Experimental studies on the effects of hepatocyte-based liver support

Abstract: Liver transplantation is limited by lack of donors and consequently there is a need to develop liver support/replacement therapies, such as hepatocyte transplantation (HcTx) and artificial livers. In the present thesis, the effects of these therapies were investigated. First, we established a technique of isolating fetal hepatocytes (FH). In cultures we found that, in contrast to adult hepatocytes, FH had high proliferative activity and they were susceptible to retroviral transduction. In vitro they were less sensitive than adult hepatocytes to growth factors involved in liver repair in response to injury such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), and both transforming growth factors alpha (TGF-α) and beta (TGF-β1). We also found that proliferation was suppressed by cyclosporine in FH cultures. Allogeneic FH were transplanted to Nagase analbuminemic rats in order to evaluate the functional effect of FH in these albumin deficient rats. Significant amounts of serum albumin were found if FH were transplanted under a hepatic regenerative stimulus, and the levels were comparable with those found in rats transplanted with adult hepatocytes.In fulminant hepatic failure (FHF) rats, intrasplenic HcTx as well as treatment with a hepatocyte-based bioartificial liver (BAL) resulted in prolonged survival, improved metabolic and coagulation parameters, lowered TGF-β1 levels and regeneration of the remnant liver. Liver regeneration after HcTx was associated with lowering of elevated plasma HGF and TGF-β1 levels and accelerated expression of HGF and its receptor c-met mRNA in the native liver. BAL treatment resulted in an increased binding of transcription factors engaged in the modulation of hepatocyte proliferation and liver-specific gene expression. In anhepatic rats, intrasplenic HcTx delayed the onset of encephalopathy and prolonged animal survival. Transplanted rats had lower blood ammonia levels. improved blood coagulation. and lowered TGF-β1 levels. These data indicate that hepatocyte-based therapy can not only provide metabolic support in acute liver failure and genetic defects of liver metabolism by increasing the available functional liver mass, but also has a beneficial effect on the function and healing of the remnant liver.Regulation of tissue-specific functions in mitogen-stimulated hepatocytes is poorly understood. Since hepatocyte proliferation seems to be controlled by a balance between growth stimulators and inhibitors, we hypothesized that after liver injury, elevated blood TGF-β1 levels may reflect au adaptive response to maintain differentiated functions in hepatocytes affected by high levels of HGF. We explored this possibility by studying the effect of TGF-β1 on the expression of liver-enriched transcription factors and genes which remain under their regulatory activity, in mitogen-stimulated primary rat hepatocytes. We found that TGF-β1, in addition to mito-inhibitory effects, plays a role in maintaining differentiated functions. Knowledge of the requirements for recovery of hepatic function and growth of the liver may be crucial in designing novel liver support strategies.