SCs, first identified in 1961 by Katz and Mauro in the muscles of frog and rat [71], represent the adult stem cell population of skeletal muscles. SCs are mononucleated cells located between the basal lamina and sarcolemma of the skeletal muscle fibres. In mature healthy muscles, SCs are in a quiescent unipotent state and enter the cell cycle as multipotent precursor cells (MPCs) to contribute to the formation of new muscle fibres in response to muscle injury, disease or during muscle physical activity (exercise or stretching) [72]. After activation, SCs proliferate extensively generating myoblasts that fuse rapidly to form multinucleated myofibres. When SCs are ablated, no other cell types can start skeletal muscle regeneration, indicating the need of resident SCs to recruit or guide any other possible contributor to muscle differentiation [73]. The localisation in specific anatomical structures and the exposure to the skeletal muscle niche signalling direct SC self-renewal. SCs undergo both symmetrical and asymmetrical cell division, mainly depending on the location of the daughter cells with respect to the myofibre. A small proportion of SCs do not undergo terminal differentiation, but repopulate the reserve pool of quiescent cells to mediate further rounds of muscle regeneration. The identification in different cell states (quiescence, activation or proliferation) for both murine and human SCs is based on the expression of numerous molecular markers [74].

In mice, paired box transcription factor Pax7 [75], adhesion molecule M-cadherin, sialomucin surface receptor CD34, integrin α7β1, transmembrane Heparan sulphate proteoglycan syndecans 3 and 4 (Hspg3 and Hspg4), C-X-C chemokine Receptor type 4 (CXCR4) [76] and cytoskeleton protein desmin [77] are used as markers. In human CD56 (also known as Neural Cell Adhesion Molecule, NCAM) [78], although not SC specific, and the nuclear transcription factor Pax7 [75] are usually combined with the nuclear laminin A/C to identify SCs in their in vivo niche [78].

In the human adult skeletal muscle, a subpopulation of SCs that co-express vascular system markers has been identified as myoendothelial cells (MyoECs). These cells can be expanded for long-term in vitro and support muscle regeneration superior to myoblasts. MyoECs, located between the muscle fibres, represent approximately 0.4% of the muscle-resident stem cells, sharing myogenic (CD56⁺, CD34⁻, CD144⁺) and endothelial features (CD56⁻, CD34⁺, CD144⁺) [79]. The strong myogenic commitment and feasible isolation and expansion of SC-derived myoblasts in vitro pointed out these cells as the first candidates for stem cell therapy in muscular diseases. However, pioneering studies in mice models showed poor survival and migration abilities for transplanted SCs [80].

Pericytes, the mural cells of the blood microvessels, have been discovered and described in 1871 as a contractile cell population surrounding the endothelial cells of small blood vessels. Pericytes were initially described as regulators of the blood flow, although confusion still exists about their identity, ontogeny and progeny. They differentiate towards adipocytes, bones, cartilage and muscles [89]. Several molecular markers for pericytes have been characterised, depending on their presence in different tissues [90, 91]. Notably, their expression pattern is dynamic in developmental stages, in vitro culturing and after pathological insults, so that no single pericyte-specific marker has been identified. Among the general accepted markers for murine pericytes are CD140b and Nerve/Glial antigen 2 (NG2), crucial for survival and development, alanyl membrane aminopeptidase CD13, desmin and alpha-Smooth Muscle Actin (αSMA). However, in mice, quiescent pericytes are not positive for αSMA while its expression level is upregulated in tumours and inflammatory environments. In human skeletal muscles, pericytes are highly positive for NG2, Alkaline Phosphatase (ALP), annexin V, desmin, αSMA, vimentin and CD140b and negative for M-cadherin (CD146), NCAM, cytokeratins and neurofilaments (except for nestin), endothelial markers (CD31, CD34, KDR) or haematopoietic markers (CD45) [88]. Myogenic differentiation capacity is not restricted to muscle-resident pericytes, since pericytes isolated from human adult adipose tissue or placenta can contribute to myotubes in vitro and to dystrophin-expressing fibres in vivo [92]. Recently, a new expression profile has been described for the simultaneous purification of pericytes, MSCs and blood vessel-derived stem cell subpopulations from human skeletal muscles [93].

Mesoangioblasts (MABs) are vessel-associated progenitors, located in the interstitial space between the muscle fibres. MABs have been first isolated from the dorsal aorta of E9.5 mouse embryos [94] and in adult muscles from mice [95, 96], dogs [97] and humans [88]. Embryonic MABs are positive for CD34, c-Kit and Flk-1 but negative for Oct4, Nkx2.5 and Myf5 [94]. MABs of embryonic origin can differentiate to multiple derivatives of the mesodermal lineages in vitro and in vivo [98]. In mice and dogs, adult MABs are isolated from adult skeletal muscles based on their expression for ALP, Sca-1, integral membrane NG2, CD140a and CD140b (also known as PDGFRα and β isoforms, respectively) [88, 95, 99]. Adult MABs have a multipotent differentiation potential towards myo-, osteo-, chondro- and adipogenic lineages [100]. Human MABs are well studied both in vitro and in vivo. They express the adhesion molecule CD146, CD140b and NG2 but do not express haematopoietic or SC markers, including CD45, CD34, CD56, CD144 and Pax7 [88]. The human counterpart for the murine Sca-1 marker is still missing, although members of the human Ly6 proteins have been suggested as correspondent to Sca-1 with homologous functions. Intriguingly, a comparison among human MABs, MSCs and pericytes has identified some similar markers (CD10, CD13, CD44, CD73, CD90), opening new questions regarding their origin [101]. Human MABs easily proliferate in vitro and spontaneously differentiate into MyHC-positive myotubes. Moreover, they express immunomediated cytokines and receptors, indicating immunomodulatory properties by inhibiting the T-cell proliferation in vitro [102]. The ability to regulate immune responses by MABs opens new clinical applications.

Another muscle-resident stem cell population, known as MDSCs, has been identified from mouse skeletal muscle and seems to be different from late-stage myogenic populations, like SCs and myoblasts, by their multipotent differentiation potential. Besides their myogenic capacities, they can differentiate in vitro and in vivo towards adipo-, osteo-, chondrogenic, haematopoietic, endothelial, smooth muscle, cardiac muscle and neural lineages [107]. Moreover, MDSCs secrete high levels of VEGF, promoting vascularisation and consequently facilitating tissue regeneration. MDSCs have a high expression of Sca-1, low levels of the fibroblast marker vimentin and low levels of desmin. These cells are negative for CD45, M-cadherin. Moreover, the low expression of the Major Histocompatibility Complex (MHC)-1 is a promising feature towards more successful stem cell-based therapy applications [108]. Intramuscular delivery of MDSCs, 4 days after induced injury, showed enhanced angiogenesis and reduced scar tissue formation. Interestingly, 1 week after injection, high levels of VEGF and high expression levels of antioxidant, glutathione (GSH) and superoxide dismutase were detected, increasing their survival after administration [109]. Aldehyde dehydrogenase 1A1 (ALDH) is a ubiquitously detoxifying enzyme involved in the metabolism of aldehydes and retinoic acid. ALDH⁺ cells are found in the bone marrow, umbilical cord and peripheral blood, although a cell population has been characterised with a high ALDH activity in human skeletal muscles [110]. These cells represent a small amount of the total mononucleated cells (2–4%). MDSCs have a higher survival ability compared to SCs and myoblasts after transplantation, probably due to their resistance to oxidative stress and high proliferation activity in vivo.

PW1-expressing interstitial cells (PICs; also known as Paternally expressed gene 3, Peg3) are characterised by the localisation in the interstitium among muscle fibres. These cells are not embryonically related to SCs, since they do not derive from Pax3⁺ myogenic progenitor cells. PICs have a Pax7⁻/Sca-1⁺ and CD34⁺ marker phenotype [112]. Recently, a high expression of PW1 has been shown in MABs of mice, dogs and humans, and the expression levels correlate with their myogenic and migratory capacities. Silencing PW1 significantly inhibits their myogenic differentiation capacity and their ability to cross the vessel wall, so consequently, their engraftment into damaged muscle tissue [113]. Therefore, PW1 may be used in the future as a biomarker to identify optimal donor populations for cell therapy in pathologic conditions.

SP cells are a rare subpopulation source of precursors associated to skeletal muscles. SP cells are characterised by the complete exclusion of the DNA-binding dye Hoechst 33342, due to their abundant expression of the Abcg2 transporter. They show heterogeneity inside their population. 80% of the total SP cells are positive for the vascular endothelial marker CD31, while 2–10% are blood derived and show positiveness for the immune marker CD45. However, a small fraction has been characterised as highly positive for Abcg2, CD31 and CD45, especially during muscle damage and the early phase of regeneration. Recently, another SP cell subgroup has been identified, representing 5% of the total population, which are negative for both CD31 and CD45, and may express Pax7, Sca-1 and syndecan 4 [114, 115]. In coculture with myoblasts, SP cells are able to fuse in vitro to form myotubes. Interestingly, in vivo engraftment experiments show high myogenic differentiation capacity in regenerating muscles after acute tissue damage [116]. Moreover, several studies have shown their ability to restore dystrophin levels when injected intravenously into mdx mice [117].

In 2010, a new muscle stem cell population located in the interstitium has been discovered. Fibro-/Adipogenic Progenitors (FAPs) are non-myogenic, mesenchymal progenitors that differentiate towards both adipo- and fibrogenic cells, indicating a potential contribution to adipose and fibrotic deposition in diseased muscles [118, 119]. These cells were isolated as CD34⁺/Sca-1⁺/CD31⁻/CD45⁻/Lin⁻/integrin α7⁻ [118] or Sca-1⁺/CD140a⁺/integrin α7⁻ [119]. They exert beneficial paracrine effects on the muscle fibres. FAPs were used in preclinical models of MDs with histone deacetylase inhibitors (HDACi), able to promote in vitro and in vivo myogenic differentiation. Transplantation of FAPs in more advanced dystrophic muscles enhanced the regeneration by interacting with the muscle-resident SCs. Several studies have shown that FAPs from dystrophic muscles of mdx mice retain a bipotency in their phenotype and functionality, either by supporting regeneration at early stages of disease progression or by differentiating in fat and fibroblasts [64, 65]. The human FAP counterpart has been isolated as CD140a-positive cells in healthy and diseased muscles, although further studies are needed to investigate the molecular and functional characterisation to develop novel pharmacological treatments or to understand their ability as a cell therapy source for muscular diseases in human settings.

MSCs are non-haematopoietic multipotent stem cells that can be found in several tissues, including bone marrow, umbilical cord, blood, placenta, liver, adipose tissue, muscle and synovial membrane. The bone marrow is the principal source [120, 121]. MSCs can renew and differentiate into multiple mesenchymal lineages, including adipo-, chondro- and osteocytes. MSCs are characterised by the expression of several markers (CD73, CD90 and CD105), although not specific for MSCs, and by the lack of haematopoietic markers (CD45, CD34 and CD14 or CD11b, CD19 and HLA-DR) [122].

Another cell population with myogenic potential has been identified expressing CD133 [129–131]. CD133⁺ cells are a small subpopulation of the mononucleated cells present in the peripheral blood [129], expressing the stem cell marker CD133. Some cells have also been identified as positive for CD133 in human skeletal muscle [131]. The function of CD133 is still unclear. However, it could be a useful marker to purify haematopoietic and endothelial progenitors. A drawback of these cells is CD133 loss in vitro after expansion, hampering cell tracking after expansion or in vivo administration. CD133⁺ cells cocultured with murine myoblasts have the capacity to form MyHC-expressing myotubes [129].

