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Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model.

Torrente Y, Camirand G, Pisati F, Belicchi M, Rossi B, Colombo F, El Fahime M, Caron NJ, Issekutz AC, Constantin G, Tremblay JP, Bresolin N - J. Cell Biol. (2003)

Bottom Line: The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs).Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin.This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Sciences, Stem Cell Laboratory, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy. torrenteyvan@hotmail.com

ABSTRACT
Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

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Muscle migration of HMDSCs in dystrophic muscle is age dependent. The radioactivity was significantly increased 2 h after the i.v. injection of HMDSC clone G13 labeled with [3H]glycerol in muscles of 2-mo-old mdx/mdx mice receiving HMDSCs versus 1-yr-old dystrophic animals injected with the HMDSCs (P < 0.001). The radioactivity levels in the diaphragm and pectoralis muscles of 2-mo-old mdx/mdx mice were respectively six- and fivefold higher than in muscles injected with HMDSCs treated with anti–L-selectin (*, P < 0.001). Muscle radioactivity levels in older (1-yr-old) mdx/mdx mice injected with clone G13 are not significantly different from those in mdx/mdx mice injected with HMDSCs treated with anti–L-selectin. (b) Expression of L-selectin ligands on endothelium from dystrophic muscle by using intravital microscopy. Fluorescently labeled anti–E-selectin, –P-selectin, and –MAdCAM-1 antibodies were injected in mdx mice, and positivity was determined on the vascular endothelium of the dystrophic muscles. Dystrophic muscles from 2-mo-old mdx/mdx mice expressed MAdCAM-1 at venular sites, but not E- and P-selectin, while aging dystrophic vessels did not express L-selectin ligands on their surface.
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fig5: Muscle migration of HMDSCs in dystrophic muscle is age dependent. The radioactivity was significantly increased 2 h after the i.v. injection of HMDSC clone G13 labeled with [3H]glycerol in muscles of 2-mo-old mdx/mdx mice receiving HMDSCs versus 1-yr-old dystrophic animals injected with the HMDSCs (P < 0.001). The radioactivity levels in the diaphragm and pectoralis muscles of 2-mo-old mdx/mdx mice were respectively six- and fivefold higher than in muscles injected with HMDSCs treated with anti–L-selectin (*, P < 0.001). Muscle radioactivity levels in older (1-yr-old) mdx/mdx mice injected with clone G13 are not significantly different from those in mdx/mdx mice injected with HMDSCs treated with anti–L-selectin. (b) Expression of L-selectin ligands on endothelium from dystrophic muscle by using intravital microscopy. Fluorescently labeled anti–E-selectin, –P-selectin, and –MAdCAM-1 antibodies were injected in mdx mice, and positivity was determined on the vascular endothelium of the dystrophic muscles. Dystrophic muscles from 2-mo-old mdx/mdx mice expressed MAdCAM-1 at venular sites, but not E- and P-selectin, while aging dystrophic vessels did not express L-selectin ligands on their surface.

Mentions: To quantify the inhibition of the binding of HMDSC clone G13 to the muscle blood vessels produced by the L-selectin antibody, we labeled these cells by growing them for 4 h with 200 μCi/ml [3H]glycerol. This quantification method was optimized to detect a small number of accumulated cells. The cells were divided in two pools, one was treated with an anti–L-selectin antibody at 50 μg/ml. Anti–L-selectin was found to be functional, as assessed by binding/blocking assays with an L-selectin–transfected cell line (unpublished data). Each pool of cells (5 × 105 cells per animal) was injected intravenously in two groups of mdx/mdx mice. Group 1 was 2 mo old (n = 5), and group 2 was 1 yr old (n = 5). The radioactivity was compared 2 h after HMDSC injection. To demonstrate that the radioactivity detected in the engrafted tissues was only due to the HMDSC accumulation and not to nonspecific leaking of radioactivity, we injected cell lysates from radiolabeled cells as a control (Table II). We found a low percentage of tissue incorporation of lysate-derived radioactivity (mostly in the liver). Moreover, to exclude the potential effects of the complement, five mdx/mdx animals were injected with HMDSCs pretreated with the F(ab')2 anti–L-selectin. This treatment revealed similar tissue distribution in comparison to the animals injected with the HMDSCs treated with anti–L-selectin (Table II). After the injection of intact HMDSCs not treated with anti–L-selectin, the radioactivity was detected prevalently in muscle tissues such as TA, quadriceps, pectoral, diaphragm, and triceps brachii muscles but not in lungs, liver, and kidneys of 2-mo-old mdx/mdx mice. However, a significant decrease in muscle radioactivity (P < 0.001) was observed in 2-mo-old mdx/mdx mice injected with HMDSCs treated with anti–L-selectin (Table II). Muscles of 1-yr-old mdx/mdx injected animals revealed a marked reduction of the radioactivity (P < 0.001) and no difference between animals injected with HMDSCs and HMDSCs treated with anti–L-selectin (Fig. 5; Table II). Organs showed no significant differences in distribution between both groups of mdx/mdx mice, as shown in Table II. Note also that the level of radioactivity was much lower in organs than in muscles. We also qualitatively assessed the muscle localization of [3H]glycerol HMDSC clone G13 by autoradiography. [3H]glycerol-labeled cells were distributed around the muscle fibers and near the muscle blood vessels. The number of [3H]glycerol-labeled cells per section reflected the level of radioactivity detected in the tissue (Fig. 4, a–d). Donor [3H]glycerol-labeled cells were indeed found in the pectoral, soleus, gastrocnemius, TA, and triceps brachii of injected mice. Lungs, liver, brain, and kidneys contained only a few labeled cells (unpublished data), which corresponded with the low radioactivity detected in these tissues (Table II; Fig. 4 i). Tissues from mice not injected with HMDSCs scored negatively (Fig. 4, i and j).


Identification of a putative pathway for the muscle homing of stem cells in a muscular dystrophy model.

Torrente Y, Camirand G, Pisati F, Belicchi M, Rossi B, Colombo F, El Fahime M, Caron NJ, Issekutz AC, Constantin G, Tremblay JP, Bresolin N - J. Cell Biol. (2003)

Muscle migration of HMDSCs in dystrophic muscle is age dependent. The radioactivity was significantly increased 2 h after the i.v. injection of HMDSC clone G13 labeled with [3H]glycerol in muscles of 2-mo-old mdx/mdx mice receiving HMDSCs versus 1-yr-old dystrophic animals injected with the HMDSCs (P < 0.001). The radioactivity levels in the diaphragm and pectoralis muscles of 2-mo-old mdx/mdx mice were respectively six- and fivefold higher than in muscles injected with HMDSCs treated with anti–L-selectin (*, P < 0.001). Muscle radioactivity levels in older (1-yr-old) mdx/mdx mice injected with clone G13 are not significantly different from those in mdx/mdx mice injected with HMDSCs treated with anti–L-selectin. (b) Expression of L-selectin ligands on endothelium from dystrophic muscle by using intravital microscopy. Fluorescently labeled anti–E-selectin, –P-selectin, and –MAdCAM-1 antibodies were injected in mdx mice, and positivity was determined on the vascular endothelium of the dystrophic muscles. Dystrophic muscles from 2-mo-old mdx/mdx mice expressed MAdCAM-1 at venular sites, but not E- and P-selectin, while aging dystrophic vessels did not express L-selectin ligands on their surface.
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Related In: Results  -  Collection

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fig5: Muscle migration of HMDSCs in dystrophic muscle is age dependent. The radioactivity was significantly increased 2 h after the i.v. injection of HMDSC clone G13 labeled with [3H]glycerol in muscles of 2-mo-old mdx/mdx mice receiving HMDSCs versus 1-yr-old dystrophic animals injected with the HMDSCs (P < 0.001). The radioactivity levels in the diaphragm and pectoralis muscles of 2-mo-old mdx/mdx mice were respectively six- and fivefold higher than in muscles injected with HMDSCs treated with anti–L-selectin (*, P < 0.001). Muscle radioactivity levels in older (1-yr-old) mdx/mdx mice injected with clone G13 are not significantly different from those in mdx/mdx mice injected with HMDSCs treated with anti–L-selectin. (b) Expression of L-selectin ligands on endothelium from dystrophic muscle by using intravital microscopy. Fluorescently labeled anti–E-selectin, –P-selectin, and –MAdCAM-1 antibodies were injected in mdx mice, and positivity was determined on the vascular endothelium of the dystrophic muscles. Dystrophic muscles from 2-mo-old mdx/mdx mice expressed MAdCAM-1 at venular sites, but not E- and P-selectin, while aging dystrophic vessels did not express L-selectin ligands on their surface.
Mentions: To quantify the inhibition of the binding of HMDSC clone G13 to the muscle blood vessels produced by the L-selectin antibody, we labeled these cells by growing them for 4 h with 200 μCi/ml [3H]glycerol. This quantification method was optimized to detect a small number of accumulated cells. The cells were divided in two pools, one was treated with an anti–L-selectin antibody at 50 μg/ml. Anti–L-selectin was found to be functional, as assessed by binding/blocking assays with an L-selectin–transfected cell line (unpublished data). Each pool of cells (5 × 105 cells per animal) was injected intravenously in two groups of mdx/mdx mice. Group 1 was 2 mo old (n = 5), and group 2 was 1 yr old (n = 5). The radioactivity was compared 2 h after HMDSC injection. To demonstrate that the radioactivity detected in the engrafted tissues was only due to the HMDSC accumulation and not to nonspecific leaking of radioactivity, we injected cell lysates from radiolabeled cells as a control (Table II). We found a low percentage of tissue incorporation of lysate-derived radioactivity (mostly in the liver). Moreover, to exclude the potential effects of the complement, five mdx/mdx animals were injected with HMDSCs pretreated with the F(ab')2 anti–L-selectin. This treatment revealed similar tissue distribution in comparison to the animals injected with the HMDSCs treated with anti–L-selectin (Table II). After the injection of intact HMDSCs not treated with anti–L-selectin, the radioactivity was detected prevalently in muscle tissues such as TA, quadriceps, pectoral, diaphragm, and triceps brachii muscles but not in lungs, liver, and kidneys of 2-mo-old mdx/mdx mice. However, a significant decrease in muscle radioactivity (P < 0.001) was observed in 2-mo-old mdx/mdx mice injected with HMDSCs treated with anti–L-selectin (Table II). Muscles of 1-yr-old mdx/mdx injected animals revealed a marked reduction of the radioactivity (P < 0.001) and no difference between animals injected with HMDSCs and HMDSCs treated with anti–L-selectin (Fig. 5; Table II). Organs showed no significant differences in distribution between both groups of mdx/mdx mice, as shown in Table II. Note also that the level of radioactivity was much lower in organs than in muscles. We also qualitatively assessed the muscle localization of [3H]glycerol HMDSC clone G13 by autoradiography. [3H]glycerol-labeled cells were distributed around the muscle fibers and near the muscle blood vessels. The number of [3H]glycerol-labeled cells per section reflected the level of radioactivity detected in the tissue (Fig. 4, a–d). Donor [3H]glycerol-labeled cells were indeed found in the pectoral, soleus, gastrocnemius, TA, and triceps brachii of injected mice. Lungs, liver, brain, and kidneys contained only a few labeled cells (unpublished data), which corresponded with the low radioactivity detected in these tissues (Table II; Fig. 4 i). Tissues from mice not injected with HMDSCs scored negatively (Fig. 4, i and j).

Bottom Line: The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs).Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin.This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurological Sciences, Stem Cell Laboratory, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy. torrenteyvan@hotmail.com

ABSTRACT
Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting skeletal myoblasts directly into muscles are faced with the problem of the limited migration of these cells in the muscles. The delivery of myogenic stem cells to the sites of muscle lesions via the systemic circulation is a potential alternative approach to treat this disease. Muscle-derived stem cells (MDSCs) were obtained by a MACS(R) multisort method. Clones of MDSCs, which were Sca-1+/CD34-/L-selectin+, were found to adhere firmly to the endothelium of mdx dystrophic muscles after i.v. or i.m. injections. The subpopulation of Sca-1+/CD34- MDSCs expressing L-selectin was called homing MDSCs (HMDSCs). Treatment of HMDSCs with antibodies against L-selectin prevented adhesion to the muscle endothelium. Importantly, we found that vascular endothelium from striate muscle of young mdx mice expresses mucosal addressin cell adhesion molecule-1 (MAdCAM-1), a ligand for L-selectin. Our results showed for the first time that the expression of the adhesion molecule L-selectin is important for muscle homing of MDSCs. This discovery will aid in the improvement of a potential therapy for muscular dystrophy based on the systemic delivery of MDSCs.

Show MeSH
Related in: MedlinePlus