Progress on Cell Therapy for Skeletal Muscle Disorders

Abstract

Cell therapy remains an attractive therapeutic option for the numerous genetic and non-genetic maladies affecting skeletal muscle. Since skeletal muscle is the largest tissue in the body, delivery has been notoriously challenging, but there have been significant advances, with several ongoing clinical trials of allogeneic and autologous cell transplantation aiming to replace diseased skeletal muscle with healthy and functional myofibers and muscle stem cells. Paracrine cellular approaches intended to enhance regeneration are also ongoing. In this review, we will provide an overview of the progress and current status of these different approaches, and discuss the forecast for future phases as well as the hurdles that need to be circumvented for the widespread application of cell therapy for skeletal muscle disorders.

Graphical Abstract

1. Introduction

The human body is composed of more than six hundred skeletal muscles, representing 30–50% of total mass [1]. The major role attributed to skeletal muscles is to contract and generate force for body movement and support, but they are also critical for many other important physiological functions, including breathing, mastication, joint stabilization, maintenance of body temperature, nutrient reservoir, and metabolism [see review 2]. Muscle fibers, post-mitotic multinucleated contractile cells, represent the main functional constituent of this tissue (Fig. 1A).

Fig. 1.

Schematic representing healthy and diseased muscle bundles. Schematic representations of cross-sectional muscle bundles illustrate healthy (A) and diseased (B) skeletal muscle. Please note the different cell types identified in skeletal muscle and the overall changes potentially occurring with disease pathology. Schematic representation of diseased muscle highlights the presence of sick myofibers and the abundance of extracellular matrix (B). FAPs: Fibro-adipogenic progenitors.

2. Skeletal muscle: origin and cellular composition

Embryonic myogenesis begins with the delamination of multipotent muscle progenitor cells arising in the dermomyotome [3], a process regulated by several signaling pathways, including Wnt, Notch, and Hedgehog, among others [see review 4]. These early muscle precursor cells expressing Pax3/Pax7 migrate medially through the dorsomedial lip of the dermomyotome to form an epithelial sheet, known as the myotome [5,6]. At this site, myogenesis takes place upon the activation of the Myogenic Regulatory Factors (MRFs) Myf5 and MyoD1 [7–11], resulting in the emergence of muscle precursors, the myoblasts. The subsequent terminal differentiation of myoblasts into myocytes depends on appropriate levels of Mrf4, and myogenin [12–14, and review 4 for more detailed information]. In addition, myocytes become positive for several other muscle-specific genes, including contractile proteins and muscle creatine kinase [see review 4], and will fuse through a process involving the muscle-specific fusion proteins myomaker and myomerger [15–18 and see review 19]. As development proceeds, a secondary wave of myogenesis occurs, and a proportion of the Pax3/Pax7 muscle progenitor cell population established during late fetal development will eventually populate the adult muscle stem cell pool [5].

Adult muscle stem cells (MuSCs), named satellite cells (SCs) in 1961 by Mauro due to their satellite position under the basal lamina adjacent to the myofiber [20]. SCs represent approximately 2% of the total myonuclei content [21] and are responsible for the highly regenerative nature of adult skeletal muscle [22]. In steady-state conditions, these cells are maintained in a quiescent state, which is correlated with their sublaminar location and the expression of the transcription factor Pax7 [23,24]. In response to environmental signals, such as injury, stress, or exercise [25–28], SCs will exit the quiescent state and upon cell division, will give rise to myoblasts, which are able to fuse with existing myofibers or form new ones [23]. During the process of repair, MuSC cells also undergo self-renewal, thus replenishing the pool of quiescent SCs, which is critical for long-term muscle maintenance [27–29]. More detailed information on regulation of SC quiescence is beyond the scope of this review, but comprehensive reviews on this topic have been recently published [see reviews 30,31].

Many other cell types are also present in the adult muscle tissue, including fibroblasts, macrophages, and endothelial cells, among others (Fig. 1A). Fibroblasts produce most of the skeletal muscle extracellular matrix (ECM) [32–35], important for muscle structure in healthy muscle (Fig. 1A) and responsible for fibrosis in the diseased muscle environment (Fig. 1B), as reported [36]. The more recently identified fibro-adipogenic progenitor (FAP) cell population is also present [37]. In healthy skeletal muscles, FAPs are quiescent and involved in ECM maintenance. Upon injury, preceding SC proliferation, FAPs will expand and secrete growth factors to support SC-mediated regeneration [38–40]. FAP dysregulation has been documented for certain muscle disorders and can result in either fibrosis [39,41] or adipocyte accumulation [42]. Immune cells, in particular macrophages, neutrophils and lymphocytes, are also present in the skeletal muscle (Fig. 1A), participating actively in tissue homeostasis, muscle growth, and regeneration [see reviews 43,44]. In disease conditions characterized by multiple rounds of degeneration/regeneration, high numbers of immune cell infiltrates (Fig. 1B) exacerbate disease pathology [45–48]. Endothelial cells, part of the muscle vasculature, play important roles in the maintenance of the SC niche as well as regeneration [49], while pericytes are important for angiogenesis, vascular homeostasis, and SC maintenance [50,51].

Numerous disorders impair skeletal muscle function, such as muscular dystrophies (MDs), mitochondrial myopathies, volumetric muscle loss, sphincter incontinence, idiopathic inflammatory myopathies, cachexia and sarcopenia, among others. Below we briefly describe disease conditions for which cell therapies have been recently tested.

