Francesco Saverio Tedesco

Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells

Skeletal muscle damaged by injury or by degenerative diseases such as muscular dystrophy is able to regenerate new muscle fibers. Regeneration mainly depends upon satellite cells, myogenic progenitors localized between the basal lamina and the muscle fiber membrane. However, other cell types outside the basal lamina, such as pericytes, also have myogenic potency. Here, we discuss the main properties of satellite cells and other myogenic progenitors as well as recent efforts to obtain myogenic cells from pluripotent stem cells for patient-tailored cell therapy. Clinical trials utilizing these cells to treat muscular dystrophies, heart failure, and stress urinary incontinence are also briefly outlined.

Transplantation of Genetically Corrected Human iPSC-Derived Progenitors in Mice with Limb-Girdle Muscular Dystrophy

Genetically corrected mesoangioblasts from human iPSCs derived from limb-girdle muscular dystrophy patients produce muscle fibers expressing the therapeutic gene in a mouse model of the disease. Muscle Progenitors Find Their Way Home Muscular dystrophies are genetic disorders primarily affecting skeletal muscle that result in greatly impaired mobility and, in severe cases, respiratory and cardiac dysfunction. There is no effective treatment, although several new approaches are entering clinical testing including cell therapy. Cell therapy aims to replace lost muscle fibers by transplanting healthy donor muscle progenitor cells or cells from dystrophic patients that have been genetically corrected in vitro. Mesoangioblasts are progenitor cells from blood vessel walls that have shown potential as a cell therapy in animal models of muscular dystrophy. In a new study, Tedesco et al. explore whether genetically corrected mesoangioblasts from patients with limb-girdle muscular dystrophy 2D (LGMD2D) have potential as an autologous cell therapy to treat this disease. The authors quickly found that they could not derive a sufficient number of mesoangioblasts from LGMD2D patients because the muscles of the patients were depleted of these progenitor cells. To overcome this problem, the authors reprogrammed fibroblasts or myoblasts from the LGMD2D patients to obtain human induced pluripotent stem cells (iPSCs) and induced them to differentiate into mesoangioblast-like cells that were then genetically corrected in vitro using a viral vector expressing the defective gene SGCA, which encodes α-sarcoglycan. After intramuscular or intra-arterial injection of these genetically corrected, iPSC-derived mesoangioblasts into mice with LGMD2D (immune-deficient Sgca-null mice), the cells homed to damaged mouse skeletal muscle, engrafted, and formed muscle fibers expressing α-sarcoglycan. Using mouse iPSC-derived mesoangioblasts, the researchers showed that the transplanted engrafted cells imbued muscle with greater strength and enabled the dystrophic mice to run for longer on a treadmill than dystrophic mice that did not receive the cells. This strategy offers the advantage of being able to produce unlimited numbers of genetically corrected progenitor cells, which perhaps could be used in the future as cell therapy for treating LGMD2D and other forms of muscular dystrophy. Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne’s muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan–null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan–null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.

Collagen VI regulates satellite cell self-renewal and muscle regeneration.

Adult muscle stem cells, or satellite cells play essential roles in homeostasis and regeneration of skeletal muscles. Satellite cells are located within a niche that includes myofibers and extracellular matrix. The function of specific extracellular matrix molecules in regulating SCs is poorly understood. Here we show that the extracellular matrix protein collagen VI is a key component of the satellite cell niche. Lack of collagen VI in Col6a1−/− mice causes impaired muscle regeneration and reduced satellite cell self-renewal capability after injury. Collagen VI null muscles display significant decrease of stiffness, which is able to compromise the in vitro and in vivo activity of wild-type satellite cells. When collagen VI is reinstated in vivo by grafting wild-type fibroblasts, the biomechanical properties of Col6a1−/− muscles are ameliorated and satellite cell defects rescued. Our findings establish a critical role for an extracellular matrix molecule in satellite cell self-renewal and open new venues for therapies of collagen VI-related muscle diseases.

Complement-Mediated Demyelination in Patients with IgM Monoclonal Gammopathy and Polyneuropathy

We investigated the role of complement in the pathogenesis of the demyelinating polyneuropathy that occurs in some patients with IgM monoclonal gammopathy. Seven patients with chronic sensorimotor polyneuropathy and IgM monoclonal gammopathy were examined. In six patients, the monoclonal protein recognized an epitope shared by myelin-associated glycoprotein and two peripheral-nerve glycolipids, whereas in one patient, IgM bound to an unidentified myelin antigen. Direct and indirect immunofluorescence and immunoperoxidase assays showed colocalization along the myelin sheaths of peripheral-nerve fibers of monoclonal protein with complement components C1q, C3d, and C5. In addition, terminal-complement complex that was not associated with S protein was detected in myelin sheaths. It appeared that alterations in myelin geometry caused by the separation of myelin lamellae corresponded to sites at which terminal-complement complex was deposited. We conclude that demyelination in polyneuropathy associated with IgM monoclonal gammopathy may be mediated by complement.

Serum-Resistant Strains of Borrelia burgdorferi Evade Complement-Mediated Killing by Expressing a CD59-Like Complement Inhibitory Molecule

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab′)2 anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8β.

