**2. Muscle homeostasis**

#### **2.1. Satellite cells are the secret of skeletal muscle regeneration**

The secret of skeletal muscle staggering regenerative capacity is found in the specific compo‐ nents of its cell niche. The muscle tissue is composed of long and slender cells that form muscle fibers grouped in bundles (Figure 1). Adjacent to these myofibers, a heterogeneous pool of subsarcolemmal progenitor and stem cells known as muscle satellite cells (SC), respectively committed to myogenic differentiation or to self-renewal, guarantee a fast and efficient regenerative process after trauma [7]. These cells, activated by injury [8], work hierarchically to maintain the *in situ* pool of cells (Figure 1) and to reconstruct damaged tissue in less than one month by differentiating into new myotubes.

#### **2.2. Injury and inflammation — The role of inflammation**

After trauma an inflammatory infiltrate can be observed when neutrophils, macrophages, satellite cells and later myoblasts work chronologically together cleaning up damaged fibers and reconstructing new functional myotubes. Neutrophils are the first cells to arrive at the site of injury, followed by macrophages three hours after damage [6]. Through the com‐ bined action of free radicals, growth factors and chemotactic factors these inflammatory cells contribute both to injury and repair [9]. Without the neutrophil-related oxidative and pro‐ teolytic modifications of damaged tissue, phagocytosis of debris would not be possible [10]. Macrophages are the major housecleaners that remove remaining debris of fibers. Further‐ more, macrophages produce proteases to lyse the sarcolemma membrane, which allows acti‐ vation and proliferation of SC [11]. Dismantling of the extracellular matrix is key to SC activation, and the up-regulation of metalloproteinase is required for muscle regeneration [12]. Macrophage infiltration is also important for SC activation and proliferation by activat‐ ing NF-κB via TWEAK ligand [13]. Quiescent SCs are still found between the basal mem‐ brane and sarcolemma until the third day after injury. Subsequently, they are slowly replaced by cells with large nuclei, nucleoli, and cytoplasmatic processes filled with ribonu‐ cleoprotein granules. These myoblasts display an initial exponential growth phase and after the seventh day they start to form myotubes with centrally placed nuclei and peripheral my‐

Stem cell therapy as a treatment for skeletal muscle diseases is becoming a reality and it represents a promising alternative for muscle regeneration and for treating SUI in a more

In this chapter, the homeostasis and maintenance of skeletal muscle is explained in order to understand the basis behind muscle regeneration. As different types of stem cells have been demonstrated to form fibers and to develop into skeletal muscle, cell sources for a muscle cell therapy is discussed. Some of them have also been applied successfully in preclinical and clinical studies that are going to be described. Finally, we are going to highlight the parts important for the translational effort into clinics including biomaterials, cell delivery, imaging,

The secret of skeletal muscle staggering regenerative capacity is found in the specific compo‐ nents of its cell niche. The muscle tissue is composed of long and slender cells that form muscle fibers grouped in bundles (Figure 1). Adjacent to these myofibers, a heterogeneous pool of subsarcolemmal progenitor and stem cells known as muscle satellite cells (SC), respectively committed to myogenic differentiation or to self-renewal, guarantee a fast and efficient regenerative process after trauma [7]. These cells, activated by injury [8], work hierarchically to maintain the *in situ* pool of cells (Figure 1) and to reconstruct damaged tissue in less than

After trauma an inflammatory infiltrate can be observed when neutrophils, macrophages, satellite cells and later myoblasts work chronologically together cleaning up damaged fibers and reconstructing new functional myotubes. Neutrophils are the first cells to arrive at the site of injury, followed by macrophages three hours after damage [6]. Through the com‐ bined action of free radicals, growth factors and chemotactic factors these inflammatory cells contribute both to injury and repair [9]. Without the neutrophil-related oxidative and pro‐ teolytic modifications of damaged tissue, phagocytosis of debris would not be possible [10]. Macrophages are the major housecleaners that remove remaining debris of fibers. Further‐ more, macrophages produce proteases to lyse the sarcolemma membrane, which allows acti‐ vation and proliferation of SC [11]. Dismantling of the extracellular matrix is key to SC activation, and the up-regulation of metalloproteinase is required for muscle regeneration [12]. Macrophage infiltration is also important for SC activation and proliferation by activat‐ ing NF-κB via TWEAK ligand [13]. Quiescent SCs are still found between the basal mem‐ brane and sarcolemma until the third day after injury. Subsequently, they are slowly replaced by cells with large nuclei, nucleoli, and cytoplasmatic processes filled with ribonu‐ cleoprotein granules. These myoblasts display an initial exponential growth phase and after the seventh day they start to form myotubes with centrally placed nuclei and peripheral my‐

complete and definitive manner.

