The human hepatic cell line HepaRG as a possible cell source for the generation of humanized liver TK-NOG mice

Figures & data

Figure 1. In vitro differentiation of HepaRG® cells. (A) Phase-contrast photographs of HepaRG® cells at the proliferative stage (D1: low-density culture), and the differentiative stage (D7: confluent culture; D21 and D35: differentiation culture with 1.7% DMSO). D1, 7, 21 and 35 in HepaRG® stage indicate the number of days after seeding. Bar = 100 μm. (B) The relative expression of 26 human drug metabolism-related mRNAs on D1, 7, 21 or 35 in the HepaRG® cells was assessed by qPCR. Each bar represents the average of two independent determinations, and the standard error is shown.

Figure 2. Reconstitution of human liver structures from differentiated HepaRG® cells in vivo. Hepatocyte-like colonies (upper panel) and biliary-like colonies (lower panel) in a TK-NOG mouse liver that was transplanted with differentiation D35 HepaRG® cells were subjected to immunohistochemical analysis. Serial liver sections were stained for H&E, HLA, hAlb and CK-19. Bar = 100 μm.

Table 1. Colony-forming ability of various differentiation stages of HepaRG® cells in TK-NOG livers.

						Hepatocyte-like colony		Biliary-like colony
Experiment No.	HepaRG® stage	Mouse ID No.	BW (g)	hAlb (μg/mL)	Area (cm^2)	Number	Colonies/cm^2	Number	Colonies/cm^2
1	D35	2	15.2	ND	3.6	0	0	0	0
		3	24.0	ND	4.3	6	1.4	5	1.2
		4	26.5	ND	3.7	1	0.3	0	0
		5	25.7	ND	4.0	0	0	1	0.3
		8	27.0	ND	4.0	7	1.8	1	0.3
2	D1	2	28.6	ND	3.3	0	0	0	0
		3	28.0	ND	4.9	0	0	31	6.3
	D7	1	27.9	6.9	4.5	24	5.3	43	9.5
		5	28.9	ND	4.0	3	0.7	23	5.7
	D21	9	26.9	14.2	3.8	40	10.5	26	6.8
		14	26.5	ND	2.9	21	7.4	37	13.0
	D35	10	25.2	ND	2.8	2	0.7	1	0.4
		12	27.7	ND	3.0	4	1.3	4	1.3

D1, 7, 21 and 35 in HepaRG® stage indicate the number of days after seeding. BW: body weight. The hAlb level of each animal is shown. ND: not detected by ELISA. Area: observed cross-sections were measured and indicated as centimeter square (cm²). Colonies per centimeter square (colonies/cm²): number of colonies per area of observation.

Table 2. Engraftment of cryopreserved human hepatocytes in TK-NOG livers.

Cryopreservedhepatocytes	Mouse ID No.	BW (g)	hAlb (μg/mL)	RI (%)
HEP187170	73	24.7	1459	20.9
	78	27.5	684	12.0
	79	27.9	1061	16.3
	712	21.0	2778	36.0
HEP187170	714	18.6	2818	36.4
	102	22.0	4803	59.1
	113	21.5	3355	42.6
	114	20.6	5115	62.7

The amount of hAlb and extent of human liver reconstitution in TK-NOG mice were measured 12 weeks after transplantation of cryopreserved human hepatocytes (HEP187170). BW: body weight. The extent of human liver reconstitution was estimated as a function of the hAlb concentration, which was shown to correlate with the extent of human liver reconstitution. RI: reconstitution index.

Figure 3. Expression of functional liver markers in reconstituted livers from TK-NOG mice. Hepatocyte-like colonies in a TK-NOG mouse liver that was transplanted with differentiation D21 HepaRG® cells (upper panel) and human hepatocyte colonies in a TK-NOG mouse liver that was transplanted with cryopreserved human hepatocytes (HEP187170; 26 years, female) (lower panel) were assessed for functionality by histochemical and immunohistochemical analyses. Serial liver sections were stained for H&E, PAS, CYP3A4 and MRP2. Bar = 100 μm.

Figure 4. Tumorigenic potency of HepaRG® cells in NOG mice. (A) The growth potential of HepaRG® cells was evaluated in NOG mice. A total of 1 × 10⁶ cells were subcutaneously transplanted into NOG mice. D1 and D35 in HepaRG® stage indicate the number of days after seeding. A total of 1 × 10⁴ HepG2 cells were used as positive control for s.c. transplantation. (B) Histologic and immunohistochemical analyses of D1 HepaRG® xenografts in NOG mouse. Twelve weeks after transplantation, the xenografts were processed for H&E staining, HLA, hAlb and CK-19 staining. Bar = 500 μm, Bar = 50 μm.