3. Skeletal muscle disorders

Most developments in the field of cell therapy for skeletal muscle have aimed at the treatment of MDs, which encompass more than 40 genetically inheritable diseases causing progressive muscle weakness and degeneration [see review 52]. Duchenne muscular dystrophy (DMD) is the most prevalent MD, affecting 1 in 5,000 male births. This X-linked recessive disorder is caused by mutations in the DYSTROPHIN (DMD) gene [53], leading to severe reduction or absence of this major muscle protein, whose main function is to act as a molecular bridge by connecting the intracellular actin cytoskeleton to the transmembrane dystroglycan complex, providing sarcolemma stability during muscle contraction [54,55]. The muscle wasting phenotype is progressive and severe with patients being usually wheelchair bonded by their teenage years [56], and succumbing to the disease before reaching their thirties mainly because of cardiorespiratory complications [57,58]. Because of its prevalence and devastating phenotype, DMD has been historically the main target for the development of skeletal muscle cell-based therapies.

Facioscapulohumeral muscular dystrophy (FSHD), with an incidence of 1 in 15,000 births, [59,60], is an autosomal dominant disease due to defective epigenetic repression of the D4Z4 macrosatellite repeat array [61]. This epigenetic defect leads to the loss of silencing of the transcription factor DUX4, which triggers expansion of FAPs, and eventually fibrotic degeneration of adult skeletal muscles [62]. The most affected muscles are the facial, shoulder, and forearm muscles, and life span is normal, as the diaphragm is largely spared and the heart is not affected. As treatments targeting DUX4 are likely to slow or stop progression, regeneration of lost muscle will be required to reverse the disease.

Limb girdle muscular dystrophies (LGMDs) comprise a genetically diverse group of MDs, affecting mostly hips and shoulder muscles [63–65]. Cell therapy is currently being developed for LGMDR2/LGMD2B, which results from mutations in the DYSFERLIN (DYSF) gene coding for the DYSF protein. This protein is involved in muscle repair and sarcomere stability [66]. Clinical manifestations appear between late teenage years and the 30s, with faster disease progression being associated with early onset [67,68]. Cell therapy is also under investigation for LGMDR1/LGMD2A, which is caused by mutations in the CALPAIN 3 (CAPN3) gene. CAPN3 is a skeletal muscle-specific isoform of the Calpain cysteine protease family [69]. While the exact role of CAPN3 is still unclear, this enzyme directly binds to titin, and thus, it has been suggested to be involved in assembly and maintenance of the sarcomere [70,71]. Clinical manifestations range from severe forms with rapid progression and onset as early as 2 years of age to milder forms showing slower progression and late onset [68,72].

Other skeletal muscle disorders of interest for this review are mitochondrial myopathies. These are progressive muscle diseases due to the malfunctioning of the mitochondrial oxidative phosphorylation (OXPHOS) system [73], which can result from mutations in nuclear genes involved in OXPHOS or mutations in the mitochondrial DNA (mtDNA). Heteroplasmic mutations in mtDNA account for approximately 20% of the mitochondrial myopathies [74]. This muscle disorder is characterized by the presence of red ragged fibers and muscle lactic acidosis, and main clinical manifestations are muscle fatigue and weakness. Exercise intolerance is also described for half of the patients. Myopathy incidence for mitochondrial mutations is high for patients in their 50s, illustrating the progressive nature of these disorders [75 and see review 76].

Sphincter incontinence encompasses both anal and urethral sphincter muscle weakening. Fecal incontinence (FI) is defined as the uncontrolled release of fecal material for at least one month after the age of 4 years old. This condition may affect up to 18% of the population [77], with 55% within the elderly [77] and 4% to 38% in women after vaginal birth [see review 78]. This condition significantly impacts life by causing emotional distress, social isolation, and physical complications [79]. The prevalence for urinary incontinence (UI) ranges from 10 – 40% in post-partum women [80]. Similar to FI, this disorder impacts patient’s physical, social, psychological well-being, and effective treatments are warranted [81].

4. The beginning of cell therapy for skeletal muscle diseases

Research on cell therapy for skeletal muscle started in the late 80s with a seminal proof-of-concept study showing that mouse primary myoblasts could engraft and rescue dystrophin expression in a mouse model of DMD [82]. This major discovery triggered numerous clinical trials in DMD, mostly using myoblasts isolated from the muscle biopsies of first-degree relatives [83–91], while one study in 21 DMD boys utilized both, myoblasts from relatives or non-related donors [92]. Although there were no safety concerns, most of the outcomes from these early clinical trials focused on the intramuscular (IM) transplantation of myoblasts were disappointing as engraftment and functional (when measured) results were overall poor and variable. While some studies reported significant rescue of DMD expression [87,89,91], many others found no or marginal DMD expression [83,84,86]. Several reasons could account for these discrepancies, including i) differences in myoblast preparation; ii) number of cells injected; iii) method of injection; iv) use or not of immunosuppression; and v) DMD detection methods, among several others. For instance, one study [92] documented force improvement in 43% of the 69 muscle groups tested in 13 patients, but there was no inclusion of a control group for functional measurements nor data for DMD expression. In a clinical trial of 5 immunocompetent DMD boys, Tremblay et al. found lower numbers of DMD+ myofibers at 6 months post-transplant compared to 1-month counterparts, which was attributed to rejection, thus suggesting the need for immunosuppression even when siblings were donors [87,89,91]. However, the use of immunosuppression, such as cyclosporine or cyclophosphamide, also sparked controversy [83–86,88,90,92] as these drugs, without the inclusion of proper controls, may have confounded the interpretation of functional data, since they have been shown to improve the dystrophic phenotypes in dog and mouse models [93,94]. This is particularly relevant when force improvements were not reported to be associated with the presence of DMD+ myofibers. The study by Mendell and colleagues [84] involving 12 DMD boys had two cohorts, with or without cyclosporine. The best outcome was 10.3% DMD+ fibers in one of the immunosuppressed patients, but this was not accompanied by force improvement.

