**5. Non–biological translational work**

The ability to regenerate muscle tissue from patient derived cells would have profound impact on many human diseases. Cell therapy is within reach as a novel treatment option for incon‐ tinence, reflux, vocal cord dysfunction and other muscle-related pathologies. However, the carrier used for cell delivery and the techniques used to inject the cells are still being optimized.

#### **5.1. Cell delivery**

between 50% and 88% in a follow-up of 12 months [138-141]. Even if the designs between studies differ, the combination of cell therapy with electrical stimulation or/and pelvic floor exercises may explain the variation between the values. In fact, a cell therapy with the application of myoblasts alone seems to provide a 50% improvement [139, 141], improving to 78.4% if electrical stimulation is added [138] and reaching 88% with pelvic floor exercises [140]. This approximate comparison can encourage future clinical studies to combine other therapies and exercises with cell therapies in order to optimize the outcome. Myoblasts have also been combined with fibroblasts mixed in a collagen solution. The results were impressive: 79% of treated women and 65% of the men reached continence [142, 143]. As a Lancet publication of this group was retracted, these results should be handled with precaution and should be confirmed by other groups [144]. Other muscle-derived cells have been injected in patients with SUI. Since 2008, MDSC have been applied in several clinical trials [145, 146] with improvement rates of 53% after 1-year follow-up with 10 million cells injected, 63% with 20 million and 67% with 50 million. The efficiency of the cell therapies seems to be dose-depend‐ ent. This was confirmed by Kaufman et al. in a 6-month dose escalating study, where im‐ provements increased with the dose of injected cells. The best results were obtained with 200 million MDSC injected [146]. Interestingly, no serious adverse effects were observed even when numbers of UCBSC as high as 400 million were applied [147]. In this latter case, 72% of

Abbreviations: MDC, muscle-derived cells; MDSC, muscle-derived stem cells; UCBSC, umbilical cord blood stem cells.

**biomaterial**

none

sphincter none at least 12

Bladder neck calcium alginate <sup>12</sup> 81% improved

sphincter none 3 to 24 63% improved

sphincter none <sup>12</sup> 50% improved

none 1.5

**Time point Months**

collagen <sup>12</sup> 79% continent

collagen <sup>12</sup> 65% continent

autologous serum 12 to 18 88% improved

**Outcomes**

13% improved

17% improved

38% continent

12% continent 42% improved 46% no efficacy

78.4% improved 13.5% cured 8.1% unchanged

none <sup>6</sup> improvement Yamamoto

**Measurements Reference Year**

50% continent Bent et al. <sup>2001</sup>

13% continent Carr et al. <sup>2008</sup>

9% continent Lee et al. <sup>2010</sup>

25% continent Sèbe et al. <sup>2011</sup>

Mitterberger et al. <sup>2007</sup>

Mitterberger et al. <sup>2008</sup>

Kajbafzadeh et al. <sup>2008</sup>

Gerullis et al. 2012

et al. <sup>2012</sup>

Blagange et al. 2012

**Cell type Source Patients / n Injection Target organ Delivery**

urethral

urethral

Transurethral

**Table 2.** Clinical trials for treating stress urinary incontinence based on cell therapy.

External

External sphincter

External

External

External Urethra sphincter Submucosa space

External Urethra sphincter

UCBSC allogenic Women / 39 Transurethral Submucosa none <sup>12</sup> 72% improved

fibroblasts autologous Woman / 123 Transurethral Urethra autologous serum

fibroblasts autologous men / 63 Transurethral Urethra autologous serum

Chrondrocytes autologous Women / 32 Trans/peri-

690 Regenerative Medicine and Tissue Engineering

MDSC autologous Women / 8 Trans/peri-

Myoblasts autologous Women / 12 Transurethral

MDC autologous Men / 222 Transurethral

ADSC autologous Men / 3 Transurethral

Myoblasts autologous Women / 38 Intrasphincteric

Girls / 1

Myoblasts autologous Boys / 7

Myoblasts and

Myoblasts and

It has been demonstrated for more than a decade that cells injected in a saline solution carrier are able to ectopically form contractile muscle [149]. However, further studies have reported very poor cell survival rates (5-20%) associated with myogenic cell implantation without embedding into protein based carriers that support cell settling into their new niche [150, 151].

Species-specific cues play an important role in cell affinity to carriers. A previous study demonstrated advantages using collagen rather than matrigel coated dishes, boosting cell growth and differentiation potential [73]. In contrast, another study with porcine satellite cells demonstrated cell preference to matrigel coated dishes and growth decrease on collagen layers[152]. Moreover, three-dimensional (3D) matrigel coated PLGA (poly lactic-co-glycolic acid) scaffolds were capable of improving cell survival when compared to direct cell injection [153]. However, the same study failed to demonstrate a comparative improvement of matrigel coated PLGA with other cell carriers. Furthermore, matrigel has not presented advantage *in vivo a*s a carrier for myogenic cells when compared to hyaluronic acid-photoinitiator (HA-PI) complex. It rather downgraded the quality of muscle structure and decreased the total number of new myofibers after cell injection [154].

Collagen is a main component of the natural extracellular matrix of skeletal muscle, it is therefore expected that satellite cells would have their functionality up-scaled in a collagen rich environment [155]. Combined with electrical stimulation collagen induces three-dimen‐ sional expansion of muscle precursor cells *in vitro* and in syngeneic recipient muscle [156]. Cell cycle analyses of engrafts implanted into a 3D collagen sponge highlighted the increment of cell fractions in proliferating phases, with 80% of cell survival [157]. In addition, the use of parallel aligned collagen nanofibers yielded good proliferation and enabled the generation of aligned cell layers [158]. Finally, grafts of myoblasts seeded into three-dimensional collagen scaffolds and implanted into injured sites in mice demonstrated improvement in muscle healing, innervation and vascularization [159]. Altogether these recent studies confirm that collagen is a very promising matrix for satellite cell ingrowth and an ideal carrier for the transplantation of myogenic cells.

#### **5.2. Imaging techniques for guided cell implantation in vivo**

The success of cell transplantation into a specific site *in vivo* is directly dependent of 3 key points: cell source, cell carrier and injection technique. The first two were previously discussed in this chapter. We dedicate this section to the discussion of injection techniques used so far to inject myogenic cells into a specific injury site. The application of myogenic cells was already used for the treatment of male and female patients suffering from urinary incontinence, the involuntary loss of urine that represents a hygienic and social problem [160]. Transurethral ultrasound guided injections of autologous cells isolated from limb skeletal muscle biopsies were so far the method of choice [161, 162]. This method is also standard for the injection of bulking agents like collagen in the clinical practice [163]. Finally, ultrasound guidance was also used to monitor percutaneous trans-coronary-venous transplantation of autologous myoblasts in infarcted myocardium [164, 165].

Recently magnetic resonance imaging (MRI) has gained attention as a useful tool for guidance during injection of drugs and potentially of cells [166]. Pulsed focused ultrasound is a new ultrasound technique that associated with magnetic resonance guidance was recently sug‐ gested as a new imaging modality that may be utilized to target cellular therapies by increasing homing to areas of pathology [167]. It has also been demonstrated to increase drug uptake into a specific target in the prostate [168] and brain [169]. This same technique has been shown to facilitate the delivery of neural stem cells into a specific site in the brain [170]. Overall, the most successful deliveries of myogenic cells have been done either operatively in 3D scaffolds or in collagen carrier that facilitates cell settling into the new cell niche. Ultrasonography is still the most adaptable and widely used imaging technique allowing visualization of the injury zone and real time needle guidance. However, new approaches combining MRI and ultrasono‐ graphic pulses are very promising methods that need to be further studied and adapted for cell injection in different anatomic sites. Moreover, MRI is used in tracking stem cells after injection [171, 172]. In fact, it is important not only to inject the cells at the right place but also to ensure that cells are not migrating to other parts and pursuing their role in regenerating the tissue of interest. Additionally, developments in MRI technology, especially in scanning technics, offer the possibility to follow the differentiation process of injected MPCs and their fate in making fibers [173].

