Am J Physiol Heart Circ Physiol 301: H663–H671, 2011.

First published June 10, 2011; doi:10.1152/ajpheart.00337.2011. Review

In vivo bioluminescence for tracking cell fate and function

Patricia E. de Almeida,1,2 Juliaan R. M. van Rappard,4 and Joseph C. Wu1,2,3
Departments of 1Medicine and 2Radiology and 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford
University School of Medicine, Stanford, California; and 4Leiden University School of Medicine, Leiden, The Netherlands
Submitted 4 April 2011; accepted in final form 1 June 2011

de Almeida PE, van Rappard JR, Wu JC. In vivo bioluminescence for

tracking cell fate and function. Am J Physiol Heart Circ Physiol 301: H663–H671,
2011. First published June 10, 2011; doi:10.1152/ajpheart.00337.2011.—Tracking
the fate and function of cells in vivo is paramount for the development of rational
therapies for cardiac injury. Bioluminescence imaging (BLI) provides a means for
monitoring physiological processes in real time, ranging from cell survival to gene
expression to complex molecular processes. In mice and rats, BLI provides
unmatched sensitivity because of the absence of endogenous luciferase expression
in mammalian cells and the low background luminescence emanating from animals.
In the field of stem cell therapy, BLI provides an unprecedented means to monitor
the biology of these cells in vivo, giving researchers a greater understanding of their
survival, migration, immunogenicity, and potential tumorigenicity in a living
animal. In addition to longitudinal monitoring of cell survival, BLI is a useful tool
for semiquantitative measurements of gene expression in vivo, allowing a better
optimization of drug and gene therapies. Overall, this technology not only enables
rapid, reproducible, and quantitative monitoring of physiological processes in vivo
but also can measure the influences of therapeutic interventions on the outcome of
cardiac injuries.
bioluminescence imaging; heart; gene therapy; stem cell therapy; in vivo cell
tracking

THIS ARTICLE is part of a collection on Assessing Cardiovas- Histopathological examination has been the main approach
cular Function in Mice: New Developments and Methods. for ex vivo evaluation of the distribution, the engraftment, and
Other articles appearing in this collection, as well as a full the differentiation of injected cells. However, histopathology
archive of all collections, can be found online at http://ajpheart. precludes the evaluation and monitoring of these parameters in
physiology.org/. real time and in vivo. Molecular and cellular imaging has
Cell-based therapies have rapidly emerged as a potential provided various techniques for identifying and tracking trans-
therapeutic approach for heart disease. After the initial work to planted cells in cardiovascular research (15). Magnetic reso-
characterize putative endothelial progenitor cells (1) and their nance imaging (MRI), computed tomography (CT), positron
potential to promote cardiac neovascularization and to attenu- emission tomography (PET), and single photon emission com-
ate ischemic injury, a decade of intense research has examined puted tomography (SPECT) offer deep tissue penetration and
several novel approaches to promote cardiac repair in adult high spatial resolution (64, 70, 94a). However, in small animal
life. A variety of adult stem and progenitor cells from different studies, these techniques are more costly and time consuming
sources have been examined for their potential to promote to implement compared with optical imaging. Among the
cardiac repair and regeneration: bone marrow-derived cells optical imaging tools, bioluminescence imaging (BLI) is a
(55, 72), circulating and mobilized CD133⫹ and CD34⫹ stem promising technique that is especially useful in small animal
and progenitor cells (36), mesenchymal stem cells (56, 65, 83), models and does not require the use of radionuclides with their
cardiac resident stem cells (43, 62), and skeletal myoblasts (54, associated hazards. BLI is a high throughput technique that can
97). However, many questions such as the optimal type and provide unmatched sensitivity because of the absence of en-
dogenous luciferase expression in mammalian cells and the
number of progenitor cells to be administered, the route of
low background luminescence emanating from animals. BLI
administration, and the best time to administer cells after injury
can be very useful in evaluating the delivery efficiency of
remain unresolved. This is particularly true in vivo where it is
therapeutic genes and their expression levels in vivo. By the
difficult to assess cellular activity in the heart in real time.
use of different cellular promoters to drive the expression of a
Therefore, it would be helpful to have practical tools to luminescent reporter gene, BLI allows monitoring of the trans-
accurately track cell location and their functional status over planted cells’ differentiation status as well as their location and
time in vivo. functional characteristics in vivo (39, 51, 72, 78, 94).
Overall, BLI of reporters cloned into promoter/enhancer
Address for reprint requests and other correspondence: J. C. Wu, Stanford
sequences or engineered into fusion proteins has demonstrated
Univ. School of Medicine, Lokey Stem Cell Research Bldg., 265 Campus Dr., the modality’s ability to monitor fundamental processes such
Rm. G1120B, Stanford, CA, 94305-5454 (e-mail: [email protected]). as transcriptional regulation, signal transduction cascades, pro-
http://www.ajpheart.org 0363-6135/11 Copyright © 2011 the American Physiological Society H663
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H664 IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION

Fig. 1. Diagram illustrating steps required for biolumines-

cence imaging (BLI). A: therapeutic cells are transduced
with a luciferase (Luc) reporter construct and injected into
an animal. B: alternatively, luciferase-expressing cells can
be isolated from a luciferase transgenic animal and injected
to the experimental subject. C: after systemic substrate
injection, a charge-coupled device camera can be used to
localize the luciferase photon signals in vivo. Pseudo-col-
ored images that represent signal intensity are overlaid with
grayscale reference images of the animal to facilitate local-
ization of the signal. Cell survival, expansion, and homing,
as well as the expression of therapeutic genes, can be
monitored using this strategy.

tein-protein interactions, protein degradation, oncogenic trans- BLI is based on the detection of light emitted by cells that
formation, cell trafficking, and targeted drug action under in express light-generating enzymes such as luciferase. In biolu-
vivo spatial registration. In this review, we will discuss recent minescent reactions, luciferase generates visible light through
advances and applications of BLI, specifically as they apply to the oxidation of enzyme-specific substrates such as D-luciferin
cardiovascular research. for terrestrial organisms (29, 30) and coelenterazine for marine
organisms (31, 53). Luciferases from different organisms can
Principles of Bioluminescence be distinguished by their abbreviations: lux (bacterial), luc
In nature, numerous luminous species exist in more than 700 (firefly), and lcf (dinoflagellate). To track cells in vivo by BLI,
genera, of which 80% are marine species (93). Luciferase the cells of interest need to be genetically modified to express
enzymes have been cloned from both marine (e.g., Renilla luciferase. The animal recipient of the cells receives the lu-
luciferase) and terrestrial (e.g., firefly and click beetle lu- ciferase substrate either intraperitoneally or intravenously and
ciferase) eukaryotic organisms and are commonly used as is placed in a light-tight dark box where luminescence is
reporters for in vitro and in vivo studies. They emit long detected (Fig. 1). In a bioluminescent reaction, the generation
wavelengths of bioluminescence (⬎600 nm) in the red and of light depends on several factors. Firefly luciferase requires
near-infrared regions of the spectrum and are efficiently trans- ATP, Mg2⫹, and oxygen to catalyze the oxidation of its
mitted through mammalian tissues (20, 86). These wavelengths substrate D-luciferin and generates CO2, AMP, inorganic py-
can avoid absorbing and scattering environment of mammalian rophosphate, oxyluciferin, and a yellow-green light at a wave-
tissues (69), thus can be efficiently detected outside a small length that peaks at 562 nm (Fig. 2). By comparison, Renilla
animal’s body using BLI. luciferase catalyzes the oxidative decarboxylation of coelen-

Fig. 2. Representation of the bioluminescence reaction.

A cell expressing the firefly luciferase reporter gene
produces the luciferase enzyme that in the presence of
oxygen, ATP, and Mg2⫹ catalyzes D-luciferin into oxy-
luciferin, CO2, inorganic pyrophosphate (PPi), and light.
Light can then be detected, collected, and quantified by
a charge-coupled device camera.

