Cell labeling is finding increasing applications in fields such as cellular
biology and medical imaging. Analysis of the distribution and cellular
migration or cellular trafficking is essential for many physiological
and pathological processes (Wallace et al., 1993). Conventional
methods based on marker proteins labeled with fluorescence probes or with
radioisotopes (111In, 123I, or 51Cr)
can be used to study lymphocyte trafficking, but these methods are limited
by the isotope half-life, the tracer transfer rate into cells and the
toxicity of the labeling process (Wallace et al., 1993). MR Imaging
can be used to follow labeled cells using an endogenous or exogenous contrast
agent. The agent must be specific for a given cell type, must not affect
the antigenic properties of the cell, must induce a specific local signal
distinguishable from neighboring tissues and must persist on or inside
the labeled cell for an adequate time. The best contrast agent for studying
cellular migration is superparamagnetic particles (Bulte et al.,
1996; Weissleder et al., 2000), also known as superparamagnetic
iron oxide (SPIO) or ultra small super- paramagnetic iron oxide (USPIO).
These particles have a remarkable r2 relaxivity which makes
them particularly effective on T2- weighted imaging. The most
widely used product is dextran-coated superparamagnetic particles (Dodd
et al., 1999; Yeh et al., 1995; Jung et al., 1995).
Cellular uptake by spontaneous endocytosis is not very efficient but can
be enhanced by the use of a peptide sequence of HIV-1 transactivator protein
(Tat). This protein is internalized by cells when present in the extracellular
medium (Vives et al., 1997; Rusnati et al., 2000). Tat peptide
can be conjugated with superparamagnetic particles, resulting in nanoparticles
with great stability and cellular permeability (Josephson et al.,
1999). The conjugate can also be labeled with a fluorochrome (FITC), enabling
it to be visualized by both flow cytometry and high-resolution MRI (Lewin
et al., 2000). Other methods used to coat magnetic nanoparticles
include monoclonal antibodies (Bulte et al., 1999), transfection
agents including dendrimers (Bulte et al., 2001a) and lipofection
agents (Hoehn et al., 2002).
Recent progress in the isolation of stem cells from various tissues and
organs, along with an improved understanding of their function has extended
applications of stem cell therapies not only to hematopoietic diseases
but also to cardiovascular and neurological diseases (Orlic et al.,
2001; Strauer et al., 2003; Cai et al., 2002). The development
of such new stem cell-based therapies requires a quantitative and qualitative
assessment of stem cell distribution to target organs (homing), differentiation
outcome and engraftment (Strauer et al., 2003). Magnetic Resonance
(MR) imaging would be well suited for this task, because it can enable
both whole-body examinations and subsequent detailed depictions of host
organs with near-microscopic anatomic resolution and excellent soft-tissue
contrast (Barkhausen et al., 2001; Choi et al., 1997). In
addition, MR imaging allows repetitive investigations without known side
effects and without risking radiotoxic damage to the transplanted cells.
