COMPARISON OF HUMAN MONOCYTE-DERIVED NEO-HEPATOCYTE WITH HUMAN PRIMARY HEPATOCYTES AS A PERSPECTIVE FOR AUTOLOGOUS CELL THERAPY

Abstract

P671* Aims: There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. Apart from bioreactors, as mechanical devices supporting liver function, first cases of human primary hepatocyte transplantations are reported. We have generated neo-hepatocytes out of terminally differentiated peripheral blood monocytes after undergoing a step of dedifferentiation which show specific in vitro functions comparable to those of primary hepatocytes Methods: Monocytes were isolated from human peripheral blood and after a six day treatment with MCSF and IL-3 were then exposed to a hepatocyte differentiation media containing FGF4 for 10 to 20 days. Neo-hepatocytes were analyzed for Albumin. Urea formation was shown using an urea-GLDH-method kit. Phase I biotransformation activity of neo-hepatocytes was verified using 7-ethoxycoumarin O-de-ethylase. The main phase II reactant, uridine di-phosphate (UDP)-glucuronyltransferase, was measured using 4-MU as a substrate protein expression, albumin production, urea metabolism for detoxification, UDP-glucuronidation and Phase I metabolism. All functions were compared with those of primary human hepatocytes. Results: 14-20 days after treatment with FGF-43 (HCM: hepatocyte conditioning medium) cells displayed a typical hexagonal shape and formed confluent layers with cell-cell contacts closely resembling those in primary human hepatocytes. RT-PCR analysis revealed that these “neo-hepatocytes”, expressed hepatocyte markers, e. g. α-fetoprotein, carbamyl phosphate synthetase I and coagulation factor II. An in-depth functional analysis of the neo-hepatocytes revealed a sustained albumin synthesis and a detoxifying capacity (urea production) similar in quantity and duration to that in genuine human hepatocytes. Furthermore, we found activity of 7-ethoxycoumarin O-deethylase in neo-hepatocytes, an enzyme that is catalyzed by a broad range of P450 isoenzymes such as CYP1A2, CYP2A6, CYP2B6, CYP2C8-9, CYP2E1, and CYP3A3-5. This was demonstrated by metabolization of ethoxy coumarin into 7OH-coumarin after stimulation with 3-methylcholanthren. In contrast, phase II metabolization through UDP-glucuronidation of 4-methyl-umbelliferone (4-MU) appears to be identical in primary human hepatocytes and neo-hepatocytes. Conclusions: These data convincingly show that neo-hepatocytes possess unique hepatocyte-specific cellular functions, namely synthesis, detoxification, phase I and II metabolism, which are quantitatively and qualitatively comparable to those of primary human hepatocytes in vitro. These cells could be clinically applied in an autologous setting for treatment of end stage liver diseases or to improve liver function prior to organtransplantation without the need for immunosuppressants or the fear of rejection