Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/10—Musculoskeletal or connective tissue disorders
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

• the present invention relates to the muscle secretome.
• Secreted proteins constitute an important class of active molecules regulating physiological and pathological processes. In higher organisms protein secretion is complex and tightly regulated, reflecting the functionality of a cell in a given environment. Besides endocrine organs specialised in the secretion of specific molecules, there is increasing evidence that many other tissues including skeletal muscle secrete factors with local and systemic effects. Skeletal muscle accounts for about 40% of body mass, is responsible for movement and is an important metabolic organ. In adults, it has a very low basal rate of cellular turnover but retains a remarkable capacity to adapt to normal physiological demands during growth and training, and to regenerate in response to injury or disease. Secreted signalling molecules including growth factors and cytokines have been shown to modulate activation, proliferation and differentiation of satellite cells.
• the inventors have shown that differentiating human muscle cells release not only a plethora of soluble secreted proteins through conventional secretory mechanisms but also complex protein/nucleic acid cargos via membrane microvesicle (MVs) shedding.
• the soluble "secretome” contains 254 proteins/peptides with signalling potential including 45 previously implicated in myogenesis. Many more have proven roles in modulating a range of other cell types, implying a much broader role for myoblasts in regulating skeletal muscle homeostasis and remodelling in vivo.
• muscle-derived MVs act in vivo as "physiological liposomes" delivering protein/RNA cargo to target cells acting in concert with soluble signalling molecules to modulate complex intercellular signalling networks during muscle regeneration.
• the present invention relates to an isolated nanovesicle secreted by a muscle cell.
• the present invention also relates to an isolated microparticle secreted by a muscle cell.
• the invention also relates to the use of said nanovesicle and/or said microparticle as a diagnostic biomarker for muscular diseases.
• Said nanovesicle and said microparticle have proteomic profiles which are specific to the muscle. These specific proteomic profiles facilitate their isolation and their identification.
• the present invention also relates to the use of said nanovesicle and/or said microparticle for delivering a molecule of interest into a target cell.
• the present invention relates to two morphologically distinct populations of isolated small bilayer membrane vesicles which are muscle secreted:
• - nanovesicles which are cup-shaped vesicles, 69 ( ⁇ 20) nm in diameter and pelleting at 100,000 g;
• nanovesicles and microparticles according to the invention may be isolated by filtration and ultracentrifugation.
• the nanovesicles and microparticles according to the invention may be frozen and stored at -80°C without losing their ability to transfer material to the target cell.
• said nanovesicle or said microparticle is secreted by a muscle cell selected from the group consisting of a skeletal muscle cell, a cardiomyocyte, a smooth muscle cell, and a myoblast.
• said nanovesicle or said microparticle is secreted by a skeletal muscle cell.
• said muscle cell overexpresses a molecule of interest.
• overexpressing it is meant any means known in the art to enhance the amount of protein expressed by a given cell.
• the overexpression of the molecule of interest may be achieved by transfecting the muscle cell with an expression vector encoding molecule of interest.
• overexpression is obtained by transfection of an exogenous DNA.
• Suitable transfection methods are classical methods known to the skilled person, such as calcium phosphate transfection, transfection using liposomes (also known as lipofection) or electroporation. It falls within the ability of the skilled person to select the appropriate transfection method for a given muscle cell.
• the term "overexpression” also covers the overexpression of an endogenous molecule, i.e. a molecule which is naturally expressed by the muscle cell. The overexpression may be achieved by the introduction of additional copies of the gene encoding said molecule or in the stimulation of the expression of the endogenous molecule.
• the muscle cell can be placed under culture conditions known to enhance the expression of said endogenous molecule.
• the nanovesicles or micropaticles are typically harvested from the cell supernatant 48-72 hours post- transfection.
• the muscle cell overexpresses 2 to 5 different molecules of interest
• molecules of interest may be selected from the group consisting of peptides, proteins, mRNA, miRNA, viral vectors... Nanovesicles and/or microparticles according to the invention as delivery vehicles
• the present invention also relates to an in vitro method for delivering a molecule of interest into a target cell by contacting said target cell with a nanovesicles and/or a microparticle according to the invention comprising said molecule of interest.
• nanovesicles and/or microparticles of the invention can be used to efficiently deliver said molecule of interest to a target cell.
• the inventors have shown that the molecule of interest retains its functionality once it has been transferred into the target cell.
• the present invention also relates to an in vivo method for delivering a molecule of interest into a target cell by contacting said target cell with a nanovesicles and/or a microparticle of the invention comprising said molecule of interest.
• target cells are involved in the muscle regeneration such as myoblasts, myotubes, muscle fibres, inflammatory cells or neighbouring fibroblasts.
• the nanovesicles and/or a microparticle of the invention may also be administered subcutaneously, and thereby target dermal fibroblasts.
• the contacting of said several different nanovesicles and/or microparticles may be simultaneous or sequential.
• protein delivery with nanovesicles and/or microparticles according to the invention is an original method to introduce rapidly a function in a targeted cell, without involvement of the transcription machinery or any viral integration processes, which represent a very serious oncogenic risk for clinical use.
• the nanovesicles and/or microparticles according to the invention could be used in virtually any cell type including resting or fully differentiated cells, without any tumorigenic risk. They can deliver proteins or RNA with an effect limited in time by the half-life of the molecule. Such vectors do not exist at present.
• the method of the invention does not rely of transcription and translation within the target cell, does not perturb the cell metabolism.
