Cellular dynamics in the muscle satellite cell niche

Abstract

Satellite cells, the quintessential skeletal muscle stem cells, reside in a specialized local environment whose anatomy changes dynamically during tissue regeneration. The plasticity of this niche is attributable to regulation by the stem cells themselves and to a multitude of functionally diverse cell types. In particular, immune cells, fibrogenic cells, vessel-associated cells and committed and differentiated cells of the myogenic lineage have emerged as important constituents of the satellite cell niche. Here, we discuss the cellular dynamics during muscle regeneration and how disease can lead to perturbation of these mechanisms. To define the role of cellular components in the muscle stem cell niche is imperative for the development of cell-based therapies, as well as to better understand the pathobiology of degenerative conditions of the skeletal musculature.

See the Glossary for abbreviations used in this article.

Glossary.

C/EBP

CCAAT/enhancer-binding protein

DTX

diphtheria toxin

ECM

extracellular matrix

FAP

fibro-adipogenic progenitor

FGF

fibroblast growth factor

FSP

fibroblast-specific protein

Fzd

Frizzled

IGF

insulin-like growth factor

IL

interleukin

MAPK

mitogen-activated protein kinase

MCP

macrophage inflammatory protein

MIP

monocyte chemotactic protein

Myf

myogenic factor

MyHC

myosin heavy chain

MyoD

myogenic differentiation

Par

partitioning defective

Pax

Paired box

PDGFR

platelet-derived growth factor receptor

Rbpj

recombining binding protein-J

Sca

stem cell antigen

Sdc

syndecan

SMA

smooth muscle actin

Introduction

Homeostatic adult skeletal muscle has a relatively uniform architecture. Bundles of longitudinally aligned muscle fibres surrounded by sheets of extracellular matrix (ECM) are attached to tendons and bone through myotendinous junctions [1]. A dense network of blood vessels supplies the tissue with nutrients and oxygen. Quiescent muscle stem cells, or satellite cells, are found underneath the ECM sheet attached to the muscle fibre plasma membrane [2]. Adult muscle tissue also contains several types of interstitial and vessel-associated cells that show little to no mitotic activity under resting conditions [3,4,5]. After injury, these cells begin to proliferate and, in conjunction with infiltrating immune cells, disperse throughout the muscle tissue (Fig 1A,B; [3,4,5,6]). The cellular dynamics during muscle regeneration are highly complex and occur with distinct temporal and spatial kinetics. In the course of muscle regeneration, satellite cells become activated and some will eventually upregulate transcription factors that trigger the myogenic differentiation programme [7]. Once differentiated into myocytes, the cells will align and form new syncytial muscle fibres or fuse to existing fibres. On completion of this regenerative response, the tissue returns to its homeostatic state and the resident cell populations re-enter a resting state.

Figure 1.

Overview of tissue histology during mouse skeletal muscle regeneration. (A) A time course of histological changes in regenerating skeletal muscle. H&E staining of uninjured TA muscles and regenerating TA muscles at 5, 10 and 30 days after intramuscular cardiotoxin injection. Regenerating muscles are reduced to mostly mononuclear cells at day 5, but are able to re-establish multinucleated myofibres by day 10. Notably, the nuclei of uninjured myofibres are located at the periphery, whereas those of regenerating muscle fibres are centrally located. Scale bar, 50 μm. (B) Longitudinal view of whole tissue preparations of uninjured (left) and regenerating (right) skeletal muscle. Immunostaining for the extracellular matrix protein laminin (green) labels the basal lamina surrounding myofibres and capillaries. In regenerating conditions, the proliferation of satellite cells can be observed by the increase in the number of Pax7 (red) expressing cells (arrows). DAPI staining of nuclei (blue) reveals accessory cells in the satellite cell niche. Scale bar, 50 μm. H&E, haematoxylin and eosin; TA, tibialis anterior.

Despite there being many differentiating cells, the total number of satellite cells remains constant through multiple rounds of regeneration [8]. This equilibrium is due to the ability of satellite cells to self-renew, which provides progeny for differentiation while uncommitted mother cells are retained [9]. Satellite cell commitment to myogenic differentiation is mediated by the myogenic regulatory factors Myf5 and MyoD. Cre/lox reporter systems show that a subpopulation of about 10% of satellite cells has never expressed Myf5 [10]. Such satellite stem cells can self-renew through asymmetrical cell divisions that give rise to Myf5-positive satellite cells in response to the demand for committed myogenic progenitors. Asymmetric satellite cell division is controlled by the Par complex, which allows activation of p38α/β MAPK and upregulation of MyoD in the committed daughter cell [11]. Self-renewing satellite cells express higher levels of Pax7 than do cells that are primed for differentiation [12]. Moreover, a subpopulation of MyoD-expressing satellite cells can downregulate this factor to resist differentiation and re-enter quiescence [13]. These mechanisms of self-renewal allow the satellite cell pool to be maintained over multiple rounds of injury and repair, and are ultimately responsible for the outstanding regenerative capacity of muscle tissue.

