Regulatory Landscapes of Muscle Satellite Cells: From Mechanism to Application

Abstract

Muscle satellite cells (SCs), also known as muscle stem cells, are crucial for the regeneration, maintenance, and growth of skeletal muscles. SCs possess a distinctive capability to self-renew and differentiate, rendering them highly promising candidates for regenerative therapies and emerging cellular agriculture applications, including cultured meat production. This review explores the mechanisms that govern SC activation, proliferation, and commitment, and emphasizes their functional heterogeneity across anatomical regions. Region-specific gene expression, including that of homeobox (Hox) genes, contributes to the positional identity and myogenic potential. Understanding these regulatory landscapes is essential for optimizing SC expansion and improving their applications in muscle repair, stem cell-based therapies, and cellular manufacturing systems.

Introduction

Skeletal muscles constitute approximately 40% of the adult human body weight and play a pivotal role in movement, posture, and metabolism (1). Satellite cells (SCs), located between the basal lamina and sarcolemma, serve as the principal stem cells for skeletal muscle regeneration and growth (2). Upon exposure to injury, exercise, or developmental cues, quiescent SCs are activated and undergo cell cycle entry, followed by clonal expansion and commitment to the myogenic lineage (3, 4). A subset of these cells engages in self-renewal, subsequently reverting to a quiescent state to replenish the SC pool and maintain their long-term regenerative potential (4, 5). While SCs are essential for postnatal muscle growth and therapeutic applications, they rapidly lose their intrinsic characteristics when isolated and cultured in vitro (6, 7). Furthermore, SCs are not a homogeneous population. They show intrinsic diversity based on their anatomical origin within the same individual, and this variation is further increased during in vitro culture owing to differing cellular responses and fate decisions (8-10). The muscles forming the head, trunk, and limbs arise from distinct mesodermal lineages, with each region governed by its own regulatory program involving regionally expressed genes, such as Pitx2, Tbx1, Lbx1, and Hox genes (10-13). These molecular signatures persist throughout adulthood and affect cell proliferation, differentiation, and stemness (14). Therefore, the recognition of anatomical and molecular heterogeneity in SCs is crucial not only for the accurate interpretation of experimental data but also for enhancing our understanding of their biological behavior and therapeutic potential.

This review provides insights into the intrinsic and extrinsic regulatory mechanisms of muscle SCs, highlighting their anatomical diversity and the significance of this heterogeneity in basic biological and applied bioengineering research.

Potential of Muscle SCs

Skeletal muscle constitutes approximately 40% of the human adult body weight (2) and is a significant constituent of lean body mass in livestock animals (15). Skeletal muscle primarily consists of muscle fibers, which lack the capacity for proliferation, but increase in muscle size via the growth of muscle SCs and undergo regeneration through the proliferation and differentiation of muscle SCs (2). SCs, also known as muscle stem cells, are small cells first identified in frog skeletal muscle fibers (16) and are located between the basal lamina and plasma membrane of the muscle fiber (Fig. 1). Their peripheral position on the muscle fiber led to their designation as “SC” (2, 16). This location also contributes to their characteristic wedged morphology, as observed in cross-sectional views of the muscle tissue (16). When observed in vitro under an inverted microscope, SCs exhibit a spindle-like morphology with slight sphericity (17).

Fig. 1.

Representative images of the satellite cell (SC) niche. SCs are located on the periphery of the muscle fiber. The positioning of the SCs between the sarcolemma and basal lamina is shown. SCs are surrounded by various cell types that can significantly influence their characteristics. These include endothelial cells, mesenchymal stem cells, pericytes, and connective tissue cells.

SCs interact with various other cell types, such as endothelial cells, mesenchymal stem cells, pericytes, and connective tissue cells, thereby influencing their proliferation and differentiation (18-22). In addition, SCs are regulated by mechanical and electrical signals and through interactions with the extracellular matrix (ECM) (4). However, the complexity of these external cues, combined with the lack of paracrine interactions in the in vivo environment, poses significant challenges in replicating these conditions in vitro, hindering the maintenance of SC characteristics during culture.

In vivo, SCs are maintained in a quiescent state until activation, after which they rapidly proliferate and differentiate (3, 23). A subset of these cells enters a quiescent state and undergoes asymmetric division to uphold the SC pool (24). However, owing to the absence of key regulatory signals in vitro, SCs rapidly enter the cell cycle and undergo premature differentiation or senescence, limiting their proliferative capacity (7, 25, 26). In particular, during prolonged in vitro cultivation, a decrease in both the expression of PAX7 and differentiation potential of bovine SCs was observed (7). The reduced SC stemness was compensated for through chemical treatment, with p38 inhibition promoting self-renewal and sustained PAX7 expression during long-term in vitro culture. These findings highlight the pivotal role of signaling pathways in preserving the characteristics of SCs.

Muscle SCs possess the intrinsic ability to regenerate muscle tissue, making them a key model in muscle development, disease, and aging research. Currently, SCs are considered as one of the most promising cell sources for cultured meat production (27, 28). However, preserving their functional characteristics during extended in vitro culture remains challenging, largely because of their substantial heterogeneity and gradual loss of stemness over time (9). Hence, ongoing studies aim to optimize in vitro culture conditions to support long-term maintenance while enhancing their proliferative and differentiation potential.

Intrinsic Regulatory Mechanisms of Muscle SCs

Muscle SC exist primarily in a quiescent state unless stimulated (24, 29). In the quiescent state, they remain in the G0 phase, refraining from proliferation and differentiation, and maintaining low metabolic rates (2, 29). Although quiescent SCs are transcriptionally inactive, certain genes are highly expressed during this stage. Specifically, regulators of G-protein signaling 2, p57, and sprouty1 (Spry1), which are negative regulators of cell cycle-related genes, were upregulated only in quiescent SCs, whereas they were downregulated in activated SCs (30). Forkhead box protein 3 (FOXO3), a member of the forkhead family of transcription factors, blocks cell-cycle progression and is highly expressed in quiescent SCs (31). MyoD inhibitory-related genes, such as bone morphogenetic protein (BMP) 6, BMP4, HeyL, and Notch are also upregulated in quiescent SCs. However, some genes are actively expressed in both the quiescent and activated states. Pax7, a well-known SC marker, is highly expressed in both phases (32).

SCs are activated by myogenic stimuli including muscle trauma, muscle development, and exercise-induced muscle damage (33, 34). Upon activation, SCs enter the cell cycle, transition from G0 to G1 phase, and initiate active proliferation (34). Pax7 and myogenic regulatory factors (MRFs) stringently regulate the myogenic SC lineage (Fig. 2) (35). MRFs include MyoD, Myf5, Myogenin, and Mrf4 (36). Activated SCs are characterized by increased MyoD expression and stable Pax7 expression (37). After approximately 24 hours of in vitro culture, most SCs exhibit this activated state, expressing both MyoD and Pax7 (38). Pax7 is responsible for maintaining the active proliferation and survival of SCs and functions upstream of MyoD (39). MyoD also fosters SC proliferation (40). These committed cells, known as myoblasts, multiply actively and undergo either asymmetric or symmetric division (Fig. 3) (19, 41, 42). An elevated ratio of symmetric divisions facilitates enlargement of the SC pool, whereas an increase in asymmetric divisions supports the generation of myogenic progenitors (37, 43). Myf5, the expression of which is regulated by Pax7, plays a key role in determining the division pattern of activated SCs (44). When the methyltransferase Carm1 methylates the arginine residue of Pax7, recruiting histone methyltransferase complexes to the Myf5 locus, thereby promoting transcription (44-46). Consequently, Myf5 is expressed in a subset of SCs, with Myf5-positive cells tending to undergo differentiation, whereas Myf5-negative cells are more likely to return to a quiescent state (5). In addition, Myf5 regulates myoblast proliferation (47). However, unlike other MRFs such as MyoD, Myogenin, and Mrf4, Myf5 expression alone does not directly influence myoblast differentiation (47, 48).

Fig. 2.

Myogenic lineages and gene expression. Quiescent satellite cells (SCs) express PAX7, whereas lack MYOD expression. Upon activation, these cells undergo active proliferation and begin to coexpress both PAX7 and MYOD. Upon proliferation, SCs have the potential to either undergo self-renewal or proceed toward differentiation, depending on MYF5 expression. The orange table presents genes that are expressed in SCs at each state, along with representative transcription factors.

Fig. 3.

Asymmetric and symmetric stem cell division. Satellite cells can self-renew via two types of stem cell division. Asymmetric cell division along the apico-basal axis yields one daughter cell that is committed to differentiation, whereas the other is focused on self-renewal. Symmetric cell division along the planar axis results in the formation of two daughter cells that either undergo differentiation or maintain self-renewal.

MyoD plays a critical role in the transition from an actively proliferating myoblast state to an early differentiation state known as myocytes (37). In the presence of persistent differentiation signals, Pax7 expression diminishes, whereas MyoD expression is increased (Fig. 3) (49). MyoD has the capacity to induce both the proliferation and differentiation of SCs (37). Investigations on the expression pattern of MyoD in relation to SC cell cycle progression showed that its levels were very low during the G0 phase, which is indicative of quiescence. However, MyoD expression increases during the G1 phase, marking a transition out of quiescence (37, 50, 51). Increased levels of MyoD expression decrease during the G1/S transition and then gradually increase again from the S phase (50, 51). Elevated MyoD expression stimulates skeletal muscle differentiation (52). As differentiation begins, the upregulation of p16 and p18, which inhibit CDK6, along with p21 and p57, which inhibit G1 CDKs, promotes transition into the G0 phase (53-56). This is followed by sequential activation of Myogenin and Mrf4 expression (57). Myogenin acts together with MyoD to drive differentiation during the early stages of myocyte differentiation (58). Mrf4 is intricately involved in the fusion and hypertrophy of myofibers, and its expression drives myofiber maturation (59-61). Four MRFs, MyoD, Myf5, Myogenin, and Mrf4, do not function independently, but interact with various genes in complex and coordinated ways (19). Therefore, myogenesis is not a simple process dependent on a few factors but a complex phenomenon involving intricate molecular networks and gene interactions.

