Adult stem cells named MuStem : phenotype, myogenicity, immunomodulation and immunological context of in vivo delivery

La dystrophie musculaire de Duchenne (DMD) est une pathologie récessive liée au chromosome X qui résulte d’une mutation sur le gène de la dystrophine aboutissant à l’absence complète de la protéine. Elle correspond à la plus fréquente des dystrophies musculaires et reste aujourd’hui sans traitement curatif. L’UMR a fait la preuve de concept de l’administration systémique d’une population de cellules souches adultes résidentes du muscle, les cellules MuStem canines chez le chien dystrophinopathe, modèle cliniquement pertinent de la DMD. L’objectif de la thèse a consisté à caractériser la population humaine (hMuStem) en terme de phénotype, myogénicité, immunomodulation et de contexte immunologique d’administration in vivo. La population hMuStem se compose de progéniteurs myogéniques précoces d’origine mésenchymateuse-périvasculaire. Elle se définit par une forte capacité proliférative, une oligopotence et une participation à la régénération musculaire après administration dans un muscle lésé. Elles présentent des propriétés immunomodulatrices en interagissant avec l’immunité adaptative et innée par inhibition de la prolifération lymphocytaire et du complément via un ensemble de molécules de surface et/ou de facteurs sécrétés. Enfin, un traitement immunosuppressif restreint à la période d’injection in vivo de la population allogénique s’avère nécessaire mais suffisant pour éviter une réaction immune de l’hôte. L’ensemble de ces résultats aboutit à une meilleure compréhension de l’identité et des modalités d’action de la population MuStem.Duchenne Muscular Dystrophy is a X-linked recessive disorder that results from mutation in the dystrophin gene leading to a total lack of the protein. It is the most frequent muscular dystrophy with no curative treatment. The lab made a proof of concept of the systemic delivery of a muscle-derived adult stem cell population called MuStem cells in dystrophic dog, the clinically relevant DMD model. The aim of my Ph.D. was to characterize the human population (hMuStem) in terms of phenotype, myogenicity, immunomodulation and immunological context of in vivo delivery. hMuStem cell population is composed of myogenic progenitors with mesenchymal/perivascular imprint. It exhibits a high proliferative capacity, an oligopotency and a participation to muscle regeneration after transplantation into injured muscle. It displays immunomodulatory properties by interacting with adaptive and innate immunity with inhibition of lymphocyte proliferation and complement thanks to expression of surface molecules and/or secreted factors. At last, an immunosuppressive regimen restricted to the allogeneic injection period is necessary but sufficient to avoid host immune response. Collectively, these results allow a better understanding of identity and action modalities of MuStem cell population

Les exosomes, des messagers intercellulaires naturels aux mécanismes polyvalents pour le traitement des myopathies?

International audienceDans la souris mdx, l’injection d’exosomes permet d’améliorer les phénotypes pathologiques musculaires, cardiaques et squelettiques, et ce grâce à plusieurs mécanismes non exclusifs. Les exosomes, qui font partie des vésicules extracellulaires, mesurent une centaine de nanomètres et sont produits par de très nombreux types cellulaires. Leurs marqueurs de surface et leurs contenus sont variables en fonction des cellules productrices et des conditions environnementales. Libérés par exocytose, les exosomes sont ensuite internalisés par endocytose ou fusion membranaire. Ils présentent une action pléiotropique et jouent un rôle primordial de messagers intercellulaires en conditions normales et pathologiques. Dans la souris mdx, l’absence de dystrophine entraîne une perte de l’intégrité membranaire, un déséquilibre de l’homéostasie, une dégénérescence progressive et une inflammation musculaire, le tout se traduisant par une baisse de force, une fatigabilité, et le développement d’une insuffisance cardiaque.Dans une première étude [1], l’injection intrapéritonéale d’exosomes produits à partir de cellules souches mésenchymateuses humaines et murines, de cellules dendritiques immortalisées murines, de myotubes murins, ou d’extraits de sérum murin, permet de limiter l’afflux de calcium intracellulaire, de diminuer l’activation de protéases, et de stabiliser les complexes existants de protéines associées à la dystrophine (sarcoglycans, dystroglycans). En restaurant partiellement la myo-architecture et l’intégrité membranaire des cellules musculaires squelettiques des souris mdx, les exosomes bloquent l’influx de marqueurs exogènes (tel le colorant bleu Evans) et la fuite de protéines endogènes (comme les CPK et la LDH). Leur activité immunomodulatrice diminue l’infiltration leucocytaire et l’inflammation, et réduit l’expansion ultérieure de la fibrose. Ces effets sur des cibles multiples se traduisent par une augmentation de la force et de l’endurance des souris.Dans une autre étude sur le même sujet [2], une injection intraveineuse unique d’exosomes produits par des cellules dérivées de progéniteurs cardiaques (cardiosphères) améliore les fonctions musculaires cardiaques et squelettiques des souris mdx. Les exosomes font régresser les lésions cardiaques, augmentent la fraction d’éjection myocardique, améliorant la capacité maximale d’exercice et diminuant la fibrose myocardique. Ils stimulent la régénération musculaire squelettique, augmentent la prolifération des cellules satellites, diminuent les réponses inflammatoires, et augmentent in fine la force isométrique des muscles squelettiques. Les bénéfices sont liés aux modifications transcriptomiques exercées par les micro-ARN (miR) contenus dans les exosomes, en particulier miR148a, ainsi qu’aux mécanismes d’immunomodulation. Les exosomes sont aussi efficaces que les cellules productrices elles-mêmes et constituent probablement leur principal mécanisme d’action

Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion of human bone marrow mesenchymal stem cells

Background We recently developed and characterized a standardized and clinical grade human Platelet Lysate (hPL) that constitutes an advantageous substitute for fetal bovine serum (FBS) for human mesenchymal stem cell (hMSC) expansion required in cell therapy procedures, avoiding xenogenic risks (virological and immunological) and ethical issues. Because of the progressive use of pathogen-reduced (PR) labile blood components, and the requirement of ensuring the viral safety of raw materials for cell therapy products, we evaluated the impact of the novel procedure known as THERAFLEX UV-Platelets for pathogen reduction on hPL quality (growth factors content) and efficacy (as a medium supplement for hMSC expansion). This technology is based on short-wave ultraviolet light (UV-C) that induces non-reversible damages in DNA and RNA of pathogens while preserving protein structures and functions, and has the main advantage of not needing the addition of any photosensitizing additives (that might secondarily interfere with hMSCs). Methodology / Principal findings We applied the THERAFLEX UV-Platelets procedure on fresh platelet concentrates (PCs) suspended in platelet additive solution and prepared hPL from these treated PCs. We compared the quality and efficacy of PR-hPL with the corresponding non-PR ones. We found no impact on the content of five cytokines tested (EGF, bFGF, PDGF-AB, VEGF and IGF-1) but a significant decrease in TGF-beta 1 (-21%, n = 11, p<0.01). We performed large-scale culture of hMSCs from bone marrow (BM) during three passages and showed that hPL or PR-hPL at 8% triggered comparable BM-hMSC proliferation as FBS at 10% plus bFGF. Moreover, after proliferation of hMSCs in an hPL- or PR-hPL-containing medium, their profile of membrane marker expression, their clonogenic potential and immunosuppressive properties were maintained, in comparison with BM-hMSCs cultured under FBS conditions. The potential to differentiate towards the adipogenic and osteogenic lineages of hMSCs cultured in parallel in the three conditions also remained identical. Conclusion / Significance We demonstrated the feasibility of using UV-C-treated platelets to subsequently obtain pathogen-reduced hPL, while preserving its optimal quality and efficacy for hMSC expansion in cell therapy applications

Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion pf human bone marrow mesenchymal stem cells

Pathogen reduction through additive-free short-wave UV light irradiation retains the optimal efficacy of human platelet lysate for the expansion pf human bone marrow mesenchymal stem cells. 43rd Annual Meeting of EBM

A standardized and characterized clinical grade human platelet lysate for optimized expansion of human bone marrow mesenchymal stem cells

A standardized and characterized clinical grade human platelet lysate for optimized expansion of human bone marrow mesenchymal stem cells. 43rd Annual Meeting of the European Society for Blood and Marrow Transplantation (EBMT

Human MuStem cells, a promising therapeutic candidate for muscular dystrophies with immunomodulatory properties

