Differentiation of activated satellite cells in denervated muscle following single fusions in situ and in cell culture

Satellite cells represent a cellular source of regeneration in adult skeletal muscle. It remains unclear why a large pool of stem myoblasts in denervated muscle does not compensate for the loss of muscle mass during post-denervation atrophy. In this study, we present evidence that satellite cells in long-term denervated rat muscle are able to activate synthesis of contractile proteins after single fusions in situ. This process of early differentiation leads to formation of abnormally diminutive myotubes. The localization of such dwarf myotubes beneath the intact basal lamina on the surface of differentiated muscle fibers shows that they form by fusion of neighboring satellites or by the progeny of a single satellite cell following one or two mitotic divisions. We demonstrated single fusions of myoblasts using electron microscopy, immunocytochemical labeling and high resolution confocal digital imaging. Sequestration of nascent myotubes by the rapidly forming basal laminae creates a barrier that limits further fusions. The recruitment of satellite cells in the formation of new muscle fibers results in a progressive decrease in their local densities, spatial separation and ultimate exhaustion of the myogenic cell pool. To determine whether the accumulation of aberrant dwarf myotubes is explained by the intrinsic decline of myogenic properties of satellite cells, or depends on their spatial separation and the environment in the tissue, we studied the fusion of myoblasts isolated from normal and denervated muscle in cell culture. The experiments with a culture system demonstrated that the capacity of myoblasts to synthesize contractile proteins without serial fusions depended on cell density and the availability of partners for fusion. Satellite cells isolated from denervated muscle and plated at fusion-permissive densities progressed through the myogenic program and actively formed myotubes, which shows that their myogenic potential is not considerably impaired. The results of this study suggest that under conditions of denervation, progressive spatial separation and confinement of many satellite cells within the endomysial tubes of atrophic muscle fibers and progressive interstitial fibrosis are the important factors that prevent their normal differentiation. Our findings also provide an explanation of why denervated muscle partially and temporarily is able to restore its functional capacity following injury and regeneration: the release of satellite cells from their sublaminal location provides the necessary space for a more active regenerative process.     

Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell

Satellite cells are committed myogenic progenitors that give rise to proliferating myoblasts during postnatal growth and repair of skeletal muscle. To identify genes expressed at different developmental stages in the satellite cell myogenic program, representational difference analysis of cDNAs was employed to identify more than 50 unique mRNAs expressed in wild-type myoblasts and MyoD-/- myogenic cells. Novel expression patterns for several genes, such as Pax7, Asb5, IgSF4, and Hoxc10, were identified that were expressed in both quiescent and activated satellite cells. Several previously uncharacterized genes that represent putative MyoD target genes were also identified, including Pw1, Dapk2, Sytl2, and NLRR1. Importantly, many genes such as IgSF4, Neuritin, and Klra18 that were expressed exclusively in MyoD-/- myoblasts were also expressed by satellite cells in undamaged muscle in vivo but were not expressed by primary myoblasts. These data are consistent with a biological role for activated satellite cells that induce Myf5 but not MyoD. Lastly, additional endothelial and hematopoietic markers were identified supporting a nonsomitic developmental origin of the satellite cell myogenic lineage.     

An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats

Summary Satellite cells were traced autoradiographically during the regeneration of skeletal muscle in young Sprague-Dawley rats. Approximately 31% of the satellite cells in uninjured muscles appeared labelled after three injections of tritiated thymidine; none of the myonuclei were labelled in the same muscles. Four to six days after transplanting the radioactive muscles to non-radioactive littermates, regenerating myotube nuclei in the host appeared labelled. Thus, this study confirms that satellite cells in young rats can differentiate into multinucleated myotubes following muscle injury.Supported by NIH grant No. 5 S01-RR05356-13I wish to acknowledge the excellent technical assistance of Ms. Amy Erisman     

The depletion of skeletal muscle satellite cells with age is concomitant with reduced capacity of single progenitors to produce reserve progeny

Satellite cells are myogenic progenitors that reside on the myofiber surface and support skeletal muscle repair. We used mice in which satellite cells were detected by GFP expression driven by nestin gene regulatory elements to define age-related changes in both numbers of satellite cells that occupy hindlimb myofibers and their individual performance. We demonstrate a reduction in satellite cells per myofiber with age that is more prominent in females compared to males. Satellite cell loss also persists with age in myostatin-null mice regardless of increased muscle mass. Immunofluorescent analysis of isolated myofibers from nestin-GFP/Myf5^nLacZ/+ mice reveals a decline with age in the number of satellite cells that express detectable levels of βgal. Nestin-GFP expression typically diminishes in primary cultures of satellite cells as myogenic progeny proliferate and differentiate, but GFP subsequently reappears in the Pax7⁺ reserve population. Clonal analysis of sorted GFP⁺ satellite cells from hindlimb muscles shows heterogeneity in the extent of cell density and myotube formation among colonies. Reserve cells emerge primarily within high-density colonies, and the number of clones that produce reserve cells is reduced with age. Thus, satellite cell depletion with age could be attributed to a reduced capacity to generate a reserve population.     

