Mesenchymal Stromal Cells (MSC) possess innate immunomodulatory properties, which can be significantly enhanced through co-culture with peripheral blood mononuclear cells (PBMC), making them attractive tools for the treatment of autoimmune and inflammatory diseases. Leveraging a multi-omics approach encompassing RNA sequencing, flow and mass cytometry, secretome analysis, completed by functional evaluations, we investigated the mechanisms underpinning PBMC conditioning of MSC in vitro and their benefits in an animal model of Myasthenia gravis. MSC derived from human adipose tissue were left untreated in resting state (rMSC), conditioned by PBMC (cMSC), or activated by the pro-inflammatory molecule interferon (IFN)-γ (γMSC), then compared for their gene expression profiles, phenotypes and functional capacities. RNA sequencing identified 244 differentially expressed genes in cMSC compared to rMSC, highlighting key immune mediators such as CCL2, CCL11, DPP4, ICAM1, IL6, PDCD1LG2, TNFRSF11B, TNIP1, TNIP3 and ZC3H12A and pinpointing genes involved in matrix remodeling, paracrine and autocrine communications. Comparatively, 2089 genes were differentially expressed between rMSC and γMSC, highlighting host defense, anti-viral response, NFκB signaling pathways modulated by IFN-γ. Flow and mass cytometry analyses revealed upregulation of the surface markers CD26, CD54, and CD273 and intracellular molecules IDO1 and PTGS2 in cMSC. In contrast, IFN-γ activation predominantly increased HLA-related markers while also enhancing the homogeneity of the populations. Together, these results underlined the treatment dependence of transcriptomic and phenotypic signatures. Secretome profiling identified 6 categories of modulated proteins, out of which 22 molecules potentially involved in PBMC conditioning and 40 implicated in cMSC-mediated immunomodulation. Functionally, cMSC induced modulation in PBMC subsets, raising the proportions of lymphocyte populations (CD4 Treg, CD8, B memory), underlining the multimodal effect of conditioning. Also, both a direct cell-cell contact and cMSC supernatants significantly suppressed activated T-cell proliferation in vitro. To confirm immunomodulation efficacy in vivo, cMSC were administrated to our humanized mouse model of Myasthenia Gravis and the treatment significantly halved disease severity from 2 weeks post-injection. This integrative study establishes distinct conditioning signatures, suggests molecular mechanisms, and underscores the therapeutic potential of cMSC, offering a robust framework for advancing cell-based therapies in autoimmune diseases.

