Table 3.

Protocols for in vitro hAEC differentiation towards hepatocytes, ordered by culture time. Observed changes in gene and protein expression are included

Culture time & surface	Differentiating medium	Gene up-regulation	Gene down-regulation	Protein expression	Cell activity	Ref.
14 days Matrigel	IMDM, 10% Fetal bovine serum (FBS), 100 mM non-essential amino acids (NEAA), 2 mM L-glutamine, 10 ng/ml EGF, 10 ng/ml bFGF, 20 ng/ml HGF, 1 μM dexamethasone (dex), 20 ng/ml oncostatin M (OSM), 55 μM β-mercaptoethanol, 1% ITS Premix	A1AT, AFP, ALB, CK19, CYP: 1A2, 2B6, 3A4, 7A1	NANOG, OCT-4, SSEA4	ALB	Indocyanine green uptake, glycogen storage, albumin and urea secretion	[113]
18 days plastic	DMEM/B27, 10ng/mL FGF2, 20ng/ml HGF, 100ng/ml Activin A, 20ng/ml OSM, 100μm/L Sodium Taurocholate Hydrate, 20ng/ml BMP4	AFP, CYP7A1, FOXA1, TAT	OCT-4	A1AT, ALB, HNF-4α	Indocyanine green and LDL uptake, CYP450 inducible activity	[14]
20 days Collagen type-I	DMEM/IDMEM, 10% FBS, 20ng/ml EGF, 10-7M dex, 100ng/ml Activin-A, 0,5% m Hu-alb (Human albumin)	A1AT, ALB, CAR, C/EBPa, C/EBPb, CK8, CK18, CK19, c-MET, CYP: 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A7, 7A1, HNF-1α, HNF-4α, OATP, PPAR, PXR, RAR, RXR,	BRCP, OCT-4	Not given	Not given	[114]
~21 days L-ECM	IDMEM, 5% FBS, 10ng/ml EGF, 10ng/ml FGF2, 10ng/ml HGF, 10−6 M dex, 100ng/ml Activin A	ALB, CYP: 2B6, 2D6, 3A4, 3A7, UGT1A1	Not given	ALB, CYP2E1, CYP3A1	Ammonia metabolism, CYP450 inducible activity	[115]
~28 days plastic	IMDM, 10% FBS, 1 mM NEAA, 4 mM L-glutamine, 10 ng/ml EGF, 0.1 μM dex, 1 mM pyruvate	A1AT, ALB, AFP, CCND1, CYP7A1	NANOG, OCT-4, P21, P53, SOX-2	CYP3A4, CYP7A1,	Increased ERK 1/2 phosphorylation	[85]
~35 days Collagen type-I	DMEM/F12, 10% FBS, 10ng/ml EGF, FGF-4, HGF, 0,1μM dex, 10% HepG2 cell-conditioned medium, 0,1μM insulin	ABCA2, ABCB11, ASS1, CYP3A4, CYP7A1	EPHX1, SLC27A	ALB, HNF-4α	LDL and glycogen presence, Indocyanine green uptake, urea synthesis	[116]