Improving muscle function with stem cells: understanding how this may help myasthenia gravis

MSCs are a type of stem cells derived from the mesoderm and are widely distributed in tissues such as bone marrow, the umbilical cord, and the placenta. In addition to these tissues, MSCs are primarily localized in the perivascular region. Crisan and colleagues identified functional progenitor cell reservoirs within the vascular wall using tissue-specific markers (Crisan et al., 2008). Furthermore, their homing process was found to be significantly dependent on the vascular network.

MSCs possess multidirectional differentiation potential (Keating, 2006). Lineage-specific differentitation of MSCs is regulated by specific factors in the microenvironment, such as secreted Bone Morphogenetic Protein (BMP)9 to induce osteogenic differentiation (Mostafa et al., 2019).

MSCs have multiple biological functions (Figure 2): Tissue microenvironmental homeostasis is maintained through the secretion of immunomodulatory factors (e.g., TGF-β, PGE2) (Kyurkchiev et al., 2014) and pro-angiogenic factors (e.g., VEGF, FGF) in a synergistic manner (Krakora et al., 2013). At the same time, metabolic disorders can be alleviated through regulation of the insulin signaling pathway and key genes involved in glucose and lipid metabolism (e.g., GLUT4, PPARγ). MSCs can improve the metabolic disorder status (Ji et al., 2015). These multidimensional regulatory capabilities endow them with unique therapeutic potential in immune repair, angiogenesis and metabolic imbalance intervention.

MSCs are also characterized by low immunogenicity and are capable of evading host immune surveillance through HLA-G–mediated immune escape mechanisms, thereby significantly reducing allograft rejection (Park et al., 2024). Its functional realization relies heavily on the dual modes of action: ① Immunomodulation through direct contact with immune cells via molecules such as PD-L1 on the membrane surface; ②Secretion of paracrine factors such as TSG-6 and PGE2 which are involved in inflammation regulation and tissue regeneration. Notably, recent studies have found that MSCs can also activate skeletal muscle satellite cells by secreting IGF-1, which significantly promotes muscle regeneration and injury repair (Baht et al., 2020).

As basic research and clinical translation of MSCs continue to advance, the lack of standardization in isolation and culture systems, phenotypic identification standards, and functional validation methods has become bottlenecks restricting widespread clinical application. In this context, the International Society for Cellular Therapy (ISCT) established criteria for the identification of MSCs in 2006, which explicitly require that the following core characteristics be met (Orbay et al., 2012): i Adherence to plastic surfaces under in vitro culture conditions; ii Specific expression of surface markers including CD73, CD90, and CD105, with the simultaneous absence of hematopoietic markers (CD14, CD34, CD45) and MHC class II molecules; iii Potential to differentiate into mesodermal cells (osteogenic, lipogenic, chondrogenic and myogenic). During the isolation and purification of MSCs, researchers can screen for lineage-specific surface markers such as Platelet-Derived Growth Factor Receptor α (PDGFRA) and Stem Cell Antigen-1 (Sca-1), and combine the above molecular characteristics in Fluorescence-Activated Cell Sorting (FACS) or Magnetic-Activated Cell Sorting (MACS) to obtain high-purity MSC populations (Uezumi et al., 2010).

The maintenance of a quiescent state by MSCs in tissues such as skeletal muscle, bone and adipose tissue is a central mechanism for the long-term preservation of their stem cell pool. This quiescent state represents a reversible, long-term exit from the cell cycle that serves dual functions in maintaining tissue homeostasis: supporting circulating progenitor cells and acting as a strategic reserve for tissue repair. Notably, quiescent MSCs exhibit remarkable self-renewal potential, a characteristic that declines rapidly during ex vivo culture but is restored following in vivo transplantation. This dynamic plasticity underscores a close link between the maintenance of stemness and regulation by the microenvironment (de Morree and Rando, 2023).

Genetic studies have shown that the quiescent state of MSCs requires the active participation of multiple signaling pathways. Once these pathways are disturbed, stem cells can be prematurely activated, which results in the loss of self-renewal capacity and ultimately leads to depletion of the stem cell reservoir and reduced regenerative potential in vivo, thereby accelerating the aging process.

For example, the balance between proliferation and differentiation of MSCs can be regulated, at least in part, through the Wnt/β-catenin axis. Activation of this axis has been shown to induce MSCs to upregulate the expression of MyoD, myogenin, and other myogenic regulators, thereby promoting myogenic differentiation (Zhou et al., 2015).

