Titel Deelnemers "Korte inhoud" "Combined Notch and PDGF Signaling Enhances Migration and Expression of Stem Cell Markers while Inducing Perivascular Cell Features in Muscle Satellite Cells" "Mattia Francesco Maria Gerli, Louise Anne Moyle, Sara Benedetti, Giulia Ferrari, Ekin Ucuncu, Martina Ragazzi, Chrystalla Constantinou, Irene Louca, Hiroshi Sakai, Pierpaolo Ala, Paolo De Coppi, Shahragim Tajbakhsh, Giulio Cossu, Francesco Saverio Tedesco" "Satellite cells are responsible for skeletal muscle regeneration. Upon activation, they proliferate as transient amplifying myoblasts, most of which fuse into regenerating myofibers. Despite their remarkable differentiation potential, these cells have limited migration capacity, which curtails clinical use for widespread forms of muscular dystrophy. Conversely, skeletal muscle perivascular cells have less myogenic potential but better migration capacity than satellite cells. Here we show that modulation of Notch and PDGF pathways, involved in developmental specification of pericytes, induces perivascular cell features in adult mouse and human satellite cell-derived myoblasts. DLL4 and PDGF-BB-treated cells express markers of perivascular cells and associate with endothelial networks while also upregulating markers of satellite cell self-renewal. Moreover, treated cells acquire trans-endothelial migration ability while remaining capable of engrafting skeletal muscle upon intramuscular transplantation. These results extend our understanding of muscle stem cell fate plasticity and provide a druggable pathway with clinical relevance for muscle cell therapy." "Pluripotent Stem Cells for Gene Therapy of Degenerative Muscle Diseases" "Mariana Loperfido, Heather B Steele-Stallard, Francesco Saverio Tedesco" "Human pluripotent stem cells represent a unique source for cell-based therapies and regenerative medicine. The intrinsic features of these cells such as their easy accessibility and their capacity to be expanded indefinitely overcome some limitations of conventional adult stem cells. Furthermore, the possibility to derive patient-specific induced pluripotent stem (iPS) cells in combination with the current development of gene modification methods could be used for autologous cell therapies of some genetic diseases. In particular, muscular dystrophies are considered to be a good candidate due to the lack of efficacious therapeutic treatments for patients to date, and in view of the encouraging results arising from recent preclinical studies. Some hurdles, including possible genetic instability and their efficient differentiation into muscle progenitors through vector/transgene-free methods have still to be overcome or need further optimization. Additionally, engraftment and functional contribution to muscle regeneration in pre-clinical models need to be carefully assessed before clinical translation. This review offers a summary of the advanced methods recently developed to derive muscle progenitors from pluripotent stem cells, as well as gene therapy by gene addition and gene editing methods using ZFNs, TALENs or CRISPR/Cas9. We have also discussed the main issues that need to be addressed for successful clinical translation of genetically corrected patient-specific pluripotent stem cells in autologous transplantation trials for skeletal muscle disorders." "Skeletal muscle is enriched in hematopoietic stem cells and not inflammatory cells in cachectic mice" "Emanuele Berardi" "Cachexia, a debilitating syndrome characterized by skeletal muscle wasting, is associated to many chronic diseases and diminishes the quality of life and survival of patients. Tumor-derived factors and proinflammatory cytokines, including TNF-alpha, IL-6 and IL-1 beta, mediate cachexia. In response to elevated cytokine levels, increased proteasome-mediated proteolysis and auto-phagocytosis result in muscle wasting. The histologic features of muscle cachexia are not fully elucidated. Therefore, we analysed alterations of different cell populations in cachectic muscle." "Dental pulp stem cells promote wound healing and muscle regeneration" "Maurilio Sampaolesi" "© Springer International Publishing AG, part of Springer Nature 2018. Stem cells (SC) are unspecialized cells that can self-renew and generate specialized progeny through differentiation. These cells are found in almost all multicellular organisms and are capable of renewing themselves through cell division. Moreover, under certain stimuli, they can differentiate into tissue-spe- cific cells. Therefore, stem cells serve as a reservoir and repair system capable of replacing differentiated cells lost either naturally through apoptosis or as a result of trauma or disease. Consequently, the potential of stem cells to renew and dif- ferentiate makes them attractive candidates for regenerative medicine. These basic stem cell properties differ among various sources of stem cells, and they can be classified based on their origin and/or differentiation potential. The capacity to differentiate into specialized cell types and be able to originate a mature cell type is referred to as potency. Stem cells can be classified depending on their differen- tiation potential. Totipotent stem cells can differentiate into embryonic and extra- embryonic cell types; such cells can construct a complete, viable organism. Pluripotent stem cells produce all cells of an organism and have the capacity to form representative tissues of all three germ layers of the developing embryo: Ectoderm, mesoderm and endoderm. Multipotent stem cells can self-renew and differentiate only in a closely related family of cells from the same germ layer tissues, while unipotent stem cells exhibit limited development potential, giving rise to only a single cell type. Stem cells can be also classified according to the type of cells. Embryonic Stem Cells (ESC) are cells derived from blastocyst, Adult Stem Cells (ASC) refer to any cell found in a developed organism that has the ability to divide and create another cell like itself or even to create a cell more differentiated than itself, and Induced Pluripotent Stem Cells (iPSC) are repro- grammed somatic cells with pluripotent capabilities. Several types of adult stem cells have been isolated from teeth, including Stem Cells from Human Exfoliated Deciduous Teeth (SHED), Periodontal Ligament Stem Cells (PDLSC), Dental Follicle Precursor Cells (DFPC), Stem Cells from Apical Papilla (SCAP) and Dental Pulp Stem Cells (DPSC). These post-natal populations have mesenqui-mal-like qualities such as the capacity for self-renewal and the potential to dif- ferentiate into multiple tissues including adipose, bone, endothelial and neural-like tissue. Dental Pulp Pluripotent-like Stem Cells (DPPSC) are also isolated from the dental pulp of the third molars, express pluripotency markers, and show embryonic-like behaviour differentiating into tissues of the three embryonic layers. Mesoderm-derived cell types are osteogenic cells, chondrogenic cells, adipogenic cells, skeletal muscle cells, smooth muscle cells, cardiac muscle cells and endo- thelial cells. To date, there exist two commonly used methods to induce vascular cell differentiation from human pluripotent stem cells: Embryoid body (EB) for- mation and monolayer-directed differentiation. The two major cellular compo- nents of blood vessels are Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC). A better understanding of the cellular and molecular mechanisms that control VSMC differentiation is essential to help develop new approaches to both prevent and treat several related diseases. Another important mesoderm-derived tissue for regenerative medicine is skeletal muscle, which is responsible for the voluntary movement of the body. Many diseases that affect the muscula- ture belong to the group of muscular dystrophies (MD). Development of reliable and reproducible in vitro cellular models to study these tissues is needed, yet it has been problematic due to intrinsic peculiarities of them." "The Myotube Analyzer: how to assess myogenic features in muscle stem cells" "Marlies Corvelyn, Domiziana Costamagna, Jean-Marie Aerts, Anja Van Campenhout, Kaat Desloovere" "BACKGROUND: The analysis of in vitro cultures of human adult muscle stem cells obtained from biopsies delineates the potential of skeletal muscles and may help to understand altered muscle morphology in patients. In these analyses, the fusion index is a commonly used quantitative metric to assess the myogenic potency of the muscle stem cells. Since the fusion index only partly describes myogenic potency, we developed the Myotube Analyzer tool, which combines the definition of the fusion index with extra features of myonuclei and myotubes obtained from satellite cell cultures. RESULTS: The software contains image adjustment and mask