The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Adult stem cells, present in tissues such as skeletal muscle, comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Muscle stem cells, known as satellite cells, reside in a quiescent state in an anatomically distinct compartment, or niche, ensheathed between the membrane of the myofiber and the basal lamina. Recently, procedures for isolating satellite cells were developed and experiments testing their function upon transplantation into muscles revealed an extraordinary potential to contribute to muscle fibers and access and replenish the satellite cell compartment. However, these properties are rapidly lost once satellite cells are plated in culture. Accordingly, the focus of this proposal is to elucidate the role of extrinsic factors in controlling muscle stem cell fate, in particular self-renewal. Our approach employs a bioengineered culture platform comprised of arrays of hydrogel microwells in which specific proteins are presented to stem cells. Critical to the approach is single cell analysis, as the behavior of slow proliferating stem cells may be masked by more rapidly proliferating progenitors. Moreover, by contrast with bulk cultures, single cell analyses enable the dynamic behavior of single stem cells to be tracked during a critical time period, the first few divisions in culture. The Specific Aims are (1) To identify extrinsic factors with a role in muscle stem cell fate in vitro. The proliferation kinetics and phenotype of single muscle satellite cells in arrays of bioengineered microwells will be tracked using time-lapse microscopy. Candidate proteins known to be associated with the niche and a library of ectodomain and transmembrane proteins will be assayed for their potential to alter satellite cell proliferation and phenotype. We will test the hypothesis that slow proliferation kinetics is a hallmark of maintenance of muscle stem cell function. (2) To elucidate mechanisms of muscle stem cell self-renewal. The frequency of self-renewal by three paradigms will be evaluated: asymmetric division leading to maintenance of stem cell number, symmetric division leading to expansion, and reversion from a committed stem cell state. We will test the hypothesis that extrinsic factors can alter the choice of self-renewal mechanisms. (3) To assess muscle stem cell function using a non-invasive in vivo assay. A novel in vivo bioluminescence imaging technology based on luciferase expression will be used to determine if cultured muscle stem cells exposed to proteins are as capable of engraftment, self-renewal and expansion in response to injury as freshly isolated stem cells. Together, these studies will provide insight into the role of extrinsic factors in the stem cell microenvironment on stem cell function and suggest novel therapeutic approaches to muscle degenerative diseases and muscle aging. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to gain an understanding of the mechanisms that regulate the behavior of adult muscle stem cells in normal development. In the organism, muscle stem cells respond to damage signals by increasing their numbers while retaining their stem cell properties, an attribute that is lost as soon as the cells are cultured. To harness the therapeutic potential of adult muscle stem cells in the treatment of dystrophies and muscle wasting associated with aging, an understanding of the factors that induce quiescence, self-renewal, and expansion of the stem cell is critical. A means for growing muscle stem cells in tissue culture without loss of stem cell properties is imperative and is the ultimate aim of this research.