US20110003008A1 - Mesenchymal stem cell particles - Google Patents

Mesenchymal stem cell particles Download PDF

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US20110003008A1
US20110003008A1 US12/918,122 US91812209A US2011003008A1 US 20110003008 A1 US20110003008 A1 US 20110003008A1 US 91812209 A US91812209 A US 91812209A US 2011003008 A1 US2011003008 A1 US 2011003008A1
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exosome
disease
mesenchymal stem
ligand
protein
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Sai Kiang Lim
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Agency for Science Technology and Research Singapore
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    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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Definitions

  • the present invention relates to the fields of development, cell biology, molecular biology and genetics. More particularly, the invention relates to a method of deriving mesenchymal stem cells from embryonic stem cells.
  • Stem cells unlike differentiated cells, have the capacity to divide and either self-renew or differentiate into phenotypically and functionally different daughter cells (Keller, Genes Dev. 2005; 19:1129-1155; Wobus and Boheler, Physiol Rev. 2005; 85:635-678; Wiles, Methods in Enzymology. 1993; 225:900-918; Choi et al, Methods Mol Med. 2005; 105:359-368).
  • MSCs Mesenchymal stem cells
  • BM bone marrow
  • adipose tissues ad
  • Mesenchymal stem cells have been used in clinical and preclinical applications to treat a wide range of diseases 1,2 including musculoskeletal tissue bioengineering 3,4 and heart disease 5,6 .
  • the therapeutic capacity of MSCs to treat a wide spectrum of diseases in clinical and preclinical applications to treat a wide range of diseases [A1,A2] e.g. GVHD [A1] in musculoskeletal tissue bioengineering [A3,A4] and heart disease [A5,A6] has been attributed to their potential to differentiate into many different reparative cell types.
  • the biological property may comprise a biological activity of a mesenchymal stem cell conditioned medium (MSC-CM).
  • the biological activity may comprise cardioprotection.
  • the particle may be capable of reducing infarct size.
  • Reduction of infarct may be assayed in a mouse or pig model of myocardial ischemia and reperfusion injury.
  • the particle may be capable of reducing oxidative stress.
  • the reduction of oxidative stress may be assayed in an in vitro assay of hydrogen peroxide (H 2 O 2 )-induced cell death.
  • the particle comprise a vesicle.
  • the particle may comprise an exosome.
  • the particle may contain at least 70% of proteins in an mesenchymal stem cell conditioned medium (MSC-CM).
  • the proteins may be selected from the list shown in Table D1 or may be gene products of the genes shown in Table D2.
  • the particle may comprise a complex of molecular weight >100 kDa.
  • the complex of molecular weight >100 kDa may comprise proteins of ⁇ 100 kDa.
  • the particle may comprise a complex of molecular weight >300 kDa.
  • the complex of molecular weight >100 kDa may comprise proteins of ⁇ 300 kDa.
  • the particle may comprise a complex of molecular weight >1000 kDa.
  • the particle may have a size of between 2 nm and 200 nm.
  • the particle may have a size of between 50 ⁇ m and 150 nm.
  • the particle may have a size of between 50 nm and 100 nm.
  • the size of the particle may be determined by filtration against a 0.2 ⁇ M filter and concentration against a membrane with a molecular weight cut-off of 10 kDa.
  • the size of the particle may be determined by electron microscopy.
  • the particle may comprise a hydrodynamic radius of below 100 nm. It may comprise a hydrodynamic radius of between about 30 nm and about 70 nm. It may be between about 40 nm and about 60 nm, such as between about 45 nm and about 55 nm.
  • the mesenchymal stem cell particle may comprise a hydrodynamic radius of about 50 nm. The hydrodynamic radius may be determined by laser diffraction or dynamic light scattering.
  • the particle may comprise a lipid selected from the group consisting of: phospholipid, phosphatidyl serine, phosphatidyl inositol, phosphatidyl choline, shingomyelin, ceramides, glycolipid, cerebroside, steroids, cholesterol.
  • the cholesterol-phospholipid ratio may be greater than 0.3-0.4 (mol/mol).
  • the particle may comprise a lipid raft.
  • the particle may be insoluble in non-ionic detergent, preferably Triton-X100.
