Morgan E. Carlson

Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells

Adult skeletal muscle robustly regenerates throughout an organism’s life, but as the muscle ages, its ability to repair diminishes and eventually fails. Previous work suggests that the regenerative potential of muscle stem cells (satellite cells) is not triggered in the old muscle because of a decline in Notch activation, and that it can be rejuvenated by forced local activation of Notch. Here we report that, in addition to the loss of Notch activation, old muscle produces excessive transforming growth factor (TGF)-β (but not myostatin), which induces unusually high levels of TGF-β pSmad3 in resident satellite cells and interferes with their regenerative capacity. Importantly, endogenous Notch and pSmad3 antagonize each other in the control of satellite-cell proliferation, such that activation of Notch blocks the TGF-β-dependent upregulation of the cyclin-dependent kinase (CDK) inhibitors p15, p16, p21 and p27, whereas inhibition of Notch induces them. Furthermore, in muscle stem cells, Notch activity determines the binding of pSmad3 to the promoters of these negative regulators of cell-cycle progression. Attenuation of TGF-β/pSmad3 in old, injured muscle restores regeneration to satellite cells in vivo. Thus a balance between endogenous pSmad3 and active Notch controls the regenerative competence of muscle stem cells, and deregulation of this balance in the old muscle microniche interferes with regeneration.

Loss of stem cell regenerative capacity within aged niches.

This work uncovers novel mechanisms of aging within stem cell niches that are evolutionarily conserved between mice and humans and affect both embryonic and adult stem cells. Specifically, we have examined the effects of aged muscle and systemic niches on key molecular identifiers of regenerative potential of human embryonic stem cells (hESCs) and post‐natal muscle stem cells (satellite cells). Our results reveal that aged differentiated niches dominantly inhibit the expression of Oct4 in hESCs and Myf‐5 in activated satellite cells, and reduce proliferation and myogenic differentiation of both embryonic and tissue‐specific adult stem cells (ASCs). Therefore, despite their general neoorganogenesis potential, the ability of hESCs, and the more differentiated myogenic ASCs to contribute to tissue repair in the old will be greatly restricted due to the conserved inhibitory influence of aged differentiated niches. Significantly, this work establishes that hESC‐derived factors enhance the regenerative potential of both young and, importantly, aged muscle stem cells in vitro and in vivo; thus, suggesting that the regenerative outcome of stem cell‐based replacement therapies will be determined by a balance between negative influences of aged tissues on transplanted cells and positive effects of embryonic cells on the endogenous regenerative capacity. Comprehensively, this work points toward novel venues for in situ restoration of tissue repair in the old and identifies critical determinants of successful cell‐replacement therapies for aged degenerating organs.

Relative roles of TGF‐β1 and Wnt in the systemic regulation and aging of satellite cell responses

Muscle stem (satellite) cells are relatively resistant to cell‐autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P‐Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor‐beta (TGF‐β) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age‐dependent myogenic activity of sera TGF‐β1, and its potential cross‐talk with systemic Wnt. We found that sera TGF‐β1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF‐β1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF‐β1 were inhibitory and young sera suppressed myogenesis if TGF‐β1 was activated. Our data suggest that platelet‐derived sera TGF‐β1 levels, or endocrine TGF‐β1 levels, do not explain the age‐dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF‐β neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF‐β1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF‐β receptor kinase inhibitor, which attenuated TGF‐β signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF‐β1‐dependent block on muscle regeneration, identify physiological modalities of age‐imposed changes in TGF‐β1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.

Molecular aging and rejuvenation of human muscle stem cells

Very little remains known about the regulation of human organ stem cells (in general, and during the aging process), and most previous data were collected in short‐lived rodents. We examined whether stem cell aging in rodents could be extrapolated to genetically and environmentally variable humans. Our findings establish key evolutionarily conserved mechanisms of human stem cell aging. We find that satellite cells are maintained in aged human skeletal muscle, but fail to activate in response to muscle attrition, due to diminished activation of Notch compounded by elevated transforming growth factor beta (TGF‐β)/phospho Smad3 (pSmad3). Furthermore, this work reveals that mitogen‐activated protein kinase (MAPK)/phosphate extracellular signal‐regulated kinase (pERK) signalling declines in human muscle with age, and is important for activating Notch in human muscle stem cells. This molecular understanding, combined with data that human satellite cells remain intrinsically young, introduced novel therapeutic targets. Indeed, activation of MAPK/Notch restored ‘youthful’ myogenic responses to satellite cells from 70‐year‐old humans, rendering them similar to cells from 20‐year‐old humans. These findings strongly suggest that aging of human muscle maintenance and repair can be reversed by ‘youthful’ calibration of specific molecular pathways.

Aging of signal transduction pathways, and pathology

The major cell signaling pathways, and their specific mechanisms of transduction, have been a subject of investigation for many years. As our understanding of these pathways advances, we find that they are evolutionarily well-conserved not only individually, but also at the level of their crosstalk and signal integration. Productive interactions within the key signal transduction networks determine success in embryonic organogenesis, and postnatal tissue repair throughout adulthood. However, aside from clues revealed through examining age-related degenerative diseases, much remains uncertain about imbalances within these pathways during normal aging. Further, little is known about the molecular mechanisms by which alterations in the major cell signal transduction networks cause age-related pathologies. The aim of this review is to describe the complex interplay between the Notch, TGFbeta, WNT, RTK-Ras and Hh signaling pathways, with a specific focus on the changes introduced within these networks by the aging process, and those typical of age-associated human pathologies.

Differentiation rather than aging of muscle stem cells abolishes their telomerase activity.

A general feature of stem cells is the ability to routinely proliferate to build, maintain, and repair organ systems. Accordingly, embryonic and germline, as well as some adult stem cells, produce the telomerase enzyme at various levels of expression. Our results show that, while muscle is a largely postmitotic tissue, the muscle stem cells (satellite cells) that maintain this biological system throughout adult life do indeed display robust telomerase activity. Conversely, primary myoblasts (the immediate progeny of satellite cells) quickly and dramatically downregulate telomerase activity. This work thus suggests that satellite cells, and early transient myoblasts, may be more promising therapeutic candidates for regenerative medicine than traditionally utilized myoblast cultures. Muscle atrophy accompanies human aging, and satellite cells endogenous to aged muscle can be triggered to regenerate old tissue by exogenous molecular cues. Therefore, we also examined whether these aged muscle stem cells would produce tissue that is “young” with respect to telomere maintenance. Interestingly, this work shows that the telomerase activity in muscle stem cells is largely retained into old age wintin inbred “long” telomere mice and in wild‐derived short telomere mouse strains, and that age‐specific telomere shortening is undetectable in the old differentiated muscle fibers of either strain. Summarily, this work establishes that young and old muscle stem cells, but not necessarily their progeny, myoblasts, are likely to produce tissue with normal telomere maintenance when used in molecular and regenerative medicine approaches for tissue repair.

Corrigendum: Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells.

This corrects the article DOI: 10.1038/nature07034