STEM CELL TRACKING TECHNIQUES

SUBMITTED BY:
M.VINOTH,
MVM 09003,

STEM CELL TRACKING TECHNIQUES

Introduction
Stem cells can be classified as embryonic stem (ES) cells, adult stem cells. Being with
the potential of differentiation, stem cell has been increasingly attractive in regenerative
therapies such as the treatment of Parkinsons disease, Alzheimer`s disease, myocardial
infarction, leukemia, diabetes, and other degenerative disorders. After the systemic or local
transplantation, stem cells and progenitor cells may be able to migrate and repopulate in
pathologic sites to bring tremendous therapeutic effect. However, risk may happen for the
misbehavior following the stem cell transplantation, for example, teratoma formation .
Therefore, in vivo tracking the fate of the transplanted stem cells over time is a vital step in
determining the efficacy of the implant. For a long time, studies on stem cell mobility
conventionally require histological investigation to determine viable engraftment of the
transplanted cells. However, the inability to visualize cell populations in the same animal
over time has been a major bottleneck in the in vivo evaluation of stem cell therapies because
groups of animals have to be sacrificed at different time point for histology purpose.
In recent years, the emerging and advances of non-invasive in vivo stem cell imaging
has significantly

contributed to the real-time tracking of grafted stem cells as well as

monitoring their proliferation, migration and persistence in live animals .Stem cells carrying
contrast agents (direct labeling) can be detected by optical, magnetic resonance imaging
(MRI) or radionuclide imaging methods, such as single photon emission computed
tomography (SPECT) and positron emission tomography (PET) (Norman et al.,1992;
Hawrylak et al.,1993). In contrast to direct labeling techniques, reporter gene techniques for
stem cell labeling offer an attractive alternative because imaging signals are generated only
from viable cells of interest. By sensitive imaging devices such as the optical coupled device,
MRI, SPECT or PET, stem cells with the stably transfected imaging reporter genes can be

detected with suitable imaging probes to visualize their distribution, to longitudinally monitor
1. Biocompatible, safe, and nontoxic
2. No genetic modification or perturbation to the stem cell
3. Single-cell detection at any anatomic location
4. Quantification of cell number
5. Minimal or no dilution with cell division
6. Minimal or no transfer of contrast agent to nonstem cells
7. Noninvasive imaging in the living subject over months to years
8. No requirement for injectable contrast agent
the cell survival and even to follow the successful differentiation of stem cells to mature
functional cells in stem cell therapies (Cao et al.,2006)

Characteristics of an Ideal Imaging Technology for Stem Cell Tracking

During Clinical Trials

Successful in vivo imaging requires that a contrast agent associated with a stem cell exert an
"effect size" sufficient for detection by imaging hardware. Although the most attractive
contrast agents for tracking are endogenous ones (ie, normal components of the stem cell),
their effect size is extremely small.

The 8 characteristics of an ideal imaging technology for stem cell tracking are presented.
First, and foremost, the exogenous contrast agent must be biocompatible, safe, and nontoxic.
This is especially important when nanotechnology solutions to the tracking problem are
considered, because most solid-state devices will be composed of materials that do not have
proven long-term safety in vivo.
Another consideration is the need for genetic modification of the stem cell or perturbation of
its genetic program by the contrast agent itself. Several imaging techniques, such as
enzymatic conversion of an injected substrate and receptor-based binding, require stable
integration of transgenes. This strategy may be combined with genetic manipulation of stem
cell populations to enhance the viability, differentiation, and coupling of these cells with the
myocardium. These types of manipulations add significant cost, regulatory roadblocks, and
the potential to induce genetic abnormalities, including uncontrolled growth and malignancy.
(Hacein-Bey-Abina et al.,2003) Although exogenous genes have the distinct advantage of not
being diluted by cell division and have the potential to induce cell survival or suicide on
demand it is unclear at present if the extra step of genetic manipulation will become routine in
human clinical stem cell trials.
Ideally, imaging technology used for stem cell tracking would have single-cell sensitivity and
would permit quantification of exact cell numbers at any anatomic location. Single-cell
sensitivity is especially important in a new field such as that of stem cells because the pattern
of migration of stem cells, even after local injection, is unknown, and there is a distinct
possibility that single stem cells scattered diffusely throughout the body might be effective
therapeutics for certain disease states.
Regardless of the level of sensitivity finally achieved, quantification of cell number can be
especially difficult when we consider the effects of contrast agent dilution during cell
division, the propensity of some contrast agents to be transferred to nonstem cells, and certain
technical limitations . The criteria of ultra-high high sensitivity, quantification, and full-body
scanning render many clinically available imaging modalities inadequate at present.
The ideal imaging technology would permit tracking of injected stem cells for months to
years because clinical trials undoubtedly will require long-term follow-up of tissue function
or host survival. Finally, injectable contrast agents, such as enzyme substrates, add complexity
and cost to stem cell tracking procedures

Imaging Modalities
Several imaging modalities have been used for the investigation of stem cell fates and
function and a number of useful tools have been developed for cell fate imaging by each of
these . Images of bone marrow and immune cell trafficking have been obtained by magnetic
resonance imaging (MRI), positron emission tomography (PET), and single photon emission
computed tomography (SPECT) (Bennink et al.,2009). Each of these is used clinically for
diagnostic applications. In vivo bioluminescence (BLI) and fluorescence imaging may have
their greatest application in the study of small animal models, and have been used to assess
the trafficking patterns of cells in vivo. There may be clinical applications of these tools, but
their use in small animal models will be invaluable as they are applied to the study of stem
cell biology. The availability of specialized MRI, PET, and SPECT scanners for small animal
imaging has increased dramatically with improved performance and capabilities. Their
increased use in animal models will likely lead to translational approaches, and they hold
great promise for advances in a number of fields where there are clinical needs. There are a
number of optical imaging approaches that have been developed for sensing and imaging in
the living body, including diffuse optical tomography (DOT), optical coherence tomography
(OCT), and others, but only in vivo BLI and fluorescence have been used to study cell
trafficking patterns. (Meral Beksac, 2009)

Recent advances in nanotechnology for stem cell tracking

Detection Methods for In Vivo Stem Cell Tracking

Magnetic Resonance Imaging(MRI)
Optical Imaging
o Bioluminescence imaging
o Fluorescence imaging
Nuclear Imaging
o Positron Emission Tomography(PET)
o Single Photon Emission Computed Tomography (SPECT)

 X-Ray computed microtomography (microCT)

Labels
In order to be tracked using any of the imaging modalities, cells of interest must be labeled
for detection. A number of studies have used exogenous fluorescent dyes to label cells outside
the body prior to transplantation. Although these dyes can have a relatively intense signal, a
disadvantage of such techniques is that these dyes can be short-lived, and as labeled cells
divide, the progeny cells, depending on the dye, may not be labeled and thus the signal is lost
over time due to dilution through cell division. Some fluorescent dyes do not produce
sufficient signal to be detectable by cameras placed outside the body, necessitating euthanasia
of the animal and tissue sampling for analysis. For these reasons, the in vivo imaging
techniques, by and large, have required the application of genetic labels. These labels are
genes that must be introduced into cells and encode proteins that interact with reporter
probes, applied substrates (bioluminescence), or exogenous excitation light sources
(fluorescence) to generate a signal that can be localized from outside the body.(zhao et
al.,2010)

Genetic labels have been developed for all of the modalities used in stem cell trafficking
studies.

Labels for PET,SPECT,MRI

The most commonly used genetic label for PET imaging is the herpes simplex virus
thymidine kinase (HSV1-tk). At the time of imaging, [18F]-FHBG or [124I]-FIAU is
administered to the animal as a radioactive reporter probe that has specificity for this TK
enzyme. The probe is transported into cells and is phosphorylated by the TK protein only in
the genetically labeled cells. The phosphorylated probe becomes trapped in the cell and
accumulates there, preferentially flagging the genetically labeled cells for detection. The
human dopamine D2 receptor (hD2R), the human somatostatin receptor (hSSTR2), and the
human transferrin receptor (hTfR) have been used with different imaging modalities and
function simply as transmembrane receptors that actively transport their corresponding
reporter probes into the genetically labeled cells. In this manner, like HSV1-tk, they also

accumulate

the

probe

within

the

labeled

cells

to

flag

them

for

detection.

Schematic overview of molecular imaging with reporter gene strategies. A vector

containing a DNA reporter construct with the reporter gene(s) driven by a specific
promoter. Transcription and translation lead to the expression of mRNA and reporter
protein, respectively. After administration of a corresponding reporter probe
systemically, the reporter probe will be catalyzed by specific cells that have the reporter
proteins. The signals occurred in this amplification process can be detected by a
sensitive imaging device. Examples of reporter genes and their specific reporter probes
are delineated by imaging modality. Fluc, Firefly luciferase; PET, positron emission
tomography; SPECT, single photon emission computed tomography; hNIS, human

sodium/iodide symporter; MRI, magnetic resonance imaging; CCD, charged coupled

device; BLI, bioluminescence imaging.

Labels for bioluminescence optical imaging

Fluorescence and bioluminescence optical imaging modalities require the generation of light
by the cells of interest.Genetic labels for BLI are genes that encode luciferases.The reporter
probes for these proteins are substrates that are oxidized and chemically consumed by the
luciferases in reactions that generate light. Fluorescence imaging detects cells that express
fluorescent proteins. By analogy to PET and SPECT, the reporter probe that must be
delivered to these genetically labeled cells is exogenous light of the appropriate wavelength
to serve as an excitation source.
In choosing a label for optical imaging, a few key parameters must be considered.
Specifically, absorption and substrate biodistribution significantly influence the behavior of
the experimental system. In the intact animal, absorption of light by tissue, and in particular
absorption by hemoglobin, attenuates the detectable signal generated by cells of interest. Red
and infrared light (wavelengths >600 nm) suffers less signal attenuation in the body due to
absorption than does light with shorter wavelengths (<600 nm). This is an advantage to
firefly luciferase (Fluc; derived from the North American Firefly Photinus pyralis) and click
beetle red luciferase (CBRluc; derived from Pyrophorus plagiophalam) over Renilla
luciferase (Rluc; derived from the sea pansy Renilla reniformis)both Fluc and CBRluc
show emission peaks at approx 620 nm at body temperature, whereas the emission peak of
Rluc is at 480 nm, in a region where absorption by hemoglobin is relatively high. The
biodistribution of the administered substrate is also an important consideration for
interpreting BLI data. To obtain quantitative information from BLI data, the substrate must
not be limiting in the oxidation reaction that generates light. Therefore, proper imaging
technique requires appropriate timing from the administration of substrate to the acquisition
of data. The optimal time from administration to acquisition depends upon both the route of
administration and the rate of clearance of the substrate in the body. The substrate for Fluc
and CBRluc, D-luciferin, is relatively stable in the body and has a relatively long circulation
time. In contrast, the substrate for Rluc, coelenterazine, is rapidly cleared from the body and
binds to serum proteins( Zhao, H et al.,2005). Coelenterazine, therefore, can only be
administered intravenously and data acquisition must be complete within a few seconds to a

