**2. Extracellular matrix in regulation of muscle stem cell niche**

the plasma membrane of myofibers (sarcolemma), and an external, reticular lamina. Extracel‐ lular matrix surrounding muscle fibers is composed of collagens (dominated by collagen IV), laminins, fibronectin, and proteoglycans, formed by glycosaminoglycans bound to a protein core. Initially, it was considered as a structure that provides mechanical support for bearing force transmission [2]. The ECM gives mechanical structure to myofibers during contractions, provides the tissue with elasticproperties, andparticipates in the transmission offorce from the myofiber to tendon. It also serves as a basic mechanical support for nerves and vessels present in skeletal muscle tissue, and determines the spatial barrier between endothelium and muscle cell surface. A great progress in cell biology, molecular biology and genetics, gives new insight into skeletal muscle biology, and now it becomes evident that cells adhere to and connect with the ECM not only for structural stability but also for signaling. The integrins, heterodimeric transmembrane receptors comprising unrelated alpha and beta subunits, play critical roles in converting extracellular signals to intracellular responses (outside-in signaling) as well as in extracellular matrix interactions based upon intracellular changes (inside-out signaling) [3]. They bind to ECM or cell surface ligands and link the actin microfilament system with ECM, providing a connection between the ECM, the cytoskeleton, and signaling molecules. Integ‐ rins are considered as sensors of tensile strain at the cell surface, and together with the cytos‐ keleton form a mechanically sensitive organelle. Despite the large overall number of integrin receptor complexes, skeletal muscle integrin receptors are limited to seven alpha subunits, all associatedwiththebeta1integrinsubunit.Integrinsignaltransmissiondependsontheactivation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, localized at focal adhesions. Integrin engagement causes the formation of transient signaling complex, initiated by the recruitment of Src-family protein SH2 to the FAK Tyr-397 autophosphorylation site, and by serving as a signaling element in cytoskeleton-associated networks [4]. Integrin-linked kinase (ILK), initially considered as a kinase, but, in fact, incapable to perform phosphorylation due to pseudoactive domain, mediates interactions of integrins with numerous cellular proteins and regulates focal adhesion assembly, cytoskeleton organization, and signaling [5]. The major enzymes responsible for the ECM breakdown under physiological conditions are matrix metalloproteinases (MMPs, or matrixins), which belong to a family of zinc-dependent and calcium-activated neutral endopeptidases, comprising secreted and membrane-associated members. MMPs are involved in degradation of the ECM and basement membrane; however, they also cleave a variety of other ECM-related proteins, including cytokines, chemokines, and growth factors [6]. There is some specificity of certain MMPs toward collagen types, that is, MMP-2 and 9 (gelatinases) primarily degrade type IV collagen and other compounds of the ECM in muscle, whereas MMP-1 and 8 (collagenases) traditionally are thought to break down types I and III collagen, being more relevant for tendon. MMP activities are regulated by tissue inhibitors of matrix metalloproteinases (TIMPs). Four TIMPs, responsible for the inhibition of over20MMPs,areidentified;ofthese,TIMP-1andTIMP-2arecapableofinhibiting,ofallMMPs, preferably MMP-2 and 9, respectively [1]. In addition to MMP-dependent mechanisms, TIMPs can alter cell growth and survival in an MMP-independent manner, mediated by integrins. A good example is TIMP-2, which regulates beta1 integrin expression and the size of myotubes formedduringmyoblastdifferentiation[7].MMPsplay animportantrole inskeletalmuscle cell growthanddifferentiation,astheyareengagedinreleaseandactivationofcytokinesandgrowth

4 Composition and Function of the Extracellular Matrix in the Human Body

Fetal stage is crucial for skeletal muscle development, when muscle fibers are formed by fusion of mesodermal progenitor cells, myoblasts. During postnatal period, the number of myofibers remains constant; however, the size of each myofiber can increase by fusion with muscle stem cells, called satellite cells. Skeletal muscle is one of the most adaptive tissues in the body, and the adult regenerative myogenesis after muscle injury depends on satellite cells. These cells are normally quiescent, but in response to overloading or muscle damage, they become activated; that is, they begin to proliferate, and their progeny myoblasts terminally differen‐ tiate and fuse with one another or with existing myofibers to restore the contractile muscle apparatus and normal tissue architecture [8].

