Magnetic Resonance in Medicine 51:938 –944 (2004)
Improved Route for the Visualization of Stem Cells
Labeled With a Gd-/Eu-Chelate as Dual (MRI and
Fluorescence) Agent
Simonetta Geninatti Crich,1 Luigi Biancone,2 Vincenzo Cantaluppi,2 Debora Duò,2
G. Esposito,1 Simona Russo,2 Giovanni Camussi,2 and Silvio Aime1*
A simple labeling procedure of stem/progenitor cells based on the
use of Gd-HPDO3A and Eu-HPDO3A, respectively, is described.
The Gd-chelate acts as T1-agent for MRI visualization, whereas
the corresponding Eu-chelate acts as reporter in fluorescence
microscopy. Owing to their substantial chemical equivalence, the
two chelates are equally internalized in EPCs (endothelial progenitor cells), thus allowing their visualization by both techniques. The
lanthanide chelates are entrapped in endosomic vesicles and the
labeled cells retain biological activity with preservation of viability
and pro-angiogenesis capacity. Hyperintense spots in MR have
been observed for Gd-labeled EPCs injected under mice kidney
capsule or grafted on a subcutaneous Matrigel plug up to 14 days
after transplantation.
Magn Reson Med 51:938 –944, 2004.
© 2004 Wiley-Liss, Inc.
Key words: endothelial progenitor cells; magnetic resonance
imaging; cell labeling; Gd/Eu-complexes; fluorescence microscopy
Pluripotent stem cells hold a great therapeutic potential
for their capacity of regenerating damaged tissues in the
presence of a number of pathologies (1–3). In particular,
several therapeutic applications have been envisaged for
blood-derived endothelial progenitor cells (4 – 6) (EPCs).
Infusion of ex vivo expanded EPCs may be used to increase
neovascularization of ischemic sites. Moreover, EPCs may
be used for in vitro tissue engineering of implantable devices such as valves, prostheses, or vascular stents. The
possibility of their in vivo visualization will allow monitoring the fate of the transplanted cells. Radioactive labeling has allowed the assessment of tissue distribution of
transplanted EPCs, but unfortunately displayed poor spatial resolution (7). Current MRI technology displays a superb spatial resolution (to ⬍100 ␮m) and is the technique
of choice for attaining the observation of a very small
number of cells. However, the cells have to be labeled
prior to transplantation with suitable relaxation agents.
Up to now, of the two classes of MRI contrast agents (8),
namely, paramagnetic Gd-chelates and iron-oxide particles, the labeling of cells has been pursued mainly using
1
Department of Chemistry IFM, University of Torino, and Center for Molecular
Imaging (CIM), University of Torino, Torino, Italy.
2
Department of Internal Medicine and Research Center for Experimental
Medicine (CeRMS) University of Torino, Torino, Italy.
Grant sponsors: MIUR (PRIN and FIRB project), Associazione Italiana per la
ricerca sul cancro (AIRC), Progetto S. Paolo (to G.C.), Bracco Imaging SpA.
*Correspondence to: S. Aime, Department of Chemistry IFM, University of
Torino, via P. Giuria 7, Torino, 10125, Italy. E-mail: [email protected]
Received 28 August 2003; revised 15 December 2003; accepted 15 December 2003.
DOI 10.1002/mrm.20072
Published online in Wiley InterScience (www.interscience.wiley.com).
© 2004 Wiley-Liss, Inc.
the latter class. Often, in order to pursue an efficient uptake in nonphagocytic cells, the outer surface of the ironoxide particles has to be substantially modified, making
the labeling procedure quite cumbersome. For instance,
successful uptakes have been obtained by targeting transferrin receptors with antibodies conjugated to the dextran
cover of iron-oxide cores (9) or by using an HIV-Tat peptide derivative (10); this allows Tat-mediated internalization of the magnetic nanoparticles. Another approach is
based on the use of DNA transfection reagents (11–16)
such as lipofectamins that allow the internalization of
enough iron-oxide particles for the visualization of the in
vivo tracking of implanted embryonic stem cells in a rat
model of ischemic stroke.
The use of iron-oxide particles may have some drawbacks. First, since they act, primarily, as T2-(negative)agents producing dark spots in the MR images, they are
particularly effective for applications dealing with anatomical districts (e.g., brain) characterized by an intense
MR signal intensity. Therefore, problems might arise when
the detection of grafted cells involves a region endowed
with a low intrinsic MR signal. Although not yet fully
addressed, there might be toxicological problems associated with the metabolic fate of iron-oxide particles. In a
typical experiment the amount of cell-internalized iron
may be as high as 10 times the amount of endogenous iron.
Moreover, it has been recently shown that the distribution
and morphology of endosomes filled with iron-oxide particles are highly sensitive to the effects of the external
static magnetic field (17). In fact, such magnetic compartments are deformed along the field and attract each other
to form chaplets within the cytoplasm. Until now, only
one Gd-based agent has been used to label stem cells. It is
a system containing a dextran polymer backbone which
contains 9 –12 Gd-chelates per dextrane (18). It also bears
a tetramethyl rhodamine functionality for fluorescence microscopy detection. Gd3⫹ ions inside such a large macromolecule (MW 16.6 kDa) appear to act essentially as T2
agents, thus compromising the basic advantage of T1-Gd
agents, that is, to generate hyperintense spots in T1weighted images.
For these reasons we thought to explore alternative
routes to label stem cells based on the use of a welltolerated, small-sized paramagnetic Gd (III) chelate. Moreover, the close analogy between Gd3⫹ and Eu3⫹ ions suggests the development of a dual probe thanks to the fluorescent properties of the latter ion (19,20). Indeed, Gd3⫹
and Eu3⫹ chelated by the same ligand display the same
chemical/biological behaviors and therefore they may be
938
Stem Cell Labeling With Gd/Eu Chelates
used to detect the localization and migration of stem cells
by both MRI and fluorescence microscopy, respectively.
MATERIALS AND METHODS
Gd-HPDO3A complex (white powder) and HPDO3A ligand were kindly provided by Bracco S.p.A. (Milano, Italy). Eu-HPDO3A was synthesized by adding Eu2O3
(1.5 mmol) to an aqueous solution of the ligand (3 mmol).
The reaction mixture was stirred at 70°C for about 8 hr.
Formation of the complex was followed by measuring 1H
NMR-spectra up to the complete disappearance of the
signals of the free ligand. The presence of an excess of free
Eu2O3 ions can be easily removed by forming the insoluble
hydroxide at basic pH followed by centrifugation and filtration of the resulting suspension.
Physicochemical Characterization
1
H-NMR spectra were obtained on a Bruker 600 Avance
spectrometer. The longitudinal water proton relaxation
rate was measured on the Stelar Spinmaster spectrometer
(Stelar, Mede (PV) Italy) operating at 20 MHz, by means of
the standard inversion-recovery technique (16 experiments, 2 scans). A typical 90° pulse width was 3.5 ␮s and
the reproducibility of the T1 data was ⫾0.5%.
Cell Lines
EPCs were isolated from peripheral blood mononuclear
cells from healthy donors as described by Vasa et al. (21).
Cells were plated on culture flasks in endothelial cell basal
medium-2 (Clonetics, Biowhittaker, Walkersville, MD)
supplemented with EGM-2 MV single aliquots consisting
of 5% FBS, VEGF, FGF-2, EGF, and insulin-like growth
factor-1 (complete medium). The endothelial phenotype
was confirmed by the expression of vWF, Tie-2, VEGFR-2,
VEGFR-3, but not of VEGFR-1 by FACS, Western blot and
by functional evaluation of in vitro and in vivo angiogenic
properties in Matrigel (22).
