Gabi Shefer

Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway

We show that muscle satellite cells, traditionally considered as committed myogenic precursors, are comprised of Pax7-expressing progenitors that preserve a mesenchymal repertoire extending beyond a mere myogenic potential. Mouse satellite cells from freshly isolated single myofibers, cultured individually in serum-rich growth medium, produced myogenic and non-myogenic clones. Only the myogenic clones expressed muscle-specific transcription factors and formed myotubes. Pax7 was initially expressed in all clones, but subsequently was associated only with the myogenic clones. Some cells in the non-myogenic clones expressed α-smooth muscle actin and nestin whereas others differentiated into mature adipocytes. This type of cell composition mirrors characteristics of mesenchymal stem cell progeny. Overall, individual myofibers persistently gave rise to both clonal phenotypes, but the ratio of myogenic to non-myogenic clones randomly varied among fibers. This randomness indicates that clonal dichotomy reflects satellite cell suppleness rather than pre-fated cell heterogeneity. We conclude that satellite cells possess mesenchymal plasticity, being able to commit either to myogenesis or to a mesenchymal alternative differentiation (MAD) program.

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 betagal. 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.

Isolation and Culture of Skeletal Muscle Myofibers as a Means to Analyze Satellite Cells

Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing, and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.

Reduced Satellite Cell Numbers and Myogenic Capacity in Aging Can Be Alleviated by Endurance Exercise

Background Muscle regeneration depends on satellite cells, myogenic stem cells that reside on the myofiber surface. Reduced numbers and/or decreased myogenic aptitude of these cells may impede proper maintenance and contribute to the age-associated decline in muscle mass and repair capacity. Endurance exercise was shown to improve muscle performance; however, the direct impact on satellite cells in aging was not yet thoroughly determined. Here, we focused on characterizing the effect of moderate-intensity endurance exercise on satellite cell, as possible means to attenuate adverse effects of aging. Young and old rats of both genders underwent 13 weeks of treadmill-running or remained sedentary. Methodology Gastrocnemius muscles were assessed for the effect of age, gender and exercise on satellite-cell numbers and myogenic capacity. Satellite cells were identified in freshly isolated myofibers based on Pax7 immunostaining (i.e., ex-vivo). The capacity of individual myofiber-associated cells to produce myogenic progeny was determined in clonal assays (in-vitro). We show an age-associated decrease in satellite-cell numbers and in the percent of myogenic clones in old sedentary rats. Upon exercise, there was an increase in myofibers that contain higher numbers of satellite cells in both young and old rats, and an increase in the percent of myogenic clones derived from old rats. Changes at the satellite cell level in old rats were accompanied with positive effects on the lean-to-fat Gast muscle composition and on spontaneous locomotion levels. The significance of these data is that they suggest that the endurance exercise-mediated boost in both satellite numbers and myogenic properties may improve myofiber maintenance in aging.

Reconstruction of Cell Lineage Trees in Mice

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.

Angiotensin 1-7 as Means to Prevent the Metabolic Syndrome: Lessons From the Fructose-Fed Rat Model

We studied the effects of chronic angiotensin 1-7 (Ang 1-7) treatment in an experimental model of the metabolic syndrome, i.e., rats given high-fructose/low-magnesium diet (HFrD). Rats were fed on HFrD for 24 weeks with and without Ang 1-7 (576 µg/kg/day, s.c., Alzet pumps). After 6 months, Ang 1-7–treated animals had lower body weight (−9.5%), total fat mass (detected by magnetic resonance imaging), and serum triglycerides (−51%), improved glucose tolerance, and better insulin sensitivity. Similar metabolic effects were also evident, albeit in the absence of weight loss, in rats first exposed to HFrD for 5 months and then subjected to short-term (4 weeks) treatment with Ang 1-7. Six months of Ang 1-7 treatment were associated with lower plasma renin activity (−40%) and serum aldosterone (−48%), less hepatosteatatitis, and a reduction in epididymal adipocyte volume. The marked attenuation of macrophage infiltration in white adipose tissue (WAT) was associated with reduced levels of the pP65 protein in the epididymal fat tissue, suggesting less activation of the nuclear factor-κB (NFκB) pathway in Ang 1-7–treated rats. WAT from Ang 1-7–treated rats showed reduced NADPH-stimulated superoxide production. In single muscle fibers (myofibers) harvested and grown ex vivo for 10 days, myofibers from HFrD rats gave rise to 20% less myogenic cells than the Ang 1-7–treated rats. Fully developed adipocytes were present in most HFrD myofiber cultures but entirely absent in cultures from Ang 1-7–treated rats. In summary, Ang 1-7 had an ameliorating effect on insulin resistance, hypertriglyceridemia, fatty liver, obesity, adipositis, and myogenic and adipogenic differentiation in muscle tissue in the HFrD rats.

