A controlled cortical impact injury device with an impactor tip (5-mm diameter), which is controlled by gas pressure (Amscience, USA), was used to generate an animal model of brain trauma, as previously described (Chen et al., 2003, Kim et al., 2007). Following the induction of anesthesia through intraperitoneal injection of 10% chloral hydrate (Fluka, USA), the left scalp was incised, and a small hole (6-mm diameter) was made between the bregma and lambda. The exposed dura was stuck by the pneumatic impactor at approximately 2 mm lateral to the central suture at a depth of 8 mm, a speed of 7 m/s and a contact time of 100 ms. The scalp was sutured, and the rats were allowed to rest for 24 h.
Meanwhile, the sham-operated rats underwent craniostomy and suture.
C. Synthesis and radiolabeling of mesenchymal stem cells with
111In-tropolone
One to two milligrams of tropolone (Sigma, St. Louis, MO, USA) was dissolved in 1 mL of normal saline, and then 80 μL of tropolone solution was mixed with 37 to 111 MBq (1-3 mCi) of 111InCl3 (physical half-life=2.83 days, γ-energy=245 and 173 keV; PerkinElmer, Waltham, MA, USA) in 0.05 N HCl. The reaction mixture was incubated for 15 min at room temperature (pH 7.2) (Gunter et al., 1983). Before labeling, the BMSCs were washed with PBS, centrifuged at 1000 rpm for 3 min and resuspended in 1 mL PBS. 111In-tropolone was then added to the BMSC suspension and incubated at room temperature for 20 min. After incubation, BMSCs were centrifuged at 1000 rpm for 3 min, and supernatant and cell pellets were collected separately to calculate the labeling efficiency. For in vivo monitoring in acutebrain trauma model and controls, 111In-BMSCswere resuspended in 1.0 mL of normal saline and injected via the lateral tail vein using 25-gauge needles.
D. In vitro stability and cell viability of mesenchymal stem cells with
111In-tropolone
To examine the retention rates, 111In-BMSCs were divided into three 60-mm culture dishes, which included 4 mL of culture medium, and incubated at 37°C in the presence of 5% CO2 for 1, 3, 6, 24, and 48 h. The supernatant was collected and the culture medium was replaced at each time point. After 48 h of incubation, cells were detached and centrifuged at 1000 rpm for 3 min. The radioactivity of the cell pellets and supernatant at each time point was counted with a dose calibrator. The retention percentage of 111In-BMSCs was calculated by dividing the activity in BMSCs by the total activity. To investigate the viability of 111 In-BMSCs, the numbers of residual viable cells were measured by 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) assay (Roche Applied Science, Mannheim, Germany). Approximately 1.0 × 104 rat BMSCs were plated in a 96-well plate and incubated at 37°C in 5% CO2 for 48 h. XTT and phenazine methosulfate were then added at a 50:1 ratio. The plates were incubated at 37°C for 4 h, and the absorbance was measured at 450 nm, subtracted from 590 nm, using a microplate reader (Bio-Rad Lab, Hercules, CA, USA).
E. Dose-dependent effect of
111In on the growth of BMSCs
To evaluate dose-dependent influence of 111In-tropolone labeling on the growth of BMSCs, cells labeled with 4 different doses of 111In-tropolone (0.4, 1.1, 4.4 and 11.1 Bq/cell) were divided into 5 dishes (3.0 × 104 cells/60 mm culture dish) respectively, and their growth was monitored for 14 days. As a control, unlabled BMSCs (3.0 × 104 cells/60-mm culture dish) were used. We counted the numbers of both 111In-BMSCs and unlabled BMSCs on the day of labeling and on the 3th, 6th, 10th, and 14th days. This experiment was performed in triplicate for accuracy.
F. In vivo tracking of
111In-BMSCs in by gamma camera animal models
Static planar images were acquired after intravenous injection of 111In-BMSCs (1.0 × 106 cells for each rat) with a dual head gamma camera (MultiSPECT2; Siemens, Erlangen, Germany) equipped with medium energy collimators. Rats were placed in a prone position and scanned for 10 min from the anterior and posterior projection. The matrix size was 256×256. A 7.4-MBq 111In-BMSCs were intravenously injected in normal rats (n=3), sham-operated rats (n=2) and brain trauma models (n=3), and then the brain uptake was compared on the 2nd day images. For the comparison of biodistribution, on both anterior and posterior images, polygonal and circular region of interests (ROI) were drawn for each organ and right thigh (background), respectively, and then geometric means were used for calculation of percentage uptake/whole body.
G. PKH 26 labeling of mesenchymal stem cells
For detection of mesenchymal stem cells in brain, the cells were labeled using a PKH 26 red fluorescence cell linker kit (Sigma, St. Louis, MO, USA) according to the manufacture's instruction (Lee-MacAry et al., 2001). Briefly, cells were detached and centrifuged at 1300 rpm for 3 min in a 15 mL conical tube, and then supernatant was discarded. One milliliter of diluent C and 4.0 × 106 M of PKH 26 dye were added to the cell pellet and incubated at room temperature for 5 min with gentle inversion. Subsequently, the mixture was incubated with 2 mL of serum for 1 min to stop the staining reaction. Stained cells were centrifuged at 1300 rpm for 10 min and washed with 10 mL of complete medium for injection or further labeling of 111In. Radiolabeling of BMSCs and intravenous injection in rat models were done at 30 min after PKH 26 dye labeling.
H. Tissue preparation with DAPI staining for confocal microscopy
Animals were sacrificed on the second day of injection, and the brain tissues were fixed overnight with 4% paraformaldehyde dissolved in 0.1 M PBS. They were embedded in paraffin block and then sectioned on a sliding microtome to obtain a 5-μm-thick coronal section. For deparaffinization, brain tissue slides were incubated for 20 min at 55°C and rinsed thrice with 1 × PBS (Berger et al., 1997). Deparaffinized brain tissue slides were incubated with 4, 6-diamidino-2-phenylindole (DAPI, Fluka, Switzerland) for 15 min at room temperature to counterstain nuclei. Brain tissue slides were then washed twice with 1 × PBS and mounted using Prolong Antifade Kit (Molecular Probes, Eugene, OR, USA).
Fluorescent signals were captured from the sham-operated control brains and traumatic
brains injected with PKH 26-labeled BMSCs or PKH 26-111In colabeled BMSCs, and analyzed using an LSM 510 confocal laser scanning microscope (Carl Zeiss, Germany).
I. In vitro BrdU labeling for
111In-BMSCs
To detect synthetic proportions, BrdU (Sigma, St. Louis, MO, USA) was labeled with
111In-BMSCs in vitro. The halogenated pyrimidine analogue BrdU can be incorporated into newly synthesized DNA in place of thymindine and can be quickly detected using a monoclonal antibody against BrdU (Philip et al., 1997). BMSCs labeled with each dose of
111In-tropolone (0.4, 1.1, 4.4 and 11.1 Bq/cell) were divided into 5 dishes (3.0 × 104 cells/60-mm dish) respectively, and grown until the 14th day. 111In-lableled BMSCs were incubated with 10 µM BrdU at 37°C in 5% CO2 for 1 h, washed with PBS, and harvested. Thereafter, the cells were resuspended ice cold PBS, 10% fetal bovine serum, and 1% sodium azide and incubated for overnight with anti-BrdU antibody (Abcam, USA). And then, cells were washed with cold PBS and incubated for 30 min at 4oC in the dark with AlexaFluor488-conjugated anti–mouse IgG antibody (Invitrogen, Carlsbad, CA, USA) in 3% bovine serum albumin (BSA)/PBS. The stained cells were analyzed by FACS with CellQuest software (Beckton Dickinson). In addition, to evaluate the cell density and morphological changes of
111In-labeled BMSCs, cells were observed using a light microscope (Olympus Optical Co, Ltd, Tokyo, Japan).
J. Annexin V-FITC/PI double staining for
111In-BMSCs
To investigate the apoptosis/necrosis of 111In-BMSCswhich tested for higher doses of In-111 (11.1 and 33.3 Bq/cell) at early (3 and 12 h) and late (7 days) stages, double staining with Annexin V-FITC and PI for flow cytometric analysis was performed using commercial kit (BD Pharmingen™, USA) according to the manufacture's instruction (Vermes et al., 1995). In brief, 111In-BMSCsand untreated BMSCs were detached with 0.25% trypsin/0.1%
EDTA, and then the cells were washed twice with cold PBS, and resuspended in binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2). Annexin V-FITC was added resulting in a final concentration of 1 mg/ml Annexin V. Then 10 mg/ml PI was added. The mixture was incubated for 15 min at room temperature in the dark and then analyzed by flow cytometry within 1h.
K. Cytochemical staining with SA-β-galactosidase for
111In-BMSCs
To investigate the premature senescence of BSMCs, we stained cells with X-gal (InnoprotTM, Spain) (Kazuaki et al., 1999). 111In-BMSCs(1.1, 4.4 and 11.1 Bq/cell) were seeded on 12 well plates at the density of 5,000 cells/cm2 and incubated under 37°C in 5%
CO2 until 14th day. For comparison, nonlabeled BMSCs were also grown. After removing culture medium and rinsing cells, cells were fixed by incubation with 1 mL of working fixing solution for 5 min at room temperature. And then, BMSCs were stained with X-gal solution (pH 6) at 37oC in 5% CO2 for 24 h, protected from light, and examined using a light microscope. The development of premature senescence was determined by the presence of
blue-stained cells. For positive staining, we prepared the cells which starved for 1 week without media change at 37oC in 5% CO2, and stained with the same protocol.
L. Statistical analysis
All data are presented as mean ± standard error. A paired t-test and Student-Newman-Keuls post hoc test was used to compare cell numbers between the control and 111In-labeled BMSCs. A p value of less than .05 was considered to be significant. Error bar means standard error.
II. RESULTS
A. Radiolabeling efficiency and viability of
111In-BMSCs
The labeling efficiency of 111In-BMSCs was 65.6 ± 5.3% (n=9), containing approximately 38 Bq/cell. The in vitro retention rates of 111In-BMSCs at 1, 3, 6, 24 and 48 h were 85.3%, 75.7%, 67.1%, 48.2% and 45.1%, respectively (Fig. 1). The XTT assay revealed that there was no significant difference in the number of viable cells between the
111In-BMSCs and control cells (98% of control, p=.22) at 48 h after labeling.
Fig. 1. Retention rate of 111In-BMSCs. Retention rate of 111In-BMSCs slowly decreased over time. Error bar means standard error.
B. Dose-dependent growth of
111In-BMSCs
A growth curve of 111In-BMSCs showed the dose-dependent effect of 111In-tropolone.
For cells labeled with the lowest and second lowest doses (0.4 and 1.1 Bq/cell, Fig. 2), their growth was not significantly different from that of controls until the 14th day (p <.001 on from the 3rd to 10th day, p=.004 on the 14th day, ANOVA). However, higher doses (4.4 and 11.1 Bq/cell) significantly inhibited all proliferation and they could not catch up with controls until the 14th day of labeling (p <.005 on from the 3rd to the 14th day, Student-Newman-Keuls post hoc test).
Fig. 2. Growth curve of 111In-BMSCs. A growth curve of 111In-BMSCs shows the dose-dependent effect of 111In-labeling. This experiment was performed in triplicate with error bars showing standard error. Concentrations of 111In were presented as Bq/cell. Bq =
becquerel, *p < 0.05, †p < 0.001, ‡p = 0.004.
