Do porcine Sertoli cells represent an opportunity for Duchenne muscular dystrophy?

Abstract Sertoli cells (SeC) are responsible for the immunoprivileged status of the testis thanks to which allogeneic or xenogeneic engraftments can survive without pharmacological immune suppression if co‐injected with SeC. This peculiar ability of SeC is dependent on secretion of a plethora of factors including maturation factors, hormones, growth factors, cytokines and immunomodulatory factors. The anti‐inflammatory and trophic properties of SeC have been largely exploited in several experimental models of diseases, diabetes being the most studied. Duchenne muscular dystrophy (DMD) is a lethal X‐linked recessive pathology in which lack of functional dystrophin leads to progressive muscle degeneration culminating in loss of locomotion and premature death. Despite a huge effort to find a cure, DMD patients are currently treated with anti‐inflammatory steroids. Recently, encapsulated porcine SeC (MC‐SeC) have been injected ip in the absence of immunosuppression in an animal model of DMD resulting in reduction of muscle inflammation and amelioration of muscle morphology and functionality, thus opening an additional avenue in the treatment of DMD. The novel protocol is endowed with the advantage of being potentially applicable to all the cohort of DMD patients regardless of the mutation. This mini‐review addresses several issues linked to the possible use of MC‐SeC injected ip in dystrophic people.


| INTRODUC TI ON
Sertoli cells (SeC) are major component of the seminiferous tubules of the testis where they contribute to the development of germ cells and protect germ cells from the attack by the host immune system. 1,2 Indeed, newly synthesized markers on germ cell surface would be recognized as non-self by the immune system, as this latter becomes mature before spermatogenesis starts. SeC exert their double role (a) by creating a physical barrier (the blood-testis barrier, BTB) made of adjacent SeC linked together with tight junctions, isolating the lumen of seminiferous tubules from the interstitial fluid, and (b) by secreting a plethora of trophic and immunomodulatory factors. 3,4 This latter ability of SeC has prompted researchers to use them in many experimental models of diseases in which supplying trophic factors, abating inflammation or modulating the immune system activity might result in reversion or attenuation of the pathology. 2,5

| S ERTOLI CELL S
The immunoprivileged status of the testis is long known. In 1767, during his studies on transplantation, John Hunter observed that rooster testes transplanted into the abdominal cavity of a hen maintained normal structure over time. 6 Subsequent investigation identified SeC as the cell type mainly responsible for the immunological properties of the testis. Indeed, allogeneic and/or xenogeneic pancreatic islets, 7,8 adrenal chromaffin cells 9 and dopaminergic neurons 10 were successfully protected by SeC in the absence of pharmacological immune suppression in different experimental models. Similarly, skin 11 and heart 12 grafts showed prolonged survival when co-injected with SeC. Moreover, grafts of SeC alone exerted trophic effects in the central nervous system in animal models of Huntington's disease and amyotrophic lateral sclerosis. 13,14 Intriguing results about SeC derive from their use as encapsulated cells in several pre-clinical studies. Alginate-based microcapsules containing SeC (MC-SeC) have been successfully employed as a single intraperitoneal (ip) injection in experimental models of type 1 and type 2 diabetes, acute hepatic failure, skin graft and Huntington's disease. [15][16][17][18][19][20][21] In an animal model reproducing the human Laron syndrome, in which mutations in the growth hormone receptor (GHR) lead to reduced production of IGF-1 and subsequent dwarfism, intraperitoneally injected MC-SeC promoted body growth through the release of IGF-1 into the circulation. 22 The trophic and immunomodulatory properties of SeC are dependent on the complex SeC secretory activity resulting in a cocktail of factors whose formulation is difficult to dissect, being affected by the biological status of the cells and likely by environmental cues.
While the composition of SeC' secretory product still awaits to be identified, it includes maturation factors, hormones, growth factors, cytokines and immunomodulatory factors (Table 1).
However, the immunomodulatory effect of SeC is obtained by a multimodal mechanism. SeC secrete (still unidentified) factors thatblock T lymphocyte proliferation and interleukin (IL)-2 production, 23,24 and SeC induce apoptosis of lymphocytes mediated by the interaction of FasL expressed on their surface and Fas receptor (CD95) expressed on T cells. 25 SeC secrete inhibitors of the complement cascade and granzyme, a cytolytic molecule released by cytotoxic T cells. 26,27 Moreover, the secretion of specific factors, such as TGF-β, IDO (indoleamine 2,3-dioxygenase), activin A and JAG1, concurs to immunomodulation favouring the emergence of tolerogenic cells, including M2 (anti-inflammatory) macrophages, and Th2 and Tregs. 15,28,29