Self-renewal and the three basic states of satellite cells—quiescence, proliferation and differentiation—are predominantly regulated by extrinsic factors in the local environment, the so-called stem cell niche [14]. These environmental cues include growth factors, cytokines, adhesion molecules and ECM contributed by the various cell types present in regenerating muscle tissue. In addition, satellite cells and their committed progeny actively participate in the remodelling of the niche during regenerative myogenesis. The diverse non-satellite cell types in muscle tissue can be categorized into cells with myogenic potential and into cells with accessory function for muscle regeneration [3,4,5]. Ablation studies have clearly shown an essential role of many accessory cell types during adult myogenesis [3,5,6]. By contrast, the physiological relevance of non-satellite cell types with myogenic potential is less clear [4]. Importantly, these cell types have unique characteristics that render them suitable for cell therapy approaches directed at treating skeletal muscle diseases.

In this article, we review the literature and present an overview of the cellular dynamics in the muscle stem cell niche in homeostasis, during regeneration and in disease. We introduce the niche under quiescent conditions, which we follow by a discussion of the cell types that constitute and modulate the niche during muscle regeneration. Subsequently, we elaborate on the deregulation of the niche under pathogenic conditions, and lastly we discuss potential importance of the niche for the recruitment of non-satellite cells with myogenic potential.

The quiescent niche

Under homeostatic conditions, adult muscle tissue has slow turnover [15]. Thus, after a proliferative phase during juvenile development, most satellite cells remain quiescent in the absence of an injury stimulus [7]. Importantly, the composition of the niche and the receptors that allow satellite cells to sense extrinsic signals are fundamentally different in quiescence than in the activated state [14,16]. In the quiescent niche, a few cell types are found in the proximity of satellite cells, for instance vessel-associated cells and muscle fibres (Fig 1B). This environment remains essentially static and imposes signals that promote the quiescent stem cell state.

Several studies have identified the Notch receptors as being critical for the maintenance of satellite cell quiescence [17,18]. Notch proteins are transmembrane receptors that are activated on exposure to ligands presented by juxtaposed cells [19]. The Notch pathway is highly pleiotropic and has important functions throughout development. In adult skeletal muscle, genetic loss of Rbpj, a downstream factor in the Notch pathway, leads to spontaneous satellite cell activation and premature differentiation. Since the satellite cell niche contains few heterogeneous cell types under quiescent conditions, regulatory Notch signals are most likely to be presented by the myofibres.

Quiescent satellite cells express high levels of integrin α7 and β1, as well as dystroglycan [20,21]. These receptors could transduce signals from the laminin-rich ECM that covers the satellite cells on their host muscle fibres [21,22]. Under homeostatic conditions, satellite cells also express M-cadherin and the glycoprotein CD34, which are involved in adhesion to myofibres [23,24]. Moreover, quiescent satellite cells are decorated with the heparan sulphate proteoglycans Sdc-3 and Sdc-4, which serve as co-receptors for integrins and sequester soluble growth factors and ECM in the immediate cellular microenvironment [25,26]. Interestingly, Sdc-3 also binds to Notch in satellite cells, and this interaction is required for self-renewal and reversible quiescence [27].

In contrast to the quiescent state, during muscle regeneration the composition of the niche is in a flux that is regulated by a spectrum of cell types (Fig 2). Satellite cell activation is coupled to the upregulation of specific receptors that integrate these niche signals to trigger the appropriate cellular responses. In the subsequent sections we discuss the different cell types involved in regulation of the niche during muscle repair.

Figure 2.

Schematic representation of the various cell types involved in muscle regeneration. Within the complexity of regenerating muscles, satellite cells are subject to a distinct environment determined by the spatial and temporal presence of cytokines, growth factors and other cell types.

