99. Dedifferentiation of Myotubes and Redifferentiation into an Osteogenic Lineage: A Cre-Lox System Study

Abstract

Top of pageAbstract Prior studies in our laboratory have shown that the number of muscle-derived stem cells (MDSCs) within skeletal muscle is very low|[mdash]|approximately 1% the number of satellite cells. Current muscle cell isolation techniques fail to utilize terminally differentiated myofibers, which constitute the bulk of cellular mass. New approaches based on the dedifferentiation of myofibers to a progenitor cell would provide access to a vast source of autologous cells for repair or regeneration of muscle, bone, and other tissues. The aims of this project were to 1) determine if BMP4 can induce muscle dedifferentiation in vitro and 2) explore if muscle dedifferentiation contributes to in vivo bone formation. We opted to use BMP4 as the dedifferentiating factor because of its close association with msx1 during development. We hypothesized that we could optimize our yield of MDSCs by using BMP4 in an ex vivo dedifferentiation technique involving mature muscle biopsies. We used a Cre-Lox system as a molecular tool to tag differentiated myotubes. We isolated muscle-derived cells (MDC-Cre) from a normal mouse and transfected them with a Cre-expressing retrovirus. We then co-cultured the cells with another population of MDCs isolated from 129-Gt(ROSA)26Sortm1Sho (Lox-|[beta]|geo) reporter mice (MDC-Lox). Fusion of MDC-Cre and MDC-Lox cells results in Cre-mediated cleavage of Lox sites and induction of |[beta]|-galactosidase (|[beta]|-gal) expression and neomycin resistance in resulting myotubes. We observed |[beta]|-gal expression exclusively in multinucleated myotubes (not in mononucleated cells), which demonstrates the specificity of our Cre-Lox system. Moreover, selection with G418 (1 mg/mL) for 6 days and subsequent stimulation with BMP4 (200 ng/mL) for 6 days revealed double-staining of mononucleated cells for |[beta]|-gal and alkaline phosphatase (ALP), an early osteogenic marker. To examine if BMP4 can stimulate dedifferentiation of myofibers in vivo, we injected the gastrocnemius muscles of Lox-|[beta]|geo mice with either 1|[times]|105 MDC-Cre cells or the same number of nontransfected MDCs. After 2 weeks, we injected AAV-BMP4 into the same site; we harvested the muscles 2 weeks later. Our results showed |[beta]|-gal[+] cells in the areas of calcified cartilage and bone and no |[beta]|-gal[+] cells outside the injected area. Our results suggest the fusion and redifferentiation of MDCs to an osteogenic lineage in vivo. Our Cre-Lox system effectively tags differentiated myotubes and enables us to track their fate after BMP4 treatment. Data suggest that, upon BMP4 stimulation, myotubes may dedifferentiate to a more primitive state and can contribute to formation of several tissue types. Specifically, we show evidence of redifferentiation of |[beta]|-gal[+] cells to the osteogenic lineage by expression of ALP in vitro and formation of ectopic bone in vivo. Our findings suggest that BMP4 is likely able to induce dedifferentiation of mature muscle fibers and thereby facilitate the isolation of a large population of MDSC-like cells for use in cell therapy applications.