A longstanding goal in regenerative medicine is to reconstitute functional tissues or organs after injury or disease. Attention has focused on the identification and relative contribution of tissue ...specific stem cells to the regeneration process. Relatively little is known about how the physiological process is regulated by other tissue constituents. Numerous injury models are used to investigate tissue regeneration, however, these models are often poorly understood. Specifically, for skeletal muscle regeneration several models are reported in the literature, yet the relative impact on muscle physiology and the distinct cells types have not been extensively characterised.
We have used transgenic Tg:Pax7nGFP and Flk1GFP/+ mouse models to respectively count the number of muscle stem (satellite) cells (SC) and number/shape of vessels by confocal microscopy. We performed histological and immunostainings to assess the differences in the key regeneration steps. Infiltration of immune cells, chemokines and cytokines production was assessed in vivo by Luminex®.
We compared the 4 most commonly used injury models i.e. freeze injury (FI), barium chloride (BaCl2), notexin (NTX) and cardiotoxin (CTX). The FI was the most damaging. In this model, up to 96% of the SCs are destroyed with their surrounding environment (basal lamina and vasculature) leaving a "dead zone" devoid of viable cells. The regeneration process itself is fulfilled in all 4 models with virtually no fibrosis 28 days post-injury, except in the FI model. Inflammatory cells return to basal levels in the CTX, BaCl2 but still significantly high 1-month post-injury in the FI and NTX models. Interestingly the number of SC returned to normal only in the FI, 1-month post-injury, with SCs that are still cycling up to 3-months after the induction of the injury in the other models.
Our studies show that the nature of the injury model should be chosen carefully depending on the experimental design and desired outcome. Although in all models the muscle regenerates completely, the trajectories of the regenerative process vary considerably. Furthermore, we show that histological parameters are not wholly sufficient to declare that regeneration is complete as molecular alterations (e.g. cycling SCs, cytokines) could have a major persistent impact.
Satellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFPHi cells ...are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFPLo cells. Pax7-nGFPHi can give rise to Pax7-nGFPLo cells after serial transplantations. Proliferating Pax7-nGFPHi cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFPLo cells perform random DNA segregation. Therefore, quiescent Pax7-nGFPHi cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFPHi cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically.
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► Pax7Hi quiescent stem cells constitute a metabolically low “dormant” subpopulation ► Both Pax7Hi and Pax7Lo quiescent stem cells are serially transplantable ► Only proliferating Pax7Hi cells segregate old DNA strands to the renewing stem cell ► CO-FISH demonstrates that template DNA strand cosegregation engages all chromosomes
Asymmetric DNA segregation occurs during cell division in a subpopulation of adult muscle stem cells expressing high levels of Pax7. Daughter cells retaining template DNA strands express stem cell markers.
Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. ...However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.
Satellite cells assure postnatal skeletal muscle growth and repair. Despite extensive studies, their stem cell character remains largely undefined. Using pulse-chase labelling with BrdU to mark the ...putative stem cell niche, we identify a subpopulation of label-retaining satellite cells during growth and after injury. Strikingly, some of these cells display selective template-DNA strand segregation during mitosis in the muscle fibre in vivo, as well as in culture independent of their niche, indicating that genomic DNA strands are nonequivalent. Furthermore, we demonstrate that the asymmetric cell-fate determinant Numb segregates selectively to one daughter cell during mitosis and before differentiation, suggesting that Numb is associated with self-renewal. Finally, we show that template DNA cosegregates with Numb in label-retaining cells that express the self-renewal marker Pax7. The cosegregation of 'immortal' template DNA strands and their link with the asymmetry apparatus has important implications for stem cell biology and cancer.
During embryonic development, skeletal muscles arise from somites, which derive from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation ...of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We show that primary and secondary skeletal myogenesis can be recapitulated in vitro from the PSM-like cells, providing an efficient, serum-free protocol for the generation of striated, contractile fibers from mouse and human pluripotent cells. The mouse ES cells also differentiate into Pax7(+) cells with satellite cell characteristics, including the ability to form dystrophin(+) fibers when grafted into muscles of dystrophin-deficient mdx mice, a model of Duchenne muscular dystrophy (DMD). Fibers derived from ES cells of mdx mice exhibit an abnormal branched phenotype resembling that described in vivo, thus providing an attractive model to study the origin of the pathological defects associated with DMD.
The growth and repair of skeletal muscle after birth depends on satellite cells that are characterized by the expression of Pax7. We show that Pax3, the paralogue of Pax7, is also present in both ...quiescent and activated satellite cells in many skeletal muscles. Dominant-negative forms of both Pax3 and -7 repress MyoD, but do not interfere with the expression of the other myogenic determination factor, Myf5, which, together with Pax3/7, regulates the myogenic differentiation of these cells. In Pax7 mutants, satellite cells are progressively lost in both Pax3-expressing and -nonexpressing muscles. We show that this is caused by satellite cell death, with effects on the cell cycle. Manipulation of the dominant-negative forms of these factors in satellite cell cultures demonstrates that Pax3 cannot replace the antiapoptotic function of Pax7. These findings underline the importance of cell survival in controlling the stem cell populations of adult tissues and demonstrate a role for upstream factors in this context.
Genetic regulatory networks governing skeletal myogenesis in the body are well understood, yet their hierarchical relationships in the head remain unresolved. We show that either
Myf5 or
Mrf4 is ...necessary for initiating extraocular myogenesis. Whereas
Mrf4 is dispensable for pharyngeal muscle progenitor fate,
Tbx1 and
Myf5 act synergistically for governing myogenesis in this location. As in the body,
Myod acts epistatically to the initiating cascades in the head. Thus, complementary pathways, governed by
Pax3 for body, and
Tbx1 for pharyngeal muscles, but absent for extraocular muscles, activate the core myogenic network. These diverse muscle progenitors maintain their respective embryonic regulatory signatures in the adult. However, these signatures are not sufficient to ensure the specific muscle phenotypes, since the expected differentiated phenotype is not manifested when satellite cells are engrafted heterotopically. These findings identify novel genetic networks that may provide insights into myopathies which often affect only subsets of muscles.
Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel ...population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.
Multiple cell types arise from cells in the dermomyotome of the somite that express Pax3 and Pax7, and myogenesis is regulated by Notch signaling. The asymmetric cell fate determinant Numb is thought ...to promote differentiation of skeletal muscle and other lineages by negatively regulating Notch signaling. We used transgenesis to overexpress Numb spatiotemporally in Pax3(+)/Pax7(+) somitic stem and progenitor cells in mouse embryos using a spatiotemporally regulated enhancer element from the Myf5 locus that can target muscle progenitor cells prior to cell commitment. Molecular analyses as well as examination of dermal and skeletal muscle cell fates in vivo show that although Numb is thought to be associated with muscle differentiation, unexpectedly the common stem/progenitor pool size for these lineages is increased in Numb-transgenic embryos. Prospective isolation of the relevant transgenic cells and analysis by quantitative reverse-transcription polymerase chain reaction demonstrated that, in this context, canonical Notch targets are not significantly downregulated. These findings were corroborated using a Notch reporter mouse during the formation of somites and prior to lineage segregation. Thus, we propose that Numb can regulate the self-renewal of dermal and muscle progenitors during a lineage progression.
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