**6. Multipotent and pluripotent stem cells for the treatment of MDs**

Satellite cells are quiescent mononucleated myogenic cells, located between the sarcolemma and basement membrane of terminally-differentiated muscle fibres [36]. For a long time, adult muscle was considered as a static tissue. Furthermore, due to its histological nature, it was considered formed by spatially-oriented post-mitotic multinucleated muscle fibres. Since 1961, the discovery of satellite cells by Katz and Mauro, together with vast studies about their biological role, revealed the existence of plasticity potential in adult skeletal muscle tissue. Their name derived from the distinctive location, wedged between the basal lamina and sarcolemma of myofibres, in a separate place from the fibre. With an estimated number between 1×1010 to 2×1010 satellite cells per person, these cells represent the main source of muscle progenitors within the adult skeletal muscle tissue [36]. Indeed, they are unipotent stem cells that, in the case of acute muscle damage and during muscle regeneration/degener‐ ation, are able to re-enter the cell cycle and contribute to muscle repair by offering new myogenic progenitors capable to fuse and form new fibres. Pax7 (a paired box transcription factor) was the first identified marker required for myogenic specification of satellite cells. However, in the last decade, several surface markers were identified within the pool of satellite cells. These include CD34, M-cadherin, syndecan-3/4, c-met and the chemokine receptor CXCR4, as well as α7β1-integrin (a transmembrane domain protein), Pax3, barx2, myocyte nuclear factor (MNF) (the latter three are known transcription factors) and caveolin-1 (a scaffolding protein within caveolar membrane) (Table 1). Since these markers are all expressed by satellite cells and not by post-mitotic myonuclei of fibres, their identification within the muscle microenvironment outside the fibres has been relatively easy. These markers were investigated both *in vitro* and *in vivo* for the marked regenerative ability potential of satellite cells [13]. Pre-clinical studies of DMD in the mdx mouse model, which recapitulates the pathophysiological features of human DMD such as the absence of dystrophin (Figure 1), contributed to the increase in knowledge of the therapeutic potential of satellite cells. It was demonstrated that the satellite cells derived from a single fibre of a healthy donor and transplanted into a muscle of mdx mouse can actively contribute to the repopulation of the satellite cell pool of dystrophic muscle, as well as to the regeneration of new dystrophin expressed fibres. Several combinations of markers have been used to identify donor satellite cells and their myogenic contribution in dystrophic mice, including Pax7/CD34 [37] and CD34/ integrin-α [38]. In 2004, a distinct subpopulation of satellite cells, named skeletal myogenic precursors (SMPS), positive for Cxcr4 and β1 integrin and negative for CD45 and Sca1 (a known maker of hematopoietic progenitor cells), were discovered, highlighting the heterogenic nature of satellite cells [39] (Table 1). SMPS showed a robust regenerating ability, as well as a strong capability of repopulating the satellite cell poll when injected in the muscles of mdx mice. Additionally, their potential role in counteracting muscle wasting was confirmed by a strong improvement of muscular contractile properties such as contractile force, observed in treated dystrophic muscles compared to the healthy controls [7].


**Table 1.** Cell markers identifying adult stem cells currently used in cell therapy.

Although satellite cells are the main source of myogenic renewal in adult skeletal muscle tissue, in recent years, other adult stem cells have been discovered. A subpopulation of muscle precursors associated with skeletal muscle tissue, named muscle side population (SP) cells, is a rare source of multipotent stem cells that contribute to muscle regeneration, upon trans‐ plantation. SP cells are characterized by a complete permeability to the Hoechst 33342 dyes, derived from their high expression level of Abcg2 transporter (Table 1). The myogenic potential ability of SP cells has been tested by *in vitro* co-culture with myoblast cells. In these conditions, SP cells were able to fuse with myoblast to form mature myotubes. At the same time, *in vivo* experiments confirmed their involvement in myogenic differentiation (see below). Interestingly, as observed in satellite cells, SP cells also show certain heterogeneity inside their population. Analysis of the expression pattern of specific markers revealed that 80% of SPs are positive for the vascular endothelial marker CD31, while 2-10% of total muscle SPs are bloodderived and positive for the immune marker CD45. In the case of muscle damage and during the followed early phase of regeneration, a fraction of SP cells have been identified as highly positive for CD45, Abcg2 and CD31 (the latter two suggested a possible intervention in modulation of both vascularization and immune system) [40]. Furthermore, a third fraction of SP cells, representing 5% of total population, were recently identified. These cells are charac‐ terized by the absence of both CD45 and CD31 expression, while they may express Pax7, Sca1 and Syndecan4 [40-42] (Table 1). Interestingly, although in physiological conditions this subpopulation of muscle resident SP cells represents the smallest fraction within the rest of population, if engrafted in a regenerating muscle (pre-treated with cardiotoxin to induce acute tissue damage), they showed the highest myogenic differentiation potential [13].

demonstrated that the satellite cells derived from a single fibre of a healthy donor and transplanted into a muscle of mdx mouse can actively contribute to the repopulation of the satellite cell pool of dystrophic muscle, as well as to the regeneration of new dystrophin expressed fibres. Several combinations of markers have been used to identify donor satellite cells and their myogenic contribution in dystrophic mice, including Pax7/CD34 [37] and CD34/ integrin-α [38]. In 2004, a distinct subpopulation of satellite cells, named skeletal myogenic precursors (SMPS), positive for Cxcr4 and β1 integrin and negative for CD45 and Sca1 (a known maker of hematopoietic progenitor cells), were discovered, highlighting the heterogenic nature of satellite cells [39] (Table 1). SMPS showed a robust regenerating ability, as well as a strong capability of repopulating the satellite cell poll when injected in the muscles of mdx mice. Additionally, their potential role in counteracting muscle wasting was confirmed by a strong improvement of muscular contractile properties such as contractile force, observed in treated

dystrophic muscles compared to the healthy controls [7].

