Characterization, Isolation and Angiogenesis of Endothelial Cells Derived from Skeletal Muscle.

We report here the characterization of endothelial cells from skeletal muscles by fluorescence-activated cell sorting on the basis of cell surface antigen expression for Sca-1, CD34 and CD31. Sca-1 is a marker of hematopoietic stem cells, although it can be expressed by other cells including endothelial cells. CD34 is also a marker of hematopoeitic stem cells and endothelial cells. Satellite cells of the skeletal muscles express CD34 but are negative for Sca-1 and CD31. CD31 or PECAM is a well recognized endothelial marker expressed by endothelium of multiple organs, however, its expression by microvascular endothelial cells of the skeletal muscle has not been previously characterized. Reports by others have described myo-endothelial progenitor cells in the interstitial spaces of the skeletal muscles based on the expression of CD34. These cells are Sca-1+ but negative for CD31 and other endothelial markers but are capable of myogenic and endothelial differentiation. Because hematopoietic cells express some of these markers, we used CD45 to exclude all hematopoietic cells in the muscles. We used a cocktail of antibodies to identify and characterize endothelial cells derived from skeletal muscles. For this whole body mouse muscles (n=20 C57BL6 mice, age 2-18 months) were harvested, digested and analyzed by flow cytometry. We found a very distinct and homogenous population identified as Sca-1+/CD34+/CD31+/CD45−. This population represents 5–10% of all mononuclear cells derived from the muscles. Triple staining of skeletal muscle using these markers highlights muscular vessels. In order to further characterize these cells, we FACS-sorted this population as well as Sca-1+/CD34+/CD31− and Sca-1−/CD34+/CD31− cells and analyzed then on cytospin by staining for endothelial as well as non-endothelial markers. While the majority of the cells within the Sca-1+/CD34+/CD31+ are positive for vWF (a marker of mature endothelial cells), the other two populations (Sca-1+/CD34+/CD31-and Sca-1−/CD34+/CD31−) were negative for vWF. Sca-1+/CD34+/CD31+ cells are also dim for Flk-1 and Tie-2. Interestingly, a minor population within this phenotype (Sca-1+/CD34+/CD31+) express syndecan-4 and CXCR4, markers important for migration and mobilization of stem cells. Studies to determine if this minor population contains more primitive endothelial progenitor cells are ongoing. In addition, Sca-1+/CD34+/CD31+ are negative for all the myogenic markers (Pax3, Pax7, MyoD, Myf5, Myf6 and Myogenin). In order to determine if muscle ECs can be expanded, Sca-1+/CD34+/CD31+ were sorted and cultured on different extracellular matrices: collagen, matrigel and fibronectin using DMEM and 10% FCS supplemented with 10ng/mL of VEGF. ECs from muscle grow better on fibronectin and can be passaged for more than 10 cell doublings. When seeded on matrigel, muscle ECs can make vascular tubes. Finally, when sorted Sca-1+/CD34+/CD31+ derived from ROSA26 mice are transplanted intramuscularly in the tibialis anterior muscle, 2 weeks after transplant bgal (lacZ)-positive cells can be detected in multiple vessels, demonstrating the angiogenic potential of these cells. Therefore, Sca-1+/CD34+/CD31+ are “bona fide” endothelial cells of the skeletal muscles. Understanding the biology of these cells is crucial to develop treatment for peripheral vascular disease and other ischemic diseases of the muscles.