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IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION H665
terazine in the presence of dissolved oxygen to yield oxylu- emitted. A mutation of a single amino acid has been shown to
ciferin, CO2, and blue light that peaks at 480 nm (49). Biolu- cause red-shifted luminescence and improve in vivo perfor-
minescence generated by the luciferase reporter reaction is mance. Recently, a red-shifted mutant of luciferase from Pho-
captured by a cooled charge-coupled device camera that can tinus pyralis was created and described to have an emission
detect very low levels of visible light emitted from internal maximum of 612 nm at pH 7.0, a narrow emission bandwidth,
organs (19). Charge-coupled device camera-imaged biolumi- and to be thermostable (with a half-life 8.8 h at 37°C vs. 0.26
nescence can then be superimposed on photographic images of h for the wild-type luciferase). This Photinus pyrallis mutant,
the mouse to detect quantitatively and repetitively the biolu- called Ppy RE-TS reporter, has been successfully used in small
minescent signal from a given location (95). Imaging can be animals to visualize cancer progr ession and shown to have
conducted 10 to 15 min after intraperitoneal injection of superior in vivo imaging performance compared with the
D-luciferin (reporter probe), with relatively stable light emis- wild-type photinus pyralis luciferase (6, 7).
sion levels for 30 to 60 min, depending on the experimental The choice of reporter gene reporter probe pair to be used
conditions. The sensitivity of detection depends on the wave- should ultimately be based on the specific biological process to
length of light emission, expression levels of the enzyme in the be monitored, the duration and intensity of the signal needed,
target cells, the location of the source of bioluminescence in and the tissue to be imaged (21, 100) (Fig. 3). As longer
the animal, the efficiency of the collection optics, and the wavelengths of light penetrate mammalian tissues with less
sensitivity of the detector (95). absorbance, luciferase from Photinus pyralis (⬎600 nm) is
Reporter genes commonly used for BLI. To track cells in more readily detected (17). Moreover, its substrate D-luciferin
vivo, reporter genes and reporter probes must be able to reveal remains in circulation longer than other substrates because it is
cellular and molecular processes throughout an entire study poorly catalyzed by mammalian tissues (101). However, Pho-
period, be highly sensitive to small changes in cell function and tinus pyralis luciferase has the disadvantage of having a longer
distribution over time, and must not alter the labeled biological coding sequence (1,653 bp) compared with marine luciferases
process itself. A variety of reporter genes have been introduced such as Renilla and Gaussia. Renilla and Gaussia luciferases
and validated for different imaging modalities, including var- have coding sequences of 936 and 558 bp, respectively, mak-
ious luciferases for BLI (5, 67, 74). Luciferase (Luc) is the only ing them more suitable for studies that require compact trans-
one that produces light without requiring an external excitation gene sequences, such as gene transfer and gene expression
source, and they offer inherently low background signals studies. Another advantage of Renilla and Gaussia luciferases
because animal tissues do not emit significant amounts of light.
over Photinus pyralis is the fact that they do not require ATP
Since the first report of the cDNA encoding Luc in 1985 (23),
as a cofactor during bioluminescent reaction and thus can be
DNA sequences have been optimized so that the Luc gene can
used for imaging cells independent of their metabolic state (5,
be expressed at high levels and its product localized in the
66). One particular characteristic of Gaussia luciferase is that
cytoplasm of the cells. For all reporter systems, the intensity of
it is naturally secreted (96) and therefore can be used as a
light is proportional to the amount of luciferase expressed in
each individual cell or the number of cells in which a gene has reporter for quantitative assessment of cells in vivo by mea-
been transferred. suring its concentration in blood (61). However, Renilla and
Of the many available luciferase enzymes, only a subset Gaussia luciferases produce shorter wavelengths of light
have been developed and used as reporter genes. Luciferase (peaking at 480 nm), which are not transmitted through tissues
from the North American firefly Photinus pyralis is the most as effectively as Photinus pyralis (100). Overall, the utility of
common choice, but other luciferases such as the sea pansy Renilla and Gaussia for in vivo BLI can benefit from improve-
Renilla reniformis, the click beetle Pyrophorus plagiophala- ments in sensitivity (46 – 48).
mus, and the copepod Gaussia princeps have also been inves- Codon-optimized humanized Gaussia (2, 79) has improved
tigated (48, 79). Gaussia and Renilla luciferase enzymes emit its sensitivity in mammalian cells, but the pharmacokinetics of
in the blue/green region of the UV visible spectrum, where its reporter probe coelenterazine are somewhat limiting in vivo.
light is strongly absorbed and scattered by tissues. Conse- Coelenterazine is prone to quick inactivation, including degra-
quently, the imaging performance suffers from poor sensitivity dation through autoxidation. This substrate is also costly with
and spatial resolution. On the contrary, Photinus pyralis and low solubility. Furthermore, coelenterazine binds to serum
click beetle luciferase emit ⬃60% of their light at ⬎600 nm, proteins, is cleared rapidly from the bloodstream (101), and
which enables great tissue penetration. Photinus pyralis has decays rapidly with time (5). Therefore, when imaging in vivo
emission at 620 nm when collected at 37°C, making it among with coelenterazine, the signal needs to be acquired immedi-
the longest emitting luciferases at mammalian body tempera- ately after substrate administration. On the other hand, this
tures and the most sensitive for in vivo applications (100). short half-life can be advantageous in cases in which sequential
Bacterial luciferases (Lux) such as from Photorhabdus lu- imaging of two luciferases, such as Gaussia princeps and
minescens emit blue light that has been used for BLI of Photinus pyralis, is required to monitor two biological pro-
bacterial infections. They are unique in that their lux operon cesses in tandem in the same animal (5).
cassette codes for the luciferase and also for enzymes that In summary, the development of reporter gene variants with
produce the substrate required for luminescence reaction, better emission spectra, brightness, and stability can ultimately
thereby eliminating the need for exogenous substrate. Trans- improve sensitivity and the overall performance of luciferases
ferring this operon to mammalian cells would be advantageous in BLI imaging studies. Moreover, cloning new luciferases
for in vivo imaging; however, limited research has been done from different organisms, such as those from Luciola italica
to determine whether this is possible (16). Mutations in lu- and Cratomorphus distinctus, can certainly improve current
ciferases can change the wavelength of the luminescence applications and make novel ones possible (8, 88).

