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Defective lysosomal acidification is responsible for a large range of multi-systemic disorders associated with impaired autophagy. Diseases caused by mutations in the VMA21 gene stand as exceptions, specifically affecting skeletal muscle (X-linked Myopathy with Excessive Autophagy, XMEA) or liver (Congenital Disorder of Glycosylation). VMA21 chaperones vacuolar (v-) ATPase assembly, which is ubiquitously required for proper lysosomal acidification. The reason VMA21 deficiencies affect specific, but divergent tissues remains unknown. Here, we show that VMA21 encodes a yet-unreported long protein isoform, in addition to the previously described short isoform, which we name VMA21-120 and VMA21-101, respectively. In contrast to the ubiquitous pattern of VMA21-101, VMA21-120 was predominantly expressed in skeletal muscle, and rapidly up-regulated upon differentiation of mouse and human muscle precursors. Accordingly, VMA21-120 accumulated during development, regeneration and denervation of mouse skeletal muscle. In contrast, neither induction nor blockade of autophagy, in vitro and in vivo, strongly affected VMA21 isoform expression. Interestingly, VMA21-101 and VMA21-120 both localized to the sarcoplasmic reticulum of muscle cells, and interacted with the v-ATPase. While VMA21 deficiency impairs autophagy, VMA21-101 or VMA21-120 overexpression had limited impact on autophagic flux in muscle cells. Importantly, XMEA-associated mutations lead to both VMA21-101 deficiency and loss of VMA21-120 expression. These results provide important insights into the clinical diversity of VMA21-related diseases and uncover a muscle-specific VMA21 isoform that potently contributes to XMEA pathogenesis.
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Enfermedades Musculares , ATPasas de Translocación de Protón Vacuolares , Humanos , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Músculo Esquelético/metabolismo , Genes Ligados a X , Autofagia/genéticaRESUMEN
Muscle stem cells (MuSC) are considered as a reliable source of therapeutic cells to restore diseased muscles. However in most cases, injected MuSC-derived myoblasts are rapidly destroyed by the host immune response, which impairs the beneficial effect. By contrast, human mesenchymal stromal cells (MSC), have been reported to exhibit potent immune regulatory functions. Thus, we investigated, in vitro, the multipotent differentiation- and immunosuppressive capacities of human myoblasts and compared these features with those of human MSC. Myoblasts shared numerous cell surface markers with MSC, including CD73, CD90, CD105 and CD146. Both cell type were negative for HLA-DR and CD45, CD34 and CD31. CD56, a myogenic marker, was expressed by myoblasts exclusively. Myoblasts displayed multipotent potential capabilities with differentiation in chondrocytes, adipocytes and osteoblasts in vitro. Myoblasts also inhibited allogenic T cell proliferation in vitro in a dose dependent manner, very similarly to MSC. This effect was partly mediated via the activation of indolamine 2,3 dioxygenase enzyme (IDO) after IFNγ exposure. Altogether, these data demonstrate that human myoblasts can differentiate in various mesenchymal linages and exhibit powerful immunosuppressive properties in vitro. Such features may open new therapeutic strategies for MuSC-derived myoblasts.
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Adipocitos/metabolismo , Proliferación Celular/fisiología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Células Madre Mesenquimatosas/citología , Adulto , Células de la Médula Ósea/citología , Diferenciación Celular/fisiología , Condrocitos/metabolismo , Humanos , Inmunomodulación/inmunología , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Persona de Mediana Edad , Mioblastos/metabolismo , Osteoblastos/metabolismoRESUMEN
Stem cell therapy holds significant potential for skeletal muscle repair, with in vitro-generated human muscle reserve cells (MuRCs) emerging as a source of quiescent myogenic stem cells that can be injected to enhance muscle regeneration. However, the clinical translation of such therapies is hampered by the need for fetal bovine serum (FBS) during the in vitro generation of human MuRCs. This study aimed to determine whether fresh allogeneic human platelet-rich plasma (PRP) combined or not with hyaluronic acid (PRP-HA) could effectively replace xenogeneic FBS for the ex vivo expansion and differentiation of human primary myoblasts. Cells were cultured in media supplemented with either PRP or PRP-HA and their proliferation rate, cytotoxicity and myogenic differentiation potential were compared with those cultured in media supplemented with FBS. The results showed similar proliferation rates among human myoblasts cultured in PRP, PRP-HA or FBS supplemented media, with no cytotoxic effects. Human myoblasts cultured in PRP or PRP-HA showed reduced fusion ability upon differentiation. Nevertheless, we also observed that human MuRCs generated from PRP or PRP-HA myogenic cultures, exhibited increased Pax7 expression and delayed re-entry into the cell cycle upon reactivation, indicating a deeper quiescent state of human MuRCs. These results suggest that allogeneic human PRP effectively replaces FBS for the ex vivo expansion and differentiation of human myoblasts and favors the in vitro generation of Pax7High human MuRCs, with important implications for the advancement of stem cell-based muscle repair strategies.