editing functions for preprocessing and semi-automatic segmentation, while other functions can be used to determine the features of nuclei and myotubes. The fusion index and a set of five novel parameters were tested for reliability and validity in a comparison between satellite cell cultures from children with cerebral palsy and typically developing children. These novel parameters quantified extra nucleus and myotube properties and can be used to describe nucleus clustering and myotube shape. Two analyzers who were trained in cell culture defined all parameters using the Myotube Analyzer app. Out of the six parameters, five had good reliability reflected by good intra-class correlation coefficients (> 0.75). Children with cerebral palsy were significantly different from the typically developing children (p " "Botulinum Toxin Treatment of Adult Muscle Stem Cells from Children with Cerebral Palsy and hiPSC-Derived Neuromuscular Junctions" "Domiziana Costamagna, Marlies Corvelyn, Robin Duelen, Jorieke Deschrevel, Nathalie De Beukelaer, Maurilio Sampaolesi, Ghislaine Gayan-Ramirez, Anja Van Campenhout" "Botulinum neurotoxin type-A (BoNT) injections are commonly used as spasticity treatment in cerebral palsy (CP). Despite improved clinical outcomes, concerns regarding harmful effects on muscle morphology have been raised, and the BoNT effect on muscle stem cells remains not well defined. This study aims at clarifying the impact of BoNT on growing muscles (1) by analyzing the in vitro effect of BoNT on satellite cell (SC)-derived myoblasts and fibroblasts obtained from medial gastrocnemius microbiopsies collected in young BoNT-naïve children (t0) compared to age ranged typically developing children; (2) by following the effect of in vivo BoNT administration on these cells obtained from the same children with CP at 3 (t1) and 6 (t2) months post BoNT; (3) by determining the direct effect of a single and repeated in vitro BoNT treatment on neuromuscular junctions (NMJs) differentiated from hiPSCs. In vitro BoNT did not affect myogenic differentiation or collagen production. The fusion index significantly decreased in CP at t2 compared to t0. In NMJ cocultures, BoNT treatment caused axonal swelling and fragmentation. Repeated treatments impaired the autophagic-lysosomal system. Further studies are warranted to understand the long-term and collateral effects of BoNT in the muscles of children with CP." "Adult stem cells and skeletal muscle regeneration" "Domiziana Costamagna, Gabriele Ceccarelli, Maurilio Sampaolesi" "Satellite cells are unipotent stem cells involved in muscle regeneration. However, the skeletal muscle microenvironment exerts a dominant influence over stem cell function. The cell intrinsic complexity of the skeletal muscle niche located within the connective tissue between fibres includes motor neurons, tendons, blood vessels, immune response mediators and interstitial cells. All these cell types modulate the trafficking of stimuli responsible of muscle fibre regeneration. In addition, several types of stem cell have been discovered in skeletal muscle tissue, mainly located in the interstitium. The majority of these stem cells appear to directly contribute to myogenic differentiation, although some are mainly implicated in paracrine effects. This review focuses on one of these classes of stem cells known as adult stem cells, which following their identification in the last decade have been used for therapeutic purposes, mainly in animal models of chronic muscle degeneration. Emerging literature identifies other myogenic progenitors generated from pluripotent stem cells as potential candidates for the treatment of skeletal muscle degeneration. However, adult stem cells still represent the gold standard for future comparative studies." "Adult stem cells and skeletal muscle regeneration" "Domiziana Costamagna, Emanuele Berardi, Maurilio Sampaolesi" "Satellite cells are unipotent stem cells involved in muscle regeneration. However, the skeletal muscle microenvironment exerts a dominant influence over stem cell function. The cell intrinsic complexity of the skeletal muscle niche located within the connective tissue between fibres includes motor neurons, tendons, blood vessels, immune response mediators and interstitial cells. All these cell types modulate the trafficking of stimuli responsible of muscle fibre regeneration. In addition, several types