Upon activation and proliferation, a subset of SCs reverts to a quiescent state, thereby contributing to the long-term maintenance of the SC pool. In addition to transient proliferation, self-renewal ensures long-term muscle regenerative capacity. Neonatal myoblast transplantation into adult Myf5^nlacZ/＋ mice revealed that the transplanted myoblasts integrated into the stem cell pool, similar to SCs (62). Sacco et al. (63) demonstrated that a single muscle SC, isolated using FACS and transplanted into irradiated muscles of scid-mdx mice, was capable of extensive proliferation, contributing to muscle regeneration, and exhibiting autonomous self-renewal capacity.

The processes of symmetric and asymmetric division are essential for maintaining a balance among quiescence, proliferation, and differentiation. Of equal significance is the ability of SCs to return to a quiescent state after activation—a process essential for the long-term preservation of the SC pool. Like other stem cells, muscle SCs maintain their pool through symmetric and asymmetric cell division (2). Symmetric division (planar) refers to the process by which a cell divides into two identical daughter cells, differentiated or self-renewing (64). In contrast, asymmetric division (apical-basal) is a type of cell division in which one cell differentiates, whereas the other undergoes self-renewal (2, 64, 65). The asymmetric division of SCs is influenced by several factors. One of the most well-known factors is Myf5 (Fig. 4). During asymmetric division, SCs lacking Myf5 expression remain at the basal surface, whereas Myf5-expressing cells localize above Myf5-negative SCs and lose contact with the basal lamina and ECM (5). Accordingly, Pax7^＋/Myf5⁻ cells undergo self-renewal, whereas Pax7^＋/Myf5^＋ cells are destined for myogenic differentiation.

Fig. 4.

Patterning and molecular specification of epaxial and hypaxial muscles from the dermomyotome. (A) A schematic representation of the early somite, which is derived from the paraxial mesoderm, demonstrates its differentiation into the dermomyotome and sclerotome. The dermomyotome subsequently gives rise to epaxial (trunk) and hypaxial (limb) muscle precursors. Epaxial muscles develop adjacent to the neural tube, whereas hypaxial muscles contribute to the musculature of the limbs and body wall. (B) Molecular signal regulating of muscle lineage specification. Wnt1 and Wnt3 from the dorsal neural tube, along with BMP4 from the surface ectoderm, induce the formation of the dorsal dermomyotome and promote epaxial muscle development. In contrast, Wnt7 promotes the formation of hypaxial muscles without BMP4, a condition maintained by Noggin, which is secreted by the notochord and floor plate and functions as a BMP4 antagonist.

Extrinsic Regulatory Mechanisms of Muscle SCs

SCs reside within a microenvironment that provides essential extrinsic signals for regulating their fate (2, 4, 19-22). Myogenesis is a complex and intricately coordinated process affected by various external signaling pathways. Thus, fully understanding myogenesis necessitates insights into both intrinsic regulation and the extrinsic signals that shape SCs fate. This section provides brief overview of the extrinsic signaling pathways closely linked to the intrinsic regulation of SCs.

One of the most prominent extrinsic regulators is the NOTCH signaling pathway. The Notch signaling pathway also plays a key role in regulating asymmetric division (5). Delta is a ligand that binds to the Notch receptor on adjacent cells, thereby activating Notch signaling in those cells (66, 67). Notch signaling is downregulated in cells expressing Delta, allowing these cells to undergo differentiation (68). Therefore, SCs with downregulated Notch3 and upregulated Delta1 enter a commitment state (5). In contrast, those exhibiting upregulated Notch3 and down-regulated Delta1 preserve the SCs pool by maintaining their self-renewal capacity.

The forkhead box O (FOXO) transcription factors, which operate downstream of the PI3K/AKT pathway, act as upstream regulators of Notch signaling (69, 70). The FOXO family are involved in numerous cellular processes including proliferation differentiation, survival, and metabolism (71). Additionally, FOXO bind to similar DNA sequences and can exert overlapping functions, they also have distinct roles and exhibit differential expression patterns depending on cellular context. FOXO3, acting downstream of the PI3K/AKT pathway, regulates the Notch signaling pathway, and promotes SC self-renewal (31, 72). SCs isolated from the muscle of Foxo3^−/−mice showed downregulation of Notch1, Notch3, and Notch4, along with reduced expression of downstream genes (Hes1, Hes2, Hes6, and HeyL). Consequently, these cells exhibited impaired self-renewal and underwent differentiation (31). Furthermore, self-renewing SCs were not detected among the SCs extracted 1 month after tamoxifen-induced muscle injury. In contrast, activation of the Notch signaling pathway by Foxo3 ultimately led to the upregulation of Pax7 expression and concurrent downregulation of MyoD, contributing to the reversion to a quiescent state (5, 73). Foxo1 primarily functions as a negative regulator of myogenic differentiation (70). Loss of Foxo1 function attenuates the inhibitory effect of Notch signaling on myogenesis. As a result, MyoD expression increases, and myogenic differentiation is promoted.

