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Blood First Edition Paper, prepublished online October 5, 2006; DOI 10.1182/blood-2006-04-018564.
STEM CELLS IN HEMATOLOGY VCAM-1 expression on dystrophic muscle vessels has a critical role in the recruitment of human blood-derived CD133+ stem cells after intra-arterial transplantationFrom the Stem Cell Laboratory, Department of Neurological Sciences, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Ospedale Maggiore Policlinico, Centro Dino Ferrari, University of Milan, Italy; the Department of Pathology, Division of General Pathology, University of Verona, Italy; the Fondazione IRCCS Ospedale Maggiore Policlinico, Institute of Nuclear Medicine, Milan, Italy; the Radiology Unit, Fondazione IRCCS Ospedale Maggiore Policlinico of Milan, Italy; the Department of Pediatrics, Pathology, and Microbiology-Immunology, Dalhousie University, Halifax, NS, Canada; and the Department of Sciences Applied to Complex Systems, Polytechnic University of Marche, Ancona, Italy.
Recently our group demonstrated the myogenic capacity of human CD133+ cells isolated from peripheral blood when delivered in vivo through the arterial circulation into the muscle of dystrophic scid/mdx mice. CD133+ stem cells express the adhesion molecules CD44, LFA-1, PSGL-1,  4-integrins, L-selectin, and chemokine receptor CCR7. Moreover these cells adhere in vitro to VCAM-1 spontaneously and after stimulation with CCL19. Importantly, after muscle exercise, we found that the expression of VCAM-1 is strongly up-regulated in dystrophic muscle vessels, whereas the number of rolling and firmly adhered CD133+ stem cells significantly increased. Moreover, human dystrophin expression was significantly increased when muscle exercise was performed 24 hours before the intra-arterial injection of human CD133+ cells. Finally, treatment of exercised dystrophic mice with anti-VCAM-1 antibodies led to a dramatic blockade of CD133+ stem cell migration into the dystrophic muscle. Our results show for the first time that the expression of VCAM-1 on dystrophic muscle vessels induced by exercise controls muscle homing of human CD133+ stem cells, opening new perspectives for a potential therapy of muscular dystrophy based on the intra-arterial delivery of CD133+ stem cells.
Attempts to repair muscle damage in patients with Duchenne muscular dystrophy (DMD) by transplanting myogenic progenitors directly into muscles are hampered by the problem of cell survival and the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions through the systemic circulation is a potential alternative approach to treat this disease, but injected cells may become trapped intravenously in other organs (eg, liver, spleen, lung) so that only a small portion enters the muscle microvasculature and migrates to dystrophic muscle. Our recent work supports the idea that stem cells can reach the site of muscle regeneration and can contribute to muscle repair and to replenishment of the satellite cell pool after intra-arterial injection, suggesting that this technique might be particularly suited for treating muscle dystrophy.1,2 This protocol succeeded primarily because of the widespread distribution of donor stem cells through the muscle capillary network, a distinct advantage of this strategy over previous approaches. Elucidation of the mechanisms involved in the muscle homing of stem cells will aid the development of a potential therapy for muscular dystrophy based on the systemic delivery of stem cells. It has been shown recently that VLA-4-VCAM-1 interactions efficiently mediate rolling and arrest in vivo in blood vessels from bone marrow or under pathologic conditions characterized by an activated endothelium.3-5 In the present study, we focused our attention on the molecular mechanisms involved in human CD133+ cells homing to dystrophic muscle and efficiently mediating their transplantation.
