650? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 3 March 2010
New horizons for studying
human hepatotropic infections
Ype P. de Jong,1,2 Charles M. Rice,1 and Alexander Ploss1
1Center for the Study of Hepatitis C, The Rockefeller University, New York, New York.
2Division of Gastroenterology, Mount Sinai School of Medicine, New York, New York.
More than 800 million people are chroni-
cally infected with hepatitis B and C viruses
(HBV and HCV, respectively) and malaria,
causing over 1.5 million deaths annually
(1, 2). Persistent HBV and HCV infections
can lead to cirrhosis and/or hepatocellular
carcinoma (HCC). Basic research, as well
as the development of drugs and vaccines
targeting human hepatotropic patho-
gens, has been handicapped by the lack of
robust in vitro and in vivo platforms that
mimic human liver biology and disease
susceptibility. The narrow species tropism
of HBV and HCV restricts preclinical stud-
ies to chimpanzees or human liver–chime-
ric mice. Large primate studies are often
limited by financial and ethical concerns,
and humanized mice are currently cum-
bersome and low-throughput. In culture,
HCC-derived cell lines and immortalized
hepatocytes have been useful for study-
ing various aspects of HBV and HCV life
cycles, but these cells differ phenotypically
and functionally from human hepatocytes
in vivo. Primary hepatocytes, which can be
coaxed to maintain liver-specific functions
by culture with fibroblasts, hold promise
as a more physiologically relevant in vitro
substrate for HCV infection (3). Addressing
fundamental questions about hepatotropic
pathogen biology in vivo, however, requires
a suitable small animal model to comple-
ment and guide more challenging and
expensive studies, including clinical trials.
Human liver–chimeric mouse models
for human hepatotropic infections
Currently, the most advanced in vivo sys-
tems for modeling human hepatotropic
infectious disease are human liver–chime-
ric mice. A high degree of chimerism (up
to 99%) can be achieved by transplanting
human hepatocytes into immunodefi-
cient mice in which liver injury has been
induced to ablate the endogenous murine
hepatocytes (Figure 1). The best-charac-
terized model to date is the immunode-
ficient urokinase-type plasminogen acti-
vator (uPA) mouse, in which an albumin
(Alb) promoter directs high-level toxic
expression of uPA (Alb-uPA mice) (4, 5).
The hepatotoxicity creates a permissive
environment for the expansion of func-
tional, transgene-free human hepatocytes.
Human liver–chimeric immunodeficient
Alb-uPA mice are susceptible to hepato-
tropic human pathogens (4, 5) and have
been used in studies ranging from viral
evolution to preclinical testing of antivi-
ral compounds (6, 7). Working with the
Alb-uPA model, however, poses serious
challenges (Table 1). The selective pressure
needed to achieve and maintain a high level
of human chimerism can only be achieved
in homozygous Alb-uPA immunodeficient
recipients, which suffer from infertility.
Therefore large, costly, hemizygous breed-
er cohorts, or rescue of breeder pairs by
transplantation with normal hepatocytes,
is required to propagate these mice (8).
The severe liver injury of homozygous Alb-
uPA immunodeficient pups necessitates
engraftment surgery in the first weeks of
life — a procedure that is complicated by
the susceptibility of these animals to fatal
hemorrhaging (9). Because of these factors,
along with the need for high-quality adult
human hepatocytes, only modest numbers
of experimental mice can be generated, and
at great expense.
To overcome some of these challenges,
alternative liver injury models have been
developed, including a less toxic Alb-uPA
line (10), mice characterized by induc-
ible uPA transgene expression (11), or
mice in which the major urinary protein
(MUP) promoter is used to delay uPA
expression (12). Recently, two groups
have reported successful engraftment of
human hepatocytes into fumaryl acetoac-
etate hydrolase–deficient (FAH-deficient)
mice bred with mice of a Rag2–/–Il2rgnull
immunodeficient background (FRG mice)
(13, 14) (Table 1). FAH is the last enzyme in
the tyrosine breakdown pathway (Figure 2),
and its deficiency leads to lethal type 1
hypertyrosinemia in humans and liver fail-
ure in mice. Treatment with 2-(2-nitro-4-
dione (NTBC) prevents the accumulation
of toxic metabolites and hepatotoxicity
(15), allowing liver injury to be induced at
will by withdrawal of the drug.
Using mice of the FRG background, Bis-
sig et al. report consistently high, on average
42%, human liver chimerism after transplan-
tation of human adult hepatocytes (Table 1)
(16). The trick to achieving this high chime-
rism seems to be the use of 3–5 times more
human adult hepatocytes than in previous
studies (13, 14). The impressively large num-
ber of successfully transplanted animals
revealed a clear correlation between the level
Conflict?of?interest: C.M. Rice holds equity in Apath
LLC, and AlphaVax Inc. and serves as an advisor for
Genentech, GlaxoSmithKline, iTherX, Merck, Novartis
Vaccines and Diagnostics, Pfizer, and Vertex.
Citation?for?this?article: J Clin Invest. 2010;
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 3 March 2010
of chimerism and human serum albumin
— providing a convenient, minimally inva-
sive test to monitor engraftment (16). The
authors then tested whether the engrafted
animals were susceptible to HBV and HCV
infection. Similar to previous studies with
immunodeficient Alb-uPA mice (5, 17), FRG
mice were shown to be susceptible to HBV
infection, irrespective of the level of human
chimerism. Hallmarks of viral replication,
including the presence of serum HBV DNA,
covalently closed circular HBV DNA, and
HBV core antigen in liver tissue, were readily
observed. In contrast, successful HCV infec-
tion could only be achieved if more than
10% of hepatocytes were of human origin,
again in line with previous observations in
immunodeficient Alb-uPA mice (4, 5). Inter-
estingly, above this 10% threshold of chime-
rism, no clear correlation between hepato-
cyte engraftment levels and serum viremia
was observed. One can only speculate as
to why HCV requires substantially higher
human chimerism — the short in vivo half-
life of the virus may require rapid uptake
by new permissive target cells, or successful
propagation may necessitate densities of
human hepatocytes sufficient for cell-to-cell
spread. Importantly, HCV infection could
be initiated using cell culture–grown or
patient-derived virus, with the highest vire-
mia level achieved using a patient-derived
isolate. Furthermore, HCV antigens could
be detected in the livers of infected animals,
traditionally a highly technically difficult
Potential of human liver–chimeric
mice in preclinical drug development
The susceptibility of humanized FRG
mice to hepatotropic infections holds
promise for preclinical drug efficacy test-
ing. The authors demonstrate a modest
decrease in HBV viremia after short-term
treatment with the reverse transcriptase
inhibitor adefovir dipivoxil, similar to
the slow kinetics of viral decline seen in
humans treated with this drug (16). For
HCV, the current treatment is pegylated
IFN-a (peg-IFN) and ribavirin, a combi-
nation therapy that is often poorly toler-
ated and unsuccessful. Several drug can-
didates targeting viral and host proteins
are in preclinical or clinical development,
and a major question is whether the virus
Creation and future improvements of human liver–chimeric FRG mice. In the model described in the current issue by Bissig et al. (16), enclosed
in the shaded rectangle, hepatocytes are isolated from adult human liver tissue and injected into FRG mice that are cycled off the protective drug
NTBC. Human hepatocyte engraftment levels are then monitored by serial human albumin (hAlb) measurements in the serum of transplanted
mice. Over 2–3 months, the human hepatocytes expand and can repopulate up to 97% of the FRG mouse liver, with the remainder of liver-resident
cells likely of murine origin. This robust system can be used to study HBV and HCV infections in vivo and can serve as a scaffold for more complex
humanized mouse models. The engrafted FRG mice reported by Bissig et al. could potentially be combined with mice bred to posses a human
immune system, which are generated by transplantation of human CD34+ stem cells (HSCs) (23). Use of donor hepatocytes and HSCs derived
from fetal liver may allow FRG mice animals to be repopulated with the immune system and liver tissue of an individual human donor, facilitat-
ing studies of immunopathogenesis and vaccine testing. Another use for the FRG model will be to study hepatocyte differentiation from human
induced pluripotent stem (iPS) cells (24, 25) derived from dermal fibroblasts or from embryonic stem cells (26). Steps depicted by solid lines are
currently feasible; the dashed lines refer to steps that are under investigation but have not yet proven workable. Figure modified with permission
from Hepatology (27), Nature Reviews Immunology (28), and Cell Host & Microbe (22) and based on concepts discussed in ref. 29.
652? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 3 March 2010
can be eliminated using solely a cocktail
of specific antivirals. Identification of
the most effective drug combinations,
and the ability to monitor resistance, is
an area where model animals could have
great utility. Bissig et al. show that a
combination of peg-IFN and Debio 025
— an HCV inhibitor targeting the host
factor cyclophilin A — is as effective as
peg-IFN plus ribavirin over a short-term
treatment period. Overall, the results of
the Bissig et al. study recapitulate many
aspects of HBV and HCV infection in
humans, illustrating the promise of this
new model for basic studies of human
hepatitis viruses and preclinical drug
The next steps
The development of more robust human
liver–chimeric mice is an important step
forward, but further refinements are
needed. Despite prolonged high vire-
mia, none of the commonly observed
sequelae associated with HBV or HCV
infections in humans, namely fibrosis or
HCC, were observed in the current study
(16). (Patho)physiological processes may
require crosstalk between hepatocytes
and other liver-resident cells, and, while
human hepatocytes are abundant in
highly engrafted chimeric mice, non-
parenchymal cells are of murine-recipient
origin. Kupffer cells and liver sinusoidal
endothelial cells, for example, appear to
be critical to the ability of Plasmodium
sporozoites (the transmission form of
the malaria parasite) to infect the liver,
as permissive hepatocytes are not directly
accessible to the sporozoites (19). While
infection of human liver–chimeric mice
with virulent and live-attenuated Plasmo-
dium species has been reported (20, 21),
infection frequencies were low and may in
the future possibly be improved by addi-
tion of other human non-parenchymal
liver cell subsets.