Additional challenges associated with myoblast transfer included massive cell death after injection and limited migration. This led to debate regarding the injection method, as some groups favored 50 to 100 injections per muscle to promote further injury to induce myoblast fusion with endogenous myofibers and counter their poor migration capacity, while others preferred a few injections to avoid sensitizing the immune system. Subsequent clinical trials using the immunosuppressant agent tacrolimus were reported to produce superior engraftment [95–97]. A study by Skuk and colleagues [97] documented 35% DMD+ myofibers in one patient’ gastrocnemius using a high-density injection protocol. This same patient also received myoblast transfer in the biceps muscle, but donor-derived myofibers were not found. The authors described this muscle to contain much more adipose tissue and fibrosis, which may be the reason for the poor outcome compared to the gastrocnemius.

During the next 30 years following these initial myoblast transfers in DMD, many strategies have been developed to circumvent encountered challenges. Despite the relative disappointment with the early myoblast clinical trials, intense research continued over the years to improve the efficiency of myoblast transplantation, which included preclinical studies using larger animal models [98–101], optimization of injection strategy [102–104] as well as the development of more sensitive and accurate detection methods, among other parameters [105–107]. For instance, using the non-human primate (NHP) model, Tremblay and colleagues optimized the transplantation protocol by assessing the relationship between cell number and cell ischemic necrosis [108], the importance of needle size, number of cells and volumes to inject [104].

Based on the outcomes of DMD clinical trials demonstrating the limited migratory ability of myoblasts, it was conjectured that these cells would be more suitable for localized muscle disorders, and therefore clinical trials were devised to test the efficacy of myoblast transfer in localized muscle wasting pathologies, such as UI and FI, which produced more encouraging results [109–124]

In this review, we discuss cell products recently tested or currently being tested in patients (Tables 1 and 2), with an emphasis on cell replacement therapies (Fig. 2B and Fig. 3; Table 1), but noting that paracrine cell therapies have also been assessed for therapeutic benefit (Fig. 2C and Table 2).

Table 1.

List of recent and ongoing clinical trials involving cell replacement therapy.

Muscle disorder	Cell type	Cell product	Muscle targeted	Injection route & (number of cells)	Immuno-suppression	Clinical trial number	Status	Phase	Publication
DMD	Myoblasts	Allogeneic myoblasts	ECR muscle	IM (30 million per cm3)	Yes (tacrolimus)	NCT02196467	Unknown	I/II	NA
	MABs	Autologous MABs	EDB muscle	IM (not specified)	No	EUCT number: 2024-51486014-00	Ongoing, recruitment ended	I/II	NA
	Chimeric cells	Chimeric cells (DT-DECO1)	Potentially all muscles	Intraosseous 2 million/kg	No	EUCT number: 2024-51900427-00	Authorized, recruitment pending	I/II	NA
	iPSC-derived myogenic progenitors	Allogeneic iPSC- derived myogenic progenitors (MyoPAXon)	EDB muscle	IM (25 to 200 million)	Yes (tacrolimus)	NCT06692426	Recruiting	I	NA
LGMDR1 and LGMDR2	MuSC-outgrowths	Autologous gene edited MuSC-outgrowths (GenPHSat)	Biceps	IM (not specified)	No	NCT05588401	Unknown	I/IIa	NA
Mitochondrial myopathies	MABs	Autologous MABs	Tibialis Anterior	IA (170 million)	No	NCT05063721	Completed	I	[158]
			Biceps Brachii	IA (3 injections of 170 million)	No	NCT05962333	Recruiting	II	NA
UI	Myoblasts	Autologous myoblasts (Iltamiocel)	Urethral muscles	IM (150 million)	No	NCT01893138	Completed	III	[126]
				IM (150 million)	No	NCT03104517 CELLEBRATE	Active, not recruiting	III	NA
		Autologous myoblasts (Innovacell)	Urethral muscles	IM (0.2 million)	No	EUCT number: 2010-02186734	Completed	I/II	[132]
	MuSC-outgrowths	Autologous MuSC-outgrowths (Satori-01)	Urethral muscles	IM (not specified)	No	NCT04729582	Not yet recruiting	I/IIa	NA
FI	Myoblasts	Autologous myoblasts (Iltamiocel)	Anal sphincter	IM (250 million)	No	NCT01600755	Completed	I/II	[127]
				IM (300 million)	No	NCT05776277 DigniFI	Active, not recruiting	III	NA
		Autologous myoblasts (Innovacell)	Anal sphincter	IM (1 or 50 million)	No	NCT04976153	Completed	II	[131]
				IM (not specified)	No	NCT04976153 Fidelia	Recruiting	III	NA
	MFFs	Autologous MFFs	Anal sphincter	IM (not specified)	No	NCT05396456	Recruiting	NA	NA
Tongue dysphagia	Myoblasts	Autologous myoblasts (Iltamiocel)	Tongue muscle	IM (150 million)	No	NCT02838316	Completed	I	[130]
				IM (300 million)	No	NCT02838316	Active, not recruiting	II	NA

DMD: Duchenne muscular dystrophy; ECR: extensor carpi radialis; IM: intramuscular; LGMD: limb girdle muscular dystrophy; MuSC: muscle stem cell; IA: intraarterial; MABs: mesoangioblasts; MFFs: muscle fiber fragments; iPSCs: induced pluripotent stem cells; MPs: myogenic progenitors; EDB: extensor digitorum brevis; UI: urinary incontinence; FI: fecal incontinence.

Table 2.

List of recent and ongoing clinical trials involving paracrine cell therapy.

Muscle disorder	Cell type	Cell product	Muscle targeted	Injection route & (number of cells)	Immuno-suppression	Clinical trial number	Status	Phase	Publication
DMD	CDCs	Allogeneic CDCs (CAP-1002)	Heart and upper limb muscles	IA (75 million)	Oral glucocorticoids, H1 and H2 blockers few hours before cell infusion.	NCT02485938 HOPE-1	Completed	I	[175]
			Potentially all muscles	IV (4 injections of 150 million)		NCT03406780 HOPE-2	Completed	II	[176]
						NCT04428476 HOPE-2	Active, not recruiting	Open label extension	NA
						NCT05126758 HOPE-3	Active, not recruiting	III	NA
	WJ-MSCs	Allogeneic WJ-MSCs (EN001)	Potentially all muscles	IV (0.5 million/kg or 2.5 million/kg)	Hydrocortisone, lorazepam, ondansetron and chlorpheniramine were used as premedication	NCT05338099	Completed	I	[180]
				IV (to be defined)		NCT06328725	Not yet recruiting	II	NA
FSHD	ULSCs	Allogeneic ULSCs	Potentially all muscles	IV (2 injections of 100 million)	Not specified	NCT07086521	Recruiting	I	NA

DMD: Duchenne muscular dystrophy; IA: intraarterial; FSHD: facioscapulohumeral muscular dystrophy; CDCs: cardiosphere-derived cells; WJ-MSCs: Wharton’s Jelly-derived mesenchymal stem cells: ULSCs: umbilical cord-lining stem cells; IV: intravenous.

Fig. 2.

Modalities of cell-based therapies for skeletal muscle. Schematic representations show a diseased muscle bundle (A) and changes resulting from cell replacement therapy with the presence of donor-derived myofibers and donor-derived SCs (B), or from paracrine-based cell therapy, in which transplanted non-myogenic cells secrete factors beneficial for muscle regeneration (C). FAPs: Fibro-adipogenic progenitors.

Fig. 3.

Recent and ongoing cell replacement clinical trials for skeletal muscle disorders. Schematic highlights the skeletal muscle disorders currently being tested, the cell type used, the injection method, and the muscle(s) targeted. DMD: Duchenne muscular dystrophy; ECR: extensor carpi radialis; IM: intramuscular; LGMD: limb girdle muscular dystrophy; MuSC: muscle stem cell; IA: intraarterial; MABs: mesoangioblasts; MFFs: muscle fiber fragments; iPSCs: induced pluripotent stem cells; MPs: myogenic progenitors; EDB: extensor digitorum brevis.

5. Muscle cell therapies

5.1. Cell replacement therapies

5.1.1. Culture expanded primary muscle cells

On the DMD front, a phase I/II trial was opened in 2014 to study the outcomes of myoblast transfer from unrelated healthy donors in the extensor carpi radialis (ECR) muscles of 10 DMD patients using the high-density injection protocol (30 million cells per cm³). Muscle force measurements were proposed for the 3- and 6-month post-injection timepoints (the latter only if improvements were confirmed at 3 months). To date, no results have been communicated for this study as the last update was released in 2021 when recruitment was active (NCT02196467).

Localized muscle disorders, such as UI, FI or tongue dysphasia, have been the major focus for the clinical use of myoblasts. The company Cook MyoSite has been exploring the clinical application of autologous myoblasts, referred as Iltamiocel, for these three conditions with success (Fig. 3). Using a production facility in compliance with current Good Manufacturing Practice (cGMP), they employ a pre-plating method for myoblast isolation [125] from vastus lateralis patient’s biopsies, which are then expanded and frozen down for future autologous transplantation. Since phase I/II and III clinical trials for UI have shown safety and potential clinical benefits in affected women [120,121,124], Iltamiocel was subsequently tested in a double-blind, randomized, stratified, placebo-controlled trial (NCT01893138) [126], in which two hundred ninety-seven patients were randomized between Iltamiocel (n = 199) and placebo (n = 98). Of note participants in the placebo group could receive the actual treatment as open labeled after 1 year. Patients received 150 million cells obtained from a 250 mg biopsy through a transurethral procedure consisting of nine IM circumferential urethral injections. Only 2 patients did not complete the 12-month follow-up, and 3 cases (1%) were reported as serious adverse events (SAEs) related to the treatment or study procedures, among which, 2 consisted of hypersensitivity reactions. While the number of patients achieving the primary endpoint of 50% reduction in incontinence episodes (IE) was not statistically different between treated and placebo cohorts, the number of participants who received prior UI surgery and achieved ≥ 75% IE reduction at 12 months trended higher compared to controls. These encouraging data prompted a new clinical study design: a double-blind, randomized, controlled phase III clinical trial looking at the reduction of IE in women with postsurgical persistent UI. Based on clinicaltrials.gov, this trial is currently active, and the recruitment has been completed (CELLEBRATE, NCT03104517).

Iltamiocel has also been investigated as a therapeutic option for FI, and results of the phase I/II study have been published [127]. During this non-randomized study, 48 patients with FI were injected with a single dose of 250 million autologous myoblasts. Cells were dispensed through 8 to 12 IM injection sites parallel to the anal canal circumferentially into the external anal sphincter. While no SAEs were reported during the 12-month follow-up period, there were adverse events linked to the biopsy procedure (n = 37), myoblast injection (n = 10), and the cell product (n = 2). Functional measurements showed a reduction of the number of fecal incontinence episodes (FIE) and a reduction of the number of days with FIE. Overall, more than 50% of the patients achieved at least 50% reduction in the number of FIE. Based on the safety and pre-efficacy outcomes of this phase I/II study [127], this was immediately followed by a phase III study (NCT05776277), named DigniFI, consisting of a two-stage, randomized, controlled clinical trial aimed at testing safety and efficacy of Iltamiocel injections in women affected by FI with an history of obstetric anal injuries. This clinical trial is active, and the recruitment completed: 200 patients will receive a single dose of 300 million myoblasts.

In addition to incontinence, Iltamiocel has been explored as a therapeutic option for tongue dysphagia following chemoradiation treatment for oropharyngeal squamous cell cancer. Studies in mice and sheep have shown the ability of myoblasts to engraft in tongue muscle and improve strength and function [128,129]. The first-in-human study, published in 2022, reported safety and possible functional improvement. In this study, ten male patients received 150 million myoblasts injected in the intrinsic muscles of the tongue at four different locations. No SAEs were reported during the 2-year follow-up. At the six-month timepoint, tongue strength was significantly improved in the myoblast-injected cohort compared to the placebo group, and this was maintained up to 24 months [130]. A phase II randomized clinical trial, in which patients will receive a higher dose of 300 million cells, is in progress, and 18 patients have already been enrolled (NCT02838316).

The company Innovacell is also applying transfer of skeletal muscle-derived cells/myoblasts for FI and UI. Results from a randomized, placebo-controlled FI study have been recently published [131]. Two hundred forty-four patients have been divided into 3 cohorts: i) placebo; ii) low dose (1 million cells); and iii) high dose (50 million cells). Pelvic floor electrical stimulation therapy, which is supposed to stimulate muscle growth and is used as a treatment for FI was performed before and after cell injection. Data from patients were collected for up to 12 months post-treatment. In terms of safety, 2 SAEs were reported, one related to the biopsy and one to the pelvic floor electrical stimulation, and 25 adverse effects were described related to injection. In terms of efficacy, the high dose group displayed significant reduction in FIE compared to control counterparts. A deeper analysis of the data revealed that the subgroup of patients affected by FI for less than 10 years showed more benefits from the treatment compared to patients affected for a longer period of time, suggesting that the status of muscle pathology is a key parameter to take into account when predicting successful outcomes. Recruitment for the confirmatory phase III is currently ongoing in Europe (EUCT Number: 2010-021463-32 and NCT04976153).

Regarding UI, results of a phase I/II open, non-randomized, single-center study (EUCT number: 2010-021867-34) were published in 2022 [132]. Thirty-eight female patients received a dose of 0.2 million cells, using an ultrasound-directed transurethral injection tool, in the external urethral sphincter. There were no adverse events related to the biopsy, but during the 2-year follow-up, 6 SAEs were reported, all described as cystitis. Whereas no changes were observed in urodynamic urethral properties, recovery in IE score, quality of life, as well as patient’s and clinician’s perception of improvement were documented. Of note, in this same study, a cohort of patients received pelvic floor muscle stimulation in parallel to cell injection, and showed better urodynamic values and maintenance of UI improvements for the 2-year duration of the study. A larger patient cohort along with a placebo control group should help to confirm these results.

A less orthodox alternative cell-based strategy for the treatment of skeletal muscle pathologies consists of the use of chimeric cells, a result from the fusion of a healthy donor cell with the patient’s cell, which have the potential to provide a healthy copy of the gene of interest and be tolerated by the recipient [133]. Siemionov and colleagues fused myoblasts from a healthy donor with myoblasts from a DMD patient, and then reported engraftment and functional improvement upon the IM transplantation of these chimeric cells into the gastrocnemius muscle of SCID/mdx mice [134]. Subsequently, these investigators tested systemic-intraosseous transplantation of these cells in mice, as these chimeric myoblasts were postulated to home to the bone marrow and function as a reservoir of DMD+ myogenic cells able to travel all over the body to repair muscle damage, a theory that has been questioned [135]. Long-term improvement was reported in different muscles, but engraftment was scant as indicated by the very few human donor-derived spectrin+ myofibers shown [136]. Upon approval by the hospital exemption framework in the European Union, patients were injected with these cells (DT-DECO1). Three DMD patients received injections of chimeric cells in the iliac crest bone marrow at a dose of 2 million cells per kg. Intraosseous injections of chimeric myoblasts appeared to be safe as no adverse effects were reported for the 3 patients during the 12-month follow-up, and no immune response against the donor HLA was observed [137,138]. Several functional assessments were improved but expression of DMD in patient biopsies was not reported. With the goal of eventually obtaining approval from the European Medicines Agency (EMA), a phase I/II study was initiated in 2024, which is currently enrolling pediatric patients (EUCT number: 2024-519004-27-00).

