Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.
ABSTRACT Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the context of the living organism, and methods of analyzing these processes in vivo are essential for development of effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to noninvasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraftment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally irradiated, syngeneic recipients. The transplantation of autologous pancreatic Langerhans islets and of allogeneic heart revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance studies across various disciplines involving transplantation.
- [Show abstract] [Hide abstract]
ABSTRACT: Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.(1) developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.Journal of Visualized Experiments 01/2012;
- Type 1 Diabetes - Pathogenesis, Genetics and Immunotherapy, 11/2011; , ISBN: 978-953-307-362-0
- InTech. 01/2011;
Molecular Imaging Using Labeled Donor Tissues
Reveals Patterns of Engraftment, Rejection, and
Survival in Transplantation
Yu-An Cao,1Michael H. Bachmann,1Andreas Beilhack,2Yang Yang,3Masashi Tanaka,5
Rutger-Jan Swijnenburg,5Robert Reeves,1Cariel Taylor-Edwards,2Stephan Schulz,2Timothy C. Doyle,1
C. Garrison Fathman,2Robert C. Robbins,5Leonore A. Herzenberg,3,4Robert S. Negrin,2,4and
Christopher H. Contag1,4,6
Tissue regeneration and transplantation of solid organs involve complex processes that can only be studied in the
context of the living organism, and methods of analyzing these processes in vivo are essential for development of
effective transplantation and regeneration procedures. We utilized in vivo bioluminescence imaging (BLI) to nonin-
vasively visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that
constitutively expresses luciferase. Dynamic early events of hematopoietic reconstitution were accessible and engraft-
ment from as few as 200 transplanted whole bone marrow (BM) cells resulted in bioluminescent foci in lethally
revealed the tempo of transplant degeneration or immune rejection over time. This imaging approach is sensitive and
reproducible, permits study of the dynamic range of the entire process of transplantation, and will greatly enhance
studies across various disciplines involving transplantation.
Keywords: Molecular imaging, Transplantation, Tissue regeneration, Stem cells.
(Transplantation 2005;80: 134–139)
tion of functional organs by cell and tissue transplantation.
Imaging strategies that noninvasively reveal cell viability and
functional information in the context of the living body can
accelerate effective development of new methods that would
One of the cornerstone technologies in the field of molecular
imaging, in vivo BLI, is based on the observations that light
in the living body (1–3). A variety of gene transfer tools have
been developed to deliver the luciferase reporters to cells
trafficking patterns of lymphocytes within the body (4, 5), to
expressing stem cells over time (6), and to examine stem cell
engraftment in cardiac tissue (7).
To assess survival and regeneration using BLI, it is es-
sential that the transplant constitutively and uniformly ex-
presses luciferase throughout the study period. To date, gene
transfer efficiency remains as an obstacle, especially for pri-
mary cells, tissues and intact organs, and stable reporter ex-
pression in particular cell types over time can be variable.
Thus, current methods of introducing reporter genes are not
sette controlled by a promoter that is largely constitutively
expressed meet these criteria and would provide a source of
uniformly labeled biological materials for transplantation
transgenic mouse line that incorporates a luciferase reporter
driven by a constitutive promoter, and demonstrate in a se-
ries of transplantation experiments the ease and success of
The transgene in this line is comprised of a CMV-?-
actin promoter (8) and a biscistronic gene consisting of two
reporter genes, firefly luciferase and enhanced green fluores-
cence protein (eGFP), separated by a 2A ribosome slippage
site from foot and mouth disease virus (FMDV), to allow
gous mice in this FVB.luc? line, designated as L2G85, indi-
cated that both the bioluminescent and fluorescent signals in
the heterozygote were lower than those in homozygote (Fig.
1B and C). While GFP is expressed mainly in skin (Fig. 1D),
This work was funded in part through a Small Animal Imaging Resource
Program (SAIRP) grant from the National Cancer Institute (NCI, grant
Health (RO1 DK58664, P20 CA86312–01, RO1 CA80006–01, PO1
CA49605–14) and the Juvenile Diabetes Foundation (4–2001–9).
eda, CA. Other authors have no competing interests.