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H666 IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION

Fig. 3. Emission wavelengths for the most commonly used reporter luciferase enzymes in BLI and their advantages and disadvantages. V, violet; B, blue; Y,
yellow; O, orange; R, red.

Vector-mediated expression of reporter genes. A fundamen- over time via the detection of reporter gene expression by BLI
tal requirement for BLI is the expression of a reporter gene (27, 35).
(e.g., luciferase) by the cells or tissues to be imaged, which
requires the introduction of genetically encoded imaging re- Applications of BLI in Cardiovascular Research
porters into cells cultured in vitro (50). Alternatively, lu-
ciferase-expressing cells can be obtained from luciferase trans- Because physiological processes are dynamic in time and
genic mice (Fig. 1). This can be achieved by using a reporter space, end-point assays do not always provide a comprehen-
vector that incorporates a luciferase gene driven by a promoter sive understanding of biology in vivo. In cardiovascular re-
that allows luciferase to be constitutively expressed by all cells search and many other scientific areas, BLI has been used for
in the animal’s body (11, 83). noninvasive visualization of a variety of biological processes in
Delivery of bioluminescent reporter genes to cells has been real time. In this section, we will review the many applications
achieved through several means, but lentiviral-based gene of BLI in cardiovascular research.
transfer has been the method of choice because of its effective Monitoring expression of therapeutic genes in the heart.
gene delivery and high expression levels of transgenes in Gene therapy is a rapidly evolving field in cardiovascular
mammalian cells in culture as well as in vivo (22). Lentiviruses medicine. This technology allows for the correction of func-
have the capability to deliver target genes to both dividing and tional gene loss and enables the expression of a therapeutic
nondividing cells and are capable of inserting genetic informa- gene in a target tissue (52). Nevertheless, gene therapy studies
tion into the host genome, ensuring prolonged gene expression continue to be plagued by suboptimal delivery of genes, poor
with a more limited host immune response (80). The safety of survival of cells carrying the therapeutic gene(s), and the
the lentiviral vectors has been further improved with the inability to evaluate the levels of gene expression in vivo (28).
generation of self-inactivating vectors and the use of minimal Thus the information gathered from BLI studies in small
packaging systems. The efficiency of gene expression has been animals can be used to design solid clinical trials that will help
improved by the introduction of a relatively strong internal improve gene therapy for cardiac repair. In cardiovascular
promoter such as cytomegalovirus. This promoter drives the research, BLI has been used to address a variety of questions
expression of the reporter gene and guarantees that the reporter that range from determining the effects of transgene expression
expression is always “on” under all conditions, in all tissue (e.g., BCL2) on cardiomyoblast survival and cardiac repair
types. Moreover, lentiviral vectors can be used to simultane- (40) to testing the efficiency of anti-inflammatory drugs on the
ously induce the expression of multiple genes in a cell. Cou- expression of specific genes (e.g., inducible nitric oxide syn-
pling the expression of a gene with a luciferase reporter gene thase) (99). Additionally, BLI has been used to optimize vector
provides a simple yet effective mechanism for studying the systems (33) and treatment regimens (71) for delivery of
regulation of gene expression and monitoring it by BLI. This therapeutic genes (e.g., hypoxia-inducible factor-1␣) to the
provides exciting opportunities for transcriptional targeting, heart.
double reporter labeling for BLI monitoring, as well as gene The applications of BLI in research are constantly expanding
therapy. For example, by linking the expression of luciferase to with the development of new therapeutic modalities. For in-
the cardiac-specific promoter myosin light chain 2v, it is stance, BLI enabled tracking the activity of a short hairpin
possible to monitor cells undergoing cardiac differentiation RNA plasmid in knocking down inhibitory factors of angio-