Immune cells are critical effectors of the satellite cell niche

Acute sterile muscle injuries trigger a precisely orchestrated inflammatory process aimed at the removal of damaged cells, coordination of the regenerative response and, ultimately, restoration of tissue homeostasis (Fig 3A; Table 1). The onset, development and resolution of inflammation involve diverse interactions between leukocytes and local cell types, including satellite cells (Fig 3B). In resting conditions, adult skeletal muscle contains different types of resident leukocyte. The most abundant are mast cells and macrophages. These resident cell types, in conjunction with ‘patrolling’ circulatory monocytes, act as sensors for distress and secrete a number of chemoattractive molecules following muscle injury [28,29]. Particularly, damage-activated mast cells almost instantly begin to secrete TNF-α, histamine and tryptase and then initiate the de novo synthesis of other cytokines, such as interleukin (IL)-6 [30]. At low physiological concentrations, TNF-α, tryptase and IL-6 promote activation and proliferation of satellite cells [31,32,33]. Moreover, inhibition of mast cell activity leads to reduced leukocyte extravasation and impairs muscle repair [34]. Thus, immune cells contribute substantially to the satellite cell niche in the earliest stages of muscle regeneration.

Figure 3.

Participation of non-myogenic cell types in muscle regeneration. (A) The relative presence of immune, fibrotic, vascular and myogenic cell types after muscle injury. (B–D) Immunofluorescence micrographs of tissue sections from regenerating mouse muscles. In their niche, Pax7-positive satellite cells (green) are in close proximity to various non-myogenic cell types (red): (B) CD11b-positive leukocytes; (C) Sca1-positive interstitial cells; and (D) VE-Cad-positive endothelial cells. ECM is shown in orange and nuclei are labelled with DAPI (blue). Scale bar, 10 μm.

Table 1. Cell types in the muscle satellite niche.

Cell type	Markers	Presence (days after injury)	Effect on myogenic cells	References
Granulocyte	CD11b+, CD31+, Gr1+, CD43+	0–3	Indirectly stimulate myogenesis through FAPs	[37,38]
Monocyte	CD11b+, CD31+, Ly6C(±)	0–3	Promote satellite cell activation and proliferation (Ly6C+) or differentiation (Ly6C−)	[28,41,42]
M1 macrophage	CD11b+, CD31+, CD68+, iNOS+	Peak at 2–3, return to baseline after 7–9	Promote myoblast proliferation, repress myogenic differentiation	[43,44,45,46,49,50,51,52]
M2 macrophage	CD11b+, CD31+, CD68+, CD206+, CD163+	Peak at 3–4, return to baseline after 8–10	Regulate entry into myogenic differentiation, promote myotube formation	[43,44,47,48,49,50,51,52,59]
FAP	CD34+, Pdgfrα+, Sca1+(high)	Peak at 3–4, return to baseline after 7–9	Promote myogenic differentiation	[37,61,62,63,65]
Fibroblast	Tcf4+, SMAα+, vimentin+, desmin+, FSP1+	Peak at 5–7, persist throughout regeneration	Promote myoblast proliferation, enhance self-renewal	[72]
Vascular cell	VE-Cad+, SMAα+, CD31+	Peak at 5–7, return to baseline after ∼28	Promote myoblast proliferation, enhance self-renewal	[73,74,75,76,77,78]
Satellite cell and myoblast	Pax7+, MyoD+*, α7β1-int+, Sdc4+, VCAM-1+	Peak at 5–7, return to baseline after ∼28	Enhance self-renewal of satellite cells	[10,56,57,82]
Differentiating myocyte	Myogenin+, α7β1-int+, Sdc4+, desmin+	Appear around day 4–5 and peak by day 10	—	[124,125]
Newly formed myofibre	MyHC+	Return to baseline numbers after ∼28 (with most myofibres remaining centrally nucleated)	Enhance self-renewal	[10,17,18,85]

^*Myoblasts only.

The initial burst of cytokines and chemokines produced by resident leukocytes, which include TNF-α and MIP-2, along with cellular and extracellular contents released by the damaged tissue, lead to the rapid attraction of circulating granulocytes [35,36]. These consist mainly of neutrophils and, to a lesser extent, eosinophils [37]. Neutrophils promote the proinflammatory environment that is necessary for the clearance of cellular debris. Under certain conditions, this cell type has been suspected to transiently aggravate tissue damage [38]. Neutrophils also secrete the chemokines MIP-1α, MCP-1 and others that favour the recruitment of monocytes [39,40]. Beyond the first day after injury, monocytes gradually become the predominant leukocytes in the exudate. Globally, monocytes are divided in two categories: the classic monocytes (Ly6C⁺) that are predominantly present during the first few days after injury and the non-classical monocytes (Ly6C⁻) that slowly replace Ly6C⁺ cells as regeneration progresses [41]. Although the origin of this switch in monocyte subpopulations is still debated, distinct functions for both cell types have been established [41,42]. Indeed, Ly6C⁺ monocytes promote the recruitment of other monocytes by secreting proinflammatory cytokines, such as TNF-α and IL-1β, whereas Ly6C⁻ monocytes express high levels of anti-inflammatory molecules and growth factors [41]. Importantly, the switch of monocyte subtypes not only influences the general course of inflammation but also is important in the satellite cell niche. The proinflammatory environment established by Ly6C⁺ monocytes promotes the proliferation of myogenic cells and reduces their differentiation and fusion capacity. On the other hand, the anti-inflammatory signals from Ly6C⁻ monocytes have opposite effects and stimulate differentiation [41]. Therefore, the emergence of Ly6C⁺ monocytes before Ly6C⁻ monocytes is important to ensure appropriate proliferation of myogenic cells and to prevent their premature differentiation.

