Table 1.

Characteristics of Mature Human Hepatocytes

Characteristic	Description
Morphology
Epithelial morphology	Polygonal in shape; microvilli on cell surface
Polarization	Tight junctions (ZO1) in apical tip of the lateral membrane; adherens junctions (E-cadherin) at lateral membrane; adaptor proteins (α- and β-catenin) at basolateral membrane
Polyploidization	Presence of 2 or more nuclei in fraction of the cells
Gene expression
Serum proteins	ALB, TF, TTR
Hepatocytic TFs	HNF4A, HNF1A, CEBPA/B
Metabolism enzymes	CYP3A4, CYP7A1, G6P
Absence of biliary markers	HNF1B, CK7, SOX9
Absence of stem cell markers or hepatic immature markers	LGR5, OCT4, CD133, AFP, CYP3A7
In vitro testing
Phase 1 CYP activities	Metabolism of phenacetin (CYP1A2), bupropion (CYP2B6), tolbutamide (CYP2C9) diclofenac (CYP2G9), diclofenac (CYP3A4)
Phase 2 transferase activities	Metabolism of bilirubin (UGT1A1), dopamine (SULT1A1), p-aminobenzoic acid (NAT1), 1-chloro-2,4-dinitrobenzene (GSTs)
Phase 3 transporter activities	Transport of indocyanine green (OATP1B1), rhodamine 123 (MDR1), 5(6)-carboxy-2,070-dichlorofluorescein (MRP2)
Bile acid synthesis	Formation of cholic acid and chenodeoxycholic acid (CYP8A1, AKR1D1, AKR1C4)
Glycogen storage	Periodic acid–Schiff staining for glycogen
Serum protein synthesis	Secretion of albumin, α−1 antitrypsin, fibronectin, transferrin, apoprotein, transthyretin, complement factors, coagulation factors
Cholesterol metabolism	Formation of 7α-hydroxycholesterol (CYP7A1)
Lipid uptake	Transport of low-density lipoprotein
Urea metabolism	Synthesis of urea in cell extract
Coagulation factors	Factors II, V, VII, IX, X
Other clinically relevant enzymes	OCT, A1AT, BSEP, UGT1A1, G-6-Pase, FAH, ATP7B
In vivo testing
Engraftment and repopulation	Human-specific markers or reporters to track transplanted HLCs in the liver (human-specific gene expression)
Restoration of liver function	Extended survival; presence of human proteins in the serum. Recovery of repopulating cells for genome-wide and functional metabolic studies
Tumorigenicity	Tumorigenesis in long-term transplantation experiments including several generations of re-transplantation