Proper muscle regeneration depends on the cross-talk between the satellite cells and their microenvironment (cell niche). According to the stem cell niche concept, the structural and biochemical stimuli emanating from surrounding environment determine the fate of stem cells present in tissues. Muscle satellite cells exist in highly specific niches, consisting of the basement membrane of myofibers, different types of resident cells (i.e., fibroblasts, adipocytes, etc.), vascular and neural systems, and extracellular matrix [9]. Each of these niche elements exerts profound effects on satellite cell functioning. Satellite cells reside between the basal lamina and the apical sarcolemma of myofibers, covered in laminin. They bind to collagen type IV and laminin through integrins, which also connect with collagen type VI and several proteoglycans, that is, perlecan and decorin. The ECM protein, nidogen (or entactin), supports cross-links between laminin and collagens. Basal lamina directly contacts satellite cells and separates them from muscle interstitium. It also acts as a mechanical barrier to prevent migration of satellite cells and their loss from normal muscle, and could be involved in repressing satellite cell mitosis and differentiation in the absence of muscle injury [2]. On the other site of the satellite cell niche, the myofiber sarcolemma links to the basal lamina, more particularly to laminin, through the dystroglycan complex [10]. Myofibers influence satellite cell behavior as a result of the physical interactions and by the secretion of paracrine factors. Nerves and associated neuromuscular apparatus exert their effects through the control of myofiber activity. Fibroblasts primarily contribute to matrix formation and, as adipocytes, secrete paracrine factors. Circulating blood transports hormones and other systemic factors; endothelial cells lining blood vessels serve as a source of growth factors, whereas immune cells, infiltrating muscle tissue upon injury, transiently affect satellite cells through the secretion of cytokines [11].

An important function of muscle progenitor cell niche is maintaining the balance between quiescence and activation. The quiescent satellite cells sense the stiffness of their niche through integrins and express various matrix proteins to maintain the stable ECM structure. Within the ECM, growth factors and other bioactive molecules are sequestered, supporting the "quiet" state [10]. Communication between the ECM and satellite cells is essential in the regulation of cellular events crucial for muscle growth and repair, such as gene expression, cell proliferation, adhesion, and differentiation of activated satellite cells. In response to muscle injury, compo‐ nents of the basal lamina are degraded by matrix metalloproteinases, and growth factors and signaling molecules are liberated, which is essential for regulation of processes ongoing in activated satellite cells. Presence of the ECM is required for muscle stem cells to respond to growth factors [12]. Proteoglycans expressed on the surface of satellite cells function as lowaffinity receptors and bind to the secreted, inactive growth factor precursors, including hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor isoforms (IGF-1, IGF-2), originating from myofibers, satellite cells, muscle-residing cells, or serum. All these growth factors play crucial roles in myogenesis, and in vivo exist in matrix-associated form. Some ECM molecules, that is, decorin [13], fibronectin [14], and laminin [15], can bind to and suppress the activity of myostatin, a negative regulator of muscle cell proliferation and differentiation. Through interactions with these growth factors, the extracellular matrix regulates the ability of skeletal muscle satellite cells to proliferate or differentiate. Differences in the expression of proteoglycans alter satellite cell responsiveness to the growth factor, that is, overexpression of glypican-1 (heparin sulfate proteoglycan) in satellite cells increases their responsiveness to FGF-2, whereas underexpres‐ sion diminishes cell proliferation and differentiation [12]. Taken together, the major compo‐ nents of basal lamina orchestrate muscle satellite cell development by presentation of mitogenic and myogenic factors. Muscle cells play an active role in creating their own microenvironment via ECM remodeling. Supporting this idea, numerous studies prove changes in expression and/or secretion of proteoglycans, metalloproteinases, adhesion molecules, and growth factors in regenerating muscle tissue and differentiating myoblasts [8, 16–18]. Activated satellite cells dynamically remodel their niche via transient high expression of fibronectin, and knockdown of this protein expression in satellite cells markedly impaired the ability to repopulate the niche [19].

When satellite cells move to the injured site, the surrounding ECM should be degraded for allowing cell migration. Matrix metalloproteinases degrade extracellular matrix components such as collagens, elastin, fibronectin, laminin, and proteoglycans. MMPs play an important role in creating cell niche in regenerating muscle and are essential for satellite cell activation, migration, and differentiation. Expression of matrix metalloproteases is up-regulated upon satellite cell activation, whereas transcripts for proteinase inhibitors are high in quiescent cells [20]. Migration of satellite cells underneath the basement membrane requires the expression of MMP-2, 3, 7, 9, and 10. The specific inhibition of these MMPs decreases the migration velocity and increases the sustainability of moving direction of myoblasts in vitro [21]. Among the MMPs expressed in skeletal muscle, MMP-2 and 9 appear particularly critical. MMP-2 is secreted by satellite cells and regenerating myofibers, whereas MMP-9 is expressed by leukocytes and macrophages. Upon injury, the release of the nitric oxide synthase (NOS) from damaged basal lamina leads to nitric oxide (NO) production, which in turn up-regulates protein level and activity of MMP-2 and 9. Activated proteases degrade collagen IV, facilitating satellite cell migration across the basement membrane to injured regions [22]. The most important details concerning the ECM structure and cues emanating from cellular elements of muscle satellite cell niche are summarized in **Figure 1**.