C. In vivo tracking of
111In-BMSCs by gamma camera in trauma models and controls
Planar gamma camera images revealed that most of 111In-BMSCs migrated to the liver at 24 h after injection in all three groups (Fig. 3), whereas the brain uptake was minimal. In the sham-operated rats and normal rats, brain uptake was not discernable visually. Unlike normal (0.3%) and sham-operated rats (0.5%), mild brain uptake (1.4%) was visible in trauma models injected with 111In-BMSCs. Other than brain uptake, the radio-uptake values of each organ among three groups were similar on gamma camera images.
Fig. 3. In vivo distribution of 111In-BMSCs in acute brain trauma rats, sham-operated control rats and normal rats at 24 h after intravenous transplantation. Most of the injected radioactivity was taken up by the liver (36%, 37%, and 35.1%, respectively normal, sham-operated, brain trauma), spleen (3%, 3.4%, and 4.2%) and lungs (2.1%, 2.4%, and 1.5%) in all three groups. The uptake of 111In-BMSCs in brain trauma rats was relatively prominent than those in sham-operated rats or normal rats.
D. Histological analysis of transplanted BMSCs in animal model of trauma
Migration of BMSCs was analyzed by fluorescent detection of cell on the margin of traumatic brain regions (Fig. 4A). In sham-operated controls, there were no PKH 26-positive (red) cells (Fig. 4B), whereas BMSCs labeled with PKH 26 only were found in the traumatic brains (Fig. 4C). As shown in Fig. 4D, PKH 26 and 111In colabeled BMSCs were alsodetected in the traumatic brains. The ratio of BMSCs stained with both PKH 26 and DAPI to total cells in the traumatic brain was 0.38 for PKH 26 labeling and 0.40 for PKH 26 and 111In colabeling. These confocal microscopy images revealed that BMSCs labeled with 111In migrated to the traumatic brains at 24 h after injection and their migratory ability seemed not to be affected by 111In-labeling. In trauma models, contralateral brain tissues did not contain BMSCs (data are not shown).
Fig. 4. Confocal laser scanning microscope image of transplanted BMSCs in the margin of traumatic regions. Rats were sacrificed at 24 h after injection and the existence of BMSCs in the margin of traumatic brain regions was identified by PKH 26-positive cells.
Diagram of brain section (A). Sham-operated control shows no BMSCs in their brain (B), whereas BMSCs labeled with PKH 26 only (C) or colabeled with 111In (D) are double-stained with PKH 26 and DAPI. These images were taken at ×100 (big box) and ×400 (small box) magnification (PKH 26, red; DAPI, blue; scale bar, 50 μm).
E. Cell cycle analysis by flow cytometry
Light microscopic images for the changes in cell density and morphology after 111 In-labeling were presented in the lowest and highest doses (Fig. 5A). BMSCs labeled with the lower dose (0.4 Bq/cell) of 111In demonstrated no significant change in density or morphology compared with controls. However, in case of those labeled with the higher dose (11.1 Bq/cell) of 111In, there was a remarkable decrease in the number of cells with changes in morphology.
After labeling BMSCs with 111In, FACS analysis with BrdU and anti-BrdU antibody was performed to discriminate cells in synthetic cycles (S-G2-M) from those in non-synthetic cycles (G0-G1) (Fig. 5B). BMSCs labeled with 0.4 Bq/cell of 111In had a similar cell cycle pattern with control from the 3rd to the 14th day. Meanwhile, on the 3rd day, BMSCs labeled with more than 1.1 Bq/cell of 111In showed less BrdU positive cells than control by FACS (36.1%, 27.4%, and 28.3% for 1.1, 4.4 and 11.1 Bq/cell, respectively). After that, BMSCs labeled with 111In of 0.4 and 1.1 Bq/cell were recovered and had similar cell cycle
patterns to controls. On the contrary, BMSCs labeled with 111In of 4.4 and 11.1 Bq/cell could not recover and showed single peak (G0-G1) on the 14th day.