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genic genes in the heart (32). The advances in reporter gene BLI has demonstrated the poor survival of bone marrow
technology and dual reporter labeling have enabled a simulta- mononuclear cells (72), cardiac resident stem cells (43), mes-
neous monitoring of distinct physiological events via BLI. enchymal stem cells (83), adipose stromal cells (82), ESC-
Dual-reporter labeling can address questions such as how the derived cardiac cells (10), and ESC-derived endothelial cells
expression of a gene affects the survival or differentiation of a (45) by 8 wk after cell injection. Conversely, the tumorigenic
specific cell type or how it alters the expression of another potential of ESCs can also be uncovered by BLI. Lee et al. (42)
gene. However, challenges in designing dual-reporter systems, demonstrated that a minimum of 100,000 human ESCs are
such as finding adequate means for separating the two biolu- necessary to form teratoma in the heart. Overall, BLI studies
minescent signals while retaining sensitivity, remain (60, 87). have provided significant insights into the biology of stem cells
Monitoring survival and homing of therapeutic cells. Stem in vivo and how issues such as poor survival and potential
cell-based therapies are expected to have an enormous impact tumorigenicity must be overcome before translation into the
in the treatment and cure of various diseases and disorders in clinic.
the future. These cells, given the right conditions, have the Monitoring immune rejection of heart transplant and cell
ability to differentiate into the constituent cells of various grafts. Allogeneic heart transplantation is the most commonly
organs. When stem cells are used in therapies, it would be ideal used therapy for end-stage heart disease. Transplant loss due to
to track their migration or differentiation process in vivo. The immune rejection remains a significant problem in clinical
therapeutic use of bone marrow stem and progenitor cells heart transplantation despite current immunosuppressive ther-
initially was popular and has been evaluated furthest in the apies (73). Acute rejection of heart transplants is an immune
clinic setting. More recently, circulating stem and progenitor response mediated by the coordinated infiltration and function
cells, resident cardiac stem cells, and mesenchymal stem cells of host alloantigen-specific T cells in the allograft (57). In this
have also been used in translational studies for clinical appli-
context, BLI has allowed monitoring of cardiac allograft over
cations (15). Although small animal studies have provided
time and revealed its rejection by day 12 after transplantation.
promising results, significant questions regarding the biology
A decrease in the intensity of bioluminescence signals was
of transplanted cells and their ability to promote cardiac repair
remain (92) (Table 1). detected as early as day 4, suggesting acute graft rejection (78).
BLI is an important tool that can answer some of these The correlation of bioluminescence intensity to other measures
pressing questions by uncovering the dynamics of cell expan- of heart function such as beating score, fractional shortening,
sion, migration, and survival upon transplantation into the heart and lymphocyte infiltration has provided additional means to
(90, 102). Several studies have indicated that cardiomyocytes assess function and to determine the possible mechanisms
purified from embryonic stem cells (ESCs) can benefit cardiac responsible for rejection.
function following myocardial ischemia (10, 12, 13, 41, 59) Acute rejection can also be a major issue in human ESC-
with a low risk of tumor formation. However, one of the central based therapy (25, 84). BLI has provided significant informa-
issues in cell therapy is the lack of long-term survival of these tion for the development of therapeutic strategies to prevent the
therapeutic cells in vivo. BLI has shown that more than 90% of rejection of stem cells. An immunosuppressant cocktail con-
transplanted adult stem cells die within the first 3 wk of sisting of tacrolimus and sirolimus has been shown by BLI to
delivery (43, 45, 83). This could be the reason for the short mitigate the rejection of human ESCs (77). Recently, Pearl et
duration of improvement of cardiac function observed in mul- al. (63) used BLI to demonstrate that a cocktail of costimula-
tiple studies focused on cell therapy for cardiac repair. Thus the tory blockade agents (anti-CD40 ligand, cytotoxic T-lympho-
problem of donor cell death continues to be troublesome and cyte antigen 4-immunoglobulin, and anti-lymphocyte function-
may limit the overall efficacy of stem cell-based therapy, associated antigen-1) induced long-term allogeneic and xeno-
making further studies to improve cell fate monitoring via geneic human ESCs and induced pluripotent stem cell
imaging technologies essential. engraftment. Overall, these studies elucidate the importance of
Failure of long-term survival of therapeutic cells has been in vivo imaging for the development of therapies that may one
demonstrated for a variety of cell types (43, 45, 83) (Fig. 4). day enable stem cell therapy to become feasible.

Table 1. Cells types that have been investigated for their potential to promote cardiac repair using BLI
Cell Type Information Gained from BLI References

Bone marrow mononuclear cells Comparison of different cell types for treatment of MI 83
Timing of cell delivery on acute vs. chronic MI 76
Systemic homing to injured heart 72
Adipose tissue-derived stem cells (ASCs) Long-term survival of cells in injured heart 3
Homing and survival of fresh versus cultured cells to injured heart 4
Mesenchymal stem cells (MSCs) Comparison of ASCs vs. MSCs for treatment of MI 82
Growth factor-treated MSCs for treatment of MI 24
Human CD34⫹ cells Cell fate in the heart using a MI model 89
Rat cardiomyoblasts Improvement of engraftment and survival with collagen matrix 38, 39
Embryonic stem cells Cell survival, proliferation, and migration 9, 42, 77
Skeletal myoblasts Cell fate in MI model 83
ESC-derived endothelial cells (ESC-EC) Cell fate in MI model 44, 45
Effects of nicotine on the therapeutic effects ESC-ECs 98
Resident cardiac stem cells Cell fate and function in MI model 43
BLI, bioluminescence imaging; MI, myocardial infarction.

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Fig. 4. Monitoring survival and homing of cells via BLI. A: skeletal myoblasts (SkMb), bone marrow-derived mononuclear cells (MN), mesenchymal stem cells
(MSC), and fibroblasts (Fibro) were injected intramyocardially after myocardial infarction. All cell types demonstrated a decrease in bioluminescence signal
intensity starting at 4 wk postinjection. Yellow arrows indicate homing of MN to femur, spleen, and liver. Red arrows indicate cells retained in the heart and
lungs. Values in y-axis are in photons·s⫺1·cm⫺2·sr⫺1. Abbreviations in x-axis: d, day; w, week. Reprinted with permission (83). B: BLI demonstrating preferential
homing of bone marrow mononuclear cells to the ischemic myocardium in a model of ischemic reperfusion injury (I/R). Bioluminescence was also detected in
the spleen and bone marrow (yellow and red arrows, respectively). A progressive decrease in bioluminescence signal was observed starting at day 14
postinjection. Reprinted with permission (72). C: cardiac resident stem cells were injected in the heart following myocardial infarction. Robust bioluminescence
activity was detected on day 2 but mostly disappeared by 8 wk postinjection. Values in x-axis represent days post-cell injection. Reprinted with permission (43).

Limitations to help define the requirements for imaging instrumentation.

However, the overall low spatial resolution (3–5 mm range)
BLI has been successfully used to obtain semiquantitative and limited tissue penetrance restrict the use of BLI to small
measurements of biological processes because of a strong animal studies (69).
correlation between the number of cells and the biolumines- Changes in tissue oxygenation can also alter biolumines-
cence signal detected both in vitro (75) and in vivo (68). cence signal. In rat gliosarcoma for instance, bioluminescence
However, a simple quantification of light emission may not signal has been shown to decrease by ⬃50% at 0.2% oxygen
provide a true representation of biological processes. This is (58). Thus, for reliable BLI measurements, it is important to
because the firefly luciferase reaction is a complex interaction understand the effects of local niche in which the luciferase-
of a variety of molecules (e.g., ATP, Mg2⫹, oxygen, and expressing cells of interest reside. This is especially the case in
luciferin) and because the intensity of the bioluminescence cardiovascular studies involving hypoxia (e.g., myocardial in-
signal depends on multiple factors. In particular, the number of farction). Similarly, BLI quantification has to be carefully
metabolically active luciferase-transfected cells, the concentra- interpreted in studies that involve surgical procedures. Changes
tion of luciferin, ATP and oxygen levels, the spectral emission in tissue thickness because of the presence of inflammation,
of bioluminescence probes, and the depth and optical proper- edema, sutures, and animal growth can alter light absorption
ties of tissues are known to alter the intensity of biolumines- and scattering as well as the bioluminescence signal.
cence signal (69). Another issue that should be considered
during quantitative BLI is the limited and wavelength-depen- Conclusions and Future Directions
dent transmission of light through animal tissues. Light sources
closer to the surface of the animal appear brighter compared Many different organisms, ranging from bacteria and fungi
with deeper sources because of tissue attenuation properties to fireflies and fish, are endowed with the ability to emit light.
(91). It is estimated that for every centimeter of depth, there is The discovery of new luminescence reporters and the use of
a 10-fold decrease in bioluminescence signal intensity (18). genetically modified reporters may further strengthen reporter
Mathematical models can be used to predict in vivo imaging gene expression in mammalian cells, thus improving the
signal levels and spatial resolution as a function of depth and sensitivity and expanding the applications of this imaging