Another obvious caveat of the current
human liver–chimeric models is their
immunodeficient background. Chronic
inflammation is thought to be a major
contributor to fibrosis during persis-
tent HBV and HCV infection. Thus, dual
engraftment of these animals, resulting
in a human liver and a human immune
system, is not only critical to monitor
immune responses to infection, but also
to more faithfully mimic disease patho-
genesis (22). Furthermore, since viral
hepatitis has become the leading mor-
bidity in HIV-infected individuals in the
Western world, modeling such coinfec-
tions in dually reconstituted mice would
be another clinically important applica-
tion (Figure 1).
In summary, human liver–chimeric mice
can serve as valuable tools in preclinical
drug efficacy, toxicity, and pharmacokinet-
ic applications but also facilitate the study
of human hepatotropic pathogens. After
more than a decade of working with frail
immunodeficient Alb-uPA mice, the FRG
model reported by Bissig and colleagues
lends itself to become the cornerstone for
a wide variety of investigations and will
likely provide the platform for further
Comparison between FRG mice and immunodeficient Alb-uPA human liver–chimeric mice
Hepatitis C: HCVcc (genotype 2a)
Hepatitis C: patient samples
? Plasmodium spp.
Inducible upon NTBC withdrawal
Not yet available
3%–97% (average, 42%)
Murine HCC after long-term
7–21 days after birth
Up to 99%
Infertility of homozygous
transgenic mice; coagulopathy
+ Not tested
iPS, induced pluripotent stem; HCVcc, cell culture–derived HCV.
Tyrosine metabolic pathway affected by FAH
deficiency, and the step in this pathway that is
inhibited by NTBC.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 120 Number 3 March 2010
The authors thank Catherine Murray (The
Rockefeller University) for editing the
manuscript. This work was supported by
grants from the Gates Foundation through
the Grand Challenges in Global Health ini-
tiative (to all authors) and funded in part
by the Greenberg Medical Research Insti-
tute, the Ellison Medical Foundation, the
Starr Foundation, the Ronald A. Shellow
Memorial Fund, the Richard Salomon
Family Foundation (to C.M. Rice), and the
NIH through the NIH Roadmap for Medi-
cal Research, grant 1 R01 DK085713-01
(to C.M. Rice and A. Ploss). C.M. Rice is an
Ellison Medical Foundation Senior Scholar
in Global Infectious Diseases.
Address correspondence to: Alexander Ploss
or Charles M. Rice, Center for the Study of
Hepatitis C, The Rockefeller University, 1230
York Avenue, Box 64, New York, NY 10065.
Phone: 212.327.7066; Fax: 212.327.7048;
E-mail: firstname.lastname@example.org (A. Ploss).
Phone: 212.327.7046; Fax: 212.327.7048;
E-mail: email@example.com (C.M. Rice).
1. Alter MJ. Epidemiology and prevention of hepatitis
B. Semin Liver Dis. 2003;23(1):39–46.
2. Shepard CW, Finelli L, Alter MJ. Global epidemiol-
ogy of hepatitis C virus infection. Lancet Infect Dis.
3. Ploss A, et al. Persistent hepatitis C virus infection
in microscale primary human hepatocyte cultures.
Proc Natl Acad Sci U S A. In press.
4. Mercer DF, et al. Hepatitis C virus replication
in mice with chimeric human livers. Nat Med.
5. Meuleman P, et al. Morphological and biochemical
characterization of a human liver in a uPA-SCID
mouse chimera. Hepatology. 2005;41(4):847–856.
6. Kaul A, Woerz I, Meuleman P, Leroux-Roels G,
Bartenschlager R. Cell culture adaptation of hepati-
tis C virus and in vivo viability of an adapted variant.
J Virol. 2007;81(23):13168–13179.
7. Meuleman P, Leroux-Roels G. The human liver-
uPA-SCID mouse: a model for the evaluation of
antiviral compounds against HBV and HCV. Anti-
viral Res. 2008;80(3):231–238.
8. Brezillon NM, et al. Rescue of fertility in homozy-
gous mice for the urokinase plasminogen activa-
tor transgene by the transplantation of mouse
hepatocytes. Cell Transplant. 2008;17(7):803–812.
