Controlled and reversible induction of differentiation and activation of adult human hepatocytes by a biphasic culture technique

Figure 1 Experimental design. HC were alternately cultured in AM or DM. Environmental switch was carried out on d 5 and 10 after plating. Incubation code A confers to initial incubation in DM, code B to initial incubation in AM. Experimental analysis of HC was carried out on d 5, 10, 15, and 20, if not otherwise indicated.

Figure 2 Expression kinetics of growth factor receptors. A: MET expression; B: EGFR expression. HC were permanently cultured either in a AM or in a DM. The receptor level was detected by flow cytometry and expressed as relative fluorescence units (RFU). mean±SD (n = 6), ^aP<0.05 between groups.

Figure 3 Confocal analysis of distribution pattern of growth factor receptors on d 0 and 5 after culture onset. A: MET distribution; B: EGFR distribution. HC were cultured either in DM or in AM. Indocarbocyanine staining, ×100/1.3 oil immersion objective. Each figure is representative for three separate experiments.

Figure 4 Dynamics of growth factor receptor expression. A: MET kinetics; B: EGFR kinetics. HC were plated on a 2D collagen matrix and incubated alternately in AM or DM. Code A (2D start_diff) is related to initial 5-d incubation in DM, followed by 5-d incubation in AM and subsequent incubation in DM. Code B (2D start_activ) concerns the incubation order AM-DM-AM. The receptor level was detected by flow cytometry and expressed as RFU. mean±SD (n = 6), ^aP<0.05 between groups.

Figure 5 Western blot analysis of MET protein content. HC were incubated alternately in AM or DM. Medium was changed on days 5 and 10. Incubation code A indicates initial 5-d incubation in DM, followed by 5-d incubation in AM and subsequent incubation in DM. Incubation code B is related to initial incubation in AM, followed by incubation in DM and, subsequently, in AM. A431 cells served as positive controls. The blots are representative for three separate experiments.

Figure 6 CK 18 and 19 expression, several time points after cell plating. A: Kinetic part I is related to initial 5-d incubation in DM, followed by 5-d incubation in AM and subsequent incubation in DM; B: Kinetic part II is related to the incubation order AM-DM-AM. HC were seeded on a 2D collagen matrix and incubated alternately in AM or DM. CK level was detected by flow cytometry and expressed as RFU. mean±SD (n = 6); ^aP<0.05 between groups.

Figure 7 Western blot analysis of CK 18 protein content. HC were incubated alternately in AM or DM. Medium was changed on d 5 and 10. Code A indicates initial 5-d incubation in DM, followed by 5-d incubation in AM and subsequent incubation in DM. Code B is related to initial incubation in AM, followed by incubation in DM and, subsequently, in AM. β-actin served as the internal control. The blot is representative for three separate experiments.

Figure 8 Dynamics of EGFR distribution depending on modulation of the culture environment. A: Receptor localization along the plasma membrane after switching the extracellular milieu from de-differentiation (activation) to differentiation inducing conditions (d 10); B: The reverse order (DM-AM) evoked a distinct receptor translocation into the cytoplasm (d 10); C: Receptor re-localization onto the cell surface was induced again after a further 10-d incubation in DM (d 20). Confocal microscopy, ×100/1.3 oil immersion objective. Each figure is representative for three separate experiments.

Figure 9 Western blot analysis of milieu dependent shift of MET phosphorylation (MET_pho). Environmental switch has been carried out on d 5 and 10. Left lanes indicate HC cultivation within a 3D collagen matrix (sandwich), right lanes confer to a 2D culture system (collagen matrix). Incubation code A indicates initial 5-d incubation in DM, followed by 5-d incubation in AM and subsequent incubation in DM. Incubation code B is related to initial incubation in AM, followed by incubation in DM and subsequent incubation in AM. The blots are representative of three separate experiments.