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IN VIVO BIOLUMINESCENCE FOR TRACKING CELL FATE AND FUNCTION H669
modality (7, 14). Additionally, alternative methods for 9. Cao F, Lin S, Xie X, Ray P, Patel M, Zhang X, Drukker M, Dylla SJ,
transduction of reporter gene may reduce the risk of anom- Connolly AJ, Chen X, Weissman IL, Gambhir SS, Wu JC. In vivo
visualization of embryonic stem cell survival, proliferation, and migra-
alous and inappropriate transcription of unwanted sequences tion after cardiac delivery. Circulation 113: 1005–1014, 2006.
in mammalian cells (37). 10. Cao F, Wagner RA, Wilson KD, Xie X, Fu JD, Drukker M, Lee A,
BLI allows real-time monitoring of survival and homing of Li RA, Gambhir SS, Weissman IL, Robbins RC, Wu JC. Transcrip-
various therapeutic cells that are currently being investigated to tional and functional profiling of human embryonic stem cell-derived
promote cardiac repair. By the use of cardiac-specific promot- cardiomyocytes. PLoS One 3: e3474, 2008.
ers linked to reporter genes, BLI can be used to monitor cell 11. Cao YA, Bachmann MH, Beilhack A, Yang Y, Tanaka M, Swijnen-
burg RJ, Reeves R, Taylor-Edwards C, Schulz S, Doyle TC, Fath-
differentiation in vivo. It also provides excellent opportunities man CG, Robbins RC, Herzenberg LA, Negrin RS, Contag CH.
to evaluate strategies designed to improve the survival of Molecular imaging using labeled donor tissues reveals patterns of en-
therapeutic cells or transplanted hearts. The ability to monitor graftment, rejection, and survival in transplantation. Transplantation 80:
physiological processes in vivo in real time is extremely 134 –139, 2005.
beneficial to evaluate the success or failure of the various 12. Caspi O, Huber I, Kehat I, Habib M, Arbel G, Gepstein A, Yankel-
son L, Aronson D, Beyar R, Gepstein L. Transplantation of human
therapies designed to promote cardiac repair. As most basic embryonic stem cell-derived cardiomyocytes improves myocardial per-
cardiovascular research is performed in rodent models, BLI formance in infarcted rat hearts. J Am Coll Cardiol 50: 1884 –1893, 2007.
can efficiently reveal problems and provide insight into solu- 13. Caspi O, Lesman A, Basevitch Y, Gepstein A, Arbel G, Habib IH,
tions for validating and optimizing novel therapies for the Gepstein L, Levenberg S. Tissue engineering of vascularized cardiac
treatment of heart disease. Overall, this technology can furnish muscle from human embryonic stem cells. Circ Res 100: 263–272, 2007.
great insights that can drive the development of better clinical 14. Caysa H, Jacob R, Muther N, Branchini B, Messerle M, Soling A. A
redshifted codon-optimized firefly luciferase is a sensitive reporter for
trials in cardiovascular medicine. bioluminescence imaging. Photochem Photobiol Sci 8: 52–56, 2009.
15. Chen IY, Wu JC. Cardiovascular molecular imaging: focus on clinical
ACKNOWLEDGMENTS translation. Circulation 123: 425–443, 2011.
16. Close DM, Patterson SS, Ripp S, Baek SJ, Sanseverino J, Sayler GS.
Because of space limitations, we were unable to cite all the important
Autonomous bioluminescent expression of the bacterial luciferase gene
papers relevant to bioluminescence imaging and cardiovascular research. We
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