Resting-state MSCs exhibit several unique cellular properties: a smaller cell volume (10–15 μm in diameter), an approximately 60% reduction in basal metabolic rate, a decrease in mitochondrial content to 30%–40% of that in proliferating cells, and a marked downregulation of RNA and protein synthesis rates were observed. This metabolic dormancy is associated with three biological advantages: enhanced resistance to cellular stress, preservation of genomic stability, and the establishment of phenotypic compartmentalization from activated descendant cells. Upon tissue injury, an activation program is rapidly initiated by these cells, the G0–G1 transition is completed within 24–48 h, and both tissue repair and the stem cell pool are regenerated through asymmetric division (de Morree and Rando, 2023).

MSCs have been shown to significantly enhance muscle repair through paracrine mechanisms, both in vitro and in vivo (Chiu et al., 2020). However, a clear distinction in surface markers can be observed between muscle stem cells (MuSCs, i.e., satellite cells) and mesenchymal stem cells (MSCs): MuSCs are characterized by the expression of VCAM-1, while MSC markers such as Sca-1 are generally absent (Liu et al., 2015). Despite differences in origin and phenotype, both cell types are considered to play crucial roles in skeletal muscle repair and are thought to partially share regulatory mechanisms governing quiescence and activation. Under physiological conditions, both MuSCs and MSCs are predominantly maintained in a quiescent state. Following skeletal muscle injury, factors such as inflammatory signals within the microenvironment are capable of triggering the activation and proliferation of these cells. Once activated, MuSCs primarily undergo proliferation and are subsequently directed to differentiate into myoblasts, which then fuse to form new muscle fibers or repair damaged ones, thereby acting as the principal executors of muscle regeneration. In contrast, activated MSCs contribute primarily through paracrine actions (i.e., secretion of various cytokines and growth factors) and immunomodulatory functions, thereby establishing a favorable microenvironment that facilitates the proliferation and differentiation of MuSCs and indirectly promotes regeneration (Theret et al., 2021; Molina et al., 2021). Additionally, MSCs are also capable of differentiating into other mesenchymal lineage cells, including fibroblasts and adipocytes. For example, studies have indicated that MSCs may influence the regeneration process through the secretion of specific extracellular vesicles (e.g., MSCs-ApoEVs) (Ye et al., 2023). In the later stages of repair, the majority of activated MuSCs are differentiated and consumed, with only a small number undergoing self-renewal and subsequently returning to a quiescent state to replenish the stem cell pool (Fu et al., 2022); meanwhile, activated MSCs are thought to contribute to the reestablishment of tissue homeostasis through differentiation or apoptosis.

There is often confusion in the classification of Mesenchymal Stem Cells (MSCs), Mesenchymal Progenitor Cells (MPCs), and fibro/adipogenic progenitors (FAPs) in existing studies. It is widely recognized that MPCs represent the early developmental stage of MSCs, in which the former are progenitor cells characterized by high proliferative capacity, whereas the latter are mature stem cells possessing multidirectional differentiation potential. Although the two terms are often used interchangeably in the literature, they essentially represent distinct differentiation states within the same cell lineage (Sarugaser et al., 2009).

Fibro/Adipogenic Progenitors (FAPs) are considered a tissue-specific population of mesenchymal stem/progenitor cells residing in skeletal muscle. They share multiple characteristics with the extensively studied bone marrow–derived mesenchymal stem cells (MSCs), including adherent growth in vitro, expression of similar surface markers (such as PDGFRα⁺ and Sca-1⁺), and the potential to differentiate into fibroblasts and adipocytes (Kim and Braun, 2020; Uezumi, 2023). However, FAPs are specifically localized within the interstitial spaces of skeletal muscle, and their functions and behaviors are strongly regulated by the muscle microenvironment. In healthy muscle, they are primarily involved in maintaining extracellular matrix homeostasis. Following injury, they are rapidly activated and transiently contribute to the regenerative function of muscle stem cells (MuSCs) through paracrine signaling, before being cleared via apoptosis. Dysregulation of their fate may result in pathological fibrosis or fatty infiltration (Joe et al., 2010).

Therefore, although homology is exhibited in the in vitro identification criteria, FAPs should be more accurately described as muscle-resident MSC-like cells characterized by distinct tissue localization and specialized functions, rather than being merely equated with bone marrow MSCs.

Two types of stem cell subpopulations are present in the skeletal muscle microenvironment: satellite cells (muscle stem cells, or MuSCs) and MSCs with myogenic differentiation potential. Satellite cells are crucial progenitor cells involved in skeletal muscle repair. During muscle regeneration, MSCs are considered to play a pivotal role in modulating the activation, proliferation, and differentiation of satellite cells. This regulatory mechanism is regarded as essential for the restoration of functional muscle tissue. Specifically, local injection of MSCs at the injury site has been shown to significantly enhance the proliferation and differentiation of satellite cells (MuSCs) via the secretion of bioactive factors, thereby facilitating the targeted regeneration of skeletal muscle fibers and the subsequent formation of functional muscle tissue (Sandonà et al., 2021).

The anatomical localization of myosatellite cells and myogenic MSCs differs markedly. Satellite cells, as skeletal muscle-specific stem cells, are strictly localized in the anatomical gap between the basal layer of muscle fibers and the myofibrillar membrane, whereas MSCs are primarily localized in the interstitial region and exhibit high-density aggregation at the muscle-ligament junction (Uezumi et al., 2010; Uezumi et al., 2021). Notably, subpopulations of MSCs exhibited a distinct tendency toward regionalized distribution. Some cells were localized in close proximity to motor nerve axons, whereas others selectively encircled the periphery of the neuromuscular junction (NMJ). This spatial specificity suggests that they may participate in regulating NMJ function through physical proximity or paracrine signaling. Based on this spatial distribution property, it was concluded that MSCs constitute essential cellular components for maintaining the structural integrity of the NMJ and muscle mass (Kurosawa et al., 2021). It was experimentally confirmed that transplantation of exogenous MSCs can improve neuromuscular conduction function by promoting nerve axon regeneration and NMJ structural remodeling (Kim et al., 2012).

Skeletal muscle abnormalities have been associated with metabolic diseases. Skeletal muscle, as the largest metabolic regulatory organ in the human body, is responsible for maintaining metabolic homeostasis through the regulation of substrate utilization processes, including glucose and fatty acid uptake and oxidation. Under conditions of elevated insulin levels or physical activity, glucose uptake and utilization are enhanced in skeletal muscle; whereas during prolonged exercise or fasting, energy production is more dependent on fatty acid oxidation (Fan et al., 2024). Such adaptive regulation of substrate utilization is essential for the maintenance of blood glucose stability, lipid balance, and overall energy metabolism. Pathological remodeling of skeletal muscle can be induced by metabolic disorders such as obesity, aging, and diabetes (Nunan et al., 2022). The balance of energy metabolism in skeletal muscle cells is disrupted by these metabolic disorders through the activation of excessive mitochondrial fission pathways, which consequently inhibit the proliferative potential of stem cells and their capacity for tissue repair (Hong et al., 2022; Jheng et al., 2012). Notably, ectopic deposition of intramuscular adipose tissue has been reported to be positively associated with the development of obesity, which in turn exacerbates skeletal muscle dysfunction through chronic inflammation and lipotoxicity, thereby establishing a vicious cycle of metabolic disturbances (Wang et al., 2024).

Skeletal muscle mass loss is commonly observed in pathological states such as aging, chronic diseases, and autoimmune disorders. Although skeletal muscle has the potential for endogenous regeneration, which can compensate for approximately 20% of muscle mass loss (Liu et al., 2018), this capacity is limited. When the injury exceeds this threshold, pathological outcomes, including progressive loss of muscle strength and motor dysfunction, are likely to occur. The process of skeletal muscle regeneration is composed of two sequential phases: a degenerative phase, defined by myofiber necrosis and infiltration of inflammatory cells, and a regenerative phase, during which satellite cells differentiate into multinucleated myotubes that subsequently form new myofibers. Quantification of Central Nucleated Fibers (CNFs) serves as a key indicator in the assessment of the regenerative process (Iyer et al., 2020). Furthermore, the restoration of innervation is essential for functional reconstruction (Chargé and Rudnicki, 2004).

MSCs, as adult stem cells with multidirectional differentiation potential, have been widely applied in regenerative medicine (Chiu et al., 2020). They are particularly effective in promoting the repair of the bone and skeletal muscle systems, as well as in ligament and tendon regeneration (Park et al., 2024; Chamberlain et al., 2017).

Although abnormal differentiation of myogenic MSCs contributes to an increase in fibroblasts and adipocytes under pathological conditions, the depletion of these cells nonetheless severely disrupts skeletal muscle homeostasis. It has been demonstrated that short-term depletion of MSCs significantly reduces skeletal muscle mass and impairs functional grip strength (Uezumi et al., 2021). Evidence suggests that ablation of fibro/adipogenic progenitors (FAPs) compromises skeletal muscle function: Cre-dependent, diphtheria toxin-mediated FAP depletion leads to impaired expansion of muscle stem cells (MuSCs) and reduced regeneration following injury; under homeostatic conditions, FAP depletion causes muscle atrophy and a decline in the MuSC pool (Murphy et al., 2011; Farup et al., 2015). Kurosawa et al. demonstrated through spectral tracing that MSCs preserve skeletal muscle homeostasis by dynamically regulating the matrix–myocyte interaction network. Their absence results in impaired myofiber regeneration, innervation abnormalities, and ultimately a collapse of the mechanisms responsible for maintaining muscle mass and functional reconstruction (Uezumi et al., 2021).

MSCs have emerged as a potential therapeutic strategy for autoimmune diseases, such as myasthenia gravis, owing to their unique immunomodulatory capacity (Konen et al., 2022). In experimental autoimmune myasthenia gravis (EAMG) animal models, MSC transplantation not only significantly enhances neuromuscular junction signaling efficiency but also reduces abnormally elevated acetylcholine receptor antibody concentrations, restores the balance of immune cell subpopulations, and confers additional regulatory benefits, thereby providing a theoretical foundation for clinical application (Tang et al., 2024; Sudres et al., 2017; Yu et al., 2010). More strikingly, recent research by Jean-Thomas Vilquin's team applied MSCs co-cultured with PBMCs to a humanized mouse model of myasthenia gravis and demonstrated that this treatment reduced disease severity by half as early as 2 weeks post-injection (Bayer et al., 2025).

Although the effectiveness of MSCs in the treatment of EAMG has been partially confirmed, their underlying mechanisms of action remain to be elucidated in greater depth. The mechanisms of MSCs in the treatment of MG will be elaborated from the perspectives of immunity, the neuromuscular junction, and acetylcholine.

Myasthenia gravis (MG), as an autoimmune disease closely associated with splenic immune function, has shown clear therapeutic efficacy from allogeneic mesenchymal stem cell (MSC) transplantation. Previous studies have demonstrated that infusion of allogeneic MSCs can alleviate the pathological progression of MG by restoring the imbalance of T-cell subsets. Nevertheless, it remains unclear whether these beneficial effects are mediated through the regulation of the spleen, which serves as the central immune organ. Of note, research in liver cirrhosis has employed In-oxine labeling to trace infused allogeneic MSCs, and these studies revealed their specific homing properties, thereby suggesting that similar mechanisms might also contribute to the therapeutic effects of MSCs in MG (Gholamrezanezhad et al., 2011)^. By the 10th day after transplantation, the concentration of MSCs in the spleen was markedly increased relative to baseline levels, suggesting that their preferential homing to the spleen may contribute to immunomodulation.

In addition to the humoral environment, the skeletal muscle microenvironment of patients with MG is characterized by a chronic inflammatory state. The main manifestations include T- and B-lymphocyte infiltration and the sustained release of pro-inflammatory factors. The inflammatory response represents a critical contributor to the pathogenesis of systemic and ocular MG (Molin et al., 2017). This inflammatory state not only inhibits myogenesis but also drives aberrant differentiation of MSCs into adipocytes via activation of the JNK signaling pathway, ultimately resulting in intramuscular adipose infiltration accompanied by fibrosis (Du et al., 2010). Notably, sustained IL-6-mediated activation of the STAT3 pathway modifies the functional behavior of MSCs, predisposing them to fibrotic processes rather than normal tissue repair (Ren et al., 2022; Malecova et al., 2018).

In this pathological context, the inhibition of muscle regeneration caused by the inflammatory microenvironment was reversed by MSCs through the secretion of anti-inflammatory mediators such as IDO, IL-10, and IL-1RA, which synergistically downregulated TNF-α and IL-6 while up-regulating pro-repair factors such as IGF-1 (Ji et al., 2015). This process effectively mitigated persistent attacks induced by immune complex deposition at neuromuscular junctions (Kanatli et al., 2017; Gradolatto et al., 2014). IDO is a hydrolytic enzyme related to tryptophan, and T cell activation primarily requires tryptophan. Moreover, MSCs eliminate activated T cells through the tryptophan metabolic pathway, which synergizes with factors such as ProstaGlandin E2 (PGE2) and TGF-β to remodel immune homeostasis (Ren et al., 2009).