• the method according to the invention does not activate any interferon response and is therefore a more specific method for delivering a molecule of interest to a target cell.
• the nanovesicles and/or microparticles Due to the low amounts of material delivered by the nanovesicles and/or microparticles according to the invention and to their non-genetic nature, the nanovesicles and/or microparticles appear to be useful for applications where low and transient presence of molecules may lead to striking biological effects.
• a nanovesicle or a microparticle according to the invention is virus free.
• nanovesicles or microparticles could also be used as packaging for viral particles, since they could physiologically contain AAV or HIV particles.
• the advantage of such a role would be to target the viral particles towards the cells targeted by the nanovesicles or microparticles.
• These nanovesicles or microparticles could be targeted towards a limited number of cell types by including in their membrane proteins that will recognize specific receptors on specific cell types.
• the invention relates to the in vitro use of the nanovesicles and/or microparticles according to invention.
• the invention relates to the in vivo use of the nanovesicles and/or microparticles according to invention.
• the invention relates to the use of a nanovesicle and/or a microparticle according to the invention for non-therapeutic applications.
• the nanovesicles and/or microparticles according to the invention can be used for introducing a molecule of interest into a target cell in vitro in order to study the physiological effect of said molecule of interest.
• the invention can be used for introducing a molecule of interest into a target cell in vitro in order to study the physiological effect of said molecule of interest.
• Another example is the delivery of a cellular protein regulating cell expansion differentiation or death, in an in vitro cellular model.
• the invention also relates to a method for inducing or potentiating cell differentiation by delivery of transcription factors
• the invention also relates to a nanovesicle and/or a microparticle according to the invention for use in therapy.
• the invention also relates to a nanovesicle and/or a microparticle according to the invention for use in the treatment and/or the prevention of sarcopenia, or of muscular dystrophies such as dysferlinopathies, (Limb-Girdle muscular dystrophy (LGMD)).
• LGMD Garnier muscular dystrophy
• the nanovesicle and/or the microparticle of the invention is the active ingredient in a pharmaceutically acceptable formulation suitable for administration to the subject.
• a pharmaceutically acceptable carrier for the active ingredient.
• the specific carrier will depend upon a number of factors (e.g., the route of administration).
• the "pharmaceutically acceptable carrier” means any pharmaceutically acceptable means to mix and/or deliver the targeted delivery composition to a subject.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and is compatible with administration to a subject, for example a human.
• Administration to the subject can be either systemic or localized. This includes, without limitation, dispensing, delivering or applying the active ingredient (e.g. in a pharmaceutical formulation) to the subject by any suitable route for delivery of the active compound to the desired location in the subject, including delivery by intramuscular injection, subcutaneous/intradermal injection, intravenous injection, transdermal delivery.
• Muscle nanovesicles and muscle microparticles according to the invention as diagnostic tools are provided.
• the present invention relates to a method for diagnosing or monitoring a muscular disease in a subject, comprising the step of detecting the presence or absence of one or more biomarkers within a nanovesicle and/or a microparticle according to the invention obtained from a biological sample of said subject, wherein said one or more biomarkers are associated with said muscular disease.
• the method may further comprise the step of comparing the result of the detection step to a control (e.g., comparing the amount of one or more biomarkers detected in the sample to one or more control levels), wherein the subject is diagnosed as having the disease if there is a measurable difference in the result of the detection step as compared to a control.
• Another aspect of the invention is a method for aiding in the evaluation of treatment efficacy in a subject suffering from a muscular disease, comprising the step of detecting the presence or absence of one or more biomarkers within a nanovesicle and/or a microparticle according to the invention obtained from a biological sample of said subject, wherein the biomarker is associated with the treatment efficacy.
• the method may further comprise the step of providing a series of biological samples over a period of time from the subject. Additionally, the method may further comprise the step or steps of determining any measurable change in the results of the detection step (e.g., the amount of one or more detected biomarkers) in each of the biological samples from the series to thereby evaluate treatment efficacy.
• any measurable change in the results of the detection step e.g., the amount of one or more detected biomarkers
• a method according to the invention may comprise the step or steps of isolating these muscle nanovesicles and/or microparticles using specific surface antigens such as the ones described in Table S12 by means of specific antibodies (FACS or MACS).
• Said subject is a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice, rats, guinea pig. etc.)
• a rodent e.g. mice, rats, guinea pig. etc.
• said subject is a human.
• the biological sample obtained from the subject is a sample of bodily fluid.
• suitable body fluids are blood and urine.
• the biological sample obtained from the subject may be a culture of muscle cells isolated from the subject.
• said muscular disease may be any pathological conditions resulting from an impairment of the muscle regeneration, such as fibrosis, adipogenesis or chronic inflammation.
• said muscular disease is selected from the group consisting of neuromuscular diseases and sarcopenia.
• said one or more biomarkers associated with a muscular disease are:
• nucleic acid or protein i) a species of nucleic acid or protein
• nucleic acid a nucleic acid, peptide or protein variant
• nucleic acid may be a mRNA or a miRNA.
• peptides or proteins may be one of the proteins listed in Table SI 3.
• the present invention relates to methods for detecting, diagnosing, monitoring, treating or evaluating a muscular disease in a subject comprising the steps of, isolating a nanovesicle and/or a microparticle according to the invention from a biological sample obtained from a subject, and analyzing one or more biomarkers contained within the nanovesicle and/or the microparticle.
