Human MuStem cells are competent to fuse with nonhuman primate myofibers in a clinically relevant transplantation context: A proof-of-concept study.

We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.

Human muscle stem cell responses to mechanical stress into tunable 3D alginate matrices.

Preclinical data acquired for human muscle stem (hMuStem) cells indicate their great repair capacity in the context of muscle injury. However, their clinical potential is limited by their moderate ability to survive after transplantation. To overcome these limitations, their encapsulation within protective environment would be beneficial. In this study, tunable calcium-alginate hydrogels obtained through molding method using external or internal gelation were investigated as a new strategy for hMuStem cell encapsulation. The mechanical properties of these hydrogels were characterized in their fully hydrated state by compression experiments using Atomic Force Microscopy. Measured elastic moduli strongly depended on the gelation mode and calcium/alginate concentrations. Values ranged from 1 to 12.5 kPa and 3.9 to 25 kPa were obtained for hydrogels prepared following internal and external gelation, respectively. Also, differences in mechanical properties of hydrogels resulted from their internal organization, with an isotropic structure for internal gelation, while external mode led to anisotropic one. It was further shown that viability, morphological and myogenic differentiation characteristics of hMuStem cells incorporated within alginate hydrogels were preserved after their release. These results highlight that hMuStem cells encapsulated in calcium-alginate hydrogels maintain their functionality, thus allowing to develop muscle regeneration protocols to improve their therapeutic efficacy.

Specific and label-free endogenous signature of dystrophic muscle by Synchrotron deep ultraviolet radiation.

Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.

Human MuStem cells repress T-cell proliferation and cytotoxicity through both paracrine and contact-dependent pathways.

BACKGROUND: Muscular dystrophies (MDs) are inherited diseases in which a dysregulation of the immune response exacerbates disease severity and are characterized by infiltration of various immune cell types leading to muscle inflammation, fiber necrosis and fibrosis. Immunosuppressive properties have been attributed to mesenchymal stem cells (MSCs) that regulate the phenotype and function of different immune cells. However, such properties were poorly considered until now for adult stem cells with myogenic potential and advanced as possible therapeutic candidates for MDs. In the present study, we investigated the immunoregulatory potential of human MuStem (hMuStem) cells, for which we previously demonstrated that they can survive in injured muscle and robustly counteract adverse tissue remodeling. METHODS: The impact of hMuStem cells or their secretome on the proliferative and phenotypic properties of T-cells was explored by co-culture experiments with either peripheral blood mononucleated cells or CD3-sorted T-cells. A comparative study was produced with the bone marrow (BM)-MSCs. The expression profile of immune cell-related markers on hMuStem cells was determined by flow cytometry while their secretory profile was examined by ELISA assays. Finally, the paracrine and cell contact-dependent effects of hMuStem cells on the T-cell-mediated cytotoxic response were analyzed through IFN-γ expression and lysis activity. RESULTS: Here, we show that hMuStem cells have an immunosuppressive phenotype and can inhibit the proliferation and the cytotoxic response of T-cells as well as promote the generation of regulatory T-cells through direct contact and via soluble factors. These effects are associated, in part, with the production of mediators including heme-oxygenase-1, leukemia inhibitory factor and intracellular cell adhesion molecule-1, all of which are produced at significantly higher levels by hMuStem cells than BM-MSCs. While the production of prostaglandin E2 is involved in the suppression of T-cell proliferation by both hMuStem cells and BM-MSCs, the participation of inducible nitric oxide synthase activity appears to be specific to hMuStem cell-mediated one. CONCLUSIONS: Together, our findings demonstrate that hMuStem cells are potent immunoregulatory cells. Combined with their myogenic potential, the attribution of these properties reinforces the positioning of hMuStem cells as candidate therapeutic agents for the treatment of MDs.

3D in vitro models of skeletal muscle: myopshere, myobundle and bioprinted muscle construct.

Typical two-dimensional (2D) culture models of skeletal muscle-derived cells cannot fully recapitulate the organization and function of living muscle tissues, restricting their usefulness in in-depth physiological studies. The development of functional 3D culture models offers a major opportunity to mimic the living tissues and to model muscle diseases. In this respect, this new type of in vitro model significantly increases our understanding of the involvement of the different cell types present in the formation of skeletal muscle and their interactions, as well as the modalities of response of a pathological muscle to new therapies. This second point could lead to the identification of effective treatments. Here, we report the significant progresses that have been made the last years to engineer muscle tissue-like structures, providing useful tools to investigate the behavior of resident cells. Specifically, we interest in the development of myopshere- and myobundle-based systems as well as the bioprinting constructs. The electrical/mechanical stimulation protocols and the co-culture systems developed to improve tissue maturation process and functionalities are presented. The formation of these biomimetic engineered muscle tissues represents a new platform to study skeletal muscle function and spatial organization in large number of physiological and pathological contexts.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

BACKGROUND: Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). METHODS: hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. RESULTS: HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. CONCLUSIONS: Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle.

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Multi-harmonic Imaging in the Second Near-Infrared Window of Nanoparticle-Labeled Stem Cells as a Monitoring Tool in Tissue Depth.

In order to assess the therapeutic potential of cell-based strategies, it is of paramount importance to elaborate and validate tools for monitoring the behavior of injected cells in terms of tissue dissemination and engraftment properties. Here, we apply bismuth ferrite harmonic nanoparticles (BFO HNPs) to in vitro expanded human skeletal muscle-derived stem cells (hMuStem cells), an attractive therapeutic avenue for patients suffering from Duchenne muscular dystrophy (DMD). We demonstrate the possibility of stem cell labeling with HNPs. We also show that the simultaneous acquisition of second- and third-harmonic generation (SHG and THG) from BFO HNPs helps separate their response from tissue background, with a net increase in imaging selectivity, which could be particularly important in pathologic context that is defined by a highly remodelling tissue. We demonstrate the possibility of identifying <100 nm HNPs in depth of muscle tissue at more than 1 mm from the surface, taking full advantage of the extended imaging penetration depth allowed by multiphoton microscopy in the second near-infrared window (NIR-II). Based on this successful assessment, we monitor over 14 days any modification on proliferation and morphology features of hMuStem cells upon exposure to PEG-coated BFO HNPs at different concentrations, revealing their high biocompatibility. Successively, we succeed in detecting individual HNP-labeled hMuStem cells in skeletal muscle tissue after their intramuscular injection.