  • the particle may be such that proteins of the molecular weights specified substantially remain in the complexes of the molecular weights specified, when the particle is treated with a non-ionic detergent.
  • the particle may be sensitive to cyclodextrin, preferably 20 mM cyclodextrin.
  • the particle may be such that treatment with cyclodextrin causes substantial dissolution of the complexes specified.
  • the particle may comprise ribonucleic acid (RNA).
  • RNA ribonucleic acid
  • the particle may have an absorbance ratio of 1.9 (260:280 nm).
  • the particle may comprise a surface antigen selected from the group consisting of: CD9, CD109 and thy-1.
  • a method of producing a particle according to any preceding claim comprising isolating the particle from a mesenchymal stem cell conditioned medium (MSC-CM).
  • MSC-CM mesenchymal stem cell conditioned medium
  • the method may comprise separating the particle from other components based on molecular weight, size, shape, composition or biological activity.
  • the weight may be selected from the weights set out above.
  • the size may be selected from the sizes set out above.
  • the composition may be selected from the compositions set out above.
  • the biological activity may be selected from the biological activities set out above.
  • the method may comprise obtaining a mesenchymal stem cell conditioned medium (MSC-CM). It may comprise concentrating the mesenchymal stem cell conditioned medium.
  • the mesenchymal stem cell conditioned medium may be concentrated by ultrafiltration over a >1000 kDa membrane.
  • the method may comprise subjecting the concentrated mesenchymal stem cell conditioned medium to size exclusion chromatography.
  • a TSK Guard column SWXL, 6 ⁇ 40 mm or a TSK gel G4000 SWXL, 7.8 ⁇ 300 mm column may be employed.
  • the method may comprise selecting UV absorbant fractions, for example, at 220 nm, that exhibit dynamic light scattering.
  • the dynamic light scattering may be detected by a quasi-elastic light scattering (QELS) detector.
  • the method may comprise collecting fractions which elute with a retention time of 11-13 minutes, such as 12 minutes.
  • a pharmaceutical composition comprising a particle as described together with a pharmaceutically acceptable excipient, diluent or carrier.
  • such a particle or such a pharmaceutical composition for use in a method of treating a disease.
  • the present invention in a 6 th aspect, provides use of such a particle in a method of treatment of a disease in an individual.
  • the disease may be selected from the group consisting of: cardiac failure, bone marrow disease, skin disease, burns and degenerative diseases such as diabetes, Alzheimer's disease, Parkinson's disease and cancer.
  • the disease may be selected from the group consisting of: myocardial infarction, a cutaneous wound, a dermatologic disorder, a dermatological lesion, dermatitis, psoriasis, condyloma, verruca, hemangioma, keloid, skin cancer, atopic dermatitis, Behcet disease, chronic granulomatous disease, cutaneous T cell lymphoma, ulceration, a pathological condition characterised by initial injury inducing inflammation and immune dysregulation leading to chronic tissue remodeling including fibrosis and loss of function, renal ischemic injury, cystic fibrosis, sinusitis and rhinitis or an orthopaedic disease.
  • the particle may be used to aid wound healing, scar reduction, bone formation, a bone graft or bone marrow transplantation in an individual.
  • the particle may be used (i) in the regulation of a pathway selected from any one or more of the following: cytoskeletal regulation by Rho GTPase, cell cycle, integrin signalling pathway, Inflammation mediated by chemokine & cytokine signaling pathway, FGF signaling pathway, EGF receptor signaling pathway, angiogenesis, plasminogen activating cascade, blood coagulation, glycolysis, ubiquitin proteasome pathway, de novo purine biosynthesis, TCA cycle, phenylalanine biosynthesis, heme biosynthesis; (ii) in the regulation of processes including any one or more of the following: cell structure and motility, cell structure, cell communication, cell motility, cell adhesion, endocytosis, mitosis, exocytosis, cytokinesis, cell cycle, immunity and defense, cytokine/chemokine mediated immunity, macrophage-mediated immunity, granulocyte-mediated immunity, ligand-mediated signaling, cytokine and chemokine mediated signal
  • a delivery system for delivering a particle comprising a source of particle together with a dispenser operable to deliver the particle to a target.
  • FIG. 3 Oxidative Stress. Viability of CEM cells in either CM or Non-CM and treated with H 2 O 2 . (* p ⁇ 0.05, A). Conditioned medium protects cells from death induced by H 2 O 2 .