minute after injection. D-Luciferin can be injected intraperitoneally or intravenously

(Bhaumik et al.,2002). The biodistribution of D-luciferin, after intraperitoneal injection,
peaks at 1520 min and the bioluminescence signal stays relatively stable for another 1520
min before degrading as a result of substrate clearance. After intravenous injection of Dluciferin the peak of light emission occurs at about 1 min and decreases rapidly (Bhaumik et
al.,2004).
Labels for Fluorescence imaging
Fluorescence imaging, like detection of any optical signal through mammalian tissues, also
requires consideration of light absorption by tissues In this case, the wavelengths of both the
excitation source and emitted light must be considered. The most commonly used genetic
label for fluorescence imaging is enhanced green fluorescent protein (eGFP). The advantage
to using fluorescent labels is the relative ease of detecting their activity in histological
samples by fluorescence microscopy, as well as in single-cell suspensions by flow cytometry.
These other modalities can provide a satisfying cross-verification to data uncovered using in
vivo imaging. The excitation and emission peaks for eGFP occur well below 600 nm and
light at these wavelengths is subject to severe signal attenuation by hemoglobin. Imaging of
reporters with optical signatures in the blue and green region of the spectrum can be severely
constrained by absorption. Whole-body imaging of labeled cells in deep tissues can be
difficult, but signals from relatively superficial sites, such as skin and subcutaneous tissues,
or from deep sites after removal of overlying tissues, can offer high-resolution images.
Several fluorescent proteins with longer emission wavelengths are available (yellow
fluorescent protein, dsRed, and others) but these, by and large, have excitation peaks below
600 nm. For these reasons, the use of these labels for deep tissue in vivo fluorescence imaging
has been shown to require high levels of reporter gene expression from a large number of
cells. (Zaheer et al.,2009)
Magnetic Resonance Imaging
Magnetic resonance imaging is a relatively new technology. The first MR image was
published in 1973(Lauterbur et al.,1973).The body is largely composed of water molecules.
Each water molecule has two hydrogen nuclei or protons. When a person goes inside the
powerful magnetic field of the scanner, the magnetic moments of some of these protons
changes, and aligns with the direction of the field.

In an MRI machine a radio frequency transmitter is briefly turned on, producing an

electromagnetic field. The photons of this field have just the right energy, known as the
resonance frequency, to flip the spin of the aligned protons in the body. As the intensity and
duration of application of the field increase, more aligned spins are affected. After the field is
turned off, the protons decay to the original spin-down state and the difference in energy
between the two states is released as a photon. It is these photons that produce the
electromagnetic signal that the scanner detects. The frequency the protons resonate at
depends on the strength of the magnetic field. This causes the nuclei to produce a rotating
magnetic field detectable by the scanner and this information is recorded to construct an
image of the scanned area of the body (Squire LF, Novelline RA 1997)
Given its extraordinary 3-dimensional capabilities and high safety profile, magnetic
resonance imaging (MRI) is the imaging modality used by most research studies to track stem
cells in vivo. At this point in time, MRI imaging techniques can be divided into those
generating primarily T1 contrast and those generating primarily T2/T2* contrast.
T1 contrast
T1 contrast agents are those that utilize the lanthanide gadolinium (Gd3+), which changes the
relaxivity of protons from associated water molecules and increases the signal on T1weighted images. Unfortunately, with presently available field strengths, Gd 3+-based contrast
requires 50- to 500-mol/L concentrations of low-molecular-weight Gd3+-containing
molecules or attachment to bulky scaffolds such as dendrimers and dextrans to increase the T1
effect. however, that Gd3+-containing scaffolds, loaded via pino/endocytosis into stem cells,
permit tracking for up to 6 weeks.( Bulte et al., 2002; Modo et al., 2002)
T2/T2* contrast
T2/T2* contrast is by far the most widely used technique for stem cell imaging studies using
MRI. This is a consequence of the observation made by the Weissleder group in the early
1990s that superparamagnetic iron oxide nanoparticles (also known as monocrystalline iron
oxide nanocrystals [MIONs], ultrasmall superparamagnetic iron oxide [USPIOs]) congeal in
endosomes after endocytosis, resulting in magnification of their susceptibility effects. More
recent formulations from this group utilize Tat peptides to improve cell loading, which can
now be accomplished in <1 hour (Josephson et al.,1999) .Because of the magnification effect,

MIONs can be used to track extremely small numbers of stem cells, on the order of
thousands, at high field strengths, for up to several weeks. T2/T2* contrast agents ( DaldrupLink et al 2009) and their technical improvements( Lewin et al., 2000) already have been
applied by many groups for stem cell tracking in vivo. Most superparamagnetic formulations
appear to be biocompatible, safe, and nontoxic, and some already have been approved by the
US Food and Drug Administration.

