Hindawi Publishing Corporation
Journal of Transplantation
Volume 2011, Article ID 202915, 14 pages
MolecularImaging: A PromisingToolto Monitor
Molecular Imaging Laboratory, MGH/MIT/HMS Athinoula A. Martinos Center for Biomedical Imaging,
Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129, USA
Correspondence should be addressed to Anna Moore, firstname.lastname@example.org
Received 31 May 2011; Accepted 29 July 2011
Academic Editor: Antonello Pileggi
Copyright © 2011 Ping Wang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Replacement of insulin production by pancreatic islet transplantation has great potential as a therapy for type 1 diabetes mellitus.
At present, the lack of an effective approach to islet grafts assessment limits the success of this treatment. The development of
molecular imaging techniques has the potential to fulfill the goal of real-time noninvasive monitoring of the functional status and
viability of the islet grafts. We review the application of a variety of imaging modalities for detecting endogenous and transplanted
beta-cell mass. The review also explores the various molecular imaging strategies for assessing islet delivery, the metabolic effects
on the islet grafts as well as detection of immunorejection. Here, we highlight the use of combined imaging and therapeutic
interventions in islet transplantation and the in vivo monitoring of stem cells differentiation into insulin-producing cells.
Type 1 diabetes mellitus (T1DM) is characterized by an ab-
solute deficiency of insulin secretion with hyperglycemia as
a consequence. T1DM is one of the most common diseases
of childhood. 13,000 new cases are diagnosed each year in
North America . The first major breakthrough in the
treatment of T1DM was the isolation of insulin and use of
its synthetic forms. Although insulin has changed the clinical
course of TIDM from an acutely fatal disease to a chronic
one with severe long-term complications, it does not cure
transplantation offers superior glycemic control for T1DM
and prevents or even reverses secondary complications, in-
cluding nephropathy . The elevated risk of surgical com-
plications and the relative invasiveness of the procedure,
however, makes the practice of solid organ transplantation
rare in T1DM patients. Since 1999, when the first accounts
of consistent success in restoring normoglycemia using
islet transplantation appeared, this less invasive procedure
has become an important alternative treatment for T1DM
The first attempt to transplant an islet cell xenograft
was performed in 1893, 29 years before the isolation of
insulin. A 15-year-old diabetic patient was transplanted
and the patient died after 3 days . In 1967, a method
of isolating islets using collagenase was described . This
launched the earliest islet transplantation in animal models
in 1972 . The first clinical islet allograft was performed
in 1974 . The next 25 years have witnessed attempts to
achieve normoglycemia in type 1 diabetic patients using
islet transplantation, with limited success. In 1999, the Ed-
monton protocol revived interest in this procedure by re-
porting reproducible success in terms of insulin indepen-
dence through islet transplantation . All of the patients
maintained insulin independence after 1 year of followup
.This new protocol relies on a prednisone-free immuno-
suppressive regimen and improved islet delivery through
intraportal infusion of freshly isolated islets, followed by a
second or third infusion of additional islets . With this
protocol, from 1999 to 2008, almost 400 patients received
allogeneic islet transplants . However, with more follow-
ups, it became apparent that insulin independence was only
2 Journal of Transplantation
receiving islet transplantation remained insulin independent
at one year after-transplant . Unfortunately, only less
than 10% of the recipients remain insulin independent for
up to 5 years .
It is believed that many factors contribute to the loss of
graft function. Early losses are primarily linked to damage
sustained during the isolation procedure or in the graft mi-
mediated inflammatory reaction (IBMIR) . Subsequent
losses are usually more progressive and involve several im-
munological related factors. The presence of allogeneic re-
jection has been strongly suggested by the poorer clinical
tion and, conversely, by favorable results achieved with more
potent immunosuppression treatment . The recurrence
of autoimmunity also is a major limiting factor in long-
term survival of islet grafts. Some studies have shown that
monocytic infiltration of the graft occurs as early as 14
days after transplantation, with a preferential loss of insulin-
secreting beta cells . After islet transplantation, elevated
islet cell autoantibody titers (to glutamic acid decarboxylase
or GAD65) persisted . Immuosuppressive drug toxicity
is another immune-related assault on the graft. It has been
demonstrated that rapamycin, at a concentration usually
used to prevent islet grafts rejection, is able to reduce the rate
of beta cell proliferation not only in transplanted rat islets
but also in host murine islets, suggesting that the progressive
islet grafts dysfunction observed under immunosuppressive
therapy may result in part from an impairment in beta cells
It is clear by now that an effective approach to islet grafts
assessment following transplantation is urgently needed.
Successful monitoring of the graft would allow us to test
the viability and functionality of the graft. Such monitoring
would also provide a better understanding of the various
mechanisms involved in graft loss. It would also permit
us to design and implement prompt intervention and
more carefully tailored treatment. However, currently the
majority of methods for assessment of the islet grafts are
still indirect. These include indicators of metabolic control,
such as fasting and stimulated glucose levels, oral glucose
tolerance testing (OGTT), C-peptide levels, HbA1c levels,
mean amplitude glycemic excursions (MAGE), and insulin
secretion. In addition, immune events and complications
associated with islet transplantation are indirectly tested as
well. These include allo- and autoimmune antibodies as well
as signs of toxicity or impairment of liver function .