RESUMEN
BACKGROUND: The capacity of skeletal muscles to regenerate relies on Pax7+ muscle stem cells (MuSC). While in vitro-amplified MuSC are activated and lose part of their regenerative capacity, in vitro-generated human muscle reserve cells (MuRC) are very similar to quiescent MuSC with properties required for their use in cell-based therapies. METHODS: In the present study, we investigated the heterogeneity of human MuRC and characterized their molecular signature and metabolic profile. RESULTS: We observed that Notch signaling is active and essential for the generation of quiescent human Pax7+ MuRC in vitro. We also revealed, by immunofluorescence and flow cytometry, two distinct subpopulations of MuRC distinguished by their relative Pax7 expression. After 48 h in differentiation medium (DM), the Pax7High subpopulation represented 35% of the total MuRC pool and this percentage increased to 61% after 96 h in DM. Transcriptomic analysis revealed that Pax7High MuRC were less primed for myogenic differentiation as compared to Pax7Low MuRC and displayed a metabolic shift from glycolysis toward fatty acid oxidation. The bioenergetic profile of human MuRC displayed a 1.5-fold decrease in glycolysis, basal respiration and ATP-linked respiration as compared to myoblasts. We also observed that AMPKα1 expression was significantly upregulated in human MuRC that correlated with an increased phosphorylation of acetyl-CoA carboxylase (ACC). Finally, we showed that fatty acid uptake was increased in MuRC as compared to myoblasts, whereas no changes were observed for glucose uptake. CONCLUSIONS: Overall, these data reveal that the quiescent MuRC pool is heterogeneous for Pax7 with a Pax7High subpopulation being in a deeper quiescent state, less committed to differentiation and displaying a reduced metabolic activity. Altogether, our data suggest that human Pax7High MuRC may constitute an appropriate stem cell source for potential therapeutic applications in skeletal muscle diseases.
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Células Musculares , Células Satélite del Músculo Esquelético , Humanos , Ácidos Grasos , Metaboloma , Músculo EsqueléticoRESUMEN
OBJECTIVE: Low-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In this study, we evaluated the role of NOX4 on joint homoeostasis after destabilisation of the medial meniscus (DMM) in mice. METHODS: Experimental OA was simulated on cartilage explants using interleukin-1ß (IL-1ß) and induced by DMM in wild-type (WT) and NOX4 knockout (NOX4-/-) mice. We evaluated NOX4 expression, inflammation, cartilage metabolism and oxidative stress by immunohistochemistry. Bone phenotype was also determined by micro-CT and histomorphometry. RESULTS: Whole body NOX4 deletion attenuated experimental OA in mice, with a significant reduction of the OARSI score at 8 weeks. DMM increased total subchondral bone plate (SB.Th), epiphysial trabecular thicknesses (Tb.Th) and bone volume fraction (BV/TV) in both NOX4-/- and wild-type (WT) mice. Interestingly, DDM decreased total connectivity density (Conn.Dens) and increased medial BV/TV and Tb.Th only in WT mice. Ex vivo, NOX4 deficiency increased aggrecan (AGG) expression and decreased matrix metalloproteinase 13 (MMP13) and collagen type I (COL1) expression. IL-1ß increased NOX4 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in WT cartilage explants but not in NOX4-/-. In vivo, absence of NOX4 increased anabolism and decreased catabolism after DMM. Finally, NOX4 deletion decreased synovitis score, 8-OHdG and F4/80 staining following DMM. CONCLUSION: NOX4 deficiency restores cartilage homoeostasis, inhibits oxidative stress, inflammation and delays OA progression after DMM in mice. These findings suggest that NOX4 represent a potential target to counteract for OA treatment.
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NADPH Oxidasa 4 , Osteoartritis , Animales , Ratones , Modelos Animales de Enfermedad , Inflamación , NADPH Oxidasa 4/deficiencia , NADPH Oxidasa 4/genética , Osteoartritis/genética , Especies Reactivas de Oxígeno , Ratones NoqueadosRESUMEN
[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].
