A Tale of Two Livers – Developing an Innovative Drug Screening Tool: January
15, 2012
New drugs undergo rigorous safety and efficacy testing in animals before they are
tried in people. However, due to differences in the ways humans and animals process
drugs in the liver, animal models cannot predict side effects that are unique to
humans. To bring animal models closer to mirroring human physiology, researchers
have recently been developing a humanized mouse model that harbors human liver tissue.
These humanized animal models provide insight into how disease processes
and new therapies might behave in people.
Reconstructing human liver tissue
Humanized mouse models are difficult to produce and maintain. For survival, transplanted
human liver cells must establish intricate signaling molecule exchanges with mouse
tissues, as well as evade attack by the mouse’s immune system. Massachusetts
Institute of Technology (MIT) Professor Sangeeta N. Bhatia, (who teaches in both
the Health Sciences and Technology and the Electrical Engineering and Computer Science
Programs), theorized that integrating human liver cells with a supportive and protective
microenvironment, prior to their implantation, would improve the survival and the
functioning of humanized liver tissue transplanted into a normal mouse.
A deep understanding of cell-cell interactions, along with a sophisticated level
of tissue engineering, was required to generate the humanized livers for implantation.
In engineered liver tissue, the microenvironment consists of a polymer scaffold
and supporting cells. After testing several combinations of ingredients, Bhatia’s
research team uncovered a recipe for tissue they dubbed human ectopic artificial
liver (HEAL). In the first step, they grow (in vitro) human hepatocytes,
the predominant liver cell type, along with mouse connective tissue cells called
fibroblasts. After one week, the hepatocyte/fibroblast cell clusters
are mixed with human endothelial cells (cells that line the inner surface
of blood vessels), and the combination is encapsulated within photo responsive polyethylene
glycol diacrylate (PEG-DA) polymer scaffolds. When exposed to light, PEG-DA
polymer chains interlink to form a mesh. “You shine a light on it, and it
entraps the cells,” says Bhatia. The PEG-DA scaffold contains biologically
active molecules that facilitate interactions between cells in three-dimensional
space. Fibroblasts provide cues to stabilize hepatocytes while they
grow in vitro, and endothelial cells secrete signals needed for the
recruitment of blood vessels that are critical to the survival of the construct
after implantation.
The liver processes drugs similar to how the gut processes food, by breaking the
drugs down into smaller components called metabolites (and some metabolites
may be very harmful to people). During the drug breakdown process, a mouse liver
might not produce all the same metabolites that a human liver would. “You
don’t want to see a [toxic] drug metabolite for the first time in humans,
one that you hadn’t seen in animal testing,” says Bhatia. The resulting
innovation came about by challenging the current thought that one needed to replace
the mouse liver with a human one. The realization that it was enough to implant
a human liver that could function side-by-side with the mouse liver was the revelation.
Alice Chen, a graduate student in Bhatia’s MIT lab, turned this idea into
reality by grafting these human liver “organoids” into normal
mice to see whether the new tissues could produce human-specific drug metabolites.
Bhatia’s research team determined that HEALs express relevant human drug-metabolizing
enzymes, proving their potential utility for drug metabolism studies. Subsequent
experiments confirmed that HEAL-humanized mice could produce human-specific drug
breakdown products that do not appear in regular mice. HEAL-humanized mice have
also proven useful for probing drug-drug interactions, a critical determinant for
drug safety and efficacy. Such assessments could be used to prevent harmful and/or
ineffective drugs from advancing to clinical testing—thereby saving time and money
while also reducing unnecessary human suffering.
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Liver cells from a human donor (primary hepatocytes), human endothelial cells, and
mouse fibroblasts are encapsulated together in a polymer skeleton and then implanted
in a mouse. The implant forms a so-called human ectopic artificial liver (HEAL).
Mice with HEALs are “humanized” because they harbor a human liver in
addition to their own liver. HEALs provide insight into human biology, predicting
how new drugs might behave in humans. (Figure adapted from PNAS. 2011;108(29):11842–7.)