In vivo MR image (top) 7 days after injection of 1.5 105 BMSCs labeled with 2 l
MPIOs / ml medium into a rat brain with a solid tumor established a week prior to
BMSC injection. Regions marked by dashed lines and arrow point to areas of
hypointense signal, indicating the presence and migration of BMSC containing MPIOs.
The numbers refer to the schematic drawing in the inset, showing the injection sites of
tumor and BMSCs .

Representative transmission electron microscopy of BMSCs labeled for 24 h with 2 l

MPIOs /ml medium. N, nucleus; arrow indicates an iron microsphere (Nohroudi et
al.,2010)
Advantage
MRI scan is harmless to the patient. It uses strong magnetic fields and non-ionizing radiation,
unlike

CT

scans

and

traditional

X-rays

use

ionizing

radiation.High

spatial

resolution,Outstanding anatomic imaging,MRI meets the requirements of penetration depth

and clinical availability (Himmelreich et al.,2008)
Disadvantage
The problems with superparamagnetic particles include dilution of contrast with cell division;
difficulty in quantification because of susceptibility artifact; and the potential transfer of
contrast to non stem cells, such as macrophages, after stem cell death. (Himmelreich et
al.,2008)
A significant clinical problem common to all MRI methods is that certain implantable
devices, such as pacemakers and defibrillators, are currently contraindications to scanning.
Although a recent report suggests that patients with pacemakers can be scanned safely at 1.5 T
(Martin et al.,2004)
X-RayBased Methods
X-ray computed tomography (CT) is a medical imaging method employing tomography
created by computer processing. Digital geometry processing is used to generate a

threedimensional image of the inside of an object from a large series of two-dimensional Xray images taken around a single axis of rotation.( Herman, G. T et al.,2009)
CT produces a volume of data which can be manipulated, through a process known as
"windowing", in order to demonstrate various bodily structures based on their ability to block
the X-ray beam. Although historically the images generated were in the axial or transverse
plane, orthogonal to the long axis of the body, modern scanners allow this volume of data to
be reformatted in various planes or even as volumetric (3D) representations of structures

Plain films and computed tomography (CT) are the most readily available clinical imaging
modalities. Unfortunately, contrast generation requires extremely high concentrations of highdensity/highatomic number materials such as iodine, gadolinium, or metals. To render a stem
cell or collection of stem cells visible by using even a solid metal, the volume of metal
associated with the cell volume must be equal to or greater than the inverse of its density. For
example, it would take approximately one eighth of the cell volume in solid iron to generate a
signal above background during CT scanning. Such contrast is difficult to achieve, rendering
x-raybased methods unlikely to play a direct role in stem cell tracking at the present time.
(Frangioni et al.,2009)

MicroCT -in vivo Cell Tracking

Microtomography uses x-rays to create cross-sections of a 3D-object that can be used to
recreate a virtual model without destroying the original model. The term micro is used to
indicate that the pixel sizes of the cross-sections are in the micrometer range.These pixel
sizes have also resulted in the terminology micro-computed tomography, micro-ct, microcomputer tomography, high resolution x-ray tomography, and similar terminologies. All of
these names generally represent the same class of instruments.
This also means that the machine is much smaller in design compared to the human version
and is used to model smaller objects. In general, there are two types of scanner setups. In one
setup, the X-ray source and detector are typically stationary during the scan while the
sample/animal rotates. The second setup, much more like a clinical CT scanner, is gantry
based where the animal/specimen is stationary in space while the X-ray tube and detector

rotate around. These scanners are typically used for small animals (in-vivo scanners),
biomedical samples, foods, microfossils, and other studies for which minute detail is desired.
The first X-ray microtomography system was conceived and built by Jim Elliott in the early
1980s. The first published X-ray microtomographic images were reconstructed slices of a
small tropical snail, with pixel size about 50 micrometers. (Elliott et al.,1982) Many believe
that the technology did not really take off until the advent of the cone beam reconstruction
algorithm originally authored by Lee Feldkamp

MicroCT is similar to conventional CT systems usually employed in medical diagnoses and

industrial applied research, but unlike these systems, which typically have a maximum spatial
resolution of about 0.5 mm, advanced microCT is capable of achieving a spatial resolution up
to 0.3 m, about three orders of magnitude lower. Use of synchrotron X-rays has several
advantages compared to laboratory or industrial X- ray sources, such as high spatial
resolution and a wide range of greyscale values (corresponding to different X-ray absorption
coefficients) within and among datasets (Ashbridge et al.,2003).
More recently Synchrotron Radiation (SR) microCT systems were made available also for
imaging small animals in vivo, such as for the examination of living rats or mice . The great
advantage of such systems is to enable longitudinal studies, thus reducing the effect of
biological variability in the cohort. The first in vivo longitudinal study reported alterations of
bone micro-architecture in the hind limb loaded female rats(Boyd et al.,2006). In vivo
microCT was also used to monitor microarchitectural changes in ovariectomized rats at the
tibial metaphyses. It is a non-invasive technique giving integral information about the content
of magnetic material along the beam direction as well as a relative local snapshot of the
magnetic nanoparticle distribution in relation to the number of slices . MicroCT provides high
spatial resolution images (from 10 m to 1 m) with high signal-to-noise ratio. In previous
study (Torrente, Y et al., 2009).we showed that X-ray microCT analysis is able to detect stem
cells, previously labeled with nanoparticles of iron oxide, inside skeletal muscles of
dystrophic mice after intra-arterial transplantation, providing biological insights into the early
processes of muscle stem cell homing.

This technique also offers the possibility of obtaining a quantification of the number of cells
that are able to migrate from the blood stream inside the muscle tissue, and a 3D visualization
of their distribution. One group analyzed nine muscular biopsies transplanted with three
different numbers of stem cells labeled with iron-oxide nanoparticles, at three different times
after injection. The different timing investigated did not show differences in the location of
stem cells, while the variation in stem cells number allowed us to optimize the experimental
conditions and identify 50,000 as the minimum number of detectable cells in a murine
muscle. X-ray microCT offers the possibility to detect with high definition and resolution
human cells after transplantation, and opens new possibilities for stem cell research. In the
perspective of clinical translation of stem cell research, it would be advantageous to develop
new techniques to detect donor cells after transplantation to track their fate in vivo.

Optical Imaging
Two complementary optical imaging methods, bioluminescence and fluorescence, can be
used for stem cell tracking.
Bioluminescence imaging (BLI)
Bioluminescence imaging (BLI) is a technology developed over the past decade that allows
for the noninvasive study of ongoing biological processes in small laboratory animals.
Recently, bioluminescence tomography (BLT) has become possible and several systems have
become commercially available.
Bioluminescence is the process of light emission in living organisms. Bioluminescence
imaging utilizes native light emission from one of several organisms which bioluminesce.
The three main sources are the North American firefly, the sea pansy (and related marine
organisms), and bacteria like Photorhabdus luminescens and Vibrio fischeri. The DNA
encoding the luminescent protein is incorporated into the laboratory animal either via a viral
vector or by creating a transgenic animal.
Systems derived from the three groups above differ in key ways:

Firefly luciferase requires D-luciferin to be injected into the subject prior to imaging.
The peak emission wavelength is about 560 nm. Due to the attenuation of blue-green

light in tissues, the red-shift (compared to the other systems) of this emission makes
detection of firefly luciferase much more sensitive in vivo.

Renilla luciferase requires its substrate, coelenterazine, to be injected as well. As

opposed to luciferin, coelenterazine has a lower bioavailability .Additionally, the peak
emission wavelength is about 480 nm.

Bacterial luciferase has an advantage in that the lux operon used to express it also
encodes the enzymes required for substrate biosynthesis. Unfortunately, this system
has not yet been adapted for mammalian cell expression .This luciferase reaction has a
peak wavelength of about 490 nm.

While the total amount of light emitted from bioluminescence is typically small and not
detected by the human eye, an ultra-sensitive CCD camera can image bioluminescence from
an external vantage point.

Bioluminescence utilizes light generated by the enzyme luciferase to detect cells in vivo. Four
published studies, 3 in mice (Wang X et al., 2003,Tang Y et al., 2003,Cao YA et al, 2004)and
1 in rats, (Wu JC et al., 2003)utilized bioluminescence to track the distribution and
engraftment of stem cells in vivo. Unfortunately, luciferase genes and substrates described to
date generate only visible (400 to 700 nm) light, which has very high absorption and scatter in
living tissue. This precludes use of the technique in animals larger than rats, and even in mice
false-negative scanning can occur, dependent on cell depth.( Rice BW et al., 2001)
Bioluminescence also requires the stable expression of nonhuman genes, and the injection of
high concentrations of potentially immunogenic, nonhuman substrates, such as luciferin and
coelenterazine. It is therefore unlikely that this technique will be used clinically.

Location of bioluminescent foci following transplantation of transgenic luc+

hematopoietic stem cells (HSCs)

Fluorescence imaging
Fluorescence imaging utilizes organic (eg, green fluorescent protein, small-molecule
polymethines) or organic/inorganic hybrids (eg, quantum dots) as exogenous contrast agents
for in vivo imaging ( Frangioni et al.,2003). Because of high photon absorption and scatter at
visible wavelengths, only near-infrared (NIR) (700 to 1000 nm) fluorophores have clinical
potential. The major problem with NIR fluorescence is that even with tomographic imaging
methods, detection is limited to only 4 to 10 cm of tissue (Ntziachristos et al and SevickMuraca et al). Hence, clinical use of NIR fluorescence likely will be limited to near-surface

applications, such as intraoperative imaging. A major advantage of NIR fluorescence is its

compatibility with conventional microscopy, (Zaheer et al.,2009) permitting single-cell
detection of stem cells in pathological specimens. Ex vivo histological detection of stem cells
undoubtedly will be required in clinical trials. Major disadvantages of NIR fluorescence are
the dilution of the agent with each cell division and the possibility of uptake by nonstem cells
after stem cell death.

Ultrasound
Because cardiologists likely will conduct the majority of clinical studies of stem cells in
cardiovascular applications, tracking by echocardiography would be extremely convenient.
Contrast for echocardiography is generated by acoustic interfaces such as water/gas (eg,
microbubbles, perfluorocarbons). Although a single unit of contrast is on the order of 0.25 to
1 m in diameter, the generated acoustic perturbation appears much larger. Echocardiography
therefore has the potential to detect a single cell loaded with a single unit of
contrast(Klibanov et al.,2008).Nevertheless, methods to accumulate contrast intracellularly
are not yet robust, and effects on cell motility, etc, are not known. An additional problem is
that echogenic contrast agents cast an acoustic "shadow" below the first unit of contrast
detected, thus precluding accurate quantification of cell number. Such contrast agents are
subject to dilution during cell division and transfer to nonstem cells after cell death. Finally,
spatial resolution of ultrasound is limited, and many anatomic sites are inaccessible.(frangioni
et al.,2008)

Single-Photon Emission Computed Tomography

Single photon emission computed tomography (SPECT, or less commonly, SPET) is a
nuclear medicine tomographic imaging technique using gamma rays. It is very similar to
conventional nuclear medicine planar imaging using a gamma camera. However, it is able to
provide true 3D information. This information is typically presented as cross-sectional slices
through the patient, but can be freely reformatted or manipulated as required.
SPECT imaging is performed by using a gamma camera to acquire multiple 2-D images ,
from multiple angles. A computer is then used to apply a tomographic reconstruction
algorithm to the multiple projections, yielding a 3-D dataset. This dataset may then be

manipulated to show thin slices along any chosen axis of the body, similar to those obtained
from other tomographic techniques, such as MRI, CT, and PET.
SPECT is similar to PET in its use of radioactive tracer material and detection of gamma
rays. In contrast with PET, however, the tracer used in SPECT emits gamma radiation that is
measured directly, whereas PET tracer emits positrons which annihilate with electrons up to a
few millimeters away, causing two gamma photons to be emitted in opposite directions. A
PET scanner detects these emissions "coincident" in time, which provides more radiation
event localization information and thus higher resolution images than SPECT (which has
about 1 cm resolution). SPECT scans, however, are significantly less expensive than PET
scans, in part because they are able to use longer-lived more easily-obtained radioisotopes
than
High-energy gamma rays emitted by radioactive atoms as

PET.
99m

Tc, 111In, and 123I are detected by

rotating a collimated gamma camera around the subject and reconstructing a 3-dimensional
image. Three strategies for in vivo stem cell detection have been described: direct loading
with a radiometal,( Gao et al.,2007) enzymatic conversion and retention of a radioactive
substrate (reviewed in Gambhir et al), and receptor-mediated binding.
Direct loading is problematic given the tradeoff between half-life and long-term exposure to
ionizing radiation and given the possibility of transfer of the radiometal from stem cells to
nonstem cells.
Enzymatic conversion/retention has been used for both single-photon emission CT (SPECT)
and positron emission tomography (PET) substrates. The significant advantages of this
strategy include the ability to follow stem cells indefinitely after stable integration of the
transgene, the absence of marker dilution by cell division, and the ability to destroy stem cells
by administration of a suicide drug specific for the enzyme. The disadvantages of this strategy
include the need to genetically manipulate the stem cell ex vivo and the need to administer a
substrate intravenously for each imaging session.
Receptor-mediated targeting requires stable expression of a receptor not found elsewhere in
the body and intravenous injection of a radioactive receptor ligand. (Simonova et al.,2006)

Positron Emission Tomography

Positron emission tomography (PET) is a nuclear medicine imaging technique which

produces a three-dimensional image or picture of functional processes in the body. The
system detects pairs of gamma rays emitted indirectly by a positron-emitting radionuclide
(tracer), which is introduced into the body on a biologically active molecule. Images of tracer
concentration in 3-dimensional or 4-dimensional space within the body are then reconstructed
by computer analysis. In modern scanners, this reconstruction is often accomplished with the
aid of a CT X-ray scan performed on the patient during the same session, in the same
machine.

PET utilizes coincident detection of 2 anti-parallel 511-keV gamma rays emitted after
positron annihilation. Tradeoffs exist between the higher energy of the photons, coincident
detection, and detector efficiency, but overall, PET has a higher sensitivity than SPECT and
permits more accurate quantification of cell number. Although the 3 strategies mentioned
above for SPECT can be used for stem cell tracking with PET, the most advanced by far is the
stable integration of a mutant herpes simplex type 1 thymidine kinase (TK) into stem cells
and periodic intravenous injection of the TK substrate 18FHBG (Wu JC et al., 2000)Although
it permits tracking and quantification of stem cells over the course of months, this strategy
requires genetic manipulation of the stem cells, an infrastructure for 18F chemistry, a PET
scanner, and radiation exposure (albeit it intermittent) to the stem cells and subject.
Additional caveats for SPECT- and PET-based tracking of stem cells include nonspecific
uptake of the radiotracer by normal tissue, relatively low efficiency of collimated SPECT
cameras, and photon attenuation by tissue. Although tissue photon attenuation can be
corrected in some cases, for example by employing hybrid nuclear medicine/CT systems, it
reduces sensitivity, and prevents accurate quantification of stem cell number (Chin BB et
al.,2003). Whether used for attenuation correction or not, hybrid CT systems have the major
advantage that they permit co registration of anatomic (CT) and physiological (SPECT or
PET) images.
Another (often overlooked) issue is the inherent limits of radioactive methods for stem cell
detection. A typical patient dose of 10 to 20 mCi is equivalent to only 3.5 to 7x10 12
radioactive molecules of contrast agent. In typical clinical nuclear medicine imaging, 109
radioactive molecules per milliliter