All of these indirect parameters only provide information
on the late stages of graft rejection. Since the mechanisms
behind islet function represent a finelytuned network of
regulated interactions and feedback loops, alterations in C-
peptide and insulin release do not become apparent until
most islets have already been destroyed [20–22]. The only
direct morphological assessment of transplant fate in the
clinic is obtained through histological biopsy. However, it
could not be widely applied due to the small size of islet
grafts and their relatively low frequency dispersed in a large
organ such as the liver. Moreover, this approach is invasive.
Therefore, it is critical to establish a noninvasive method to
monitor the fate of islets directly in a clinical setting.
Molecular imaging is a rapidly emerging biomedical
research discipline. Considerable efforts have been directed
in recent years toward the development of noninvasive high-
resolution in vivo imaging technologies including optical
imaging, nuclear imaging, and magnetic resonance imaging
(MRI). At the same time, various molecular imaging probes
with greater specificity and targeting potential have been
designed and tested (antibodies, ligands, or substrates that
cellular compartments) . The development of molecular
time non-invasive monitoring of the functional status and
viability of the islet grafts after transplantation. The present
paper explores the various preclinical and clinical molecular
imaging strategies for the tracking of graft fate, islet delivery
strategies, as well as detection of immunorejection. We also
review the use of combined imaging and therapeutic ap-
proaches in islet transplantation  and the in vivo mon-
itoring of embryonic stem cells differentiation into insulin-
2.Imaging Beta Cells as a Key Step towards
Progress towards the goal of direct assessment of graft in-
tegrity, viability, and function would rest on previous ex-
perience in the area of pancreatic islet imaging as a tool for
measurement of islet mass and integrity. Imaging beta cells
represents a daunting challenge both from a biological and a
technological perspective, owing to the innately complex
structure and distribution of pancreatic islets within the
function. Pancreatic islets are small structures (100–400μm
in diameter) that are dispersed throughout the pancreas and
constitute only 2% of the pancreatic volume . A variety
of imaging modalities have shown promise exclusively in
2.1. Bioluminescence and Fluorescence Optical Imaging. In
2003, Hara et al.  generated transgenic mice that express
green fluorescent protein (GFP) under the control of the
mouse insulin I gene promoter (MIP). Histological studies
showed that the MIP-GFP mice had normal islet architecture
with coexpression of insulin and GFP in islet beta cells. GFP-
expressing beta cells could be then imaged in vivo allowing
for beta cell mass determination. Our group described
the ex vivo imaging of beta-cell apoptosis with a near-
infrared (NIR) probe (Cy5.5-labeled annexin V) in 2005
. Following, we described the synthesis and testing of an
NIR probe for imaging beta-cells in pancreatic islets, which
was based on the beta-cells-specific ligand streptozotocin
(STZ) labeled with Cy5.5. We observed a bright fluorescence
signal consistent with intracellular accumulation of the
probe, which was mediated by the glucose transporter 2
(GLUT2 transporter) . Another recent example of a
Journal of Transplantation3
fluorescent probe for detecting beta cells is a near-infrared
fluorescent exendin-4 analogue with specificity for the
Glucagon-like peptide 1 (GLP-1) receptor on beta cells. Fol-
lowing intravenous administration into mice, pancreatic is-
lets were readily distinguishable from exocrine pancreas,
achieving target-to-background ratios within the pancreas
of 6:1 using intravital microscopy . Bioluminescence
imaging of beta-cell mass was applied by Virostko et al.
 in a recent study where a transgenic mouse model
expressing luciferase under control of the mouse insulin
I promoter (mouse insulin promoter-luciferase-Vanderbilt
University (MIP-Luc-VU)) was used. This model enabled
non-invasive assessment of changes in beta-cell mass after
islet transplantation based on changes in bioluminescence
2.2. Nuclear Imaging. An attractive approach to image beta
cell is nuclear imaging. The major advantage of nuclear im-
aging is its high sensitivity. It relies on radionuclide-labeled
contrast agents that target beta cells based on cell-specific
antigens, receptors, metabolites, or pharmacologic agents.
Nuclear imaging has been utilized by our group to es-
timate beta-cell mass using an111In-labeled monoclonal
antibody targeting the beta-cell surface antigen IC2 . In
as well as in vivo in a diabetic mouse model. Other examples
of agents that target beta cells directly through surface
antigens include125I-labelled monoclonal antibody R2D6
directed against gangliosides on the plasma membranes
of pancreatic beta cells  as well as phage-display-de-
rived peptides . In order to overcome some of the weak-
antibodies (SCAs) were developed. It was reported that
removal of the Fc portion to produce an antibody fragment
reduced the nonspecific binding to beta cells [34, 35].
