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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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FACscan® immunophenotyping of the muscle-derived cells isolated using MACS® columns. The MDSCs obtained by six preplatings were tested for CD34 and Sca-1 antigen expression. Three distinct populations of MDSCs were obtained: Sca-1+/CD34− (a, first screen), Sca-1+/CD34+ (a, second screen), and Sca-1−/CD34+ (a, third screen). In b, the donor cells were detected by PCR for the Y chromosome in the hind limb muscles (lane 1), blood (lane 2), and bone marrow (lane 3), but not in samples from organs such as the liver (lane 4), spleen (lane 5), lung (lane 6), and brain (lane 7). 3 wk after i.m. transplantation of H2d unselected MDSCs in mdx mice (H2b), engrafted tissues were analyzed by FACS® for the presence of positive H2d (donor) cells (c). The positive H2d cells from the blood tissue (boxed region indicated in the second quadrant of the blood row) of the injected animals represent ∼0.1% of the total bloodstream. A similar percentage (∼1%) of donor cells was also detected in the bone marrow tissues after 3 wk (c, bone marrow row). We observed <5% donor cells in the controlateral hind limb muscles. The H2d expression in the bloodstream correlates with the expression of Flk-1 and stem cell marker Sca-1, whereas in the bone marrow, donor cells coexpressed the CD90 (12%) and Sca-1 (85%) antigens (c). The muscle tissue also contained some donor cells positive for Flk-1, CD34, and Sca-1 markers. A few donor cells (H2d positive) were also positive for CD34 (45%), Sca-1 (20%), or Flk1 (3%), as indicated by double immunostaining.
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fig1: FACscan® immunophenotyping of the muscle-derived cells isolated using MACS® columns. The MDSCs obtained by six preplatings were tested for CD34 and Sca-1 antigen expression. Three distinct populations of MDSCs were obtained: Sca-1+/CD34− (a, first screen), Sca-1+/CD34+ (a, second screen), and Sca-1−/CD34+ (a, third screen). In b, the donor cells were detected by PCR for the Y chromosome in the hind limb muscles (lane 1), blood (lane 2), and bone marrow (lane 3), but not in samples from organs such as the liver (lane 4), spleen (lane 5), lung (lane 6), and brain (lane 7). 3 wk after i.m. transplantation of H2d unselected MDSCs in mdx mice (H2b), engrafted tissues were analyzed by FACS® for the presence of positive H2d (donor) cells (c). The positive H2d cells from the blood tissue (boxed region indicated in the second quadrant of the blood row) of the injected animals represent ∼0.1% of the total bloodstream. A similar percentage (∼1%) of donor cells was also detected in the bone marrow tissues after 3 wk (c, bone marrow row). We observed <5% donor cells in the controlateral hind limb muscles. The H2d expression in the bloodstream correlates with the expression of Flk-1 and stem cell marker Sca-1, whereas in the bone marrow, donor cells coexpressed the CD90 (12%) and Sca-1 (85%) antigens (c). The muscle tissue also contained some donor cells positive for Flk-1, CD34, and Sca-1 markers. A few donor cells (H2d positive) were also positive for CD34 (45%), Sca-1 (20%), or Flk1 (3%), as indicated by double immunostaining.

Mentions: To isolate the MDSCs, we used a previously described approach that had proven successful for the isolation of stem and progenitor cells from the muscle, namely preplatings (Qu et al., 1998; Torrente et al., 2001). 500,000 MDSCs from male F1 Balb/c-C57BL10J mice (H2db) were injected intramuscularly in 10 female mdx/mdx mice (H2b). Using donor cells from an F1 donor, thus expressing the host and the donor major histocompatibility complexes (H2db), prevented a graft versus host disease. The presence of donor-derived cells in blood and bone marrow was still detectable by FACS® analysis for H2d. The donor cells were also evident by PCR for the Y chromosome in the injected tibialis anterior (TA) muscle, controlateral hind limb muscles, blood, and bone marrow, but not in sample organs such as the liver, spleen, lung, and brain (Fig. 1 b). The animals seemed in good health throughout the experimental period (3 wk). At 1 and 3 wk after i.m. injection of MDSCs, the flow cytometric analysis of peripheral blood nucleated cells revealed the presence of <1% positive H2d (donor) cells. A similar percentage (0.1–0.5%) was also detected in the bone marrow tissues after 3 wk. Recipient blood and bone marrow tissues were analyzed for the presence of donor MDSCs by double staining for H2d and one of the following markers: Sca-1, Flk-1, CD34, CD90, and CD45. 3 wk after MDSC i.m. engraftment, whole-body perfusion with saline solution was done to exclude contamination of circulating cells in tissues. Muscle tissues were dissociated mechanically and enzymatically and analyzed by FACS® for H2d donor antigen. We observed <5% of donor cells in the controlateral hind limb muscle tissues, and the cells expressing H2d also expressed the stem cell markers Sca-1 and CD34 (Fig. 1 c). This analysis indicated a selective engraftment by donor cells of mesodermal origin.


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)

FACscan® immunophenotyping of the muscle-derived cells isolated using MACS® columns. The MDSCs obtained by six preplatings were tested for CD34 and Sca-1 antigen expression. Three distinct populations of MDSCs were obtained: Sca-1+/CD34− (a, first screen), Sca-1+/CD34+ (a, second screen), and Sca-1−/CD34+ (a, third screen). In b, the donor cells were detected by PCR for the Y chromosome in the hind limb muscles (lane 1), blood (lane 2), and bone marrow (lane 3), but not in samples from organs such as the liver (lane 4), spleen (lane 5), lung (lane 6), and brain (lane 7). 3 wk after i.m. transplantation of H2d unselected MDSCs in mdx mice (H2b), engrafted tissues were analyzed by FACS® for the presence of positive H2d (donor) cells (c). The positive H2d cells from the blood tissue (boxed region indicated in the second quadrant of the blood row) of the injected animals represent ∼0.1% of the total bloodstream. A similar percentage (∼1%) of donor cells was also detected in the bone marrow tissues after 3 wk (c, bone marrow row). We observed <5% donor cells in the controlateral hind limb muscles. The H2d expression in the bloodstream correlates with the expression of Flk-1 and stem cell marker Sca-1, whereas in the bone marrow, donor cells coexpressed the CD90 (12%) and Sca-1 (85%) antigens (c). The muscle tissue also contained some donor cells positive for Flk-1, CD34, and Sca-1 markers. A few donor cells (H2d positive) were also positive for CD34 (45%), Sca-1 (20%), or Flk1 (3%), as indicated by double immunostaining.
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Related In: Results  -  Collection

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fig1: FACscan® immunophenotyping of the muscle-derived cells isolated using MACS® columns. The MDSCs obtained by six preplatings were tested for CD34 and Sca-1 antigen expression. Three distinct populations of MDSCs were obtained: Sca-1+/CD34− (a, first screen), Sca-1+/CD34+ (a, second screen), and Sca-1−/CD34+ (a, third screen). In b, the donor cells were detected by PCR for the Y chromosome in the hind limb muscles (lane 1), blood (lane 2), and bone marrow (lane 3), but not in samples from organs such as the liver (lane 4), spleen (lane 5), lung (lane 6), and brain (lane 7). 3 wk after i.m. transplantation of H2d unselected MDSCs in mdx mice (H2b), engrafted tissues were analyzed by FACS® for the presence of positive H2d (donor) cells (c). The positive H2d cells from the blood tissue (boxed region indicated in the second quadrant of the blood row) of the injected animals represent ∼0.1% of the total bloodstream. A similar percentage (∼1%) of donor cells was also detected in the bone marrow tissues after 3 wk (c, bone marrow row). We observed <5% donor cells in the controlateral hind limb muscles. The H2d expression in the bloodstream correlates with the expression of Flk-1 and stem cell marker Sca-1, whereas in the bone marrow, donor cells coexpressed the CD90 (12%) and Sca-1 (85%) antigens (c). The muscle tissue also contained some donor cells positive for Flk-1, CD34, and Sca-1 markers. A few donor cells (H2d positive) were also positive for CD34 (45%), Sca-1 (20%), or Flk1 (3%), as indicated by double immunostaining.
Mentions: To isolate the MDSCs, we used a previously described approach that had proven successful for the isolation of stem and progenitor cells from the muscle, namely preplatings (Qu et al., 1998; Torrente et al., 2001). 500,000 MDSCs from male F1 Balb/c-C57BL10J mice (H2db) were injected intramuscularly in 10 female mdx/mdx mice (H2b). Using donor cells from an F1 donor, thus expressing the host and the donor major histocompatibility complexes (H2db), prevented a graft versus host disease. The presence of donor-derived cells in blood and bone marrow was still detectable by FACS® analysis for H2d. The donor cells were also evident by PCR for the Y chromosome in the injected tibialis anterior (TA) muscle, controlateral hind limb muscles, blood, and bone marrow, but not in sample organs such as the liver, spleen, lung, and brain (Fig. 1 b). The animals seemed in good health throughout the experimental period (3 wk). At 1 and 3 wk after i.m. injection of MDSCs, the flow cytometric analysis of peripheral blood nucleated cells revealed the presence of <1% positive H2d (donor) cells. A similar percentage (0.1–0.5%) was also detected in the bone marrow tissues after 3 wk. Recipient blood and bone marrow tissues were analyzed for the presence of donor MDSCs by double staining for H2d and one of the following markers: Sca-1, Flk-1, CD34, CD90, and CD45. 3 wk after MDSC i.m. engraftment, whole-body perfusion with saline solution was done to exclude contamination of circulating cells in tissues. Muscle tissues were dissociated mechanically and enzymatically and analyzed by FACS® for H2d donor antigen. We observed <5% of donor cells in the controlateral hind limb muscle tissues, and the cells expressing H2d also expressed the stem cell markers Sca-1 and CD34 (Fig. 1 c). This analysis indicated a selective engraftment by donor cells of mesodermal origin.

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