are required to generate detectable signal above

background (Anthony Parker. To detect a single stem cell, 0.01% of the injected dose would
have to be concentrated in/on the cell, which is a formidable technical challenge.
Resolution in PET is less than that which can be achieved by MRI, but the cross-sectional
information and three-dimensional (3D) reconstruction capability offer the potential to be
more informative than the typical planar projection data obtained using optical imaging
techniques (Kim et al.,2004)

Compare PET and SPECT

SPECT is similar to PET in its use of radioactive tracer material and detection of gamma
rays. In contrast with PET, however, the tracer used in SPECT emits gamma radiation that is
measured directly, whereas PET tracer emits positrons which annihilate with electrons up to a
few millimeters away, causing two gamma photons to be emitted in opposite directions. A
PET scanner detects these emissions "coincident" in time, which provides more radiation
event localization information and thus higher resolution images than SPECT (which has
about 1 cm resolution). SPECT scans, however, are significantly less expensive than PET
scans, in part because they are able to use longer-lived more easily-obtained radioisotopes
than PET.
The PET and SPECT radionuclide imagine techniques allow the imaging of radiolabeled
markers and their interaction with biochemical processes in living animals. Due to their
nanomolar (<10-9 M) sensitivity, PET and SPECT are able to measure biological process at
very low concentrations. The mass of radiotracer injected is extremely small and does not
impact the biological system under study. Technological developments of both PET and
SPECT have led to the implementation of specialized systems for small animals imaging,
with a better spatial resolution (<2 mm) and consequent advancement in the field of cell
tracking in animals model in vivo.
In general a radioisotope with a relatively long decay half life is use to enable the tracking of
cells over period of several hours, or even days (111In T = 2.8days). Numerous cell tracking
experiments have been performed using cells labeled with a radioactive markers.
111

In-labeled cells have been widely used in humans in localizing areas of inflammation by

imaging the leukocyte distribution . Furthermore,

111

In-labeled cells have been applied in

various experimental settings in animals to determine migration of dendritic cells ,

biodistribution of transplanted hepatocytes and even homing of injected mesenchymal stem
cells (MSCs) in animal models .

Micro-single-photon-emission-computed-tomography

(microSPECT/CT) small animal imaging system and an FDA-approved radiotracer (111In

oxyquinoline), has been use to demonstrate that monocyte recruitment to atherosclerotic
lesions. With a noninvasive, dynamic, and three-dimensional fashion in live animals it is
possible to track the monocytes recruitment to the atherosclerotic lesions in apolipoprotein Edeficient (ApoE- /- ) mice. The long half-life of

111

In (2.8 days) enabled the detection of

monocytes for up to seven days after adoptive transfer, and the high-resolution anatomical
data derived from CT allowed localization of hotspots of monocyte infiltration in a submillimeter range. Non invasive radionuclide imaging is also well suited to dynamically track
the biodistribution and trafficking of mesenchymal stem cells to both target and non target
organs. MSCs isolated from bone marrow have the ability to differentiate into multiple cell
lineages including osteocytes, chondrocytes, and cardiac myocytes. The recent ability to label
MSCs with radiotracers provides a method to serially assess the biodistribution of these stem
cells after intravenous administration with the use of radio-nuclide imaging as well as to
determine the homing potential of MSCs to sites of injury (Kraitchman et al.,2005)The use of
the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in
vivo trafficking of allogeneic mesenchymal stem cells (MSCs) labeled with a radiotracer and
MR contrast agent to acute myocardial infarction was dynamically determined and
redistribution of the labeled MSCs after intravenous injection from initial localization in the
lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48
hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was
already visible in SPECT/CT images in the first 24 hours after injection and persisted until
seven days after injection and was validated by tissue counts of radioactivity. Nevertheless,
SPECT- and PET-based tracking of stem cells include nonspecific uptake of the radiotracer
by normal tissue, relatively low efficiency of collimated SPECT cameras, and photon
attenuation by tissue. Therefore, as concerns 111In oxyquinoline labeling of human stem cells,
the effect of radiation dose on human cell lines need to be carefully observed. A critical factor
is to determine whether the

111

In oxyquinoline labeling affects viability, functionality,

migration and proliferative capacity in distinct cell populations as well as species.