Cell Labeling
EPCs were cultured in tissue culture flasks and allowed to
grow to 60 –70% confluence. Then the cells were washed
and incubated with the required amount of Gd-HPDO3A
in the complete medium. At the end of the uptake experiments, the cells were washed five times with 50 ml RPMI,
detached with trypsin/EDTA solution, and then washed
three times by centrifugation at 1000g for 5 min.
Determination of Gd3⫹ Content in Cells
At the end of the uptake experiment (see above), labeled
cells were collected in 250 ␮l PBS and destroyed by adding the same volume of HCl 37% and left at 120°C overnight. Upon this treatment, all Gd3⫹ is solubilized as the
free aquo-ion. Then the water proton T1 of these solutions
was measured at 20 MHz and 25°C, and the Gd3⫹ concentrations determined from a standard curve obtained using
standard GdCl3 solutions (0.01–2 mM). The method was
double-checked by ICP measurements.
939
The protein concentration was determined from cell
lysates by the method of Bradford (Sigma, St. Louis, MO)
using bovine serum albumin as standard. One mg protein
corresponds to about 4 ⫻ 106 cells.
Determination of Intracellular Relaxivity
At the end of the uptake (16 hr, 37°C), labeled cells (⬃2
million) were transferred to the NMR tube. The supernatant was carefully removed and the T1 of the intact cells
was measured at 20 MHz and 25°C. Then the pellets were
treated with HCl 37% (50:50) at 120°C overnight, in order
to evaluate quantitatively the amount of Gd3⫹ as free aquoion. Intracellular relaxivity was determined using the following equation:
r 1p (intracellular) ⫽ (R *1obs – R 1obsc)/[Gd 3⫹],
where R*1obs are the observed relaxation rates (measured at
c
room temperature) for the Gd-containing cells, R1obs
the
observed relaxation rates for untreated cells, and [Gd3⫹] is
the intracellular mM concentration of Gd3⫹.
Cell Viability
To evaluate cell viability, EPCs were cultured in 96-well
flat-bottom microtiter plates (Falcon Labware, Oxnard,
CA) at a concentration of 5 ⫻ 104 cells/well in DMEM/10%
FBS in the presence of increasing concentrations of GdHPDO3A (10 –100 mM) for 24 hr at 37°C. The cell viability
was evaluated using the sodium 3⬘-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene
sulfonicacid hydrate (250 ␮g/ml XTT)-based assay. The
reduction of XTT was monitored by determination of the
absorption values at 620 nm in an automated ELISA
reader. The viability assay was performed by doublestaining the cells with 0.46 mM fluorescein diacetate
(FDA) as an inclusion dye and 14.34 mM propidium iodide as an exclusion dye for living cells.
Confocal Fluorescence Microscopy
Uptake of Eu-HPDO3A was analyzed by confocal microscopy. Confocal microscopy was performed on a Leica TCS
SP2 model confocal microscope (Heidelberg, Germany)
using a 63⫻ magnification lens and excitation was obtained by an argon laser at 351 and 363 nm (20).
Angiogenesis Assays
In vitro formation of vessel-like tubular structures was
studied on growth factor-reduced Matrigel (8.13 mg/ml;
Becton Dickinson Labware, Bedford, MA) diluted 1:1 in
ice-cold DMEM under a Nikon Diaphot inverted microscope in a Plexiglas Nikon NP-2 incubator at 37°C. Briefly,
the 5 ⫻ 104/well cells were seeded onto Matrigel-coated
wells in 10% FCS/DMEM. After the cells had attached, the
FCS-containing media was removed and 0.5 ml serum-free
DMEM was added. Image analysis was performed by digital saving of images at an interval of 30 min with a MicroImage analysis system (Casti Imaging srl, Venice Italy).
In vivo, angiogenesis was assayed as blood vessel formation from 0.5 ⫻ 106 EPCs embedded into a solid gel of
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Geninatti Crich et al.
basement membrane (23). As standard procedure, EPCs
were resuspended in 0.5 ml of Matrigel in liquid form at
4°C, and then injected into the dorsal subcutaneous tissue
of 8-week-old SCID mice.