For transplanted stem cells to be visualized and tracked with MR imaging,
they must be labeled with MR imaging contrast agents and the development
of these dedicated labeling techniques is currently being broadly investigated
(Schoepf et al., 1998; Weissleder et al., 1997; Hinds et
al., 2003; Kraitchman et al., 2003; Zhao et al., 2002;
Frank et al., 2003; Daldrup-Link et al., 2003). Initial
cell-labeling techniques were hampered by limited concentration of internalized
contrast agent which resulted in a limited sensitivity of MR Imaging to
show the labeled cells. To compensate for this limited sensitivity, experimental
cell-tracking studies were performed by using MR imagers with very high
magnetic field strengths of up to 14 Tesla (Bulte et al., 2001b)
alternatively, the contrast agent-labeled cells were directly injected
into the organ of interest (Orlic et al., 2001) in which the cells
had a small distribution volume, ensuring that contrast agent concentration
remained relatively high in the examined field of view. With clinical
1.5-Tesla MR equipment and clinically applicable contrast agents, it has-to
our knowledge-not been possible to trace the in vivo distribution
of intravenously injected hematopoietic cells to more than one final target
organ or to depict the migration of the transplanted cells to several
subsequent target organs over time (Schoepf et al., 1998). Recently
some studies show that it is possible to use 1.5 Tesla MR imaging to monitor
the distribution of iron oxide labeled hematopoietic progenitors in different
organs (Daldrup-Link et al., 2005). Newer and optimized labeling
techniques have improved the efficiency of stem cell labeling and allowed
the labeling of the cells with contrast agents that have been approved
by the US. Food and Drug Administration or are currently being investigated
in clinical trials (Frank et al., 2003; Daldrup-Link et al.,
2003). Thus, the purpose of this study was to evaluate the use of clinical
1.5-T MR imaging equipment to depict the in vivo distribution of
iron oxide-labeled human hematopoietic progenitor cells in irradiated
mice. The MR imaging method by which we are exploring this concept is
new and has an extensive potential in studying hematopoiesis. The results
of this investigation can be later used to mark and follow stem cells
in vivo and help better understand and monitor stem cell transplantation.
MATERIALS AND METHODS
This study was conducted in the Hematology department of Tarbiat Modares
University in Iran.
Isolation of hematopoietic CD34+ stem cells: Suspensions
of human hematopoietic progenitor cells were prepared from Umbilical Cord
Blood (UCB), which was collected from the umbilical cord vein after normal
delivery of a full-term infant after obtaining informed constant. The
Medical Ethics Review board of hospital approved the protocol for collecting
the UCB for research purpose. The samples were collected in heparin-flushed
syringes, stored at 4°C and processed within 8 h of collection. Low-density
cells were isolated with density centrifugation (d = 1.077 g mL-1).
After centrifugation at 400x for 30 min, the mononuclear cells were collected
and washed once in PBS. Subsequently, red cells were lysed with a lysing
reagent. After erythrolysis the cell samples were washed twice in the
PBS. The CD34+ MNC fraction was directly isolated with superparamagnetic
microbead selection using high-gradient magnetic field and mini MACS column
(Miltenyl Biotech, Gladbach, Germany). The efficacy of the purification
was verified by flow cytometry counter staining with anti-CD34-FITC and
anti-CD133-PE antibody. In the cell fraction containing purified cells,
CD34+ cells ranged from 80 to 95%.
Ex vivo expansion of hematopoietic stem cells: UCB CD34+
cells were seeded at a concentration of 1x105 cells mL-1
in hematopoietic stem cell expansion medium (Sigma; S-0189, StemlineTM)
supplemented with 50 ng mL-1 recombinant human thrombopoietin
(Sigma®; T-1568) and stem cell factor (Sigma®;
S-7901). Cultures were incubated at 37°C in a humidified atmosphere
containing 5% CO2. The cell count was monitored daily and cell
concentration was readjusted by the addition of fresh medium.
Cell labeling with superparamagnetic iron oxide: Different ratios
of Fe-Pro complex were examined to determine the optimum concentration
of these reagents. The commercially available ferumoxide suspension, ENDOREM®
(Guerbet) contains particles approximately 80 to 150 nm in size and has
a total iron content of 11.2 mg mL-1. Ferumoxide at a concentration
of 100 μg mL-1 and protamine sulfate at a concentration
of 6 μg mL-1 were put into a mixing tube containing serum-free
media. Protamine sulfate was prepared as a fresh stock solution of 1 mg
mL-1 in distilled water at the time of use. The solution was
mixed for 10 min with continuous shakings. For cells suspension, FE-Pro
complexes were applied directly to the cells and then an equal volume
of the respective complete medium was added to the cells in a final concentration
of 50 μg ferumoxide and 3 μg protamine sulfate per ml of medium.
The cells were then incubated overnight.