Once monocytes have invaded the tissue, they begin to differentiate into macrophages. Macrophages can be divided into several subtypes. Analogous to monocytes, this classification of macrophages during muscle regeneration can be simplified into an initial wave of proinflammatory, or M1, macrophages that is followed by a second wave of anti-inflammatory, or M2, macrophages. These macrophage subsets, however, are not mutually exclusive, and, at a given time point, distinct subtypes can be found in the same regenerating area [43]. Depletion models of different types of acute sterile injury have shown that suppression of M1 macrophages leads to persistence of necrotic cells, impaired myoblast proliferation, increased fibrosis and fat accumulation [44,45,46]. By contrast, inhibition of the transition from M1 to M2 macrophages in mice negative for IL-10 or the transcription factor C/EBPβ resulted in reduced myogenin expression and fibre growth [47,48]. Therefore, M1 and M2 macrophages stimulate, respectively, the early and the late phases of myogenesis. These results are supported by the observation that in injured human muscle, M1 macrophages are found close to proliferating myogenic cells and M2 macrophages interact with differentiating myocytes [43]. The proximity is important for macrophages to mediate myogenic effects and is favoured by attractive reciprocal chemotactic signals [49]. Indeed, direct physical contact, for instance through VCAM1–VLA4, allows macrophages to inhibit apoptosis and promote survival of myogenic cells [50]. Treatment of myogenic cells with macrophage-conditioned medium revealed that the effects of M1 and M2 macrophages are also mediated by paracrine signalling [43,51]. IL-1β, IL-6 and TNF-α secreted by M1 macrophages are particularly important to induce proliferative effects on myogenic cells, whereas IL-4 and IGF-1 released by M2 macrophages promote their differentiation [43,52,53]. Moreover, macrophages also secrete different ECM proteins according to the stage of macrophage differentiation. For example, M2 macrophages secrete a more mature form of fibronectin and a higher amount of ColVI than do M1 macrophages [54,55]. The ECM proteins secreted by M2 macrophages are important components of the muscle stem cell niche and promote self-renewal of satellite cells [56,57].

The regulatory function of immune cells for myogenesis is sensitive to perturbation and efficient muscle repair depends on their precise coordination. For instance, M2 macrophages can secrete anti-inflammatory molecules and pro-resolving mediators [58]. The anti-inflammatory properties of these molecules allow M2 macrophages to efficiently decrease the oxidative activity and the cytolytic muscle damage caused by neutrophils and M1 macrophages [58,59]. Thus, even slight imbalances in immune cell populations due to sustained and successive inflammatory signals in diseased muscles can disrupt the cellular dynamics in the niche and provide inappropriate environmental cues to satellite cells (see below). Nonetheless, if appropriately synchronized and controlled, immune cells serve as key effectors in the muscle stem cell niche to guide satellite cells through the regeneration process.

Fibrogenic cells remodel the niche during regeneration

During muscle regeneration the extracellular environment in the stem cell niche is dynamically rearranged [60]. The functions of various ECM components being deposited in regenerating tissue are only beginning to be elucidated. Structurally, this transitional fibrillar ECM serves to preserve the gross integrity of the tissue until degenerated fibres have been cleared and innervated young muscle fibres have been formed in the correct anatomical position. The main source of these ECM proteins during muscle regeneration is fibrogenic mesenchymal stromal cells, such as fibroblasts and FAPs.

FAPs are mesenchymal stem cells resident in skeletal muscle that have the ability to differentiate into fibroblasts, adipocytes and possibly into bone and cartilage cells, although not into satellite cells or muscle fibres [61,62,63]. This cell type is marked by the expression of the mesenchymal stem cell surface markers, CD34, Sca-1 and PDGFRα, and by the absence of the haematopoietic markers CD45 and CD31 and of the satellite cell marker integrin α7 [3]. Under quiescent conditions FAPs localize close to blood vessels [64]. On muscle injury, these cells are activated, expand and take over the interstitium, where they have a promyogenic function (Fig 3C; [63]). The number of FAPs increases rapidly and peaks 3–4 days after injury, then returns to baseline levels after 7–9 days (Fig 3A).

