Volume 64, Issue 6, Pages 1315-1326 (June 2016) Stage-specific regulation of the WNT/β-catenin pathway enhances differentiation of hESCs into hepatocytes  Thomas Touboul, Shujuan Chen, Cuong C. To, Sergio Mora-Castilla, Karen Sabatini, Robert H. Tukey, Louise C. Laurent  Journal of Hepatology  Volume 64, Issue 6, Pages 1315-1326 (June 2016) DOI: 10.1016/j.jhep.2016.02.028 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2016 64, 1315-1326DOI: (10. 1016/j. jhep. 2016 Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Schematic representation of the differentiation protocols used to differentiate hESCs into fetal hepatocytes. (A) the method developed in the current study. (B) the previously developed protocol per Touboul et al. LY: Ly 294002; SB: SB 431542; CHIR: CHIR99021; CE: Compound E; RA: Retinoic Acid. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Establishment of stage 2 differentiation conditions: Differentiation of DE to posterior foregut. (A) qRT-PCR analysis of gene expression of SOX17, SOX2, HHEX, and HNF4 in undifferentiated hESCs, DE, and DE treated for 3days in: media without additives (Ø) or in presence of increasing doses of Activin A, Noggin and/or IWR-1. Gene expression levels were normalized to DE. (B) Immunocytochemistry showing expression of the PFG markers HNF4, SOX17, HNF1β after 3days of treatment of DE cells with IWR-1. (C) qRT-PCR analysis shows expression of HNF4 and HHEX in hESCs, DE cells were treated with IWR-1 or combination of IWR-1/NOG. Gene expression levels were normalized to DE. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Establishment of stage 3 differentiation conditions: Induction of hepatic gut from PFG cells. (A) Effect of BMP4, IWR-1, RA and SB on hepatic commitment of PFG cells. PFG cells were exposed for 4days to various combinations of BMP4, IWR-1, RA and SB. Hepatic and non-hepatic gene expression in the different conditions and in undifferentiated hESCs was quantified by qRT-PCR analysis and normalized to stage 2 IWR-1-treated cells (St 2). (B) Generation of hepatic gut by exposure of stage 2 cells to BMP4/SB/IWR-1 for 4days was confirmed by immunocytochemistry. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Establishment of stage 4 differentiation conditions: Generation of hepatoblasts through activation of WNT/β-catenin pathway. (A) Establishment of conditions for differentiation to hepatoblast. Hepatic gut cells were grown for 4days in presence of different combinations of HGF, VEGF, TGF-β, and CHIR. Gene expression of hepatic and cholangiocytes markers were analyzed by qRT-PCR and normalized to stage 3 cells. (B) Expression of proliferative hepatoblasts markers was detected by immunocytochemistry after treatment with CHIR/TGF-β/HGF/VEGF for 4days. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Establishment of stage 5 differentiation conditions: Differentiation of hepatoblasts into hepatocytes. (A) Hepatoblasts were exposed for 4days to HGF in combination with oncostatin m (OSM), CE, and SB. qRT-PCR analysis of hepatic (ALB, AFP, AAT, and CEBPA) and cholangiocyte (SOX9) gene expression. Gene expression data were normalized to stage 4. (B) ELISA analysis showing secretion of albumin into the culture medium by hESCs-derived hepatocytes (compared to undifferentiated hESCs). Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Importance of the hepatoblast stage for later cell maturation. (A) Effect of WNT/β-catenin activation on the generation of hepatoblasts. FACS analysis performed on stage 3 cells and on stage 4 cells (stage 3 cells exposed for 4days to: HGF/TGF-β/SB/CHIR; HGF/TGF-β/SB/IWR-1; HGF/CE/SB). Each column represents the average of 3 independent biological replicates. ∗p<0.02; ∗∗p<0.03. (B) and (C) qRT-PCR analysis of hESCs-derived hepatocytes generated by culturing stage 3 (St3→ St5) or stage 4 (St3→ St4→ St5) for 14days in HGF/CE/SB. Also represented gene expressions of human fetal and adult hepatocytes respectively abbreviated HFH and HAH. Gene expression was normalized to freshly isolated human adult hepatocytes (HAH) (B) Gene expression analysis of the common hepatic markers ALB and AFP in hESC-derived hepatocytes. (C) Expression of the transcripts for the nuclear receptors AhR and CAR, CYP family members in hESC-derived fetal hepatocytes. ∗p<10−3; ∗∗p<2×10−2; ∗∗∗p<10−4; ∗∗∗∗p<10−2, ∗∗∗∗∗p<10−8, ∗∗∗∗∗∗p<10−6. (D) Immunocytochemistry for hepatic markers in the hESC-derived hepatocytes generated using the 5-stage protocol. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Comparison of stage 5 hepatocyte-like cells with fetal and adult liver. Transcripts that are differentially expressed between undifferentiated hESC and human fetal liver and/or undifferentiated hESC and human adult liver are shown (t test, adjusted p value<0.00001, maximum number of counts across all samples>20, absolute fold-change between groups>4). The differentially expressed transcripts were clustered into co-expressed groups using CLICK. Results are shown in the heatmap on the left, as well as the mean expression graphs on the right. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions

Fig. 8 Functionality of hESC-derived hepatocytes generated using the 5-stage protocol. (A) Activity of CYP family members CYP3A, CYP1A1, and CYP2B6 in untreated hepatocytes-like cells (HLC) and after exposure to RIF, TCDD and PB. Human fetal hepatocytes (HFH) and freshly isolated human adult hepatocytes (HAH) were used as positive controls. The values are indicated as relative light unit (RLU) per million cells and represent the average of 3 independent biological replicates. ∗p<10−5; ∗∗p<10−8; ∗∗∗p<10−11; ∗∗∗∗p<10−3; ∗∗∗∗∗p<10−7. (B) Production of urea in culture medium by the hESC-derived hepatocytes generated using the 5-stage protocol (HLC) at 24 and 48h. HAH and HFH were used as positive controls. The values represent the average of 3 independent biological replicates. (C) Oil Red O (ORO) staining and BODIPY 493/503 show storage of lipids by HLCs. Periodic acid-Schiff (PAS) staining show glycogen storage by the HLCs. Uptake of LDL shown by LDL-Dylight staining. Journal of Hepatology 2016 64, 1315-1326DOI: (10.1016/j.jhep.2016.02.028) Copyright © 2016 European Association for the Study of the Liver Terms and Conditions