• the one or more biomarkers are analyzed qualitatively and/or quantitatively, and the results are compared to results expected or obtained for one or more other subjects who have or do not have the muscular disease.
• the presence of a difference in the nucleic acid, peptide and protein content of nanovesicle and/or the microparticle of the subject, as compared to that of one or more other individuals, can indicate the presence or absence of, the progression of the muscular disease, or the susceptibility to a muscular disease in the subject.
• Nanovesicles and microparticles of the invention have proteomic profiles which are specific to the muscle.
• the specific surface molecules carried by the nanovesicles and microparticles of the invention may be used to identify, isolate and/or purify the nanovesicles and microparticles.
• Typical specific surface molecules carried by the nanovesicles are listed in Table SI 2.
• Typical specific surface molecules carried by the microparticles are listed in Table S12.
• Suitable purification methods include, but are not limited to immunoprecipitation, affinity chromatography, FACS and magnetic beads coated with specific antibodies or aptamers.
• the present invention also relates to an antibody microarray which could be used to detect specifically variations in the muscle secretome.
• said antibody microarray comprises a solid support with a plurality of antibodies immobilized on the solid support, wherein said plurality of antibodies is able to bind to at least 30, 40, 60, 70, 80, 90, 100, 110, 120, 130, or 140 different proteins listed in one of the tables Tl, T2, and T3.
• said antibody microarray comprises a solid support with a plurality of antibodies immobilized on the solid support, wherein said plurality of antibodies is able to bind to at least 30, 100, 200, 300, 400, or 443 different proteins listed in T4.
• said plurality of antibodies is able to bind to at least 45, 148, 143 and 443 different proteins selected from tables Tl, T2, T3 and T4 respectively.
• Table Tl may be used to detect modifications in muscle development and formation.
• Table T2 may be used to detect molecules involved in the muscle extracellular matrix as well as the detection and quantification of fibrosis in disease situations.
• Table T3 may be used to measure deregulation in muscle homeostasis.
• Table T4 may be used to assess the whole muscle secretome.
• said plurality of antibodies is able to bind to all the proteins listed one of the tables Tl , T2, T3 or T4.
• Antibody microarray is a well known technology, for producing an antibody microarray of the invention, the skilled person may use for example the teaching of WO 2007/130549 or WO0127611. In the following, the invention will be illustrated by means of the following examples and figures.
• Figure 1 Characterization of the secretome of differentiating human myoblasts
• This virtual secretome was compared with the "experimental secretome" of differentiating human myoblasts obtained by analysing the culture medium by combined proteomics strategies.
• computational analysis classified 257 proteins (27%) as classical or leaderless secreted molecules.
• the remaining unclassified proteins (708; 73%) were of various intracellular origins.
• (c) To understand how "soluble" secreted proteins might be related to specialized cellular functions and/or processes, we performed detailed functional analysis in silico.
• the functional analysis in (d) revealed selective enrichment of 45 soluble secreted proteins crucial for myogenesis and myoblast differentiation. Values represent the -Log (p-value). The p-value was calculated after Right-tailed Fisher's exact test; only significantly overrepresented categories (p ⁇ 0.05) are shown.
• Figure 2 Potential extracellular pathways and/or networks involved in muscle differentiation.
• a protein-protein interaction network was generated using Ingenuity Pathway Analysis (IPA) on 45 extracellular proteins crucial for myogenesis and myoblast differentiation following serum starvation (see Table S6 for protein descriptions, HUGO gene symbols and methods).
• IPA Ingenuity Pathway Analysis
• the intensity of "light grey” and “dark grey” indicates the degree of down or upregulation respectively (based on expression data from MAP assay or immunoblot).
• a solid line indicates a direct interaction while a dashed line indicates an indirect interaction.
• Figure 3 Differentiated human muscle cells secrete 2 distinct types of microvesicles.
• the dashed line represents 434 proteins previously identified in MVs produced by other cells;
• (c) 901 non-redundant proteins were identified by LC-MS/MS.
• Inset Venn diagram shows proteins identified in exosome and microparticles and the overlap between fractions.
• MS/MS spectral count To further assess selective enrichment, the relative abundance of proteins in the two MV fractions was evaluated by MS/MS spectral count; fold change comparison of the 2 proteomes are shown,
• Figure 4 Identification and characterization of the muscle-derived microvesicles RNA cargo.
• FIG. 1 Muscle-derived microvesicles can dock, fuse and deliver functional protein to target cells.
• LC-MS/MS is an extremely powerful tool to identify a large number of proteins simultaneously over a wide dynamic range.
• low abundance and low molecular weight proteins, such as cytokines and growth factors, overlooked by both 2DE and LC-MS/MS are detected by antibody array.
• the 965 identified proteins were further submitted to a similar computational method as for our transcriptome dataset for predicting secretory mechanisms.
• Phobius predictor was used to sort protein sequences with only one TM region from those whose single TM domain overlapped with the signal peptide (Kail, Krogh et al. 2004). Subsequently, putative classically secreted proteins (Signal Peptide positive/Transmembrane domain negative) were scanned for the presence of an ER retention signal. Those containing in their sequence the extended KDEL motif were discarded (PROSITE PS00014/ER_TARGET) (Hulo, Bairoch et al. 2008). Finally, for each remaining protein, the annotation information of the corresponding entry in the GO annotation (Ashburner, Ball et al. 2000), Uniprot (http ://www.uniprot.
