In vitro transformation of peripheral monocytes to putative hepatopoetic progenitors with hepatocyte-like glucagon responsiveness

Introduction: Since shortage in donor organs and side effects by the obligatory live-long immunosuppressive therapy necessitate the search for alternatives to liver transplantation, considerable effort is focussed at establishing cell based therapies. Transformed peripheral monocytes have been described as a possible source of hepatocyte-like cells. Aim of this study was to establish the generation of peripheral blood monocyte-derived hepatocytoid cells to enable their further use in transplantation experiments.

Methods: Peripheral monocytes of healthy donors underwent gradient centrifugation and adherence separation. These cells were incubated for 7 days in RPMI 1640 and serum containing M-CSF, IL-3 and mercaptoethanol, followed by 14 days culture in the presence of FGF-4. Marker expression was investigated by FACS analysis. Protein content of adherent cells was determined. The ability to release glucose in response to glucagon was examined and compared to HepG2 cells.

Results: Freshly isolated peripheral monocytes showed expression of the monocyte marker CD14 in 61% of the cells. The pan-leucocyte marker CD45 was expressed by >80% of isolated cells. The stem cells antigen CD90 was expressed by 2% of these cells. FACS analysis revealed an increase in CD90-expression to 44% and a slight decrease in CD14 expression. Culture in the presence of FGF-4 led to the development of an epitheloid appearance. Analysis of protein content showed a ˜7 fold increase of adherent cell protein. Glucose release was increased to 168±9% of control in HepG2 cells by glucagon stimulation. The effect of glucagon in transformed monocytes was 153% on day 14 and 159% of control on day 21, respectively.

Discussion: These results show that the generation of hepatocytoid cells from monocytes was successful. A glucagon-inducible glucose release was observed, pointing towards a hepatocytoid potential. Further hepatocyte specific functions need to be evaluated. We here provide evidence for a functional approximation of monocyte derived cells to HepG2 concerning the metabolic effects of glucagon stimulation. The experimental transplantation of these cells to assess their possible benefit in animal models of acute and chronic liver failure is currently being established.

1) Morphologic transformation of monocytes during culture period

2) Changes in Protein content[Abbildungen 1 und 2]

3) Changes in CD14 and CD90 Expression[Abbildung 3]

4) Glucagon-responsiveness[Abbildung 4]