**6. Outlook: Concerns and improvements**

#### **6.1. Effects of age**

ment of the regulations and guidelines concerning different aspects: manufacturing, quality

Therefore, the regulations and guidelines have been reviewed and adapted for some of them in order to be applied in the field of cell therapy. This paves the road for regenerating the

Besides, chemical drugs, medical devices and biotechnology drugs, advanced therapies are developed and offer tailored solutions for patients. These therapies are based on genes, cells

Cell therapy for skeletal muscle is one of many therapies that are in translational phase and can be applied in near future on treating patients. As it is involving individuals' health and the cell product is delivered to human, safety concerns are raised. In fact, cell therapy product – as an investigational or marketed one- needs to meet requirements as any medicinal product or medical device. The goal is to deliver a consistent, safe, good quality and well-defined product. Therefore, Good Manufacturing Practice (GMP) is requested for the development of cell-based product, or its production for the market, and it consists on guidelines and regula‐ tions that advertise quality principles for manufacturing biological products. These rules are covering all the processes from the biopsy up to the final product. It involves several aspects: Quality management, buildings and facilities, the equipment, the personnel, the documenta‐ tion, the materials management, the processes in production, the monitoring, the packaging

Advanced therapies are new technology. Hence, protocols, guidelines and regulations that are used for existing medicinal product cannot be transposed literally for cell therapies and need

In cell therapy, the starting material represents a critical part that takes account of donor eligibility criteria including age, tissue quality, source accessibility and viral testing. For skeletal muscle cell therapy, as described above, the sources are multiple and the efficiency of

As soon as the biopsy is received in the manufacturing site, the GMP requirements have to be followed. Hence, quality management should be applied at all production steps: processing,

Manufacturing cell product necessities safe and certified raw materials and components for cell culture and preparation. In addition, upon reception to the GMP facility, the materials need to be tested in-house regarding quality and safety. Only then, the products can be released and accepted into the production area by the responsible for quality in the facility. It is highly recommended by the regulations to use supplements – as cytokines and growth factors- from human origin and therefore some adaptations are needed in the production protocols coming

assurance, quality control and pre-clinical studies [179].

and labeling, the storage and distribution, the laboratory controls.

most of them is good in regenerating muscle in the case of SUI.

adaptations. However, the goals stay the same: safety, quality and efficacy.

sphincter muscle by using stem cells.

**5.4. Production of cell–therapies**

694 Regenerative Medicine and Tissue Engineering

**5.5. Manufacturing process**

testing, release, storage and transport.

or tissues.

A decline of approximate 30% in muscle strength and 40% in muscle volume occurs between the second and seventh decades of life [182]. Also the total number of MPCs and their proliferation potential in culture gradually decrease in an age-dependent manner [183] due to apoptosis [184]. Additionally cell fate is tightly defined by the interactions with the microenvironment and the host age is of key importance, as the stem cell regenerative capacity reduces in aged niches [185]. We have reported that although human MPCs can be successfully isolated and grown from patients of all ages and genders (figure 3), both elderly and male donors provide unstable and slower growing cells *in vitro* with de‐ creased contractile output *in vivo* [186]. Hence, a combination of stem cell and gene therapy might be needed in older patients [187, 188].

**Figure 3. Muscle progenitor cells identification** *in vitro* **and muscle formation after transplantation** *in vivo***.** Myogenic cells isolated from the *Rectus abdominis* of patients undergoing abdominal surgery, grown in culture and characterized by FACS, immunohistochemistry *in vitro*. Tissue formation was evaluated *in vivo* by Hematoxilin and Eo‐ sin staining and immunohistochemistry. Function was assessed by electromyography. A: FACS analyses of cells in P2 expressing Pax 7, MyOD, desmin and upon differentiation induction Myosin Heavy Chain (MyHC). An IgG Isotype con‐ trol (red curve) was used to determine the background, whereas positive cells are plotted as a green curve. Immunocy‐ tochemistry of cells in culture expressing, MyOD (B), MyHC (C), desmin (D), sarcomeric α-actinin (E) (green -Phalloidin 488, blue – DAPI, red - mM anti-IgG Cy3). Muscle cells injected subcutaneously in nude-mice revealed muscle forma‐ tion *in vivo* (F, G, H) and contraction upon eletrical stimulation (I). HE stained (G) and labelled with sarcomeric α-acti‐ nin-Cy3 and PKH67 (H). Muscle function significantly improved over time (I), with contraction strength still increasing after 4 weeks.\*p=0.015

#### **6.2. Overcoming pitfalls by reactivating muscle metabolism, tissue vascularization and innervation**

In the context of muscle reconstruction, gene therapy is not aimed at rectifying a genetic mutation, but at boosting the myogenic potential and ultimately the muscle functionality of the injected autologous muscle cells. Two key factors have been demonstrated to improve the quality of satellite cells for transplantation: a better vascularization [189] and endurance exercise [190]. We have previously described that an angiogenic modification of muscle precursors can overcame some of the limitations of aged muscle cells [189]. For future application expanding the knowledge produced on this study and therapeutically combining it with the intrinsic adaptation effects of endurance exercise would be of major interest. In this context, studies using muscle-specific PGC-1α transgenic animals demonstrated that ectopic expression of PGC-1α in muscle seems sufficient to evoke a trained phenotype avoiding muscle atrophy [191]. Upon activation, PGC-1α in turn controls many, if not all of the adaptations of skeletal muscle to endurance exercise [192]. Hereafter, PGC-1α muscle-specific transgenic animals exhibit high endurance, oxidative muscle fibers, an increase in mitochondrial biogen‐ esis and oxidative metabolism, augmented muscle capillarization and a remodeling of the neuromuscular junction [193, 194].

Although innervation of the newly implanted tissue is also essential to engineer a functional muscle tissue there is few approaches that could effectively promote nerve ingrowth after transplantation. Some studies described a spontaneous nerve ingrowth from the neighbor tissues into the newly transplanted sites [195, 196], but non-invasive methods to induce nerve ingrowth after newly formed muscle engrafts are still to be investigated. We have recently proposed that magnetic stimulation supports regeneration of injured muscle with activating resident stem cells or supporting integration of newly implanted myoblasts [197, 198]. Exposition of injured limb and co-cultures of muscle cells and neurons to magnetic fields was sufficient to trigger synapses, induce acetylcholine receptors clustering and cause typical muscular metabolic adaptations verified during endurance exercise [197]. Notwithstanding, magnetic stimulation mimicked the effects of exercise inducing PGC-1α up-regulation, induces myogenic cells differentiation and increases nerve fibers and acetylcholine receptor clustering after cell transplantation [198]. New efforts in establishing functional innervation, proper vascular network and the development of a high endurance resistance muscle are going to be the three main pillars supporting future translational studies and bringing myogenic cell transplantation from bench to bedside.
