Pierre Rocheteau

A Critical Requirement for Notch Signaling in Maintenance of the Quiescent Skeletal Muscle Stem Cell State

Notch signaling plays a key role in virtually all tissues and organs in metazoans; however, limited examples are available for the regulatory role of this pathway in adult quiescent stem cells. We performed a temporal and ontological assessment of effectors of the Notch pathway that indicated highest activity in freshly isolated satellite cells and, unexpectedly, a sharp decline before the first mitosis, and subsequently in proliferating, satellite cell‐derived myoblasts. Using genetic tools to conditionally abrogate canonical Notch signaling during homeostasis, we demonstrate that satellite cells differentiate spontaneously and contribute to myofibers, thereby resulting in a severe depletion of the stem cell pool. Furthermore, whereas loss of Rbpj function provokes some satellite cells to proliferate before fusing, strikingly, the majority of mutant cells terminally differentiate unusually from the quiescent state, without passing through S‐phase. This study establishes Notch signaling pathway as the first regulator of cellular quiescence in adult muscle stem cells. STEM CELLS 2012; 30:243–252.

Skeletal muscle stem cells adopt a dormant cell state post mortem and retain regenerative capacity

The accessibility to stem cells from healthy or diseased individuals, and the maintenance of their potency are challenging issues for stem cell biology. Here we report the isolation of viable and functional skeletal myogenic cells from humans up to 17 days, and mice up to 14 days post mortem, much longer beyond previous reports. Muscle stem cells are enriched in post mortem tissue, suggesting a selective survival advantage compared with other cell types. Transplantation of mouse muscle and haematopoietic stem cells regenerates tissues robustly. Cellular quiescence contributes to this cell viability where cells adopt a reversible dormant state characterized by reduced metabolic activity, a prolonged lag phase before the first cell division, elevated levels of reactive oxygen species and a transcriptional status less primed for commitment. Finally, severe hypoxia, or anoxia is critical for maintaining stem cell viability and regenerative capacity. Thus, these cells provide a useful resource for studying stem cell biology.

Comparative Study of Injury Models for Studying Muscle Regeneration in Mice

Background A longstanding goal in regenerative medicine is to reconstitute functional tissus 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. Methods 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®. Results 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. Conclusions 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.

Sepsis induces long-term metabolic and mitochondrial muscle stem cell dysfunction amenable by mesenchymal stem cell therapy

Sepsis, or systemic inflammatory response syndrome, is the major cause of critical illness resulting in admission to intensive care units. Sepsis is caused by severe infection and is associated with mortality in 60% of cases. Morbidity due to sepsis is complicated by neuromyopathy, and patients face long-term disability due to muscle weakness, energetic dysfunction, proteolysis and muscle wasting. These processes are triggered by pro-inflammatory cytokines and metabolic imbalances and are aggravated by malnutrition and drugs. Skeletal muscle regeneration depends on stem (satellite) cells. Herein we show that mitochondrial and metabolic alterations underlie the sepsis-induced long-term impairment of satellite cells and lead to inefficient muscle regeneration. Engrafting mesenchymal stem cells improves the septic status by decreasing cytokine levels, restoring mitochondrial and metabolic function in satellite cells, and improving muscle strength. These findings indicate that sepsis affects quiescent muscle stem cells and that mesenchymal stem cells might act as a preventive therapeutic approach for sepsis-related morbidity.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

