br Experimental Procedures br Acknowledgments br Introductio

Experimental Procedures
Acknowledgments
Introduction The ability to characterize, isolate, and transplant human muscle stem hydroxycarboxylic acid receptors will lay the foundation for translational efforts to regenerate or engineer human muscles. However, the absence of approaches to expand limited amounts of available tissue ex vivo with retention of stem cell properties (Montarras et al., 2005), and difficulty developing xenograft model systems to test in vivo function (Boldrin et al., 2010; Silva-Barbosa et al., 2005; Zhang et al., 2014) make it difficult to study human muscle regeneration. Consequently, endogenous human muscle stem cells have not been characterized definitively, precluding the development of clinical applications. Muscle regeneration in mice is mediated by satellite cells that are anatomically defined based on their position between the fiber plasma membrane and the basal lamina. A subset of mouse satellite cells fulfill criteria of adult stem cells in that they engraft, proliferate, respond to injury by regenerating mature muscle, reoccupy the muscle satellite cell niche, and self-renew (Collins et al., 2005; Kuang et al., 2007; Montarras et al., 2005; Sacco et al., 2008; Sherwood et al., 2004). In contrast, satellite cell progeny can be propagated in vitro and show some capacity for differentiation, but after even brief culture cannot engraft efficiently when isolated from mouse (Montarras et al., 2005) or human (Brimah et al., 2004; Cooper et al., 2001). To date, most attempts to transplant adult human muscle cells have used cultured derivatives of endogenous or induced muscle cells, and few used freshly isolated or prospectively identified cells. Culture-expanded human myoblasts (Skuk et al., 2010) and CD133+ cells (Meng et al., 2014) can engraft and generate functional satellite cells after xenotransplantation of large numbers of cells, suggesting the potential for regenerative applications. Despite advances, the results of clinical trials (Miller et al., 1997; Partridge, 2002) and xenotransplantation experiments (Bareja et al., 2014; Castiglioni et al., 2014; Darabi et al., 2012; Ehrhardt et al., 2007; Miller et al., 1997; Partridge, 2002; Pisani et al., 2010; Silva-Barbosa et al., 2008) collectively show low transplantation efficiency, and satellite stem cell functions of self-renewal or expansion in vivo after injury have not been demonstrated from endogenous satellite cells. Thus, there is currently no established approach for directly isolating and transplanting endogenous bona-fide skeletal muscle stem cells from adult humans. Mouse satellite cells have been well characterized (reviewed in Yin et al., 2013), providing a strong foundation for human translation. Although available evidence suggests that human and mouse satellite cells have similar morphological characteristics and surface marker expression (Boldrin et al., 2010; Kadi et al., 2004; Mackey et al., 2009), significant differences have been identified. For example, it has been suggested that the canonical satellite cell transcription factor Pax-7 (Seale et al., 2000) is not absolutely restricted to or expressed in all human satellite cells (Reimann et al., 2004). Moreover, surface marker expression is not identical between mouse and human satellite cells (Boldrin and Morgan, 2012) and there is no accepted set of surface markers upon which to base human satellite cell isolation. Whereas satellite cell frequency and function is heterogeneous in murine muscles (Collins et al., 2005; Kuang et al., 2007; Ono et al., 2010; Pavlath et al., 1998; Zammit, 2008), little is known about heterogeneity in human muscles. Human satellite cell heterogeneity could in theory constrain transplantation of particular recipient muscles. Finally, while direct transplantation of individual muscle fibers from mice preserves very robust satellite cell function (Collins et al., 2005; Hall et al., 2010) and bypasses flow cytometry, human fiber experimentation has not kept pace. Human fibers have been successfully cultured (Bonavaud et al., 2002) but are significantly longer than in the mouse, making them difficult to handle and deterring experimentation with them (Boldrin and Morgan, 2012). Recently, the feasibility of transplanting cultured human fiber fragments has been demonstrated (Marg et al., 2014).