FAPs are major contributors to the deposition of several extracellular proteins—for instance, certain collagen isoforms that are abundant in the supportive transitional ECM during muscle regeneration [65]. This cell type also secretes high levels of IL-6, which promotes the differentiation of myogenic cells [63]. Vice versa, myotubes seem to inhibit the differentiation of FAPs into adipocytes [62]. Regulation of cellular crosstalk also involves eosinophils that are recruited during the early stages of regeneration [37]. Eosinophils release the cytokines IL-4 and IL-13, which induce the proliferation of FAPs and simultaneously block their adipogenic differentiation. In mice deficient for IL-4 and IL-13, adipocytes accumulated after muscle injury and reparative myogenesis was impeded. Remarkably, FAPs seem to remove necrotic debris from regenerating muscles more efficiently than macrophages [37]. The physiological contribution of these two cell populations to debris clearance, however, remains to be investigated, since their abundance and temporal regulation in regenerating muscle probably differs.

Fibroblasts are elongated cells with extended cell processes and a fusiform or spindle-like shape that are identifiable by high expression levels of the intermediate-filament-associated proteins vimentin, desmin, FSP1 and α-SMA [66,67]. This cell type is of a non-vascular, non-epithelial and non-inflammatory nature. Fibroblasts are heterogeneous, with expression profiles that differ depending on the tissue source [68]. A major function of this cell type is the deposition of fibrillar ECM, such as collagen and fibronectin, and basement membrane constituents [68,69,70]. Moreover, fibroblasts can actively remodel the ECM by secretion of matrix metalloproteinases [71].

Under homeostatic conditions in adult skeletal muscle, fibroblasts reside in the interstitium between myofibres [72]. After muscle injury, they quickly start to proliferate and become highly abundant. Tissue fibroblast content is greatest at about 5 days after muscle damage and coincides with the peak of satellite cell proliferation (Fig 3A). DTX-driven ablation of a subpopulation of fibroblasts from regenerating muscles, achieved with a tamoxifen-inducible Cre allele driven by the Tcf4 promoter, resulted in premature satellite cell differentiation and impaired regeneration [72]. Similar to FAPs, Tcf4-positive fibroblasts express PDGFRα, and it remains to be determined whether the Tcf4-Cre allele is also expressed in the former cell type [61,62,63,72]. Within the muscle fibroblast population, the Tcf4-Cre allele is only active in about 40% of cells. Despite this low percentage of fibroblasts that were depleted by DTX with this Cre driver, a notable phenotype was observed [72]. These results emphasize the critical role of ECM-producing cell types in the stem cell niche during regenerative myogenesis. A possible interpretation of this study is that fibroblasts allow for transient expansion of satellite cells during muscle regeneration while preventing their differentiation. Interestingly, such a role for fibroblasts during muscle regeneration would be opposed to the effects of FAPs, which seem to have prodifferentiation effects [63].

DTX-mediated ablation of satellite cells with a tamoxifen-inducible Pax7-Cre driver revealed that a proper satellite cell response to an injury stimulus is reciprocally required for a normal fibroblast response [72]. The number of fibroblasts in satellite cell-depleted muscles was reduced by ∼50% at the peak of regeneration, 5 days after injury, when the first centrally nucleated fibres are normally formed. This effect on fibroblasts might be due to the absence of a trophic signal from proliferating satellite cells or from the young muscle fibres that cannot be established in satellite cell-depleted muscle. Taken together, the complex role of mesenchymal fibrogenic cells in the muscle stem cell niche only begins to be understood and future studies will be required to explore their interplay with myogenic cells in more detail.

Endothelial and periendothelial cells in the niche

Skeletal muscle is laced with a dense microvasculature, and most quiescent satellite cells are found in close proximity to these vessels [73]. During muscle injury, the number of capillaries in the tissue initially increases and then returns to baseline about 4 weeks after injury (Fig 3D; [74,75]). In co-culture experiments, endothelial cells promote the proliferation of satellite cell-derived myoblasts. Reciprocally, differentiating myogenic cells are proangiogenic and increase the formation of capillary-like structures [73]. Endothelial cells secrete a variety of mitogenic and/or anti-apoptotic factors, such as VEGF, that influence muscle cells [76]. Intriguingly, differentiating myogenic cells also secrete VEGF and their proangiogenic function mainly depends on this factor [77]. This finding suggests an intricate feed-forward mechanism through which VEGF in the stem cell niche co-regulates both myogenesis and angiogenesis.

