Improvement of hepatocyte transplantation efficiency in the mdr2-/- mouse model by glyceryl trinitrate.

INTRODUCTION: Hepatocyte transplantation could be an alternative to liver transplantation for the treatment of metabolic diseases. However, rodent models have shown that engraftment of transplanted cells in the liver is low and requires deposition of cells in hepatic sinusoids. Splanchnic vasodilatators improved hepatocyte engraftment in a rat model. Therefore, we investigated the effect of glyceryl trinitrate (GTN) on the efficacy of cell engraftment and on liver repopulation in the mdr2-knockout mouse, a model for progressive familial intrahepatic cholestasis type 3. METHODS: Congenic normal mdr2 (+/+) hepatocytes were isolated by two-step collagenase perfusion and transplanted into mdr2(-/-) mice livers through the portal vein in the presence or absence of GTN. Liver repopulation was assessed by immunohistochemistry, and transplanted hepatocyte function was assessed at different times after transplantation by measurement of biliary lipid secretion and quantification of fibrosis. RESULTS: The number of engrafted cells in GTN-treated mice was significantly higher than that in control mice, and transplanted hepatocytes were found in a greater number of distal sinusoids. Levels of phospholipid secretion were significantly higher than those in the control group 3 months after hepatocyte transplantation (18.3 ± 2.3 vs. 5.2 ± 3.9 nmol/min/100 g, P < 0.0001), and the ratio of phospholipids to bile salt was greater (6.8 ± 1.3 vs. 3.2 ± 1.6, P = 0.03). The percentage area of liver fibrosis was also significantly reduced in GTN-treated mice (5.7% ± 2.3% vs. 12.4% ± 2.9%, P = 0.016). CONCLUSION: The use of GTN improves hepatocyte engraftment and correction of metabolic disease in mdr2 (-/-) mice. This approach might be beneficial in hepatocyte transplantation for the treatment of patients with liver diseases.

Nitric oxide donor drugs improve the distribution and engraftment of transplanted hepatocytes in the liver.

BACKGROUND: Hepatocyte transplantation could be an alternative to liver transplantation for the treatment of metabolic diseases, however this therapy is still limited by the loss of transplanted cells in the portal radicles before their entry into the sinusoids to engraft. Therefore, we investigated the effect of glyceryl trinitrate on hepatic sinusoids and on the efficacy of cell engraftment in a syngenic mice model. METHODS: We first assessed the effect of GTN portal infusion on the parenchymal spreading of colored microspheres. Hepatocytes transplantation in a syngenic mice model was then performed concomitantly with GTN infusion. The distribution of transplanted hepatocytes and their ultimate engraftment were analysed. RESULTS: After GTN perfusion 27% of microspheres shifted from the portal to the sinusoidal zone. Transplanted hepatocytes distribution changed significantly in the portal and parenchymal zones from respectively 53 +/- 2% and 46.8 +/- 2% in control animals to 32.5 +/- 2.4% and 67.5 +/- 2.4% in GTN-treated animals. At days 7 and 15, we noted a significantly better engraftment in GTN group vs. controls (60 +/- 4 vs. 37 +/- 2 transplanted hepatocytes in 20 fields x 400). CONCLUSIONS: Portal perfusion of GTN improved the access of microspheres and transplanted hepatocytes to the sinusoidal bed and also improved the percentage of cell engraftment in the liver. These results suggest that drug dilatation of portal pedicles prior to transplantation increases the efficiency of hepatocyte grafting.

Increased engraftment of hepatic progenitors after activation of the hepatocyte growth factor signaling pathway by protein transduction.

Cell transplantation has become a major focus in biomedical research. However, efficient engraftment in solid tissues remains a challenge. Hepatocyte growth factor (HGF) signaling increases survival, proliferation, migration, and invasion of many cell types through Met, its cell surface receptor. Therefore, activation of this signaling pathway may improve the ability of many cells to be transplanted. We constructed a constitutively activated form of Met (Tpr-Met) fused to the protein transduction domain of HIV-TAT to activate the HGF/Met pathway for a few hours following cell injection. Matrix-assisted refolding was used to renature TAT-Tpr-Met protein, which was efficiently delivered into cells and recapitulated several biological functions of Met in vitro. Furthermore, treatment of hepatic progenitors with this molecule for one hour before transplantation significantly improved engraftment efficiency (31% untreated cells, 58% treated cells). These findings suggest that the transient transfer of Tpr-Met may provide a new approach to increase the proportion of successfully engrafted cells.