680 Regenerative Medicine and Tissue Engineering

regulatory affairs, and manufacturing.

**2.1. Satellite cells are the secret of skeletal muscle regeneration**

one month by differentiating into new myotubes.

**2.2. Injury and inflammation — The role of inflammation**

**2. Muscle homeostasis**

**Figure 1. The muscle niche is the secret of skeletal muscle astounding regenerative capacity.** Attached to bones, skeletal muscle are organs composed of skeletal muscle tissue, connective tissue, nerves and blood vessels. Each indi‐ vidual skeletal muscle is composed by hundreds or thousands bundles of muscle fibers that are single cylindrical mus‐ cle cells. (A) The connective tissue surrounding each muscle is called epimysium, and its projections that separe muscle bundles are called perimysium. (B) The connective tissue between single muscle fibers is called endomysium and serv‐ ers as the muscle satellite cells (SCs) niche. SCs are subsarcolemmal cells that can be activated to regenerate new mus‐ cle fibers. (C) Skeletal Muscle tissue is not only formed by muscle fiber, but also by acellular matrix, cellular components, blood and lymphatic vessels and nerves. Altogether, these muscle niche components play a distinct role on muscle regeneration and on muscle progenitor cell regulation.

ofibrils. On the periphery of these newly formed myotubes a new population of subsarco‐ lemmal quiescent cells replenishes the SC pool [6]. Finally, mature myofiber nuclei do not display mitotic figures throughout the regeneration process, demonstrating that the dam‐ aged fiber cannot heal itself without the activation of satellite cells.

#### **2.3. The role of the muscle niche in muscle regeneration**

Components of the muscle niche are important for skeletal muscle regeneration and satellite cell activation. The basal lamina is the common anatomic site of satellite cells and also contributes to cell fate. The basal lamina is rich in α7β1 integrin which acts directly in the anchorage, adhesion and quiescence of satellite cells [14]. These integrin functions also comprise the migration and proliferation of developing myoblasts [15], the forma‐ tion and integrity of neuromuscular junctions [16], as well as the binding of muscle fibers. Another integrin, VLA-4, is expressed as myotubes form and influences the alignment and fusion of myoblasts [17]. Finally, the calcium-dependent cell adhesion protein M-cadher‐ in is a morphoregulatory molecule facilitating myoblast fusion and cell adhesion to its adjacent myofibers [18, 19].

The surrounding acellular matrix (ACM) contains a number of components that can influence the behavior and regulate the growth of muscle progenitor cells. The ACM is a source of hepatocyte [20] and fibroblast [21] growth factors, which act on the activation of satellite cells, proliferation and inhibition of differentiation. Another factor produced by the ACM is the endothelial growth factor, which promotes satellite cell activation and cell survival after injury [22]. Finally, the aged ACM is capable of impairing the regenerative potential of satellite cells and inducing fibrosis by activating the Wnt signaling pathway [23].

Fibroblasts are the main source of collagen in the muscular interstitial space [24]. They continuously promote the formation of the basal lamina during myogenesis [25] and after muscle injury proliferate hand in hand with Pax7 positive satellite cells, orchestrating the fine balance between muscle reconstruction and fibrosis formation [26]. These fibroblasts prevent premature activation and differentiation of muscle progenitor cells, thereby avoiding depletion of the pool of satellite cells. Accordingly, satellite cells are sufficient to regulate the ingrowth of fibroblasts and fibrosis formation [26]. Fibroblasts are also involved in myosin switch from fetal to adult muscle, specially promoting Myosin Heavy Chain type 1 expression (slow twitch) in several limb muscles in the fetal mouse and in the soleus in the adult muscle [27].