| DUCHENNE MUSCUL AR DYS TROPHY AND REL ATED THER APEUTI C APPROACHE S
Duchenne muscular dystrophy (DMD) is an X-linked recessive disease due to mutations in the dystrophin gene (DMD), the biggest gene of the human genome for which about 4700 different mutations have been reported. [30][31][32][33] Dystrophin is an essential component of the dystrophin-associated protein complex (DAPC), a multiprotein complex located at the sarcolemma and responsible for the mechanical link between the intracellular cytoskeleton and the extracellular matrix; dystrophin ensures the structural and functional integrity of myofibres during contraction. DMD gene mutations translating into absence of dystrophin or expression of functionally inefficient protein lead to the Duchenne phenotype, in which loss of the integrity of DAPC causes myofibre degeneration and progressive loss of muscle efficiency, wheelchair dependency before teenage years, and premature death by cardiac and respiratory failure. 31,34 Morphologically, DMD muscles are characterized by infiltration with immune cells and chronic activation of inflammatory signalling pathways due to continuous degeneration/regeneration cycles, with the final result that fibrous and fatty tissues progressively overtake functional myofibres. 35 Therapeutic approaches to DMD have been experiencing multiple obstacles against their success (Table 2). Firstly, DMD gene is a Exon skipping is an approach to overcome specific regions with deletions, duplications or small mutations in the DMD gene pointing to recovery of the reading frame and production of truncated but functional forms of dystrophin, and translating into a switch from the DMD pathology to the milder phenotype known as Becker muscular dystrophy (BMD). 45,46 Specifically designed antisense oligonucleotides (AON), which are 20-30 nucleotides in length, are used to obtain skipping of different exons resulting in truncated but in-frame transcripts. [47][48][49] The modified AON, 2′-O-methyl-p phosphorothioate oligonucleotides (PS) and phosphorodiamidate morpholino oligomers (PMO) have shown high stability and efficacy, and low toxicity.
One major limit of AON is that they are only useful for DMD pa-  cell types for cell therapy in DMD patients. Mesoangioblasts are endowed with myogenic potential and ability to cross the blood vessel wall, and their use has resulted in improvement of muscle morphology in several experimental models of muscular dystrophy. 45,61,62 Intra-arterial injection of allogeneic human mesoangioblasts isolated from adult skeletal muscle is currently under phase I clinical trial (EudraCT #2011-000176-33).
The existence of the dystrophin paralogue, utrophin, has fostered another approach to rescue homeostasis in the muscles of DMD patients. Utrophin shares a very high degree of sequence identity with dystrophin and even associates with members of the DAPC, thus mimicking the role of dystrophin in dystrophin-negative myofibres. 30,63 In healthy adult muscle fibres, dystrophin and utrophin show different expression patterns, with dystrophin being expressed along the entire sarcolemma and utrophin confined to the myotendinous and the neuromuscular junctions (NMJs). 64,65 However, utrophin is expressed at high levels at the sarcolemma during development, when dystrophin is not expressed yet. 66 Indeed, necrosis of mdx limb muscles begins only when the high neonatal levels of utrophin become reduced to adult levels. 64 Since forced expression of utrophin in dystrophic myofibres can restore assembly of DAPC members at the sarcolemma and prevent the dystrophic pathology, 67,68 up-regulation of utrophin in muscles represents a still active field of investigation in DMD treatment. 69 In this regard, the small molecule, SMT C1100 (Ezutromid), which has shown promising results in a phase I clinical trial, 70 was stopped after a phase II clinical trial (NCT02858362) since it failed to reach its primary and secondary objectives.
Several alternative approaches to treat DMD are currently under investigation (Table 3) (Table 3).

