Aims: Shortage of donor organs and side effects by the obligatory live-long immunosuppressive therapy lead the focus of scientific efforts at establishing cell based therapies. Transformed peripheral monocytes have been described as a possible source of hepatocyte-like cells (Ruhnke et al. 2005a; Ruhnke et al. 2005b). Generated from a CD90- positive precursor, these cells have been characterized for expression of hepatocyte-specific mRNAs, generation of urea as well as phase-I and phase-II metabolism. Yet, there is considerable variation between monocytes inter- and intraindividually. In addition, more data on hepatocyte-specific functions are needed. Aim of this study was to establish the generation of peripheral blood monocyte-derived hepatocytoid cells with tolerable fluctuation in the parameters investigated and additional functional characterization. Methods: Peripheral monocytes of healthy donors were transformed 7 days in conditioning medium followed by up to 28 days culture in differentiation medium. Marker expression was investigated by FACS analysis. Protein content, specific activities of ALT, GGT, DPP IV and LDH were determined. Glucose secretion was investigated after stimulation with glucagon and forskolin. Additionally, CREB phosphorylation was investigated by western blotting. Toxicity of paracetamol was determined by LDH-release and caspase assays. All results were compared to HepG2 cells. Results and Conlusions: (i) The measured parameters exhibited low variations inter- and intraindividually. (ii) In transformed monocytes, glucagon and activation of adenylyl-cyclase by forskolin enhanced glucose-secretion. Glucagon increases CREB phosphorylation in transformed monocytes on day 14, indicating activation of the specific signalling pathway by glucagon. (iii) Paracetamol showed a dose dependent toxicity. Thus, there is evidence for active CYP P450 metabolism. (iv) These data indicate hepatocyte-specific functions in transformed monocytes. Work in progress and future perspectives: A) Comparison of transformed monocytes with primary human hepatocytes, focussed on biochemistry and functional aspects (e.g. specific enzyme activities, glucagon-response, functional clotting factors, signalling). B) Establishing reporter-gene assays for hepatocyte specific, regulated genes. C) Validation of cellular imaging for transplantation experiments. D) Transplantation of transformed monocytes in animal models of acute and chronic liver failure
Literatur: Ruhnke, M.; Nussler, A. K.; Ungefroren, ; Hengstler, J. G..; Kremer, B.; Hoeckh, W. et al. (2005a): Human Monocyte-Derived Neohepatocytes: A Promising Alternative to Primary Human Hepatocytes for Autologous Cell Therapy.Miscellaneous Article. In: Transplantation, 79, 9, S. 1097–1103. Ruhnke, M.; Ungefroren, ; Nussler, A.; Martin, F.; Brulport, M.; Schormann, W. et al. (2005b): Differentiation of In Vitro-Modified Human Peripheral Blood Monocytes Into Hepatocyte-like and Pancreatic Islet-like Cells. In: Gastroenterology, 128, 7, S. 1774–1786.
cell therapy - hepatocyte function - transformed monocyte