Histology: After incubation with FE-Pro, cells were washed three
times to remove excess FE-Pro; and then transferred for cytospin slide
preparation. Cells were fixed with methanol and washed and incubated for
30 min with 2% potassium Ferrocyanide (Potassium hexacyanoferrate ii trihydrate,
ACS reagent, 98.5-102.0%, Sigma® P3289) in 3.7% hydrochloric
acid. They were washed again and counter stained with nuclear fast red.
Determination of labeling efficiency: FE-Pro labeling efficiency
was determined by manual counting of stained and unstained cells on cytospinned
slides with Perl staining. The percentage of labeled cells was determined
from the average count in 5-10 high-powered fields.
Determination of mean iron concentration per cell: Ferumoxide
at a concentration of 50 μg mL-1 was mixed with protamine
sulfate at a concentration of 3 μg mL-1 and incubated
with cells overnight to determine the intracellular iron concentration.
After labeling, cells were washed twice with PBS and a specific number
of labeled and unlabeled cells were collected for atomic absorption assay.
Histological evaluation of spleen: For histopathologic examination,
spleens were taken into paraffin block and sliced with microtome in the
thickness of 5 μm for hematoxilin and eosin staining.
In order to reveal homing of CD34+ cells in the spleen of
injected mice, spleens of mice were removed after 1-2 days, blocked in
paraffin and examined using Prussian blue staining.
Functional and phenotypic analysis of FE-Pro labeled cells: Both
labeled and unlabeled CD34+ cells were analyzed with spleen
colony assay and histological staining. Labeled cells were injected at
a concentration of 1x105 to 5x106 cells into tail
vein in order to obtain the best dose needs for colony assay. Then each
group received 6x105 CD34+ cells and compared with
control group that expected the same number of unlabeled cells. After
10 to 15 days, mice were killed by cervical dislocation and assessed for
colony forming unit-spleen (CFU-S) generation. The spleens were put into
the methanol for 30 sec and then were counted for colonies.
Animal and animal procedure: 6-8 week-old (20 to 30 g) female
Balb/c mice were obtained and housed under pathogen-free conditions and
fed with autoclaved food and water. All animal experiments were approved
by the Animal Care Committee of Tarbiat Modares University School of Medicine.
Mice received a fatal dose of 7.5 Gy total body gamma irradiation using
a Co-60 source (Theratron II, 780C, Canada).
Ciprofloxacin (85 mg L-1), Polymixine B (70 mg L-1)
and Amphotericin B (80 mg L-1) were added to the drinking water
after mice were irradiated.
Human CD34+ cells were counted and resuspended in Stemline
media containing 2% FCS and a cell dose of 6x105 cells/mouse
was transplanted into mice for colony assay. Also a cell dose of 0.5x107
to 1.5x107 cells/mouse was transplanted into mice for
MRI assay by tail vein injection 10 to 12 h after irradiation in a volume
of 300 μL medium/ FCS 2%per mouse.
MR imaging: MR imaging of the animals was performed in each group
before and 24 (n = 6), 36 (n = 6), 48 (n = 6) and 72 (n = 6) h after injection
of labeled cells or contrast agent. Imaging was performed with a 1.5-Tesla
MR imaging unit (SN 23344, Symphony, SIEMENS) and a knee coil. Pulse sequences
comprised coronal T2-weighted three-dimensional fast field-echo, 32/10
sequences with a flip angle of 25° and an effective section thickness
of 650 μm. MR images were acquired with a field of view of 100-75
mm, a 384 x 384 x 16 bit pixel matrix and an in-plane spatial resolution
of 200x150 μm. Average signal intensities of bone marrow before and
after cell injection were measured by one investigator (H.E.D.), who was
blinded to the applied labeling procedure. Signal to noise ratios (SNRs)
were calculated by dividing signal intensity data of the target organ
by the image (background) noise (random fluctuations in signal intensity),
which was measured in the background anterior to the depicted object.
