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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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Myogenic differentiation of Sca-1+/CD34− MDSCs in cultures. Characterization of Sca-1+/CD34− MDSCs from Rosa26 and Des-LacZ transgenic mice for the L-selectin expression (a). L-selectin expression by Sca-1+/CD34− MDSCs (a, first screen) was decreased when these cells differentiated in myoblasts (a, second screen) and lost when they formed myotubes (a, third screen). For cloning, single cells within the Sca-1+/CD34− MDSCs (b, inset) were plated in proliferative conditions described in the Materials and methods section. Sca-1+/CD34−/L-selectin+ cells (HMDSCs) obtained from clone G13 differentiated well into myotubes (b and c) expressing the late myogenic marker MyHC (d). Few round single cells near myotubes were positive for the expression of m-cadherin (e) and desmin (f) markers. Panel g corresponds to the merging of e and f. Metalloproteinase activity of the HMDSCs (h, lane 2) was also tested and compared with that of myoblasts (h, lane 1). (i) Immunoblotting analysis of slow MyHC and m-cadherin by HMDSCs obtained from clone G13. The first lane of all immunoblottings corresponds to a homogenate of clone G13. 3T3 fibroblast and G8 myoblast cell lines were used as controls. Utrophin immunoblotting indicated that the same total protein concentrations were present in all specimens.
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fig3: Myogenic differentiation of Sca-1+/CD34− MDSCs in cultures. Characterization of Sca-1+/CD34− MDSCs from Rosa26 and Des-LacZ transgenic mice for the L-selectin expression (a). L-selectin expression by Sca-1+/CD34− MDSCs (a, first screen) was decreased when these cells differentiated in myoblasts (a, second screen) and lost when they formed myotubes (a, third screen). For cloning, single cells within the Sca-1+/CD34− MDSCs (b, inset) were plated in proliferative conditions described in the Materials and methods section. Sca-1+/CD34−/L-selectin+ cells (HMDSCs) obtained from clone G13 differentiated well into myotubes (b and c) expressing the late myogenic marker MyHC (d). Few round single cells near myotubes were positive for the expression of m-cadherin (e) and desmin (f) markers. Panel g corresponds to the merging of e and f. Metalloproteinase activity of the HMDSCs (h, lane 2) was also tested and compared with that of myoblasts (h, lane 1). (i) Immunoblotting analysis of slow MyHC and m-cadherin by HMDSCs obtained from clone G13. The first lane of all immunoblottings corresponds to a homogenate of clone G13. 3T3 fibroblast and G8 myoblast cell lines were used as controls. Utrophin immunoblotting indicated that the same total protein concentrations were present in all specimens.

Mentions: To verify whether L-selectin was involved in the adhesion of MDSCs to blood vessels, we treated Sca-1+/CD34− MDSCs with antibodies to L-selectin before their i.v. injection. This treatment reduced >70% of the adhesion of these cells to the muscle blood vessels. This demonstrated that L-selectin is involved in the migration of MDSCs to the muscles. Adhesion fractions of injected MDSCs were determined by counting the number of interacting cells in each muscle vessel per number of cells that passed through the same vessel during an injection. The subpopulation of MDSCs (Sca-1+/CD34−/L-selectin+) capable of adhesion to muscle blood vessels represents <3% of all MDSCs and will thus be referred to as homing MDSCs (HMDSCs). To increase the percentage of HMDSCs, we cloned the Sca-1+/CD34− MDSCs obtained from newborn Des-LacZ mice. Among the 30 clones produced, two (named G13 and F9) expressed the L-selectin on all cells and CD44 adhesion markers on 30% of these cells (unpublished data). The morphology of these cells was similar to medium-sized blast cells. In a myogenic differentiation medium, G13 and F9 clones lost the L-selectin expression (Fig. 3 a), indicating a specific correlation between the loss of this adhesion molecule and the lineage commitment of these stem cells. These clones produced cells expressing either myosin heavy chain (MyHC) (markers of late myogenic differentiation) or desmin and m-cadherin (markers of quiescent muscle satellite cells), indicating two characteristics of the myogenic potential of the HMDSCs (Fig. 3, d–g). These data were confirmed by immunoblot analysis. Multinucleated MyHC-positive myotubes were also observed after a 14-d culture of Sca-1+/CD34−/L-selectin− MDSC clones. These results indicate that L-selectin expression in the Sca-1+/CD34− MDSC subpopulation does not correlate with the capacity to differentiate into muscle cells. Metalloproteinases are a family of molecules that are critical in cell migration and remodeling of the ECM. We observed that HMDSCs expressed higher levels of MMP-2 and MMP-9 than myoblasts obtained after HMDSC commitment (Fig. 3 h). These data support the notion that the high migratory capacity of HMDSCs is the result of a combination of factors.


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)

Myogenic differentiation of Sca-1+/CD34− MDSCs in cultures. Characterization of Sca-1+/CD34− MDSCs from Rosa26 and Des-LacZ transgenic mice for the L-selectin expression (a). L-selectin expression by Sca-1+/CD34− MDSCs (a, first screen) was decreased when these cells differentiated in myoblasts (a, second screen) and lost when they formed myotubes (a, third screen). For cloning, single cells within the Sca-1+/CD34− MDSCs (b, inset) were plated in proliferative conditions described in the Materials and methods section. Sca-1+/CD34−/L-selectin+ cells (HMDSCs) obtained from clone G13 differentiated well into myotubes (b and c) expressing the late myogenic marker MyHC (d). Few round single cells near myotubes were positive for the expression of m-cadherin (e) and desmin (f) markers. Panel g corresponds to the merging of e and f. Metalloproteinase activity of the HMDSCs (h, lane 2) was also tested and compared with that of myoblasts (h, lane 1). (i) Immunoblotting analysis of slow MyHC and m-cadherin by HMDSCs obtained from clone G13. The first lane of all immunoblottings corresponds to a homogenate of clone G13. 3T3 fibroblast and G8 myoblast cell lines were used as controls. Utrophin immunoblotting indicated that the same total protein concentrations were present in all specimens.
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Related In: Results  -  Collection

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fig3: Myogenic differentiation of Sca-1+/CD34− MDSCs in cultures. Characterization of Sca-1+/CD34− MDSCs from Rosa26 and Des-LacZ transgenic mice for the L-selectin expression (a). L-selectin expression by Sca-1+/CD34− MDSCs (a, first screen) was decreased when these cells differentiated in myoblasts (a, second screen) and lost when they formed myotubes (a, third screen). For cloning, single cells within the Sca-1+/CD34− MDSCs (b, inset) were plated in proliferative conditions described in the Materials and methods section. Sca-1+/CD34−/L-selectin+ cells (HMDSCs) obtained from clone G13 differentiated well into myotubes (b and c) expressing the late myogenic marker MyHC (d). Few round single cells near myotubes were positive for the expression of m-cadherin (e) and desmin (f) markers. Panel g corresponds to the merging of e and f. Metalloproteinase activity of the HMDSCs (h, lane 2) was also tested and compared with that of myoblasts (h, lane 1). (i) Immunoblotting analysis of slow MyHC and m-cadherin by HMDSCs obtained from clone G13. The first lane of all immunoblottings corresponds to a homogenate of clone G13. 3T3 fibroblast and G8 myoblast cell lines were used as controls. Utrophin immunoblotting indicated that the same total protein concentrations were present in all specimens.
Mentions: To verify whether L-selectin was involved in the adhesion of MDSCs to blood vessels, we treated Sca-1+/CD34− MDSCs with antibodies to L-selectin before their i.v. injection. This treatment reduced >70% of the adhesion of these cells to the muscle blood vessels. This demonstrated that L-selectin is involved in the migration of MDSCs to the muscles. Adhesion fractions of injected MDSCs were determined by counting the number of interacting cells in each muscle vessel per number of cells that passed through the same vessel during an injection. The subpopulation of MDSCs (Sca-1+/CD34−/L-selectin+) capable of adhesion to muscle blood vessels represents <3% of all MDSCs and will thus be referred to as homing MDSCs (HMDSCs). To increase the percentage of HMDSCs, we cloned the Sca-1+/CD34− MDSCs obtained from newborn Des-LacZ mice. Among the 30 clones produced, two (named G13 and F9) expressed the L-selectin on all cells and CD44 adhesion markers on 30% of these cells (unpublished data). The morphology of these cells was similar to medium-sized blast cells. In a myogenic differentiation medium, G13 and F9 clones lost the L-selectin expression (Fig. 3 a), indicating a specific correlation between the loss of this adhesion molecule and the lineage commitment of these stem cells. These clones produced cells expressing either myosin heavy chain (MyHC) (markers of late myogenic differentiation) or desmin and m-cadherin (markers of quiescent muscle satellite cells), indicating two characteristics of the myogenic potential of the HMDSCs (Fig. 3, d–g). These data were confirmed by immunoblot analysis. Multinucleated MyHC-positive myotubes were also observed after a 14-d culture of Sca-1+/CD34−/L-selectin− MDSC clones. These results indicate that L-selectin expression in the Sca-1+/CD34− MDSC subpopulation does not correlate with the capacity to differentiate into muscle cells. Metalloproteinases are a family of molecules that are critical in cell migration and remodeling of the ECM. We observed that HMDSCs expressed higher levels of MMP-2 and MMP-9 than myoblasts obtained after HMDSC commitment (Fig. 3 h). These data support the notion that the high migratory capacity of HMDSCs is the result of a combination of factors.

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