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

Multiple mechanisms may account for bone marrow (BM) cell incorporation into myofibers following muscle damage. Here, we demonstrated that CD45−:Sca-1+:CD34− cells may play a role in the regeneration of mdx4cv skeletal muscles, an animal model for Duchenne muscular dystrophy. To understand the origin of CD45−:Sca-1+:CD34− cells in skeletal muscle, we reconstituted lethally irradiated wild type (wt) 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 a small percentage of BM-derived (GFP+) CD45−:Sca-1+:CD34− cells in the skeletal muscles of these GFP+ BM transplant recipients. These BM-derived cells are localized in the perivascular tissue by immunostaining and their frequency increases with time. We were interested in the potential of these cells for clinical application for muscle diseases. Thus, we FACS-sorted CD45−:Sca-1+:CD34− cells from GFP transgenic mouse skeletal muscles and transplanted them in the tibialis anterior (TA) muscle of mdx4cv mice. In ten days we found significant muscle engraftment. In addition, we studied the response of this population upon acute muscle injury. For this, we injected cardiotoxin in the right TA muscle of mdx4cv and wt mice, followed by BrdU administration in drinking water for three days. After 3.5 days, mice were sacrificed and the right and left (control) TA muscles were harvested and muscle CD45−:Sca-1+:CD34− cells were FACS-sorted, fixed and stained for BrdU and Myf-5. In the injured muscle (right TA), more than 70% of these cells were BrdU+ and more than 50% were Myf-5+, compared to baseline levels (close to zero) in the left TA. This indicates that this population can undergo proliferation and myogenic commitment upon muscle injury. To understand how these BM cells migrate to the muscle and once in the muscle how they mobilize, we investigated the in vitro chemotatic response of GFP+ (BM derived) CD45−: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−:Sca-1+:CD34− cells in dystrophic muscle than wt muscle, which may be explained by higher expression levels of SDF-1 in dystrophic muscles. In an effort to determine the identity of these cells when ex vivo cultured, we cultured them in several stem cell media, including a low-serum medium containing specific cytokines for the isolation and expansion of multipotent adult progenitor cells (MAPCs). MAPCs can be isolated from skeletal muscle and BM and can differentiate into multiple tissue cells. Strikingly, we found that MAPCs were enriched up to 40 folds by sorting this population from skeletal muscle. The frequency of BM-derived muscle MAPCs also increases with time post-transplantation in dystrophic muscles. These BM-derived muscle MAPCs displayed the typical MAPC immunophenotypes, displayed a normal diploid karyotype and were capable to differentiate into endothelial cells, hepatocytes and neurons.

Taken together, our results suggest that dystrophic muscles recruit BM cells that localize in perivascular tissues and can be defined as CD45-:Sca-1+:CD34-. This population when cultured enriches for MAPCs and can participate in muscle regeneration in dystrophic muscles.