  • Infarct area sections from pigs treated with Non-CM (B), CM (C), or saline (D) were stained for 8-OHdG, a product of nuclear oxidative stress. Quantification of 8-OHdG positive nuclei was assessed at 200 ⁇ magnification and is depicted in Figure E.
  • CM reduces oxidative stress.
  • FIG. 4 TGF- ⁇ Signaling and Apoptosis.
  • Beta-tubulin was assessed as a loading control, and no differences were found in beta-tubulin expression between the groups (E).
  • FIG. 6 Chemically defined medium conditioned by hESC-MSC HuES9.E1 cells for three days was filtered through a 0.22 ⁇ filter.
  • the conditioned medium (CM) was concentrated 25 times by filtering through a membrane with a 10 kD MW cut-off.
  • the concentrated CM was then centrifuged through a membrane with either a 100 kD or a 300 kD MW cut-off resulting in a 4:1 (vol:vol) filtrate to retentate ratio.
  • the unfractionated CM, filtrate and retentate samples were loaded in the volume ratio of 5:4:1
  • FIG. 7 Identity of components in the filtrate after filtration through a membrane with a 100 kD MW cut-off.
  • NCM non-conditioned medium
  • CM Conditioned medium
  • the filtrate after CM was filtered through membrane with a 100 kD MW cut-off (100 kD filtrate) and NCM was separated on a SDS-PAGE. The gel was stained with silver to visualize protein bands;
  • CM was filtered through a 100 kD MW cut-off membrane to produce a retentate to filtrate volume ratio of 4:1.
  • the retentate, filtrate, and the different protein supplements in the chemically defined medium namely insulin-transferin-selenoprotein, FGF2, EGF and PDGF AB were separated on a SDS-PAGE
  • the retentate and filtrate was loaded in a volume ratio of 4:1
  • FIG. 8 Relative sizes of biological materials and pore sizes in membranes. Reprint from Spectrum ⁇ Laboratory.
  • FIG. 9 Relative AAR in mice after ischemia/reperfusion.
  • Myocardial infarction was induced by left coronary artery (LCA) occlusion by suture ligation for 30 minutes, and reperfusion was initiated by removal of suture.
  • mice Five minutes before reperfusion, mice were treated with tail vein injection of 20 ⁇ l unfractionated MSC-CM (10-220 nm), 20 ⁇ l of ⁇ 100 or 1,000 kD fraction, 4 ⁇ l of >1000 kD retentate or saline. 24 hours later, the hearts were excised. Before excision, the Area At Risk (AAR) was determined by religating the LCA and then perfusing Evans blue through the aorta. AAR was defined as the area not stained by the dye and was expressed as a percentage of the left ventricular wall area.
  • AAR Area At Risk
  • FIG. 10 Effects of conditioned media and fractionated conditioned media on relative infarct size in mice after ischemia/reperfusion. After excision of the heart, infarct size was assessed 24 hours later using TTC and expressed as a percentage of AAR.
  • FIG. 11 Physical entities of between 50-100 nm in diameter were observed using electron microscopy.
  • FIG. 12 Size fractionation of CM after treatment with Triton X-100, CM was treated with a final 0.5 or 1.0% (v/v) TritonX-100 for 30 minutes and then fractionate by filtration through a membrane with MW cut-off of 100 kD to generate a filtrate:retentate volume ratio of 4:1.
  • FIG. 13 Proteins found from MS/MS analysis that are common to exosomes from different cell types 44 .
  • FIG. 14 Cardioprotective Properties of CM Fractions. Myocardial infarct size quantification in mice treated with saline, HEK293 conditioned medium, unfractionated hESC-CM, CM filtered with MWCO of 100 kDa, 300 kDa, 500 kDa, and 1000 kDa or CM concentrated 50 times against membrane with MWCO of 100 kDa fraction of CM or unfractionated CM.
  • FIG. 15 Immunoprecipitation. CM immunoprecipitated with anti-CD81 or mouse IgG as negative control. The immunoprecipitate and supernatant were analysed by western blot hybridization using antibody against CD9, Alix, Tsp-1, pyruvate kinase and SOD1.
  • FIG. 16 Ultracentrifugation of CM.
