Volume 25, Issue 6, Pages 1420-1433 (June 2017) Human ESC/iPSC-Derived Hepatocyte-like Cells Achieve Zone-Specific Hepatic Properties by Modulation of WNT Signaling  Seiji Mitani, Kazuo Takayama, Yasuhito Nagamoto, Kazuo Imagawa, Fuminori Sakurai, Masashi Tachibana, Ryo Sumazaki, Hiroyuki Mizuguchi  Molecular Therapy  Volume 25, Issue 6, Pages 1420-1433 (June 2017) DOI: 10.1016/j.ymthe.2017.04.006 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 HLCs Were Cultured with Various Forms of Cell Type-CM (A) The procedure for differentiation of human iPSCs (Dotcom) into HLCs via definitive endoderm cells and hepatoblast-like cells is presented schematically. Details on the hepatic differentiation procedure are described in the Materials and Methods. HLCs were cultured with various forms of cell type-CM (HepG2 cells, PHHs, iPS-HLCs, HuCCT1 cells, HUVECs, TMNK-1 cells, LX-2 cells, or Swiss3T3 cells) for 5 days. (B) Gene expression levels of CYP3A4, CYP7A1, and UGT1A1 in HLCs cultured with the indicated CM were examined by real-time RT-PCR. (C) Gene expression levels of CYP1A2, CYP2C9, CYP3A4, CYP7A1, MDR1, MRP2, GSTA1, and UGT1A1 in HLCs were examined by real-time RT-PCR. (D) CYP3A4 activity was measured in HLCs. (E) The efficiency of hepatocyte or cholangiocyte differentiation was measured by estimating the percentage of ASGR1- or CK19+ cells using FACS analysis, respectively. In real-time RT-PCR analysis and CYP3A4 activity measurement, levels in the HLCs cultured with fresh medium were taken as 100. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (compared with fresh medium). See also Figures S1 and S2. Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 HLCs Were Converted into Zone 1 HLCs by Culturing with Cholangiocyte-CM (A) Human iPSC (Dotcom)-derived HLCs were cultured with HuCCT1-CM, HepG2-CM, or PHH-CM for 5 days, and zone-specific characteristics were examined. (B and C) Urea production capacity (B) and urea production-related genes (CPS1 and ARG1) (C) in HLCs were examined. (D) CPS1 protein expression levels (green) were examined by immunohistochemical analysis. Nuclei were counterstained with DAPI (blue). Scale bars represent 50 μm. (E and F) Gluconeogenesis capacity (E) and gluconeogenesis-related genes (PCK1 and G6PC) (F) in HLCs were examined. In real-time RT-PCR analysis, expression levels in HLCs cultured with fresh medium were taken as 100. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (compared with fresh medium). See also Figures S3 and S4. Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 HLCs Were Converted into Zone 3 HLCs by Culturing with Hepatocyte-CM (A) Human iPSC (Dotcom)-derived HLCs were cultured with HuCCT1-CM, HepG2-CM, or PHH-CM for 5 days, and zone-specific characteristics were examined. (B and C) Glutamine production capacity (B) and glutamine production-related genes (GS and GLT1) (C) in HLCs were examined. (D) Protein expression levels of GS (green) were examined by immunohitochemical analysis. Nuclei were counterstained with DAPI (blue). Scale bars represent 50 μm. (E–G) CYP1A2 activity (E), CYP1A2 induction potency (F), and gene expression levels of CYP1A2 and AhR (G) in HLCs were examined. In real-time RT-PCR analysis, expression levels in HLCs cultured with fresh medium were taken as 100. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (compared with fresh medium). See also Figures S3 and S4. Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 The WNT Signaling Pathway in HLCs Was Activated or Inactivated by Culturing with Hepatocyte- or Cholangiocyte-CM, Respectively Human iPSC (Dotcom)-derived HLCs were cultured with HuCCT1-CM, HepG2-CM, or PHH-CM. (A) Protein expression levels of β-catenin in HLCs were examined by western blotting. Quantitative analysis of western blotting was performed by using ImageJ software (NIH). (B) Gene expression levels of WNT target genes (AXIN2 and Cyclin D1) in HLCs were examined by real-time RT-PCR. Expression levels of each gene in HLCs cultured with fresh medium were taken as 100. (C) Gene expression levels of canonical WNT ligands (WNT3A, WNT7A, WNT8A, and WNT8B) and WNT inhibitors (DKK1, DKK4, and WIF-1) in HuCCT1 cells, human iPS-derived cholangiocyte-like cells (sorted Aquaporin 1 [AQP1]+ cells), non-purified HLCs (a mixture of ASGR1+ cells and CK19+ cells; BULK), purified HLCs (sorted ASGR1+ cells), HepG2 cells, and PHHs were examined by real-time RT-PCR. All data are presented as means ± SE (n = 3). **p < 0.01 (compared with fresh medium). (D) Human liver tissues were subjected to immunostaining with anti-WIF-1 (green), anti-WNT7B (green), and anti-WNT8B (green) antibodies. Nuclei were counterstained with DAPI (blue). Scale bars represent 100 μm. PV, portal vein. *p < 0.05 (compared with fresh medium). See also Figure S5. Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 HLCs Were Converted into Zone 3 HLCs by Culturing with Hepatocyte-CM through WNT7B and WNT8B (A) HepG2 cells were transfected with si-control, si-WNT7B, si-WNT8B, or si-WNT7B plus siWNT8B. At 2 days after siRNA transfection, transfected cells were used for the preparation of CM. Human iPSC (Dotcom)-derived HLCs were cultured with si-control-, si-WNT7B-, si-WNT8B-, or si-WNT7B plus si-WNT8B-transfected HepG2 cell-CM (si-control-HepG2-CM, si-WNT7B-HepG2-CM, si-WNT8B-HepG2-CM, or si-WNT7B,WNT8B-HepG2-CM, respectively) for 5 days, and zone-specific characteristics were examined. (B and C) Urea production capacity (B) and urea production-related genes (CPS1 and ARG1) (C) in HLCs were examined. (D and E) Gluconeogenesis capacity (D) and gluconeogenesis-related genes (PCK1 and G6PC) (E) in HLCs were examined. (F and G) Glutamine production capacity (F) and glutamine production-related genes (GS and GLT1) (G) in HLCs were examined. (H, I, and J) The CYP1A2 activity (H), CYP1A2 induction potency (I), and gene expression levels of CYP1A2 and AhR (J) in HLCs were examined. In real-time RT-PCR analysis, expression levels in HLCs cultured with fresh medium were taken as 100. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (compared with si-control-HepG2-CM). See also Figure S6. Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 6 HLCs Were Converted into Zone 3 HLCs by WNT7B Transduction (A) Human iPSCs (Dotcom) were differentiated into HLCs and then transduced with 2,000 VP/cell of LacZ- or WNT7B-expressing Ad vectors for 1.5 hr. After transduction, HLCs were cultured with fresh medium for 5 days. At day 30 of differentiation, the zone-specific characteristics were examined. (B and C) Urea production capacity (B) and urea production-related genes (CPS1 and ARG1) (C) in HLCs were examined. (D and E) Glutamine production capacity (D) and glutamine production-related genes (GS and GLT1) (E) in HLCs were examined. In real-time RT-PCR analysis, expression levels in HLCs cultured with fresh medium were taken as 100. (F and G) HLCs were transduced with various amounts of WNT7B-expressing Ad vector for 1.5 hr. After transduction, HLCs were cultured with fresh medium for 5 days. At day 30 of differentiation, urea (F) and glutamine (G) production capacities were examined. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (fresh medium versus HepG2-CM, PHH-CM; Ad-LacZ versus Ad-WNT7B). Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 7 HLCs Were Converted into Zone 1 HLCs by Culturing with Cholangiocyte-CM through WIF-1 (A) HuCCT1 cells were transfected with si-control or si-WIF-1. At 2 days after siRNA transfection, the transfected cells were used for the preparation of CM. Human iPSC (Dotcom)-derived HLCs were cultured with si-control- or si-WIF-1-transfected HuCCT1 cell-CM (si-control-HuCCT1-CM or si-WIF-1-HuCCT1-CM, respectively) for 5 days, and zone-specific characteristics were examined. (B and C) Urea production capacity (B) and urea production-related genes (CPS1 and ARG1) (C) in HLCs were examined. (D and E) Gluconeogenesis capacity (D) and gluconeogenesis-related genes (PCK1 and G6PC) (E) in HLCs were examined. (F and G) Glutamine production capacity (F) and glutamine production-related genes (GS and GLT1) (G) in HLCs were examined. (H, I, and J) The CYP1A2 activity (H), CYP1A2 induction potency (I), and gene expression levels of CYP1A2 and AhR (J) in the HLCs were examined. In real-time RT-PCR analysis, expression levels in HLCs cultured with fresh medium were taken as 100. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (compared with si-control-HuCCT1-CM). See also Figure S6. Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 8 HLCs Were Converted into Zone 1 HLCs by WIF-1 Transduction (A) Human iPSCs (Dotcom) were differentiated into HLCs and then transduced with 2,000 VP/cell of LacZ- or WIF-1-expressing Ad vectors for 1.5 hr. After transduction, HLCs were cultured with fresh medium for 5 days. At day 30 of differentiation, the zone-specific characteristics were examined. (B and C) Urea production capacity (B) and urea production-related genes (CPS1 and ARG1) (C) in HLCs were examined. (D and E) Glutamine production capacity (D) and glutamine production-related genes (GS and GLT1) (E) in HLCs were examined. In real-time RT-PCR analysis, expression levels in the HLCs cultured with fresh medium were taken as 100. (F and G) HLCs were transduced with various amounts of WIF-1-expressing Ad vector for 1.5 hr. After transduction, HLCs were cultured with fresh medium for 5 days. At day 30 of differentiation, urea (F) and glutamine (G) production capacities were examined. All data are presented as means ± SE (n = 3). *p < 0.05; **p < 0.01 (fresh medium versus HuCCT1-CM; Ad-LacZ versus Ad-WIF-1). Molecular Therapy 2017 25, 1420-1433DOI: (10.1016/j.ymthe.2017.04.006) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions