1. Volume 64, Issue 5, Pages 1068-1075 (May 2016)
Transplantation of a human iPSC-derived hepatocyte sheet increases survival in mice with acute liver failure 
Yasuhito Nagamoto, Kazuo Takayama, Kazuo Ohashi, Ryota Okamoto, Fuminori Sakurai, Masashi Tachibana, Kenji Kawabata, Hiroyuki Mizuguchi 
Journal of Hepatology 
Volume 64, Issue 5, Pages (May 2016)
DOI: /j.jhep
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

2. Journal of Hepatology 2016 64, 1068-1075DOI: (10. 1016/j. jhep. 2016
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

3. Fig. 1
Experimental protocol for human iPS-HLC sheet transplantation onto the recipient mouse liver surface. (A) The procedure for differentiation of human iPS cells (Dotcom) into HLC via mesendoderm cells, definitive endoderm cells, and hepatoblast-like cells. In the hepatic differentiation, not only the addition of growth factors but also stage-specific transient transduction of both FOXA2- and HNF1α-expressing Ad vectors (Ad-FOXA2 and Ad-HNF1α, respectively) was performed. Further details of the iPS-HLC differentiation procedure are described in the Supplementary materials and methods. The human iPS-HLCs were seeded on a temperature-responsive culture dish (TRCD) on day 35. Two days after seeding, human iPS-HLC sheets were harvested as a contiguous cell sheet. (B–E) The gene expression levels of αAT (B), ALB (C), CYP3A4 (D), and CYP1A2 (E) in human iPS-HLCs were measured by real-time RT-PCR. On the y axis, the gene expression levels in primary human hepatocytes (PHHs; three lots of PHHs were used), which were cultured for 48h after plating (PHH48hr), were taken as 1.0. (n=3 each) (F) The efficiency of hepatocyte differentiation was measured by estimating the percentage of ALB- (open) and ASGR1-positive cells (closed) using FACS analysis (n=3 each). (G) Phase-contrast micrographs of the vertical sections of a human iPS-HLC sheet. (H, I) Immunofluorescence staining of the hepatocyte markers (CK18: green (H); αAT: red (I)) in the human iPS-HLC sheet. Nuclei were counterstained with DAPI (blue). (J) The human iPS-HLC sheet-harvesting steps and transplantation procedures. The human iPS-HLC sheets were harvested in a contiguous cell sheet using a CellShifter support. The harvested human iPS-HLC sheet was placed onto the liver surface of the recipient mouse, and then the CellShifter was removed while leaving the human iPS-HLC sheet on the liver surface of the recipient mice. These operations were repeated. (K) An image of the human iPS-HLC sheets transplanted on the liver surface with the CellShifter. The scale bars represent 40μm.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

4. Fig. 2
Distribution of the transplanted human iPS-HLCs in mice. Human iPS cells (Dotcom) were differentiated into the iPS-HLCs as described in Fig. 1A, and then passaged onto a TRCD to generate the human iPS-HLC sheet. The human iPS-HLC sheet or iPS-HLCs in suspension were transplanted into the two-thirds partially hepatectomized mice (PHx mice). The distribution of transplanted human iPS-HLCs in the recipient mice was examined by semi-quantitative PCR. One day after transplantation, the liver, spleen, stomach, small intestine, large intestine, brain, heart, lung, kidney, and pancreas were harvested and genomic DNAs were extracted. The genomic DNAs of each group (human iPS-HLC sheet-transplanted mice: sheet-mice; human iPS-HLC intrasplenic transplanted mice: intrasplenic-mice; sham-operated mice: sham-mice; and human iPS-HLC (before transplantation)) were pooled, respectively. Amplified DNA fragments of the human ALU sequences are shown in the upper panel (h), and amplified DNA fragments of the mouse c-mos are shown in the lower panel (m). The genome of human iPS-HLC before transplantation was used as a control. (sheet-mice: n=5; others: n=3).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