  • CM was concentrated five times by filtering through a membrane with MWCO of 500 kDa.
  • the retentate, and the unfiltered CM were ultracentrifuged at 200,000 g for 2 hour. The supernatant and the pellet were analysed by western blotting for the presence of CD9.
  • Lane 1-3 different protein concentration of CM.
  • Lane 4 and 5 The pellet (P) and supernatant (S) after ultracentrifugation of unfiltered CM.
  • Lane 6 Retentate after filteration of CM through a membrane with MWCO of 500 kDa.
  • Lane 7-8 The pellet (P) and supernatant (S) after ultracentrifugation of retentate.
  • Lane 9 Filtrate after after filteration of CM through a membrane with MWCO of 500 kDa.
  • FIG. 17 Fractionation on sucrose gradient density.
  • a sucrose density gradient was prepared by layering 14 sucrose solutions of concentrations from 22.8-60% (w/v) in a SW60Ti centrifuge tube with the most concentrated solution at the bottom of the ultracentrifuge tube. The sample was loaded on top of the gradient and ultracentrifuged for 16.5 hours at 200,000 ⁇ g, 4° C. in a SW60Ti rotor (Beckman Coulter Inc.). 13 fractions were collected from top to the bottom of the sucrose gradient. The density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were then analysed by western blot analysis and probed for pyruvate kinase, CD9, CD81 and SOD1.
  • Protein standard molecular weight markers were fractionated on a similar gradient and the distribution of the markers in the different fractions of the sucrose gradient were denoted at the bottom of the figure. a) CM was fractionated, b) CM was treated with lysis buffer before being fractionated on the sucrose gradient.
  • FIG. 18 Trypsinization of CM.
  • CM was digested with trypsin for 0, 0.5, 2, 10 and 20 mins.
  • the partially digested CM was analysed for the presence of CD9 and SOD1.
  • FIG. 19 Analysis of RNA in the CM.
  • RNA was extracted from MSCs and MSC-CM. The purified RNA was denatured and separated on a glyoxal agarose gel and urea-PAGE, respectively.
  • Equal volume of CM was treated with PBS, cyclodextrin, lysis buffer or phospholipase A2. The untreated and treated CM were extracted for RNA. The RNA was separated on urea-PAGE.
  • RNA from CM was treated with RNAse III and the treated RNA was separated in parallel with untreated RNA on a urea-PAGE. RNA in the gels was visualized by staining with ethidium bromide.
  • FIG. 20 Density of RNA in the CM. After fractionation of CM, CM pretreated with lysis buffer and RNA MW standards on a sucrose density gradient equilibrium centrifugation as described in FIG. 4 , each fraction was extracted for RNA. a) the relative RNA yield for each fraction in each sample was plotted against fraction number. The relative concentration was normalized to the highest RNA concentration in each gradient which was arbitrary set as 100%. b) The RNA extracted from each fraction was separated on a urea-PAGE.
  • FIG. 21 RNA was not in CD81+ exosomes.
  • CD81 immunoprecipitation which also precipitated CD9 was performed as described in FIG. 2 .
  • the immunoprecipitate and the supernatant were extracted for RNA and the extracts were separated on a urea-PAGE and visualized by ethidium bromide staining.
  • FIG. 22 Microarray analysis of miRNA in the CM.
  • RNA samples from MSC and the CM were hybridized to microarray chips containing probes for miRNA transcripts listed in Sanger miRBase Release 10.1. The hybridization was performed using two biological replicates of each RNA samples.
  • FIG. 23 HPLC fractionation of CM and NCM.
  • CM and NCM were fractionated on a HPLC using BioSep 54000, 7.8 mm ⁇ 30 cm column.
  • the components in CM or NCM were eluted with 20 mM phosphate buffer with 150 mM of NaCl at pH 7.2.
  • the elution mode was isocratic and the run time was 40 minutes.
  • the eluent was monitored with an UV-visible detector set at 220 nm. The % area under each peak was integrated from the UV-visible detector.
  • the present invention is based on the demonstration that human ESC-derived mesenchymal stem cells (MSCs) mediate cardioprotective effects through secreted large complexes of ⁇ 50-100 nm in diameter. Such complexes or particles may therefore be used for therapeutic means, including for cardioprotection, in place of the cells themselves.