Therapeutic efficacy is exerted by heterologous MSC infusion through a multidimensional immunomodulatory network (Pistoia and Raffaghello, 2017): 1 aberrant T cell activation is inhibited, and regulatory T cell (Treg) differentiation is selectively induced; 2 CD4⁺T cell-mediated B-cell activation and plasma cell differentiation are blocked (Maccario et al., 2005), while the expression of the critical B-cell activation factor BAFF is downregulated, thereby preventing autoantibody production pathways and ameliorating MG pathological progression (Sudres et al., 2017); 3 tissue repair is promoted through the secretion of IL-33, which recruits Tregs to the site of injury (Kuswanto et al., 2016). 4 Pro-inflammatory factors such as IFN-γ, TNF-α, IL-1β can evoke MSCs to modulate the infiltrating T cells by creating nitric oxide (NO)-mediated immunosuppressive microenvironment. This is achieved through inducing high expression of chemokines and iNOS (Ren et al., 2008).

This multidimensional regulatory mechanism enables MSCs to maintain immune homeostasis in the skeletal muscle of patients with myasthenia gravis (MG). On the one hand, MSCs attenuate muscle fiber damage by suppressing excessive immune responses; on the other hand, they promote myofibrillar repair by activating satellite cell function. Such a unique dual effect—combining anti-inflammatory and pro-regenerative properties—provides a theoretical foundation for alleviating skeletal muscle weakness and delaying secondary pathological manifestations in MG.

The core pathological feature of myasthenia gravis (MG) is skeletal muscle weakness caused by impaired signal transmission at the neuromuscular junction. Skeletal muscle tissue is composed of highly differentiated multinucleated muscle fibers, and its contractile function is dependent on signaling at the NeuroMuscular Junction (NMJ) - Acetylcholine (ACh) released by motor neurons reaches the muscle membrane through diffusion, and the transmitter triggers action potential conduction by binding to membrane-specific receptors, ultimately triggering the myofilaments to contract. This sophisticated regulatory system ensures the precise response of skeletal muscle to neural commands (Lo et al., 2020).

Modern studies have demonstrated that MSCs play a crucial role in neuromuscular junction reconstruction and nerve regeneration (Kim et al., 2012). Using an Amyotrophic Lateral Sclerosis (ALS) model, Krakora's team demonstrated that intramuscularly transplanted MSCs release glial cell-derived neurotrophic factor (GDNF) in response to stimulation by the inflammatory microenvironment. As a critical regulator of pre- and postsynaptic differentiation, GDNF has been shown to significantly increase neuromuscular junction density and enhance motor neuron survival by preserving synaptic plasticity (Krakora et al., 2013). In skeletal muscle injury models, transplantation of MSCs has been shown to accelerate repair by reducing collagen deposition, upregulating myogenic marker expression, and promoting neuromuscular junction regeneration. In addition, Ciliary Neurotrophic Factor (CNTF) and Brain-Derived Neurotrophic Factor (BDNF) secreted by MSCs synergistically exert neuroprotective effects: CNTF specifically promotes motor neuron survival and axon regeneration, while BDNF optimizes neural signaling by modulating synaptic plasticity (Ropper et al., 2017).

Chronic impairment of neuromuscular junction transmission is often followed by secondary denervation, which results in the progressive worsening of skeletal muscle atrophy. The characteristic alterations are typified by a reduction in the cross-sectional area of both type I and type II muscle fibers (Snijders et al., 2014). Satellite cells, which represent the primary cellular population responsible for skeletal muscle regeneration, are activated upon injury and subsequently undergo a regenerative cascade involving proliferation, differentiation, and fusion, ultimately contributing to myofiber repair through the incorporation of nascent myonuclei (Chargé and Rudnicki, 2004).

Skeletal muscle atrophy is inhibited by MSCs through the regulation of satellite cell activity and the suppression of myonuclear apoptosis via a paracrine mechanism. Homeostasis of the satellite cell pool is maintained by MSCs primarily through the downregulation of the pro-apoptotic protein Bax, while concurrently up-regulating the anti-apoptotic protein Bcl-2 and activating the phosphorylated Akt (p-Akt) signaling pathway, thereby effectively counteracting muscle wasting (Li et al., 2016). Notably, MSCs and their exosomes (MSC-EXO) alleviate the dysregulation of protein metabolism in skeletal muscle atrophy through activation of the AMPK/ULK1-mediated autophagy pathway (Perry et al., 2016). Mechanistic studies have demonstrated that transplantation of human umbilical cord–derived mesenchymal stem cells (hucMSCs) enhances autophagic activity by activating the AMPK/ULK1 signaling pathway and specifically downregulating the expression of Atrogin-1 and MuRF1, thereby effectively suppressing the pathological progression of skeletal muscle atrophy (Song et al., 2023).

