Replicative Potential and Telomere Length in Human Skeletal Muscle: Implications for Satellite Cell-Mediated Gene Therapy

Abstract

In this study, we have evaluated the ability of human satellite cells isolated from subjects aged from 5 days to 86 years to proliferate in culture. Cells were cultivated until they became senescent. The number of cell divisions was calculated by counting the number of cells plated in culture compared to the number of cells removed following proliferation. Telomere length, which is known to decrease during each round of cell division, has been used to analyze the in vitro replicative capacity and in vivo replicative history of human satellite cells at isolation. The rate of telomere shortening in myonuclei of these muscle biopsies was also examined. Our results show that both proliferative capacity and telomere length of satellite cells decreases with age during the first two decades but that the myonuclei of human skeletal muscle are remarkably stable because telomere length in these myonuclei remains constant from birth to 86 years. The lack of shortening of mean terminal restriction fragments (TRF) in vivo confirms that skeletal muscle is a stable tissue with little nuclear turnover and therefore an ideal target for cell-mediated gene therapy. Moreover, our results show that it is important to consider donor age as a limiting factor to obtain an optimal number of cells. One of the gene transfer methods currently being explored is cell-mediated gene therapy. This technique involves the genetic manipulation of cells followed by their in vitro amplification. Skeletal muscle due to its stability and the presence of mononucleate myogenic precursor cells (satellite cells) has been suggested to be an ideal target for gene therapy. Satellite cells can be isolated from donor or host tissue, amplified in vitro, and then reinjected into host muscle where they will become incorporated into growing or regenerating muscle fibers. The success of cell-mediated gene therapy is related to the capacity of cells to proliferate during the stage of in vitro amplification and their ability to fuse with host muscle fibers subsequent to their transplantation. In this study, we have used telomere length to measure the in vitro replicative capacity of satellite cells. We have shown that both replicative capacity and telomere length decline rapidly during the first two decades of life but the myonuclei are stable because telomere length remains constant in adult muscle.