Taisuke Yamazaki

Cloning and characterization of liver progenitor cells from the scattered cell clusters in primary culture of porcine livers.

The scattered cell clusters that can differentiate into hepatocytes or biliary epithelial cells have been isolated from primary cultures of adult porcine livers. We have generated 11 clonal cell lines from this system and identified liver progenitor cells (LPCs) among the clonal lines. These clonal lines expressed c-kit, HNF-1, HNF-6, and/or CK19 mRNA. An immunocytochemical study of the clonal lines indicated that clonal line CL-11 expressed liver epithelial cell markers CK14, vimentin, CK18, and BD-1. The expression of albumin and α1-antitrypsin (α1-AT) mRNA was only upregulated in CL-11 among the clonal lines when they were grown as aggregates. Under these conditions, CL-11 also exhibited ammonia metabolic activity and several indicators that suggest hepatocytic differentiation, including the upregulation of liver-specific genes such as dipeptidyl peptidase IV, CYP1A1, and CYP3A4 mRNA, and the downregulation of biliary cell markers such as γ-glutamyltrans-peptidase (GGT), CK19, and HNF6 mRNA. After culturing CL-11 in Matrigel, the expression of GGT and HNF6 mRNA was upregulated. These results indicate that CL-11 has dual potential: the ability to differentiate as hepatocytes or as bile duct cells. The isolation of scattered cells could provide a simple method to generate LPC lines from adult livers.

EFFECT OF DEXAMETHASONE ON BINDING ACTIVITY OF TRANSCRIPTION FACTORS NUCLEAR FACTOR-κB AND ACTIVATOR PROTEIN-1 IN SW982 HUMAN SYNOVIAL SARCOMA CELLS

SummaryPreviously, we showed that dexamethasone (DEX) inhibited the expression of inflammatory cytokines, matrix metalloproteinase-1, and cyclooxygenase-2 messenger ribonucleic acid in SW982 cells. In this study, the effect of DEX on the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) was examined in SW982 cells by electrophoretic mobility shift assay (EMSA). Both NF-κB and AP-1 deoxyribonucleic acid binding activities were detectable in SW982 cells by EMSA, and they were induced by interleukin-1β treatment. DEX inhibited NF-κB binding activity at 10 μM as well as at 100 μM, although the inhibition was only partial. However, DEX had little effect on AP-1 activity. These results suggest that DEX reduces the expression of inflammatory cytokines and other proteins in SW982 cells by inhibiting NF-κB.

Presence of side-population cells in an immortalized nontumorigenic human liver epithelial cell line

Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verapamil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.

Isolation of Hepatic Progenitor Cells from Human Liver with Cirrhosis Secondary to Biliary Atresia Using EpCAM or Thy-1 Markers:

We sought to determine whether hepatic progenitor cells can be isolated from cirrhotic liver using epithelial cell adhesion molecule (EpCAM) or Thy-1 markers. Liver tissue with cirrhosis secondary to biliary atresia (BA) was collagenase digested, and nonparenchymal cells (NPCs) were cultivated for 24 h. Noncirrhotic NPCs derived from patients with carbamyl phosphate synthetase and ornithine transcarbamylase deficiencies were used as controls. Flow cytometric analysis demonstrated that the percentages of EpCAM- and Thy-1-positive cells were significantly higher in NPC populations derived from BA liver than in those derived from control liver. Reverse transcription polymerase chain reaction analysis revealed that EpCAM-positive sorted cells expressed EpCAM, Thy-1, albumin, and CK-19, whereas Thy-1-positive sorted cells expressed Thy-1, albumin, and CK-19. These findings indicate that EpCAM- or Thy-1-positive hepatic progenitor cells can be more efficiently isolated from BA liver than from control liver and suggest that the properties of EpCAM-positive cells are somewhat different from those of Thy-1-positive cells.

Repopulation of Human Origin Hepatocyte Progenitor-Like Cell Line, THLE-5b, in the SCID Mouse Liver under p21-Mediated Cell Growth-Arresting Conditions:

The in vivo repopulation of hepatocytes depends on donor cell growth potential and recipient conditioning. We herein demonstrate the successful cell transplantation of a human hepatocyte cell line, THLE-5b, into the SCID mouse liver by means of a rather mild conditioning using a 55% hepatectomy and p21 transfection. Adult human liver-derived cells, THLE-5b, are SV40 T antigen-immortalized epithelial cells. A phenotypic examination of THLE-5b showed they expressed hepatic stem cell markers such as EpCAM, OCT3/4, and Thy-1, thus indicating the immature nature of the cells. A three-dimensional aggregate culture of THLE-5b showed a higher expression level of liver-specific genes such as albumin, α1-antitrypsin, and CYP3A4, thus suggesting that THLE-5b possess the capability to differentiate into hepatocytes. In a cell transplantation experiment, the cell cycle regulator p21 was transfected with adenoviral vector into the SCID mouse liver. On the next day, 8 × 105 cells of GFP-transfected THLE-5b were injected intrasplenically, together with the intraperitoneal administration of anti-asialo GM1 antibodies. The following day, a partial hepatectomy was performed. The GFP-THLE-5b cells were observed to have migrated and become integrated into the liver parenchyma 14 days after transplantation. The present protocol is thus considered to be a novel experimental model to elucidate the mechanism of hepatocyte repopulation and to develop efficient stem cell therapy in the liver.

Subgrouping of equine transferrin types by peptide mapping

quantitative variants between T+ and Tcomponents composed of Tf types, the Tf types could be classified into three groups as Tf-D・F・H, Tf-O・R and Tf-X subgroups. To certify furthermore the results, the equine Tf types were subgrouped from peptide cleavage patterns produced by peptide mapping using four proteases (α-chymotrysin, V8 protease, papain and endoprotease Asp-N). The results obtained are as follows: 1) When Tf・D, F and H types were treated by the four proteases, these three Tf types had almost the same peptide cleavage patterns. Although there were respectively some different band patterns between each peptide cleaved in Tf・O and R types, they had more mutually similar points than those of the Tf-D・F・H subgroup. This means that Tf・O and R types may be mutually specialized, but it was guessed that they can be basically classified into the same subgroup in view of having many of the same peptide cleavage components. The four peptide patterns which were cleaved donkeys Tf components, were each very similar with those of equine Tf・O and R types. 2) T+ and Tcomponents of Tf・D and R types were each cleaved by V8 protease and α-chymotrypsin, and both components of the Tf・D type showed almost same peptide pattern, and there were distinct differences when the two components of the Tf・R type were digested by the latter protease. From analysis by peptide mapping, Tf・O and R types might be newly subgrouped, but it was again recognized that the peptide structures of the Tf・O and R types were each distinctly different from those of the Tf-D・F・H subgroup. Accordingly, the equine Tf types could be classified into two groups as Tf-D・F・H and Tf-O・R types by peptide mapping. Then the T+ and Tcomponents in the Tf・R type had basically different peptide structure, respectively.

Side Population Cells From an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties.

The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.