The core pathological mechanism of myasthenia gravis (MG), an autoimmune disease, is closely associated with immune-mediated attack on the acetylcholine receptor (AChR). The pathogenesis involves two principal mechanisms: 1 competitive binding of autoantibodies to the AChR, which interferes with neuromuscular transmission, and 2 antibody-mediated endocytic degradation of AChRs, resulting in reduced receptor density at the postsynaptic membrane. Notably, disease severity is more directly determined by the extent of reduced AChR expression at the muscle endplate than by circulating antibody levels alone (Sudres et al., 2017). Current therapeutic approaches primarily aim to increase acetylcholine concentrations within the synaptic cleft and suppress autoantibody production, for example, by delaying acetylcholine degradation with anticholinesterase agents.

It has been demonstrated that MSCs regulate the cholinergic system through multiple mechanisms. Acetylcholine exerts various physiological effects through muscarinic receptors (mAChRs) and nicotinic receptors (nAChRs), the latter being specifically responsible for regulating skeletal muscle contraction. MSCs promote acetylcholine synthesis and release by upregulating Choline Acetyltransferase (ChAT) and Vesicular Acetylcholine Transporter (VAChT) expression. Concomitantly, the secretion of Prostaglandin E2 (PGE2) activates relevant signaling pathways, thereby enhancing ACh synthesis and release (Zhang et al., 2022). Undifferentiated MSCs inherently possess the capacity to store acetylcholine, which provides the fundamental basis for their regulation of neuromuscular function (Hoogduijn et al., 2006).

Literature studies have demonstrated that, in animal models of MG, the injection of MSCs significantly reduces serum anti-acetylcholine receptor antibody (AChR-Ab) levels (Yu et al., 2010) and restores AChR density at motor endplates (Sudres et al., 2017). On the basis of these findings, it is hypothesized that MSCs may alleviate MG symptoms by enhancing acetylcholine (ACh) synthesis and release, thereby competitively inhibiting the pathogenic effects of circulating AChR-Ab. This proposed mechanism requires further experimental validation.

Aging-related skeletal muscle degeneration is accompanied by increased ectopic fat infiltration and fibrosis (Marcus et al., 2010; Uezumi et al., 2014; Chen et al., 2022), and this pathological process is closely associated with the senescence of MSCs. Senescent MSCs are characterized by reduced autophagy, diminished proliferative capacity, and enhanced lipogenic differentiation, which result in abnormal intramuscular fat accumulation and an imbalance in matrix remodeling (Hansen et al., 2018; Ma et al., 2018). It has been demonstrated that young MSCs preserve stem cell properties under microenvironmental stress via autophagy, whereas senescent MSCs exhibit dysfunction of the autophagy–lysosome system, which induces the accumulation of reactive oxygen species and aberrant activation of p53 signaling, ultimately leading to the collapse of musculoskeletal homeostasis (Ma et al., 2018).

The senescence of MSCs has been shown to mediate the disruption of skeletal muscle homeostasis through key molecular pathways. Bone morphogenetic protein 3b (Bmp3b), an essential factor for maintaining skeletal muscle mass and stabilizing the neuromuscular junction (NMJ), is abundantly expressed in young MSCs but becomes progressively downregulated with aging (Uezumi et al., 2021; Kurosawa et al., 2021). This factor has been reported to delay the progression of sarcopenia by suppressing aberrant differentiation of MSCs (Uezumi et al., 2021). In addition, extracellular vesicles (EVs) secreted by senescent bone marrow-derived MSCs have been shown to suppress the myogenic differentiation of satellite cells, thereby leading to impaired myotube fusion and regenerative failure (Dai et al., 2022).

Late-onset MG shows a high incidence among elderly individuals aged 70–74 years. We speculate that its development may be linked to senescent MSCs; nevertheless, this possibility needs to be substantiated by large-scale clinical studies. We intend to explore this direction in future studies.

Currently, clinical research on MSCs for neuromuscular diseases has provided considerable evidence; however, findings from registered clinical trials in myasthenia gravis (MG) have not yet been disclosed, thereby underscoring a notable research gap.

