**3. Muscle regeneration**

muscle progenitor to the quiescent and activated satellite cells (Figure2). Its induction in muscle-derived stem cells induces satellite cell specification by restricting alternate

**Figure 2. Myogenic cell characterization and culture.** Myogenic cell lineage can be identified in each differentiation state and pursue tightly regulated proliferation and differentiation cycles. From the embryonic state until the terminal differentiation into muscle fibers an intricate network of transcription factors regulates the fate of muscle progenitor cells. These cells can be isolated from any skeletal muscle tissue, grown in culture and reimplanted into a damaged

Specific molecular markers have been demonstrated to distinguish between activated and quiescent SC. Quiescent satellite cells express the transcription factor Pax7, after activation in

developmental programs [51].

684 Regenerative Medicine and Tissue Engineering

muscle to promote muscle regeneration.

### **3.1. Cell sources for skeletal cell therapy**

Several types of cell populations have been identified as potentially efficient in muscle regeneration, especially in cell therapy. They are able to self-renew, proliferate and form muscle fibers. Among these cells some are muscle derived and some are from other origin.

#### **3.2. Muscle–derived cells**

Muscle satellite cells, which are squeezed between the plasma membrane and basement membrane of muscle fibers, are the natural source of muscle regeneration during homeostasis or after injury in postnatal stages [63]. They are specifically expressing the paired box tran‐ scription factor pax7 [51] and have been shown to be efficient in the muscle regeneration process. One study illustrated that as little as seven satellite cells were able to generate more than 100 muscle fibers in irradiated muscle [64]. Though, satellite cells isolated from different muscles are not equivalent: they produce muscle fibers with variable contractile abilities depending on the muscle of origin [65]. This can be explained by the fact that a satellite cell pool does not seem to consist of a homogeneous population of cells [66-70]. Once activated, satellite cells are triggered toward proliferation and differentiation by giving rise to muscle precursor cells that fuse and form skeletal muscle fibers [71]. The two techniques used to isolate muscle precursor cells are selection of single fibers that are cultured or mechanical processing of muscle biopsies and enzymatic treatment with a mixture of collagenase and dispase [64, 72-74]. The first method is claimed to be less aggressive and to better preserve the cells.

Another type of cells is isolated from muscle biopsies through a series of preplating stages. These cells are also recognized to have a myogenic profile and are capable to fuse and form skeletal muscles fibers. They are known as muscle-derived stem cells (MDSC) with character‐ istics of non-committed progenitor cells [75, 76] and are most probably originating from blood vessel walls [77]. Similarly, other cells types isolated from the muscle compartment such as mesoangioblasts and pericytes are involved in the muscle regeneration but are of nonmyogenic origin. These are vessel-associated progenitors, not expressing myogenic markers such as Myf5 and MyoD even though they can differentiate to myotubes and fuse to form fibers [78-80]. More cell types with non-myogenic profile are found in the skeletal muscle and have recently been demonstrated to form fibers. Hence, skeletal myogenic precursors or muscle stem cells sorted by FACS are capable to reconstitute fibers in rodent models [72, 81]. The first type of cells is characterized by expression of β1-integrin (adhesion protein) and CXCR4 (SDF-1 receptor), the second type by α7-integrin (adhesion protein) and CD34 markers. Side popula‐ tions are also isolated from muscle tissues and are expressing specific surface markers [82]. They are distinct from satellite cells and have been used successfully in muscle regeneration in rodent models [83-87]. Surprisingly, more types of cells of the skeletal muscle tissue can contribute to muscle regeneration. In fact, recently, a new type of myogenic cells, localized in the area of the interstitium between muscle fibers, has been characterized and is known as PW1-interstitial cells (PICs). They are characterized as positive for cell stress mediator PW1 but negative for Pax7; though they possess myogenic profile *in vitro* and lead to muscle regeneration *in vivo*, which includes the generation of satellite cells [88]. Hence, various types of cells isolated from skeletal tissue either mechanically or by flow cytometry are capable to regenerate muscle. In addition to the muscle there are more sources of stem/precursor cells isolated from other compartments.

#### **3.3. Sources outside of the skeletal muscle compartment**

Mesenchymal stem cells (MSC) are procured from bone marrow biopsies and are multipotent stem cells that give rise also to skeletal muscle fibers and participate to restore the satellite cell niche [89]. These cells are well characterized and involved in many different applications due to their multipotency as it is the case for adipose-derived stem cells (ADSC). The latter are easily harvested by liposuction, cultured *in vitro* and injected to restore muscle in the case of SUI [90, 91]. Embryonic stem cells, induced pluripotent stem cells and umbilical cord blood have been demonstrated to be good alternatives for skeletal muscle regeneration [74, 92, 93]. However, precaution should be taken when these types of cells are considered for further development in clinics, as different types of viruses are used during the process of myogenic induction. In addition, there are still potential tumorigenicity issues with this sort of cells that need to be solved before further clinical application.

Hence, the sources of stem/progenitor cells for skeletal muscle regeneration are large. Though, several important factors need to be considered when choosing the optimal source for treating patients. Autologous cell therapy avoids immunogenic reaction and therefore complications after the implantation procedure. Therefore, autologous satellite/muscle precursor cells are advantageous for muscle regeneration. They are committed to muscle restoration and therefore the most convenient cells for applications in cell therapy. Their dedication to one lineage offers an advantage over other, previously discussed sources, which are multipotent and hence differentiate also into non-muscular tissue cells. Furthermore, satellite / muscle precursor cells can be isolated in a simple procedure and are easily expanded in a GMP facility. They produce enough cells to be injected after 2-3 weeks, which is much faster than the 5 - 6 weeks required for muscle derived stem cells. For allogenic application, mesenchymal and adipose derived stem cells represent valid alternatives when satellite cells cannot be extracted from the skeletal muscle.