1Departments of Pediatrics, Radiology, and Microbiology & Immunology,
Stanford School of Medicine, Stanford, California.
2Department of Medicine, Stanford School of Medicine, Stanford,
3Department of Genetics, Stanford School of Medicine, Stanford,
4Program in Immunology, Stanford School of Medicine, Stanford,
ter, 318 Campus Drive, Stanford, CA 94305–5427.
Received 31 August 2005. Revision requested 28 October 2004. Accepted 28
Copyright © 2005 by Lippincott Williams & Wilkins
Transplantation • Volume 80, Number 1, July 15, 2005
BLI of dissected tissues showed that luciferase is expressed in
heart, spleen, muscle, pancreas, skin, thymus, and BM (Fig.
1E and data not shown).
To enable the assessment of multilineage hematopoi-
etic reconstitution originating from a small number of BM
of luciferase to permit monitoring by BLI. We therefore
quantified the bioluminescence of purified hematopoietic
subsets. Splenocytes were sorted by flow cytometry and then
lysates in a standard luciferase assay. Although they usually
produced light less intensively immediately after sorting,
sorted cells recovered after incubation at 37°C for 4–12 hr
and exhibited varying levels of luciferase activity in all leuko-
B cells, NK1.1?NK cells, and Gr-1?Mac-1?granulocytes
(Fig. 2A). Light production of different subsets ranged from
0.02 to 0.14 photons/second/cell in the live cell assay after 12
hr in the incubator and 0.15 to 1.30 photons/second/cell in
significant levels in mature erythrocytes (Ter119?CD45?),
but low levels of activity are detected in erythrocyte precur-
The transgene is comprised of a hybrid CMV-chicken-?-actin promoter, a firefly luciferase gene, a FMDV 2A ribosomal
slippage site and GFP gene. The luciferase, 2A and GFP are in one open reading frame. Primers P1 & P2 are for amplifying
the first 750 bp of CMV-?-actin promoter and P3 & P4 are for GFP. Positive results from both PCR suggest the presence of an
(3 days) and adult (8 weeks) L2G85 mice showed a green fluorescent signal in the upper epidermal layers, that is, stratum
granulosum and stratum corneum. This signal could be confirmed by immunofluorescence microscopy, using an antiGFP
antibody, detected by a conjugated secondary antibody (Alexa 546, red). No specific green fluorescent signal and no anti
GFP reaction was observed on corresponding skin samples of adult Balb/c control mice. Nuclei were stained with DAPI
(blue). (E) A representative 30 ?m cryosection of a 3-day-old L2G85 Tg mouse is shown. Panel A: color image; Panel B:
luminescence image after application of a luciferin/ATP solution. Light emission was recorded as previously described.
Generation of luciferase-expressing transgenic mouse. (A) Transgene composition and PCR primer location.
© 2005 Lippincott Williams & Wilkins
Cao et al.
topoietic stem cells (HSCs) express luciferase at the highest
level among the different hematopoietic cell types, with 3
levels in the sorted live cells since there are excess amounts of
ATP and a CoA enzyme in the lysate assay. The consistent
pattern of activity between the two assays indicates that the
live cell assay reflects relative expression levels in each cell
To test if we could visualize the dynamics of hema-
topoietic engraftment from whole BM cells of this trans-
genic line and how the kinetics of reconstitution from
whole BM differ from that of highly purified KTLS HSCs
(10), 5 million whole BM cells were transplanted into le-
thally irradiated syngeneic recipients. Bioluminescent sig-
nal was detected from the splenic area 15 min after cell
transfer (data not shown). Images obtained at 1 day post-
2B). Three days later, four views of the animals revealed
intense signals from the bones, most likely from the mar-
row, and secondary lymphoid organs, spleen and lymph
nodes. Hematopoietic reconstitution, measured by inte-
grated whole body photon emission, rapidly progressed
(Fig. 2B). Engraftment from luc?BM peaked at 5 weeks
in multiple cell lineages were detected. (B), (C) Dynamics of hematopoietic reconstitution after BM transplantation. 5? 106
whole BM cells from L2G85 were transferred via tail vein into lethally irradiated syngeneic recipients and bioluminescent
imaging was performed at different time points (day 0 through 70). (B) representive images during hematopoietic engraft-
ment. (C) Kinetics of hematopoietic reconstitution. (D) Small numbers of transgenic BM cells can be detected as individual
foci of engraftment. Two hundred whole BM cells were transferred into lethally irradiated recipients along with 3? 105
helper non-transgenic BM cells. Bioluminescent foci were detectable 10 day after transplantation. Some of the foci ex-
panded and engrafted hematopoietic compartment at a detectable level (upper panel) and others contributed little to
hematopoietic reconstitution (lower panel).