A novel approach involving culture expanded primary muscle cells has been developed by Spuler and colleagues, which consists of subjecting human muscle fiber fragments (HMFFs) to hypothermic treatment and subsequently expanding these as MuSC outgrowths that are endowed with regenerative capacity and are amenable to genetic modification [139]. Upon transplantation in a rat model of damaged sphincter, functional recovery was documented [140]. While engraftment of human cells was not reported, the treated group showed normal histology of the urethral sphincter tissue compared to placebo. This study led to the design of a clinical trial in which patients with congenital UI (isolated epispadias) will be transplanted with autologous MuSC outgrowths, Satori-01, in the urethral sphincter (NCT04729582).

These same investigators also combined this technology with gene editing. Transplantation studies of gene edited MuSC outgrowths were reported in mice for LGMDR1/LGMD2A [141] and LGMDR2/LGMD2B [142], with engraftment outcomes averaging 30–40 DYSF+ donor-derived myofibers in hEx44mut mice that had been pre-injured with irradiation or irradiation + cardiotoxin (CTX) [142] and 20 human donor-derived myofibers in NSG-C3KO mice [141]. Based on these preclinical data, a Phase 1/2a first-in-human trial of autologous gene-edited MuSC outgrowths, referred as GenPHSats, has been initiated for LGMD (NCT05588401). The initial intervention to assess safety consists of six injections into the left biceps. If results are favorable, this will be followed by a second procedure aiming at efficacy with 36 injections into the right biceps.

5.1.2. Muscle fiber fragments (MFFs)

Transplantation studies in mice and pigs have provided evidence for the regenerative potential of freshly isolated myofibers [27,143–145] as well as MFFs [139]. The MFF approach, based on the injection of minced autologous skeletal muscle tissue, was tested more than a decade ago in a clinical trial for UI, involving 20 and 15 women with uncomplicated and complicated cases, respectively [146]. Overall outcomes were superior in the uncomplicated group, for which it was reported 25% cure and 63% improvement, whereas for the most affected patients, these dropped to 7% and 57%, respectively. The therapy was considered safe as only minor adverse events were reported [146].

More recently, Ko and colleagues postulated that uniformly sized MFFs may represent a better option for therapeutic purposes than long myofibers [147]. MFFs injected in pre-injured rat gastrocnemius muscles led to improvement in muscle strength compared to controls, however quantification of the number of MFF-derived myofibers was not reported. A phase I clinical trial has been initiated to investigate the benefits of autologous MFF transplantation for FI, bowel dysfunction, and rectal disorders, which is currently recruiting (NCT05396456).

5.1.3. Mesoangioblasts (MABs)

Numerous preclinical studies have highlighted the therapeutic potential of MABs, also known as pericytes, for skeletal muscle disorders, as these cells can be delivered systemically, give rise to donor-derived myofibers and are amenable to genetic modification to correct patients’ mutations [148–155]. Following the extensive characterization of their regenerative capacity in several mouse models of MD as well as in dystrophic dogs, a clinical trial involving the systemic delivery of HLA-matched donor MABs was conducted in DMD boys. While there were no safety concerns, efficacy was reported to be low [156], which was suggested to be a consequence of advanced disease pathology, insufficient cell dose, and/or issues with MAB extravasation due to the use of anti-inflammatory/immunosuppressant drugs. Next, Cossu’s team developed an autologous cell transplantation strategy by applying cell-mediated exon skipping to MABs from DMD patients with mutations in exon 51 of the DMD gene [157]. Lentiviral transduction of human patient MABs with a small U7 nuclear RNA engineered to skip exon 51 restored DMD expression, and upon IM or intraarterial (IA) injections in NSG-mdx-Δ51 mice, DMD expression was restored in vivo. The authors observed that once donor-derived MABs integrated within a myofiber, the U7 small RNA was able to travel from myonuclei to myonuclei to restore DMD expression, leading to significant DMD rescue that was detectable by western blot. These results led to a phase I/IIa clinical trial aimed at assessing the safety of autologous MABs genetically corrected with U7 snRNA exon 51 skipping lentiviral vector upon their IM injection in the extensor digitorum brevis (EDB) muscles of 5 non-ambulatory DMD patients (EUCT number: 2024-514860-14-00). Based on euclinicaltrials.eu, this trial is ongoing, and the recruitment completed.

The IA delivery of autologous MABs has also been recently tested in patients with mitochondrial myopathies, and the results from a phase I/II study in 3 patients carrying the mtDNA mutation m.3243A>G have just been published [158]. This clinical trial was built from previous findings by this group suggesting that MABs have a lower load of mutated mtDNA compared to skeletal muscle cells [159]. Therefore, the premise is to use low mutation load MABs as a source for muscle cell replacement. Autologous MABs were injected at 50 million/kg in the femoral artery. Because muscle wasting and cycles of degeneration/regeneration are not characteristic of this disorder, the clinical trial design involved the pre-injury of the patient’s Tibialis Anterior (TA) muscle with eccentric contraction one day prior to MAB transplantation to stimulate the homing and engraftment of infused MABs. With the goal to detect infused cells, the investigators made an attempt to stain for MABs using indocyanine green dye one day after injection, but unfortunately, the first patient for which this was tested reacted to the injection with a local grade II adverse event, so this staining was not used on the 2 other patients (NCT05063721). No additional adverse events were observed. MAB engraftment was then measured by quantifying NG2+ cells and NCAM+ myofibers at 24 hours and 4–6 weeks post-transplantation, respectively. At the first timepoint, significant increases of NG2+ cells were detected for one patient, suggesting the presence of MABs in the targeted TA. For the second timepoint, even though targeted muscles showed more NCAM+ myofibers, there was no significant difference from the control. A subsequent phase IIa clinical trial is underway to assess the efficacy of three IA administrations of autologous MABs to promote strength and reduce fatigue of the biceps brachii muscle of m.3243A>G patients (NCT05962333).

5.1.4. Pluripotent stem cell (PSC)-derived myogenic progenitors

Clinical trials involving both allogeneic and autologous transplantation of PSC-derivatives for several diseases are ongoing, including macular degeneration, Parkinson’s disease, as well as solid tumors and hematological disorders, among others, for which reported outcomes provide evidence for safety [see reviews 160–162]. Just recently the first-in-human clinical trial of induced PSC (iPSC)-derived myogenic progenitors, MyoPAXon, has been authorized for skeletal muscle (NCT06692426).

MyoPAXon, licensed by Myogenica, is an off-the-shelf cryopreserved cGMP allogeneic myogenic drug product (DP) manufactured from healthy iPSCs derived from cord blood. This DP is the result of more than a decade of proof-of-concept studies assessing the regenerative potential and safety of human PSC-derived PAX7-induced myogenic progenitors in several models of MD [163–167]. In a recent study, the authors describe the detailed cGMP manufacturing of MyoPAXon as well as its comprehensive in vitro and in vivo characterization [168]. Biodistribution and safety studies in NSG mice under full GLP conditions showed no adverse effects. Local transplantation of MyoPAXon in immunodeficient mouse models of DMD and LGMDR9/LGMD2I resulted in human donor-derived myofibers (~200) and SCs, in addition to the improvement of muscle specific force. Long-term studies in NSG mice showed persistent engraftment as hundreds of donor-derived myofibers were detected at 13 months post-injection. The feasibility and safety of transplanting large numbers of MyoPAXon were investigated in NHP recipients. Between 40 and 60 million cells were injected in CTX-injured EDB muscles, the same muscle that will be targeted in the phase I clinical trial, which produced up to 1800 donor-derived myofibers. The first-in-human dose escalation clinical trial of MyoPAXon was designed to address safety and tolerability of IM injections of MyoPAXon in the EDB muscle of a small cohort of adult DMD patients. The first patient will be injected with 25 million cells, and if the DP is well tolerated, the next patients will receive a higher dose. Tacrolimus will be used as an immunosuppressant since this is an allogeneic cell transplantation. The duration of the study is 3 months, at which time the EDB muscles will be collected for histological assessment. This clinical trial is currently enrolling (NCT06692426).

5.2. Paracrine cell therapies

These comprise cell-based therapies in which the mechanism of action relies on the secretion of factors to support muscle regeneration, not on cell replacement. Cardiosphere-derived cells (CDCs) and umbilical cord-derived mesenchymal stem cells (UC-MSC) are the main cell sources currently being tested in the clinic. CDCs have been reported to possess regenerative, anti-inflammatory, and anti-fibrotic capacities [169–171], and to improve the disease phenotype of mdx mice [172,173], as a result from the secretion of growth factors and exosomes laden with microRNAs [174]. Capricor Therapeutics has led a phase I trial, referred as HOPE, in which DMD patients were injected with allogeneic CDCs, named CAP-1002 (NCT02485938). The results of this phase I study indicated safety and highlighted the capacity of CDCs to alleviate the disease phenotype of upper limb skeletal muscles and heart [175]. However, because CDCs do not engraft, their effect is transient, thus requiring repeated injections. This was subsequently tested in a phase II clinical trial (HOPE-2; NCT03406780), in which patients received CAP-1002 every 3 months for a total of 4 doses during a 12-month period. Five hypersensitivity reactions were reported among three (38%) individuals in the CAP-1002 group. Since one of them was considered a SAE, an amendment to the protocol was implemented to include a glucocorticoid pretreatment to reduce immune reaction. After implementation of this protocol, only one hypersensitivity reaction was observed. By the 12-month time point, patients showed stabilization of upper limb function and improvement of cardiac function compared to the placebo group [176]. This was followed by an open label extension of the HOPE-2 clinical trial (HOPE-2-OLE), in which patients who had completed the 12-months follow-up were eligible to receive twenty intravenous administrations of CAP-1002, each separated by three months, over a period of approximately 60 months. Following these encouraging results, the anticipated phase 3 (HOPE-3) pivotal trial was launched in 2022 (NCT05126758). HOPE 3 is a multi-center, randomized, double-blind, placebo-controlled clinical trial including ambulatory and non-ambulatory DMD boys aimed at evaluating the efficacy and safety of CAP-1002. To date, 104 patients have been enrolled and study completion is estimated for 2027.

UC-MSCs, also referred as UC-lining stem cells (ULSC), have also been proposed as a cellular alternative to ameliorate skeletal muscle disease phenotype [177–179]. Wharton’s Jelly-derived MSCs (WJ-MSCs), produced by ENCell, have been tested in a phase I clinical trial for DMD (NCT05338099), and the outcomes recently reported [180]. Functional tests performed at 12 weeks post-injection showed no difference with baseline, questioning the therapeutic effects of this strategy [181], however, a phase II clinical trial has followed (NCT06328725). RESTEM has proposed to test allogeneic cGMP ULSC preparations in a phase I study, not yet recruiting, to assess safety and preliminary efficacy in FSHD patients (NCT07086521).