• Figure 8 Analysis of conditioned media during human primary myoblast differentiation in vitro using antibody-based assays.
• CM Conditioned media
• SPARC showed the reverse secretion pattern with greatest level at 24h but remained detectable at 72h.
• LGALS1 and SPARC were detected with the 2 others proteomics screens performed on CM collected after 72h of differentiation, whereas MIF was among the few proteins only detected by 2DE and IGF2 identified by HPLC- ESI-MS/MS with only one unique high score peptide.
• Interact ome "fibrillar matrix”, “basement membrane”, “elastic fibers” and “proteases and inhibitors” (Figure 5B), mimicking the highly organised interstitial connective tissue surrounding individual muscle fibers in vivo.
• Protein abbreviations correspond to HUGO gene symbols and are reported in Table S5 with detailed information of the associated proteins.
• the intensity of light grey and dark grey indicates the degree of down or upregulation respectively (if expression data was available from MAP assay or immunoblot).
• a solid line indicates a direct interaction while a dashed line indicates an indirect interaction.
• 203/257 "soluble" secreted proteins were successfully assigned into one or more biological functions as determined by IPA
• vesicles harbouring typical exosomal features and larger, morphologically distinct, microvesicles.
• these vesicles differ in their protein and RNA content, differentially dock and fuse with adjacent cells, and demonstrate delivery of a functional protein cargo to target cells following microvesicle uptake.
• the intercellular signalling networks invoked during in vivo muscle regeneration may employ soluble signalling molecules acting in concert with muscle-derived microvesicles delivering protein/RNA cargo to target cells Results/Discussion
• MAP multi-analyte Luminex based immunoassay
• 2DGE 2D gel electrophoresis
• HPLC-ESI-MS/MS gel-free tandem mass spectrometry
• a putative muscle differentiation interactome model was generated after seeding with key muscle markers such as the myogenic regulatory factors Myogenin (MYOG) and MYOD1.
• MYOG myogenic regulatory factors
• Figure 2 shows these proteins coalesce into networks based on "differentiation”, “matrix remodelling & migration” and “fusion", all events essential to myodifferentiation.
• Important factors include IL6 which mediates hypertrophic muscle growth by controlling satellite cell recruitment and fusion (Serrano, Baeza-Raja et al. 2008), LGALSl (galectin-1) enhancing myoblast fusion (Watt, Jones et al.
• MMP2 essential for matrix remodelling during muscle growth and regeneration (Yagami-Hiromasa, Sato et al. 1995) and members of the TGF- ⁇ family and insulin-like growth factors (TGFB1, Myostatin (MSTN), IGF1, IGF2) which will either inhibit or promote muscle differentiation and hypertrophy (Langley, Thomas et al. 2002; Jacquemin, Butler-Browne et al. 2007).
• the remaining 150 secretory proteins have not been described in a muscle context and either had unassigned functions (54) or were associated with housekeeping (43) or specific processes such as the immune response, vascularisation, connective tissue and innervation (54) (Fig. Id; Fig 10, Table S6).
• VEGF-C and Placenta growth factor which act on blood vessels (Roy, Bhardwaj et al. 2006), GDF15 on inflammatory cells (Bootcov, Bauskin et al. 1997), and CTGF on neighbouring fibroblasts (Leask and Abraham 2004). Therefore these secreted proteins potentially interact with neighbouring muscle and non-muscle cells playing a crucial role in orchestrating muscle differentiation and remodelling.
• PEF Placenta growth factor
• microvesicle secretion two major mechanisms of microvesicle secretion have been described, each leading to the release of distinct cargo-loaded vesicles into the extracellular space: (i) microparticles (or shedding vesicles) generated from the direct budding of the plasma membrane; and (ii) exosomes, nanovesicles of endocytic origin released into the extracellular environment upon fusion of multivesicular endosomes with the plasma membrane.
• culture supernatants were subjected to differential centrifugation (Miguet, Pacaud et al. 2006; Thery, Ostrowski et al. 2009).
• Fig. 3a Two distinct populations of small bilayer membrane vesicles were isolated (Fig. 3a). The first were cup-shaped vesicles, 69 ( ⁇ 20) nm in diameter, sedimenting at 100,000 g, selectively enriched for tetraspanin surface proteins (CD81, CD82, CD63 and CD9), Hsp70 (HSPA8) , with a buoyant density between 1.11 and 1.14 g/ml, all features characteristic of exosomes (Fig. l la,b).
• the second population were polymorph microvesicles with electron dense cores, 80 to 290 nm in diameter, which pelleted at 20,000 g and were tetraspanin-negative except for CD81, but enriched in CLIC1 and galectin-1 (LGALS1) (Fig. 11a), and will hereafter be called microparticles (MPs).
• MPs microparticles
• the specific protein cargo of these two types of vesicles was analyzed using the same mass spectrometry-driven proteomics strategy employed for secretome mapping (i.e. HPLC-ESI-MS/MS). A total of 764 unique proteins were identified from MPs and 564 from exosomes (Fig. 3c and Table S7-S8).
• ER resident proteins (CALU, CALR, HSP90B1, HSPA5), TCP-1 chaperonins (TCP1, CCT2), actin and tubulins (ACTB, TUBA1B), ribosomal subunits (RPL4, RPL10, RPS5, RPS17), translation initiation factors (EIF3A, EIF5), poly(A) binding protein (PABPC1), and proteasome sub-components (PSMA1, PSMB1)).