Circulating and locally produced soluble factors participate in the signaling pathway that regulates satellite cell activity. During exercise and stretching muscle fibers liberate hepatocyte growth factor (HGF) through nitric oxide stimulation and induce activation of satellite cells [28]. HGF can also activate satellite cells by activating the sphingolipid signaling cascade upon disruption of the laminin-integrin adhesion in the event of trauma [29]. Furthermore, the insulin-like growth factor 1 (IGF-1), a potent mitogen produced locally during muscle hypertrophy and injury, can induce activation, proliferation and differentiation of satellite cells [8, 30]. In contrast, mysotatin, a growth differentiation factor and member of the TGF-beta protein family secreted by adult skeletal muscle, is capable of inhibiting activation and selfrenewal of quiescent cells [31]. Finally, a hormone produced by the thyroid gland and responsible for inducing hypercalcemia named Calcitonin [32], has been associated with delay of satellite cell activation [33]. Together all these components and products of the muscle niche are key regulators of all the development and regeneration processes of skeletal muscle.

#### **2.4. Satellite cells are also required for exercise related muscle turn–over**

Exercise is capable of activating muscle gene transcription within seconds and these molecular responses can last for hours even after exercise cessation [34]. During endurance exercise, muscle consume large amounts of oxygen to generate energy by breaking down carbohydrates and posteriorly fat [35]. Muscle fibers are not in a smooth continuous muscle contraction during exercise, but rather act as a series of small groups of fibers contracting at the same time [36]. This occurs due to stimulation of neuromuscular junctions of terminal branches of axons whose cell body is in the anterior horn of the spinal cord. Altogether, these nerve and muscle components comprise the motor unit [37] and conduce impulses that enable sharp muscle contraction within milliseconds [38]. A signaling pathway is then activated by rapamycin kinase (mTOR) leading to hypertrophic changes in muscle mass [39]. The opposing effect is found during starvation when the AMP-activated protein kinase (AMPK) is switched on to up-regulate energy-conserving processes and ultimately induce muscle atrophy [39]. How‐ ever, exercise is sufficient to increase the pool of stem cells reversing the effects of atrophy after prolonged limb immobilization [40].

After a trauma or during exercise nitric oxide is liberated and modulates the activation of satellite cells [41, 42]. Another evidence of this cell addition during exercise is the decrease of telomeres length detected in marathon runners, which correlates to their running hours [43]. Endurance exercise has been reported to stimulate the production of free radicals like nitric oxide [44], which has been shown to again induce activation of satellite cells thereby increasing muscle turn-over [28]. On the other hand, during muscle atrophy caused by limb immobili‐ zation an apoptotic decrease of myonuclei occurs [45] associated with a decrease in mitotic activity of satellite cells [46]. These findings underline the involvement of satellite cells in the regulation of muscle mass during exercise.

#### **2.5. Markers for satellite cells**

ofibrils. On the periphery of these newly formed myotubes a new population of subsarco‐ lemmal quiescent cells replenishes the SC pool [6]. Finally, mature myofiber nuclei do not display mitotic figures throughout the regeneration process, demonstrating that the dam‐

Components of the muscle niche are important for skeletal muscle regeneration and satellite cell activation. The basal lamina is the common anatomic site of satellite cells and also contributes to cell fate. The basal lamina is rich in α7β1 integrin which acts directly in the anchorage, adhesion and quiescence of satellite cells [14]. These integrin functions also comprise the migration and proliferation of developing myoblasts [15], the forma‐ tion and integrity of neuromuscular junctions [16], as well as the binding of muscle fibers. Another integrin, VLA-4, is expressed as myotubes form and influences the alignment and fusion of myoblasts [17]. Finally, the calcium-dependent cell adhesion protein M-cadher‐ in is a morphoregulatory molecule facilitating myoblast fusion and cell adhesion to its

The surrounding acellular matrix (ACM) contains a number of components that can influence the behavior and regulate the growth of muscle progenitor cells. The ACM is a source of hepatocyte [20] and fibroblast [21] growth factors, which act on the activation of satellite cells, proliferation and inhibition of differentiation. Another factor produced by the ACM is the endothelial growth factor, which promotes satellite cell activation and cell survival after injury [22]. Finally, the aged ACM is capable of impairing the regenerative potential of satellite cells