For the kidney subcapsular implant, under general anesthesia induced by methoxyflurane (Metofane; ScheringPlough Animal Health, Atlanta, GA), a left lobotomy was
performed, the right kidney was exposed, and 2 ⫻ 106
labeled EPCs were injected in the subcapsular space. The
care of the animal was in accordance with institutional
guidelines.
MRI
All MR images were acquired on a Bruker Avance 300 (7 T)
equipped with a microimaging probe. The system is endowed with two birdcage resonators with 30 and 10 mm
inner diameter, respectively.
For MRI of cell pellets, cells were incubated with the
contrast agent, extensively washed, detached, and again
washed as described above. The cells were pelleted within
a 200 ␮l end-sealed plastic pipette tip that was then inserted in 2% agarose within a 50-ml tube. The imaging
protocol consisted of a coronal T1-weighted spin echo
(TR/TE/NEX 140/4.4/36, FOV 2.8 cm, 1 slice 1 mm). The
ex vivo and in vivo imaging of Matrigel plugs was
achieved with a T1-weighted spin echo (TR/TE/NEX 100/
4.4/8, FOV 3.0 cm, 1 slice 1 mm and TR/TE/NEX 120/4.4/
42, FOV 2.7 cm, 1 slice 1 mm, respectively). Imaging of
mouse kidneys was performed using a T1-weighted spin
echo protocol (TR/TE/NEX 180/4.4/56, FOV 2.6 cm, 1 slice
0.7 mm). After imaging, Matrigel plugs were excised and
processed for optical microscopy. The care of the animal
was in accordance with institutional guidelines. In vivo
imaging was performed under general anesthesia induced
by methoxyflurane (Metofane; Schering-Plough Animal
Health).
RESULTS AND DISCUSSION
Gd-HPDO3A (Prohance, Bracco Imaging) (Fig. 1) is a commercially available, MRI nonspecific agent. Once administered intravenously, it quickly equilibrates between the
intra- and extravascular compartments. It is a neutral,
highly hydrophilic chelate endowed with a very high thermodynamic stability (log K⬃23.2) (24) which ensures
against any release of harmful Gd3⫹ ions under in vivo
conditions. Its relaxivity, r1p (where r1p is the relaxation
enhancement of solvent water protons at 1 mM concentration of the paramagnetic chelate) is 4.2 mM-1s-1 in water at
25°C.
From pharmacological studies, Gd-HPDO3A appears to
be one of the best-tolerated xenobiotics, as it yielded an
LD50 ⬎10 mmol/kg in rats (24).
As anticipated above, the model system for the stem
cells used in this work is represented by ex vivo expanded
blood-derived human EPCs. In a typical experiment, 2 ⫻
106 EPCs were incubated in media containing different
concentrations of Gd-HPDO3A. As there is no specific
mechanism available for the uptake of such neutral and
highly hydrophilic molecules, their cell entrapment occurs mainly through pinocytosis.
FIG. 1. The internalization of GdHPDO3A into EPCs. The uptake
was carried out on ⬃2 million cells incubated in the presence of
increasing concentrations of the complex at 37°C for 24 hr.
The amount of internalized Gd-HPDO3A has been determined by measuring the relaxivity of the cytoplasmatic
extracts mineralized at 120°C overnight, in the presence of
HCl (6 M). From the known relaxivity of aquo-Gd3⫹ ion
(r1p ⫽ 13.5 mM-1 s-1), one may easily determine the
amount of total Gd taken up in a given experiment. In Fig.
