Volume 142, Issue 3, Pages 602-611 (March 2012) Direct and Indirect Contribution of Human Embryonic Stem Cell–Derived Hepatocyte- Like Cells to Liver Repair in Mice  Dong–Hun Woo, Suel–Kee Kim, Hee–Joung Lim, Jeonghoon Heo, Hyung Soon Park, Gum–Yong Kang, Sung–Eun Kim, Hyun–Ju You, Daniel J. Hoeppner, Youngchul Kim, Heechung Kwon, Tae Hyun Choi, Joo Hee Lee, Su Hee Hong, Kang Won Song, Eun–Kyung Ahn, Josh G. Chenoweth, Paul J. Tesar, Ronald D.G. McKay, Jong–Hoon Kim  Gastroenterology  Volume 142, Issue 3, Pages 602-611 (March 2012) DOI: 10.1053/j.gastro.2011.11.030 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Derivation and functional characterization of purified HL cells. (A) Propagation of polygonal cells from lithium-treated EBs attached on collagen I–coated plates. Higher magnification of cells migrated from EBs (upper right panel). Fluorescence micrograph of polygonal cells coexpressing albumin and keratin 18 (lower right panel). (B) Proportions of multiple human ES and iPS cell line–derived cells expressing both albumin and keratin 18 after differentiation of lithium-treated EBs in the absence (Cont.) or presence of growth factors (OD = OSM + DEX; HD = HGF + DEX; HOD = HGF + OSM + DEX). * P < .01 vs control. (C) γ-Glutamyl transpeptidase (left panel) and periodic acid–Schiff staining (right panel) of differentiated cells. Insets show higher magnification of cells. (D) Levels of urea secretion measured at various time points throughout the differentiation period. (E) Levels of secreted albumin in medium from human ES cells, ICGlow cells, and ICGhigh cells. HepG2 and primary human hepatocytes were used as positive controls. * P < .05 versus ICGlow cells. (F) Quantitative polymerase chain reaction analysis of albumin and GSTA1 and 2 messenger RNA in ICGlow and ICGhigh cells. Scale bars = 50 μm. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Engraftment and function of ICGhigh cells in injured liver parenchyma. (A) Schematic representation of the experimental design. (B) In vivo bioluminescence imaging after intrasplenic injection of ICGlow or ICGhigh cells. (C) Immunohistochemical staining of liver sections with anti-human hepatocytes (Hep Par 1) and anti-human albumin antibodies at day 3 after transplant of ICGhigh cells. (D) Thirty-five days after transplant, liver sections of ICGhigh cell-grafted mice were stained with anti-human albumin antibody. A higher magnification image of the boxed area is shown on the right. (E) Western blot detection of human albumin in blood serum of mice at day 35 after transplant. (F) Human albumin levels in the blood of mice at different time points over the entire 35-day period of transplantation. Scale bars = 100 μm. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Host liver regeneration after transplant of ICGhigh cells. (A) Relative ratio of increase in total albumin level is expressed as fold increases over the sham operation 35 days after transplant. Red bars reflect the ratio of increased albumin level by host liver regeneration, and blue bars indicate the contribution of human albumin. * P < .05 vs sham-operated mice. (B) BrdU labeling of ICGlow and ICGhigh cell-grafted tissues at day 2 after transplant. (C) Immunohistochemical double staining of liver sections with anti-mouse albumin (msALB) and anti-BrdU antibodies after transplant of ICGhigh cells. (D) Immunofluorescent labeling of liver sections with anti-human and anti-mouse PECAM antibodies (huPECAM and msPECAM). White dotted lines indicate the area of central veins. Scale bars = (B) 100 μm, (C and D) 50 μm. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Host liver regeneration after injection of ICGhigh cell secretome. (A) Schematic representation of the experimental design. (B) Hematoxylin staining of liver sections after 3 days of secretome (Scrt) administration. Sham-operated mice received an equal volume of medium. (C) BrdU labeling of liver tissues after 3 days of secretome administration. (D) Immunohistochemical staining of liver sections with anti-mouse PECAM antibody after injection of secretome. Scale bars = 100 μm. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Quantitative comparison of liver repair after secretome injection and cell transplantation. (A) Quantification of host cell proliferation by counting BrdU-positive cells after secretome injection and cell transplantation. In the case of cell transplantation, only mouse albumin/BrdU double-positive cells were counted. (B) Quantification of revascularization by measuring the area of each PECAM-immunoreactive cell. (C) Serum alanine aminotransferase levels 3 days after cell grafting or secretome injection. * P < .05 vs sham, +P < .05 vs ICGhigh cells, #P < .05 vs ICGhigh cell-Scrt. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Wound healing responses of the injured liver tissue after administration of ICGhigh cell secretome. (A and B) Immunohistochemical staining of liver sections with (A) anti-desmin and (B) anti-F4/80 antibody after 3 days of secretome administration. (C) Fibrin(ogen) deposition in injured liver at day 3 after indicated treatments. (D–F) Immunoreactive areas for (D) desmin and (E) F4/80 and (F) areas of fibrin deposition are expressed as a percentage of the total image area. * P < .01 vs sham. Scale bars = 100 μm. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Schematic representation of the hypothetical role of human ES cell–derived grafts in endogenous tissue regeneration and cell replacement in an acute model of liver injury. Gastroenterology 2012 142, 602-611DOI: (10.1053/j.gastro.2011.11.030) Copyright © 2012 AGA Institute Terms and Conditions