ESCs have been isolated from the inner cell mass of the murine [136] and human [133] blastocyst. The therapeutic applications of ESCs have been strongly debated in the scientific community, mainly because of ethical concerns and immune rejection issues. Moreover, their use needs to be carefully evaluated, since ESCs can lead to teratoma formation [137]. Few studies have described the use of human ESCs in muscle transplantation. One study documented the transplantation of mesenchymal precursors derived from human ESCs in cardiotoxin-injured tibialis anterior (TA) muscles of scid/beige mice. Although only a small cell proportion could be tracked in the muscle, a possible future role for ESCs in muscle cell transplantation could be suggested, since no tumorigenic effect was observed [138]. Several groups have documented the differentiation of myogenic progenitors from ESCs. Darabi et al. used inducible Pax3 and Pax7 overexpressing murine ESCs, injected intramuscularly or systemically administered in mdx mice, resulting in engraftment with dystrophin-expressing myofibres and improvements in the muscle function [139]. Similar outcomes were observed in mice affected with FSHD [140]. Zheng et al. showed that human ESCs exposed to serum in the presence of EGF directed differentiation towards myogenic precursors. Subsequent addition of the DNA-demethylating agent 5-azacytidine increases even more their myogenic potential. in vitro terminal differentiation and fusion into myotubes was not observed. However, transplantation of human ESC-derived cells into injured TA reached terminal differentiation, as well as localisation in the SC compartment [141]. Barberi et al. differentiated human ESCs to obtain mesenchymal precursors for myogenic progenitor cells. After transplantation in the hindlimb muscle of immunodeficient mice, long-term survival and myofibre commitment were reported [138]. Nowadays, several preliminary studies have been performed with PSC-derived myogenic cells, showing their capacity to form muscles in vivo. However, their long-term engraftment into adult muscle and their functional contribution still have to be elucidated, as well as their depleted muscle stem cell pool repopulation.

Human iPSCs can be generated from adult somatic cells by introducing a defined set of transcription factors to reprogram them towards an embryonic-like pluripotent state [134, 142–145]. Human iPSCs have been derived from fibroblasts [146] and Peripheral Blood Mononuclear Cells (PBMCs) of DMD and Becher Muscular Dystrophy (BMD) patients [147]. Myogenic lineage induction from murine ESCs and iPSCs has been described [140, 148], although terminal differentiation of human ESCs, as well as human iPSCs, is still facing some difficulties. Murine and human iPSCs have been provided to counteract muscle wasting in MDs [116]. So far, in vitro and in vivo studies have shown that myogenic precursors derived from iPSCs could form chimeric myotubes in coculture with C2C12 myoblasts. In addition, iPSC-derived myogenic precursors transplanted in dystrophic muscles contribute to an improvement in the contractile properties. The iPSC technology has been used to allow the generation of genetically corrected human DMD iPSCs by using a human artificial chromosome able to carry the full-length dystrophin gene. This strategy is an alternative to induce truncated forms of dystrophin (quasi-, mini-, micro-dystrophin genes) in mouse and human iPSCs [149]. Recently, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-modified DMD human fibroblasts reprogrammed to iPSCs have been tested for skeletal and cardiac muscle differentiation abilities both in vitro and in mdx mice [150].

AAV has already been used in many clinical trials since its broad tropism and infectivity, long-term expression and site-specific integration (NCT01344798; Table 3.4) [157]. Non-selective infection and organ-restricted transduction have pushed researchers to look for better vectors, based on different serotypes able to transduce more efficiently muscle tissue or to offer the best compromise between skeletal muscle expression levels and ability to escape human immune response [158]. A mini-dystrophin gene (rAAV2.5-CMV-mini-dystrophin gene vector) has been tested for safety (phase I) in the treatment of progressive muscle weakness due to DMD (NCT00428935) [159]. Analogously, a double-blind randomised study (NCT00494195) reported the administration of intramuscular injection of rAAV.tMCK.haSG gene vector in LGMD2D patients as being safe. In addition, AAV8 has been used to repress chronic inflammation and muscle degeneration specifically into mdx skeletal muscle, overexpressing an endogenous inhibitor of NF-κB signalling pathway under the truncated Muscle Creatine Kinase (tMCK) promoter. Short hairpin RNA carrier AAV9 has been successfully developed to target the major subunit of NF-κB (NF-κB/p65) and ameliorate muscle pathologic phenotype in mdx mice [160]. Finally, AAV9 mini-dystrophin gene delivery combined to the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-Binding Domain (8K-NBD) peptide promoted a higher mini-dystrophin expression in skeletal muscle compared to AAV9 alone [161].