The p38α/β family of mitogen-activated protein kinases (MAPKs) is essential at multiple levels for the transition from proliferation to differentiation in SCs (74). Through phosphorylation, p38 indirectly suppresses Pax7 expression via epigenetic regulators, while simultaneously enhancing the transcriptional activity of MyoD and MEF2, thereby promoting muscle differentiation and inducing cell cycle exit (74-76). The p38α/β MAPK signaling pathway is also recognized for its role in regulating the asymmetric division of SCs (77). When p38α/β MAPK is activated by the Par complex in only one of the daughter cells, that cell rapidly differentiates into a myoblast, whereas the non-activated daughter cell retains its stemness and undergoes self-renewal.

The Wnt7a exerts its effects via the Frizzled-7 (Fzd7) receptor and enhances muscle regeneration by stimulating symmetric cell division through Vangl2, a constituent of the planar cell polarity pathway (78). In myofibers extracted from Myf5-Cre/RoSA26-YFP mouse muscle and cultured in vitro, treatment with recombinant Wnt7a increased the ratio of symmetric division from 30% to 67%. This Wnt7a-induced symmetric division is further stimulated by the ECM glycoprotein fibronectin, which binds to the Fzd7 and Syndecan-4 complex, amplifying the signaling pathway (79). The ECM, including components like collagen VI, laminin, and fibronectin, serves as a scaffold that not only provides structural support but also modulates SC polarity, adhesion, and signal reception (2, 4). Mechanical and biochemical properties of the ECM play a crucial role in guiding SC behavior. The absence or alteration of niche signals in vitro often leads to spontaneous differentiation or senescence, highlighting the importance of maintaining appropriate extrinsic conditions for SC function.

Furthermore, fibroblast and hepatocyte growth factors are ligands of receptor tyrosine kinase (RTK) that influence SC activation and proliferation (80, 81). Spry1 acts as a negative feedback regulator in the RTK signaling pathway, particularly in response to growth factor signaling (82) and promotes cell cycle withdrawal (2, 83). These findings indicated the crucial function of Spry1 in promoting a reversible quiescent state.

Altogether, these extrinsic regulatory mechanisms interact closely with intrinsic transcriptional programs to regulate the fate of SCs, which can maintain the SC pool and enable regeneration.

Regulatory Mechanisms Governing Tissue Development across Anatomical Regions

Hox genes play a critical role in anterior-posterior specification across vertebrates and contribute significantly to developmental processes (84). These genes are organized into four major clusters, HoxA, HoxB, HoxC, and HoxD, each located on a different chromosome (85). During embryonic development, Hox genes exhibit sequential expression along the body axis, from the anterior to the posterior (86). This colinear and sequential activation is critical for the correct spatial and temporal development of body structures (87, 88). A total of 39 Hox genes have been categorized into 13 paralogous groups (89, 90), numbered 1 through 13, with lower numbers associated with the development of more anterior regions (84). For instance, Hoxa13–Hoxd13 specifically influence limb bud formation in mice (91). In addition, Hox genes retain their developmental patterns throughout postnatal growth and into adulthood. Jin et al. (14) demonstrated that adult pig muscles from different anatomical regions, such as the head, forelimb, trunk, and hind limb, showed distinct patterns of HOX expression, suggesting a role in maintaining regional muscle identity. The head, trunk, and limb muscles originate from different locations in the embryo and are influenced by different transcription factors owing to their unique developmental origins. These region-specific cues guide the proliferation of precursor cells at defined locations and their subsequent differentiation into specialized muscle types (8, 92). During embryonic development, the paraxial mesoderm is located on either side of the neural tube (93). The paraxial mesoderm forms somites, which give rise to the muscles of the trunk and limbs as well as many connective tissues (13, 93, 94). In contrast, head muscles follow a different segmental pattern (10). The most anterior paraxial mesoderm, which gives rise to the cranial paraxial mesoderm (CPM) responsible for head muscle development, differentiates into head mesoderm instead of forming somites (10, 95).

During maturation, somites differentiate into dermomyotomes and sclerotomes (Fig. 4) (96). The dermomyotome, occupying the dorsal half of the somite, further differentiates into the myotome, which is responsible for muscle formation, and dermatome, which gives rise to the dermis (97, 98). The initial observable indication of myogenesis in the embryo is the emergence of primitive myoblasts in myotomes (93, 94). In myotomes, MRFs play a fundamental role in orchestrating the formation, localization, proliferation, and differentiation of muscle cells across all anatomical muscle regions (99). The expression of MRFs is influenced by different regulatory factors depending on their anatomical location, although some factors are shared across regions. For example, trunk muscles originate from myoblasts in the dorsomedial lip of the dermomyotome (epiaxial muscles), which is adjacent to the neural tube (97, 100). Epaxial muscle myoblasts are activated by Wnt1a and Wnt3a, which are secreted from the roof plate of the neural tube, along with low levels of sonic hedgehog (Shh) derived from the floor plate of the neural tube (101-103). Wnt1a, Wnt3, and Shh induce Pax3 expression in somites to activate Myf5 (104), which is highly expressed in the epaxial regions (105). Eventually, Pax3 regulates the expression of Dmrt2 to activate Myf5 expression, while Dmrt2 regulates Myf5 by activating the epaxial enhancer, a specific DNA region that regulates Myf5 expression in the epaxial muscles (99, 106). Myf5, which is activated by Pax3, activates MyoD, leading to subsequent activation of myogenin (107). Myoblasts do not express MRFs during migration. However, upon reaching their designated locations, they begin to proliferate and gradually upregulate MRFs (108). Eventually, myoblasts align, fuse, and grow according to gene expression, forming back muscles that firmly connect to the developing spine (109).