Myasthenia gravis (MG) is an autoimmune disorder primarily caused by autoantibodies that target the acetylcholine receptor (AChR) at the neuromuscular junction (NMJ). The classical experimental autoimmune myasthenia gravis (C-EAMG) mouse model has long been used by immunizing mice with acetylcholine receptor from Torpedo fish (T-AChR), combined with complete Freund’s adjuvant (CFA). This mixture is administered via subcutaneous injections into the hind footpads and back, but CFA often causes strong inflammatory reactions, including lesions at the injection sites. Our objective was to develop a new EAMG model (N-EAMG) that is more compliant with animal welfare. C57Bl/6 mice were immunized twice weekly by intraperitoneal (i.p.) injection of T-AChR with a poly(I:C) and lipopolysaccharide (LPS) adjuvant mix. Control mice were injected with either physiological saline or the adjuvant mix alone. Various doses and injection schedules were tested, and the new model was compared with C-EAMG. Clinical symptoms were scored, antibody subtypes against T-AChR and mouse AChR were measured, and NMJ morphology and functionality were evaluated. We demonstrate that the N-EAMG model is at least as effective as the C-EAMG model. Moreover, similar to the C-EAMG model, the N-EAMG model is characterized by the production of T-AChR and m-AChR antibodies. This model also exhibited alterations in transmission at the NMJ due to antibody attack, resulting in a decrease in AChR surface area and increased AChR fragmentation. Symptoms were similar in both models but appeared more rapidly in the N-EAMG model. In addition, investigating the sensitization mechanism, we showed that i.p. injections of T-AChR with the poly(I:C)/LPS adjuvant mix, led to the recruitment in monocytes and changes in the two peritoneal macrophage subpopulations that were able to phagocytose T-AChR. These observations suggest that macrophage subtypes, albeit with varying efficiency, present the T-AChR to immune cells, leading to a specific immune response and the development of anti-AChR antibodies. In conclusion, our results demonstrate that this novel EAMG model is as effective as the C-EAMG model and offers several advantages. In particular, this model is more suitable for animal welfare and can replace the classical model in preclinical and fundamental research.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle fatigability due to acetylcholine receptor (AChR) autoantibodies. To better characterize juvenile MG (JMG), we analyzed 85 pre- and 132 post-pubescent JMG (with a cutoff age of 13) compared to 721 adult MG patients under 40 years old using a French database. Clinical data, anti-AChR antibody titers, thymectomy, and thymic histology were analyzed. The proportion of females was higher in each subgroup. No significant difference in the anti-AChR titers was observed. Interestingly, the proportion of AChR+ MG patients was notably lower among adult MG patients aged between 30 and 40 years, at 69.7%, compared to over 82.4% in the other subgroups. Thymic histological data were examined in patients who underwent thymectomy during the year of MG onset. Notably, in pre-JMG, the percentage of thymectomized patients was significantly lower (32.9% compared to more than 42.5% in other subgroups), and the delay to thymectomy was twice as long. We found a positive correlation between anti-AChR antibodies and germinal center grade across patient categories. Additionally, only females, particularly post-JMG patients, exhibited the highest rates of lymphofollicular hyperplasia (95% of cases) and germinal center grade. These findings reveal distinct patterns in JMG patients, particularly regarding thymic follicular hyperplasia, which appears to be exacerbated in females after puberty.

Culture-adapted human mesenchymal stromal cells (hMSCs) are appealing candidates for regenerative medicine applications. However, these cells implanted in lesions as single cells or tissue constructs encounter an ischemic microenvironment responsible for their massive death post-transplantation, a major roadblock to successful clinical therapies. We hereby propose a paradigm shift for enhancing hMSC survival by designing, developing, and testing an enzyme-controlled, nutritive hydrogel with an inbuilt glucose delivery system for the first time. This hydrogel, composed of fibrin, starch (a polymer of glucose), and amyloglucosidase (AMG, an enzyme that hydrolyze glucose from starch), provides physiological glucose levels to fuel hMSCs via glycolysis. hMSCs loaded in these hydrogels and exposed to near anoxia (0.1% pO 2 ) in vitro exhibited improved cell viability and angioinductive functions for up to 14 days. Most importantly, these nutritive hydrogels promoted hMSC viability and paracrine functions when implanted ectopically. Our findings suggest that local glucose delivery via the proposed nutritive hydrogel can be an efficient approach to improve hMSC-based therapeutic efficacy.

Stem cell-based therapies are a promising approach for the treatment of degenerative muscular diseases; however, clinical trials have shown inconclusive and even disappointing results so far. Noninvasive cell monitoring by medicine imaging could improve the understanding of the survival and biodistribution of cells following injection. In this study, we assessed the canine sodium iodide symporter (cNIS) reporter gene as an imaging tool to track by single-photon emission computed tomography (SPECT/CT) transduced canine myoblasts after intramuscular (IM) administrations in dogs. cNIS-expressing cells kept their myogenic capacities and showed strong (99) Tc-m-pertechnetate ((TcO4-)-Tc-99 m) uptake efficiency both in vitro and in vivo. cNIS expression allowed visualization of cells by SPECT/CT along time: 4 h, 48 h, 7 days, and 30 days after IM injection; biopsies collected 30 days post administration showed myofiber's membranes expressing cNIS. This study demonstrates that NIS can be used as a reporter to track cells in vivo in the skeletal muscle of large animals. Our results set a proof of concept of the benefits NIS-tracking tool may bring to the already challenging cell-based therapies arena in myopathies and pave the way to a more efficient translation to the clinical setting from more accurate pre-clinical results.