Extensive nuclear imaging studies have been performed
targeting receptors expressed on beta-cell surface. The vesic-
ular monoamine transporter 2 (VMAT2) is a monoamine
transporting integral membrane protein expressed by rodent
and human beta cells . Tetrabenazine (TBZ) and Dihy-
drotetrabenazine (DTBZ) specifically bind to the synaptic
VMAT2 . A clinical study showed a reduction in pan-
creatic uptake of11C-DTBZ in long-standing T1D patients
compared to the uptake in the pancreas of healthy control
subjects . DTBZ compounds labeled with
developed to overcome the short half-life of11C-labeled
DTBZ (T1/2 = 20min). Preclinical studies showed high
pancreatic uptake of18F-DTBZ in healthy rats with favorable
biodistribution leading to improved target-to-background
ratios . Recently another new DTBZ derivative, 18F-FP-
(+)-DTBZ, was tested. The result showed that this com-
pound had higher pancreatic uptake and lower uptake in the
nontarget tissues, especially the liver . The glucagon-like
peptide-1 (GLP-1) receptor is a potential target for beta cells
imaging as well. It is triggered after binding of the agonists
Exendin-3 and Exendin-4 . The first preclinical study
using123I-labeled Exendin showed high uptake in the pan-
creas and in subcutaneous insulinomas . Furthermore,
it has been shown that the uptake of111In-DTPA-Lys40-
Exendin-3 correlates with beta-cell mass in a linear manner
in diabetic rats and that the pancreas can be visualized
by SPECT imaging on a dedicated microSPECT scanner
[43, 44]. Radiolabeled pharmacologic agents targeting other
receptors also have been investigated. They include ligands
to sulfonylurea receptors, such as glyburide or tolbutamide
analogs [45–47]. 18F-L-DOPA  targeting dopamine
receptor may also be a suitable approach for the detection
Various tracers based on radiolabeled glucose have also
been explored for targeting beta cells and, of those, the most
mannoheptulose, which is apparently transported into cells
mainly at the intervention of GLUT-2 [49–51].
2.3. MR Imaging. One of the most logical modalities to
explore for imaging of pancreatic islets is MRI. MRI does
not utilize ionizing radiation, has tomographic capabilities,
can deliver the highest-resolution images in vivo, and has
unlimited depth penetration. MR imaging has an overall
low sensitivity in detecting molecular probes (10−3–10−5M)
. However, this drawback can be overcome by the appli-
cation of contrast agents that amplify the signal. New agents
such as a novel class of lanthanide complexes for labeling
beta cells were first reported at the 2003 NIH Workshop on
Imaging Pancreatic Beta cells .1H NMR spectroscopy
has been suggested for measuring choline levels, which can
One group’s studies demonstrated that in vitro1H NMR
imaging could be used to visualize islets or βTC3 cells within
their encapsulated environment. They also showed that
localizing implanted microencapsulation-based bioartificial
pancreas in vivo was feasible with the use of diffusion-
weighted imaging . C13 spectroscopy has also been ap-
plied for studying glucose-stimulated insulin secretion and
has shown promise for the investigation of beta-cell func-
tion . Another interesting approach demonstrated the
feasibility of direct imaging of beta-cell activation in the
presence of divalent manganese cations Mn2+[56, 57].
Recently, Mn2+-enhnced MRI was applied for noninvasive
detection of beta cell function after glucose infusion. Serial
inversion recovery MRI was subsequently performed to
probe for Mn2+accumulation in pancreas. This experiment
demonstrated the potential of Mn2+-enhanced MRI for
noninvasive monitoring of beta cell function .
A multimodal approach for imaging beta cells was
were generated in which beta-cells express a fusion of three
different imaging reporters. Multimodal imaging of MIP-
TF pancreatic beta-cells was demonstrated by fluorescence
microscopy, BLI, and microPET. The MIP-TF mice enabled
noninvasive monitoring of beta-cells in models of type 1 and
type 2 diabetes. This multimodality imaging animal model
might expedite studies in a broad range of diabetes research.
Despite these encouraging examples, however, in the au-
thors’ opinion, the issue of visualizing beta-cell mass in vivo
using noninvasive imaging remains an unsolved challenge.
4 Journal of Transplantation
The ultimate solution to this problem would likely require
the identification of specific beta-cell markers and targeting
ligands, as well as improvements in imaging technology
to permit the acquisition of quantitative information with
both high sensitivity and high spatial resolution. The latter
would probably rely on the development of multimodality
approaches and would demand sophisticated image analysis
tools to monitor even small changes in the signal reflective of
the dynamic nature of beta-cell mass.
the fact that isolated islets can be labeled before-transplant
using various approaches, including genetic modification
with fluorescent or bioluminescent reporters, labeling with
exogenous contrast agents, such as superparamagnetic iron
oxides for MRI or radiolabeled metabolites for nuclear im-
aging. These general strategies for the monitoring of trans-
planted islets by noninvasive imaging have proven valuable
in answering questions about graft fate, designing new ther-
apeutic interventions, and testing alternative transplant sites,
all ultimately aimed at extending graft longevity and func-
Here, we highlight some of the progress made using
different imaging modalities in acquiring new knowledge
about islet transplantation. These studies represent just the
first steps towards realizing the true potential of noninvasive
of noninvasive imaging lies in its capacity to provide in-
formation in authentic physiologic environments, in real-
time, and on a systemic level.
3.1. Bioluminescence and Fluorescence Optical Imaging. The
first evidence of the feasibility of imaging transplanted islets
noninvasively was obtained using bioluminescence imag-
ing (BLI). Several laboratories [60–62] reported proof-
of-principle studies in which isolated rodent or human
islets were genetically engineered to express luciferase and
imaged following transplantation. In islets transplanted
underneath the renal capsule of immunocompromised mice,
the magnitude of the signal was dependent on the islet dose,
indicating that the collected information could be used to
obtain accurate quantitative information about islet number
over time. Although adenovirus-directed luciferase expres-
sion attenuated, consistent with the transient nature of the
vector, lentivirus vectors could be used to direct the long-
term expression of reporter genes in transduced islets. Fur-
thermore, the functionality of transduced islets was retained
since transplanted lentivirus-transduced islets led to long
studies provided new information about islet fate following
transplantation. Luciferase signal emanating from the graft
remained stable for at least 140 days, indicating graft stability
intrahepatic transplantation model. Long-term monitoring
immunocompromised mice, suggested that, in the described
animal model, the intrahepatic islet grafts were also stable,
BLI [64, 65].
The first study of the fluorescence optical imaging of islet
grafts was published in 2003 . In this study, the graft was
our group reported that islets incubated with nanoparticle
probes (labeled with near-infrared fluorescent Cy5.5 dye)
could be detected in the near-infrared channel under the
kidney capsule in vivo after transplantation .
Despite the lack of a clinical equivalent of these modali-
ties, the information gained from BLI and fluorescence im-
aging represents a variable contribution to the field of islet
transplantation. Therefore, these studies demonstrated the
feasibility of conducting preclinical research in small animals
in order to address some basic questions about transplanted
islet biology and begin to develop alternative strategies for
the enhancement of graft function.
3.2. Nuclear Imaging. Unlike bioluminescence imaging,
nuclear imaging has a clinical equivalent and therefore is
more likely to evolve into a modality routinely used in hos-
pitals for monitoring of patients that receive islet trans-
plantation therapy. Initial reports regarding the application
of radionuclide imaging to monitor transplanted pancreatic
islet grafts employed islets genetically engineered to express
a mutant herpes simplex virus type 1 thymidine kinase
(sr39tk). The expressed enzyme can phosphorylate positron-
emitting, radionuclide-labeled thymidine analogues or acy-
cloguanosine substrates once they are delivered inside the
cell, leading to trapping of the phosphorylated product and
its detection by positron emission tomography. In one of
the early studies, islets were transduced with an Adeno-Tkm
adenovirus engineered to express sr39tk under a constitutive
promoter . Mice were subjected to microPET imaging
after injection of the sr39tk substrate [18F] FHBG. As
observed by BLI , the signal was unstable after a few
weeks, likely due to the transient expression of adenovirally
directed reporter genes. By contrast, lentiviral transduction
of pancreatic islets with sr39tk was used for the long-term
monitoring of transplanted islet fate . Islets implanted
in the liver were detectable for several weeks after trans-
plantation, suggesting the persistence of the graft. A similar
gene (interleukin-10), which could prolong the survival of
islet grafts under the kidney capsule of diabetic mice .
Although very valuable from a research perspective the
reporter nuclear imaging studies are not likely to apply in a
clinical scenario in the near future since they involve genetic
modification of the islets before-transplant.
A more clinically relevant technique for the PET imaging
of early posttransplant events involved labeling of rodent
islets with 2-[18F] fluoro-2deoxy-D-glucose (FDG) and their
subsequent implantation in the livers of syngeneic rats. The
grafts were detected for up to 6 hours . This model,
however, is only useful for the short-term monitoring of
Journal of Transplantation5
transplanted islet fate, since the persistence of the label in
the islet cells is unknown. Furthermore, the short half-life
of18F (110min) adds to the difficulty of long-term graft
monitoring. Without a clear understanding of how long
the islets retain the label if at all, it is difficult to obtain
quantitative information about islet abundance through
time, using this method. In subsequent studies performed in
large animals, only ∼50% of the administered radioactivity
was observed in the liver at the end of islet infusion, likely
due to islet damage or FDG leakage from the islets .
Nevertheless, the clinical feasibility of detecting transplanted
islets labeled with [18F] FDG was demonstrated for the first
time in 2007 . The same group reported that islets
labeled with [18F] FDG transplanted to 6 patients could be
detected during the first 1-2h. Beyond this, the radioactive
half-life and retention within the islets limit the use of this
method . Recently, clinical testing of GLP-1 receptor
was applied for imaging of human beta cells transplanted
in patient muscle, which showed its potential to assess islet
survival in clinical transplantation . However, long-term
monitoring after-transplant presents a significant problem
associated with this method.
3.3. Magnetic Resonance Imaging. Magnetic resonance imag-
ing (MRI) is a modality, which, like nuclear imaging, has
a broad clinical applicability and can be used to monitor
transplanted pancreatic islets. Whereas nuclear imaging is
characterized by high sensitivity to contrast agent abundance
concentration of contrast agent, MRI is tomographic and
can acquire images with a high spatial resolution. Therefore,
MRI, unlike nuclear imaging, can be used to study directly
the abundance and tissue distribution of transplanted islets.
The innate low sensitivity of MRI can be overcome with
the use of contrast agents, such as superparamagnetic iron
ticles have been extensively used as magnetic resonance
reporters. Their basic structure includes an iron oxide core
covered with a dextran coat  that can be functionalized
with additional imaging, targeting, or therapeutic moieties.
The presence of iron oxides in tissue is evidenced by a loss in
signal intensity on T2-weighted and T2∗-weighted MR im-
ages or, in technical terms, by a shortening of the T2 re-
laxation time of surrounding water protons.