In comparison, clinical investigations of MSCs in other neuromuscular disorders have made gradual progress: ① Amyotrophic Lateral Sclerosis (ALS): Genetically modified MSCs engineered to secrete neurotrophic factors, when administered intrathecally, have been shown to significantly attenuate the decline in the revised ALS Functional Rating Scale (ALSFRS-R) score and to prolong progression-free survival by approximately 6 months (Petrou et al., 2016). ②Multiple Sclerosis (MS): The combined administration of intravenous and intrathecal infusions of autologous MSCs has been shown to significantly reduce the annualized relapse rate in patients with active-progressive MS and to improve motor function (Petrou et al., 2020).

Although a comprehensive clinical evidence system for MSC-based therapy in MG has not yet been established, a series of clinical trials in ALS and MS has produced a relatively complete body of safety and efficacy evidence. This evidence has provided a solid methodological and biological foundation for translational research on MSCs in neuromuscular diseases such as MG, while suggesting their potential therapeutic value.

In order to deeply analyze the potential mechanism of MSCs injection therapy, various research teams have revealed the key regulatory elements through multi-dimensional experimental systems. The current consensus is that the therapeutic effects of MSCs are primarily mediated through the following mechanisms: 1 secretion of immunomodulatory factors (e.g., IL-10, TGF-β): that modulate the local inflammatory microenvironment; 2 release of pro-angiogenic factors (VEGF, bFGF) that facilitate the reconstruction of tissue blood circulation; 3 delivery of anti-apoptotic signals (e.g., Bcl-2 family proteins) through exosomes, which protect damaged cells; and 4 genetic or molecular engineering to confer targeted differentiation potential or specific protein expression capacity. These findings provide a theoretical foundation for the development of next-generation therapies based on precision-engineered MSCs.

Regarding the mechanism of action of transplanted MSCs in vivo, several perspectives have been proposed: it has been suggested that transplanted MSCs primarily function through immunomodulation rather than direct tissue regeneration (Hoang et al., 2022). Another perspective suggests that although most exogenous MSCs undergo apoptosis, a subset of transplanted MSCs may still differentiate into myoblasts (Dezawa et al., 2005). Clinical observations by another researcher indicated that MSC infusion elicited a response only when patients exhibited elevated cytotoxic activity. Therefore, the researcher proposed that in vitro–induced apoptotic MSCs might be utilized as a therapeutic strategy. After infusion, recipient phagocytes phagocytose apoptotic MSC and produce indoleamine 2,3-dioxygenase, thus realizing immunosuppression (Galleu et al., 2017).

Optimization of therapeutic parameters is crucial for determining the efficacy of MSCs. Administration of MSCs during the acute phase of injury results in minimal regenerative benefit, whereas administration at the peak of the inflammatory response markedly enhances myofiber regeneration and capillary density (Helal et al., 2016). The improvement in muscle strength is most evident when the graft volume reaches 1.0 × 10^6 cells in rat soleus muscles after severe crush trauma (Qazi et al., 2019).

Hypoxic preconditioning has been shown to enhance the regenerative efficacy of MSCs through a mechanism involving the hypoxia-inducible factor (HIF-1α)-mediated upregulation of VEGF and WNT4, thereby promoting vascularization and myogenesis (Archacka et al., 2021; Zhang et al., 2015).

Bone marrow–derived mesenchymal stem cells (BMMSCs) are constrained by invasive collection procedures, ethical concerns, and challenges in large-scale expansion. Consequently, the pursuit of alternative cell sources has emerged as a prominent research focus. Adipose-derived mesenchymal stem cells (ADSCs) represent a significant breakthrough, as they can be isolated through minimally invasive liposuction. ADSCs have emerged as an ideal progenitor cell source for regenerative medicine, owing to their accessibility (abundant adipose tissue reserves and minimal donor trauma) and robust proliferative capacity (Cannon et al., 2003; Jack et al., 2005). Studies have demonstrated that ADSCs exhibit enhanced myogenic differentiation potential compared with BMMSCs, conferring notable advantages in promoting myofiber neogenesis and structural tissue reconstruction (de la Garza-Rodea et al., 2012). In particular, the transplantation of BMMSCs has been reported to induce abnormal remodeling of the collagen matrix, which results in the formation of pathological fibrotic foci and impaired skeletal muscle contractile function. In contrast, the application of ADSCs has been shown to markedly suppress excessive collagen deposition and promote the restoration of microenvironmental homeostasis (Moussa et al., 2020). However, ADSCs still have technical bottlenecks such as fluctuating phenotypic plasticity and heterogeneity of therapeutic responses, so their potential for clinical application as an alternative to BMMSCs still needs to be carefully evaluated (Mohamed-Ahmed et al., 2021).

