Isolation and Culture of Myofiber-Derived Cells from the Extensor Digitorum Longus Muscle

Peripheral arterial occlusive disease (PAOD) involves distal artery occlusion by atherosclerotic plaques, which restricts blood flow and leads to ischemia in downstream tissues. Increased blood flow through preexisting collateral vessels leads to increased shear stress that triggers an outward remodeling of the vessel called arteriogenesis. In some cases this natural compensatory mechanism is able to sufficiently restore blood flow following arterial occlusion. However, for many individuals this process is insufficient to relieve peripheral ischemia, and patients experience intermittent claudication, or limb pain with locomotion or exercise. Without treatment, reduced blood flow can lead to tissue necrosis and potentially amputation. The efficacy of medication, such as drugs to lower cholesterol, is limited while surgical intervention is only available to a limited patient population. Supervised exercise therapy can improve important patient outcomes such as pain-free walking time and distance. Unfortunately, many patients fail to adhere to regimented exercise programs, limiting its functional efficacy. A potential alternative treatment approach is to stimulate or enhance arteriogenesis with cell therapy to recapitulate the beneficial effects of exercise therapy. Following exercise, resident muscle stem cells known as Satellite Cells (SCs) activate, repairing damaged muscle fibers and playing an important role in local angiogenesis. They and their progeny myoblasts, secrete a wide range of factors known to be involved in arteriogenesis and the recruitment of other cells involved in remodeling. As such, these cells are ideal candidates for transplantation. Before preliminary evaluation of these myogenic cells can be performed, a reliable source of these cells is required. Towards this goal, protocols were developed to obtain cells in sufficient numbers for transplantation. Whole Extensor Digitorum Longus (EDL) muscles were excised from mouse hindlimbs. Myofibers were isolated by collagenase digestion and trituration, and placed in specialized culture conditions to promote the adherence and selective growth of myogenic cells. In these conditions, SCs activate, migrate from myofibers, and proliferate. With increasing time in culture, especially following myofiber hypercontraction, SCs increasingly differentiate, limiting expansion and the availability of cells for transplantation. Future research will focus on improving culture conditions to inhibit differentiation and maintain sustained growth to improve the availability and in vivo behavior of isolated cells. Once sufficient cell numbers are obtained they will be transplanted into a mouse model of chronic peripheral ischemia to investigate their impact on collateral remodeling