| DO P ORCINE S ERTOLI CELL S REPRE S ENT AN OPP ORTUNIT Y FOR DUCHENNE MUSCUL AR DYS TROPHY ?
Having the peculiar secretory properties of SeC in mind, we treated acute and chronic dystrophic, mdx mice with a single ip injection of in muscle cells. 96 Heregulin β1 acts by binding to erbB/HER receptor, resulting in intracellular ERK activation and subsequent binding of the ets-related GABPα/β transcription factor complex to the utrophin-A promoter. 97 Three weeks after ip injection of MC-SeC, a ~2.8-fold increase in utrophin expression was observed in muscles of mdx mice, 94 which is similar to that reported after 3 months of repeated ip injections each other day of the active domain of heregulin β1 (Aa 176-246) in the same experimental model. 98 In both cases, utrophin was found localized at the sarcolemma, a condition necessary for the protein to mimic the role of dystrophin (Figure 1).
Microcapsules containing SeC-based protocol resulted efficacious also in pre-symptomatic (2-week-old) and in chronic (12month-old) mdx mice. 95 In the diaphragms of these latter mice, a significant reduction of adipose and fibrous tissue deposition (~43% and ~58% reduction, respectively), macrophage infiltrate (~70% reduction of MAC3-positive areas) and damaged myofibres (more than 80% reduction of EBD-positive myofibres) were observed 3 weeks after ip injection of MC-SeC. 95 The  porcine SeC were inserted together with pancreatic islets subcutaneously in a porous chamber in the abdominal wall of young diabetic patients, in the absence of immunosuppressive treatment, and half patients significantly diminished their insulin doses with no complications reported in a 7-year follow-up. 106,107 The use of pig cells, tissues and organs meets the general need to satisfy the increasing request for transplantation by humans who do not find sufficient availability among members of their species. Pigs represent suitable animals for human xenotransplantation because they share a similar organ physiology and size, for the relatively low costs of breeding and for the possibility to be genetically modified. One concern in xenotransplantation using pigs as a donor species is represented by porcine endogenous ret- patients' blood cells was observed. 111 The reason why PERVs are not transmitted is that they probably are not released from the transplants or they are neutralized by the host cellular defence and immune system. 109 However, porcine pancreatic islets were demonstrated to produce PERV and infect NOD/SCID (non-obese diabetic, severe combined immunodeficiency) mice after transplantation, suggesting that PERV infection is a risk to take into account when pig xenotransplantation involves immunocompromised subjects. 112 In this regard, it is noteworthy that PERV-inactivated pigs have been recently generated via somatic cell nuclear transfer using a cell line in which PERVs were inactivated by CRISPR/Cas9 technology. 113 Among the factors secreted by SeC are mitogenic factors that could potentially sustain cell growth and induce the formation of tumour masses in the recipient. However, this event has not been reported in any study involving the use of SeC, including the above-mentioned clinical study. 110 Probably, this is because SeC secrete a cocktail of molecules whose global effect results from the combination of all factors rather than being the sum of each single factor activity.
Another issue about the use of SeC is related to their immunosup-

| CON CLUS IONS
Duchenne muscular dystrophy is a lethal muscular dystrophy affecting 1 in 3600-5000 male live births worldwide. 115 The progressive muscle degeneration subsequent to lack of dystrophin creates a condition of chronic inflammation that culminates in the progressive substitution of myofibres with fibrous and adipose tissues, impaired locomotion and premature death. Several intrinsic properties of the DMD pathology have nullified the huge effort to find a cure so far. In addition, many suggested therapeutic treatments have immunosuppression as a necessary co-treatment, which means adding problem to problem, especially in a lifelong perspective. Therefore, investigation is still particularly active on DMD, and combinatorial therapeutic approaches might be envisaged.
Intraperitoneal It is noteworthy that treatment with MC-SeC has the advantage to be a universal approach to treat DMD since it is potentially appli-

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; and in the decision to publish the results.