Table 1.

Protocols to Expand Primary Human and Rodent Hepatocytes in Vitro

Article	Hepatocyte source	Types of cell culture	Signals used to expand hepatocytes	Expansion period	Gene expression of expanded hepatocytes	In vivo experiment
Hu et al, 2018	Human and mouse	3D organoid cell culture	HGF, EGF, WNT agonist (R-spondin 1, CHIR99021), TGFβ inhibitor (A83-01), ROCK inhibitor (Y27632), fibroblast growth factor 7 (FGF7), FGF10, TGFα	Mouse: 2–3 months Human: 2.5–11 months	Mouse: Afp, Alb, Hnf4a, Cyp1a2, Cyp3a11 Human: ALB, HNF4A, CYP2E1, AFP	Hepatocytes were expanded as single cells and transplanted into immunodeficient FRG mice via splenic injection. Engrafted hepatocytes expressed ALBUMIN, MRP2, CYP2E1, and secreted human serum albumin
Fu et al, 2018	Human	2D cell culture	HGF, EGF, WNT agonist (CHIR99021), TGFβ inhibitor (A83-01), ROCK inhibitor (Y27632), lysophosphatidic acid, sphingosine-1-phosphate	10 days	CK19 (cytokeratin 19), CK7 (cytokeratin 7), HNF4A, SOX9, HNF1A, CK18 (cytokeratin 18), EpCAM, CD24	Hepatocytes were transplanted via intrasplenic injection into Fah−/−Rag2−/− mice; 7.2%–16.1% engraftment was observed; the cells expressed ALBUMIN and were able to secrete ALBUMIN and alpha-1-antitrypsin
Garnier et al, 2018	Human	3D organoid cell culture	HGF, EGF, Wnt3a, TGFβ inhibitor (A83-01), forskolin, R-spondin 1, FGF10, Noggin, ROCK inhibitor (Y27632)	2 months	CK19, EpCAM, SOX9, Ki67	N/A
Katsuda et al, 2019	Human	2D cell culture	Wnt agonist (CHIR99021), TGFβ inhibitor (A83-01), ROCK inhibitor (Y27632)	14–18 days	CK19, EPCAM, CD44, CD133, CD24, integrin subunit alpha 6 (ITGA6), SOX9, Prospero Homeobox (PROX1), AXIN2	Hepatocytes were transplanted via intrasplenic injection into chronic injured livers of cDNA-μPA/SCID mouse. 10–11 weeks after transplantation, 17%–39% of transplanted cells engrafted the liver, expressed CYP2C, and secreted ALBUMIN
Katsuda et al, 2017	Rat and mouse	2D cell culture	Wnt agonist (CHIR99021), TGFβ inhibitor (A83-01), ROCK inhibitor (Y27632)	15 days	Ck19, Forkhead Box M1(Foxm1), proliferating cell nuclear antigen (PCNA), Afp, Sox9, Epcam, Cd90, Forkhead Box J1 (Foxj1), Hnf1b, Hnf6, Cd44, Itga6	Hepatocytes were transplanted via intrasplenic injection into cDNA-μPA/SCID mouse model and observed that 75%–90% of the liver comprised of transplanted rat cells. Engrafted cells secreted Albumin and expressed hepatocyte markers (Cyp1a2, Cyp3a1, Cyp7a1, Mrp2, Hnf4a)
Zhang et al, 2018	Human	2D cell culture	HGF, EGF, Wnt3a, FGF10, TGFβ inhibitor (A83-01), ROCK inhibitor (Y27632)	1 month	CK19, CK7, SOX9, CD133, CYP1A2, ALB, AAT	Hepatocytes were transplanted via intrasplenic injection into Fah−/−Rag2−/−IL2rg-/- mouse model. The engrafted cells increased survival after 4 months of transplantation, reduced serum levels of aspartate transaminase, alanine transaminase, and total bilirubin, and increased serum levels of ALBUMIN. Engrafted cells repopulated approximately 64% of the mouse liver and expressed ALBUMIN and HNF4A 4 months after transplant
Xiang et al, 2019	Human	2D cell culture	Forskolin, TGF inhibitor (SB431542), Wnt inhibitor (IWP2), Notch inhibitor (DAPT), bone morphogenetic protein inhibitor (LDN193189)	2 months	ALB, AAT, carbamoyl-phosphate synthase 1(CPS1), CYP3A4, CYP1A2, CYP2C9 CYP2D6, CYP2B6	N/A
Peng et al, 2018	Mouse	3D cell culture	HGF, EGF, Wnt agonist (CHIR99021), TNFα	6 months	Hnf4a, Yap, NF-kB, Ki67, Alb, transthyretin (Ttr), tyrosine aminotransferase (Tat), Fah	Hepatocytes were transplanted via intrasplenic injection into Fah−/−Rag2−/−IL2rg-/- mouse model. 80% repopulation was observed 103 days after transplantation, and the engrafted cells expressed FAH, pericentral genes (Glt1, Gs, Cyp2e1), and Cps1
Huch et al, 2013	Mouse	3D cell culture	HGF, EGF, R-spondin 1, Wnt3a, FGF10	8 months	Ck19, Lgr5, Sox9	Hepatocytes were expanded as 3D aggregates and were transplanted via intrasplenic injection into Fah−/−Rag2−/−IL2rg-/- mice. Transplanted cells expressed Fah and Hnf4a and improved mice survival
Huch et al, 2015	Human	3D cell culture	HGF, EGF, Wnt conditioned medium, R-spondin 1, TGFβ inhibitor (A83-01), FGF10, forskolin, bone morphogenetic protein inhibitor (Noggin), ROCK inhibitor (Y27632)	6 months	CK7, LGR5, SOX9, HNF4a, CK19, AXIN2	Hepatocytes were transplanted via intrasplenic injection into CCl4-induced liver injury mouse model. Engrafted cells expressed ALBUMIN; secreted ALBUMIN and alpha-1-antitrypsin

ROCK, rho associated kinase; TGFβ, transforming growth factor beta.