1, the amount of internalized Gd (expressed in terms of
1 mg of total protein) is plotted against the concentration
of Gd-HPDO3A in the incubation medium (incubation
time ⫽ 24 hr). As expected for this type of internalization
process, no saturation effect was detected and the amount
of Gd3⫹ taken up is linearly proportional to the concentration of Gd-HPDO3A in the incubation medium. It is
known that the pinocytosis process leads to the formation
of small endosomes which may eventually fuse into larger
lysosomes. In principle, one would expect that the relaxivity of Gd-HPDO3A inside the endosomes to be not too
different from that shown in the incubation medium
(⬃4.2 mM-1s-1 at 20 MHz, 298 K), as the endosome formation simply proceeds through the entrapment of a portion
of the extracellular fluid. Against this expectation, the
measure of r1p of internalized Gd-HPDO3A carried out on
pellets of ⬃2⫻106 EPCs showed that the intracellular relaxivity is indeed dependent on the amount of Gd taken up
by the cell (Fig. 2a). In fact, pellets obtained from incubation at low concentration yielded r1p values significantly
higher than the relaxivity found in the incubation medium, whereas when the amount of internalized Gd is
⬎0.1 ␮mol/mg of protein the observed relaxivity is
“quenched” to a value of 1.0 mM-1 s-1. It is likely that, at
low concentrations, this observed relaxation enhancement
is the result of a decreased molecular mobility. Conversely, the decrease of the observed relaxivity, as the
endosomic concentration of Gd-HPDO3A increases, may
be attributed to different causes. One may think of a sort of
shrinking effect inside endosomes for which the distances
Stem Cell Labeling With Gd/Eu Chelates
941
FIG. 2. a: The relaxivity (r1p) of intracellular Gd-HPDO3A as a function of the amount of Gd internalized into EPCs/mg of proteins. One mg
protein corresponds to about 4 ⫻ 106 cells. b: T1-weighted spin echo image (TR/TE/NEX (140/4.4/36), FOV 1.6 cm, 1 slice 0.5 mm) of an
agar phantom containing EPCs labeled with increasing amounts of Gd-HPDO3A. Each pellet (⬃5 ⫻ 105 cells) contains: A) 5 (SI ⫽ 112), B)
8 (SI ⫽ 180), C) 11 (SI ⫽ 165), D) 14 (SI ⫽ 166), e) 20 (SI ⫽ 165) nmol of Gd-HPDO3A. SI is the corresponding signal intensity. SI measured
for a corresponding number of unlabeled cells yielded a value of 43.
between Gd-containing molecules becomes short enough
to affect the electronic relaxation time. Such an effect has
been recently shown to occur on the surface of micelles
formed by Gd-chelates functionalized with a hydrophobic
chain (25). Another cause may be the extensive broadening
of the signal (mainly as the result of T*2 effects). Such a loss
of signal intensity is expected to alter the assessment of
proton T1 by the inversion recovery procedure in cell
pellets. In order to assess the effect on signal intensity,
associated with the changes in intracellular relaxivity, T1weighted MR images of a phantom, made up of pellets (5 ⫻
105 cells) containing different amounts of internalized GdHPDO3A, were recorded (Fig. 2b). As expected from the
results shown in Fig. 2a, the intensity of the signal (SI)
decreases for higher intra-endosomic concentrations of the
paramagnetic agent. Accordingly, T2 varies from 66 to
about 25 ms on going from the lowest to the highest GdHPDO3A concentration in the pellets reported in Fig. 2b.
However, the decrease of SI is not dramatic and it would
not compromise the possibility of detecting the labeled
cells over an extended range of Gd concentrations. All
above considerations are based on the assumption that
water molecules have free access to the compartment containing the paramagnetic agent. This seems a likely assumption, as the membrane delimiting the endosome vesicle should have maintained the same water permeability
of the cellular membrane from which it derives.