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Tweaking the recipe
As they already contain a supportive microenvironment, HEALs can be implanted in
the abdominal cavity or under the skin of mice, and they function independently
of the existing mouse liver. Within a week from implantation, HEALs connect with
mouse vasculature and start producing human liver proteins. The same process takes
2–6 months in other humanized mouse models, where transplanted cells need to travel
to the mouse liver and “set up shop.” In these other models, the mouse
liver must be injured to make room for transplanted cells, and mice must be immunosuppressed
to prevent human cell rejection. Ideally, “to get clean results for certain
human metabolites, you need to implant HEALs into different mouse models, including
those with intact immune systems,” says Bhatia. Because the polymer can act
as a barrier to attacks from the immune system, HEALs could potentially be used
in animals with normal immunity.
The initial results in HEAL-humanized mice are very promising, yet there is room
for improvement. Bhatia’s collaborator, Christopher Chen, Skirkanich Professor
of Innovation in Bioengineering at the University of Pennsylvania, is exploring
ways to enhance HEAL vascularization. “Our joint effort is looking at how
biomaterials, cell organization, and different types of cells might support the
hepatocytes to accelerate integration of implanted tissue and extend implant
lifetime,” he says. This research will also address challenges related to
making larger implants for therapeutic applications in people. “We’re
looking at different adhesion molecules and changing how the cells are organized
in the implant.”
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A: To the naked eye, a HEAL looks like a pink contact lens. Approximately 4/5 of
an inch in diameter, the implant comprises half a million liver cells. B: Growth
of blood vessels (red) enables implanted HEALs to grow and function in the mouse.
This miniature human liver was removed from a HEAL-humanized mouse.
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Bhatia is also developing HEAL mouse models for hepatitis C, malaria, and other
human pathogens for which no animal models are available. These new models would
allow researchers to grow human viruses—that normally don’t infect mice—in
a human liver setting to study human drug responses.
In the future, HEALs created from different donor cells could be used to study drug
effects in people who are susceptible to drug-induced liver injury, as well as people
who metabolize drugs at different rates (so-called slow versus rapid metabolizers).
A person’s metabolism rate can affect drug efficacy and safety. “Slow
metabolism can increase exposure to the drug and increase toxic side effects. Fast
metabolism can cause rapid clearance and reduce efficacy,” explains Bhatia.
To study rare liver diseases such as alpha-1-antitrypsin disease, Bhatia is constructing
HEALs from donor cells that can be harvested from blood or skin and converted to
hepatocyte-like cells in the lab. In addition, this new technique offers
the opportunity to evaluate drug-drug interaction in a living organism, something
that is not possible in static in vitro systems.
Chen and Bhatia’s research is laying the groundwork for the option to construct
an implantable artificial liver as an alternative to whole-organ transplantation
for people with liver failure. The PEG-diacrylate skeleton is suitable for organ
printing—that is, assembling organs layer by layer. “Some of the technologies
that we are developing will hopefully even translate to engineering other organs,”
says Chen.
This work is supported in part by the National Institute of Biomedical Imaging and
Bioengineering; the National Institute of Diabetes, Digestive, and Kidney Diseases;
the National Cancer Institute; Howard Hughes Medical Institute; and the National
Defense Science and Engineering; and National Science Foundation Graduate Research
Programs.
References
Chen AA, Thomas
DK, Ong LL, Schwartz RE, Golub TR, Bhatia SN. Humanized mice with ectopic artificial
liver tissues. Proc Natl Acad Sci U S A. 2011 Jul 19;108(29):11842-7.
Underhill GH,
Chen AA, Albrecht DR, Bhatia SN. Assessment of hepatocellular function within PEG
hydrogels. Biomaterials. 2007 Jan;28(2):256-70.
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Sangeeta Bhatia (Photo courtesy of Robert E. Klein/AP, (c) HHMI)
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Alice Chen
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Christopher Chen
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Last Updated On 02/13/2012