Several groups focused on superparamagnetic iron
before-transplant, followed by their noninvasive monitoring
human pancreatic islets were labeled with superparam-
agnetic iron oxide magnetic nanoparticles (MNs) mod-
ified with a near-infrared fluorescent dye (MN-NIRF) and
transplanted under the kidney capsule in immunocompro-
mised mice. MN-NIRF-labeled human pancreatic islets were
visualized for up to 188 days after transplantation in this
model demonstrating graft stability and persistence of the
since the graft could restore normoglycemia in diabetic mice
In more recent experiments, we also demonstrated the
applicability of the approach to the intrahepatic transplanta-
tion model. For islet labeling we utilized an FDA-approved
commercially available iron oxide agent (ferumoxides),
which is routinely used in the clinic for liver imaging. Similar
to MN-NIRF, it consists of superparamagnetic iron oxide
covered with a dextran coat. Human islets labeled with
ferumoxides and transplanted into the liver appeared as dis-
tinct hypointense foci representing single islets and/or islet
clusters. The persistence of the graft in immunocompro-
mised animals was demonstrated for the entire observation
period of two weeks .
The applicability of MRI for the visualization of trans-
planted islets, following their labeling with a contrast agent,
was also demonstrated using paramagnetic beads , and a
paramagnetic contrast agent, GdHPDO3A . In another
study, the strong relaxation effect of superparamagnetic iron
oxides allowed the detection of transplanted islets at a lower
magnetic field strength (1.5T), equivalent to the ones used
in hospitals today, advancing the method to a more clinically
relevant stage .
Visualizing transplanted islet in large animal models
using MRI serves as an important step before clinical ap-
plication. The first study in large animals was reported in
2007 . Human islets labeled with immunoprotective iron
oxide-loaded magnetic capsules were detected with real-time
MRI . Recently, our group reported the in vivo imaging
of autologous islet grafts in the liver and under the kidney
capsule in nonhuman primates. The renal subcapsular islet
pocket of signal loss disrupting the contour of the kidney
at the transplantation site. Islets transplanted in the liver
appeared as distinct signal voids dispersed throughout the
liver parenchyma. This study established a method for the
planted into non-human primates using a low-field clinical
MRI system .
The first imaging study applied in humans with super-
paramagnetic iron oxide nanoparticles was carried out in
2008 . Islets were labeled with superparamagnetic iron
oxide particles (SPIO, 280microg/mL) and transplanted into
patients with T1DM. All patients could stop insulin after
transplantation. Three out of four patients had normal
intensity on pretransplant images, and iron-loaded islets
within the liver. However, this clinical study did not show
any correlation between the number of labeled transplanted
islets and the number of hypointense spots on MR images.
In addition, the number of spots varied significantly over
the course of the study making it impossible to make any
conclusions regarding graft outcome. In spite of these short-
islet function is not affected by labeling.
4.Imagingof Islet Grafts Rejection
4.1. Fluorescence Optical Imaging. The earliest report uti-
lizing fluorescence optical imaging for the monitoring of
6 Journal of Transplantation
Figure 1: Transverse T2-weighted magnetic resonance images of transplanted labeled and nonlabeled human islets 14, 23, 37, 58, 97, and
188 d after transplantation under the kidney capsule in nude (nu/nu) mice. The dark area in the left kidney represents a labeled graft (red
outline). No darkening was reported for the right kidney with unlabeled graft. S: stomach; SC: spinal cord, reproduced with permissionfrom
Nature Publishing Group .
immunological effects associated with islet transplantation
relied on genetic engineering to create transgenic mice that
express proinsulin II fused with the live-cell fluorescent
reporter protein, Timer . Since Timer protein changes its
emission wavelength in the first 24hr after synthesis, it can
be used to monitor the time course of insulin synthesis. Islets
expressing this construct were transplanted into recipient
transgenic mice in which T lymphocytes were fluorescently
labeled with a fluorochrome different from Timer. The an-
imals were monitored through a body window device to
derive complementary information about insulin synthesis
and alloimmunity triggered by the engrafted islets. The value
of this method lies in the fact that, for the first time, it allows
the direct analysis not simply of islet abundance but also of
islet function in the context of immune rejection.
Recently, Fan et al. have developed a new reporter mouse
model, which has its T-cell expressing distinct “color-coded”
proteins enablingin vivo detection of differentT-cellsubsets.
With these tools, the authors found notable differences
in the T cell response in islet grafts recipients receiving
tolerance-inducing treatment compared to control group.
the islet grafts immunologic rejection at cellular level .
4.2. Bioluminescence Imaging. A comprehensive investiga-
tion into the relative effects of immune rejection on viable
islet mass was obtained by the transplantation of islets
obtained from a transgenic mouse strain, which consti-
tutively expresses firefly luciferase, underneath the renal
capsule or into the liver of syngeneic or allogeneic strep-
tozotocin-induced diabetic recipients. Whereas, in isografts,
following an almost 50% decrease in signal intensity in
the first 14 days after transplantation, there was an overall
long-term stability of luminescence intensity signals, in allo-
grafts, graft bioluminescent intensity progressively decreased
several days before the permanent recurrence of rejection-
induced hyperglycemia . In nontransgenic syngeneic
recipients transplanted with transgenic islets, high levels of
bioluminescence over the abdominal region of the liver were
detected within 24hr after . Monitoring biolumines-
cence signal from the abdomen of the recipient for more
than 90 days revealed a decline over time. With the caveat
that bioluminescence signal can be influenced by a variety of
factors, such as serum glucose levels, mouse positioning, and
that in the absence of immune rejection islet grafts survival
can be extended significantly. Even though this conclusion is
not surprising, the described method lays the groundwork
for future studies in which various immunosuppression
strategies can be tested for their potential to more closely
emulate a syngeneic immune context.