In contrast to the predominantly promitotic effects of endothelial cells on myogenic progenitors, cells in the periendothelial position, such as smooth muscle cells and fibrogenic cell types, are crucial for re-entry into quiescence on completion of regeneration [76]. Satellite cells transitioning into quiescence increase expression of the Ang1 receptor Tie-2. Forced expression of Ang1 in mouse muscles increases the number of quiescent cells and inhibition of Tie-2 prevents cell-cycle exit on completion of regeneration [78]. Importantly, periendothelial cells seem to be the major source of Ang1 during muscle regeneration. In summary, vessel cells in the stem cell niche coordinate both the acute satellite cell response and the late stages of muscle regeneration when the tissue returns to homeostasis.

Regulation of the niche by cells of the muscle lineage

In many tissues, the committed or differentiated progeny of stem cells become components of the niche where they provide regulatory signals [79]. This feature is the same in the skeletal muscle lineage and, as discussed below, involves cell–cell interactions, as well as the secretion of growth factors and regulatory ECM.

Notch signals originating from differentiating myogenic cells allow for self-renewal of muscle progenitors while suppressing activation of the commitment factor MyoD during development [18,80,81]. This Notch-dependent developmental mechanism is preserved in adult satellite cells. Experiments using the Myf5-Cre-YFP reporter system revealed that during the regenerative response of mature skeletal muscle, committed proliferating YFP+ satellite cells of the Myf5-dependent lineage provide Notch signals to the self-renewing YFP⁻ satellite stem cells [10]. Satellite stem cells contain high levels of Notch-3, while their committed Myf5/MyoD-positive progeny express the Notch ligand Delta-1. In asymmetric divisions, Delta-1 localizes to the cell interface with the YFP⁻ cell and probably activates self-renewal signals by binding to Notch-3 [10]. Thus, next to its established role in maintaining satellite cell quiescence (see above), Notch also plays a role in activated cells.

When compared with quiescent cells, activated satellite cells and their differentiated progeny express high levels of ECM components and various molecules involved in the remodelling of extracellular space [56,82]. An intriguing role of Notch signalling during myogenic development is the regulation of ECM synthesis by myogenic progenitors [83]. Genetic loss of Rbpj from myogenic cells, whose terminal differentiation is blocked due to knockout of MyoD, leads to an inability to acquire the satellite cell position underneath the basal lamina surrounding the developing muscle fibres. Importantly, the expression of several ECM components is strongly disrupted in such cells, suggesting that they actively contribute to the formation of the basal lamina. Thus, the regulation of ECM synthesis could be another Notch-related developmental process that plays a role in adult myogenesis.

The pool of Myf5-independent satellite stem cells in muscle tissue is critically controlled by Wnt7a, a lipophilic factor that is released into the stem cell niche by newly formed fibres [10,84,85]. Expansion of the satellite stem cell population is a mechanism that is essential for maintenance of the myogenic progenitor pool after muscle injury. Consequently, knockout of Wnt7a severely reduces overall satellite cell number after regeneration. The effect of Wnt7a on satellite stem cells depends on the Fzd7–Sdc4 co-receptor complex, the function of which is modulated by fibronectin that is secreted into the niche microenvironment by committed Myf5-positive satellite cells [56]. Binding of Wnt7a and fibronectin to the Fzd7–Sdc4 co-receptor complex allows for downstream GTPase signalling and the induction of symmetric expansion of satellite stem cells. Similar to the loss of Wnt7a, loss of fibronectin from regenerating muscle severely reduces the overall pool of satellite cells. Thus, the release of fibronectin into the stem cell niche represents a feedback mechanism originating from committed satellite cell progeny that, in concert with Wnt7a, modulates the self-renewing stem cell pool. Other cell types in muscle express high levels of fibronectin and, therefore, might also contribute to fine-tuning of the Wnt7a response [68,69,70].

Another ECM molecule critical to the satellite cell niche is ColVI [57]. Mutations in ColVI are the underlying cause of Bethlem myopathy and Ullrich congenital muscular dystrophy [86]. ColVI knockout mice show deficiency in muscle regeneration and mild myopathy [57,87]. Satellite cells express high levels of ColVI and secrete this factor to autoregulate the softness of their niche. In elegant grafting experiments, wild-type satellite cells ameliorated the regenerative phenotype of ColVI-deficient muscle tissue, which demonstrates a cell-autonomous requirement for this factor. Interestingly, in this mouse model, the ColVI content in the satellite cell niche can also be restored by transplantation of wild-type fibroblasts.