Transplantation • Volume 80, Number 1, July 15, 2005
emission [photons/sec/cm2] is represented according to the adjacent scale in a false color image overlay. (B) BLI of a FVB
of the abdominal region of interest. The formula best fitting to the data is a power law equation, describing a linear curve in
a log-log plot, with an R2value of 0.906. (D) Transplanted bioluminescent cardiac allograft showed a decrease in light
production over time by 12 days after transplantation. L2G85 mice were bred with a GFP transgenic line (GFPU). Cardiac
allografts from the offsprings of this breeding (FVB) were transplanted into Balb/c recipients and all acutely rejected by 12
days. Correlating with decrease in light production, immunohistochemistry shows a decline in viable cardiomyocyte
structure and an increase in inflammatory cells. In the lower panel of histology pictures, red, inflammatory cells (CD45
stain); green, cardiomyocyte (GFP); blue, nuclei (DAPI stain) (magnification?400?).
Pancreatic islet and cardiac transplantations. (A–C) Transplanted bioluminescent pancreatic islets expire at
© 2005 Lippincott Williams & Wilkins
Cao et al.
after transplantation, and remained high thereafter (Fig.
2C). To confirm the presence of significant numbers of
donor-derived hematopoietic cells in the recipients, we
sorted myeloid and lymphoid cells from peripheral blood.
In vitro luciferase assays of these sorted cells showed that
there were significant numbers of donor-derived periph-
eral blood cells and that hematopoietic reconstitution was
multilineage (data not shown). We did not observe trans-
plant rejection in these syngeneic models, as based on the
To assess whether engraftment of small numbers of
cells in BM could be detected, we transferred 200 whole
BM cells into lethally irradiated syngeneic recipients along
with 3? 105nontransgenic helper BM cells. Biolumines-
frequently at anatomic sites corresponding to the location
of the spleen, skull, vertebrae and femurs (Fig. 2D and data
not shown). Confirmation of tissue origin was obtained by
imaging from different views and in some instances, im-
mediately postmortem. Surprisingly, we detected biolumi-
nescent foci arising from engrafting cells in 12% of recip-
ients who had received 200 BM cells each. This suggests
that the foci observed may be derived from both HSCs and
hematopoietic progenitors since the HSC frequency in BM
is usually about 0.01% (11).
Transplantation of pancreatic islets into patients suf-
fering from type 1 diabetes mellitus has become increasingly
applied but ascertaining the presence and quantity of beta
cells has proven difficult (12). BLI has been used to track
lentiviral vector transduced human or mouse islets after
transplantation (13–15). Since a transgenic donor with uni-
form integration sites could potentially offer greater signal
stability, we purified Langerhans islets from L2G85 pancreas
(16) for transplantation. These islets emitted significant
these islets were transferred into a non-transgenic syngeneic
recipient via portal vein injection, high levels of biolumines-
cence over the abdominal region of the liver were detected
within 24 hr (Fig. 3B). Monitoring abdominal biolumines-
over time that was best described by a power law equation
(Fig. 3C). Islet transplantation under the kidney capsule of
syngeneic mice produced comparable results whereas alloge-
neic recipients rejected the grafts within 3 weeks (data not
occuring with high frequency in islet transplant models (17)
or to a gradual loss of luciferase gene expression in the trans-
planted islets since the bioluminescent signal remains stable
reason(s) for the decline. Nevertheless, BLI offers the possi-
bility, in conjunction with conventional assays, to rapidly
evaluate the efficacy of therapeutic strategies to improve islet
Hearts from the L2G85 mice also exhibit biolumi-
nescence that can serve as a marker for survival and rejec-
tion of transplants. When transplanted in an acute rejec-
tion model (18) into the abdominal cavity (19) of Balb/c
mice, i.e. across a major MHC mismatch barrier, all L2G85
cardiac allografts were acutely rejected by 12 days. Light
production from the cardiac allografts correlated with
their viability, as it gradually declined over time after
transplantation (Fig. 3D). L2G85 mice were bred with a
GFP transgenic line (FVB.Cg-Tg(GFPU)5Nagy) (20) to
L2G85XGFPU. Hearts of these double transgenic mice
were rejected after transplantation with kinetics identical
to those of L2G85 transplants. The course of rejection was
corroborated by immunohistochemical analysis of trans-
of inflammatory cell infiltration and a decrease in the
number of viable cardiomyocytes with time during the
acute rejection phase (Fig. 3D).
