Volume 44, Issue 6, Pages 1115-1124 (June 2006) The expression of mesenchymal, neural and haematopoietic stem cell markers in adult hepatocytes proliferating in vitro  Sarah Koenig, Petra Krause, Birgit Drabent, Ines Schaeffner, Bruno Christ, Peter Schwartz, Kirsten Unthan-Fechner, Irmelin Probst  Journal of Hepatology  Volume 44, Issue 6, Pages 1115-1124 (June 2006) DOI: 10.1016/j.jhep.2005.09.016 Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Time course of hepatocyte proliferation. Cells were cultured with base (○) or complete medium (●) containing conditioned media from hepatocytes (20%) and stromal cells (30%). Thymidine incorporation (A) was determined for 20h-intervals using three new parallel dishes each day. The inserted graph (B) depicts the mitogenic activity of 30% conditioned medium sampled from stromal cells at different culture ages. Here, thymidine uptake into hepatocytes was determined from day 3 to day 4 only. The increase in DNA during time of culture is shown in (C). Data are means±SEM from three individual cell preparations. Journal of Hepatology 2006 44, 1115-1124DOI: (10.1016/j.jhep.2005.09.016) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Micrographs of colonies derived from adult hepatocytes. Cells were cultured with complete medium. Fig. 2(A)–(D): phase contrast micrographs at a magnification of 100×. Cells after 6h (A), cell cultures on day 1 (=24h, B), day 3 (C) and day 7 (D). All images were taken from the same optical field. Scanning electron micrographs of colony cells cultured for 7 (E) and 20 days (F). Journal of Hepatology 2006 44, 1115-1124DOI: (10.1016/j.jhep.2005.09.016) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Semiquantitative RT-PCR analysis to compare the gene expression profiles of colony cells at different points in time of culture. Demonstration of hepatic, bile duct/oval cell, mesenchymal, and neural mRNA markers in proliferating hepatocytes. Journal of Hepatology 2006 44, 1115-1124DOI: (10.1016/j.jhep.2005.09.016) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Two-layer immunofluorescence staining of colony cells in situ grown for 10 days. Cells were fixed and processed on the dish. Nuclear counterstaining with DAPI (blue), original magnification ×400. A–E: immunofluorescence staining as marked. F: phase contrast micrograph of the culture shown in E. Journal of Hepatology 2006 44, 1115-1124DOI: (10.1016/j.jhep.2005.09.016) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Multi-layer immunofluorescence staining to co-localise differentiation markers in colony cells at two points in time of culture (0 and 10 days). 0 day=cytospins of the cell inoculate before culturing. Ten days=cultured cells were detached from the gel matrix as vital colony cell sheets, cytospins were prepared and subsequently stained. At the time point 10 days, the FITC and TRITC-fluorescence channels are demonstrated separately, the last column presents the overlay. Immunofluorescence staining as marked, nuclear counterstaining with DAPI (blue), original magnification ×400. Journal of Hepatology 2006 44, 1115-1124DOI: (10.1016/j.jhep.2005.09.016) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Functional re-differentiation of proliferating hepatocytes in vitro. Re-differentiation was initiated on day 6 by overlaying the whole surface of the dish with a thin collagen gel. Hepatocytes were then further cultivated in base medium without insulin and 30% HCM for 24h. (A) Phase contrast micrograph of re-differentiated cells on day 7 showing bile canalicular formations, magnification of 100×; please compare with Fig. 2(D), which shows cells that have not re-differentiated. (B) PCK gene expression, determined by the increase in enzyme activity, was induced by 10nM glucagon for 6h at day 6 and day 7. Data are means±SEM from three different cell preparations. Journal of Hepatology 2006 44, 1115-1124DOI: (10.1016/j.jhep.2005.09.016) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions