Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice

Abstract

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.

Fig 1. Production of cDNA-uPA mice.

(A) Construction of the cDNA-uPA transgene. (B) Body weight in 8-week-old mice and (C) serum ALT activity in #1C2, 2C7, 1D3, and non-transgenic control mice. Body weight of #1C2 and #2C7 mice was lower than that of #1D3 and control mice. ALT activity in #1C2 and #2C7 mice increased from the age of 6–8 weeks. The approximate values of ALT are given on the right side of the vertical axis of the graph. These values were calculated as follows: 1 ALT (IU/L) ≈ 1.58 Karmen.

Fig 2. Changes in blood h-alb concentrations.

BD85 h-heps were transplanted into (A) #1C2 and (B) #2C7 hemi and homozygotes. Solid and dotted lines show homozygotes and hemizygotes, respectively.

Fig 3. uPA and ALT activity in uPA/SCID mice and #1C2 homo- and hemizygotes.

(A) uPA activity was more than 100 times higher in 14-week-old uPA/SCID mice than #1C2 mice. #1C2 homozygotes showed higher uPA activity than hemizygotes. (B) No statistical differences in ALT activity among the three groups at 3, 7, and 10 weeks old were observed. Fourteen-week-old #1C2 homozygotes showed ALT activity that was approximately 2 times higher than that of #1C2 hemizygotes. *, p < 0.05; **, p < 0.01; #, measurement limit.

Fig 4. Histological findings in uPA/SCID and #1C2 homo- and hemizygous livers.

H&E staining of (A and C) uPA/SCID, (D and F) #1C2 homozygous and (G and I) #1C2 hemizygous mice aged 3 and 14 weeks, respectively. Oil red O staining in 3-week-old (B) uPA/SCID and #1C2 (E) homo and (H) hemizygous mouse livers. Hepatocytes of all 3 groups were degenerating in 3-week-old mice. In 14-week-old mice, degenerating hepatocytes and hyperplastic m-hep colonies were observed in uPA/SCID mice but not in #1C2 homo- and hemizygotes. Varying m-hep sizes were observed in 3-week-old #1C2 homo- and hemizygotes. Lipid droplets were more abundant in uPA/SCID mice and #1C2 homozygotes than #1C2 hemizygotes. De, degenerating hepatocytes. Hy, hyperplastic hepatocyte colony. Bar, 100 μm.

Fig 5. Changes in h-alb concentration and body weight in chimeric mice up to 4 months of age.

(A) h-alb levels in uPA/SCID chimeric mice were the highest among the 3 types of chimeric mice, and those of #1C2 homozygous mice were higher than those of hemizygous mice by 6 weeks of age. H-alb levels in uPA/SCID and #1C2 homozygous mice reached a plateau by 10 and 11 weeks of age, respectively, whereas those in #1C2 hemizygous mice continued to increase up to 17 weeks of age. (B) Body weight of #1C2 hemizygous mice was the highest, followed by #1C2 homozygous mice, and then uPA/SCID chimeric mice. (C) RI and h-alb concentrations plots showed similar correlation curves in uPA/SCID and #1C2 homo- and hemizygous chimeric mice.

Fig 6. Histopathological findings in chimeric mouse livers and kidneys.

H&E staining of left lateral lobes of (A) uPA/SCID chimeric mice, (B) #1C2 homozygous and (C) #1C2 hemizygous chimeric mice, and magnifications of the rectangular parts (D) on the left side of A and (E) on the right side of A, (F) in B, and (G) in C are shown. h-heps with a clear cytoplasm and lipid droplets occupied most areas of the liver sections (A-G). Arrows show (D) degenerating m-heps and (E) hyperplastic m-hep nodules in uPA/SCID chimeric mice. M-heps with eosinophilic cytoplasm of various sizes are shown by arrows in (F) #1C2 homozygous and (G) hemizygous chimeric mice. (H) The left lateral lobe of a #1C2 hemizygous chimeric mouse was immunostained with anti-hCK8/18 antibodies. H-heps were brown-colored, and (I) an area of rectangle was magnified. m, m-heps, h, h-heps. Kidney sections in (J, M) uPA/SCID, (K, N) #1C2 homozygote and (L, O) hemizygous chimeric mice were stained with H&E. Enlarged glomeruli and glomerulosclerosis were observed in uPA/SCID mice (J, M). No pathological findings were observed in (K, N) #1C2 homozygote and (L, O) hemizygous chimeric mouse kidneys. (M), (N) and (O) were high magnification of (J), (K) and (L), respectively. Bar, 100 μm in D-G, I and J-O. Bar, 1 mm in A-C and H.

Fig 7. Changes in h-alb levels and body weight of chimeric mice during a long-term period.

14-week-old chimeric mice with >70% RI were selected based on h-alb levels and observed until they were 28 or 29 weeks old. (A) After 16 weeks of age, h-alb levels in uPA/SCID chimeric mice gradually decreased, whereas those of #1C2 homozygous chimeric mice stabilized, but those of #1C2 hemizygous chimeric mice increased gradually until mice were at least 28 weeks old. (B) Body weight of uPA/SCID and #1C2 homozygous chimeric mice decreased gradually after 16 weeks of age, but increased in #1C2 homo chimeras until mice were 18 weeks old, and then stabilized.

Fig 8. Microarray mRNA expression analysis of phase I and II metabolic enzymes levels in chimeric mice.

mRNA expression levels of phase I and II expressed in human livers in (A and B) uPA/SCID and #1C2 homozygous mice, (C and D) #1C2 homo- and hemizygous mice, and (E and F) uPA/SCID and #1C2 mice were plotted. Phase I metabolic enzymes are CYP1A1, 1A2, 1B1, 2A6, 26A1, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7, 3A43, 39A1, 4A11, 4F2, 4F3, 7A1, 7B1, 8B1. Phase II metabolic enzymes are UGT1A6, 2A1, 2B15, 2B17, 2B4, SULT1A1, 1A3, 1B1, 1E1, 2A1, GSTA1, A4, M1, M2, M3, M4, P1, T1, Z1, and NAT1, 2. The solid and dotted lines represent unity and 4-fold differences, respectively. All values were within the 4-fold difference range.

Fig 9. HBV and HCV infections in chimeric mice.

Chimeric mice shown in Table 4 were inoculated with (A) HBV and (B) HCV, and serum HBV and HCV copy numbers were monitored up to 70 days and 40 days post-infection, respectively. HBV and HCV copy numbers did not differ among groups and increased up to approximately 50 and 20 days post-infection, respectively, reaching a plateau afterwards.