6. Current challenge, limitations, and future directions

As discussed in this review, there has been significant progress in the translation of cell-based therapies for skeletal muscle disorders in recent years, with numerous clinical trials involving several different cell products, targeting distinct muscles, and different skeletal muscle disorders. However, some major challenges still need to be taken into consideration. In the context of cell replacement therapies, an important aspect is the regenerative capacity of transplanted cells. It is apparent that not all cell products tested or under evaluation in clinical trials have been properly assessed for safety and efficacy in preclinical work. Even though studies in animal models do not fully recapitulate the environment transplanted cells will encounter when injected into patients, these provide proof-of-concept for their regenerative capacity, guidance for extrapolating cell doses, and reassurance of potential clinical benefit. Ideally, a cGMP-compatible cell product should provide robust and unquestionable engraftment results using at least two human-specific markers (for rigor) in relevant animal models of disease. In addition, functional improvement should be measured and linked to the presence of donor-derived myofibers in tested muscles. For degenerative skeletal muscle conditions, such as MDs, the detection and quantification of donor-derived SCs is critical for enabling long-term cell replacement. Rushing cell products into clinical trials without sufficient in vivo proof-of-concept foundation raises two major concerns: foremost safety of treated patients, but also the possibility of ruining the reputation of cell-based therapies in general.

Delivery has been viewed as a major challenge for the successful implementation of cell replacement therapies in skeletal muscle disorders. While the treatment of focal skeletal muscle disorders, including UI, FI, dysphasia or oculopharyngeal muscular dystrophy (OPMD), is much more feasible with IM injections than muscle disorders characterized by widespread muscle defects (DMD, LGMDs, FSHD or mitochondrial myopathies, among others), it is still possible to easily target smaller muscles to provide benefit with such diseases, such as targeting the hand muscles of non-ambulatory DMD patients so they maintain the ability to feed themselves, use computers, and/or operate a wheelchair. Larger muscles would require multiple injection sites, which may be feasible, as shown in earlier myoblast transplantation studies, as long as large numbers of the drug cell product are available. Systemic delivery would be the ideal route of administration to allow widespread distribution of injected cells to multiple skeletal muscles. Based on preclinical studies, MABs have the capacity to home efficiently to target muscles upon IA administration [148–151,153,154], but this has not been clearly demonstrated in clinical trials yet, as IA delivery of MABs in DMD patients showed limited engraftment, which could be due to reduction of MAB extravasation due to anti-inflammatory and immunosuppressive therapies, but also to other issues, such as the number of cells delivered and/or status of muscle pathology [156], whereas the studies in mitochondrial myopathies did not involve engraftment quantification [158]. IA injection of myoblasts has been tested in pre-injured rat and NHP recipients [102,182]. While widespread donor-derived myofibers were detectable in the gastrocnemius muscle after myoblast infusion in NHPs, suggesting a certain capacity for these cells to be delivered using this route, direct comparison of IM and IA injections showed vascular delivery to be suboptimal [102]. Other cell products remain to be tested using systemic routes.

Cell transplantation can elicit an immune response. Autologous cell products are not expected to trigger immune reactions; however, these personalized treatments can be costly. For instance, numerous skeletal muscle diseases are due to unique or rare genetic mutations and would require gene correction prior to autologous cell transplantation, which would involve screening as well as characterization of properly corrected clones, which further significantly augments the cost and production time. In addition, immunosuppression may be needed as there is the possibility of immune reaction against the new protein present in the modified patient cells. On the other hand, allogeneic cell transplantation presents the advantage that a single product may be used for multiple patients in addition to multiple indications. However, patients may potentially need lifelong immunosuppression and experience related side effects. To circumvent this feasibility issue, there has been significant investment by several institutions worldwide in the establishment of universal donor iPSC banks, also referred as the HLA haplobank model [183–186], which would allow for the selection of matched donors to generate graft material that would require only limited immunosuppression. For iPSC-derived drug products, there has been also discussion on the generation of universal iPSC lines, which are genetically modified to be invisible to the immune system [187]. Although attractive, this raises obvious safety concerns.

The status of muscle pathology at the time of treatment is another important parameter to be considered, in particular for progressive skeletal muscle disorders, such as MDs. At least two publications in DMD attributed negative or poor clinical trial outcomes potentially to the absence or limited presence of endogenous myofibers in transplanted muscles, which coincided with elevated fibrosis [97,156]. For instance, when cells were transplanted in a less affected skeletal muscle of the same patient (gastrocnemius vs. biceps), myoblasts were able to engraft robustly [97]. These data suggest that the presence of endogenous fibers and the muscle surrounding environment are helpful in the engraftment process, and therefore, early intervention would likely produce more encouraging outcomes. This represents a major catch 22, as, per safety guidelines, first-in-human safety-focused clinical trials are not typically authorized in pediatric patients without expectation of functional benefit. Cell transplantation in non-functional muscles of patients with advanced pathology can demonstrate safety, but would be unlikely to engraft well. Muscles with the least wasting in such patients would make the best test case to predict engraftment. Another significant challenge in some diseases, including DMD, is the recent flurry of approved gene therapies, which renders patients with a certain background of DMD-expressing fibers that may exclude them from eligibility for cell therapy on the basis of confounding influence of the prior therapy.

Nevertheless, regardless of these challenges and limitations, cell-based therapy has entered into an exciting era in the field of skeletal muscle diseases.

HIGHLIGHTS.

• No cell therapy is currently approved for skeletal muscle disorders.

• Cell replacement focuses on primary cells and iPSC-derived myogenic progenitors.

• There are 16 ongoing cell therapy clinical trials for skeletal muscle disorders.

• FDA authorized a first-in-human iPSC-based therapy for skeletal muscle disorders.