Isolation and characterization of human blood-derived CD133+ cell fraction for FACS analysis
Human circulating CD133+ cells were isolated from blood mononucleated cells, as previously described.1 The enrichment of CD133+ cells was followed by a second round of cell sorting for the CD133 antigen with a dual-laser FACSVantage SE (Becton Dickinson, Franklin Lakes, NJ). Sorted CD133+ cells were double labeled with anti-CD133-phycoerythrin (anti-CD133-PE) (Miltenyi Biotech, Bergisch Gladbach, Germany), anti-VLA4-FITC, anti-CD44-FITC, anti-LFA1-FITC, anti-L-selectin-FITC (BD Pharmingen, San Diego, CA), anti-PSGL1-FITC (MBL International, Woburn, MA), anti-CCR3-FITC, anti-CCR5-FITC, anti-CCR7-FITC, anti-CXCR3-FITC, and anti-CXCR4-FITC (R&D Systems, Minneapolis, MN). Isotype control was performed using fluorescein isothiocyanate (FITC)-conjugated mouse IgG1, R-phycoerythrin (R-PE)-conjugated rat IgG2a. To ensure the quality of our analysis, we also investigated sorted CD133- cells for the expression of molecules, as previously indicated. We also characterized the adhesion molecules and chemokines receptor expression of sorted CD133+ cells after stimulation with 5 ng TNF- In vitro adhesion assay on ICAM-1 and VCAM-1
We evaluated the functionality of LFA-1 and VLA-4 expressed by CD133+ cells using an in vitro spontaneous adhesion assay for human ICAM-1 and VCAM-1 (R&D Systems).6 After isolation, sorted CD133+ cells were resuspended in PBS, CaCl2, MgCl2 1 mM, and 10% FCS (pH 7.2) at 2.5 x 106/mL, and 25 µL of this suspension was plated on 18-well glass slides for 30 minutes. The slides were coated with recombinant 1 µg/mL ICAM-1 and 1 µg/mL VCAM-1 (R&D Systems), as previously described.6 Five microliters of CCL19/MIP3 Selectin chimera binding assays The functionality of PSGL-1 expressed on sorted blood-derived CD133+ and CD133- cells was demonstrated with a cytofluorimetric assay. Approximately 5 x 105 cells were resuspended in 100 µL standard medium (SM [DMEM, 0.1% BSA, 0.1% NaN3]) and were incubated for 30 minutes at 4°C with E- and P-selectin chimeras (R&D Systems). Cells were washed twice, resuspended in 100 µL SM, and incubated with anti-human IgG FITC (diluted 1:32; Sigma, St Louis, MO) for 20 minutes at 4°C. After 2 washes in SM, cells were pelleted and resuspended in fresh DMEM immediately before FACS analysis. Nonspecific staining was established by the addition of EDTA (10 mM) to staining reactions conduced with the selectin chimeras. In vivo staining of endothelial adhesion molecules
To characterize the adhesion molecules expressed by murine muscle vessels, we analyzed 3 groups (n = 5 each) of 3-month-old scid/mdx mice: group 1, mice under basal conditions; group 2, mice 4 to 5 hours after stimulation with 1 µg TNF- Intravital microscopy With the intravital microscopy model,7 we verified the muscle homing and vascular adhesion properties of CD133+ stem cells into the pectoral and iliopsoas muscles of scid/mdx mice after muscle exercise (n = 6) and after administration of anti-VCAM-1 mAb (n = 6). Animals were anesthetized, and the pectoral muscle was analyzed with an intravital microscope (BX50WI; Olympus, Tokyo, Japan). A dextran dye (155 kDa; Sigma) was injected to visualize the blood vessels. CD133+ stem cells (106) were fluorescently labeled with green CMFDA (5-chloromethylfluorescein diacetate; Molecular Probes) and injected by a digital pump at flow rate of 0.13 to 1 µL/s. Characterization of muscle homing of CD133+ stem cells by HMPAO labeling
We tested the muscle selectivity of human circulating CD133+ cells by labeling them with technetium Tc 99m-labeled hexamethylpropylene amine oxime (HMPAO), a radioactive marker previously described.8 Cells were incubated for 2 hours at 37°C in RPMI medium supplemented with 1 µCi/mL (0.037 MBq) HMPAO. Labeling efficiency was measured with a dose calibrator (Atomlab 100; Biodex Medical, Shirley, NY). Cell vitality was assessed by trypan blue and by double staining with propidium iodide (PI) and annexin V. HMPAO-labeled CD133+ (10 x 105) or CD133- cells were injected into the right femoral artery of 2-month-old scid/mdx mice in basal condition (n = 5 each) and after swimming (n = 5 each). We compared the distribution of radioactivity of the first series of experiments with animals pretreated intraperitoneally with 250 µg selective blocking antibodies against VCAM-1 (n = 4) and ICAM-1 (n = 4) (generous gifts from Eugene C. Butcher, Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA) 1 hour before stem cell injection. Animals were killed 24 hours later, tissue from muscle, lung, kidney, liver, and brain was collected, and radioactive content was measured in a Microfil perfusion PkH2-labeled CD133+ cells (5 x 105) were injected into the right femoral artery of scid/mdx mice in basal conditions (n = 3) and 24 hours after exercise (n = 3). After cell injection, the mice were perfused with 10 mL PBS and then 10 mL liquid silicon rubber (Microfil; Flow Tech, Carver, MA). Hind limbs of treated mice were isolated in toto and placed under refrigeration at 4°C overnight to allow polymerization. On the following day, specimens were carefully dissociated and placed in a 50% mixture of water and glycerin. At successive 24-hour intervals, the glycerin concentration was raised to 75%, then to 85%, and finally to pure glycerin. Statistical analysis using the Student t test was also performed. Immunofluorescence images were captured with a DMIRE2 microscope (Leica Microsystems, Cambridge, United Kingdom) and a Leica DC350 camera. The microscope was equipped with a 40x/0.75 NA dry objective. Images were acquired and processed with Leica Qfluoro software. Labeling of CD133+ cells with iron oxide To better characterize the migratory capacity of CD133+ cells in mouse dystrophic animal models, we used live cell tracking methods such as magnetic resonance imaging (MRI) after labeling with nanoparticles of iron oxide (Endorem; Guerbet, Sulzbach, Germany).9,10 Human CD133+ stem cells were labeled with 250 µg/mL Endorem (Guerbet) in RPMI 1640 medium enriched with 20 ng/mL EGF and 10 ng/mL bFGF for 12 or 24 hours. We tested different cell suspensions (100 000, 50 000, 20 000, 5000, 500 cells) in 1.7% gelatin. For in vivo experiments, CD133+ cells were injected intra-arterially in the scid/mdx dystrophic animal model and were visualized with MRI. Images were obtained using a 4.7-T spectrometer (Burker, KooWeeRup, VIC, Australia) equipped with a surface coil made in house. Single sagittal, coronal, and transversal images were obtained by a fast gradient-echo sequence to localize the subsequent T2-weighted transverse images, as measured by a standard turbo spin-echo sequence. The same muscles monitored by MRI were isolated and characterized by immunohistochemistry for the expression of CD133 antigen and Prussian blue staining.9 Histochemistry and immunocytochemistry of injected dystrophic muscles
For human dystrophin detection, unexercised (n = 5) and exercised (n = 6) scid/mdx mice were injected intra-arterially with 500 000 human CD133+ stem cells, as previously described.1 Sixty days later, muscle sections were characterized using the anti-human nuclear lamin A/C (Novocastra, 1:200), anti-human dystrophin (1:50; Chemicon, Temecula, CA), anti-
Characterization of adhesion molecules and chemokine receptors
To evaluate the migratory capacity of human circulating CD133+ cells, we first studied by flow cytometry the adhesion molecules and chemokine receptors expressed on their surfaces. In these experiments, we sorted CD133+ and CD133- cells, and we characterized both populations by flow cytometry analysis. Sorted CD133+ cells (99% ± 1% [mean ± SD]) coexpressed CD44 and LFA-1. In particular, we observed 2 subpopulations of CD133 on the basis of LFA-1dim and LFA-1bright expression. In addition, 83% ± 2% of sorted CD133+ cells expressed PSGL-1, 44% ± 3% expressed VLA-4, and 60% ± 5% expressed L-selectin. Chemokine receptor analysis showed very low expression of CXCR3 and CXCR4 (4% ± 3%), whereas 44% ± 5.4% of CD133+ cells were positive for CCR7. Less than 2% of sorted CD133+ cells expressed CCR3 (1.6% ± 1.3%) or CCR5 (1.7% ± 1.5%). We observed that sorted CD133- cells expressed 98% ± 1% CD44, 87% ± 1% LFA-1, 63.5% ± 1% L-selectin, 53.5% ± 1% CCR7, 19% ± 1% CXCR3, 3.5% ± 1% CXCR4, 1.2% ± 1% CCR3, and 0.09% ± 1% CCR5. In contrast to CD133+ cells, very low expression of VLA-4 (0.6% ± 0.5%) and reduced expression of PSGL-1 (7.5% ± 1%) were observed on sorted CD133- cells. Although the expression of chemokine receptors and adhesion molecules was regulated in leukocytes by the inflammatory microenvironment, human circulating CD133+ cells did not seem to be particularly influenced by this condition. In fact, after 5 and 20 hours of stimulation with TNF- Evaluation of adhesion capability of CD133+ cells in vitro
The adhesion capacity of CD133+ cells to integrin ligands was evaluated in vitro by adhesion assays to VCAM-1 and ICAM-1 before and after stimulation with MIP3
In vivo staining of adhesion molecules in dystrophic vessels
To support the results obtained with binding assays, we performed an in vivo staining study aimed at identifying the endothelial adhesion molecules potentially involved in dystrophic muscle homing. For this reason, scid/mdx mice were monitored in basal conditions (n = 5), after 1 hour of swimming exercise (n = 5), and after intravenous injection of 1 µg TNF- Intravital microscopy performed in dystrophic muscle vessels Muscle homing and vascular adhesion properties of CD133+ cells were next verified in the pectoral and gastrocnemius muscles of 3-month-old scid/mdx mice under flow conditions. For all in vivo experiments, we used blood-derived CD133+ cells obtained with magnetic column separation. Even if the purity obtained with this method was just greater than 90%, the contaminating fraction of CD133- cells did not affect our results. In fact, the characterization of adhesion molecules and the adhesion assays of sorted CD133- cells showed a low capacity of these cells to interact with the main endothelial adhesion molecules involved in the recruitment from the bloodstream described in our experiments. When injected into the aortic arch (through the right carotid), few human CD133+ cells were able to firmly adhere to the murine muscle vessels, but most interacting cells performed rolling (21 ± 5.4 for rolling interactions and 0.8 ± 0.3 for firm adhesion; mean ± SEM). We observed that some cells were mechanically trapped in blood capillaries and in proximity to narrowing vessels. This low capacity of CD133+ cells to firmly adhere to the dystrophic vessel endothelium probably resulted because the expression of P-selectin, as a rolling receptor, in the muscle vessels of scid/mdx was insufficient to mediate adhesion interactions necessary for efficient firm arrest. We thus extended these experiments injecting the CD133+ cells in scid/mdx mice, which, after 1 hour of swimming exercise, displayed increased expression of VCAM-1. Rolling events were more frequent (increase of 50%; P < .04) in vessels stimulated with exercise than in vessels in basal condition (31.5 ± 3.6) (Figure 4E). We also analyzed the quality and strength of rolling by measuring the rolling velocities (Vrolls) of interacting human CD133+ cells (Figure 4F). Median Vroll in basal conditions and after swimming was 49.9 µm/s and 24.9 µm/s, respectively. In addition, the distribution of Vroll in velocity classes showed a larger number of cells with higher Vroll under basal conditions, suggesting that different molecular mechanisms might mediate rolling interactions after swimming compared with basal conditions. Importantly, we observed a 6-fold increase in the number of CD133+ cells able to firmly adhere in capillaries, small postcapillary venules, and larger venules (5.5 ± 1.9) (Figure 4E). The increased recruitment after swimming could be explained by the natural capacity of CD133+ cells to bind VCAM-1 because of the presence on their membrane of the integrin very late antigen (VLA-4). Taken together, these results show that the activation of dystrophic vessel endothelium by exercise improves the adhesion of human CD133+ cells in vivo.
We next sought to identify the molecular mechanisms of rolling and arrest in dystrophic muscle venules after swimming. Rolling interactions were not inhibited after the administration of anti-VCAM-1 mAb. In contrast, almost 60% of firm adhesion/arrest was blocked by the use of anti-VCAM-1 mAb (Figure 4G). Median Vroll was 23.8 for control cells and 24.5 after anti-VCAM-1 administration. However, though not statistically significant, the mean ± SD value of Vroll was slightly increased after VCAM-1 blockade: 31 ± 21 for control cells compared with 39 ± 42 after mAb blockade. In addition, the distribution of Vroll in velocity classes showed a slightly larger number of cells with higher Vroll after the administration of anti-VCAM-1 mAb (Figure 4H), suggesting that VCAM-1 might contribute to roll strengthening of some CD133+ cells. An isotype-matched control antibody (anti-human Ras) had no effect on rolling or arrest (data not shown). These results clearly show an important role for VCAM-1 in the recruitment of CD133+ cells in dystrophic muscle after muscle exercise. Hemodynamic parameters for intravital microscopy experiments are depicted in Figure 4E,G. Detection of intra-arterially injected CD133+ stem cells by fluorescence and microfil perfusion To confirm the results obtained by intravital microscopy and quantify firmly adhered cells with a different method, animals injected intra-arterially with CD133+ stem cells were perfused with microfil. This technique offers the possibility of eliminating transient adhered cells, observing the 3-dimensional cast of dystrophic muscle vessels, and identifying injected cells distribution in toto. In basal conditions, the transillumination of microfil-perfused vessels of intra-arterially injected scid/mdx mice rarely showed PKH2 CD133+-labeled cells in large and small vessels of dystrophic muscles (2 ± 1.41 cells/field at 20x magnification) (Figure 5A, C). In contrast, after muscle exercise, we observed a 4- to 5-fold increase of the number of adhered cells in large vessels and distributed in the capillary network (10 ± 2.51 cells/field at 20x magnification; P < .01) (Figure 5B, D-E). MRI analysis To test the migration capacity of human circulating CD133+ stem cells and to monitor the fate of implanted cells using a noninvasive method such as MRI, we labeled these cells with a contrast agent based on dextran-coated superparamagnetic iron oxide nanoparticles clinically approved as a blood pool agent (Endorem; Guerbet). In vitro results demonstrated that CD133+ stem cell iron incorporation leads to MRI visualization. The iron oxide nanoparticles in cultured CD133+ stem cells were observed as blue spots after Prussian blue staining (Figure 6F-G). Cell counting of Prussian blue-stained cells revealed that after 12 hours of labeling with Endorem (Guerbet), the percentage of labeled cells was 44.2% ± 7.4% and that after 24 hours of labeling, the percentage was increased to 85.6% ± 8.3%. Longer labeling did not increase either the number of labeled cells or the number of iron particles inside the cells. A colorimetric assay for the quantification of cell proliferation and cell viability based on the cleavage of the tetrazolium salt WST-1 (Roche Molecular Biochemicals, Indianapolis, IN) by mitochondrial dehydrogenases in viable cells did not show any differences between nanoparticle-labeled and nonlabeled cell groups. Moreover, MRI images showed a clear hypointense signal at all concentrations of more than 500 cells (Figure 6A-E). At 24 hours after intra-arterial transplantation, we observed a hypointense signal obtained by MRI evaluation in the quadriceps, tibialis anterior, and soleus muscle tissues. No recognizable hypointense signal in the muscle of uninjected leg was detected. The hypointense signal in muscle remained visible during the first 24 hours after intra-arterial injection with no change in shape. The histology of these hypointense areas showed that a large number of Prussian blue-positive cells had entered into the muscle and that most of them coexpressed the CD133 antigen around vessels, suggesting migration from the arterial circulation (Figure 6I, J).
In vitro labeling of CD133+ stem cells with 99mTc-labeled HMPAO and evaluation of labeled cell accumulation in dystrophic scid/mdx mice To test the efficiency of labeling, CD133+ stem cells were incubated with 15 MBq HMPAO in serum-free RPMI between 30 and 120 minutes. A time-dependent incorporation of HMPAO was observed with maximum efficiency after 60 minutes, resulting in an overall labeling efficiency of 61% ± 11%. Next, we investigated whether the labeling affected cell viability or function. Up to 120 hours after HMPAO labeling, trypan blue uptake in the radiolabeled CD133+ stem cells was not significantly different from that in controls, with 97.1% ± 2.1% of cells trypan blue negative after labeling (n = 3). The viability of labeled CD133+ cells was also confirmed by cytofluorimetric analysis. In fact, annexin V and PI staining showed a vitality of 97% ± 2% (Figure 7A). Similarly, the functional capacity of CD133+ stem cells to migrate in response to vascular endothelial growth factor in a modified Boyden chamber assay was not affected by radiolabeling when radiolabeled CD133+ stem cells were compared with their unlabeled controls (data not shown). To determine the leakage of HMPAO into the supernatant, we checked the activity of HMPAO in the supernatants and in the adherently growing CD133+ stem cells. We found that 32.4% ± 5.2% of the HMPAO incorporated into CD133+ stem cells was retained after 24 hours (data not shown). We thus investigated the distribution of radioactively labeled human CD133+ stem cells after their intra-arterial injection into scid/mdx mice. In these experiments, we compared the distribution of radioactivity obtained after the injection of CD133+ cells in exercised mice pretreated with anti-ICAM-1 (n = 4) or anti-VCAM-1 (n = 4) or without pretreatment (n = 5). As control, we characterized the radioactivity distribution obtained after intra-arterial injection of radiolabeled CD133- cells in basal condition (n = 5) and after exercise (n = 5). Twenty-four hours after the injection of CD133+ cells, a high tracer accumulation was found in muscle tissues of mice without pretreatment (70.1% ± 18.3% of the injected activity). The muscle tracer distribution did not show major changes after anti-ICAM-1 mAb pretreatment (71.2% ± 9.1% of whole body activity). However, we observed reduced accumulation of CD133+ cells in other organs, such as the spleen, in which it is known that the blood vessel endothelium expresses ICAM-1. In contrast to the results obtained with anti-ICAM-1 antibody, we registered a marked reduction (approximately 10-fold) of the radioactivity in the muscle of dystrophic mice pretreated with anti-VCAM-1 mAb (Figure 7B). The specific radioactivity in isolated muscles after swimming exercise and no pretreatment compared with anti-ICAM-1 pretreatment were, respectively, 68 047.9 ± 5048.36 cpm/g tissue and 90 337.1 ± 6874.11 cpm/g tissue. After anti-VCAM-1 pretreatment, specific total muscle activity decreased significantly to 8152.4 ± 1507.36 cpm/g (P < .05). All these data suggest a critical role of VCAM-1 in the recruitment of CD133+ stem cells in exercised dystrophic muscle tissues. However, an important interaction was observed in control mice injected with CD133- cells. In fact, the radioactivity detected after the injection of these labeled cells was 79 721.5 ± 1060.66 cpm/g tissue in basal condition, whereas a decrement of 3-fold (to 31 533 ± 1342.07 cpm/g tissue) was observed after exercise. These data demonstrate that the improvement observed in the recruitment of radiolabeled cells after swimming exercise is related to the CD133+ fraction, and these observations are not affected by the contaminating negative cells. Human dystrophin expression in injected scid/mdx mice
We then investigated the in vivo ability of CD133+ stem cells to differentiate into the myogenic lineage after intra-arterial injection. As shown by radioactivity studies to contain donor cells, tissues were evaluated for the expression of human dystrophin 2 months after CD133+ stem cell engraftment. Human dystrophin-positive myofibers were counted in 5 nonadjacent cross-sections of the intra-arterially injected muscles, and the longitudinal dimension of the positive area was approximately 600 µm. In unexercised animals that received intra-arterial injection, we observed that the highest number of dystrophin-positive fibers per cross-section was detected in the quadriceps muscle of the injected leg (roughly 0.5% to 1.0% of total fibers in a given cross-section) (Figure 7E; Table 1). However, after muscle swimming/exercise, the percentage of human dystrophin increased 4-fold in all observed muscle tissues of the injected leg (Figure 7F; Table 1). In all injected muscles, many of the human dystrophin-positive fibers expressed the anti-human lamin A/C (Figure 7C-D). Moreover, human dystrophin-positive myofibers were clustered primarily near
Recently we demonstrated that human circulating cells expressing the CD133 antigen behave as a stem cell population capable of commitment to hemopoietic, endothelial, and myogenic lineages.1 The discovery of the mechanisms involved in the muscle homing of stem cells will aid in improving a potential therapy for muscular dystrophy based on the systemic delivery of such stem cells.