RESUMEN
PURPOSE: The myofibroblast, a contractile fibroblastic cell expressing α-smooth muscle actin (α-SMA), has been reported to play a role in ligament healing. The aim of this study was to evaluate the feasibility of transplanting culture-derived myofibroblasts in injured rabbit medial collateral ligaments (MCL) and in intact anterior cruciate ligaments (ACL). METHODS: Fibroblasts isolated from the iliotibial band were cultured in the presence of transforming growth factor beta-1 (TGF-ß1) for five days and analysed for α-SMA expression. In a concentration of TGF-ß1 ≥ 10 ng/ml, the differentiation rate into myofibroblast was 90%. After labelling with PKH26, α-SMA -positive cells were transplanted in intact ACL and in injured MCL of ten rabbits. RESULTS: Survival of PKH-26+ cells was seen in all intact and damaged ligaments one day after injection. The density of PKH-26+ cells had decreased at seven days postinjection in both ligaments. Double-positive PKH-26+/α-SMA+ cells were only observed in injured MCL at seven days postinjection. Moreover, we found that genetically modified fibroblasts differentiate into myofibroblasts and can be transplanted into ligaments. CONCLUSIONS: Our data demonstrate that culture-born myofibroblasts survive and maintain α-SMA expression up to one week after transplantation. This study provides the first insight into the feasibility of transplanted mechanically active cells for ligament reconstruction.
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Ligamento Cruzado Anterior/cirugía , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Ligamento Colateral Medial de la Rodilla/lesiones , Ligamento Colateral Medial de la Rodilla/cirugía , Miofibroblastos/trasplante , Actinas/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Estudios de Factibilidad , Femenino , Modelos Animales , Miofibroblastos/citología , Miofibroblastos/metabolismo , Conejos , Procedimientos de Cirugía Plástica , Trasplante Autólogo , Resultado del TratamientoRESUMEN
Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT.
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The replacement of a native hip joint by a metal-on-metal prosthesis may induce deleterious inflammatory side effects that are associated with the release of wear particles and metal ions. These events are referred to the adverse reaction to metal debris (ARMD) and the adverse local tissue reaction (ALTR). While wear particles seem involved in ARMD, the role of metal ions in ALTR and their impact on myoblasts, located in the prosthesis vicinity, has not been fully identified. To clarify this issue we investigated, using an in vitro culture system, the effect of cobalt and/or chromium ions (Co2+ and/or Cr3+ ) on human myoblast proliferation, cellular differentiation, and inflammatory marker expression. Freshly isolated human myoblasts were cultured in media supplemented with graded concentrations of Co2+ and/or Cr3+ . Co2+ induced a concentration-dependent decrease of both myoblast viability and myogenic differentiation while Cr3+ did not. Co2+ or Co2+ /Cr3+ also induced the upregulation of ICAM-1, whereas HLA-DR expression was unaffected. Moreover, allogenic monocytes induced the synergistic increase of Co2+ -induced ICAM-1 expression. We also found that Co2+ stabilized HIF-1α and increased TLR4, tumor necrosis factor-alpha (TNF-α), and interleukin 1ß (IL-1ß) expression in a dose and time-dependent manner in human myoblasts. This study showed that Co2+ , but not Cr3+ , was toxic toward myoblasts and induced, in the surviving cells, expression of inflammatory markers such as ICAM-1, TLR4, TNF-α, and IL-1ß. This suggests that Co2+ , most efficiently in the presence of monocytes, may be a key inducer of ALTR, which may, if severe and long-lasting, eventually result in prosthesis loosening.
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Cromo/efectos adversos , Cobalto/efectos adversos , Mioblastos/efectos de los fármacos , Adolescente , Adulto , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1beta/genética , Desarrollo de Músculos/efectos de los fármacos , Mioblastos/fisiología , Receptor Toll-Like 4/genética , Adulto JovenRESUMEN
Tissue-engineered grafts may be useful in Anterior Cruciate Ligament (ACL) repair and provide a novel, alternative treatment to clinical complications of rupture, harvest site morbidity and biocompatibility associated with autografts, allografts and synthetic grafts. We successfully used supercritical carbon dioxide (Sc-CO2) technology for manufacturing a "smart" biomaterial scaffold, which retains the native protein conformation and tensile strength of the natural ACL but is decellularized for a decreased immunogenic response. We designed and fabricated a new scaffold exhibiting (1) high tensile strength and biomechanical properties comparable to those of the native tissue, (2) thermodynamically-stable extra-cellular matrix (ECM), (3) preserved collagen composition and crosslinking, (4) a decellularized material milieu with potential for future engineering applications and (5) proven feasibility and biocompatibility in an animal model of ligament reconstruction. Because of the "smart" material ECM, this scaffold may have the potential for providing a niche and for directing stem cell growth, differentiations and function pertinent to new tissue formation. Sc-CO2-related technology is advanced and has the capability to provide scaffolds of high strength and durability, which sustain a lifetime of wear and tear under mechanical loading in vivo.