Next, the issue of the preservation of the chemical integrity of Ln(III)-HPDO3A upon cellular internalization
was tackled. For this experiment, Gd-HPDO3A was replaced in the incubation medium with Eu-HPDO3A
(50 mM, 16 hr, 2 ⫻ 106 cells). The latter complex displays
relatively sharp lines in its high-resolution 1H-NMR spectrum. Although the assignment of all 1H resonances was
not carried out, the lowest absorptions (at 32.5, 32.1, 27.4,
and 22.1 ppm) are likely due to the four axial protons of
the tetraazamacrocycle ring. This has been used as a reporter of the presence of Eu-HPDO3A in the cytoplasmatic
extracts recovered by sonication of EPC pellets. To quantitate the amount of Eu-HPDO3A, a weighed amount of a
related Eu(III)-complex (26) was added. The measurement
of the relative ratios of the integrated areas of the signals of
the two complexes allowed us to determine the actual
amount of Eu-HPDO3A in the specimen. This should correspond to the amount of Gd-HPDO3A, as the two chelates
have identical behavior in the internalization process. Interestingly, the obtained amounts of Eu-HPDO3A correspond nicely to those found for Gd, from relaxometric
measurement after mineralizing the EPC pellet in related
experiments. Thus, we may conclude that all entrapped
Gd is present as Gd-HPDO3A.
To determine the minimum number of Gd-HPDO3Alabeled cells detectable by MRI under in vitro conditions,
six pellets containing different ratios of labeled/unlabeled
cells were embedded in agar and their image detected by a
T1-weighted sequence. We found that the minimum detectable number of labeled cells is on the order of 2–3 ⫻
103 where each pellet contained ⬃250,000 cells.
An important issue is the biological effect associated
with the entrapment of Gd-HPDO3A in EPCs. The percentile viability of EPCs treated with Gd-HPDO3A, evaluated
by the XTT-based assay, did not differ from that of the
control for all concentrations tested (10, 25, 50, 100 mM in
the incubation media) and was greater than 98%. Moreover, no difference in cell proliferation was detected at the
scale of XTT reduction (Fig. 3a). The absence of dead cells
was confirmed by the absence of nuclear staining by propidium iodide, which was less than 1% in both treated
and untreated EPCs (not shown). Moreover, Gd-HPDO3Alabeled EPCs retained the functional ability to make vessel-like structures in vitro and in vivo Matrigel (Fig. 3b,c).
Thus, we conclude that, also when internalized into cells,
the lanthanide chelates of HPDO3A are an inert and welltolerated species.
Gd-chelate distribution cannot be observed by the microscopic techniques currently used in cellular biology.
However, its neighbor in the periodic table is Eu, which
has excellent fluorescent properties. A characteristic feature of lanthanide(III) ions is their remarkably analogous
chemistry. One would expect that Gd-HPDO3A and EuHPDO3A demonstrate identical behavior in the cell internalization process. Thus, the fluorescent response of EuHPDO3A can be exploited for histological confirmation of
cell distribution. In Fig. 4, the image of EPCs incubated
with Eu-HPDO3A obtained with a confocal microscope is
shown. The endosomic vesicles containing Eu-HPDO3A
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Geninatti Crich et al.
FIG. 3. a: The proliferation assay of EPCs (labeled with Gd-HDO3A or unlabeled) detected by
XTT-reduction assay. Data are the mean ⫾ SD of
three independent experiments. b: Micrograph
representative of in vitro angiogenesis assay on
Matrigel. EPCs labeled with 25 mM Gd-HPDO3A
form vessel-like tubular structures after 24 hr incubation at 37°C. c: Micrograph representative
of in vivo angiogenesis induced by EPCs 1 week
after subcutaneous implantation within Matrigel
in SCID mice. Prior to injection the cells were
labeled with 25 mM Gd-HPDO3A for 16 hr at
37°C. The same animals were subjected to MRI.
Three experiments were performed with similar
results.
are clearly detected in the cytoplasmatic region, around
the nucleus. Actually, EPCs can be incubated in a medium
containing both Gd-HPDO3A and Eu-HPDO3A in order to
have cells suitably labeled for the two imaging modalities.
The proposed method is one of general applicability and is
simpler than the use of the recently reported GRID (gadolinium rhodamine dextran) agent (18).