Myoblasts derived from the ventrolateral lip, which are located farthest from the neural tube, primarily form hypaxial muscles, such as those in the limb, tongue, and diaphragm (97, 100, 110-112). The ventrolateral (hypaxial) lip of the dermomyotome undergoes epithelial–mesenchymal transition and migrates to the limb bud (113). In the transcriptional mechanism of hypaxial muscle development, Wnt7, which originates from the dorsal ectoderm, acts as a direct activator of MyoD (114). BMP4 antagonizes the activity of Wnt7 and suppresses the premature expression of MyoD (96, 114). However, BMP4 expression is suppressed by noggin protein secreted from the medial lip of the dermomyotome; ultimately, Wnt7 activity is not suppressed (115). Through this process, the development of the hypaxial muscles is achieved. Sine oculis homeobox homologue (SIX) proteins, particularly SIX1 and SIX4, act as upstream regulators of MRFs and PAX, including Pax3 and Pax7 (99, 116). These proteins, in cooperation with eyes absent (EYA) 1 and EYA2, contribute to the activation of Pax3 in the hypaxial dermomyotome, from which limb muscles originate (116). Pax3 serves as a key upstream regulator of MRFs in limb (hypaxial) muscle development, although its regulatory role differs from that in epaxial (trunk) muscles. In the limbs, inhibition of Pax3 significantly reduced Myf5 expression but had no direct effect on MyoD expression, suggesting a MyoD-independent pathway (99, 117). Furthermore, Pax3 plays a critical role in cell migration rather than in limb muscle differentiation (118). Once specified, muscle progenitor cells from the hypaxial lip of the dermomyotome migrate toward the developing limb bud. Because limb muscles are located far from their origin in the somites, these progenitors must acquire high motility. These migratory cells express Lbx1, a gene that is critical for directed migration. In Lbx1-deficient embryos, myoblasts failed to reach the limb bud, resulting in the absence of limb musculature (119-121). In addition, Meox2 exerts a significant influence on the migration of muscle progenitor cells to the limbs (122). In Mox2-depleted mice, proper limb muscle differentiation failed, leading to reduced muscle mass (123). Following successful migration, these progenitor cells undergo proliferation and subsequently upregulate MRFs, initiating differentiation and progressing toward myofiber maturation.

The head muscles originate from a source different from that of the trunk and limb muscles. Head muscles primarily originate from the CPM, although a subset arises from the lateral splanchnic mesoderm (SpM) (12). The CPM primarily contributes to the proximal central region of the branchial arches, leading to the formation of masticatory (MAS) muscles (12, 99, 120, 124). In contrast, the muscles derived from the SpM contribute to the distal region of the head muscles and consequently give rise to the lower jaw muscles. Unlike the trunk and limb muscles, the development of head muscles does not involve the expression of Pax3 and Pax7 (99, 107, 125). Similar to other regions, MRFs in the head are regulated by upstream genes. In mouse MAS, early specification is controlled by genes such as Tcf21 and Capsuli (126). In Tcf21^−/−/Capsulin^−/− double mutant mice, the development of MAS muscles was severely impaired, leading to apoptosis and dysregulation of MyoD and Myf5 genes. Thus, Tcf21 and Capsulin function as key regulators of muscle progenitor cell fate in specific head muscles. Paired-like homeodomain transcription factor 2 (Pitx2) also functions as an upstream factor of MRFs during head muscle development (99, 125). Pitx2, which is expressed not only in the head muscle, but also in various muscles, including cardiac and trunk skeletal muscles, regulates the proliferation of myoblasts and the transcription of MRFs (127-129). In Pitx2 gene-modified mice, the development of the first branchial arch, mandibular muscles, and periocular muscles was impaired (129-131). Furthermore, in these Pitx2 modified mice, the transcriptional expression of Tbx1 and Tcf21, which are essential for activating MRFs during head muscle development, was reduced (130). In conclusion, genes such as Tbx1, Pitx2, Tcf21, and Capsulin, which are involved in head muscle development as upstream regulators of MRFs, may play roles analogous to PAX3 or PAX7 (99). Unlike trunk muscles, which originate from relatively uniform somites, head muscles have diverse origins, as evidenced by specific developmental defects in certain muscles due to the deficiency of these genes (Table 1) (130, 132-138).