Future applications of BLI with luciferase transgenic
mice are distributed widely across biomedical fields. While
the studies presented here demonstrate the use of this lu-
ciferase transgenic mice as a highly useful donor for track-
ing transplanted cells, tissues and organs, realization of the
the physiological behavior and function of transplanted
cells, tissue and organs, and the evaluation of new trans-
plantation protocols in an rapid and reliable fashion.
in living mammals. Nature Medicine 1998; 4: 245.
Contag CH, Contag PR, Mullins JI, et al. Photonic detection of bacte-
rial pathogens in living hosts. Molecular Microbiology 1995; 18: 593.
in living mammals using a bioluminescent reporter. Photochemistry
and Photobiology 1997; 66: 523.
Edinger M, Cao YA, Verneris MR, et al. Revealing lymphoma growth
imaging. Blood 2003; 101: 640.
Nakajima A, Seroogy CM, Sandora MR, et al. Antigen-specific T cell-
Investigation 2001; 107: 1293.
etic stem cell engraftment using in vivo bioluminescence imaging.
Blood 2003; 102: 3478.
transplantation in living animals using optical bioluminescence and
positron emission tomography. Circulation 2003; 108: 1302.
Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expres-
sion transfectants with a novel eukaryotic vector. Gene 1991; 108: 193.
Donnelly ML, Luke G, Mehrotra A, et al. Analysis of the aphthovirus
2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic
reaction, but a novel translational effect: a putative ribosomal ‘skip’.
J Gen Virol 2001; 82: 1013.
Cao YA, Wagers AJ, Beilhack A, et al. Shifting foci of hematopoiesis
during reconstitution from single stem cells. Proc Natl Acad Sci U S A
2004; 101: 221.
Morrison SJ, Qian D, Jerabek L, et al. A genetic determinant that spe-
2002; 168: 635.
Lu Y, Dang H, Middleton B, et al. Bioluminescent monitoring of islet
graft survival after transplantation. Mol Ther 2004; 9: 428.
Chen J, Kaufman DB. Bioluminescent Imaging of Pancreatic Islet
Transplants. Current Medicinal Chemistry – Immunology, Endocrine
and Metabolic Agents. 2004; 4: 301.
Transplantation • Volume 80, Number 1, July 15, 2005
15.Powers AC, Jansen ED. Current Medicinal Chemistry – Immunology.
Endocrine and Metabolic Agents 2004; 4: 339.
Lejon K, Fathman CG. Isolation of self antigen-reactive cells from in-
flamed islets of nonobese diabetic mice using CD4high expression as a
marker. J Immunol 1999; 163: 5708.
Ricordi C, Strom TB. Clinical islet transplantation: advances and im-
munological challenges. Nat Rev Immunol 2004; 4: 259.
Miura M, Morita K, Kobayashi H, et al. Monokine induced by IFN-
gamma is a dominant factor directing T cells into murine cardiac allo-
grafts during acute rejection. J Immunol 2001; 167: 3494.
Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of
hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in
rejection. Transplantation 1973; 16: 343.
Hadjantonakis AK, Gertsenstein M, Ikawa M, et al. Generating green
Mech Dev 1998; 76: 79.
© 2005 Lippincott Williams & Wilkins
Cao et al.