Therapeutic Effects Of Embryonic Stem Cell-derived Hepatocytes Transplantation On Mice With Chronic Hepatic Failure

Objective:Hepatocyte transplantation has been proposed as a method to support liver function in acute or chronic hepatic insufficiency and as a ’cell therapy’ for metabolic disease in liver. Through setting up the chronic liver failure mode in mice, differentiating embryonic stem (ES) cells into hepatocytes by induction in vitro, and transplanting hepatocytes into mice with chronic hepatic failure, the influence of hepatocyte to mice with chronic liver failure was observed and it provides a new strategy for the chronic hepatic failure.Methods:1.Establishing mice model with chronic hepatic failure:Mice with chronic hepatic failure were induced by intraperitoneal injection using50%CCl4vegetable oil solution. The evaluation of the model was performed by the examination of serum ALT, AST and the observation of the pathology of liver tissues at the corresponding time point, and recording the diet and bodyweight.2.Differentiating embryonic stem (ES) cells into hepatocytes by induction in vitro: Human embryonic stem cell line BG02were cultured on Matrigel-coated culture plates for4days, and hepatic differentiation was initiated by adding to the based differentiation solution (Activin A) for inducing5days, then the induce liquid was replaced by the hepatic differentiation solution for inducing another6days, finally, HGF and OSM were added in hepatic differentiation solution for continuing inducing differentiation for5-7days. Morphology were investigated by inverted phase contrast microscopy after the induction of hESC at the different stages. The expression of AFP, ALB were assayed by RT-PCR and Western blotting.3.Therapeutic effects of Transplanting hepatics into mice with the chronic hepatic failure:The hepatocytes with successful differentiation were transplanted into mice with chronic hepatic failure,6.0x106cells were transplanted by tail vein. Blood from heart was collected and the serum level of hepatic function index:ALT, AST, ALP, ALB was detected by auto-biochemical analyzer in the1st day,7th day and15th day after hepatocyte transplantion. At the same time, liver tissues were collected in liquid nitrogen or directly examine by pathology.Results:l.The pathological changes of hepatic failure mode&liver biochemical index:①. In normal group, mouse liver was bright red, smooth surface and sharp edges. The hepatic lobule of normal liver tissue has normal morphology, and the portal area appeared morphological integrity, no degeneration and necrosis. In mode group, liver significantly reduced, with darker color, blunt edge, and hard, the there was a large fiber exudate on the surface of liver, with the surrounding tissue adhesion. In addition, pathological section had showed that was typical cytoplasm loosing, ballooning degeneration, scattered spotty, piecemeal necrosis, inflammatory cell infiltration; and significant fibrosis change in the mode group liver cells.②. The serum level of ALT and AST in the mode group increased significantly compared with those in the normal group.2.The characteristics of differentiated cells were determined by morphology, RT-PCR, Western blotting:①. On the10th after the induction, differentiated cells treated with Activin A, FGF-1, BMP-4, HGF and OSM sequentially were morphologically larger and became spherical, oval or polygon. On the15th day after the induction, some cells had2or3nuclei, and the result suggested that the cells had a hepatocyte-like morphology.②.The differentiated cells at various stages also expressed hepatocyte specific protein ALB and AFP.3.The pathological changes after the hepatocyte transplantation:In normal group, after liver tissue sections were stained with HE, it showed lobular structure was clear, the hepatic cord was radical around central vein, and hepatic sinusoid was clear. In mode group, on the7th day, the hepatic lobule partial was damaged, with a large number of liver cells lysis, inflammatory cell infiltration, the sinusoids clogged, endothelial cell was damaged, the edge of plasma of hepatocyte nuclear not clear, a large number of vacuoles in liver cells. In mode group, on the15th day, there were a large number of inflammatory cells infiltration, and vacuole structures are still observed in liver cells. In treatment group, on the1st day, central vein center regional of hepatic lobule was dissolved, and hepatocytes partial was damaged, with a small amount of inflammatory cells infiltration. In treatment group, on the7th day, the binuclear hepatocytes increased, with inflammatory cells infiltration. In treatment group, on the15th day, there were mainly degeneration and inflammatory cells infiltration, small necrotic, congestion and bleeding relieved in hepatocytes.4.The changes of liver biochemical index after hepatocyte transplantation:The1st day after hepatocyte transplantation, liver functional index (ALT, AST, ALP) were significantly higher in treat group and mode group than the normal group (P<0.05), but no significant difference compared with mode group (P>0.05). On the7th day, other index had significant difference between mode and treat group except ALB (P<0.05). On the15th day, ALT and AST were different between mode and treat group except ALP and ALB(P<0.05), AST in the treat group had recovered basically to the normal range compare with normal group, the other parameters have improved, but has not been recovered (P>0.05).Conclusion:1. The mice model with chronic liver failure can be induced by using intraperitoneal injection of50%CCl4(carbon tetrachloride) vegetable oil solution at dosage of1ml/kg body weight, once every three days, for seven consecutive weeks.The animal mode is stable, simple operation, suitable for experimental research applications.2. Under the particular culture condition in vitro, using a simple-step induction method and by supplied with hepatocyte growth factor (HGF) and other cytokines consequently, hESCs can be induced to differentiate into hepatocytes, and express hepatocyte specific protein ALB&AFP. These hepatocytes can be used for cell transplantation.3. Transplanted hepatocytes by the mouse tail vein can siginificantly improve liver function of mice with chronic liver failure, promote the recovery of liver function, and improve the liver pathological conditions of the mice.