myofiber activity. Fibroblasts primarily contribute to matrix formation and, as adipocytes, secrete paracrine factors. Circulating blood transports hormones and other systemic factors; endothelial cells lining blood vessels serve as a source of growth factors, whereas immune cells, infiltrating muscle tissue upon injury, transiently affect satellite cells through the

An important function of muscle progenitor cell niche is maintaining the balance between quiescence and activation. The quiescent satellite cells sense the stiffness of their niche through integrins and express various matrix proteins to maintain the stable ECM structure. Within the ECM, growth factors and other bioactive molecules are sequestered, supporting the "quiet" state [10]. Communication between the ECM and satellite cells is essential in the regulation of cellular events crucial for muscle growth and repair, such as gene expression, cell proliferation, adhesion, and differentiation of activated satellite cells. In response to muscle injury, compo‐ nents of the basal lamina are degraded by matrix metalloproteinases, and growth factors and signaling molecules are liberated, which is essential for regulation of processes ongoing in activated satellite cells. Presence of the ECM is required for muscle stem cells to respond to growth factors [12]. Proteoglycans expressed on the surface of satellite cells function as lowaffinity receptors and bind to the secreted, inactive growth factor precursors, including hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor isoforms (IGF-1, IGF-2), originating from myofibers, satellite cells, muscle-residing cells, or serum. All these growth factors play crucial roles in myogenesis, and in vivo exist in matrix-associated form. Some ECM molecules, that is, decorin [13], fibronectin [14], and laminin [15], can bind to and suppress the activity of myostatin, a negative regulator of muscle cell proliferation and differentiation. Through interactions with these growth factors, the extracellular matrix regulates the ability of skeletal muscle satellite cells to proliferate or differentiate. Differences in the expression of proteoglycans alter satellite cell responsiveness to the growth factor, that is, overexpression of glypican-1 (heparin sulfate proteoglycan) in satellite cells increases their responsiveness to FGF-2, whereas underexpres‐ sion diminishes cell proliferation and differentiation [12]. Taken together, the major compo‐ nents of basal lamina orchestrate muscle satellite cell development by presentation of mitogenic and myogenic factors. Muscle cells play an active role in creating their own microenvironment via ECM remodeling. Supporting this idea, numerous studies prove changes in expression and/or secretion of proteoglycans, metalloproteinases, adhesion molecules, and growth factors in regenerating muscle tissue and differentiating myoblasts [8, 16–18]. Activated satellite cells dynamically remodel their niche via transient high expression of fibronectin, and knockdown of this protein expression in satellite cells markedly impaired

When satellite cells move to the injured site, the surrounding ECM should be degraded for allowing cell migration. Matrix metalloproteinases degrade extracellular matrix components such as collagens, elastin, fibronectin, laminin, and proteoglycans. MMPs play an important role in creating cell niche in regenerating muscle and are essential for satellite cell activation, migration, and differentiation. Expression of matrix metalloproteases is up-regulated upon satellite cell activation, whereas transcripts for proteinase inhibitors are high in quiescent cells

secretion of cytokines [11].

6 Composition and Function of the Extracellular Matrix in the Human Body

the ability to repopulate the niche [19].

**Figure 1.** Schematic representation of the complex microenvironment (niche) of satellite cells in skeletal muscle. Left part illustrates the networks and cross-linkings of major ECM proteins in the immediate environment of muscle satel‐ lite cells. Right part presents contributions of cellular components in creating the satellite cell niche. The small symbols represent humoral factors released by different types of cells (the colors used correspond with the source of appropri‐ ate bioactive factors).

The role of specific niche for muscle stem cell's self-renewal and differentiation is supported by observations, that after removal from the microenvironment, the satellite cells quickly withdraw from quiescence, begin to proliferate, and lose their myogenic properties. On the other hand, myogenic cells cultured on the ECM extracted from large thigh adult muscles manifest enhanced proliferation and differentiation in comparison to standard growth surfaces [23]. In order to study the role of specific ECM components in creating the niche of muscle stem cells, in vitro cell culture models are employed, where the environmental conditions can be easily controlled. In such experiments, primary muscle stem cells derived from muscle tissue are cultured in vitro on surfaces coated with the ECM components (i.e. collagen, laminin, fibronectin, gelatin, or Matrigel––a balanced mixture of different ECM proteins) to mimic the muscle extracellular environment. Usually, the primary muscle stem cells show distinct proliferation and differentiation pattern, as well as different muscle-specific and ECM-related gene expressions, dependent on the coating type used [9]. These experiments reveal that the loss of mitogenic and/or myogenic potential of muscle stem cells, due to their transfer from the specific niche to an ex vivo situation, could be reduced by using some ECM components/mixture coating. For example, fibronectin and laminin could be used for sorting myoblasts from fibroblasts. Such observations are of great interest and importance in tissue engineering and stem cell therapies.