Table 1.

Region-specific regulatory genes and signaling pathways in skeletal muscle development

Region	Gene	Related pathway and signaling	Features	References
Head	Tbx1	Upstream of Myf5/MyoD	Regulated by Pitx2	(132-135)
	Pitx2	Activates MRFs, Tbx1, and Tcf21 in head	Common upstream regulator of head and forelimb myogenesis	(127-131, 135)
	Capsulin	Cooperates with Tcf21	Required for MAS muscle development	(126)
	Tcf21	Upstream of Myf5/MyoD	Deletion of the gene results in apoptotic responses	(99, 126, 130, 136)
Limb (hypaxial)	Noggin	BMP antagonist→activates MyoD and Myogenin	Facilitates myogenic differentiation	(115, 137)
	BMP4	Suppresses MRFs and Wnt7, and is repressed by Noggin	Inhibits myogenic differentiation	(115, 137)
	Six1/Six4	Activates Pax3 and Pax7 and interactd with Eya1 and Eya2	Key transcriptional regulators of early myogenesis in both cranial and hypaxial muscle progenitors	(99, 116)
	Lbx1	Downstream of Pax3 and FGF8-ERK signaling	Facilitates migration	(119, 121, 138, 150, 151)
	Wnt7a	Suppressed by BMP4	Symmetric expansion enhances and sustains the SC pool during regeneration	(68, 79)
	Meox2	Acts downstream or in parallel with Pax3 and Lbx1	Facilitates migration	(122, 123)
	Pax3	Activates Dmrt2, Myf5, Lbx1, and migration-associated genes	Controls both lineage commitment and precursor migration	(106, 152)
Trunk (Epaxial)	Shh	Activates Pax3	Synergizes with Wnt for dorsal somite patterning	(104, 105, 107, 108)
	Myf5	Induced near axial body regions, downstream of Pax3	Facilitates myogenic differentiation and highly express in trunk muscle	(104, 105, 107)
	Dmrt2	Activates Myf5	Promotes early myogenic commitment	(99, 106)
	Pax3	Wnt1/Wnt3a and Shh activate the Dmrt2–Myf5 cascade	Absent only in the head muscle lineage	(106, 152)
	Mrf4	Myogenic regulation	Activated with Myf5 in trunk regions	(57)
	Wnt1, Wnt3a	Induces Pax3	Roof plate signaling	(101-103)

MRFs: myogenic regulatory factors, BMP: bone morphogenetic protein, Shh: sonic hedgehog, MAS: masticatory.

Regional Heterogeneity of SCs

SCs exhibit region-specific characteristics that reflect the anatomical and embryonic origins of the muscles in which they reside. This heterogeneity remains evident in adult SCs, with functional properties varying according to muscle fiber type and developmental lineage (8, 10). In addition, the regenerative potential and behavior of muscle SCs differs depending on the muscle region of origin (139-142). Such region-specific differences affect a wide range of SC properties, including the proliferation rate, differentiation capacity, and gene expression profiles.

Hox genes are highly conserved homeodomain transcription factors that are differentially expressed in a spatiotemporal manner along the anterior-posterior axis, providing spatial coordinates for embryonic patterning (Table 2) (84, 86, 143). The trapezius muscle exhibits high expression of Hoxa5∼7, the tibialis anterior (TA) muscle predominantly expresses Hoxa9∼13, whereas Hoxa5∼13 is scarcely detected in MAS muscles (139). Additionally, HOXB2, HOXB4, HOXB9, and HOXC8 are upregulated in the longissimus dorsi (LD), whereas HOXA11 and HOXC11 are upregulated in semimembranosus (SM) muscle SCs (142). Furthermore, Hox genes have nonhomeotic roles in SC functions (11, 144, 145). Transcriptome sequencing of SCs from adult mouse extraocular muscles (EOMs) and TA muscles by Evano et al. (146) revealed upregulation of Hox gene family members in SCs derived from the TA muscle. In contrast, SCs obtained from EOM exhibited superior self-renewal, proliferation, and differentiation potential in vitro compared with their TA-derived counterparts (147). Therefore, Hox genes may contribute to the high myogenic potential of SCs (11, 146). In the TA muscle, SCs lacking Hoxa10 exhibited a diminished capacity for postnatal muscle regeneration, whereas no such impairment was observed in the MAS muscle (139). In addition, postnatal SCs of somite-derived muscles, which have decreased Hoxa10 expression, show impaired cell division and reduced proliferation rates. These studies suggested that Hox genes may affect SC function in a region-specific manner. Upregulation of HOXA11 has been associated with decreased expression levels of MYOD in embryonic chicken limbs, C2C12 cells, and LD and SM muscles of Hanwoo SCs (142, 143, 148). These findings indicated that HOX genes possess the ability to govern muscle differentiation efficiency (142). However, the precise role of Hox genes in the development of different muscle types remains unclear (11).

Table 2.