#### **5.3. Regulation and guidelines**

The application of cell-based therapies is not only advancing scientifically but also regulations are adapting and including the new scientific discoveries for clinical use. The relevant health agencies all around the world are creating committees that are modifying the regulations in order to take account of these new categories of products that are cell-based. Stem cell based therapies are part of advanced therapies, which are therapies based on genes, or cells, or tissues [174]. Concerning this emerging branch of medicinal products, the regulations are new and still in development. They have their own classification, distinct from chemical and biologic drugs, transplantation organs and medical devices. Though, they can be sometimes included in these categories. In Europe, the European Medicines Agency (EMA) is in charge of improv‐ ing the standards and reviewing the applications for stem cell based therapies, which are part of Advanced Therapy Medicinal Products (ATMP), and they are found in regulation (EC) N° 1394/2007 [175]. The Committee for Advances Therapies (CAT) is the body responsible within EMA of this new field of science and its approval for marketing. The goals are to protect the patient from contaminated tissues/cells, to avoid the inappropriate handling of tissues/cells and to guaranty safety and efficacy of therapies. The documents are providing a regulatory framework that is coherent with existing ones, specific to biological and chemical entities for instance. Hence, before starting any clinical trial on human, several requirements are to be fulfilled. The cell-based product needs to be grounded on a sound and solid scientific work that is confirmed in pre-clinical studies, which show its quality, safety and efficacy. During this preparation phase, CAT is available for giving advice in preparing all the relevant files for obtaining clinical trials authorization or latter for marketing authorization. Guidelines are specifying aspects of pharmacovigilance, risk management planning, monitoring, labeling, safety, efficacy follow-up and traceability. The submission process should comply with these requirements in order to receive the green light for starting clinical trials or entering the market. During product development and clinical investigations guidelines have also been adapted by CAT for stem-cell based therapies for specifications on Good Manufacturing Practice (GMP) and Good Clinical Practice (GLP) [176]. In the US, the Office of Cellular, Tissue, and Gene Therapies (OCTGT) - part of the Center for Biologics Evaluation and Research (CBER) in FDAis responsible of the cellular therapies products [177]. They are regulated by human cells, tissues, and cellular and tissue-based products (HCT/Ps) under the authority of Section 361 of the Public Health Services (PHS) Act as well as Title 21 of the Code of Federal Regulations (CFR) part 1271 [178]. The OCTGT are making sure that the cell-based products meet safety, purity, potency and effectiveness qualifications. EMA and FDA are collaborating closely together in the Advanced Therapies Medicinal Product cluster. The development of regulatory frameworks is not equal in all countries and is independent from a state to another state. However, at the international level, regulatory agencies are working together in sharing and harmonizing the regulatory frameworks for cellular therapy products through the Interna‐ tional Conference of Harmonization of Technical Requirements for Registration of Pharma‐ ceuticals for Human use (ICH), the Pan-American health Organization (PAHO), WHO and Asia-Pacific Economic Cooperation (APEC). This global interaction facilitates the development of the cellular therapy field and prepares in bringing the products to the markets. As the experience is right now limited in this field, this discussion panels permits to cover the different applications and cases among the countries and therefore increase the knowledge levels among the participants and the regulatory boards. In addition, it creates convergence in the develop‐

cell fractions in proliferating phases, with 80% of cell survival [157]. In addition, the use of parallel aligned collagen nanofibers yielded good proliferation and enabled the generation of aligned cell layers [158]. Finally, grafts of myoblasts seeded into three-dimensional collagen scaffolds and implanted into injured sites in mice demonstrated improvement in muscle healing, innervation and vascularization [159]. Altogether these recent studies confirm that collagen is a very promising matrix for satellite cell ingrowth and an ideal carrier for the