Nowadays, allogeneic cell-based therapeutic approaches for regenerative medecine are limited by graft rejection. To counteract this major deleterious effect, immunosuppressive regimens are developed and given to patients, improving their lifespan but causing in return severe adverse effects. Over the last years, a number of adult stem cell populations including mesenchymal stem cells (MSC) and vessel-associated stem cells were described for the treatment of genetic muscle diseases. Those cells were shown to display immunomodulatory properties by acting, directly or through the secretion of soluble factors, on a large number of immune cell partners (Cossu et al., 2012; English et al., 2013). Duchenne Muscular Dystrophy (DMD) is a degenerative muscle disease characterized notably by an inflammatory component that negatively impacts on muscle regeneration activity. In this context, these original immune features attributed to stem cells could be a great advantage to improve cell engraftment and efficiency. In the laboratory, we have isolated a population of muscle-derived stem cells from healthy dog muscle tissue, called cMuStem cells, and made the proof of concept of their systemic delivery efficiency in the preclinical GRMD canine model of DMD (Rouger et al, 2011; Robriquet et al, 2015). Recently, we managed to isolate the same population from Paravertebralis muscle of 9 to 15-years old patients free of known muscle disease (hMuStem cells). It was defined as a mixed population composed of both myogenic progenitors and mesenchymal perivascular cells, and characterized by a large proliferation rate, an oligopotency as well as an in vivo myogenic regenerative potential. The aim of the study was to determine whether hMuStem cells also exhibit immunomodulatory properties. Interestingly, our preliminary data show the ability of hMuStem cells from different donors to modulate allogeneic T cell proliferation and to secrete various immunomodulatory molecules (prostaglandin-E2, indoleamin-2,3-deoxygenase-1, heme oxygenase-1 and inducible nitric-oxide synthase-1). Our data also suggest that hMuStem cells are able to interact with the complement system by inhibiting complement-mediated lysis of erythrocytes. This effect seems to be mediated by factor H, an alternative inhibitory complement pathway. Overall, our study is critical for the understanding of the interaction between MuStem cells and the immune system, as well as the design of safe and efficient allogeneic stem cell-based therapy for the treatment of muscular dystrophies

Immunomodulatory properties of human MuStem cells: assessing their impact on adaptive and innate immunity

Several preclinical approaches based on allogeneic stem cell delivery were shown to be attractive for the treatment of genetic muscular dystrophies. Nevertheless, a significant hurdle for their clinical translation is the immune rejection of donor cells. Immunosuppressive regimens are generally used to overcome host immunity and can allow the improvement of graft survival. Nevertheless, they are associated with a number of side effects, limiting their long term use. Recently, some tissue-specific adult stem cell populations were described to exhibit immunomodulatory properties that could increase their ability to engraft in an allogeneic recipient and improve their regenerative potential. We have previously demonstrated that allogeneic muscle-derived delayed adherent stem cells (that we called MuStem cells) are able to phenotypically and clin- ically correct the Duchenne dystrophic canine model (Rouger et al., 2011; Robriquet et al., 2015). Recently, we isolated human MuStem cells and assessed their immunomodulatory potential. We evaluated their ability to inhibit T cell prolif- eration and to modulate the complement pathway. Interest- ingly, our preliminary data showed that human MuStem cells were able to modulate allogeneic T cell proliferation and to express immunomodulatory molecules such as prostaglandin- E2, indoleamin-2,3-deoxygenase-1 and TGFb2. Moreover, MuStem cells were also able to secrete Factor H molecule suggesting a potential effect on the alternate pathway of the complement system. Overall, our study is critical for the un- derstanding of the crosstalk between MuStem cells and the im- mune system, as well as the design of safe and efficient allogeneic stem cell-based therapy for the treatment of muscle dystrophie

Proliferation of BM-hMSCs cultured in an FBS-containing medium.

<p>Eight references of FBS were tested at a dose of 10% with 1 ng/mL bFGF. Results are presented as amplification yields for means of triplicates. NS: <i>not significant</i>; ***: <i>p<0</i>.<i>001 versus</i> FBS 3 (one-way ANOVA and Bonferroni posttests).</p

Representative histogram overlays for expression of CD13 (a), CD44 (b), CD73 (c), CD90 (d), CD105 (e), CD34 (f), CD45 (g), HLA-DR (h), CD40 (i), CD80 (j) and CD86 (k) of BM-hMSCs cultured in an hPL- (red curves <i>versus</i> isotype controls in black) or PR-hPL-containing medium (green curves <i>versus</i> isotype controls in blue).

<p>Representative histogram overlays for expression of CD13 (a), CD44 (b), CD73 (c), CD90 (d), CD105 (e), CD34 (f), CD45 (g), HLA-DR (h), CD40 (i), CD80 (j) and CD86 (k) of BM-hMSCs cultured in an hPL- (red curves <i>versus</i> isotype controls in black) or PR-hPL-containing medium (green curves <i>versus</i> isotype controls in blue).</p