HOX gene profiles across muscle regions and their implications in SCs regulation

Gene	Expression location	Role in SCs	References
Hoxa5∼7	Highly expressed in trapezius	Function not specifically detailed	(11)
Hoxa9∼13	Predominantly expressed in TA	Identity; influences myogenic potential	(11)
Hoxa10	Required for postnatal muscle regeneration in TA	Essential for SC proliferation and regeneration in TA	(11)
Hoxa11	Embryonic chicken limb, C2C12 cells, LD, and SM muscles	Negatively regulates MYOD expression, and represses differentiation	(142, 143, 148)
Hoxb2	Upregulated in LD	Affects SC proliferation/differentiation	(142)
Hoxb4	Upregulated in LD	Affects SC proliferation/differentiation	(142)
Hoxb9	Upregulated in LD	Affects SC proliferation/differentiation	(142)
Hoxc8	Upregulated in LD	Affects SC proliferation/differentiation	(142)
Hoxc11	Upregulated in SM	Affects SC proliferation/differentiation	(142)

SCs: satellite cells, TA: tibialis anterior, LD: longissimus dorsi, SM: semimembranosus.

In addition to Hox genes, various genes exhibit region-specific expression patterns that contribute to the functional diversity of SCs. Some region-specific gene expression profiles persist into adulthood and influence the SC characteristics (10). In mouse SCs with high expression of the Eya2 gene, which is associated with limb development, the self-renewal ability persists for a longer period (128). Lbx2 is highly expressed in muscle cells that require long-distance migration during embryonic development such as those involved in limb formation (141). Lbx2 is highly expressed in muscle cells that require long-distance migration during embryonic development such as those involved in limb formation (119, 120, 149). Lbx2 can suppress MyoD expression (150, 151). Pax3 plays a crucial role in the development of trunk and limb muscles but is not required for head muscle development (2). Pax3 is expressed at higher levels in SCs derived from the trunk than in those derived from the limbs, reflecting its region-specific regulatory function (152). Taken together, the functional diversity of SCs is closely linked to their anatomical origin, with region-specific gene expression beyond the pivotal role of Hox genes. Consequently, this regional gene signature may serve as a key determinant of SC identity and function in different muscle groups.

SCs Dysfunction in Musculoskeletal Disorders

In musculoskeletal disorders such as muscular dystrophy (MD), sarcopenia, and muscle atrophy, the regenerative capacity of skeletal muscle is often severely disrupted (153, 154). This impairment is closely associated with the dysfunction of SCs, which play a pivotal role in muscle maintenance.

MD includes inherited diseases characterized by progressive muscle degeneration and fibrosis, particularly the Duchenne (DMD) and Becker types (155, 156). Both types are caused by mutations in the gene encoding dystrophin, a protein that stabilizes the sarcolemma, protects muscle fibers from mechanical stress, and serves as a critical linker between intracellular actin filaments and extracellular laminin (154, 155). The exact role of SCs in the pathogenesis of MD remains under active investigation (157). Previously, dystrophin protein was thought to be minimally expressed in primary myoblasts and SCs under in vitro conditions (157-159). However, the Dumont et al. (160) reported, based on RNA-seq and microarray analyses, that dystrophin transcription is markedly elevated in SCs. Moreover, dystrophin-deficient SCs exhibited a marked reduction in asymmetric divisions, accompanied by an increase in aberrant cell divisions, ultimately leading to a significant decrease in the generation of myogenic progenitors (160). Based on these findings, DMD has been proposed as a muscle stem cell disease.

Given the increasing age of the global population, age-related changes in muscle function have garnered significant attention as a major health concern (161). Sarcopenia is an age-related disorder characterized by the progressive decline of skeletal muscle mass and strength (162, 163). Symptoms usually begin to appear after age 60, and about 40% of individuals over 80 are affected by this condition (164, 165). The mechanisms underlying muscle mass loss in sarcopenia are thought to be closely associated with SCs (166, 167). In aged SCs, Pax7 expression is reduced, leading to a diminished stem cell pool and a significant delay in migratory capacity (168), along with a decline in proliferative potential (169). Recent research indicates that quiescent SCs in older muscle display a broadly suppressed chromatin state, potentially resulting in the dysregulation of signaling pathways related to muscle regeneration (65, 170). For example, the excessive activation of the p38α/β MAPK signaling pathway in aged SCs diminishes their ability to self-renew and proliferate, while simultaneously boosting the expression of genes linked to cellular senescence (65, 171). In aged mouse models, excessive activation of p38 signaling has been shown to promote symmetric rather than asymmetric division, leading to reduced self-renewal capacity, a diminished pool of SCs, and a shift in cell fate toward differentiation (65). In addition, mitochondrial dysfunction is regarded as a key driver of sarcopenia (172-174). Aging impairs the mitochondrial electron transport chain, leading to decreased ATP production and elevated levels of intracellular reactive oxygen species (175). This oxidative stress induces apoptosis in muscle cells (176). As a results, mitochondrial dysfunction contributes significantly to the impaired regenerative capacity of aged skeletal muscle.

