References Species Protocol specificity EB/monolayer Endoderm induction Hepatic specification and maturation In vitro functional assays In vivo assay Inducers Efficiency & marker Inducers Efficiency & marker     Cytokines only Gouon-Evans et al. [50] mESC   EB Act 55% Foxa2/ckit/ Cxcr4 BMP4, bFGF, HGF, Dex, TGFa, EGF, VEGF 70% Afp, 60% Alb Alb, Glycogen yes Cai et al. [25] hESC   monolayer Act, ITS 80% Foxa2/Sox17 FGF4, BMP2, HGF, OSM, Dex 70% Alb Alb, Glycogen, ICG, LDL, p450 yes Hay et al. [26] hESC   monolayer Act, Wnt3a   Serum, Insulin, HGF, OSM, up to 90% Alb Urea, Gluconeogenesis, Afp yes Agarwal et al. [40] hESC   monolayer Act, low serum 72% Cxcr4 FGF4, HGF 70%   yes Basma et al. [115] hESC   EB/monolayer Act, bFGF   FGF, DMSO, Dex 26% Asgr-1 Alb, Urea, AAT, P450 yes Si-Tayeb et al. [17] hiPSC   monolayer Act 80% BMP4 FGF2, HGF 80% Glycogen, LDL, oil red O storage, ICG, Urea yes Touboul et al. [27] hESC   monolayer Act, FGF2, BMP4, LY294002 80% Cxcr4 FGF10, RA, SB431542, FGF4, HGF, EGF 50-60% Hnf4a, Afp or Ck19, 35% Asgr1 Glycogen, p450, ICG, LDL yes Rashid et al. [31] hiPSC   EB Act   DMSO, HGF, Dex   p450, Alb, Afp, urea, LDL, ICG, Glycogen, oil red O storage   Mfopou et al. [35] hESC   monolayer Act, Wnt3a, low serum 60-80% Cxcr4/Foxa2 FGF10, BMP, Cyclo, DAPT, Exendin-4, IGF1, HGF 40-60% Afp Urea, Glycogen   Sullivan et al. [34] hiPSC   monolayer Act, Wnt3a   β-ME, DMSO, Insulin, HGF, OSM 70-90% Alb p450, Fibrinogen, Fibronectin, Transthyretin and Afp secretion   Rashid et al. [31] hiPSC   monolayer Act, FGF2, BMP4, LY294002   Act, HGF, OSM 83% Alb Alb, p450, Glycogen, LDL,   Liu et al. [41] hiPSC   monolayer Act, low serum 90% Cxcr4 FGF4, HGF, OSM, Dex 90% Afp Alb, p450 yes Li et al. [119] miPSC   monolayer Act, ITS   FGF4, HGF, OSM, Dex ~90% Afp, ~90% Alb LDL, Glycogen, P450 yes Woo et al. [39] hESC / hiPSC   EB LiCL, GSK-3 inhibitor (BIO), Wnt3a 35% Foxa2/Sox17 HGF, OSM, Dex ~69% Alb/CK18 Urea, Alb, Glycogen yes Pauwelyn et al. [37] mESC   monolayer Act, Wnt3a, low serum   FGF2, BMP4, FGF8b, FGF1, FGF4, Follistatin   Alb, Urea, Glycogen, p450   Sancho-Bru et al. [36] miPSC   monolayer Act, Wnt3a, low serum 70% Foxa2 BMP4, FGF2, FGF1, FGF4, FGF8, HGF, Follistatin 30% Hnf4a Alb, Glycogen, Urea, p450   He et al. [113] mESC   EB/monolayer Act 75% Cxcr4/cKit (monolayer) BMP4, bFGF, EGF, TGFα, VEGF, HGF, Dex 31% Alb (monolayer) Alb, Ammonia, Glycogen, LDL, ICG yes Chen et al. [38] hiPSC   monolayer Act, Wnt3a, HGF 61-64% OSM, Dex, ITS   p450, Urea, LDL, Glycogen yes Genetic Modification Kubo et al. [71] mESC Hex expression EB Act   Hex expression, BMP4   Alb, Transferrin   Inamura et al., 2011 [69] hESC / hiPSC Hex expression monolayer Act, bFGF, Hex expression 46% BMP4, FGF4, HGF, OSM, Dex   p450   Takayama et al. [70] hESC / hiPSC Sox17 and Hex expression monolayer Act, Sox17 and Hex expression 67% Cxcr4/ckit and 57% Hex BMP4, FGF4, HGF, OSM, Dex 50% p450 LDL, p450   Takayama et al. [56] hESC / hiPSC Sox17 and Hex expression monolayer Act, Sox17 and Hex expression   BMP4, FGF4, HGF, OSM, Dex 80% p450, 80% Asgr1, cmet or Alb LDL, p450, ICG, Glycogen, metabolismmediated toxicity   Epigenetic modification Hay et al. [79] hESC NaBu monolayer Act, NaBu 70% Cxcr4 DMSO, HGF, OSM, 8.3% serum 71% Alb, 65% Hepar-1 Glycogen, generation and secretion of plasma proteins, p450   Li et al. [78] mESC NaBu monolayer Act, NaBu 100% Cxcr4, 65% cKit, 63% Epcam BMP4, FGF2, HGF, EGF, TGFa, De 51% Afp, 62% Alb ICG, Glycogen yes Duan