Journal of Transplantation7
ilar studies to the ones described above in which we em-
ployed a pre-clinical model of islet transplantation at the
hepatic site where islets were monitored by MRI. Immuno-
islet loss as seen on MR images, compared to immunocom-
promised animals. Islet loss in the immunocompetent model
was especially pronounced on day 10 after transplantation
and ultimately resulted in a 20% difference in relative islet
report established a quantitative framework to describe the
rate of islet loss in an immunocompetent context. Because
of the direct comparison between the immunocompetent
and immunocompromised models, this quantitative, non-
invasive, and time-course sensitive imaging method allowed
us to isolate out the relative contribution of allorejection to
the overall decrease in transplanted islet mass in the early
posttransplant period from among a multitude of factors,
such as mechanical stress and vascular disruption .
5.Imagingof Novel Islet TransplantSites
5.1. Bioluminescence Imaging. The clinically relevant site of
islet transplantation is the liver. In research models, islets
are also routinely transplanted underneath the renal capsule.
Still, because of the suboptimal performance of islet grafts
at these two sites, there is an ongoing effort to identify and
test novel transplantation sites that provide a more suitable
vascular environment for the islets, as well as a more im-
munoprotected physiological niche. The epididymal fat pad
has emerged as an alternative transplant site, characterized
by similar outcomes to intraportal transplantation. Mouse
islets implanted into the intra-abdominal epididymal fat pad
restored normoglycemia in STZ-treated recipients . As
few as 50 islets mediated similar levels of glucose tolerance
when transplanted in the fat pad and the liver. When
transgenic luciferase-positive islets were transplanted, bio-
luminescence imaging showed stability of the grafts for
over 5 months. This was further supported by histological
examination of the grafts showing healthy, well-granulated
insulin-containing cells surrounded by healthy adipocytes
. These experiments suggest that the fat pad may be an
alternative site of islet transplantation that is characterized
by outcomes at least equivalent to the intraportal route.
5.2. Laser Scanning Microscopy (LSM). A very interesting
transplantation . The anterior chamber of the eye is a
suitable transplantation site because it provides an immune-
privileged environment and because the high amount of
autonomic nerves and blood vessels found in the iris enables
fast engraftment. Islets were isolated from transgenic mice
expressing enhanced GFP under the control of the rat
insulin-1 promoter (RIP-GFP) and used for transplantation.
Simultaneous two-photon LSM (TPLSM) of beta cells (GFP
signal) and the vascular network (intravenous Texas Red-
conjugated 70-kDa dextran) revealed that, by day 3 after
blood vessels from the iris. By day 14, blood vessels formed a
microvascular network throughout the islet grafts [86, 87].
Furthermore, the authors monitored changes in cyto-
plasmic free Ca2+concentration of the transplanted islets, as
a direct indicator of islet function. Islets were loaded with
the Ca2+indicators Fluo-4 and Fura-Red via perfusion of the
anterior chamber of the eye and imaged using LSM. Changes
in cytoplasmic free Ca2+concentration were successfully
measured following stimulation of beta-cells activity with
glibenclamide [86, 87].
Finally, the authors used LSM to noninvasively image
beta cell death in islets transplanted into the anterior cham-
ber of the eye. RIP-GFP islets were implanted into the ante-
rior chamber of the eye and, after complete engraftment and
vascularization, monitored after intravenous administration
of the fluorescently labeled apoptotic marker annexin V.
These experiments revealed a very low incidence of cell
death in islets transplanted in this site most likely due to its
immune-privileged surroundings [86, 87].
These combined studies illustrate the potential of nonin-
vasive imaging for the collection of comprehensive informa-
tion about transplanted islet biology and function on a sys-
temic level. Islet engraftment, vascularization, function, and
cell death were for the first time examined within a coherent
experimental framework, providing unique knowledge on
the subject of islet transplantation.
Most recently, the same group reported that they used
intravital multiphoton microscopy to monitor transplanted
islets in the anterior chamber of the mouse eye. This tech-
nique allowed for studies at the single-cell resolution and
enabled longitudinal, noninvasive imaging of immune re-
sponse within target tissues during islet allorejection .
5.3. Nuclear Imaging. Lu et al.  implanted islets ade-
novirally transduced with sr39tk into the axillary cavity
of recipient animals. The reason the authors selected this
transplantation site is because untrapped PET probe is
eliminated through the gut and kidney and can create
spillover background signals in the pancreas, kidney, and
sr39tk-expressing islets could be imaged after implantation
into the axillary cavity, which is far from the probe elimina-
tion pathway. In this model, there was a direct relationship
between microPET signal and implanted islet mass. In
longitudinal studies, signal decreased by approximately one-
half during the first few weeks after transplantation, followed
by stabilization over 90 days, suggesting significant islet cell
death in the immediate posttransplant period. Ex vivo anal-
ysis demonstrated that sr39tk-expressing islets transplanted
no evidence of inflammation .