Muscle function and fat content in relation to sarcopenia, obesity and frailty of old age--An overview.

BACKGROUND AND AIM In western countries, the proportion of people over age 60 is increasing faster than any other group. This is linked to higher rates of obesity. Older age, co-morbidities and obesity are all associated with frailty syndrome. In the core of both frailty and sarcopenia there are dysfunction and deterioration of the muscle and the fat tissues. This overview interlinks the phenotypes presented in older adults such as sarcopenia and frailty-alone and with relation to obesity, muscle function and fat tissue accumulation. RECENT FINDINGS Observational studies have well described the loss of muscle mass and strength through the years of adult life, both components of frailty and sarcopenia. They have shown that these changes are associated with dysmetabolism and functional deterioration, independent of common explanatory variables. In the metabolic mechanism core of this link, insulin resistance and higher ectopic fat accumulation may play a role. Basic experiments have partially validated this hypothesis. Whether there is a synergistic effect of obesity and frailty phenotype on morbidity risk is still questionable and currently under investigation; however, few cohort studies have shown that the frail-obese or sarcopenic-obese group have higher probability for metabolic complications. SUMMARY Muscle mass loss and fat accumulation in the muscle in the elderly, with or without the presence of obesity, may explain some of the functional and metabolic defects shown in the frail, sarcopenic population.

Adipose tissue renin–angiotensin–aldosterone system (RAAS) and progression of insulin resistance

This review focuses on the expression of the key components of the renin-angiotensin-aldosterone axis in fat tissue. At the center of this report is the role of RAAS in normal and excessive fat mass enlargement, the leading etiology of insulin resistance. Understanding the expression and regulation of RAAS components in various fat depots allows insight not only into the processes by which these complex patterns are modified by the enlargement of adipose tissue, but also into their impact on local and systemic response to insulin.

Estimating cell depth from somatic mutations.

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.

Moderate-intensity treadmill running promotes expansion of the satellite cell pool in young and old mice

Satellite cells, the myogenic progenitors located at the myofibre surface, are essential for the repair of adult skeletal muscle. There is ample evidence for an age‐linked decline in the number of satellite cells and performance in limb muscles. Hence, an effective means of activating and expanding the satellite cell pool may enhance muscle maintenance and reduce the impact of age‐associated muscle deterioration (sarcopaenia). Accordingly, in the present study, we explored the beneficial effects of endurance exercise on satellite cells in young and old mice. Animals were subjected to an 8‐week moderate‐intensity treadmill‐running approach that does not inflict apparent muscle damage (0° inclination, 11.5 m·min−1 for 30 min·day−1, 6 days·week−1). Myofibres of extensor digitorum longus muscles were then isolated from exercised and sedentary mice and used for monitoring the number of satellite cells, as well as for harvesting individual satellite cells for clonal growth assays. We specifically focused on satellite cell pools of single myofibres, with the view that daily wear of muscles probably affects individual myofibres rather than causing overall muscle damage. We found an expansion of the satellite cell pool in the exercised groups compared to the sedentary groups, with the same increase (~ 1.6‐fold) in both ages. The results of the present study are in agreement with our findings obtained using rat gastrocnemius, indicating the consistent effect of exercise on satellite cell expansion in limb muscles. The experimental paradigm established in the present study is useful for investigating satellite cell dynamics at the myofibre niche, as well as for broader investigations of the impact of physiologically and pathologically relevant factors on adult myogenesis.