• proteins from the plasma membrane, sub-plasma membrane, endosome and lysosome are enriched within exosomes (e.g.
• Integrins IGA4, ITGA6, ITGA7, MHC molecules (HLA-A HLA-B) and tetraspanins (CD 9, CD63, CD81, CD82), flotillin-1 (FLOT1), Alix (PDCD6IP), TSGlOl, lysosome-associated protein (LAMP2), Fig. 3d, top panel and Table S9).
• proteins identified in MPs principally involve RNA- post- translational modification, amino acid metabolism, protein synthesis, folding, post- translational modification and trafficking as well as molecular transport and protein degradation.
• the distinct functions of the proteins contained in each type of vesicle reinforce their potentially specific role.
• RNAs were afforded protection from RNase action by the microvesicle membrane, particularly in exosomes, while RNA adhering to the microvesicle exterior was completely destroyed (Fig. 4 a).
• RNA cargo would also be different.
• Hierarchical clustering of microarray data indicated that the transcriptome of myotubes, MPs and exosomes were indeed separable into distinct groups (Fig. 4c). Further analysis identified 185 core transcripts in exosomes, whereas MPs contained 4431 transcripts. The abundance of 185 exosome transcripts correlated with that in myotubes, suggesting they are a subset of myotube transcripts - although these were not simply the most abundant myotube transcripts.
• Exosomes were also internalized preferentially into mononucleated cells since a signal could be detected in myotubes only after 25 hours of incubation.
• MPs were internalized by both myotubes and mononucleated cells.
• alkaline phosphatase one of the proteins identified by our proteomic screen in both exosomes and MPs, in dermofibroblasts with no endogenous activity. Alkaline phosphatase activity was detected after 48 hrs of incubation with purified exosomes or MPs (Fig. 5c). The expression of muscle-derived microvesicle delivered alkaline phosphatase by fibroblasts is powerful evidence that the protein cargo is functional after entry into the cytoplasm of recipient cells.
• muscle regeneration is a highly synchronized process that involves a multitude of coordinated cellular responses, such as inflammation, neo-vascularisation, muscle differentiation, innervation, and requires precise cell-to-cell signalling. Deregulation of any component of the process will lead to impaired regeneration.
• human myoblasts use a combination of soluble secreted proteins as well as secreted microvesicles to regulate the behaviour of neighbouring cells during muscle regeneration and orchestrate organogenesis.
• the secreted proteins encompass classical signalling factors known to act via cell surface receptors, whilst microvesicles allow transport of molecules that can act directly intracellularly once these vesicles are internalized.
• Human skeletal muscle culture Human satellite cells were isolated as described previously (Edom, Mouly et al. 1994) in accordance with French legislation on ethics. Cells were expanded in growth medium (Ham's F10, 20% foetal calf serum (FCS) and 5 ⁇ g/ml gentamycin (Invitrogen, Paisley, UK) in 5% C0 2 , at 37°C. In all experiments, myogenicity was greater than 90% as assessed by the expression of desmin localized by immunostaining (D33; DAKO, Glostrup, DK). After six washes to remove contaminating serum proteins, confluent cultures were switched to a serum-free Dulbecco's modified Eagle's medium (DMEM, Invitrogen) which triggered differentiation.
• DMEM serum-free Dulbecco's modified Eagle's medium
• CM Conditioned media
• microparticles were sedimented by centrifugation at 20,000 g for 70 min at 4°C.The remaining supernatant was further ultracentrifuged at 100,000 g for 70 min at 4°C to pellet the exosomes. Finally, purified vesicles were washed twice in PBS and fixed in 2% PAF prior to electron microscopy or resuspended in PBS. For the density gradient experiment, isolated exosomes were floated in a iodixanol gradient as previously described (Mathivanan, Lim et al. 2010).
• Protein extracts were fractioned by SDS PAGE using 4-12% acrylamide Bis-Tris precast gels with a MES buffer system according to the manufacturer's protocol (NuPAGE Novex Bis-Tris gel; Invitrogen) before transfer to PVDF membranes.
• Binding of a secondary antibody coupled to HRP was revealed using a chemiluminescence detection reagent (Pierce Biotechnology, Rockford, IL, USA). Signals were detected on film and quantified by densitometry using Quantity One software v. 4.6.5 (Bio-Rad, Hercules, CA, USA). Human multi-analyte profile - Luminex assays. Unconcentrated CMs from differentiating myoblasts were screened for cytokines and growth factors using Luminex multi-analyte profiling (MAP) technology (Oliver, Kettman et al. 1998). The assays (Human MAP® v. 1.6) were performed by Rules Based Medicine (Houston, TX, USA). Heat-maps for visualization of expression data were produced using FIRe v. 2.2 (Garcion, Baltensperger et al. 2006).
• MAP Luminex multi-analyte profiling
• CM and microvesicle pellets were dissolved in urea-buffer and proteins were reduced, alkylated and subsequently digested with endoproteinase Lys-C (Wako, Neuss, Germany) followed by digestion with trypsin (Promega, Southampton, UK).
• the resulting peptide mixture was fractionated using an ICAT® Cation Exchange Buffer Pack (Applied Biosystems, CA, USA) following the manufacturer's instructions.Bound peptides were eluted by washing the column progressively with elution buffers of increasing ionic strength. Fractions were dried and re-dissolved in 0.1% trifluoroacetic acid.