In summary, important regulatory functions of the stem cell niche are controlled by committed satellite cell progeny or by differentiated fibres. Specifically, satellite cells actively autoregulate their immediate microenvironment. The emerging mechanisms that integrate feedback and feed-forward signals within the myogenic lineage are highly complex and further investigation will undoubtedly unravel important new concepts in basic stem cell biology.

The satellite cell niche in ageing and pathology

Chronic degenerative conditions of skeletal muscle can lead to permanent changes within the muscle stem cell niche. Evidence suggests that, under specific conditions, satellite cells can differentiate into brown fat, osteocytes and myofibroblasts [88,89,90]. Pathological deterioration of the niche or systemic changes can influence these fate decisions and disrupt normal cellular responses to injuries [91,92]. Ultimately, imbalance within the local satellite cell milieu attenuates the formation of new myofibres and leads to the eventual loss of muscle function. Therefore, understanding of pathological conditions of the muscle stem cell niche is important to treat muscle diseases.

Although ageing should not be considered a pathological state, the deterioration in muscle regeneration through this process is directly correlated with changes to local and circulating factors that influence satellite cell function [93]. With use of heterochronic parabiosis to connect the circulatory systems of young and old mice, serum from young mice reduced age-related tissue fibrosis and restored satellite cell function in old mice. Systemically, serum levels of TGF-β1 are increased in elderly humans and mice [94]. This profibrotic factor not only stimulates the expansion of tissue-resident fibroblasts but also inhibits the myogenic differentiation of satellite cells, which diminishes the regenerative capacity of ‘old’ muscle. Additionally, altered levels of osteopontin secreted by CD11b⁺ macrophages that are found in the serum of old mice are associated with reduced proliferative and differentiation capacities of satellite cells [95]. This finding suggests that immune responses to injuries can shift with age. Accordingly, subtle changes to the systemic milieu can affect the cellular response of support cells within the muscle stem cell niche and affect the efficiency of myogenic regeneration. Combined with localized FGF-2 secreted by the myofibres that disrupt the ability of aged muscle stem cells to return to quiescence in old mice, the aged niche creates an unfavourable environment for the proper function of muscle stem cells [96]. The full extent of these gradual changes is not yet known and requires further investigation, along with whether the ‘young’ stem cell niche can be therapeutically restored.

In degenerative muscle diseases, localized muscle pathologies can transform the normal wound-healing programme into a positive feedback loop that prevents the proper function of satellite cells. Notably, the attenuation of muscle repair in most forms of muscular dystrophy is correlated with a build-up of fibrotic scarring, adipose tissue and immune infiltrations [97]. The increased susceptibility of dystrophic muscle fibres to damage leads to cycles of degeneration and regeneration. In most cases, necrotic and regenerating areas occur concurrently throughout dystrophic muscles. Unlike the beneficial effects of transient ECM protein upregulation during normal regeneration, increased inflammation and persistent expression of ECM proteins reduce the differentiation of myoblasts into myofibres [98,99]. Moreover, the altered elasticity of fibrotic muscle tissue is likely to have a negative influence on the self-renewal of satellite cells [57,100]. The lack of efficient myofibre formation in diseased muscle reduces the inhibitory feedback on the fibrogenic and adipogenic differentiation of FAPs [62]. This feedback mechanism persists until anti-myogenic signals accumulate exponentially and muscle regeneration is essentially halted.

Restoration of a functional muscle stem cell niche is an important aspect of treating degenerative muscle diseases. Experimental regulation of supportive cell types during regeneration can optimize the myogenic efficiency of satellite cells [37,63,72]. Therefore, it is conceivable that these cells can be effectively manipulated in disease conditions to combat the loss of muscle stem cell function over time. In agreement with this idea, anti-fibrotic and anti-inflammatory therapies have reduced the progression of Duchenne muscular dystrophy in the short term [101,102]. Furthermore, transplantation of corrective supportive cells can recondition the niche to restore satellite cell function [57,95]. The intricacies of the regenerating environment and altered systemic milieu in specific diseases, however, have made research into this area challenging, and there are many aspects regarding the structural and temporal regulation of the muscle stem cell niche that remain unknown. Thus, future research into the regulation of the stem cell niche in regeneration and disease holds great potential for the therapeutic enhancement or restoration of muscle regeneration.

