Fig. 6
Tumorigenicity associated with iHepL cells. a Hematoxylin and eosin (H&E) staining of a liver section showing a cell mass after hepatic progenitor-like (iHepL) cell transplantation in the spleen. Tumor cells (black arrow) are less eosinophilic and the nuclei are abnormal in size and shape when compared to hepatocytes (green arrow). b Cells within the tumor that do not express green fluorescent protein (GFP) proliferated intensely and lost the characteristic E-cadherin staining. A white dotted line marking the borders of the cell mass is displayed. Co-labeling of GFP and phospho-Histone H3 (pH3) is not observed. c iHepL cells (1.5 × 106/injection) in 150 μl DPBS were implanted subcutaneously in the right flank region of mice (n = 5). Upon sacrifice, primary tumors were removed, formalin-fixed, and histologically evaluated by H&E staining and immunostaining with antibodies against pH3, Ki67, and Hnf4. Nuclei were counterstained with DAPI (blue). d Relative gene copy number analysis. Genomic DNA isolated from iHepL cells in culture, embedded tumors, and embedded control liver mouse tissue was analyzed by qPCR using primers specific for intron 1 and intron 2 of Ccnd1 gene, GFP, and fragments corresponding to lentiviral vectors pMIGR1-Hnf1a (HNF1a exo), pMIGR1-Hnf6a (Hnf6a exo), pMIGR1-Hhex (Hhex exo), pMSx-Oct4 (Oct4 exo), pMXs-Sox2 (Sox2 exo), pMXs-Klf4 (Klf 4 exo), and pMXs-cMyc (cMyc exo). Data were normalized to the level of Intron 1 of Ccnd1 gene; a fragment of intron 2 from Ccnd1 gene was used as a secondary normalization control. Then values for each sample were normalized to the value for iHepL cells. *p < 0.05; **p < 0.005 (compared to iHepL cells)