6.Combination of Imagingand
Therapeutic interventions for the enhancement of islet grafts
performance can include immunomodulatory treatments of
8 Journal of Transplantation
Day 2Day 10 Day 14
Figure 2: In vivo imaging of intrahepatically transplanted human islets. (a) Representative images of NOD.scid mice with transplanted
islets. On in vivo images, Feridex-labeled islets appeared as signal voids scattered throughout the liver. (b) Nonlabeled islets were not
detectable using the same imaging parameters. (c) In vivo time course imaging of immune rejection in immunocompetent (upper row)
and immunocompromised (lower row) animals. Representative images are shown from days 2, 10, and 14 after transplantation. Note that
the signal voids (arrows) representing labeled islets/islet clusters tend to disappear faster in Balb/c mice, reproduced with permission from
American Diabetes Association .
the graft or of the host to minimize allorejection or autoim-
munity, methods to expedite graft revascularization, and
approaches aimed at optimizing islet function or survival.
The potential of noninvasive imaging to evaluate these treat-
ments is an important part of their success. In addition,
novel technologies may provide tools combining imaging
and delivery of experimental therapies that aim to extend the
lifespan and functionality of islet grafts.
6.1. Bioluminescence Imaging. In one of the earliest reports
using BLI to evaluate a novel therapeutic strategy for trans-
planted islet immunoprotection, canine pancreatic islets
were encapsulated into biocompatible alginate beads and
transplanted into the dorsal-cervical fat pad of a nuclear
factor-kappa beta (NF-κB) luciferase transgenic mouse
model . Mice were imaged by BLI for up to 45 days
after transplantation to evaluate the long-term inflamma-
tory response provoked by the transplantation. The results
suggested that the trauma of surgery was a more significant
inflammatory trigger than host immune responses to the
capsules and that capsule size, rather than composition,
correlated with an increase in inflammatory activity in this
model. This study is valuable because it establishes the
feasibility of a method that can be used to assess the extent
to which various immunomodulatory strategies provoke an
A more recent investigation attempted to determine
whether early detection of rejection by BLI could aid in the
timing of antilymphocyte serum (ALS) treatment for pro-
longing islet grafts survival . Transgenic islets expressing
the firefly luciferase were transplanted under the kidney cap-
sule of streptozotocin-induced diabetic allogeneic immuno-
competent mice. The animals received anti-lymphocyte
therapy whose effect on islet survival was monitored by BLI.
Imaging was proven useful in designing an optimal timing of
therapy administration resulting in a close to 60% reduction
Journal of Transplantation9
in grafts loss from rejection. Interestingly, the same success
could not be achieved using blood glucose levels as a guide
for the timing of therapy.
6.2. Nuclear Imaging. Nuclear imaging has been used to
determine the feasibility of PET reporter gene (PRG) and
PET reporter probe (PRP) technology for tracking islet
grafts survival and quantifying the expression of poten-
tial therapeutic genes . The authors generated a dual
gene—expressing recombinant adenovirus rAD-vIL10-ITK,
which coexpresses viral interleukin-10 (vIL-10) and HSV1-
sr39tk. vIL-10 was chosen because it protects transplanted
islets from immunological attack by regulating autoimmune
activity. Islets from nondiabetic NOD mice were infected
with rAD-vIL10-ITK or rAD-ITK, transplanted under the
right kidney capsule of diabetic NOD mice, injected with
[18F] FHBG, and scanned by microPET. PET signals in
mice transplanted with islets infected with rAD-ITK (rAD-
ITK mice) were decreased at 3d and had almost reached
basal values at 14d after transplantation. Imaging revealed
a relative extension of islet grafts survival in the presence of
IL-10 treatment , illustrating the potential utility of anti-
inflammatory therapy in islet transplantation.
6.3. Magnetic Resonance Imaging. Magnetic resonance imag-
ing also has been explored for the pre-clinical assessment of
therapeutic interventions in the context of islet transplanta-
and described the application of immunoprotective magne-
tocapsules containing ferumoxides, as carriers of pancreatic
islets . Islet-containing magnetocapsules were infused
and engrafted into the liver. MRI could monitor this process
in real time because of the incorporation of ferumoxides into
A similar multifunctional rationale aimed at exploring
the possibility for concurrently imparting imaging and ther-
apeutic capabilities to transplanted pancreatic islets has also
been behind some of our most recent work . Taking
advantage of the propensity of pancreatic islets to avidly take
up dextran-coated superparamagnetic iron oxide nanopar-
ticles, we designed a probe that consisted of a dextran-
coated iron oxide core, conjugated to small interfering RNA
(siRNA). Considering the potential of siRNA as a novel
class of small molecule drugs capable of selectively silencing
the expression of essentially any gene of choice with single
nucleotide specificity, we speculated that we could explore
the mechanism of RNA interference in the context of
islet transplantation. As proof of concept, we showed that
siRNA tagged to magnetic nanoparticles could accumulate
in pancreatic islets in quantities sufficient for detection by
MRI in vitro and for silencing target genes (green fluorescent
protein [GFP] was used as a model gene) .
A more recent study by our group used a dual-purpose
therapy/imaging nanoparticle probe to target the apoptotic-
related gene caspase-3. We demonstrated that our “two-
in-one” MN-siCaspase-3 imaging probe could silence the
apoptotic-related gene, providing significant protection to
the grafts from early loss after transplantation, and at the
same time served as an MRI label to assess the in vivo post-
transplant fate of the grafts noninvasively (Figure 3) [24, 91].
