Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model

Abstract

We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah(-/-)) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse liver is repopulated by human hepatocytes, and sustained expression of lentiviral vector transduced gene can be observed. We further report the development of a hepatocyte transplantation method involving a transcutaneous, intrahepatic injection in neonatal mice. Human hepatocytes engraft over the entire injected lobe with an expansion pattern similar to those observed with intrasplenic transplantation.

Fig. 1.

Engraftment and repopulation of human hepatocytes in adult fah^−/−/rag-2^−/−/il-2rg^−/− mice. Shown is immunohistochemistry for FAH (A–D) and human-specific CK-18 (E and F). (A) One month after transplantation, FAH-positive human hepatocytes were found over the entire liver acinus. The arrow points to a central vein, and arrowheads point to bile ducts of the portal field. (B, C, and E) Repopulation 2 (B and E) and 3 (C) months after transplantation. (D and F) Negative control without primary antibodies for FAH (D) and CK-18 (F). (Magnification: A, ×200; B–D, ×100; E and F, ×400.)

Fig. 2.

Exclusion of cell fusion. (A) DAPI (blue) and Cy3 (red) visualization to show nuclei and mouse pan centromeric probe. (B) DAPI (blue) and FITC (green) visualization to show nuclei and human pan centromeric probe. (C) Localization of human (FITC, green) and mouse (Cy3, red) pan centromeric probe (without DAPI). Overlapping yellow signals do not colocalize with DAPI (A and B) and therefore are not nuclear. (Magnification: ×400.)

Fig. 3.

Detection of human proteins in the blood of transplanted animals. (A) Human C3a detected in the plasma. (B) Human albumin detected in the serum. A and B are blood samples from the same animals at the same time points. Green and blue columns are from fah^−/−/rag-2^−/−/il-2rg^−/−, and red columns are from fah^−/−/rag-2^−/− mice.

Fig. 4.

Expression of lentivirally transduced GFP 2 months after transplantation. Sections have been stained with DAPI only, and green fluorescence originates from transduced human hepatocytes. (Magnification: A, ×100; B, ×630.)

Fig. 5.

Human hepatocyte transplantation into neonatal fah^−/−/rag-2^−/−/il-2rg^−/− mice. (A) Human hepatocyte clusters (FAH staining) are found over the entire left lower lobe. (B and C) Serial sections of a transplanted human hepatocyte cluster stained with FAH (B) and H&E (C). The arrow points to a small vein. (D and E) Staining for human-specific CK-18 (D) with the typical cytoskeleton staining pattern and FAH (E) with the cytoplasmic staining pattern. (F) Merger of D and E shows colocalization of double staining. (Magnification: A, ×50; D–F, ×400; B and C, ×630.)