High angiogenic potential represents the main feature of
EPCs and holds promise for their use in therapeutic settings of local revascularization (1–5). In an in vivo mouse
model of angiogenesis, we implanted EPCs subcutaneously within a Matrigel plug. After 7 days, Matrigel plugs
were vascularized by a capillary network formed by EPCs
and connected to the circulation. Our task was therefore
the assessment of whether Gd-HPDO3A-labeled EPCs (5 ⫻
105 cells) implanted within Matrigel (0.5 ml) were detectable under in vivo conditions. One day after implantation,
hyperintense spots, well distributed in the Matrigel, were
clearly detected in the resulting MR image (Fig. 5a). The
histologic examination showed that the cells were dis-
persed within the gel without evidence of capillary formation. In contrast, 7 days after implantation a fine network
of bundles was seen in vivo within the Matrigel (Fig. 5b,c).
The histologic examination showed large capillary structures transposing the gel plugs. Thus, MRI images parallel
histologic findings: the hyperintense signal persisted for
14 days. As a control, Matrigel-embedded unlabeled cells
were implanted under the same conditions and were always negative for an MRI signal.
Moreover, 1 ⫻ 106 labeled or unlabeled EPCs were injected under the right kidney subcapsular space of SCID
mice and images were taken after 24 hr. As shown in Fig.
6, labeled EPCs were visible at the site of injection. This
result supports the view that the use of Gd-labeled cells
may be particularly useful in organs, like the kidney, endowed with an intrinsic low MRI signal.
In summary, the labeling procedure herein discussed for
EPCs appears to be of general applicability, as all cell types
tested to date showed an analogously efficient uptake of
Gd(III)-chelates via the pynocytotic route, with no appar-
FIG. 4. a: Micrograph representative of intracellular EPC uptake of Eu-HPDO3A after 16 hr of incubation at 37°C with 50 mM Eu-HPDO3A. b: Serial
confocal optical sections of the cells showing that
the fluorescent Eu-HPDO3A accumulates in the
endosomal vesicles around nuclei.
Stem Cell Labeling With Gd/Eu Chelates
943
FIG. 5. a: Ex vivo T1-weighted spin echo
image (TR/TE/NEX 100/4.4/8, FOV 3.0 cm,
1 slice 1 mm) of Gd-labeled EPCs dispersed into a subcutaneous Matrigel plug
excised one day after the implantation. b,c:
In vivo T1-weighted spin echo image (TR/
TE/NEX 120/4.4/42, FOV 2.7 cm, 1 slice
1 mm) of Gd-labeled EPCs dispersed into a
subcutaneous Matrigel plug 7 days after
implantation.
ent cytotoxicity. The compartmentalization of lanthanide(III)-HPDO3A complexes into membrane-bound cytoplasmatic perinuclear endosomes is likely to prevent
any impact on relevant cellular processes, while maintaining an efficient accessibility to cytoplasmatic water molecules. Actually, the internalization of GdDTPA into tumor
cells in culture was reported for human glioblastoma cells
(27) and MCF7 (28). Consistent with our results, entrapment of GdDTPA in these cells takes place because of the
high concentration of the paramagnetic agent in the incubation media. The detection of hyperintense spots in MR
images of EPCs, 14 days after their grafting on Matrigel
structures, shows that Gd-complexes can be competitive
with iron-oxide particles for in vivo tracking of stem cells.
Finally, it has been shown that the related Eu-HPDO3A
complex is an excellent probe for fluorescence microscopy
that allows a good match with the corresponding MR im-
FIG. 6. T1-weighted spin echo image (TR/TE/NEX 180/4.4/56, FOV
2.6 cm, 1 slice 0.7 mm) of transplanted Gd-labeled EPCs (⬃1 million
cells) under the right kidney capsula. The image was obtained 1 day
after transplantation.
ages based on the distribution of the Gd-chelate for an
efficient localization of the transplanted cells.
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