of stem cell have been discovered in skeletal muscle tissue, mainly located in the interstitium. The majority of these stem cells appear to directly contribute to myogenic differentiation, although some are mainly implicated in paracrine effects. This review focuses on one of these classes of stem cells known as adult stem cells, which following their identification in the last decade have been used for therapeutic purposes, mainly in animal models of chronic muscle degeneration. Emerging literature identifies other myogenic progenitors generated from pluripotent stem cells as potential candidates for the treatment of skeletal muscle degeneration. However, adult stem cells still represent the gold standard for future comparative studies." "Human Amniotic Fluid Stem Cells Modulate Muscle Regeneration After Cardiotoxin Injury in Mice" "Silvia Zia, Mattia Quattrocelli, Ester Di Flippo, Francesca Bosisio, Maurilio Sampaolesi, Jan Deprest, Jaan Toelen" "Amniotic fluid stem cells (AFSc) are a very heterogeneous subtype of stem cells with a broad multi potential. They could be used to treat congenital malformations or diseases. Recently, mesoangioblasts, resident pericytes of skeletal muscles, were shown to undergo muscle differentiation in vitro and in vivo. In this study we focused on the identification of an AFS subtype with pericytic characteristics and evaluate its myogenic potential. We identified monoclonal AFSc lines expressing alkaline phosphatase activity (ALP) and the canonical pericytic markers neural-glial-2 chondroitin sulphate proteglycan (NG2), platelet derived growth factor receptor α and β (PDGFR-α, -β) and α smooth muscle actin (α-SMA). These cells were able to integrate into the newly formed myotubes when co-cultured with the C2C12 cells. To test the paracrine effects of these AFSC on muscle regeneration, we assessed their affects in a transwell assay with acutely injured myotubes. AFSc were able to modulate the expression of specific growth factors involved in muscle regeneration, such as Transforming Growth Factor β (Tgfβ), Interferon γ (Ifnγ), Hepatocyte Growth Factor (Hgf) and Matrix Metalloproteinase 2 (Mmp2). When AFSc were injected in injured muscles they ameliorated muscle repair as measured by the reduction of centronucleated fibers and fibrosis. Interestingly, the transcriptional program of growth factor response in vitro is observed in large part in the in vivo xenograft experimental model, with the extension of Myostatin and Matrix Metalloproteinase 9 (Mmp9). Our data suggest that AFSc subtype with pericytic characteristics have the ability to modulate muscle regeneration in vitro and in vivo." "Human dental pulp pluripotent-like stem cells promote wound healing and muscle regeneration" "Domiziana Costamagna, Alessio Rotini, Aernout Luttun, Maurilio Sampaolesi" "BACKGROUND: Dental pulp represents an easily accessible autologous source of adult stem cells. A subset of these cells, named dental pulp pluripotent-like stem cells (DPPSC), shows high plasticity and can undergo multiple population doublings, making DPPSC an appealing tool for tissue repair or maintenance. METHODS: DPPSC were harvested from the dental pulp of third molars extracted from young patients. Growth factors released by DPPSC were analysed using antibody arrays. Cells were cultured in specific differentiation media and their endothelial, smooth and skeletal muscle differentiation potential was evaluated. The therapeutic potential of DPPSC was tested in a wound healing mouse model and in two genetic mouse models of muscular dystrophy (Scid/mdx and Sgcb-null Rag2-null γc-null). RESULTS: DPPSC secreted several growth factors involved in angiogenesis and extracellular matrix deposition and improved vascularisation in all three murine models. Moreover, DPPSC stimulated re-epithelialisation and ameliorated collagen deposition and organisation in healing wounds. In dystrophic mice, DPPSC engrafted in the skeletal muscle of both dystrophic murine models and showed integration in muscular fibres and vessels. In addition, DPPSC treatment resulted in reduced fibrosis and collagen content, larger cross-sectional area of type II fast-glycolytic fibres and infiltration of higher numbers of proangiogenic CD206+ macrophages. CONCLUSIONS: Overall, DPPSC represent a potential source of stem cells to enhance the wound healing process and slow down dystrophic muscle degeneration."