402 Muscle Cell and Tissue

**Table 1.** Cell markers identifying adult stem cells currently used in cell therapy.

Although satellite cells are the main source of myogenic renewal in adult skeletal muscle tissue, in recent years, other adult stem cells have been discovered. A subpopulation of muscle precursors associated with skeletal muscle tissue, named muscle side population (SP) cells, is a rare source of multipotent stem cells that contribute to muscle regeneration, upon trans‐ plantation. SP cells are characterized by a complete permeability to the Hoechst 33342 dyes, derived from their high expression level of Abcg2 transporter (Table 1). The myogenic potential ability of SP cells has been tested by *in vitro* co-culture with myoblast cells. In these conditions, SP cells were able to fuse with myoblast to form mature myotubes. At the same time, *in vivo* experiments confirmed their involvement in myogenic differentiation (see below). Interestingly, as observed in satellite cells, SP cells also show certain heterogeneity inside their population. Analysis of the expression pattern of specific markers revealed that 80% of SPs are positive for the vascular endothelial marker CD31, while 2-10% of total muscle SPs are bloodIn 2010, a new population of muscle interstitial stem cells were identified. These are charac‐ terized by their ability to undertake both fibrogenic and adipogenic differentiation [43, 44]. These mesenchymal fibro-adipogenic precursors (FAPs) show an intriguingly functional crosstalk with satellite cells. The nature of this relationship is mutually exclusive within the homeostatic processes of skeletal muscle and, indeed, the presence of FAPs during muscle regeneration enhances the myogenic potential of satellite cells. At the same time, the presence of satellite cells derived from new myofibres inhibits the adipogenic differentiation of FAPs [45]. FAPs were also investigated in the pre-clinical models of MDs. In particular, the treatment of FAPs with histone deacetylase inhibitors (HDACi) increased their *in vitro* myogenic differentiation (Figure 2), while, if transplanted in advanced dystrophic muscle (old mdx mouse), FAP cells can enhance the regenerative potential of resident satellite cells [46].

Another important source of muscle progenitors associated with vasculature, named mesoan‐ gioblasts (MABs), were investigated for their therapeutic potential in the treatment of muscu‐ lar dystrophies (Figure 2). Mesoangioblasts were originally isolated from embryonic aorta of a quail and mouse and, later on, in the adult skeletal muscle of a mouse, dog and human. MABs are multipotent stem cells positive for CD34, SMA, Pdgfrα, Pdgfrβ, Ng2, AP and, accordingly withsuchexpressionpattern,canundertakeseveraldifferentiationfates (Table1).Theseinclude myogenic, osteogenic, chondrogenic and adipogenic. Studies of *Scga*-null mice (a limb-girdle muscular dystrophy mouse model) and GRMD (golden retriever muscular dystrophy) dogs showedthatintra-veininjectionofMABs canrestoreboththehistological structureandfunction of large areas of dystrophic muscles [11, 12]. These promising results provided important knowledge regarding the therapeutic use of MABs for the treatment of muscular dystrophies. Recently, these efforts were finalized in a phase I/II clinical trial of donor mesoangioblasts transplantation from HLA-identical donors in five DMD patients, nearing completion (Eu‐ draCT Number: 2011- 000176-33). This clinical trial will provide useful information regarding the safety for the systemic delivery of stem cells in dystrophic patients, as well as the assess‐ ment of the ability of MABs to increase the dystrophin expression (Figure 2).

The scaling up of the research on pluripotent stem cells of the last decades has offered the interesting prospective to adopt this new precious source of stem cell in the treatment of MDs.

Embryonic stem (ES) cells are pluripotent stem cells, originally isolated from the inner cell mass of the blastocyst in a mouse in 1981 and from human in 1998. Their efficient pluripo‐ tency arises from the ability of ES cells to differentiate in all three germ layers - meso‐

derm, ectoderm and endoderm. It was demonstrated in the early 1990s that, if cultured *in vitro*, murine ES cells can develop aggregates of cells (embryoid bodies) and differentiate in skeletal muscle cells expressing myogenic markers in the same muscle-specific determina‐ tion genes order observed during embrional development: myf5, myogenin, myoD and myf6 [47]. Later on, both *in vitro* and *in vivo* studies confirmed their myogenic differentiation potential [13]. Nonetheless, the possibility of a therapeutic adoption of ES cells met the criticisms of both civil and scientific communities (see below). In 2006, Yamanaka publish‐ ed a revolutionary, paradigm-shift study. For the first time, a fate conversion of somatic cells (fibroblasts) into pluripotency was demonstrated [48]. As a result of this study, Yamanaka won the Nobel Prize in 2012 and began a new era for pluripotent stem cells-based therapeu‐ tic approach of chronic illness. So far, the myogenic potential of the induced pluripotent stem (iPS) cells, either from mouse or human origin, have been provided to counteract muscle degeneration in MDs [13] (Figure 2). In particular, *in vitro* and *in vivo* analyses showed that myogenic precursors generated from iPS cells could produce chimeric myotubes if cocultured with C2C12 myogenic cell line. Furthermore, if transplanted in dystrophic mus‐ cles, their contractile properties could also be improved [49].
