Massimiliano Cerletti

Restoring Systemic GDF11 Levels Reverses Age-Related Dysfunction in Mouse Skeletal Muscle

Help the Aged Muscle function declines with age, as does neurogenesis in certain brain regions. Two teams analyzed the effects of heterochronic parabiosis in mice. Sinha et al. (p. 649) found that when an aged mouse shares a circulatory system with a youthful mouse, the aged mouse sees improved muscle function, and Katsimpardi et al. (p. 630) observed increased generation of olfactory neurons. In both cases, Growth Differentiation Factor 11 appeared to be one of the key components of the young blood. A circulating growth factor promotes youthful muscles and brains in aged mice. Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral “rejuvenating” factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.

A Special Population of Regulatory T Cells Potentiates Muscle Repair

Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.

Highly Efficient, Functional Engraftment of Skeletal Muscle Stem Cells in Dystrophic Muscles

Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.

Identification of inducible brown adipocyte progenitors residing in skeletal muscle and white fat

Brown fat is specialized for energy expenditure and has therefore been proposed to function as a defense against obesity. Despite recent advances in delineating the transcriptional regulation of brown adipocyte differentiation, cellular lineage specification and developmental cues specifying brown-fat cell fate remain poorly understood. In this study, we identify and isolate a subpopulation of adipogenic progenitors (Sca-1+/CD45−/Mac1−; referred to as Sca-1+ progenitor cells, ScaPCs) residing in murine brown fat, white fat, and skeletal muscle. ScaPCs derived from different tissues possess unique molecular expression signatures and adipogenic capacities. Importantly, although the ScaPCs from interscapular brown adipose tissue (BAT) are constitutively committed brown-fat progenitors, Sca-1+ cells from skeletal muscle and subcutaneous white fat are highly inducible to differentiate into brown-like adipocytes upon stimulation with bone morphogenetic protein 7 (BMP7). Consistent with these findings, human preadipocytes isolated from subcutaneous white fat also exhibit the greatest inducible capacity to become brown adipocytes compared with cells isolated from mesenteric or omental white fat. When muscle-resident ScaPCs are re-engrafted into skeletal muscle of syngeneic mice, BMP7-treated ScaPCs efficiently develop into adipose tissue with brown fat-specific characteristics. Importantly, ScaPCs from obesity-resistant mice exhibit markedly higher thermogenic capacity compared with cells isolated from obesity-prone mice. These data establish the molecular characteristics of tissue-resident adipose progenitors and demonstrate a dynamic interplay between these progenitors and inductive signals that act in concert to specify brown adipocyte development.

Notch signaling in the regulation of stem cell self-renewal and differentiation.

Stem cells are rare and unique precursor cells that participate in the building and rebuilding of tissues and organs during embryogenesis, postnatal growth, and injury repair. Stem cells are distinctively endowed with the ability to both self-renew and differentiate, such that they can replenish the stem cell pool while continuing to produce the differentiated daughter cells that are essential for tissue function. Stem cell self-renewal/differentiation decisions must be carefully controlled during organogenesis, tissue homeostasis, and regeneration, as failure in stem cell maintenance or activation can lead to progressive tissue wasting, while unchecked self-renewal is a hallmark of many cancers. Here, we review evidence implicating the Notch signaling pathway, an evolutionarily conserved cell fate determinant with widespread roles in a variety of tissues and organisms, as a crucial regulator of stem cell behavior. As discussed below, this pathway plays varied and critical roles at multiple stages of organismal development, in lineage-specific differentiation of pluripotent embryonic stem cells, and in controlling stem cell numbers and activity in the context of age-related tissue degeneration, injury-induced tissue repair, and malignancy.

Short-term calorie restriction enhances skeletal muscle stem cell function.

Calorie restriction (CR) extends life span and ameliorates age-related pathologies in most species studied, yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle after transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.

Dystrophic phenotype of canine X-linked muscular dystrophy is mitigated by adenovirus-mediated utrophin gene transfer.

