1127. Perivascular CD45-:Sca-1+:CD34- Cells Are Derived from Bone Marrow Cells and Participate in Dystrophic Skeletal Muscle Regeneration

Abstract

Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that mouse CD45|[minus]|:Sca-1+:CD34|[minus]| cells may play a role in the maturation of skeletal muscles and regeneration of mdx4cv dystrophic skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45|[minus]|:Sca-1+:CD34|[minus]| cells in mouse skeletal muscle, we reconstituted lethally irradiated wild type or mdx4cv mice with unfractionated BM cells from transgenic mice ubiquitously expressing green fluorescence protein (GFP). 1, 2, and 6 months post-transplantation, we analyzed the skeletal muscle mononuclear cells from the recipients by flow cytometry for GFP, CD45-PerCP, Sca-1-PE, and CD34-APC. To our surprise, we found BM-derived (GFP+) CD45|[minus]|:Sca-1+:CD34|[minus]| cells in the skeletal muscles of these GFP+ BM transplant recipients. We also demonstrated that these BM-derived cells were localized in the perivascular tissue by immuno-staining and that their frequency increased with time. We were interested in the potential clinical application of these cells for muscle diseases. Thus, we sorted CD45|[minus]|:Sca-1+:CD34|[minus]| cells by fluorescence activated cell sorting (FACS) skeletal muscle mononuclear cells and cultured them in several stem cell media (recipes), including a low-serum medium containing specific cytokines for isolating multipotent adult progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and differentiate to form myotubes in vitro and in vivo. Strikingly, we found that MAPCs were enriched up to 40 folds by sorting this population from skeletal muscle mononuclear cells. Concomitantly with the increase in frequency of BM-derived muscle CD45|[minus]|:Sca-1+:CD34|[minus]| cells, frequency of BM-derived muscle MAPCs also increased with time post-transplantation in dystrophic muscles. Furthermore, BM-derived muscle MAPCs displayed similar phenotypes of endogenous muscle MAPCs, suggesting a potential mechanism of BM cell migration to dystrophic skeletal muscles. To understand how these BM-derived cells migrate to the muscle and once in the muscle how they mobilize, we investigated the in vitro chemotatic response of GFP+ (BM derived) muscle MAPCs and CD45|[minus]|:Sca-1+ cells isolated from muscles of GFP+ BM transplant recipients. We found that these cells were highly chemoattrated to stroma derived factor, SDF-1, a chemo-attractant for cells expressing CXCR4. We also observed higher frequency of BM-derived CD45|[minus]|:Sca-1+:CD34|[minus]| cells in mdx dystrophic muscle than wild type muscle, which may be explained by higher expression levels of SDF-1 in mdx dystrophic muscles. Taken together, our results suggest that dystrophic muscles recruit BM cells that localize in perivascular tissues and can be defined as CD45|[minus]|:Sca-1+:CD34|[minus]|. This population when cultured enriches for MAPCs and can participate in muscle regeneration in dystrophic muscles.