Muscle-derived mesenchymal stem cells (mdMSCs) have been recognized as a highly promising therapeutic approach because of their distinct clinical applicability and functional characteristics. Utilizing minimally invasive muscle micro-biopsy techniques, mdMSCs can be obtained in a highly scalable manner, while avoiding the risk of immune rejection and substantially reducing patient trauma, thereby providing greater clinical advantages compared with bone marrow- or adipose tissue-derived MSCs. Functional studies have demonstrated that mdMSCs possess significant angiogenic, immunomodulatory, and anti-fibrotic capacities through paracrine mechanisms. Their secretome is enriched with pro-repair factors, which effectively suppress inflammatory responses and promote angiogenesis in in vitro models (Guesdon et al., 2025).

Modern studies have shown that researchers have successfully explored other sources of MSCs for therapeutic application such as umbilical cord/amniotic fluid: extra-embryonic tissue-derived MSCs (e.g., umbilical cord/amniotic fluid) are favored because of their embryonic stem cell-like phenotypic profile and lesser ethical concerns (Kim et al., 2012; Shareghi-Oskoue et al., 2021). Tonsil-derived mesenchymal stem cells (T-MSCs), obtained from surgical waste such as tonsils, have emerged as a focus of regenerative medicine research because of their distinctive immunomodulatory properties and remarkable proliferative capacity (Cho et al., 2015; Park et al., 2019). This innovative cell source offers an ethically compliant, scalable, and high-quality solution to advance mesenchymal stem cell–based therapeutic strategies.

Modern bioengineering often incorporates therapeutic molecules (e.g., VEGF, IL-10) into MSCs via viral transduction or gene-editing methods to achieve targeted therapeutic effects (von Bahr et al., 2012). MSCs retain stemness markers (Oct4+, Nanog+) and exhibit robust proliferative capacity, and it has been demonstrated that MSCs cultured over successive passages preserve karyotypic stability and trilineage differentiation potential (Debnath and Chelluri, 2019). This property, together with the CXCR4/SDF-1 axis–mediated homing ability, renders MSCs an ideal biocarrier. However, it should be noted that long-term infusion of allogeneic MSCs may pose a risk of metabolic disorders, as postmortem pathological analyses have revealed an abnormal 32% increase in mitochondrial respiratory chain complex activity (von Bahr et al., 2012).

Previous studies have demonstrated that the primary factor limiting the efficacy of MSCs is closely associated with their survival microenvironment. Following xenotransplantation, MSCs undergo apoptosis in response to alterations in the microenvironment, including changes in extracellular matrix composition, oxygen partial pressure, and nutrient availability, ultimately resulting in a marked reduction in the number of viable cells. To overcome this bottleneck, various engineering strategies have been developed, such as the maintenance of MSC stemness through sulfated alginate encapsulation, which markedly enhances their survival during skeletal muscle regeneration (Gionet-Gonzales et al., 2023). Implantation of an epicardial patch loaded with MSCs on the cardiac surface after coronary artery bypass grafting significantly improves the prognosis of postoperative cardiac function in patients by ameliorating the dysregulation of mitochondrial proteomic profile (Agrawal and Thankam, 2021).

The development of novel delivery systems has provided innovative strategies to optimize the therapeutic efficacy of MSCs. For instance, 3D bioprinting technology has been employed to fabricate cardiac patches that co-encapsulate bone marrow–derived MSCs (BM-MSCs) with genetically engineered MSCs expressing hepatocyte growth factor (HGF). Following implantation into the infarcted myocardial region, this system has been shown to extend MSC survival by more than threefold and to promote both neovascularization and myocardial tissue repair (Park et al., 2020). In addition, the combined application of ultrasound-targeted microbubble disruption (UTMD) technology with platelet-derived growth factor-BB (PDGF-BB) pretreatment has been shown to enhance the homing efficiency of MSCs through upregulation of stromal cell–derived factor-1 (SDF-1) and to inhibit apoptosis of grafted cells, thereby improving the therapeutic efficacy of ischemic myocardial repair (Sun et al., 2023).

At the mechanistic level, proteomic tracking utilizing a mutant methoxy-tRNA synthetase (MetRS^L274G) demonstrated that MSCs transplanted into ischemic hearts underwent extensive proteomic remodeling, encompassing pathways such as the complement–coagulation cascade, fibrinogen activation, regulation of apoptosis, and actin cytoskeleton remodeling. Among them, complement factor H (CFH) and actin depolymerization-associated proteins (e.g., cofilin, fibronectin) play a key role in cardiac repair by regulating cell migration, proliferation, and neovascularization (Han et al., 2020).