• Bioinformatics methods for analysis of specialized functions or processes To further evaluate the specific functional profiles of the various "soluble" secreted or membrane vesicle-associated proteins, we performed detailed functional analysis using Ingenuity Pathway Analysis System (IPA 5.0, http://www.ingenuity.com/ ; Ingenuity Systems®, Redwood, CA). The functional analysis identified the molecular/cellular functions that were most significantly represented in our three datasets. Fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to the data set is due to chance alone.
• Ingenuity Pathway Analysis System IPA 5.0, http://www.ingenuity.com/ ; Ingenuity Systems®, Redwood, CA.
• IPA Ingenuity Pathway Analysis
• Electron microscopy Human myotubes plated on plastic (Thermanox, Nalge Nunc, Rochester, NY , USA) coverslips were fixed in 2.5% glutaraldehyde in 0.1M phosphate buffer, pH 7.4 and further post-fixed in 2% Os0 4 . They were gradually dehydrated in acetone including a 2% uranyl en-bloc staining step in 70% acetone, and embedded in Epon resin (EMS, Fort Washington, PA, USA). Ultrathin sections were counterstained with uranyl and lead citrate. MVs were processed as described in Thery et al., 2006 (Thery, Amigorena et al. 2006). Observations were made using a CM120 transmission electron microscope (Philips) at 80 kV and images recorded with a Morada digital camera (Olympus Soft Imaging Solutions GmbH, Miinster, Germany).
• RNA was extracted using the RNeasy® Micro kit (Qiagen Ltd., Crawley, UK) with on-column DNase according to manufacturer's instructions. Isolated RNA were quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and integrity assessed on an Agilent 2100 Bioanalyser (Agilent Technologies, Inc., Santa Clara, CA, USA).
• RNA aliquots were subsequently incubated at 37°C for 15 minutes with either 100 ⁇ g/ml RNase (Fermentas, Glen Burnie, MD, USA) or nuclease free water alongside aliquots of cell-derived RNA acting as a positive control; the integrity of treated samples was reassessed using the Agilent 2100 Bioanalyser. Microarray analysis. Total RNA from 3 independent 72h differentiated cell cultures and MVs recovered from duplicate cultures at 72h differentiation were isolated using the RNeasy® kit (Qiagen Ltd.,Crawley, UK) according to manufacturer's instructions.
• RNA samples were quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and cellular RNA quality checked on an Agilent 2100 Bioanalyser (Agilent Technologies, Inc., Santa Clara, CA, USA). Samples were labelled using the Illumina TotalPrep RNA Amplification kit (Applied Biosystems, Foster City, CA, USA) then hybridised to Illumina human whole genome BeadChips (cell samples: HumanWG-6 v3; MVs: HumanHT12 v3; Illumina, Inc., San Diego, CA, USA ) according to manufacturer's instructions.
• the raw data were pre-processed using GenomeStudio (Illumina, Inc, San Diego, CA, USA) to determine mean signal values (AVG_Signal) and detection calls (Detection Pval). Details regarding the generation of the in silico/wktual secretome from the transcriptome of 72h differentiating cultures are provided in Fig. 6 and 7. To assess overrepresentation of functionally related gene and terms in MVs transcripts, functional annotation clustering was performed using DAVID (David Bioinformatics Resources 6.7; http://david.abcc.ncifcrf.gov/) (Huang da, Sherman et al. 2009)).
• MV transcripts were compared against a population background of all transcripts expressed in 72h myotubes.
• Protein transfer experiment alkaline phosphatase assay. Differentiating human myoblasts and dermofibroblasts (isolated from the skin of a 19 year old donor) were incubated with purified exosomes and microparticles for 48h at 37°C. After 3 washes in PBS, cells were fixed in 100% cold methanol and alkaline phosphatase activity revealed using the SIGMAFastTM BCIP ® /NBT ALP kit according to manufacturer instructions. Coverslips were scanned and images acquired using a Leica DMR microscope (Leica Microsystems GmbH).
• MIC-1 a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc Natl Acad Sci U S A 94(21): 11514-11519.
• 393 transcripts were identified as strong candidates for being secreted into the extracellular space via either classical or unconventional pathways.
• CDS9 IL N. .1905 Homo sapiens CD59 molecule, complement regulatory protein (COS9), transcript variant 995.3 0.00001 ⁇ SP[
• CFH ILMN 83 ⁇ 7 Homo sapiens complement factor H (CFH), transcript variant 1 , mR A. 988.1 0.0002: ⁇ SP!
• transcript variant 10 10. mRNA.
• B P1 ILMN .6483 Homo sapiens bone morphogenatic protein 1 (BMP1), transcript variant 8MP1-3, mRNA 738.1 0.0004; ⁇ SPj
• QPCT ILMN 6S10 Homo sapiens gtutaminyt-pepUde cyclotransferase (QPCT), mRNA. 727.S 0.00001
• ECM2 ECM2
• COL4A5 ILMN. .13514 Homo sapiens eoBagen. typo IV. alpha 5 (COL4A5). transcript variant 1 , mRNA.. 606.4 0.00007 +SP!
• LTBP2 ILMN. .139258 Homo sapiens latent transforming growth factor beta binding protein 3 (LTBP3), mRNA. 515 0.00855
• ANG ILMN Homo sapiens angiogenic ribonuclease, RNase A family, 5 (ANG), transcript variant 2, 477.1 0.00001 V mRNA.
• COL11A1 ILM .162953 Homo sapiens collagen, type XI, alpha 1 (COL11A1). transcript variant B. mRNA. 421.7 0.0042!
