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  • br Results br Discussion Our analysis of satellite

    2018-11-02


    Results
    Discussion Our analysis of satellite cell frequency in muscles of the head, trunk, and limbs suggests considerable homogeneity among different healthy human muscle types. However, when considering muscle volume, higher satellite cell content of the temporalis may relate to physiological differences of that muscle and also suggests satellite cell content may be heterogeneous in other muscles yet to be examined. Moreover, potential functional heterogeneity within the human PAX7-positive satellite cell compartment remains to be examined and important variations in satellite stem improve may yet be identified. In the two outliers we found, recent physical activity or injury may have influenced satellite cell activation and frequency as has been shown to occur in humans (Crameri et al., 2004; Mackey et al., 2009). It will be important to determine whether activated human satellite cells retain satellite stem cell properties. Our analyses demonstrate that human skeletal muscles derived from either somitic or cranial mesoderm retain an abundance of satellite cells in adulthood. It appears feasible to obtain enriched populations of relatively large numbers of satellite cells from diverse human muscles (Table S4). Our data suggest that when optimized, the yield from individual human donor muscle harvests could be expected to be adequate for regeneration of smaller recipient muscles without ex vivo expansion. The consistent engraftment that occurred after transplantation of similar numbers of fiber-associated or sorted satellite cells from six different muscle types from males and females aged 17–81 years suggests substantial diversity of potential satellite cell donor muscles. An implication of our findings is that choice of donor muscle(s) in future applications including clinical transplantation can be based on previously established criteria for human muscle expendability and donor morbidity (Mathes and Nahai, 1997) and on ease of muscle sample preparation, rather than on a muscle-specific preference based on properties of the satellite cells within it. The different approaches to transplant satellite cells each have advantages and disadvantages from the standpoint of potential clinical application. Like in mouse experiments in which transplantation of single fibers carrying <10–20 satellite cells yield up to 100 fibers without additional injury (Collins et al., 2005; Hall et al., 2010), we found that human muscle fibers retain similarly high regenerative capacity derived from tens of satellite cells (Figure 2; Table 1) that are presumably better protected within their endogenous niche and have better preservation of surface proteins because of less enzyme exposure. Fiber transplantation also is not limited by selection of arbitrary surface markers, which could in theory exclude some stem cells. Not unlike currently used techniques of grafting adipose tissue or skin, both of which carry tissue stem cells that support graft homeostasis throughout the life of the organism, transplantation of muscle grafts (Zhang et al., 2014) is a theoretically feasible approach to regenerate small muscles that have depleted regenerative capacity. However, inherent technical difficulty complicates muscle fiber grafting. Prospective isolation helped to better define and characterize adult human satellite cells. Our analysis of PAX7, CD56, and CD29 in adult human muscle biopsies demonstrated that the co-expression and staining pattern of CD56 and CD29, as opposed to either alone, faithfully marks PAX7-expressing satellite cells. We observed a distinct staining pattern of apical CD56 expression that was particular to sublaminar satellite cells, similar to that previously shown for M-CADHERIN (Reimann et al., 2004), which may be specific to quiescent satellite cells. It has been described previously (Lindström and Thornell, 2009) that CD56 expression can occur on PAX7-negative human mononuclear muscle cells that could represent activated satellite cells or myoblasts. The relative scarcity of this population in our study could reflect the fact that most human muscle is quiescent with little proliferation in the absence of injury. We observed a population of CD56+/CD29+ cells that did not stain for PAX7 on sections. It is unclear what the lineage of these cells is, but during flow cytometry, a population of CD56+/CD29+ cells was depleted because of co-expression of CD31. In addition to satellite cells, the myofiber and some non-muscle cells within muscle tissue also express CD29. This makes CD29, if used alone, inadequate for identification of human satellite cells. Therefore, when using CD29 to evaluate human satellite cells, CD29 must be used in conjunction with other markers such as PAX7 and LAMININ. Additionally, it is important to confirm that sublaminar CD29 staining on satellite cells is distinguished from staining of the fiber plasma membrane. Our analysis of biopsies and transplanted populations provides strong evidence that most adult muscle stem cell activity resides within the CD56+/CD29+ population. This study does not exclude the possibility that extralaminar PAX7 cells or rare PAX7 satellite cells that do not exhibit CD56 and CD29 co-expression have myogenic properties. Moreover, the possibility that a subpopulation of PAX7+/CD56+/CD29+ cells can be further enriched for muscle stem cell functions merits investigation.