Fibroblasts are the main source of collagen in the muscular interstitial space [24]. They continuously promote the formation of the basal lamina during myogenesis [25] and after muscle injury proliferate hand in hand with Pax7 positive satellite cells, orchestrating the fine balance between muscle reconstruction and fibrosis formation [26]. These fibroblasts prevent premature activation and differentiation of muscle progenitor cells, thereby avoiding depletion of the pool of satellite cells. Accordingly, satellite cells are sufficient to regulate the ingrowth of fibroblasts and fibrosis formation [26]. Fibroblasts are also involved in myosin switch from fetal to adult muscle, specially promoting Myosin Heavy Chain type 1 expression (slow twitch) in several limb muscles in the fetal mouse and in

Circulating and locally produced soluble factors participate in the signaling pathway that regulates satellite cell activity. During exercise and stretching muscle fibers liberate hepatocyte growth factor (HGF) through nitric oxide stimulation and induce activation of satellite cells [28]. HGF can also activate satellite cells by activating the sphingolipid signaling cascade upon disruption of the laminin-integrin adhesion in the event of trauma [29]. Furthermore, the insulin-like growth factor 1 (IGF-1), a potent mitogen produced locally during muscle hypertrophy and injury, can induce activation, proliferation and differentiation of satellite cells [8, 30]. In contrast, mysotatin, a growth differentiation factor and member of the TGF-beta protein family secreted by adult skeletal muscle, is capable of inhibiting activation and self-

aged fiber cannot heal itself without the activation of satellite cells.

and inducing fibrosis by activating the Wnt signaling pathway [23].

**2.3. The role of the muscle niche in muscle regeneration**

adjacent myofibers [18, 19].

682 Regenerative Medicine and Tissue Engineering

the soleus in the adult muscle [27].

A transcriptional network controls progression of both embryonic and adult muscle stem cells [47]. Quiescent muscle embryonic progenitor cells can be identified by the coexpression of the paired-domain transcription factors Pax3 and Pax7 (Figure 2) and are maintained as a self-renewing proliferative population [48]. During embryogenesis Pax3 is required to maintain muscle progenitor cells in the somite and further induce cell migration to the required site of skeletal myogenesis [49]. Indeed the normal expression of Pax3 seems to be decisive for the development of normal muscle, and its mutation promotes malig‐ nant growth and induces tumorigenesis in alveolar rhabdomyosarcoma tumor cells [50]. However, its down-regulation is necessary for final cell commitment to myogenesis and leads to rapid and robust entry into the myogenic differentiation program [49]. The expression of transcription factor Pax7 is detectable in cells starting from the embryonic muscle progenitor to the quiescent and activated satellite cells (Figure2). Its induction in muscle-derived stem cells induces satellite cell specification by restricting alternate developmental programs [51].

**Figure 2. Myogenic cell characterization and culture.** Myogenic cell lineage can be identified in each differentiation state and pursue tightly regulated proliferation and differentiation cycles. From the embryonic state until the terminal differentiation into muscle fibers an intricate network of transcription factors regulates the fate of muscle progenitor cells. These cells can be isolated from any skeletal muscle tissue, grown in culture and reimplanted into a damaged muscle to promote muscle regeneration.

Specific molecular markers have been demonstrated to distinguish between activated and quiescent SC. Quiescent satellite cells express the transcription factor Pax7, after activation in co-expression with MyoD [52]. This dual expression is followed by a proliferative phase, downregulation of Pax7 and terminal differentiation. If Pax3 and Pax7 down-regulation do not occur *in vitro* differentiation is blocked [53, 54]. In this context microRNAs (miRNAs) play a regula‐ tory role conferring robustness to developmental timing by posttranscriptional repression of genetic programs of progenitor and satellite cells [55]. They allow rapid gene program transitions from proliferation to differentiation, blocking PAX3 [56] and Pax7 [57] activity in progenitor and satellite cells.

This interplay during development is required to ignite the commitment of satellite cells to the myogenic program, to activate the myogenic regulatory factors Myf-5 and MyoD and to promote terminal muscle differentiation [55] [58] [59], which are decisive to subsequent myoblast cell cycle progression or exit into differentiation. Through the action of the myogenic regulatory factors (MRFs), Myf5 and MyoD, the muscle progenitor cells (Pax3+ ) and quiescent satellite cells (Pax3+ /Pax7+ ) become muscle lineage committed and activated myoblasts [60]. They express *Myf5* and *Mrf4* and rapidly give rise to Desmin+ cells, whose differentiation is regulated by myogenin, MyoD and MRF4 [61]. Completing these regulatory features, MyoD is also a main player in the intricate epigenetic cascade that controls skeletal myogenesis [62].