• transcript variant 1 mRNA. ⁇
• TNFSF15 ILMN. .2068 Homo sapiens tumor necrosis factor (Ugand) superfamily, member 15 (TNFSF1S). 351.6 0.0020! ⁇ SP
• PENK ILMN. .9859 Homo sapiens proenkephalin (PEN ), mRNA. 349 0,00132
• OLR1 ILMN. .17381 Homo sapiens oxidized low density lipoprotein (lectuv!ike) receptor 1 (OLR1), mRNA. 310.4 0.00264 (-SP)
• transcript variant 1 mRNA.
• SPP1 ILMN. .9394 Homo sapiens secreted phosphoprotein 1 (SPP1 ), transcript variant 2 (Osteopontin) , 302.8 0.00001 ⁇ SP!
• TNFSF12 ILMN. .25202 Homo sapiens tumor necrosis factor (figand) superfamily, member 12 (TNFSF12), 2949 0.0048! ⁇ SPj transcript variant 2. mRNA.
• ST3GAL4 ILMN. .19390 Homo sapiens ST3 beta-galactoside alpha-2,3-sialyllransferase 4 (ST3GAL4), mRNA. 2924 0.0039!
• GDF5 ILMN. .27925 Homo sapiens growth differentiation factor 5 (GDF5), mRNA. 280.4 0.00395 ⁇ SP!
• LAMC2 ILMN. .28991 Homo sapiens laminin, gamma 2 (LAMC2L transcript variant 2, mRNA. 257 3 0.00001
• NEGRI ILMN_20523 Homo sapiens growth regulator 1 (NEGRI), mRNA. 247 0.00527 ⁇ SPj
• IL28B rLMN_4533 Homo sapiens interieukin 288 (interferon, lambda 3) (IL28B). mRNA. 243 0.00527
• GHR ILMNJI2966 Homo sapiens growth hormone receptor (GHR). mRNA. 238.7 0.00527 ⁇ spj
• ADAMTS2 L N_20S66 Homo sapiens ADAM merattopeptidase. with thrombospondin type 1 motif, 2 233.9 o.ooooe
• WNT10A ILMN_12046 Homo sapiens wingless-type MMTV integration site family, 10A (WNT10A), 232.8 0.00527 +SP
• C6orf15 ILMN_8387 Homo sapiens chromosome 6 open reading frame 15 (C6orf15), mRNA. 228.1 0.00527 ⁇ spj
• THSD4 ILMN_25S98 Homo sapiens fhrombospondin, type 1, domain containing 4 (THS04), mRNA. 227.9 0.00527 ⁇ SPj
• FAS ILMN_9068 Homo sapiens Fas (TNF superfamify, member 6) (FAS), transcript variant 3, 2273 000007 ⁇ SPi mRNA.
• C3 ILMN_S682 Homo sapiens complement component 3 (C3), mRNA. 213.7 o.ooos: ⁇ SPi V
• FGL2 ILMNJ9861 Homo sapiens fibrinogen-like 2 (FGL2), mRNA. 212.3 0.00527
• BDNF ILMN_26926 Homo sapiens brain-derived neurotrophic factor (BDNF), transcript variant 3. mRNA. 206.7 0.00651 ⁇ spj V
• MMP14 ILMN_75I1 Homo sapiens matrix metallopepridase 14 (membrane (M P14), mRNA. 202.6 0.00527 ⁇ SP
• CCL26 ILMN 6946 Homo sapiens chemokine (C-C motif) ligand 26 (CCL26). mRNA. 200.6 0.00527 ⁇ spj
• LIFR IL NJ5930 Homo sapiens leukemia inhibitory factor receptor alpha (LIFR), mRNA. 190.3 0.00527 (-SP),
• AOAMTS7 ILMN_2S45 Homo sapiens ADAM metallopeptidase with thrombospondtn type I motrf, 7 185.4 0.00527 +SP
• ADAMTS7 mRNA
• CEL ILMN 26946 Homo caifcovyl ester lipase (bile salt-stimulared lipase) (CEL), mRNA. 182 0.00527 ⁇ SP!
• SFRP4 IL NJ3024 Homo sapiens secreted frizzled related protein 4 (SFRP4). mRNA. 176.5 O.0O463 ⁇ spj
• WNT3 ILMN_1260 Homo sapiens wingless-type MMTV integration site family, member 3 (WNT3). mRNA. 1745 000527 *spj
• APOC1 ILMN_14337 Homo sapiens apoltpoprotein C-t (APOC1), mRNA. 172.5 000527
• SGD3 ILMN 20909 Homo sapiens superoxide dismutase 3, extracellular (SOD3), mRNA. 172.5 0.00527 ⁇ spj
• PGF ILMN_27436 Homo sapiens placental growth factor (PGF), mRNA. 168.3 0.00527 ⁇ SPi V
• IFI30 ILMN.23180 Homo sapiens interferon, gamma-inducible protein 30 (IFI30). mRNA. 1668 000527 ⁇ spj •1
• BGLAP ILMNJ 7038 Homo sapiens bone gamma-carboxygrufamale (gla) protein (osteocalcin) (8GLAP), 1663 0.00527
• LA A1 IL N_138581 PREDICTED Homo sapiens alpha 1 (LAMA1), mRNA. 166.1 O.OOOOE ⁇ spj
• LFNG lLMN_t 66415 Homo sapiens LFNG O-fucosylpeptide 3"beta-N-ace!ylglua ⁇ mriiylrransferass (LFNG). 164.9 0.0095S •1 transcript variant 2, mRNA.
