Overexpression of the myogenic transcription factors MyoD and Pax7 both reliably induce iPSCs toward a myogenic fate. The first examples of iMP direct reprogramming demonstrated that activation of these inducible transgenes generated cells that reliably engraft into muscle tissue, and are capable of restoring tissue specific expression of dystrophin and a-sarcoglycan in their respective murine disease models (Darabi et al., 2012; Tedesco et al., 2012). Since these initial demonstrations, direct reprogramming protocols have been used to reliably produce iMPs with similar regenerative capacity (Tanaka et al., 2013; Quattrocelli et al., 2015; Uezumi et al., 2016; Azzag et al., 2022). Despite these successes, a number of caveats specific to direct reprogramming warrant consideration. First, many direct reprogramming strategies adopt the use of small molecules and growth factors to prime iPSCs towards a mesodermal lineage prior to myogenic transgene induction, raising the question of whether introducing transgenes for the final step of myogenic specification is truly necessary (Magli et al., 2017; Rao et al., 2018; Selvaraj et al., 2019). Correspondingly, direct reprogramming inherently skips critical developmental maturation steps and little has been done to assess the relative developmental maturity of iMPs generated using this method. We will elaborate on the importance of iMP maturity in later sections; however, the lack of evidence for maturation in these cells represents one of the most critical shortcomings of direct reprogramming. Concerns over the risks associated with random transgene integration in direct reprogramming have also drawn scrutiny. Targeting transgenes to genetic safe-harbor loci and transient overexpression systems have been developed to reduce the perceived risks associated with transgene integration but are likely precautionary and immaterial to therapeutic application (Sadelain et al., 2012; Ordovás et al., 2015; Kim et al., 2017; Kim et al., 2021; Shin et al., 2020).

Despite the drawbacks associated with direct reprogramming the FDA has recently given clinical trial approval for a first-of-its-kind muscular dystrophy treatment in which allogeneic cell banks, induced to a myogenic lineage using Pax7 overexpression, will be assessed for safety, tolerability, and engraftment capacity in DMD patients (Myogenica, 2024). Successes in these early trials have the potential to transform the landscape of myopathy-targeting therapeutics and open the door to more advanced therapeutic modalities, as we discuss below.

Unlike direct reprogramming, directed differentiations use transgene-free methods of generating iMPs, relying on a holistic consideration of the developmental conditions that give rise to adult MuSCs. The first directed differentiation protocols were adapted from human embryonic stem cell (hESC) systems, and relied on undefined medias to differentiate embryoid body outgrowths into iMPs (Barberi et al., 2007; Chang et al., 2009; Mizuno et al., 2010; Awaya et al., 2012). While heterogeneity in the resulting populations was duly noted, these early studies provided evidence of myogenic potential in human iPSCs, paving way for more sophisticated protocols.

In 2016, two seminal papers established the culture conditions for generating Pax7+ myogenic progenitors from human iPSCs, optimizing an approach that induces presomitic mesoderm (PSM) specification prior to myogenic progenitor differentiation (Chal et al., 2015; Chal et al., 2016; Shelton et al., 2016). The resulting iMPs demonstrated robust myogenic capacity, but the rationale for the timing and concentration of certain growth factors remains dubious, even today. As these remain two of the most cited protocols for the generation of iMPs, a more granular screening aimed at optimizing these conditions is warranted and overdue. Data on the transition from early fetal to adult skeletal muscle in human tissue isolates are publicly available and may provide insights that would resolve this ambiguity (Xi et al., 2020).

Today, generation of iMPs using PSM to myogenic precursor differentiation has become the gold-standard in directed differentiations. However, the resulting cells are heterogeneous and there remains potential for further optimization and refinement (Bou Akar et al., 2024). Directed differentiations can generate iMPs that stably express Pax7 and are a more developmentally accurate recapitulation of MuSC-like cells than those generated via direct reprogramming. Standardization of directed differentiation protocols will be crucial to delivering therapeutic-grade iMPs. Towards this end STEMCELL technologies has released a product (STEMdiff™ Myogenic Progenitor Supplement Kit), that reliably produces a robust population of iMPs. cGMP qualification of products like this would represent a major step towards therapeutic application.

Regardless of the method used to generate iMPs, their therapeutic potential remains constrained by population heterogeneity. Direct reprogramming protocols utilize Pax7-driven fluorescent reporters to facilitate iMP purification, but neural crest progenitors, which also express Pax7, commonly contaminate these cultures (Basch et al., 2006). Non-transgenic directed differentiation strategies lack fluorescent reporters, necessitating the identification of surface markers that correlate with myogenic Pax7 expression. Cell surface markers for the purification of adult MuSCs have been identified, but are not reliably expressed in human iMPs (Wosczyna and Rando, 2018; Mierzejewski et al., 2020). Characterization of Pax7+ iMPs has led to the identification of several combinations of cell surface markers that aid in their purification. While CD54, CD10, CD82, CD56, and CDH13, and FGFR-4 all correlate with Pax7+ expression in human iMPs, the epidermal growth factor receptor ERBB3 has thus far been demonstrated the most reliable (Choi et al., 2016; Uezumi et al., 2016; Hicks et al., 2018; Sakai-Takemura et al., 2018; Wu et al., 2018; Nalbandian et al., 2021). In combination with positive selection for CD271 and negative selection of CD57 neuroectodermal contaminates, ERBB3 allows robust purification of iMPs that are phenotypically similar to the MuSCs observed developmentally during secondary myogenesis (Hicks et al., 2018; Tey et al., 2019). To validate myogenicity after cell sorting, iMPs should be plated and immediately stained for Pax7 and MyoG. Additional confirmation of myogenic potential should be confirmed by staining for myosin heavy chain (MYH) and dystrophin following subsequent differentiation into myotubes.

3D directed differentiation of iMPs attempts to integrate the culture conditions established in 2D protocols with the spatial/environmental cues afforded by 3D organization. In one study, fully humanized multilineage embryoids containing iPSCs, growth-arrested embryonic fibroblasts and embryonic endothelial cells were grown together using a directed differentiation protocol, resulting in robust myogenic induction of the iPSCs; after only 13 days 40%–50% of cells in these embryoids were Pax7+ (Mashinchian et al., 2022). Unfortunately, the method used in this study to quantify Pax7 via RNA-FISH misrepresents the true myogenic Pax7 population, and staining with surface markers for Pax7-associated proteins significantly reduced the number of truly myogenic cells. In addition, despite acceptable engraftment and improvements to functional force production, these cells exhibited an embryonic (week 9) phenotype, indicating that they are no more mature than those produced in 2D systems.

In a more recent publication, human skeletal muscle organoids formed a largely myogenic population of cells (∼90%). Interestingly, a significant number of mesenchymal (∼4%) and neural (∼5%) cells also populated these 3D structures. While not explicitly studied, the heterogenous 3D structure was likely partially responsible for the maturation of the resulting Pax7+ iMPs, which closely resembled a fetal week 17 phenotype (Mavrommatis et al., 2023). Cell-cell communication analysis of the scRNA sequencing data generated in this work identified a number of upregulated ECM components (COL1A2, COL5A2, COL5A3, FN1) and transcription factors (FBN1, CHODL, SPRY1) that could be targeted in 2D or 3D systems to further mature iMPs.

While inducing differentiation of iMPs using 3D culture systems is relatively new, the application of differentiated iMPs within 3D systems has been widely reported (Rao et al., 2018; Selvaraj et al., 2019; van der Wal et al., 2023). It is established that 3D co-cultures can mimic the MuSC niche, enhance myofiber maturation, boost in vivo engraftment potential, and act as effective disease models (Quarta et al., 2016; Quarta et al., 20017; Bersini et al., 2018; Maffioletti et al., 2018; Rao et al., 2018; Bakooshli et al., 2019; Kim et al., 2020; Urciuolo et al., 2020; Massih et al., 2023). While recovery of mature iMPs from 3D systems may limit certain therapeutic applications (e.g., those requiring single cell suspensions), it may enhance their utility in others (e.g., traumatic skeletal muscle injury). 3D systems should be considered valuable tools for assessing iMP maturation and could help to further identify culture conditions that enhance this process.

Stimulation of myogenic cells can be achieved through either mechanical, chemical, electrical, or static tension. Early studies of C2C12-derived myofibers seeded within 3D collagen scaffolds demonstrated that electrical stimulation of these cells improved sarcomere organization and myofiber maturity (Park et al., 2008). In 2018, Rao et al. demonstrated that myofibers grown within their 3D scaffolds supported a population of self-sustaining Pax7+ iMPs (Rao et al., 2018). Despite thorough characterization of this stimulatory effect on myofiber maturity and force production, the Pax7+ iMP population was not further characterized. In a more recent study, van der Wal et al. directly compared directly differentiated iMP-derived myofibers to those of primary human myoblasts, showing that electrical stimulation of the 3D scaffolds produced comparable force production measurements (van der Wal et al., 2023). While this study provided a detailed physical and proteomic analysis of the resulting myofibers, the presence and maturity of Pax7+ cells within the constructs were not reported. In vivo, maturation of the MuSC compartment coincides with myofiber maturation following secondary myogenesis. Future studies should make use of systems like those described above to assess the effects of stimulation on the capacity of iMP-derived myofibers to support maturation of the associated iMPs.

Human MuSC development is a spatiotemporally coordinated process guided by concurrent maturation of the surrounding tissue (Chal and Pourquié, 2017). Differentiation of iPSCs to fully mature iMPs may therefore be limited by constraints inherent to culture systems. Comparing MuSCs with iMPs matured in vivo or in vitro has demonstrated that in vivo-matured iMPs more closely resembled adult MuSCs, whereas in vitro-matured iMPs retained a more fetal-like transcriptomic profile (Incitti et al., 2019). Important myogenic regulators (Stat3, Jun, Itga7, Tgfb2, Notch1, Notch3, and Jag1) and genes involved in ECM regulation were upregulated in the in vivo matured iMPs compared to those matured in vitro. Future studies aimed at targeted activation or inhibition of signaling pathways in which these factors are involved may improve maturation of iMPs in vitro. Likewise, incorporating the identified ECM modalities within engineered scaffolds may expedite the maturation of these cells when applied within certain therapeutic contexts. The key takeaway from these data is that iMP maturity in vitro may be less significant to successful therapeutic application than previously thought; instead, we may be able to rely on in vivo maturation to carry these cells towards a more MuSC-like phenotype.

VML is characterized by a loss of tissue architecture that leads to chronic fibrosis and an associated loss of function (Corona et al., 2015). Current clinical treatments rely on crude muscle flap surgeries, which are limited by donor-site availability (Greising et al., 2017). Experimental therapeutics targeting VML aim to restore tissue integrity and function through the use of 3D scaffolds seeded with myoblasts or MuSCs (Corona et al., 2013; Quarta et al., 2017; Goldman et al., 2018). Despite the obvious application of iMPs as a substitute for patient-derived MuSCs, few studies have reported successful therapeutic application of these cells (Wu et al., 2021; Pinton et al., 2023). iMP maturity likely dictates survival within the scaffold and the capacity to successfully generate functional de novo muscle.

Scaffold design is critical to targeting VML pathophysiology (Wolf et al., 2015). In previous sections we have alluded to different ECM components that might help to mature iMPs in vitro. Current scaffolds can include either synthetic or natural material compositions, with natural hydrogels generally favored due to their intrinsic biocompatibility (Fischer et al., 2020; Luo et al., 2022). Hydrogels are frequently generated using proteins like collagen or fibrinogen, or polysaccharides such as hyaluronic acid or alginate. Those composed of proteins - specifically collagen - rarely consider particular isoforms, which have diverse functional characteristics (Goldman et al., 2018; Matthias et al., 2018; Pollot et al., 2018). Future studies should aim to incorporate specific collagen isotypes, like those already identified as important for iMP maturation, into scaffolds seeded with iMPs to improve maturation and survivability (Incitti et al., 2019). Additional considerations, such as the addition of growth factors and materials that promote revascularization and reinnervation must also be considered but are outside the scope of this perspective (Wu et al., 2021).

Skeletal muscle is a highly vascularized, metabolically active tissue, making it an excellent vehicle for the delivery of biological therapeutics. The engraftment of iMPs, while imperfect, is likely sufficient to deliver effective cell-based synthetic gene products in situ. A key advantage of iMPs is their capacity to be extensively engineered prior to differentiation and/or transplantation, affording flexibility in tailoring treatments. Utilizing iMPs as a delivery platform for therapeutics like growth factors, cytokines, and biologics represents a highly promising, yet largely untapped, application. This approach could revolutionize the delivery of therapeutics by directly targeting affected tissues, enhancing treatment precision and efficacy.

Following VML, a dysregulated immune response results in persistent inflammation, elevated TGFβ expression within the damaged tissue, and resulting in fibrosis (Larouche et al., 2021). Experimental treatments have successfully alleviated fibrosis through systemic administration of anti-fibrotic agents that target TGFβ, but these treatments have dangerous off-target effects (Garg et al., 2014; Greising et al., 2019; Larouche et al., 2022). More recently, work has demonstrated that local administration of the pro-regenerative cytokine IL-10 can improve the regeneration in skeletal muscle following VML (Huynh et al., 2023). Engineering iMPs to target inflammation and fibrosis - either by locally secreting anti-TGFβ antibodies or by counteracting inflammation using pro-regenerative cytokines such as IL-10 - could enhance regeneration after VML while mitigating the risks of systemic anti-TGFβ therapies (Borok et al., 2020; Narasimhulu and Singla, 2023). The capacity to generate inducible transgenes, controlled either temporally or spatially, would further improve the precision of these treatments (Figure 1).

In another example, we imagine a system in which iMPs engineered to constitutively express and secrete hormones responsible for blood glucose-homeostasis, might alleviate hyperglycemia in diabetic patients (Xie and Fussenegger, 2018). Synthetic circuits enabling glucose-mediated insulin secretion have been developed, bypassing the need for islet cells, which remain targeted by the immune system in type 1 diabetics receiving islet transplants (Xie et al., 2016). Type 2 diabetes might also be targeted using iMP-delivered synthetic gene circuits that secrete adiponectin to improve insulin sensitivity (Ye et al., 2017). Hypothetically, these synthetic gene circuits, applied to iMPs transplanted in otherwise healthy diabetic patients, could counteract hyperglycemia without the documented shortcomings of iPSC-based pancreatic beta cell therapies (Maxwell and Millman, 2021).

Thoughtful design of inducible synthetic transgenes should allow for iMPs to deliver therapeutics either locally or systemically. While the immediate application of these systems to target VML pathology are evident, the application of iMPs as a therapeutic delivery platform targeting systemic disease has yet to be discussed in the literature. The capacity for iMPs to persist long term as engrafted myofibers makes them an attractive alternative to other cell-based therapies that are often limited by poor engraftment efficiency and survivability (Levy et al., 2020; Zhou et al., 2021). We propose that future studies aiming to address disease using cell therapeutics consider the use of iMPs as a reliable delivery platform.

Temporal regulation of synthetic gene circuits is easily achieved through the use of inducible promoters, but the spatial organization of regenerating skeletal muscle may provide additional, therapeutically relevant, cues. Spatial transgene regulation has been achieved through the engineering of synthetic receptors capable of recognizing specific ligands, resulting in customizable sense/response behaviors (Roybal et al., 2016; Scheller et al., 2018). Synthetic notch (synNotch) receptors have proven a particularly powerful tool, capable of inducing the expression of myogenic transgenes in a spatially-regulated context (Morsut et al., 2016). Application of the synNotch system in vivo has demonstrated broad utility, from serving as an intercellular contact sensor capable of fate-mapping endothelial cells during development to enhancing CAR cell therapeutic efficacy (Hyrenius-Wittsten et al., 2021; Zhang et al., 2022). iMPs engineered to express customized synNotch constructs could provide spatial, context-dependent, regulation of transgene activation/deactivation. For example, pro-fibrotic collagens 3, 4 and 6 are significantly upregulated in the fibrotic lesion following VML (Hoffman et al., 2022). A self-regulating, cell-based synNotch system that uses antiCol3-tTa to control expression of anti-TGFβ antibody expression could enable targeted delivery, limiting expression to areas only where pro-fibrotic collagens are present (Figure 1).

When skeletal muscle is insufficiently innervated exogenous electrical stimulation can improve both the maturation and force production of the myofibers, aiding in the rehabilitation of muscle strength (Gordon and Mao, 1994; Crist et al., 2024). Optogenetic switches, which allow for transdermal stimulation of skeletal muscle using visible light, represents an attractive alternative to invasive patch clamps. Optogenetic switches have already proven capable of driving skeletal muscle contraction both in vitro and in vivo (Bruegmann et al., 2015; Ganji et al., 2021). Their therapeutic application as part of an iMP-based treatment should be investigated as it may allow for partial restoration of muscle function in spinal cord injury patients; or, at the very least, assist in the rehabilitation process.