Liver regeneration and recanalization time course following reversible portal vein embolization.

BACKGROUND/AIMS: Permanent portal vein embolization (PVE) is a widely practised technique. The use of an absorbable material would be safer in clinical situations in which the embolized liver is not resected. We evaluated the efficiency of reversible PVE in terms of liver regeneration and analyzed the precise time course of portal recanalization. METHODS: Nine monkeys underwent PVE of the left and right anterior portal branches using powdered absorbable material. Repeated portograms were carried out until complete revascularization of the embolized liver. Hepatocyte proliferation rates were assessed by BrdU incorporation. Liver segment volumes were determined by CT scans performed before embolization, then 1 month and 1 year after embolization. RESULTS: Reversible PVE induced significant hepatocyte proliferation in the non-embolized segments (13.5+/-1.0%, 10.5+/-0.8% and 9.1+/-2.0% of cells on days 3, 5 and 7, respectively). One month after the embolization, the non-embolized liver volume had increased from 38.4+/-1.3% to 54.8+/-0.5% of total liver volume. Proximal and complete revascularization occurred 6-8 and 12-16 days, respectively. CONCLUSIONS: Reversible PVE efficiently induces liver regeneration. The use of absorbable material avoids long-term liver scarring. Such material may be suitable for several clinical indications, including cell transplantation.

In vivo imaging of transplanted hepatocytes with a 1.5-T clinical MRI system--initial experience in mice.

The feasibility of in vitro mature mouse hepatocyte labeling with a novel iron oxide particle was assessed and the ability of 1.5-T magnetic resonance imaging (MRI) to track labeled mouse hepatocytes in syngenic recipient livers following intraportal cell transplantation was tested. Mouse hepatocytes were incubated with anionic iron oxide nanoparticles at various iron concentrations. Cell viability was assessed and iron oxide particle uptake quantified. Labeled hepatocytes were intraportally injected into 20 mice, while unlabeled hepatocytes were injected into two mice. Liver T2 values, spleen-to-muscle relative signal intensity (RI( spleen/muscle )), and liver-to-muscle relative signal intensity (RI( liver/muscle )) on gradient-echo T2-weighted imaging after injection of either labeled or unlabeled hepatocytes were compared with an ANOVA test followed by Fisher's a posteriori PLSD test. Livers, spleens and lungs were collected for histological analysis. Iron oxide particle uptake was saturable with a maximum iron content of 20 pg per cell and without viability alteration after 3 days of culture. Following labeled-cell transplantation, recipient livers showed well-defined nodular foci of low signal intensity on MRI--consistent with clusters of labeled hepatocytes on pathological analysis--combined with a significant decrease in both liver T2 values and liver-to-muscle RI( liver/muscle ) (P = 0.01) with minimal T2 values demonstrated 8 days after transplantation. Conventional MRI can demonstrate the presence of transplanted iron-labeled mature hepatocytes in mouse liver.

Efficient hepatocyte engraftment in a nonhuman primate model after partial portal vein embolization.

BACKGROUND: Hepatocyte transplantation could be an alternative to whole liver transplantation for the treatment of metabolic liver diseases. However, the results of clinical investigations suggest that the number of engrafted hepatocytes was insufficient to correct metabolic disorders. This may partly result from a lack of proliferation of transplanted hepatocytes. In rodents, portal ligation enhances hepatocyte engraftment after transplantation. We investigated the effects of partial portal ligation and embolization on engraftment and proliferation of transplanted hepatocytes in primates. METHODS: Hepatocyte autotransplantation was performed in Macaca monkeys. The left lateral lobe was resected for hepatocyte isolation. The first group of monkeys underwent surgical ligation of the left and right anterior portal branches; in the second group, the same portal territories were obstructed by embolization with biological glue. To evaluate the proportion of cell engraftment hepatocytes were Hoechst-labeled and transplanted via the portal vein. Cell proliferation was measured by BrdU incorporation. RESULTS: Hepatocyte proliferation was induced by both procedures but it was significantly higher after partial portal embolization (23.5% and 11.2% of dividing hepatocytes on days 3 and 7) than after ligation (3% and 0.8%). Hepatocytes engrafted more efficiently after embolization than after ligation. They proliferated and participated to liver regeneration representing 10% of the liver mass on day seven and their number remained constant on day 15. CONCLUSIONS: These data suggest that partial portal embolization of the recipient liver improves engraftment of transplanted hepatocytes in a primate preclinical model providing a new strategy for hepatocyte transplantation.