Fig. 5. Light microscopic images and cell cycle graphs of 111In-BMSCs. Light microscopic images show the density and morphological changes of BMSCs labeled with the lowest and the highest doses of 111In (A, original magnification- X200). Cell cycle graphs of
111In-BMSCsare presented according to the dose and day of labeling (B). Proportions of BrdU positive cells are presented as percentage of total cell count. Concentrations of 111In were presented as Bq/cell. Bq = becquerel.
F.
Annexin V-FITC/PI double staining flow cytometryThe Annexin V-FITC and PI staining of 111In-BMSCswere performed at early (3 and 12 h) and late (7 days) stages for higher doses of 111In (11.1 and 33.3 Bq/cell, Fig.6).
Apototic (Annexin V+/PI-)/necrotic (Annexin V-/PI+) fractions were 0/0.9, 0.2/2.3 and 0.2/3.0% at 3 h, 0.1/1.2, 0.1/0.4 and 0/0.9% at 12 h and 0.4/2.8, 0.8/3.3 and 0.7/3.3% on the 7th day for 0 (control), 11.1 and 33.3 Bq/cell of 111In, respectively. FACS analysis with Annexin V-FITC and PI revealed no significant difference in apoptosis or necrosis from controls at all time points (3, 12 h and 7 days) and at all doses (11.1 and 33.3 Bq/cell). On day 7, double positive (late apoptotic) cells were observed in both 111In-BMSCs and control.
However, the proportions of double positive cells of 111In-BMSCs were 20.5 and 20.3% for 11.1 and 33.3 Bq/cell of 111In, which were similar to that of control (23.9%)
Fig. 6. Apoptotic and necrotic change of 111In-BMSCs. Apoptosis and necrosis of 111 In-BMSCswas evaluated by staining with Annexin V-FITC and propidium iodide. Horizontal
and vertical axes show the number of Annexin V-FITC (+) cells and propidium iodide (+) cells, respectively. Concentrations of 111In were presented as Bq/cell. Bq = becquerel.
G. Senescence associated β-galactosidase histochemistry
SA-β-galactosidase was used as a marker for assessing senescence in cells. After X-gal staining with various doses of 111In from 1.1 to 11.1 Bq/cell, the cytochemical assay for 111 In-BMSCswas performed until the 14th day. Senescent cells were stained blue. As shown in Fig.
7A, most of the 111In-labeled BMSCs were not stained for SA-β-galactosidase at all concentrations of 111In from the 3rd day to the 14th day. There was no difference in the number of stained cells between 111In-BMSCs and controls, which indicates that 111 In-labeling does not induce a cellular senescence. For reference, BMSCs incubated in a starved condition were stained blue in senescent cells (Fig. 7B).
Fig. 7. Cellular senescence of 111In-BMSCs. Cellular senescence of 111In-BMSCs was evaluated by staining with X-gal (A). Positive staining cells which incubated in senescent environment were stained with X-gal (B). Senescent cells are stained blue. Concentrations of
111In were presented as Bq/cell. Bq = becquerel.
IV. DISCUSSION
This study was performed to evaluate the migration of intravenously injected therapeutic mesenchymal stem cells in a brain disease model by a direct cell-labeling method with 111In-tropolone and to elucidate the effects of 111In-labeling on the growth, cell cycle and cellular death of labeled BMSCs. As a result, intravenously injected BMSCs migrated to the injured brain area, which were imaged by gamma camera with a direct cell labeling with
111In, and 111In-labeling did not induce a cellular death within the experimental dose range despite it led to cell cycle arrest transiently at lower doses and permanently at higher doses.
These results indicate that 111In-labeling could be used for monitoring of intravenously transplanted BMSCs in brain disease models without deteriorating viability significantly.
