Regenerative potential of human muscle stem cells in chronic inflammation

International audienceABSTRACT: INTRODUCTION: Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle. METHODS: As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation. RESULTS: After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group. CONCLUSIONS: In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate

Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies

Altres ajuts: Association Française contre les Myopathies (22525)Altres ajuts: Fundación Isabel GemioSkeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR

Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation

Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors.

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

BACKGROUND:In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. OBJECTIVE:to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. METHODS:an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. RESULTS:blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. CONCLUSION:a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions

Hydrophilic antioxidant compounds in orange juice from different fruit cultivars: Composition and antioxidant activity evaluated by chemical and cellular based (Saccharomyces cerevisiae) assays

Antioxidant capacity was evaluated by a cellular model (Saccharomyces cerevisiae) and chemical methods (FRAP, TEAC and total phenols by Folin-Ciocalteu assay) in the hydrophilic fraction (phenolic compounds and ascorbic acid) of orange juices (OJs) from six varieties (Midknight, Delta Seedless, Rohde Red, Seedless, Early and clone Sambiasi), harvested in two seasons. The contents of phenolic compounds and ascorbic acid analyzed, respectively, by UPLC and HPLC were 370.04 76.97 mg/L and 52.05 6.69 mg/100 mL. Variety and season significantly influenced (p < 0.05) composition and antioxidant capacity. TEAC and FRAP values correlated well with individual hydrophilic compounds (R2 > 0.991) but no correlation with cellular assay was observed. An increase in survival rates between 23% and 38% was obtained, excepting for two varieties that showed no activity (Rohde Red and Seedless). Narirutin, naringin-d, ferulic acid-d2, didymin, neoeriocitrin and sinapic acid hexose and caffeic acid-d1 were the phenolic compounds which contributed to survival rates (R2 = 0.979, p < 0.01

A novel bioengineered functional motor unit platform to study neuromuscular interaction

Background: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. Objective: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. Methods: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. Results: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. Conclusion: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions

Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2)

BACKGROUND: The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. METHODS: The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined. RESULTS: The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent K(m )values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family. CONCLUSIONS: The current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects