245. Muscle-Derived Stem Cells Display an Extended, but Not Unlimited, Expansion Capability: Implication for Muscle Regeneration

Abstract

Stem cells are frequently considered the optimal cell type for regenerative cell-based therapies; however they generally represent a small fraction of cells isolated from a biopsy or other cell source. Ex vivo cell expansion is a necessary step to obtain clinically relevant numbers of cells. In addition, stem cells are often theorized as cells with unlimited long-term expansion potential. The purpose of this study is to test the long-term expansion capability of a population of muscle-derived stem cells. We first examined the proliferation kinetics of murine muscle-derived stem cells (MDSCs) to determine if they obey Hayflick's limit. We determined that these cells can be expanded for more than 300 population doublings (PDs) with no indications of replicative senescence. Next we examined how the molecular and behavioral stem cell phenotype, including the regenerative capacity, changes over time. We find that the MDSC population continues to maintain a relatively low level of desmin expression ( 65%) throughout the expansion. We observe that up to 200 PDs the MDSCs readily differentiate to form multinucleated myotubes, however expansion beyond 200 PDs leads to a decline in the number of cells entering the post-mitotic differentiated state. Remarkably, MDSC are capable of regenerating dystrophin expressing muscle fibers upon implantation in mdx muscular dystrophy model even after 200 population doublings. However, expansion beyond 200 PDs resulted in a subsequent decline in regeneration efficiency. Observed phenotypic changes highlight the inevitable aging of cells that results from cell expansion. Several findings including loss of contact inhibition, ability to grow on soft agar and an increase in numerical chromosomal abnormalities suggests that the MDSC may have become transformed. While the MDSC demonstrate a highly extended functional lifetime for muscle regeneration, we find that this potential is not unlimited.