The results of our study are in line with those of another
recently published study showing that the use of fluorinated
alginate microcapsules increased the insulin secretion rate of
human islets and at the same time allowed detection by MRI
and CT imaging .
In the context of islet transplantation, these studies
are valuable because they lay the groundwork for future
applications, in which genes implicated in islet grafts loss
(immunological or nonimmunological) can be similarly
targeted in order to improve graft outcome. Furthermore,
the inherent imaging capabilities of the approach permit the
noninvasive tracking of therapeutic moiety conjugated to the
contrast agent and its relationship to graft fate.
7.Imagingof Metabolic Effects on IsletGrafts
One factor normally present during clinical transplantation
and influencing transplanted islet fate is glucotoxicity caused
by chronic hyperglycemia. To assess its input to the graft
outcome, our group labeled human pancreatic islets with
iron oxides and transplanted them into hyperglycemic and
normoglycemic animals . Noninvasive MRI monitored
the fate of the grafts in the two groups. We found that
in diabetic animals there was a significantly higher rate of
islet death than in the normoglycemic counterparts. The
half-life of an islet in the diabetic group was 4.8 ± 1.1
days compared with 12.6 ± 2.9 days for control nondiabetic
mice (P< 0.05). This is the first in vivo study that
confirms previous in vitro reports demonstrating that severe
transplantation depends on the degree of hyperglycemia in
the recipient [96–103].
Pluripotent stem cells have the potential to differentiate into
specialized cells of all three primary germ layers. Embryonic
stem (ES) cells and the newly developed induced pluripotent
stem (iPS) cells are an ideal source for generating insulin-
producing cells (IPCs) that could be used to treat diabetes.
Most recently, BLI was used for monitoring ES cell sur-
vival and differentiation into insulin-producing cells in a
diabetic animal model. This group generated a double trans-
genic mouse ES cell line ectopically expressing Pdx1-
aequorea coerulescens green fluorescent protein (AcGFP)
fusion protein, and rat insulin promoter- (RIP-) driven
luciferase reporter [93, 104]. Real-time noninvasive BLI was
used to monitor cell fate and function after transplantation.
They speculated that, in vivo, pancreatic endoderm-like cells
(PELCs) migrate into the streptozotocin-damaged pancreas
and differentiate into IPCs. The in vivo differentiation of
double transgenic ES cells transplanted under the renal
capsule or systemically infused cells could be imaged by BLI
as early as day 3 and until day 35 after-transplantation .
10 Journal of Transplantation
Day 1Day 3 Day 5Day 7Day 14
Days after Tx
Figure 3: (a) Representative in vivo MRI of islet transplantation showing MN-siCaspase-3-treated islets implanted under the left kidney
(inset) and parental MN-treated islets implanted under the right kidney. The dark area outlined under both kidney capsules represents the
labeled grafts (day 3 shown). (b) Semiquantitative assessment of the relative changes in graft volumes revealed protective effect in MN-
siCaspase-3-labeled grafts (∗day 7, P < 0.05;∗∗day 14, P < 0.05). (c) Fluorescence microscopy revealed higher expression of insulin and
lower expression of caspase-3 in MN-siCaspase-3-treated grafts compared with MN-labeled islet grafts on the 14th day after-transplantation
(Tx) (green, insulin; red, cleaved caspase-3; blue, DAPI nuclear stain) (magnification bar = 50mm). (d): TUNEL assay on insulin-stained
sections confirmed lower apoptotic rate and higher insulin expression in islets treated with MN-siCaspase-3 compared with islets treated
with MN on the 14th day post-Tx (red, insulin; green, TUNEL; blue, DAPI nuclear stain) (magnification bar = 50μm), reproduced with
permission from American Diabetes Association .
glucose homeostasis in the patient with diabetes. Nev-
ertheless, the processes behind islet isolation, delivery, en-
graftment, and proper functioning in a new metabolic and
immunological environment are complex and still poorly
understood. Here, we have briefly discussed the important
questions related to islet transplantation that could be tack-
led through in vivo molecular imaging. They bridge a range
of topics, including graft longevity in the context of immune
rejection and hyperglycemia, effects of autoimmunity and
strategies to alleviate immune-mediated deterioration of
graft viability and function, and the exploration of novel
Still, despite the significant value of these studies, they
reflect only a small proportion of the issues that face the field
of islet transplantation. Questions that remain unresolved or
need to be studied in more detail span from the mechanics
of islet engraftment and vascularization to the molecular
aspects allowing islets to adapt to a new immunological,
physiologic, and metabolic niche distinct from the pancreas,
the importance of factors, such as islet innervation, ambient
metabolite, ion, or signaling molecule concentrations for
proper islet function, and so forth. Furthermore, having in
mind the limited supply of donor tissue, another crucial
subject to investigate would involve the potential of trans-
planting nonislet tissue, such as stem cells or cell types that
can be induced to differentiate or transdifferentiate into
functional islet tissue.
Since molecular imaging has the advantage of delivering
quantitative temporal information in intact, authentic phys-
iologic environments and on a systems’ level, its potential
for comprehensively addressing the issues of immediate
interest to islet transplantation is apparent. Development of
Journal of Transplantation 11
probes targeting biomarkers linked to beta-cell function and
combining therapeutic and diagnostic (theragnostics) tools
for improving islet transplantation should be encouraged.
With the first feasibility steps having been already made, the
near future should see rapid progress.
This paper describes the work supported in part by
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