Dormancy and Quiescence of Skeletal Muscle Stem Cells

The skeletal muscle of vertebrates has a huge regenerative capacity. When destroyed after different types of injury, this organ can regenerate very quickly (less than 20 days following myotoxin injection in the mouse) ad integrum and repeatedly. The cell responsible for this regeneration is the so-called satellite cell, the muscle stem cell that lies on top of the muscle fibre, a giant, multinucleated cell that contains the contractile material. When injected in the muscle, satellite cells can efficiently differentiate into contractile muscle fibres. The satellite cell shows great therapeutic potential; and its regenerative capacity has triggered particular interest in the field of muscular degeneration. In this review we will focus on one particular property of the satellite cell: its quiescence and dormancy. Indeed adult satellite cells are quiescent; they lie between the basal lamina and the basement membrane of the muscle fibre, ready to proliferate, and fuse in order to regenerate myofibers upon injury. It has recently been shown that a subpopulation of satellite cells is able to enter dormancy in human and mice cadavers. Dormancy is defined by a low metabolic state, low mobility, and a long lag before division when plated in vitro, compared to quiescent cells. This definition is also based on current knowledge about long-term hematopoietic stem cells, a subpopulation of stem cells that are described as dormant based on the same criteria (rare division and low metabolism when compared to progeny which are dividing more often). In the first part of this review, we will provide a description of satellite cells which addresses their quiescent state. We will then focus on the uneven distribution of satellite cells in the muscle and describe evidence that suggests that their dormancy differs from one muscle to the next and that one should be cautious when making generalisations regarding this cellular state. In a second part, we will discuss the transition between active dividing cells in developing animals to quiescence. This mechanism could be used or amplified in the switch from quiescence to dormancy. In a third part, we will review the signals and dynamics that actively maintain the satellite cell quiescent. The in-depth understanding of these mechanisms is key to describing how dormancy relies on quiescent state of the cells. In a fourth part, we will deal with dormancy per se: how dormant satellite cells can be obtained, their characteristics, their metabolic profile, and their molecular signature as compared to quiescent cells. Here, we will highlight one of the most important recent findings: that quiescence is a prerequisite for the entry of the satellite cell into dormancy. Since dormancy is a newly discovered phenomenon, we will review the mechanisms responsible for quiescence and activation, as these two cellular states are better known and key to understanding satellite cell dormancy. This will allow us to describe dormancy and its prerequisites.

A novel paradigm links mitochondrial dysfunction with muscle stem cell impairment in sepsis

Sepsis is an acute systemic inflammatory response of the body to microbial infection and a life threatening condition associated with multiple organ failure. Survivors may display long-term disability with muscle weakness that remains poorly understood. Recent data suggest that long-term myopathy in sepsis survivors is due to failure of skeletal muscle stem cells (satellite cells) to regenerate the muscle. Satellite cells impairment in the acute phase of sepsis is linked to unusual mitochondrial dysfunctions, characterized by a dramatic reduction of the mitochondrial mass and hyperactivity of residual organelles. Survivors maintain the impairment of satellite cells, including alterations of the mitochondrial DNA (mtDNA), in the long-term. This condition can be rescued by treatment with mesenchymal stem cells (MSCs) that restore mtDNA alterations and mitochondrial function in satellite cells, and in fine their regenerative potential. Injection of MSCs in turn increases the force of isolated muscle fibers and of the whole animal, and improves the survival rate. These effects occur in the context of reduced inflammation markers that also raised during sepsis. Targeting muscle stem cells mitochondria, in a context of reduced inflammation, may represent a valuable strategy to reduce morbidity and long-term impairment of the muscle upon sepsis.

Muscle regeneration after sepsis

Severe critical illness is often complicated by intensive care unit-acquired weakness (ICU-AW), which is associated with increased ICU and post-ICU mortality, delayed weaning from mechanical ventilation and long-term functional disability. Several mechanisms have been implicated in the pathophysiology of ICU-AW, but muscle regeneration has not been investigated to any extent in this context, even though its involvement is suggested by the protracted functional consequences of ICU-AW. Recent data suggest that muscle regeneration could be impaired after sepsis, and that mesenchymal stem cell treatment could improve the post-injury muscle recovery.

16-P029 Skeletal muscle cell fate decisions and the role of Numb

cilia. At E9.5, B9D1 is expressed throughout the spinal cord, limb bubs, and, bronchial arches; tissues that required Shh signal for proper development. To further understand its molecular mechanism, we sought out other interacting proteins using tandem IP followed by mass spec analysis. A protein complex is identified and one of them was a transmembrane protein BCAP1 (B9 Complex Associated Protein 1). KO mice for BCAP1 was made and analyzed. BCAP1 KO mice were embryonic lethal at E15.5. Analysis of the BCAP1 KO embryos showed that the phenotypes are consistent with defect in Shh signaling such as polydactyly and microopthalmia. Further analysis is carried out to determine the exact role of this protein complex in Shh signaling.