Figure 8

DCLK1-expressing primary human hepatocytes shows activation of β-catenin in humanized Fah^−/−/Rag2^−/−/Il2rg^−/− (FRG) mouse livers. (a) Scheme of NTBC cycle and hepatocyte transplantation into the FRG mice. (b) RFP or RFP-DCLK1-expressing primary human hepatocytes (1 million each) transplanted into the spleen of FRG mice after removing hair (red arrow, clean area). The mice were imaged live for RFP expression 10 days post-transplantation with IVIS Spectrum Imager. Control, FRG mice at ± NTBC water cycles but without transplantation (left panel); images of a representative transplanted FRG mouse are shown in each group. Red to yellow scale indicates increase in epi-fluorescence intensities. (c) Immunohistochemistry of human-mouse chimeric FRG livers were carried out for human albumin (hAlb, ab2406) (lower panel, brown) and DCLK1 (ab109029) (upper panel, brown) 6 weeks post-transplantation. Control, FRG mice treated at ± NTBC water cycles similar to other mice but without transplantation of hepatocytes (left panel). Blue, nuclear stain. (d) Co-staining of active β-catenin (red) and DCLK1 (brown) in the chimeric (RFP and RFP-DCLK1) and control (untransplanted) FRG livers. Lower panel, hepatocytes highlighted for nuclear and cytoplasmic expressions of active β-catenin (red) in DCLK1 + cells. The controls (RFP-transplanted and untransplanted FRG livers) lack this co-expression pattern. (e) Distinct proliferation of RFP-DCLK1 + human hepatocytes in the humanized FRG livers confirmed by DCLK1 and hAlb expression (left pane). H&E staining, left panel. (f) Schematic presentation of DCLK1 signaling-mediated activation of the short β-catenin (48-kDa), which contributes to hepatocytes plasticity and neoplastic growth.