Utrophin is highly homologous and structurally similar to dystrophin, and in gene delivery experiments in mdx mice was able to functionally replace dystrophin. We performed mini-utrophin gene transfer in Golden Retriever dogs with canine muscular dystrophy (CXMD). Unlike the mouse model, the clinicopathological phenotype of CXMD is similar to that of Duchenne muscular dystrophy (DMD). We injected an adenoviral vector expressing a synthetic utrophin into tibialis anterior muscles of newborn dogs affected with CXMD and examined transgene expression by RNA and protein analysis at 10, 30 and 60 days postinjection in cyclosporin-treated and -untreated animals. Immunosuppression by cyclosporin was required to mitigate the immune response to viral and transgene antigens. RT-PCR analysis showed the presence of the exogenous transcript in the muscle of cyclosporin-treated and -untreated animals. The transgenic utrophin was efficiently expressed at the extrajunctional membrane in immunosuppressed dogs and this expression was stable for at least 60 days. We found reduced fibrosis and increased expression of dystrophin-associated proteins (DAPs) in association with muscle areas expressing the utrophin minigene, indicating that mini-utrophin can functionally compensate for lack of dystrophin in injected muscles. For this reason, utrophin transfer to dystrophin-deficient muscle appears as a promising therapeutic approach to DMD.

Skeletal Muscle Stem Cells: Effects of Aging and Metabolism on Muscle Regenerative Function

Homeostatic and regenerative replacement of skeletal muscle fibers requires the activity of a dedicated pool of myogenic stem cells, called satellite cells, that are activated by muscle injury and act as a renewable source of muscle-forming cells throughout adult life. Satellite cell function is controlled by both intrinsic and extrinsic regulatory cues, whose integration determines the success of muscle regenerative responses. Pathological deregulation of satellite cell function through perturbation of these signaling pathways appears to play an important role in age-dependent deterioration of muscle function and in muscle dystrophic disease. The regenerative activity of skeletal muscle also appears to be tightly linked to metabolism, and alterations in metabolic state can directly influence the activity of these tissue-specific stem cells. Here, we review recent and emerging insights into the molecular and biochemical signals that control satellite cell function and discuss these in the context of muscle degenerative diseases such as dystrophy and sarcopenia. Novel discoveries from this ongoing work bring new opportunities to enhance or restore muscle repair and are likely to facilitate satellite cell transplantation in clinical applications.

Early life nutrition modulates muscle stem cell number: implications for muscle mass and repair.

Suboptimal nutrition during prenatal and early postnatal development is associated with increased risk for type 2 diabetes during adult life. A hallmark of such diabetes risk is altered body composition, including reduced lean mass and increased adiposity. Since stem cell number and activity are important determinants of muscle mass, modulation of perinatal nutrition could alter stem cell number/function, potentially mediating developmentally programmed reductions in muscle mass. Skeletal muscle precursors (SMP) were purified from muscle of mice subjected to prenatal undernutrition and/or early postnatal high-fat diet (HFD)--experimental models that are both associated with obesity and diabetes risk. SMP number was determined by flow cytometry, proliferative capacity measured in vitro, and regenerative capacity of these cells determined in vivo after muscle freeze injury. Prenatally undernutrition (UN) mice showed significantly reduced SMP frequencies [Control (C) 4.8% ± 0.3% (% live cells) vs. UN 3.2% ± 0.4%, P=0.015] at 6 weeks; proliferative capacity was unaltered. Reduced SMP in UN was associated with 32% decrease in regeneration after injury (C 16% ± 3% of injured area vs. UN 11% ± 2%; P<0.0001). SMP frequency was also reduced in HFD-fed mice (chow 6.4% ± 0.6% vs. HFD 4.7% ± 0.4%, P=0.03), and associated with 44% decreased regeneration (chow 16% ± 2.7% vs. HFD 9% ± 2.2%; P<0.0001). Prenatal undernutrition was additive with postnatal HFD. Thus, both prenatal undernutrition and postnatal overnutrition reduce myogenic stem cell frequency and function, indicating that developmentally established differences in muscle-resident stem cell populations may provoke reductions in muscle mass and repair and contribute to diabetes risk.

Cell Invasion Is Affected by Differential Expression of the Urokinase Plasminogen Activator/Urokinase Plasminogen Activator Receptor System in Muscle Satellite Cells from Normal and Dystrophic Patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti–u-PA and anti–u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA–induced invasion, as well as in u-PA–induced proliferation.