Stem cell therapies for muscle disorders

PURPOSE OF REVIEW This review focuses on stem cell-based therapies to treat skeletal muscle disorders, with a special emphasis on muscular dystrophies. RECENT FINDINGS We briefly review previous attempts at cell therapy by the use of donor myoblasts, explaining the likely reasons for the poor clinical results; we then describe the use of the same cells in current promising trials for localized treatments of different diseases of skeletal muscle. Moreover, we discuss important novel findings on muscle stem/progenitor cell biology and their promise for future clinical translation. Preclinical and clinical applications of novel myogenic stem/progenitor cells are also described. SUMMARY We summarize several ongoing clinical trials for different muscle disorders and the advances in the understanding of the biology of the myogenic progenitors used in such trials. On the basis of the currently available information, a prediction of developments in the field is proposed.

Intra-arterial transplantation of HLA-matched donor mesoangioblasts in Duchenne muscular dystrophy

Intra‐arterial transplantation of mesoangioblasts proved safe and partially efficacious in preclinical models of muscular dystrophy. We now report the first‐in‐human, exploratory, non‐randomized open‐label phase I–IIa clinical trial of intra‐arterial HLA‐matched donor cell transplantation in 5 Duchenne patients. We administered escalating doses of donor‐derived mesoangioblasts in limb arteries under immunosuppressive therapy (tacrolimus). Four consecutive infusions were performed at 2‐month intervals, preceded and followed by clinical, laboratory, and muscular MRI analyses. Two months after the last infusion, a muscle biopsy was performed. Safety was the primary endpoint. The study was relatively safe: One patient developed a thalamic stroke with no clinical consequences and whose correlation with mesoangioblast infusion remained unclear. MRI documented the progression of the disease in 4/5 patients. Functional measures were transiently stabilized in 2/3 ambulant patients, but no functional improvements were observed. Low level of donor DNA was detected in muscle biopsies of 4/5 patients and donor‐derived dystrophin in 1. Intra‐arterial transplantation of donor mesoangioblasts in human proved to be feasible and relatively safe. Future implementation of the protocol, together with a younger age of patients, will be needed to approach efficacy.

EMILIN1 represents a major stromal element determining human trophoblast invasion of the uterine wall

The detection of EMILIN1, a connective tissue glycoprotein associated with elastic fibers, at the level of the ectoplacental cone and trophoblast giant cells of developing mouse embryos (Braghetta et al., 2002) favored the idea of a structural as well as a functional role for this protein in the process of placentation. During the establishment of human placenta, a highly migratory subpopulation of extravillous trophoblasts (EVT), originating from anchoring chorionic villi, penetrate and invade the uterine wall. In this study we show that EMILIN1, produced by decidual stromal and smooth muscle uterine cells, is expressed in the stroma and in some instances as a gradient of increasing concentration in the perivascular region of modified vessels. This distribution pattern is consistent with the haptotactic directional migration observed in in vitro functional studies of freshly isolated EVT and of the immortalized HTR-8/SVneo cell line of trophoblasts. Function-blocking monoclonal antibodies against α4-integrin chain and against EMILIN1 as well as the use of EMILIN1-specific short interfering RNA confirmed that trophoblasts interact with EMILIN1 and/or its functional gC1q1 domain via α4β1 integrin. Finally, membrane type I-matrix metalloproteinase (MT1-MMP) and MMP-2 were upregulated in co-cultures of trophoblast cells and stromal cells, suggesting a contributing role in the haptotactic process towards EMILIN1.

Injectable polyethylene glycol-fibrinogen hydrogel adjuvant improves survival and differentiation of transplanted mesoangioblasts in acute and chronic skeletal-muscle degeneration

BackgroundCell-transplantation therapies have attracted attention as treatments for skeletal-muscle disorders; however, such research has been severely limited by poor cell survival. Tissue engineering offers a potential solution to this problem by providing biomaterial adjuvants that improve survival and engraftment of donor cells.MethodsIn this study, we investigated the use of intra-muscular transplantation of mesoangioblasts (vessel-associated progenitor cells), delivered with an injectable hydrogel biomaterial directly into the tibialis anterior (TA) muscle of acutely injured or dystrophic mice. The hydrogel cell carrier, made from a polyethylene glycol-fibrinogen (PF) matrix, is polymerized in situ together with mesoangioblasts to form a resorbable cellularized implant.ResultsMice treated with PF and mesoangioblasts showed enhanced cell engraftment as a result of increased survival and differentiation compared with the same cell population injected in aqueous saline solution.ConclusionBoth PF and mesoangioblasts are currently undergoing separate clinical trials: their combined use may increase chances of efficacy for localized disorders of skeletal muscle.

PW1/Peg3 expression regulates key properties that determine mesoangioblast stem cell competence

Mesoangioblasts are vessel-associated progenitor cells that show therapeutic promise for the treatment of muscular dystrophy. Mesoangioblasts have the ability to undergo skeletal muscle differentiation and cross the blood vessel wall regardless of the developmental stage at which they are isolated. Here we show that PW1/Peg3 is expressed at high levels in mesoangioblasts obtained from mouse, dog and human tissues and its level of expression correlates with their myogenic competence. Silencing PW1/Peg3 markedly inhibits myogenic potential of mesoangioblasts in vitro through MyoD degradation. Moreover, lack of PW1/Peg3 abrogates mesoangioblast ability to cross the vessel wall and to engraft into damaged myofibres through the modulation of the junctional adhesion molecule-A. We conclude that PW1/Peg3 function is essential for conferring proper mesoangioblast competence and that the determination of PW1/Peg3 levels in human mesoangioblasts may serve as a biomarker to identify the best donor populations for therapeutic application in muscular dystrophies.