Almost all CD133+ cells coexpressed CD44 and LFA-1, with a particular distribution of the latter antigen in 2 subpopulations, LFA-1dim and LFA-1bright, suggesting the existence of a less activated and a more activated subpopulation of CD133+ cells. More than 40% of the CD133 cells expressed PSGL-1, VLA-4, L-selectin, and CCR7, suggesting that part of CD133+ cells might migrate in secondary lymphoid organs.11 Moreover, the expression of these chemokine receptors and adhesion molecules of CD133+ cells do not seem to be particularly influenced by exposure to TNF-
Submitted April 24, 2006; accepted June 15, 2006.
Prepublished online as Blood First Edition Paper, June 29, 2006; DOI 10.1182/blood-2006-04-018564.
Supported by the Association Française contre les Myopathies; the Italian Ministry of Health (Ricerca Finalizzata); the IRCCS Fondazione Policlinico Hospital (Progetto a Concorso); Cariverona Foundation; Fondo Incentivazione Ricerca di Base (FIRB); and Fondazione Italiana Sclerosi Multipla (FISM).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yvan Torrente, Department of Neurological Science, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy; e-mail: yvan.torrente{at}unimi.it.
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Abstract
Introduction
(R&D Systems) was added (1 µM) to the cell-containing wells for 3 minutes at 37°C. This chemokine has been shown to induce the adhesion of human lymphocytes to endothelial cells or endothelial cell adhesion ligands. Cells were fixed in 1.5% glutaraldehyde for 20 minutes. The number of adherent cells in a 0.2-mm2 field was counted using cell-counting software. 
-counter in dystrophic injected mice. (A) Viability of radiolabeled cells. Flow cytometry analysis with PI and annexin V confirmed the 97% viability observed with trypan blue staining. (B) When we blocked the VCAM-1 molecule with the anti-VCAM-1 antibody, we obtained a significant decrease in radioactivity 12 hours after intra-arterial injection in the muscles and in the different organs, such as brain, lungs, kidneys, spleen, and liver, compared with values obtained from untreated scid/mdx mice. A decrement in radioactivity in organs of mice treated with anti-ICAM-1 was also detected, whereas in the muscle we registered a significant improvement in counts per minute per gram. Animals injected with CD133- cells used as control showed a decrement in the recruitment of intra-arterial injected cells after exercise (C-F). Human dystrophin expression 60 days after intra-arterial injection of human CD133+ stem cells is shown. Error bars indicate standard deviation. (C-D) Colocalization of the human dystrophin (red), human lamin A/C nuclei (green), and Hoechst (blue) demonstrated the formation of normal human myofibers in dystrophic muscles after transplantation of the human blood-derived CD133+ cells. The number of human dystrophin-positive myofibers in quadriceps of exercised mice (D, F) was higher than muscle of unexercised mice (C, E). Low magnification revealed mature human dystrophin muscle fibers (red) near