This modification has been designed and conducted for the first time by
Statistical analysis: For this pilot study, increasing quantities
of cells -0.5x107, 1x107 and 1.5x107
cells-were injected into two animals in each contrast agent group. The
initial number of cells was chosen on the basis of results from other
studies (Daldrup-Link et al., 2005). Results of the pilot study
revealed that only injections of 1.5x107 cells caused visible
signal intensity changes in bone marrow.
The MR signals intensities before and after cell injections, quantified
as SNR data were presented as means and standard errors of the mean. To
compare differences in these quantitative MR data before and after injection
of 1.5x107 cells, a two-tailed paired Student t-test was used.
Differences in SNR data at different time points before and after injection
in the same animals were tested for significance with an analysis of variance
for repeated measurements. Statistical significance was assigned if p<0.05.
CD34+ cells isolated from UCB: CD34+ positive
cells were isolated by manual cell separating unit and expanded in the
presence of growth factors for more than three weeks. The purity of isolated
umbilical cord blood CD34+ cells was determined by flow cytometry.
It was about 80 to 95% (Fig. 1 and 2).
||Colony assay of mouse spleen for human CD34+
|*Significant difference between dosage of CD34+:
Labeling efficiency of FE-Pro and iron content per cell: Cells
evaluation demonstrated approximately 100% labeling efficiency with FE-Pro
at ratios of ferumoxide 50 μg mL-1 to protamine sulfate
3 μg mL-1 (Fig. 3). The average iron
content per cell following FE-Pro labeling of cells was 1.98±0.03
pg iron cell-1 for HSCs. There was also iron in unlabeled HSCs
ranging from 0.03 to 0.07 pg cell-1.
Trypan blue dye exclusion test showed no significant increase in cell
death compared with control cells at the end of overnight incubation.
Colony forming capacity of labeled and unlabeled CD34+:
The spleens of injected mice were counted in different groups. A cell
dose of 5 to 6x105 cells showed the best result and choiced
for final injection (Table 1). There was no different
between labeled and unlabeled cells in colony forming properties, as compared
with each other (Fig. 4).
In order to show CD34+ cells engraftment in mouse spleen,
the section of spleens stained with Prussian blue for iron-oxide particles
(Fig. 5) and Hematoxcilin and Eosin staining for colony
assessment (Fig. 6).
||The CD34+ cells isolated from human umbilical cord blood.
Cell culture after one week of the initial seeding of CD34+
cells (A, B). A final magnification for (A) is 20 and for (B) is 40
|| Flow cytometry histograms show the immunophenotype
of UCB CD34+ isolated from the human cord blood. Isolated cells were
positive for CD34+ and negative for CD133
|| Stem cells labeled with SPIO and protamine sulfate
and stained with Perl`s staining. B and C indicate that stem cells
cultured in the presence of SPIO and protamine sulfate absorbed these
contrast agents from the medium. A indicates stem cells as a control
|| Comparison of mouse spleen in normal and after transplantation.
The spleen above belongs to a mouse that was injected with CD34+ cells
(after 14 days) and the picture below shows a normal spleen
|| Prussian blue iron staining of mouse spleen. A and
B indicate colonies that include stem cells with iron particles in
their cytoplasm after transplantation with labeled CD34+ cells. (C)
Shows normal colony with no iron particle in it
|| Hematoxcilin and Eosin staining of mouse spleen. (Left)
Shows colonies present in normal spleen. (Right) Indicates CFU-S that
formed in spleen of irradiated mouse after transplantation with human
UCB CD34+ cells
|| Follow-up T2-weighted MR images of a Balb/c mouse before
(left) and 48 h after (p.i.) intravenous injection of 2×107
FE-Pro-labeled progenitor cells (right). MR images show femoral bone
marrow. Iron oxide–labeled cells show accumulation and subsequent
signal intensity decline in bone marrow 48 h after injection (right).