5. Fig. 3
Engraftment efficiency of the transplanted human iPS-HLCs. Human iPS cells (Dotcom) were differentiated into the iPS-HLCs as described in Fig. 1A, and then passaged onto a TRCD to generate the human iPS-HLC sheet. The human iPS-HLC sheet or iPS-HLCs in suspension were transplanted into the two-thirds partially hepatectomized mice (PHx mice). (A) One day before transplantation, the human iPS-HLCs were transduced with luciferase-expressing Ad vector. One day after transplantation, the luciferase expression in the livers of recipient mice, which were subjected to human iPS-HLC sheet transplantation (sheet-mice), human iPS-HLC intrasplenic transplantation (intrasplenic-mice), or sham operation (sham-mice), was measured (n=4 each). (B) Two weeks after transplantation, serum human ALB levels of the recipients were measured (sheet-mice: n=11; intrasplenic-mice: n=10; sham-mice; n=9). (C) Two weeks after transplantation, the liver that received human iPS-HLC sheet transplantation was observed macroscopically. The dotted area indicates the transplanted human iPS-HLC sheet. (D) Two weeks after transplantation, the livers of sheet-mice were analyzed by H&E staining. (E–I) Two weeks after transplantation, the livers of sheet-mice were analyzed by immunohistochemical staining. Frozen sections of these livers were stained with anti-human ALB (F and G), human αAT (E, G, H, and I), and mouse CD31 (H) -antibodies and Dylight 594-labeled L. esculentum lectin (I). Nuclei were counterstained with DAPI (blue). ∗p<0.05. The scale bars represent 40μm (D-I).
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

6. Fig. 4
Therapeutic effects of human iPS-HLC sheet transplantation on CCl4-induced acute liver injury in mice. Human iPS cells (Dotcom) were differentiated into the iPS-HLCs as described in Fig. 1A, and then passaged onto a TRCD to generate the human iPS-HLC sheet. (A) The human iPS-HLC sheet and iPS-HLCs in suspension were transplanted into mice that had been intraperitoneally inoculated with 3ml/kg carbon tetrachloride (CCl4) 1day before transplantation. The survival rate of the mice after human iPS-HLC sheet transplantation (sheet-mice, circle; n=19), human iPS-HLC intrasplenic transplantation (intrasplenic-mice, triangle; n=15), and sham operation (sham-mice, square; n=18) was examined. (B-D) One day after transplantation (2days after the CCl4 inoculation), serum ALB (B), AST (C), and ALT levels (D) of the recipients were measured (sheet-mice: n=5; intrasplenic-mice: n=7; sham-mice; n=6). ∗p<0.05. sheet-mice vs. intrasplenic-mice: p=0.037; sheet-mice vs. sham-mice: p=0.002; intrasplenic-mice vs. sham-mice: p=0.381.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

7. Fig. 5
Therapeutic effects of human iPS-HLC sheet transplantation via HGF. Human iPS cells (Dotcom) were differentiated into the iPS-HLCs as described in Fig. 1A. (A) The gene expression levels of HGF in human iPS cells (iPSCs), human iPS-HLCs, human bone marrow mesenchymal stromal cells (BM-MSCs), and PHH were measured by real-time RT-PCR. On the y axis, the gene expression level in BM-MSCs was taken as 1.0. n=3 (B) The human iPS-HLCs were passaged onto a TRCD to generate the human iPS-HLC sheet. The human iPS-HLC sheet was transplanted into mice that had been intraperitoneally inoculated with 3ml/kg carbon tetrachloride (CCl4). At 36h after transplantation, the livers of sheet-mice were analyzed by immunohistochemical staining using anti-HLA Class 1 ABC and anti-human HGF antibodies. The scale bars represent 40μm. (C) The human iPS-HLC sheet, which was transfected with si-control or si-HGF, was transplanted into the mice that had been intraperitoneally inoculated with 3ml/kg CCl4 1day before transplantation. The survival rate of the mice after transplantation with the human iPS-HLC sheet transfected with si-control (si-control, circle; n=14), human iPS-HLC sheet transfected with si-HGF (si-HGF, triangle; n=15), or sham operation (sham-mice, square; n=15) was examined. si-control vs. si-HGF: p=0.039; si-control vs. sham-mice: p=0.002; si-HGF vs. sham-mice: p=0.359.
Journal of Hepatology  , DOI: ( /j.jhep )
Copyright © 2016 European Association for the Study of the Liver Terms and Conditions