Non-satellite cells with myogenic potential

Various cell types other than satellite cells are able to fuse into differentiating muscle fibres and, in certain cases, can also acquire Pax7 expression and become satellite cells [4]. These properties are attractive for the development of cell-based therapies for muscle diseases. Examples of these cell types include side population cells, PW1-positive interstitial cells, pericytes, mesoangioblasts, CD133-positive circulating cells and integrin β4-positive interstitial cells [103,104,105,106,107,108]. The existence of these cell types raises questions of whether a non-satellite cell progenitor could compensate for the role of satellite cells during physiological myogenesis and whether an exogenous stem cell type could feed into the myogenic lineage to generate new satellite cells and replenish the pool of cells available for regenerative myogenesis. Moreover, the niche signals that are required for myogenic conversion of these cell types remain largely unknown. Several groups have started to address this interesting subject by using ablation methods that are coupled to the expression of Pax7 [72,109,110]. These studies have used either a tamoxifen-inducible Pax7-Cre with a stop-flox DTX allele or the human DTX receptor inserted into the Pax7 locus, so that the respective cells can be selectively ablated by exposure to DTX. Targeted elimination of satellite cells with DTX in these mouse models resulted in a severely impaired regenerative response of muscle tissue. Importantly, in one study, regeneration was induced in muscles that had been depleted of satellite cells 2 weeks earlier, and virtually no fibre formation was observed up to 5 weeks after injury [110]. These results indicate that no other cell type can compensate for the loss of satellite cells by direct differentiation into myofibres, and that no other cell type can replenish the satellite cell pool in the intermediate term after injury. As discussed above, however, signals originating from satellite cells and their committed and differentiated progeny are critical for the function and the recruitment of many different cell types in the niche, for instance fibroblasts, endothelial cells and FAPs [62,72,77]. Therefore, DTX-induced loss of satellite cells and the absence of newly formed muscle fibres on injury could lead to impaired recruitment of myogenic non-satellite cell types due to the absence of a trophic signal. In support of this idea, in vitro, most myogenic non-satellite cell types require co-culture and co-differentiation with myoblasts to substantially contribute to the formation of myotubes. In spite of their unclear physiological role in directly contributing to adult myogenesis and to the stem cell niche, non-satellite cells with myogenic potential seem to have tremendous therapeutic value that enables, for instance, systemic delivery or extensive ex vivo expansion [111,112,113,114,115,116,117,118,119,120,121,122]. Thus, their use for cell therapy might allow bypassing of several problems associated with the isolation and expansion of conventional myogenic cells for transplantation [123]. Importantly, an improved understanding of the niche signals required to recruit these cells to myogenesis will help to advance such therapies.

Conclusion and outlook

On injury, the stem cell niche in muscle transitions from a relatively steady state involving few cell types into an enormously complex environment with spatiotemporally regulated cascades of direct and indirect cellular interactions (Fig 4, Table 1). The sum of these interactions, combined with intrinsic stem cell programming, controls the regenerative dynamics in the tissue and ultimately allows for the re-establishment of muscle structure and function. The study of muscle regeneration has taken us away from a view that is centred on intrinsic satellite cell regulation towards an understanding that integrates the immense relevance of the niche. With the mouse as a versatile model to study the biology of skeletal muscle, it is becoming increasingly apparent how elaborately fine-tuned is the role of the different cell types involved in muscle regeneration, and how detrimental are the consequences of disease-related imbalances in these dynamics.

Figure 4.

Schematic of extrinsic signals in the muscle stem cell niche. Paracrine signals (thin arrows) regulate the recruitment, proliferation rate and differentiation (bold arrows) of each cell type.

An integrative understanding of the cellular complexity in the niche will allow for the development of therapeutic strategies targeted to normalize or adapt the global behaviour of specific cell populations rather than single signalling pathways. The field has taken great steps forward due to the development of several important genetic tools allowing the manipulation and observation of specific cell populations in muscle tissue. The further refinement of these tools and the identification of mutually exclusive cellular markers will be crucial to answering many of the outstanding questions (Sidebar A) and to a future holistic understanding of the dynamics of muscle regeneration.

Sidebar A | In need of answers.

1. How does the niche instruct fate decisions of satellite cells?

2. What are the main circulating signals that influence the satellite cell niche in systemic conditions, such as ageing, cancer cachexia and diabetes? What changes in the niche do these factors trigger?

3. Is there a specialized ECM microenvironment that instructs the maintenance of satellite cells in quiescence?

4. What are the niche signals that recruit non-satellite cell types with myogenic potential?

5. What are the critical components required to create a functional artificial niche for the expansion of uncommitted satellite cells ex vivo?

6. Is it possible to develop an experimental system that allows the observation of cellular dynamics in a completely undisturbed niche?

7. Are there differences in the composition of the satellite cell niche between mice and humans?

C Florian Bentzinger, Yu Xin Wang, Nicolas A Dumont & Michael A Rudnicki