The amount of administered iron was 30 µg through injection
of iron oxide-labeled cells
||MR signal intensities of bone marrow before and after
injection of labeled human CD34+ cells
|*Significant difference between before and after treatment:
MR imaging: After injection of ferumoxide-labeled cells, a cell
concentration-dependent signal intensity decline was observed in bone
marrow on T2- weighted MR images. Injection of 1.5x107 cells
resulted in a significant signal intensity decline in bone marrow (p<0.05)
(Table 2 and Fig. 7). A control injection
of 1.5x107 unlabeled cells did not result in detectable changes
in MR signal intensity.
In this study we used a method for cell labeling by using contrast agent
and transfection agent. After cell labeling, we successfully used clinically
applicable 1.5 Tesla MR imaging equipment to monitor homing of labeled
cells in bone marrow of lethally irradiated mice. The results showed that
by adding 3 μg protamine sulfates and 50 μg ferumoxide per ml
culture medium the best effect in labeling process of CD34+
cells was observed. In this concentration, cytoplasmic absorption of iron
oxide by CD34+ cells was estimated to be about 1.98 pg cell-1.
These findings are almost like the findings of other researchers who have
used these two agents for cell labeling (Arbab et al., 2004a).
To make a better labeling, we tried to use another transfection agent.
For this purpose we used PLL, a transfection agent, instead of Protamine
sulfate. Unfortunately we didnt obtain a reasonable result from
this agent contrary to the findings of previous studies (Arbab et al.,
2004b). In this study about 100% of CD34+ cells were labeled
with FE-Pro complex and effectively absorbed iron oxide. But when we used
PLL as a transfection agent, the number of labeled cells significantly
decreased and moreover the cytoplasmic absorption of the contrast agent
was reduced. The advantage of using protamine sulfate and ferumoxide is
that both of these agents have FDA-approval and have been used clinically.
In a recent study in which the homing of stem cells by MRI was monitored
(Daldrup-Link et al., 2005), P7228 was used as a contrast agent
and liposome as a transfection agent none of which have been approved
for clinical use by FDA.
We showed that cell labeling with contrast agent has no effect in the
CFU-S formation properties of CD34+ cells. For this purpose
labeled and unlabeled CD34+ cells were injected into the tail
vein of irradiated mice and compared after two weeks for their CFU-S formation
capacity (Table 1 and Fig. 4). The
results showed that the labeling process had no effect on the function
of labeled cells. In this study we didnt evaluate the effect of
labeling on the lineage differentiation in the spleen. Some researches
emphasize that the labeling procedure may change the lineage differentiation
of labeled stem cells and labeled CD34+ cells tend to differentiate
to myeloid and megakaryocytic lineages rather than lymphoid.
The main goal in the development of new stem cell therapies is to achieve
and prove the homing of transplanted cells to the particular tissue where
they should exert their therapeutic activity (Strauer et al., 2003).
This is of special importance if the cells are administrated systematically
(e.g., after intravenous injection, when cells pass through several intermediate
organs before reaching their final destination) rather than directly into
the target organ. Data in our xenotransplant model showed that the in
vivo distribution of systematically injected CD34+ cells
to the desired final target organ (bone marrow) can be traced using MR
imaging. The observed in vivo cell distribution corresponded in
general to the distribution of human hematopoietic cells in immunodeficient
NOD/SCID mice in previous studies (Van Hennik et al., 1999; Kerre
et al., 2001; Kollet et al., 2001). These cells disappeared
rapidly from the circulation and accumulated in bone marrow (Kerre et
al., 2001; Kollet et al., 2001). In bone marrow, an increasing
accumulation of human cells was observed up to 24 h after injection (Van
Hennik et al., 1999). This initial cell accretion was followed
by cell persistence in marrow (Kerre et al., 2001; Kollet et
In this study, we first used MR imaging to depict early processes of
cell transplantation. And then in order to show the successfully engrafted
cells, we evaluated spleens of mice for CFU-S formation. In this context,
it is very important to distinguish between homing and engraftment. Homing
is characterized by the in vivo distribution of transplanted cells
to specific target organ. This usually occurs within 24 to 48 h after
injection (Oostendorp et al., 2000). But cell engraftment is characterized
by subsequent proliferation and separation of progenitor cells and which
usually occurs over several days or weeks after transplantation (Daldrup-Link
et al., 2005).