9. Heckel JL, Sandgren EP, Degen JL, Palmiter RD,
Brinster RL. Neonatal bleeding in transgenic mice
expressing urokinase-type plasminogen activator.
10. Suemizu H, et al. Establishment of a humanized
model of liver using NOD/Shi-scid IL2Rgnull mice.
Biochem Biophys Res Commun. 2008;377(1):248–252.
11. Song X, et al. A mouse model of inducible liver
injury caused by tet-on regulated urokinase for
studies of hepatocyte transplantation. Am J Pathol.
12. Weglarz TC, Degen JL, Sandgren EP. Hepatocyte
transplantation into diseased mouse liver. Kinetics
of parenchymal repopulation and identification of
the proliferative capacity of tetraploid and octaploid
hepatocytes. Am J Pathol. 2000;157(6):1963–1974.
13. Azuma H, et al. Robust expansion of human
hepatocytes in Fah–/–/Rag2–/–/Il2rg–/– mice. Nat
14. Bissig KD, Le TT, Woods NB, Verma IM. Repopu-
lation of adult and neonatal mice with human
hepatocytes: a chimeric animal model. Proc Natl
Acad Sci U S A. 2007;104(51):20507–20511.
15. Grompe M, et al. Pharmacological correction of
neonatal lethal hepatic dysfunction in a murine
model of hereditary tyrosinaemia type I. Nat Genet.
16. Bissig K-D, et al. Human liver chimeric mice pro-
vide a model for hepatitis B and C virus infection
and treatment. J Clin Invest. 2010;120(3):924–930.
17. Dandri M, et al. Repopulation of mouse liver with
human hepatocytes and in vivo infection with hep-
atitis B virus. Hepatology. 2001;33(4):981–988.
18. Liang Y, et al. Visualizing hepatitis C virus infec-
tions in human liver by two-photon microscopy.
19. Frevert U, Usynin I, Baer K, Klotz C. Plasmodium
sporozoite passage across the sinusoidal cell layer.
Subcell Biochem. 2008;47:182–197.
20. VanBuskirk KM, et al. Preerythrocytic, live-
attenuated Plasmodium falciparum vaccine
candidates by design. Proc Natl Acad Sci U S A.
21. Morosan S, et al. Liver-stage development of Plas-
modium falciparum, in a humanized mouse model.
J Infect Dis. 2006;193(7):996–1004.
22. Legrand N, et al. Humanized mice for modeling
human infectious disease: challenges, progress, and
outlook. Cell Host Microbe. 2009;6(1):5–9.
23. Shultz LD, Ishikawa F, Greiner DL. Humanized
mice in translational biomedical research. Nat Rev
24. Sullivan GJ, et al. Generation of functional human
hepatic endoderm from human induced pluripo-
tent stem cells. Hepatology. 2010;51(1):329–335.
25. Si-Tayeb K, et al. Highly efficient generation of
human hepatocyte-like cells from induced pluripo-
tent stem cells. Hepatology. 2010;51(1):297–305.
26. Snykers S, De Kock J, Rogiers V, Vanhaecke T. In
vitro differentiation of embryonic and adult stem
cells into hepatocytes: state of the art. Stem Cells.
27. Kneteman NM, Mercer DF. Mice with chimeric liv-
ers: who says supermodels have to be tall? Hepatology.
28. Adams DH, Eksteen B. Aberrant homing of muco-
sal T cells and extra-intestinal manifestations of
inflammatory bowel disease. Nat Rev Immunol.
29. Ploss A, Rice CM. Towards a small animal model for
hepatitis C. EMBO Rep. 2009;10(11):1220–1227.
Bidirectional homing of Tregs
between the skin and lymph nodes
Hironori Matsushima and Akira Takashima
Department of Medical Microbiology and Immunology, University of Toledo College of Medicine, Ohio.
Conflict?of?interest: The authors have declared that no
conflict of interest exists.
Citation?for?this?article: J Clin Invest. 2010;
Memory T cells are disseminated to lym-
phoid and nonlymphoid tissues throughout
the body, and their migration to respective
tissues is tightly regulated by adhesion mol-
ecules and chemokine receptors (Table 1).
For example, memory T cells that infil-
trate the skin express a unique adhesion
molecule, known as cutaneous lympho-
cyte-associated antigen (CLA), which is
produced from P-selectin glycoprotein
ligand-1 through posttranscriptional car-
bohydrate modification by fucosyltrans-
ferase VII. Skin-homing memory T cells
also express specific chemokine receptors:
CCR4, CCR6, and CCR10. In contrast,
memory T cells that preferentially circulate
through lymphoid tissues express CD62
ligand (CD62L; also known as L-selectin)
and CCR7 (1).