In this study, the viability of 111In-BMSCs measured by XTT assay at 48 h was not different from that of unlabeled cells. However, the proliferation of labeled BMSCs began to decrease on the 3rd day after radiolabeling and the difference in the number of viable cells between 111In-labeled and unlabeled BMSCs reached a statistical significance on the 6th, 10th and 14th day. This proliferative inhibition of 111In-BMSCs also showed a dose-dependency.
For cells labeled with lower doses (0.4 and 1.1 Bq/cell), their growth had no significant difference from controls during the measurement period and, on the 14th day, they caught up with controls. Meanwhile, BMSCs labeled with higher doses (4.4 and 11.1 Bq/cell) had significantly lower cell numbers that controls until the 10th day (4.4 Bq/cell) and 14th day (11.1 Bq/cell). This inhibition of proliferative capacity was not due to tropolone or other chemicals that were added during the labeling process, because cold compound-treated
BMSCs proliferated similar to controls (data are not shown).
There have been conflicting reports concerning the cytotoxic effect of 111In on BMSCs.
The Trypan blue excursion test revealed that the viability of 111In-labeled swine BMSCs (40 Bq/cell) was more than 95% at 48 h after radiolabeling (Chin et al., 2003). In another study with canine BMSCs, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay revealed a slight difference in metabolic activity/proliferation between labeled cells and unlabeled cells at 48 h and after the sixth day of labeling; however, there was no dose dependency at concentrations ranging from 5 to 30 mCi (185–1110 MBq) of 111In per million cells. Moreover, 111In-labeled canine BMSCs retained the ability to differentiate into adipose cells as efficiently as unlabeled BMSCs (Kraitchman et al., 2005). On the other hand, in another recent study, while all canine BMSCs exposed to 111In-tropolone of more than 8 Bq/cell did not survive at 14 days after labeling, all other cells incubated with 111In-tropolone of less than 0.9 Bq/cell were viable (Jin et al., 2005). As with the human BMSCs, at concentrations from 30 to 260 Bq of
111In/cell, the doubling time of 111In-labeled human BMSCs (labeling efficiency=26 ± 5%) was not significantly different from that of the controls, but their proliferation was significantly inhibited at a higher radioactivity concentration (800 Bq 111In/cell) (Bindslev et al., 2006). Similarly, in this study, 4.4 and 11.1 Bq/cell had an inhibitory effect on the growth of BMSCs (p <.005). Although the threshold was different among those studies, possibly because of the different cell types and cell-handling methods, it seems obvious that, at high concentrations, 111In has an inhibitory effect on BMSCs. For comparison, the growth of 111 In-labeled H9c2 cell line derived from embryonic rat ventricle was evaluated. Just like the 111
In-BMSCs, H9c2 cells labeled with lower doses (0.4 and 1.1 Bq/cell) showed no significant difference from controls, whereas those labeled with higher doses (4.4 and 11.1 Bq/cell) were significantly inhibited until the 14th day of labeling (data are not shown). Another study with hematopoietic progenitor cells also supported this result (Brenner et al., 2004). 111 In-labeled hematopoietic progenitor cells showed impaired viability at 48 and 96 h after labeling, and their migration was decreased to 74% of the controls as early as 24 h after labeling. Moreover, they lost their colony-producing capacity (Brenner et al., 2004).
Although the data were not shown in the results of this study, a similar pattern of proliferative inhibition was observed with 111In-labeled human BMSCs. Therefore, this radiation effect of 111In-labeling should be overcome by careful dosimetry prior to clinical application, and it should be investigated in other radioisotopes, such as 18F-FDG or 99mTc, which are used to monitor therapeutic cells.
For further evaluation of the effect of 111In-labeling on BMSCs, cell cycle analysis and
For further evaluation of the effect of 111In-labeling on BMSCs, cell cycle analysis and