In previous studies mostly NOD/SCID mice have been used as animal models
but we have used Balb/c mouse as the animal model to simulate clinical
situations. To this end we have also used radiotherapy for complete suppression
of the immune system and elimination of Hematopoietic cells in our animal
model. In previous studies to evaluate the engraftment of CD34+
cells, a of radiation dose of 3 to 3.5 Gy has been used for engraftment
of human cells in NOD/SCID mouse (Van Hennik et al., 1999; Kerre
et al., 2001; Kollet et al., 2001). In order to completely
eradicate hematopoietic stem cells, we used a dose of radiation of 7.5
Gy that is lethal for mice and while completely eliminating hematopoietic
stem cells it also suppresses the mouse immune system.
We have adjusted the amount of injected cells based on body weight and
therefore we have overlooked the dosage of contrast agents and have based
our injection upon the number of cells. In clinical situations 2x108
to 2x109 mononuclear hematopoietic cells per kilogram body
weight are injected (Daldrup-Link et al., 2005), in this context
we have used a dose of 0.5-1.5x107 cells per 20 g mouse. We
purified CD34+ cells from UCB and expanded them in specific
culture medium with growth factors like SCF and TPO; these purified cells
were then injected. In former studies mononuclear cells have been totally
injected without purification (Daldrup-Link et al., 2005). We purified
CD34+ cells to eliminate the effect of other mononuclear and
stromal cells in the homing of CD34+ cells.
The results of MR imaging showed that injecting 1.5x107 cells
per mouse causes significant decrease in the intensity of T2 signal
within 48 h (Table 2 and Fig. 7).
In the only study performed in this manner the maximum signal reduction
was achieved using 3x107 mononuclear cells within 24 h (Daldrup-Link
et al., 2005). We can attribute the time difference between these
studies to two factors:
First, we have injected purified CD34+ cells to the animal.
This causes the cells effective in homing of CD34+ cells to
be eliminated from the sample. Second, we have used Balb/c mice having
experienced radiotherapy before engraftment. As some findings emphasize,
this can destroy the bone marrow microenvironment ultimately disturbing
the homing process of hematopoietic stem cells and causing delay in the
reduction of BM signal.
In this study we managed to evaluate the homing of CD34+ cells
in vivo using the MR imaging method. In this research we showed
that using proper contrast and transfection agents the hematopoietic stem
cells can be more effectively labeled for MR imaging to monitor homing
of CD34+ cells in a noninvasive way. Use of the above agents
that have also clinical applications opens a new perspective in the biology
of cell therapy to better assess the homing of stem cells after transplantation.
Other potential clinical applications of this technique could be as follows:
tracing cells in allogenic and autologous bone marrow transplantation,
assessment of specific homing of diverse varieties of stem cell subtypes
manipulated using genetic engineering methods and also evaluation of the
effect of cell therapy on differentiation of stem cells and knowing the
in vivo causes of graft rejection in certain diseases.
Nowadays, cells are used in therapy in various medical fields and MR
imaging can be used for follow-up of therapy in some disease states. Monitoring
of mesenchymal stem cells in injured myocardium (Kraitchman et al.,
2003) and homing of neurological stem cells in impaired brain tissue (Bulte
et al., 2001a) are examples of such therapies.
The authors wish to express their sincere thanks to Dr. S. Kaviani for
his helps and advice, Mrs. P. Lotfinezhad, Mr. B. Delalat, Mr. M. Shojaei
Moghadam, Miss H. Vafaeian and Ms. A. Omidkhoda for their technical assistance
and Mrs. Torabi at the Taleghani Hospital for Providing CB samples.