• IL26 ILMN_7281 Homo sapiens interieukin 26 (IL25). mRNA. 163.7 0.006SS +SP
• GPX3 ILMN .137905 Homo sapiens glutathione peroxidase 3 (GPX3), mRNA. 159.1 0.0065S ⁇ SP.
• GNRH1 ILMN_28035 Homo sapiens gonadotropin-reteasing hormone t -releasing hormone) 158.3 0.Q06SS
• CST9 IL N_4872 Homo sapiens cystatin 9 (testatin) (CST9), mRNA. 158.1 0.0066S +SPj
• transcript variant 2 mRNA.
• FMOD ILMN 29801 Homo sapiens frbromodutin (FMOD), mRNA. 153.2 0.00791 +SPi
• CYTLI ILMN_7848 Homo sapiens cytokine-tike 1 (CYTLI), mRNA. 141.3 0.00921 ⁇ spj ⁇ '
• SOD1 ILMNJ4302 Homo sapiens superoxide dismutase 1, soluble (SOD1), mRNA. 14588.3 0 (-SP).
• ANXA2 ll_MN_9658 Homo sapiens A2 (ANXA2), transcript variant 1, mRNA. 146182 0 (-SP)!
• FTH1 ILMNJ 1546 Homo sapiens ferritin, heavy polypeptide t (FTH1 ), mRNA. 5403.3 0 (-SP)]
• ADAMTS1 IL N_11081 Homo sapiens ADAM metaltopeptidase with thrombospondin type 1 motif, 1 4270.4 0 +spj
• ADAMTS1 mRNA
• CDH13 ILMN_26240 Homo sapiens cadherin 13. H-cadherin (heart) (CDH13), mRNA. 3635.6 c ⁇ SP
• HDGF ILMNJ6816 Homo sapiens hepatoma-derived growth factor (high-mobflHy group protein 1 -tike) 2668.1 0 (-SP)j
• HDGF HDGF
• GPI ILMN 7872 Homa sapiens glucose phosphate isomerase (GPI), mRNA 2377.3 0 ⁇ -sp)
• ISG15 ILMN_6174 Homo sapiens ISG15 ubiquKin-ffke modifier (ISG15). mRNA 2274.5 0 (-SP)j V
• IL1B ILMN_27277 Homo sapiens interleukm 1, beta (IL1B). mRNA. 2265.7 c (-SP)j AP2K2 ILMN 24956 Homo sapiens mtogen-activated protein kinase kinase 2 (MAP2K2), mRNA. 2215.9 0 (-SP)!
• LGALS3 ILMNJ4333 Homo sapiens lectin, galactoside-binding, soluble, 3 (gatectin 3) (LGALS3), mRNA. 1718.8 0 (-SP)I
• IL32 ILMNJ 7936 Homo sapiens irrterteutrin 32 (IL32), transcript variant 4, mRNA. 1564.4 0 (-SP)!
• FGF2 1LMNJS7999 Homo sapiens fibroblast growth factor 2 (basic) (FGF2), mRNA 1295.2 c (-SP)I ⁇
• RGMB ILMNJ3578 Homo sapiens RGM domain family, member 8 (RGMB), transcript variant 2.
• VASH1 ILMNJ905 Homo sapiens vasohibin 1 (VASH 1 ), mRNA. 601.2 0 (-SP)j
• KIAAQ564 ILMNJ6676 Homo sapiens KIAA0564 (KIAA0564), transcript variant 2, mRNA. 239.9 0 (-SP)j
• AGGF1 ILMNJ 76602 Homo sapiens angiogenic factor with G patch and FHA domains 1 (AGGF 1 ), mRNA. 427.4 o.oooo; (-sp.il
• VEGFA ILMN_5181 Homo sapiens vascular endothelial growth factor A (VEGFA), transcript variant 3, 238.2 0.00759 ⁇ spj mRNA.
• IL15 ILMNJ 6803 Homo sapiens crizofici 15 (IL15), transcript variant 1, mRNA. 186.8 000137 (-spj!
• FGF9 IL NJ771 Homo sapiens fibroblast growth factor 9 (g!ia-activaiing factor) (FGF9), mRNA 158.3 0.00527 (-SP)j
• APOM ILMNJ9748 Homo sapiens apoNpoprotein M (APOM), mRNA 154.7 0.0065S (-SP)I
• WNT2B ILMNJ8414 Homo sapiens wingless-type MMTV integration site family, member 2B (WNT2B). 158 0.00011 (-SP)j transcript variant WNT-2B2, mRNA.
• TMPO P42166 Lamina-associated polypeptide 2 isoform alpha 83.1 739
• Antibody array 1 45 molecules essential for muscle formation
• Antibody array 3 143 molecules with reported roles in skeletal muscle homeostasis, muscle formation and regeneration
• Table T4 The Human muscle "soluble” Secretome : a catalogue of 443 gene products soluble "secreted candidates"
• ILM .20566 ADAMTS-2 precursor ( . . .
• ILMN_2074 PotyfADP-ribose) gtycorr/drotase ARH3 V ILMN 19039 Aipf! a-fetoprotefn precursor
• ILMN_ 4097 Alfcofine pttosptiatnse, Ussue-nonepecfrtc Isozyme precursor
• ILMN_3tB91 Angiopotefirvrelotect protein 4 precuroor

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