One of the main challenges in cell therapy for muscle diseases is to efficiently target the muscle. To address this issue and achieve better understanding of in vivo cell fate, we evaluated the relevance of a non-invasive cell tracking method in the Golden Retriever Muscular Dystrophy (GRMD) model, a well-recognised model of Duchenne Muscular Dystrophy (DMD). Mesoangioblasts were directly labelled with 111In-oxine, and injected through one of the femoral arteries. The scintigraphy images obtained provided the first quantitative mapping of the immediate biodistribution of mesoangioblasts in a large animal model of DMD. The results revealed that cells were trapped by the first capillary filters: the injected limb and the lung. During the days following injection, radioactivity was redistributed to the liver. In vitro studies, performed with the same cells prepared for injecting the animal, revealed prominent cell death and 111In release. In vivo, cell death resulted in 111In release into the vasculature that was taken up by the liver, resulting in a non-specific and non-cell-bound radioactive signal. Indirect labelling methods would be an attractive alternative to track cells on the mid- and long-term.

Several autoimmune diseases are mediated by antibodies produced after deregulations of the immune system and directed against self antigens. Myasthenia Gravis (MG) is a rare disease in which pathogenicity is due to autoantibodies directed against the neuromuscular endplate. MG is not really cured, corticosteroids and azathioprin are commonly used, however they trigger severe side-effects, mandating the setup of novel therapies. Mesenchymal Stromal/Stem Cells (MSC) are multipotent progenitor cells that can be isolated from various human tissues and can modulate the immune system via soluble mediators and cell-cell contacts. Our team has recently validated a new animal model of MG, in which we demonstrated that the transfer of MSC conditioned by peripheral blood mononucleated cells (PBMC) improved the clinical status of the animals (Sudres et al., JCI Insight 2017). To develop this immunomodulating approach in clinical perspective, we compared the phenotypes of research-grade (RG) and clinical-grade (CG) MSC testing a series of 60 antibodies (Ab) directed against surface antigens by flow cytometry. We evaluated the variations introduced by different conditioning treatments (activation by gamma-interferon, cross-stimulation by PBMC or monocytes). Markers involved in immunomodulation (recognition, activation, function of complement, co-stimulation, immune checkpoints) were increased or decreased depending on treatment. Adhesion molecules and receptors were also differentially modified (integrins, selectins, cell-cell adhesion molecules, growth factor receptors, tetraspanins). These results suggest that the conditioning regimens act through different pathways. From this panel, we derived a second one of 31 Ab allowing simultaneous labeling of MSC at single cell level by mass cytometry (CyTOF). We defined MSC clusters which were modulated upon activation. In parallel, we evaluated the functional activity of resting or conditioned CG MSC through a cell proliferation assay, and through the quantification by ELISA of secreted immunomodulating products. The conditioning regimens differentially modulated the secretion of Prostaglandin E2 and TGFbeta1, and inhibited PBMC proliferation. This work unveiled phenotypic and functional markers of MSC along with their modulations according to different treatments, and will contribute to validate a cell therapy product for immunomodulation purposes.

Cell therapy approaches dedicated to the treatment of dystrophinopathies and involving essentially myoblasts and mesoangioblasts have produced mitigated clinical results. If several types of alternative progenitors have been developed, no standardized comparison has been carried out yet to investigate their regenerative efficacy in vivo, at least at a local level. A comparative study has therefore been designed recently aiming at giving a new impetus to this therapeutic field.

Les approches de thérapie cellulaire des dystrophinopathies basées sur l’utilisation de myoblastes ou de mésoangioblastes se sont traduites par des résultats cliniques mitigés. De nombreux candidats cellulaires alternatifs ont été décrits, mais aucune comparaison standardisée n’a pu encore établir leurs efficacités, ne serait-ce qu’en vue d’une régénération musculaire localisée. Une étude comparative a donc été décidée récemment et pourrait permettre de donner un nouvel élan à cette approche.