Fig 2: Up-regulation of the genes for ammonia metabolism via three-dimensional cultivation in the ammonia-selected hepatocyte-like cells. (A) Protocol for spheroid formation of ammonia-selected hepatocyte-like cells. (B) Phase-contrast photomicrograph of the ammonia-selected hepatocyte-like cells at 3, 6, and 9 days after spheroid formation. (C–G) qRT-PCR analysis of the genes for ALB (C), AFP (D), CPS1 (E), OTC (F) and CYP3A4 (G). Each expression level was calculated from the results of technical triplicate experiments. Average fold increase with standard deviation is shown. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. From left to right: Ammonia-selected hepatocyte-like cells at passage 12 without sphere formation, ammonia-selected hepatocyte-like cells at passage 12 with sphere formation (Day 7, Day 10), HepG2 cells, negative control (H2O).

Fig 3: Ammonia selection of iPSC-derived hepatocyte-like cells. (A) Scheme for ammonia selection of iPSC-derived hepatocyte-like cells. (B–D) Phase contrast photomicrograph of the hepatocyte-like cells generated from DILI-derived iPSC-O cells exposure to the indicated concentration of ammonia at day 0 (B), and with exposure to the indicated concentration of ammonia for 1 day (C) or 2 days (D). (E–I) qRT-PCR analysis of the genes for ALB (E), AFP (F), CSP1 (G), OTC (H) and CYP3A4 (I). Each expression level was calculated from the results of technical triplicate experiments. Average fold increase with standard deviation is shown. Statistical analysis was performed using the unpaired two-tailed Student's t test. ***P < 0.001. From left to right: Hepatocyte-like cells generated from DILI-derived iPSC-O cells without ammonia selection, ammonia-selected hepatocyte-like cells generated from DILI-derived iPSC-O cells, HepG2 cells, negative control (H2O).

Fig 4: Functional glycosylation of a-dystroglycan and characterization of dystroglycanopathy patient-specific iPSCs Current model of the core M3 functional glycan structure on a-dystroglycan and enzymes involved in its synthesis. ECM ligands, such as laminins, bind to the Xyl-GlucA disaccharide repeats (IIH6 epitope). Man, mannose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Rbo5P, ribitol-5-phosphate; Xyl, xylose; GlcA, glucuronic acid.Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60, and SSEA4.FKRP-iPSCs have a normal karyotype. In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (ß-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin), and endoderm (AFP, a-fetoprotein).Data information: Scale bars, 50 µm.

Fig 5: Generation of hepatocyte-like cells from SEES2 ESCs after exposure to ammonia. (A,B) Hepatocytes in liver that metabolize ammonia. (C) Scheme for characterization of ammonia-selected hepatocyte-like cells and ammonia metabolism. (D) Phase-contrast photomicroscopy of the hepatocyte-like cells at passage 3 during the selection with 0.02% ammonia. Panels shown in (D) are: Cells without treatment (before treatment), cells treated for 24 h (24 h), cells treated for 48 h (48 h) and passaged cells (after passage). Bar: 500 µm. (E) Growth curve of the ammonia-selected cells. (F) Immunocytochemistry of the ammonia-selected cells by using antibodies to CK7 and AFP. (G) Immunocytochemistry of the ammonia-selected cells using an antibody to CPS1. (H) Immunocytochemistry of the ammonia-selected cells using an antibody to CK7. (I) Immunocytochemistry of the ammonia-selected cells by using antibodies to CYP3A4 and ALB. (J) Immunocytochemistry of the ammonia-selected cells using an antibody to GLUL. (K) Immunocytochemistry of the ammonia-selected cells using an antibody to ALB. (L–P) qRT-PCR analysis of the genes for ALB (L), AFP (M), CPS1 (N), OTC (O) and CYP3A4 (P). Each expression level was calculated from the results of technical triplicate experiments. Average fold increase with standard deviation is shown. From left to right: ESC-derived hepatocyte-like cells without ammonia selection, ammonia-selected ESC-derived hepatocyte-like cells at passage 3–10. HepG2 cells, negative control (H2O), iPSC-derived immortalized cells (SM).