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Reconstrucción del Ligamento Cruzado Anterior/métodos , Dióxido de Carbono/química , Ingeniería de Tejidos , Animales , Fenómenos Biomecánicos , Femenino , Prueba de Estudio Conceptual , Conejos , Tendones/metabolismo , TermodinámicaRESUMEN
Cell therapy for Duchenne muscular dystrophy and other muscle diseases is limited by a massive early cell death following injections. In this study, we explored the potential benefit of heme oxygenase-1 (HO-1) expression in the survival of porcine myogenic precursor cells (MPCs) transplanted in pig skeletal muscle. Increased HO-1 expression was assessed either by transient hyperthermia or by HO-1 lentiviral infection. One day after the thermic shock, we observed a fourfold and a threefold increase in HSP70/72 and HO-1 levels, respectively. This treatment protected 30% of cells from staurosporine-induced apoptosis in vitro. When porcine MPC were heat-shocked prior to grafting, we improved cell survival by threefold at 5 days after autologous transplantation (26.3 +/- 5.5% surviving cells). After HO-1 lentiviral transduction, almost 60% of cells expressed the transgene and kept their myogenic properties to proliferate and fuse in vitro. Apoptosis of HO-1 transduced cells was reduced by 50% in vitro after staurosporine induction. Finally, a fivefold enhancement in cell survival was observed after transplantation of HO-1-group (47.5 +/- 9.1% surviving cells) as compared to the nls-LacZ-group or control group. These results identify HO-1 as a protective gene against early MPC death post-transplantation.
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Hemo-Oxigenasa 1/metabolismo , Lentivirus/genética , Mioblastos/citología , Mioblastos/trasplante , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Femenino , Citometría de Flujo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Hemo-Oxigenasa 1/genética , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/metabolismo , ARN Interferente Pequeño/genética , Estaurosporina/farmacología , Porcinos , Trasplante AutólogoRESUMEN
It is most probable that, in a near future, myogenic precursor cell transplants will have clinical applications in domains as different as orthopaedics, endocrinology, management of heart infarct, and therapies of muscle diseases. We have proposed to introduce the use of myogenic precursor cell transplantation in patients, after preliminary tests in a large animal model, the pig. Our initial effort was centred on the domain of orthopaedics. Muscle damages are frequent complications of traumas and sport accidents with serious consequences both in terms of disabilities and health economics. Often these lesions heal very poorly. A number of growth factors seemed successful as healing agents but they are difficult to deliver clinically. The goal was to use ex vivo somatic gene therapy with myogenic precursor cells modified to secrete growth factors with the aim of improving muscle healing in patients and of demonstrating the potential of this technology. To do so, we used a suitable large animal model, the pig, for exploring myogenic precursor cell transplantation strategies that could be used in patients.
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Modelos Animales , Músculo Esquelético/fisiología , Mioblastos/trasplante , Animales , Operón Lac/genética , Regeneración , Porcinos , Trasplante AutólogoRESUMEN
Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treatment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and massive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42 degrees C for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and nontreated cells (67.69% +/- 8.35% versus 58.79% +/- 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% +/- 8.22% versus 28.27% +/- 6.32%, P < 0.01) and more than threefold at 120 h (26.33% +/- 5.54% versus 8.79% +/- 2.51%, P < 0.01). Histological analysis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treatment.
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Mioblastos Esqueléticos/trasplante , Animales , Supervivencia Celular , Células Cultivadas , Femenino , Proteínas HSP70 de Choque Térmico/metabolismo , Calor , Técnicas In Vitro , Operón Lac , Desarrollo de Músculos , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Músculo Esquelético/cirugía , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Sus scrofa , Transducción Genética , Trasplante AutólogoRESUMEN
Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.