Overall, this review indicates that dysfunction of SCs is a key pathological mechanism that hampers muscle regeneration in both genetic and age-related musculoskeletal disorders. This dysfunction not only undermines the muscle’s ability to repair and regenerate, but also accelerates the gradual loss of muscle mass and function seen in conditions like MD and sarcopenia. Recognizing the essential role of SCs in the progression of these diseases may provide important insights for future therapeutic interventions and regenerative strategies.

Therapeutic and Biotechnological Applications of SCs

Recent studies have explored the therapeutic potential of SCs in the treatment of DMD. A study involved transplanting SCs transduced with a lentiviral vector that encodes full-length dystrophin into mdx mice, serving as a model for DMD (177). This led to the expression of dystrophin in regenerated fibers and the development of functional myofibers. Furthermore, stimulation with epidermal growth factor repaired cell polarity defects induced by dystrophin deficiency, subsequently enhancing muscle regeneration and improving muscle strength (178). Another study showed that glycine supplementation in mdx mice has been shown to activate the mTOR signaling pathway, thereby promoting cellular growth and enhancing dystrophin expression, which collectively contribute to improved muscle regeneration (179). In addition, the injection of glycine improves the transplantation efficiency of SCs and myoblasts. Although these studies underscore the promise of targeting SCs, current strategies remain insufficient to fully restore function in DMD models. Continued research into SC-targeted strategies, combined with adjunctive interventions such as physical activity, may enhance therapeutic outcomes (180).

As SCs are responsible for muscle regeneration in sarcopenia, they have been increasingly recognized as a promising therapeutic target (181). Exercise has traditionally been utilized as a prevalent strategy for enhancing muscle function among elderly individuals (182). It has been shown to enhance muscle regeneration by promoting the activation of SCs (183). In addition, nutritional supplementation in combination with exercise has been investigated as a strategy to improve muscle function impaired by sarcopenia. Recently, the incorporation of melatonin has been shown to preserve the SC pool by suppressing cellular senescence and mitigating mitochondrial dysfunctions, as well as by inhibiting fibrogenic conversion (184, 185). Similarly, green tea catechins have been reported to enhance the function of aged SCs by reducing oxidative stress (186). In addition to nutritional supplementation strategies, various other approaches to mitigate sarcopenia have been actively explored. Electrical stimulation has been demonstrated to improve the regenerative ability of muscle SCs in a rat model of stroke-related sarcopenia, thereby promoting muscle mass (184). Furthermore, approaches utilizing exosomes, which are cell-derived vesicles capable of delivering bioactive molecules, have emerged beyond traditional cell-based therapies (187). Mesenchymal stem cells-derived exosomes enhance the regenerative function of SCs in sarcopenia by activation of the PI3K/AKT pathway and promoting muscle regeneration (188). Taken together, these SC targeted approaches reveal therapeutic potential against sarcopenia. However, further research is needed to confirm effectiveness and improve our understanding of optimal methods for preventing age-related muscle decline.

SCs have recently emerged as a promising cell source in the development of cultured meat (27, 28). Cultured meat is an emerging alternative protein produced by isolating and cultivating SCs from livestock (189). It offers a sustainable solution to global challenges such as food insecurity, environmental degradation, and ethical concerns related to conventional animal farming (189). Current research efforts in cultured meat focus on enabling the large-scale proliferation, cost-effective expansion and efficient myogenic differentiation of SCs to produce tissue structures that closely resemble conventional meat (26, 190-192). To achieve this, various strategies are being actively explored, including methods to maintain SC stemness during long-term in vitro culture and the development of serum-free culture media to replace fetal bovine serum (6, 17, 192-194).

Thus, in addition to their therapeutic applications, SCs are now being explored as a key cellular resource in cultured meat production, and their relevance has extended beyond medical use to industrial food biotechnology.

Conclusions

SCs, a type of stem cell found in muscle tissue, are extensively used in diverse biomedical and biotechnological applications and in cultured meat development because of their remarkable ability to differentiate into muscle fibers (2, 6). Despite their potential, continuous in vitro cultivation of SCs is challenging, as they tend to lose their stemness or fail to maintain a stable stem cell pool over time (6, 7). The self-renewal ability of SCs allows them to maintain the stem cell pool, while generating a large number of progenitor cells capable of proliferating and differentiating (2, 6). However, the current technologies are insufficient to support the long-term maintenance of this stem cell pool in vitro. Recent studies have explored pharmacological and chemical strategies to delay stemness loss and maintain the SC pool, particularly in cultured meat applications (6, 7, 195, 196). However, the use of such agents increases production costs, limiting their feasibility for large-scale applications.

Despite significant progress in the understanding of the molecular and developmental regulation of muscle SCs, translating this knowledge into robust in vitro strategies remains a major challenge. To overcome these limitations, a more comprehensive understanding of the intrinsic and extrinsic factors that influence supply chain behavior is necessary. Therefore, elucidating the regulatory landscapes that govern the behavior and function is essential for maintaining their in vitro properties and enhancing their applications in regenerative medicine, disease modeling, and scalable tissue engineering.