The success of cell transplantation into a specific site *in vivo* is directly dependent of 3 key points: cell source, cell carrier and injection technique. The first two were previously discussed in this chapter. We dedicate this section to the discussion of injection techniques used so far to inject myogenic cells into a specific injury site. The application of myogenic cells was already used for the treatment of male and female patients suffering from urinary incontinence, the involuntary loss of urine that represents a hygienic and social problem [160]. Transurethral ultrasound guided injections of autologous cells isolated from limb skeletal muscle biopsies were so far the method of choice [161, 162]. This method is also standard for the injection of bulking agents like collagen in the clinical practice [163]. Finally, ultrasound guidance was also used to monitor percutaneous trans-coronary-venous transplantation of autologous myoblasts

Recently magnetic resonance imaging (MRI) has gained attention as a useful tool for guidance during injection of drugs and potentially of cells [166]. Pulsed focused ultrasound is a new ultrasound technique that associated with magnetic resonance guidance was recently sug‐ gested as a new imaging modality that may be utilized to target cellular therapies by increasing homing to areas of pathology [167]. It has also been demonstrated to increase drug uptake into a specific target in the prostate [168] and brain [169]. This same technique has been shown to facilitate the delivery of neural stem cells into a specific site in the brain [170]. Overall, the most successful deliveries of myogenic cells have been done either operatively in 3D scaffolds or in collagen carrier that facilitates cell settling into the new cell niche. Ultrasonography is still the most adaptable and widely used imaging technique allowing visualization of the injury zone and real time needle guidance. However, new approaches combining MRI and ultrasono‐ graphic pulses are very promising methods that need to be further studied and adapted for cell injection in different anatomic sites. Moreover, MRI is used in tracking stem cells after injection [171, 172]. In fact, it is important not only to inject the cells at the right place but also to ensure that cells are not migrating to other parts and pursuing their role in regenerating the tissue of interest. Additionally, developments in MRI technology, especially in scanning technics, offer the possibility to follow the differentiation process of injected MPCs and their

The application of cell-based therapies is not only advancing scientifically but also regulations are adapting and including the new scientific discoveries for clinical use. The relevant health

transplantation of myogenic cells.

692 Regenerative Medicine and Tissue Engineering

in infarcted myocardium [164, 165].

fate in making fibers [173].

**5.3. Regulation and guidelines**

**5.2. Imaging techniques for guided cell implantation in vivo**

ment of the regulations and guidelines concerning different aspects: manufacturing, quality assurance, quality control and pre-clinical studies [179].

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 sphincter muscle by using stem cells.

#### **5.4. Production of cell–therapies**

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 or tissues.

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

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

#### **5.5. Manufacturing process**

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

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, testing, release, storage and transport.

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 from the research laboratories. One of the major problems in the cell culture is to replace the fetal bovine serum (FBS). Most of the protocols are still based on this animal derived product. Recently, some efforts have been made to work with xeno-free medium by replacing FBS with human serum and platelet lysate [180]. In the case of MPCs, one of the major sources of cells for muscle cell therapy as described above, pooled human platelet lysate was demonstrated to be a good alternative to FBS [181]. Other factors are important and must be controlled as cell seeding, growth rate, differentiation process, markers expression, potency of the cells in making contractile fibers. The protocols for each step - from receiving the biopsy up to the final product -must be standardized and approved by local authorities before starting clinical trials. Standardization means that clear and details protocols should be written and followed without deviation or modifications. Quality controls are done not only for starting materials but also at critical steps in production. Quality is a key parameter that applies to all levels of the cell therapy production: building and facilities environments, equipment, production, labeling, storage and distribution. The quality unit performs all the controls to show the purity of the products, the cleanness of the environment, the maintenance of the equipment and the respect of the specifications set for obtaining a safe, effective and potent cell product. In muscle cell therapy, the cell population should have a pure or a very high percentage of cells expressing markers of skeletal cell as described above.

All the stages and elements related to the GMP facility or the production process should be documents to insure traceability of every single action. The documents should be prepared, reviewed, approved and distributed as specified in established and written procedures. All these demanding steps require qualified personnel, well-trained in working in GMP facilities. It includes good sanitation and health habits and the right skills to accomplish the work with products for cell therapy. Finally, internal and external audits are conducted regularly to verify the respect of the GMP regulations and guidelines as validated by the GMP facility and the authorities.
