before and after surgery. We performed intracellular cytokine stainings to exactly detect Th1/Th2 subsets. The proportions of interferon-γ-producing CD4+ T (Th1) cells in the preoperative state were almost equal in the two groups, and the proportion decreased on postoperative day (POD) 1 in both groups. On POD 7, the proportion of Th1 cells recovered to the preoperative level in the noninfection group, while the suppression was further reinforced in the infection group (26.8% versus 18.3%, p < 0.005). In contrast, the proportion of interleukin-4-producing CD4+ T (Th2) cells in the infection group (11.3%) was already suppressed in the preoperative state when compared with the noninfection group (17.3%, p < 0.005). Changes in HLA-DR and CD16a expression on monocytes were similar to the changes in the proportion of Th1 cells. These results indicate that the suppression of Th1 cell and monocyte functions during the early phase of the postoperative course was directly related to the occurrence of infectious complications and that several immunological impairments have already occurred in the preoperative state in cancer patients.

Bile canalicular formation in hepatic organoid reconstructed by rat small hepatocytes and nonparenchymal cells

The morphogenesis and movement of bile canaliculi (BC) are not well understood. This is because culture of hepatocytes that maintain polarity of cell membranes and possess highly differentiated functions has never been successful. We found that small hepatocytes (SHs), which are known to be hepatic progenitor cells, could proliferate and differentiate into mature hepatocytes and that BC‐like structures developed between rising/piled‐up cells. We investigated how BC‐like structures developed with maturation of SHs and whether the structures were functionally active as BC. Hepatic cells, including SHs, were isolated from an adult rat liver and cultured. Immunocytochemistry and immunoblotting for BC proteins, such as ectoATPase, 5′‐nucleotidase, dipeptidylpeptidase IV, and multidrug‐resistance associated protein 2, were examined and time‐lapse microscopy was used for the observation of BC contractions. Secretion of bilirubin into the reconstructed BC was also observed. The results of immunocytochemistry, immunoblots, and immunoelectron micrographs revealed that BC proteins were localized in the intercellular space that coincided with BC‐like structures reconstructed between rising/piled‐up cells. Tight junction‐associated protein ZO‐1 was also expressed along the BC‐like structures. Bilirubin added to the medium were secreted into BC‐like structure and accumulated without leakage. Time‐lapse microscopy showed continuous contractions of reconstructed BC. In conclusion, BC‐like structures reconstructed by SHs may be functional with membrane polarity, secretory ability, and motility. These results show that this culture system may suitable for investigating the mechanism of the formation of BC and their functions. J. Cell. Physiol. 199: 252–261, 2004© 2003 Wiley‐Liss, Inc.

A Novel Experimental Mouse Model of Peritoneal Dissemination of Human Gastric Cancer Cells: Analysis of the Mechanism of Peritoneal Dissemination Using cDNA Macroarrays

We established a new cell line, NUGC‐3P4T, with high peritoneal metastatic disseminating potential in nude mice. NUGC‐3P4T cells were derived from the human gastric carcinoma line NUGC‐3, which has low capacity for peritoneal dissemination. NUGC‐3P4T cells developed peritoneal dissemination in 10/10 (100%) mice, whereas the parental NUGC‐3 cells developed dissemination in 1/5 (20.0%) mice. The metastatic foci in the peritoneum showed essentially the same histological appearance as those induced by parental cells. The tumorigenicity, the motile activity and the adhesive activity to the laminin of NUGC‐3P4T cells were stronger than those of NUGC‐3 cells. Production of IL‐8 was significantly higher in NUGC‐3P4T than in NUGC‐3. cDNA macroarrays analysis showed that a variety of cytokines, interleukins, and other immunomodulators and their receptors were up‐ or down‐regulated at the mRNA level in NUGC‐3P4T cells, compared with NUGC‐3 cells. Thus, this unique cell line and in vivo model might be useful to study the biology of peritoneal dissemination of human gastric cancer.