[Efficacy and toxicity of transcatheter arterial chemoembolization with Cisplatin suspended in lipiodol for unresectable hepatocellular carcinoma].

In this study we evaluated the efficacy and toxicity of transcatheter arterial chemoembolization (TACE) with Cisplatin (CDDP)-Lipiodol (LIP) suspension in 24 patients with advanced hepatocellular carcinoma (HCC). Eligibility criteria were as follows; unresectable HCC, age <75 years, performance status (PS) 0-2, Child-Pugh A or B and adequate heart and renal function. When TACE was performed, the catheter was placed selectively in feeding arteries of the tumors, and CDDP-LIP suspension (20 mg/mL) was injected followed by gelatin sponge particles. The direct and total effect on tumors were evaluated 3 and 6 months after TACE, respectively. As for a direct effect, complete and partial response rates were 54.2% and 25%, respectively. As for a total effect, complete and partial response rates were 41.7% and 4.1%, respectively. Grade 3/4 drug-related toxicities were as follows: thrombocytopenia (13%), appetite loss (8%) and nausea (4%). These severe side effects disappeared within 10 days after TACE. No renal and hepatic dysfunction was encountered, and no drug-related deaths occurred. TACE with CDDP suspended in LIP may provide some clinical benefits with relatively tolerable toxicities.

Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.

Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)-transgenic mice and fluorescence-activated cell sorting.

BACKGROUND/AIMS: Recent advances in stem cell research have revealed that hepatic stem/progenitor cells may play an important role in liver development and regeneration. However, a lack of detectable definitive markers in viable cells has hindered their primary culture from adult livers. METHODS: Enzymatically dissociated liver cells from green fluorescent protein (GFP)-transgenic mice, which express GFP highly in liver endodermal cells, were sorted by GFP expression using a fluorescence-activated cell sorter. Sorted cells were characterized, and also low-density cultured for extended periods to determine their proliferation and clonal differentiation capacities. RESULTS: When CD45(-)TER119(-) side-scatter(low) GFP(high) cells were sorted, alpha-fetoprotein-positive immature endoderm-characterized cells, having high growth potential, were present in this population. Clonal analysis and electron microscopic evaluation revealed that each single cell of this population could differentiate not only into hepatocytes, but also into biliary epithelial cells, showing their bilineage differentiation activity. When surface markers were analyzed, they were positive for Integrin-alpha6 and -beta1, but negative for c-Kit and Thy1.1. CONCLUSIONS: Combination of GFP-transgenic mice and fluorescence-activated cell sorting enabled purification of hepatic progenitor cells from adult mouse liver. Further analysis of this population may lead to purification of their human correspondence that would be an ideal cell-source candidate for regenerative medicine.

Enrichment of hepatic progenitor cells from adult mouse liver.

Hepatic progenitor cells (HPCs) have been characterized in several drug-treated rodent models and in the fetal liver; however, their properties have not been fully clarified in the normal adult liver, presumably because of their relatively small population and the existence of mature hepatocytes. In an attempt to resolve this issue, we developed a new enrichment system for HPCs using their cell aggregate formation properties. Nonparenchymal cells (NPCs) derived from enzymatically digested liver cells in normal adult mouse liver were treated in a hypoxic 2-hour suspension culture under constant shaking. This procedure resulted in cell aggregate formation and almost complete elimination of mature hepatocytes. Cell aggregates were formed only in Ca(2+)-containing medium, suggesting cadherin-dependent cell-cell adhesion. In these cell aggregates, 95% consisted of vascular endothelial cells that expressed VE-cadherin. The remaining 5% consisted of rapidly proliferating, small epithelial cells that expressed alpha-fetoprotein (AFP), E-cadherin, and albumin but not cytokeratin 19 (CK19), alpha-smooth muscle actin, or VE-cadherin. These results are consistent with an immature hepatic cell phenotype. When these immature hepatic cells were cultured with 10(-7) mol/L dexamethasone and 1% dimethyl sulfoxide, the de novo expression of mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) was induced concomitantly with the induction of morphologic characteristics such as mitochondria- and peroxisome-rich cytoplasm and bile canaliculi formation. In conclusion, our methodology allows the enrichment of immature hepatic cells from the normal adult mouse. These cells are capable of growth and maturation along the hepatocyte lineage, indicating that these cells are HPCs.

Establishment of a highly efficient gene transfer system for mouse fetal hepatic progenitor cells.

Because of a donor shortage problem in liver transplantation, cell transplantation has been anticipated as a useful bridge or substitute therapy, and has necessitated the development of cell sources other than donated organs. Therefore, the use of fetal hepatic progenitor cells (HPCs) is now being focused on. In this study, we intended to establish an efficient ex vivo nonviral gene-transfer system using a newly developed isolation and culture system for mouse fetal HPCs. Fetal HPCs, characterized using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) for lineage markers, were collected from E13.5 Balb/c mice using change in size because of cell aggregation by their homophilic cell-to-cell binding occurring during suspension culture. Optimal conditions for culture and ex vivo gene transfection for fetal HPCs were determined by (3)H-thymidine incorporation and the expression efficacy of transfected red fluorescent protein (DsRed) gene in different culture media. The optimum timing for gene transfection was also evaluated. To evaluate the in vivo expression of the transferred gene, DsRed-transferred fetal HPCs were transplanted into 70% partially hepatectomized allogenic mice. The highest efficacy of DsRed gene transfection into fetal HPCs in vitro (45% +/- 12.3%) was achieved with culture media, which also enabled the highest (3)H-thymidine incorporation, containing the deleted form of hepatocyte growth factor (dHGF) and insulin, and when transfection was performed immediately after isolation. In vivo DsRed expression in fetal HPCs was maintained concomitantly with albumin expression even after HPC transplantation. In conclusion, we established a highly efficient in vitro gene transfer system for mouse fetal HPCs using a newly developed isolation and culture system.

Contribution of bone marrow cells to liver regeneration after partial hepatectomy in mice.

BACKGROUND/AIMS: We examined whether bone marrow (BM) cells can commit to liver-consisting cells during liver regeneration after partial hepatectomy, using mice transplanted with green fluorescent protein (GFP) positive BM from GFP transgenic mice. METHODS: Partial hepatectomy or sham operation was performed. Lineage marker analysis of GFP positive liver cells was by immunostaining and flow cytometry. DiI-labeled acetylated low-density lipoprotein uptake or microsphere phagocytosis was examined in vitro. Lineage marker expression in BM and peripheral blood (PB) cells, and the vascular endothelial growth factor (VEGF) concentration in the liver were also examined. RESULTS: In hepatectomized mice, significantly more GFP positive cells participated in liver sinusoid than in sham-operated mice, expressing CD31 but not albumin. The percentage of cells that incorporated acetylated low-density lipoprotein but not microspheres was 69.5+/-3.4%, while 28.3+/-2.6% incorporated both, revealing sinusoidal endothelial and Kupffer cells, respectively. Increased expression of the CD31 and CD16/CD32 on GFP positive liver cells was also detected. The elevation of the VEGF concentration during liver regeneration and the increase in the CD34 and Flk-1 expression in the liver, BM, and PB cells suggested endothelial progenitor cell mobilization. CONCLUSIONS: GFP cell-marking provided direct evidence of the BM cells participation in liver regeneration after hepatectomy, where the majority was committed to sinusoidal endothelial cells probably through endothelial progenitor cell mobilization.