Irina M. Conboy

Rejuvenation of aged progenitor cells by exposure to a young systemic environment

The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-α and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-α complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.

The Regulation of Notch Signaling Controls Satellite Cell Activation and Cell Fate Determination in Postnatal Myogenesis

We have studied the role of Notch-1 and its antagonist Numb in the activation of satellite cells during postnatal myogenesis. Activation of Notch-1 promoted the proliferation of myogenic precursor cells expressing the premyoblast marker Pax3. Attenuation of Notch signaling by increases in Numb expression led to the commitment of progenitor cells to the myoblast cell fate and the expression of myogenic regulatory factors, desmin, and Pax7. In many intermediate progenitor cells, Numb was localized asymmetrically in actively dividing cells, suggesting an asymmetric cell division and divergent cell fates of daughter cells. The results indicate that satellite cell activation results in a heterogeneous population of precursor cells with respect to Notch-1 activity and that the balance between Notch-1 and Numb controls cellular homeostasis and cell fate determination.

A Temporal Switch from Notch to Wnt Signaling in Muscle Stem Cells Is Necessary for Normal Adult Myogenesis

The temporal switch from progenitor cell proliferation to differentiation is essential for effective adult tissue repair. We previously reported the critical role of Notch signaling in the proliferative expansion of myogenic progenitors in mammalian postnatal myogenesis. We now show that the onset of differentiation is due to a transition from Notch signaling to Wnt signaling in myogenic progenitors and is associated with an increased expression of Wnt in the tissue and an increased responsiveness of progenitors to Wnt. Crosstalk between these two pathways occurs via GSK3beta, which is maintained in an active form by Notch but is inhibited by Wnt in the canonical Wnt signaling cascade. These results demonstrate that the temporal balance between Notch and Wnt signaling orchestrates the precise progression of muscle precursor cells along the myogenic lineage pathway, through stages of proliferative expansion and then differentiation, during postnatal myogenesis.

Isolation of Adult Mouse Myogenic Progenitors:Functional Heterogeneity of Cells within and Engrafting Skeletal Muscle

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

Cellular and Molecular Signatures of Muscle Regeneration: Current Concepts and Controversies in Adult Myogenesis

Adult skeletal muscle generates force in a controlled and directed manner through the contraction of highly specialized, postmitotic, multinucleated myofibers. Life-long muscle function relies on maintenance and regeneration of myofibers through a highly regulated process beginning with activation of normally quiescent muscle precursor cells and proceeding with formation of proliferating progenitors that fuse to generate differentiated myofibers. In this review, we describe the historical basis and current evidence for the identification of satellite cells as adult muscle stem cells, critically evaluate contributions of other cells to adult myogenesis, and summarize existing data regarding the origins, genetic markers, and molecular regulation of satellite cells in normal, diseased, and aged muscle.

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.

Aging, Stem Cells and Tissue Regeneration: Lessons from Muscle

With age, there is a gradual decline in the regenerative properties of most tissues due to a combination of age-dependent changes in tissue-specific stem cells and in the environmental cues that promote those cells to participate in tissue maintenance and repair. In adult skeletal muscle, where the resident dedicated stem cells (“satellite cells”) are capable of rapid and highly effective regeneration in response to injury, there is just such a loss of regenerative potential with age. Satellite cell activation and cell fate determination are controlled by the Notch signaling pathway that is initiated by the rapid increase in expression of the Notch ligand, Delta, following injury. In old muscle, this upregulation of Delta is blunted and thus satellite cell activation is markedly diminished. However, by indirectly inducing Notch activity, the regenerative potential of aged satellite cells can be restored. Furthermore, exposure of aged satellite cells to serum from young mice, either in vivo by heterochronic parabiotic pairings or in vitro, rejuvenates the satellite cell response. This restorative potential suggests that tissue-specific stem cells do not lose their ability to participate in tissue maintenance and repair. Therefore, it may be that even very old stem cells may be capable of maintaining and repairing aged tissues if provided with optimal environmental cues.

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.