Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

Once escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray–based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [3H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced MSCs

Mise en évidence d'un rôle de support stromal du macrophage vis-à-vis des cellules précurseurs myogéniques

Le muscle strié squelettique adulte est capable de régénérer, grâce aux propriétés des cellules satellites, ou cellules précurseurs myogéniques (mpc). Au cours de la régénération musculaire, le processus myogénique est supporté par des signaux issus dune part des cellules stromales et d'autre part, des monocytes/macrophages recrutés à partir de la circulation sanguine. Nous avons étudié les interrelations entre les mpc et les monocytes/macrophages humains. L'observation de coupes histologiques de muscle humain normal montre que les cellules satellites sont à proximité immédiate des capillaires (environ 12 um). In vitro, nous avons montré que les mpc, dès leur sortie de quiescence, attirent spécifiquement et préférentiellement les monocytes circulants. Nous avons identifié 5 facteurs chimiotactiques qui participent pour 77% du chimiotactisme : MP-derived chemokine (MDC), monocyte chemoattractant proteine-1 (MCP-1), fractalkine (FKN), VEGF et le système de l'urokinase. L'étude des contacts cellule:cellule entre les mpc et les macrophages nous a permis de montrer que les macrophages stimulent la prolifération des mpc par des facteurs solubles et inhibent leur apoptose par des contacts directs cellule:cellule impliquant les couples moléculaires fractalkine/CX3CR1 et VCAM-1 NLA-4. In vivo, dans les thérapies cellulaires réparatrices de l'infarctus du myocarde, les macrophages améliorent la survie des mpc implantées et semblent faciliter la migration des cellules myogéniques dans le tissu myocardique.Adult skeletal muscle s able to regenerate affer injury, through the property of satellite cells, or myogenic precursor cells (mpc). During muscle regeneration, myogenic process is supported by signaIs from stromal cells and monocytes/macrophages. We studied the relationship between human mpc and monocytes/macrophages. Normal human muscle histological observation shows that satellite cells are very close to capillaries (about 12 pm). In vitro, we showed that as soon as their exit of quiescence, mpc specifically and preferentialy attract circulating monocytes. We identified 5 chemotactic factors which are responsible for 77 % of monocytes chemotaxis: MP-derived chemokine (MDC), monocyte chemoattractant proteine-1 (MCP-1), fractalkine (FKN), VEGF and urokinase system. Study of ceIl:cell contacts between mpc and macrophages permit us to show that macrophages stimulate mpc proliferation by soluble factors and inhibit their apoptosis by direct celI:cell contacts involving fractalkine/CX3CRl and VCAM-1/VLA-4 molecular couples. In vivo, in muscle cell transplant to repair experimental heart injury, macrophages improve survival of implanted mpc and seems facilitate myogenic cells migration in the myocardic tissue. Thus, macrophage seems to be a stromal support for mpc in vitro et in vivo.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocFranceF

Dual and beneficial roles of macrophages during skeletal muscle regeneration.

International audienceMacrophages are necessary for skeletal muscle regeneration after injury. Muscle recruits inflammatory monocytes/macrophages that switch toward an anti-inflammatory profile upon phagocytosis of debris. In vitro, proinflammatory macrophages stimulate myoblast proliferation, whereas anti-inflammatory macrophages stimulate their differentiation. Thus, macrophages are involved in both phases of skeletal muscle regeneration: first, inflammation and cleansing of necrosis, and then myogenic differentiation and tissue repair

Role of thrombospondin 1 in macrophage inflammation in dysferlin myopathy

Muscle inflammation can be a prominent feature in several muscular dystrophies. In dysferlin myopathy, it is mainly composed of macrophages. To understand the origin of inflammation in dysferlin-deficient muscle, we analyzed soluble factors involved in monocyte chemotaxis released by myoblasts and myotubes from control and dysferlinopathy patients using a transwell system. Dysferlin-deficient myotubes released more soluble factors involved in monocyte chemotaxis compared with controls (p < 0.001). Messenger RNA microarray analysis showed a 3.2-fold increase of thrombospondin 1 (TSP-1) expression in dysferlin-deficient myotubes. Retrotranscriptasepolymerase chain reaction analysis, ELISA, and immunohistochemistry confirmed these results. Dysferlin mRNA knockdown with short-interfering RNA in normal myogenic cells resulted in TSP-1 mRNA upregulation and increased chemotaxis. Furthermore, monocyte chemotaxis was decreased when TSP-1 was blocked by specific antibodies. In muscle biopsies from dysferlinopathy patients, TSP-1 expression was increased in muscle fibers but not in biopsies of patientswith other myopathies with inflammation; TSP-1 was seen in some macrophages in all samples analyzed. Taken together, the data demonstrate that dysferlin-deficient muscle upregulates TSP-1 in vivoand in vitro and indicate that endogenous chemotactic factors arecrucial to the sustained inflammatory process observed in dysferlinopathies. © 2010 by the American Association of Neuropathologists, Inc.This work was supported by the Beca del Fondo de Investigacion Sanitaria PI06/0455 and by the Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas.Peer Reviewe

ADAM12 and α(9)β(1) Integrin Are Instrumental in Human Myogenic Cell Differentiation

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding α(9)β(1) integrin. We report that normal human mpc express both ADAM12 and α(9)β(1) integrin during their differentiation. Expression of α(9) parallels that of ADAM12 and culminates at time of fusion. α(9) and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/α(9)β(1) integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to α(9)β(1), inhibited overall mpc fusion by 47–48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/α(4)β(1), which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-α(9)β(1) antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12–α(9)β(1) interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and α(9)β(1) blockade inhibited more importantly formation of large (≥5 nuclei) myotubes than that of small (2–4 nuclei) myotubes. We conclude that both ADAM12 and α(9)β(1) integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth

Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

International audiencence escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray-based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase O system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [ 3 H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth

Imprinted genes that regulate early mammalian growth are coexpressed in somatic stem cellsImprinted genes that regulate early mammalian growth are coexpressed in somatic stem cells

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