Ancestral Myf5 gene activity in periocular connective tissue identifies a subset of fibro/adipogenic progenitors but does not connote a myogenic origin

Extraocular muscles (EOM) represent a unique muscle group that controls eye movements and originates from head mesoderm, while the more typically studied body and limb muscles are somite-derived. Aiming to investigate myogenic progenitors (satellite cells) in EOM versus limb and diaphragm of adult mice, we have been using flow cytometry in combination with myogenic-specific Cre-loxP lineage marking for cell isolation. While analyzing cells from the EOM of mice that harbor Myf5^Cre-driven GFP expression, we identified in addition to the expected GFP⁺ myogenic cells (presumably satellite cells), a second dominant GFP⁺ population distinguished as being Sca1⁺, non-myogenic, and exhibiting a fibro/adipogenic potential. This unexpected population was not only unique to EOM compared to the other muscles but also specific to the Myf5^Cre-driven reporter when compared to the MyoD^Cre driver. Histological studies of periocular tissue preparations demonstrated the presence of Myf5^Cre-driven GFP⁺ cells in connective tissue locations adjacent to the muscle masses, including cells in the vasculature wall. These vasculature-associated GFP⁺ cells were further identified as mural cells based on the presence of the specific XLacZ4 transgene. Unlike the EOM satellite cells that originate from a Pax3-negative lineage, these non-myogenic Myf5^Cre-driven GFP⁺ cells appear to be related to cells of a Pax3-expressing origin, presumably derived from the neural crest. In all, our lineage tracing based on multiple reporter lines has demonstrated that regardless of common ancestral expression of Myf5, there is a clear distinction between periocular myogenic and non-myogenic cell lineages according to their mutually exclusive antecedence of MyoD and Pax3 gene activity.     

Efficient in vitro myogenic reprogramming of human primary mesenchymal stem cells and endothelial cells by Myf5

Background information. The identification of a source of stem cells able to regenerate skeletal muscle was the goal of numerous studies with the aim to develop new therapeutic approaches for genetic muscle diseases or muscle injuries. A series of studies have demonstrated that stem cells derived from various tissues may have a role in the regeneration of damaged muscles, but this contribution is always very weak. Thus we established a project aiming to reprogramme non‐muscle cells into the skeletal striated differentiation pathway. Results. We transduced several human primary adult stem or progenitor cells using a recombinant lentivirus containing the coding sequence of the Myf5 gene considered as a master gene for the determination of skeletal striated muscle. These original results are the first demonstration of a myogenic conversion of human mesenchymal and endothelial cells by Myf5. Conclusions. The procedure described in the present paper could be used to develop new research protocols with the prospect of using these cells as therapeutic agents.     

Survival of satellite cells following exposure to the local anesthetic bupivacaine (Marcaine)

Summary Rat lumbrical muscles were incubated in a concentration of 10^-2 M bupivacaine for 5 or 15 min and examined after further incubation in the absence of the drug for periods totalling 1, 2, and 3h. Electron microscopy showed that muscle fibers and their component organelles and myonuclei underwent a series of irreversible degenerative changes. However, satellite cells retained their normal morphology under similar conditions. It is concluded that satellite cells are responsible for the rapid regeneration of muscles that follows degeneration induced by bupivacaine. The role of satellite cells in muscle regeneration is discussed.This investigation was supported by United States Public Health Service Research Grant NS 13296 from the National Institutes of Health. A supply of bupivacaine was kindly made available by Winthrop Laboratories, New York, N.Y., USA     

Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers

The satellite cell compartment provides skeletal muscle with a remarkable capacity for regeneration. Here, we have used isolated myofibers to investigate the activation and proliferative potential of satellite cells. We have previously shown that satellite cells are heterogeneous: the majority express Myf5 and M-cadherin protein, presumably reflecting commitment to myogenesis, while a minority is negative for both. Although MyoD is rarely detected in quiescent satellite cells, over 98% of satellite cells contain MyoD within 24 h of stimulation. Significantly, MyoD is only observed in cells that are already expressing Myf5. In contrast, a minority population does not activate by the criteria of Myf5 or MyoD expression. Following the synchronous activation of the myogenic regulatory factor+ve satellite cells, their daughter myoblasts proliferate with a doubling time of approximately 17 h, irrespective of the fiber type (type I, IIa, or IIb) from which they originate. Although fast myofibers have fewer associated satellite cells than slow, and accordingly produce fewer myoblasts, each myofiber phenotype is associated with a complement of satellite cells that has sufficient proliferative potential to fully regenerate the parent myofiber within 4 days. This time course is similar to that observed in vivo following acute injury and indicates that cells other than satellite cells are not required for complete myofiber regeneration.     

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4^Cre and Mrf4^nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.     

Skeletal muscle stem and progenitor cells: reconciling genetics and lineage

Skeletal muscle provides a unique paradigm for studying stem to differentiated cell transitions, as well as the acquisition of cellular identity. Embryological and genetic studies over the last decades have unveiled key signaling pathways and regulatory genes which are involved in this process. In the adult, regeneration from fiber-associated satellite cells as well as non-muscle cells have opened the perspective for cell therapy studies. Paradoxically, however, the lineage has remained largely elusive. Recent studies have provided clues regarding the cellular organization in this lineage. Furthermore, the complexity of the genetic networks regulating global and local myogenic programs can be correlated with location and lineage. Finally, prenatal and postnatal developmental strategies have similarities and differences which will also be highlighted.     

Functional properties of muscle-derived cells related to morphological characteristics

Satellite cells represent a specific lineage of myogenic progenitors that allow skeletal muscle postnatal growth and repair. They have been described as being heterogeneous in nature, a characteristic associated with functional disparities. Here, we aimed at determining whether the morphometric characteristics of freshly extracted turkey muscle-derived cells (MDC) could represent a distinctive criterion between them and could also be associated with their behavioural features. Morphometric analysis showed that MDC displayed wide cell size diversity, from 4 to 10 μm. Lineage marker analysis was performed on MDC sorted by their size using counterflow centrifugal elutriation and showed that the cell size was associated with the specific expression of myogenic markers, revealing different commitment levels. In vitro, the smallest MDC exhibited limited myogenic activity while larger MDC displayed a myogenic potential that increased with their size. Ultrastructural analysis revealed that the smallest MDC shared quiescent cell features, whereas the other cells displayed metabolic activity that also increased as a function of their size. Collectively, our results demonstrate that the size of freshly extracted MDC is indicative of their respective progression towards myogenic differentiation lineage. This criterion could be useful for the early separation of more or less committed cells in the myogenic programme.Gregory Jouvion and Karl Rouger contributed equally to this work.     

The effects of aging on satellite cells in skeletal muscles of mice and rats

Summary Myosatellite cells were examined and quantified at the fine structural level of resolution during aging of skeletal muscles in mice and rats. Satellite cells in the soleus and gastrocnemius muscles of animals between eight and 30 months of age appeared, according to morphological criteria, metabolically less active than those examined in immature muscles. In the soleus muscle of the mouse, satellite cells decreased in number from 4.6% at eight months of age to 2.4% at 30 months. This decrease appeared to be due to the passage of some satellite cells into the interstitial space as a result of the formation of external lamina material around the entire satellite cell surface.This study was supported by NIH grant No. 5 S01-RR05356-13Appreciation is extended to Ms. Amy Erisman and Mr. Monroe Sprague for their excellent technical assistance on this project     

Geroconversion of aged muscle stem cells under regenerative pressure

Regeneration of skeletal muscle relies on a population of quiescent stem cells (satellite cells) and is impaired in very old (geriatric) individuals undergoing sarcopenia. Stem cell function is essential for organismal homeostasis, providing a renewable source of cells to repair damaged tissues. In adult organisms, age-dependent loss-of-function of tissue-specific stem cells is causally related with a decline in regenerative potential. Although environmental manipulations have shown good promise in the reversal of these conditions, recently we demonstrated that muscle stem cell aging is, in fact, a progressive process that results in persistent and irreversible changes in stem cell intrinsic properties. Global gene expression analyses uncovered an induction of p16^INK4a in satellite cells of physiologically aged geriatric and progeric mice that inhibits satellite cell-dependent muscle regeneration. Aged satellite cells lose the repression of the INK4a locus, which switches stem cell reversible quiescence into a pre-senescent state; upon regenerative or proliferative pressure, these cells undergo accelerated senescence (geroconversion), through Rb-mediated repression of E2F target genes. p16^INK4a silencing rejuvenated satellite cells, restoring regeneration in geriatric and progeric muscles. Thus, p16^INK4a/Rb-driven stem cell senescence is causally implicated in the intrinsic defective regeneration of sarcopenic muscle. Here we discuss on how cellular senescence may be a common mechanism of stem cell aging at the organism level and show that induction of p16^INK4a in young muscle stem cells through deletion of the Polycomb complex protein Bmi1 recapitulates the geriatric phenotype.     

Motility,linear arrangement and cell-to-cell contact of myogenic cells prior to fusion

Summary Time-lapse cinematography elucidates the genesis of a uniform and approximately linearly arranged myogenic cell aggregate, stemming from two larger cell groups. The ultimate aggregate is created by continuous movement of one cell group toward the other. Following this motion, the angle between the cell groups is reduced as they approach each other.Different patterns of cell motility can be recognized. Some cells move in a preferred direction in relation to the aggregate as a whole, whereas others alter their direction of movement.The myogenic cells are aligned end-to-end and side-by-side. The latter is often accomplished in the following manner: two cells in end-to-end contact form as crescent-shaped free space with their polar extensions; a neighboring spindle-shaped cell then settles in this space. An arrangement of cells such that their greatest cytoplasmic widths lie at the same level can also be seen. During the recording period, two cells in one of the groups were replicating. One of them realized karyo- and cytokinesis in approximately 80min. The daughter cells moved apart in opposite directions, but never lost contact to the aggregate. This observation shows that contact between presumptive myoblasts and myoblasts is established.This research was supported by a grant from the Deutsche Forschungsgemeinschaft (Ba 689/1)