Figure 1.

Differentiation of human induced pluripotent stem cells toward hepatocytes. (A–F): The cells were analyzed by flow cytometry for the percentage of positive cells for CXCR4 (A), SOX17 (B), and FOXA2 (C) at day 8 during induction of definitive endoderm; for AFP (D) at day 9 after differentiation; and for ALB (E) and α1-AT (F) at day 18 after differentiation. (G, N): Definitive endodermal cells were stained with the primary antibodies goat anti-SOX 17 (green) (I) and FOXA2 (red) (J) during induction of human induced pluripotent stem (iPS) cells to definitive endoderm; the differentiated cells were stained with the primary antibodies monoclonal antibody against AFP (red) (M) and goat anti-ALB (green) (N) during differentiation of human iPS cells toward hepatocytes. (G, H, K, L): Negative controls of SOX17 (G), FOXA2 (H), AFP (K), and ALB (L) stained with isotype antibodies. All immunohistochemistry analyses were merged with 4′,6′-diamidino-2-phenylindole nucleic acid staining (G–N). (O): Relative expression of ALB, α1-AT, TAT, and HNF4α in hiHs and hPHs determined by quantitative reverse transcription-polymerase chain reaction. (P, Q): Polymerase chain reaction was used to determine expression of the liver-associated genes G-6-P and TAT (P), and liver-associated transcriptional factors and BMP signaling (Q). Scale bars = 100 μm (G–N). Details of primers and antibodies can be found in the supplemental online tables. Abbreviations: α1-AT, α1-antitrypsin; AFP, α-fetoprotein; ALB, albumin; BMP, bone morphogenetic protein; G-6-P, glucose-6-phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hiH, human induced pluripotent stem cell-derived hepatocytes at day 25; hiH1, human induced pluripotent stem cell-derived hepatocytes at day 10; hPH, human primary hepatocyte; HNF4α, hepatocyte nuclear factor 4α; iPSC, induced pluripotent stem cell; TAT, tyrosine aminotransferase.