Quantum Dots for Labeling Of Stem Cells

Recent advances in nanotechnology offer some prospects to combine the best of each
imaging technique with respect to sensitivity and specificity. There is now an array of
artificial particulate systems used as diagnostic agents capable of targeting different cells in
vivo. Those include colloidal gold, superparamagnetic iron-oxide crystals, dendrimers,
polymeric micelles and liposomes, nanotubes, nanowires, nanoshells, and quantum dots
(QDs). Quantum dots consist of semiconductor nanocrystals 25 nm in diameter, which have
highly favorable fluorescence properties (broad band absorption spectra, narrow band
emission, and high resistance to photobleaching) compared to commonly used fluorophores .
Fluorescent QDs possess several unique optical properties best suited for in vivo imaging .
Because of quantum confinement effects, the emission color of QDs can be precisely tuned
by size from the ultraviolet to the near-infrared. QDs are extremely bright and photostable.
Colhera toxin subunit B (CTB)-quantum dots conjugates were developed for labeling
mammalian cells. Several stem cell types were labeled with CTB-QD conjugates and
quantum dots were completely dispersed throughout the cytoplasm in each cell type,
presumably in vesicles . Stem cells labeled appear to maintain their differentiation potential
as well as stem cell properties .CTB-QD labeled muscle derived stem cells maintain similar
percentage of expression for surface markers indicative of stem cell phenotype, such as stem
cell antigen 1 (sca-1) and CD34. They can also form myotubes under serum deprivation,
hence maintaining their myogenic potential following labeling with CTB-QD conjugates. The
CTB-QD conjugates are likely to be suitable for long term cell tracking . Functionalized
quantum dots offer several advantages for tracking the motion of individual molecules on the
cell surface, including selective binding, precise optical identification of

cell surface

molecules, and details examination of the molecular motion. They were conjugated with
integrin antibodies to perform studies about changes in the integrin dynamics during
osteogenic differentiation of human bone marrow derived progenitors cells (BMPCs) . It was
possible to obtain a single particle tracking, by which it was possible to monitor and
determine quantum dots conjugated integrin molecules on the surface of BMPC and to
elucidate the physical constraints on the protein mobility at the cell surface. (Michalet et
al.,2005) .
QDs long-term fluorescence makes them an important new class of nanomaterials available
for stem cell tracking advance. Although QDs can be optically imaged, in vivo tracking
typically requires a whole animal imaging. None of whole animal imaging systems have been
employed so far to track QDs labeled stem cells in vivo. Moreover, QDs size and surface

coating might affect their cellular internalization, their intracellular concentration and,
consequently, the cytotoxicity of QDs.
Multimodality Contrast Agents

Because no single contrast agent/detector pair will satisfy all needs of stem cell clinical trials,
dual- and multimodality contrast agents, which combine the best features of each technology,
have been developed. Fluorophore-labeled MIONs permit injection of stem cells under
optical image guidance and identification of single cells in pathological specimens ex vivo.
Similar dual optical/MRI contrast has been described using visible-wavelength fluorophores
and Gd3+ chelators conjugated to high-molecular-weight scaffolds such as dextran. Large
nanoparticles generating simultaneous MRI, ultrasound, and fluorescence contrast have also
been described (reviewed in Wickline and Lanza) and might prove useful for multimodality
stem cell tracking.

Future Directions
Given the inherent limitations of currently available imaging technology, future research
should focus on improving sensitivity while minimizing patient exclusion, study cost, and
study complexity. Novel ideas would be welcomed in the field. Paramagnetic chemical
exchange saturation transfer (PARACEST) agents for MRI have the potential to improve MRI
sensitivity by up to 2 orders of magnitude (reviewed in Zhang et al). Terahertz and other

electromagnetic frequencies offer certain advantages, although exogenous contrast agents do

not yet exist. Solid-state nanotechnology solutions, however remote at present, are
particularly attractive because they could potentially provide noninvasive, real-time
monitoring of intracellular pH, calcium, etc, as well as anatomic location, of single stem cells.
Findings through these techniques
Transplantation of predifferentiated rather than undifferentiated hES cells would be more
suited for avoiding teratoma formation.(Li et al.,2008) in this both MRI/BLI were
used.Labeled NPC are recruited to infarcts with both parenchymal and cerebrospinal fluid
administration, but higher initial photon counts suggest that cerebrospinal fluid
administration is more efficient(kim et al.,2004) (BLI was used).After BMSC transplantation
through intravenous route , 17% of infused cells localized to the marrow space within
15 h(cui et al 2005)(PET was used).

Summary

In conclusion, x-ray techniques do not provide adequate contrast sensitivity for cardiovascular
stem cell tracking in the clinical setting. Bioluminescence is limited to small animal studies
and

NIR

fluorescence

to

near-surface

and

histological

applications.

Ultrasound/echocardiography has the potential for single-cell detection but has limited
anatomic accessibility, resolution, and quantification. High-energy photon imaging (SPECT
or PET) has high sensitivity, but for long-term tracking it requires genetic manipulation of the
stem cell, stable expression of a transgene, and multiple exposures to ionizing radiation. MRI
provides excellent 3-dimensional anatomy but is contraindicated for many patients and has
limited availability at many institutions, and some contrast techniques have low sensitivity.
Although multimodality contrast agents might improve the prospects for stem cell tracking
both in vivo and ex vivo, no currently available imaging technology is ideal.
Reporter gene imaging using PET is a better technique for monitoring long-term cell
viability, death, and proliferation, whereas MR imaging is a better technique for highresolution detection of cell location post-transplantation (li et al.,2008). Bioluminescent
imaging should be seen as the one that is possibly complementary to other modalities such as
MRI in which higher resolution is required( kim et al.,2009). To confirm the fate of stem cells
in vivo, it is crucial to continue the development and further refinement of noninvasive
imaging techniques.

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