et al. [57] hESC NaBu monolayer Act, NaBu 85% Cxcr4, Sox17, and Foxa2 HGF, BMP2, FGF4, BMP4, DMSO, serum, OSM, Dex 90% Alb ICG, drug metabolism   Ren et al. [74] mESC NaBu EB NaBu   HGF, Dex 50% Glycogen   Scaffold Fang et al. [100] mESC scaffold-alginate microbeads EB FGFa   HGF, OSM, Dex, Insulin, Transferrin, Selenium 49% Alb, 50% Ck18 Alb, Urea   Li et al. [95] mESC scaffold-polyacrylamide substrate monolayer DMSO   NaBu, cells placed on polyacrylamide substrate 70% functional hepatocytelike cells Urea, Alb   Mizumoto et al. [97] mESC, monkeyESC scaffold-hollow fibers fibers     NaBu   Ammonia, Alb   Matsumoto et al. [93] mESC scaffold-Polyurethane Foam EB     aFGF, HGF, OSM, Dex, ITS   Glycogen, Ammonia, Alb   Lee et al. [94] mESC Fibronectin / collagen1, collagen 4 / laminin monolayer             Farzaneh et al. [99] hESC scaffold-ultraweb nanofiber monolayer Act 86% Foxa2, 94% Sox17, 92% Cxcr4 FGF4, HGF, OSM, Dex 66% Afp Afp, Alb, Urea, LDL, Glycogen, ICG, PROD activit   Liu et al. [28] mESC scaffold-50% poly-l-lactic acid and 50% polyglycolic acid EB Dex, DMSO   FGF4, rmHGF, OSM, Dex, ITS   Alb, LDL, Glycogen   Shafritz et al. [104] miPSC/hESC scaffold monolayer Act, bFGF 40% Cxcr4/E-cad (mouse) RA, Act, bFGF, ITS-DMEM, Dex, HGF, DMSO, NA, AsP, Akt inhibitor treated cells 45% Alb     Miki et al. [59] hESC scaffold-hydrophilic hollow fiber microfiltration membranes monolayer Act   FGF4, BMP2, HGF, OSM, Dex 30% Asgpr Alb, Ammonia, Glycogen, p450   Haque et al. [101] mESC E-cadherin substratum monolayer Act, bFGF ~ 55% HGF, OSM, Dex 92% Alb Glycogen   Amimoto et al. [96] mESC / miPSC cellulose triacetate hollow fibers fibers     NaBu, Dex, OSM, ITS   Ammonium, Alb   Co-culture or conditional medium Cho et al. [84] mESC Co-culture with rat hepatocytes monolayer             Zhao et al. [82] hESC, primate ESC Co-culture with STO feeder cells monolayer Act   FGF4, BMP2, HGF, OSM, Dex 90% Afp Alb, Glycogen, ICG, LDL, PROD assay   Fukumitsu et al. [88] mESC Co-culture with murine fetal liver stromal stromal cell line monolayer trans retinoic acid   HGF, bFGF, Nicotinamide, lascorbic acid phosphate, ITS, Dex, OSM   Glycogen, Ammonia, Alb   Huang et al. [83] primate ESC Co-culture with human ESCderived fibroblast-like cells coculture monolayer         Haptoglobin, Urea, Alb, EROD, Glycogen, HBV infection   Tuleuova et al. [89] mESC, hESC Culture with protein microarray matrices monolayer             Ishii et al. [87] mESC, hESC Co-culture with murine fetal liver stromal cells and mesenchymal cells monolayer Act   HGF   Glycogen, Ammonia, p450   Yu et al. [81] hESC Co-culture with mitomycin treated 3T3-J2 feeder cells EB Act           Nishiofuku et al. [90] mESC, ratESC Co-culture with rat hepatic stellate cells EB             Han et al. [86] mESC Co-culture with D4T endothelial cells monolayer Act ~ 50% Foxa2/Foxa3 EGF, TGFa, Dex, VEGF, bFGF, HGF, BMP4 60%     Pal et al. [80] hESC Conditioned medium from HepG2 cells EB bFGF       Glycogen, Afp, SGOT, SGPT, GGT   PSC: pluripotent stem cell; Act: Activin A; NaBu: sodium butyrate; Afp: afp secretion; Alb: albumin secretion; LDL: low-density lipoprotein uptake; ICG: indocyanine green uptake; p450: cytochrome p450 activity; ammonia: ammonia metabolism; Urea: urea secretion and production; glycogen: glycogen storage (PAS staining); ASGPR: asiaglycoprotein receptor 1; ITS: Insulin, transferin, selenium.

Table 1: Summary of PSC-Hep differentiation protocols in serum free media.