Fig. 3

Characterization of puromycin-selected cells. A Karyotypic analysis of puromycin-selected cells at passage 10. B Histological analysis of puromycin-selected cells embedded in iPGell. The puromycin-selected cells exhibited hepatocyte-like morphology. HE stain. C, D Ultrastructural analysis. C: Desmosome (arrow), D: Microvilli (arrow). E–L Immunocytochemistry of the puromycin-selected cells with the antibodies to AE1/AE3 (E, F), E-cadherin (G, H), PCNA (I, J), and Ki67 (K, L). Panel F, H, J, and L are high-power views of Panel E, G, I, and K, respectively. M Quantitative RT-PCR analysis of the CYP1A2 gene on the puromycin-selected immortalized cells with exposure to 50 μM omeprazole (OME) for 24 h. mRNA levels were normalized using ubiquitin expression as a housekeeping gene. Statistical analysis was performed using the unpaired two-tailed Student's t-test. **P < 0.01. N Quantitative RT-PCR analysis of the CYP2B6 gene on the puromycin-selected immortalized cells with exposure to 500 μM phenobarbital (PB) for 48 h. Statistical analysis was performed using the unpaired two-tailed Student's t-test. **P < 0.01. O Quantitative RT-PCR analysis of the CYP3A4 gene on the puromycin-selected immortalized cells with exposure to 20 μM rifampicin (RFP) for 48 h. Statistical analysis was performed using the unpaired two-tailed Student's t-test. **P < 0.01. P Induction of CYP3A4 enzymatic activity in the puromycin-selected cells and HepG2 cells exposed to 20 μM rifampicin (RFP) for 48 h. Q Growth curve of the puromycin-selected immortalized cells. R Principal component analysis of gene expression on lymphangioma-derived cell (1: Lym1915-NC, 2: Lym-0 h, 3: Lym-12 h, 4: Lym-24 h, 5: Lym-72 h), pluripotent stem cells (6: SEES5P37 ESC, 7: Edom iPSCs, 8: G72-1 iPSCs, 9: G72-1Shh iPSC, 10: G72-1Vis iPSC, 11: pHAES5-1 ESC) [26,27,28,29,30,31,32], HepaSM cells (12: HepaSM), and hepatocytes (13: Saeko_hP3, 14: Saeko_hP10, 15: Ruri_hP4, 16: Ruri_hP10)