  • MSCs human ESC-derived mesenchymal stem cells
  • the Examples further demonstrate other properties of these particles or complexes.
  • proteins of such particles or complexes have MW-independent sedimentation densities of 1.016-1.215 g/ml that revert to MW-dependent densities upon treatment with a membrane lysis buffer.
  • the secretion also contains RNAs ( ⁇ 300 nts) in cholesterol-rich lipid vesicles.
  • HPLC fractionation and dynamic light scattering studies further indicate that the only detectable particles in the secretion within hydrodynamic radius (rh) range of 1-1000 nm had a rh of 45-55 nm.
  • mesenchymal stem cell particles, complexes or exosomes may be used for a variety of purposes, such as treatment or prevention for cardiac or heart diseases such as ischaemia, cardiac inflammation or heart failure. They may also be used for repair following perfusion injury.
  • MSC mesenchymal stem cell
  • the particle may be derivable from the MSC by any of several means, for example by secretion, budding or dispersal from the MSC.
  • the particle may be produced, exuded, emitted or shed from the MSC.
  • the particle may be secreted into the cell culture medium.
  • the particle may in particular comprise a vesicle.
  • the particle may comprise an exosome.
  • the particles described here may comprise any one or more of the properties of the exosomes described herein.
  • the particle may comprise vesicles or a flattened sphere limited by a lipid bilayer.
  • the particles may comprise diameters of 40-100 nm.
  • the particles may be formed by inward budding of the endosomal membrane.
  • the particles may have a density of ⁇ 1.13-1.19 g/ml and may float on sucrose gradients.
  • the particles may be enriched in cholesterol and sphingomyelin, and lipid raft markers such as GM1, GM3, flotillin and the src protein kinase Lyn.
  • the particles may comprise one or more proteins present in mesenchymal stem cells or mesenchymal stem cell conditioned medium (MSC-CM), such as a protein characteristic or specific to the MSC or MSC-CM. They may comprise RNA, for example miRNA.
  • the particle may comprise molecules secreted by the MSC.
  • Such a particle, and combinations of any of the molecules comprised therein, including in particular proteins or polypeptides, may be used to supplement the activity of, or in place of, the MSCs or medium conditioned by the MSCs for the purpose of for example treating or preventing a disease.
  • the particle may comprise a cytosolic protein found in cytoskeleton e.g. tubulin, actin and actin-binding proteins, intracellular membrane fusions and transport e.g. annexins and rab proteins, signal transduction proteins e.g. protein kinases, 14-3-3 and heterotrimeric G proteins, metabolic enzymes e.g. peroxidases, pyruvate and lipid kinases, and enolase-1 and the family of tetraspanins e.g. CD9, CD63, CD81 and CD82.
  • the particle may comprise one or more tetraspanins.
  • the particles may comprise mRNA and/or microRNA.
  • the term “particle” as used in this document may be taken to mean a discrete entity.
  • the particle may be something that is isolatable from a mesenchymal stem cell (MSC) or mesenchymal stem cell conditioned medium (MSC-CM).
  • the particle may be responsible for at least an activity of the MSC or MSC-CM.
  • the particle may be responsible for, and carry out, substantially most or all of the functions of the MSC or MSC-CM.
  • the particle may be a substitute (or biological substitute) for the MSC or MSC-CM.
  • the particle may be used for any of the therapeutic purposes that the MSC or MSC-CM may be put to use.
  • the particle preferably has at least one property of a mesenchymal stem cell.
  • the particle may have a biological property, such as a biological activity.
  • the particle may have any of the biological activities of an MSC.
  • the particle may for example have a therapeutic or restorative activity of an MSC.
  • media conditioned by MSCs comprise biological activities of MSC and are capable of substituting for the MSCs themselves.
  • the biological property or biological activity of an MSC may therefore correspond to a biological property or activity of an mesenchymal stem cell conditioned medium.
  • the particle may comprise one or more biological properties or activities of a mesenchymal stem cell conditioned medium (MSC-CM).
  • MSC-CM Mesenchymal Stem Cell Conditioned Medium
  • the conditioned cell culture medium such as a Mesenchymal Stem Cell Conditioned Medium (MSC-CM) may be obtained by culturing a mesenchymal stem cell (MSC), a descendent thereof or a cell line derived therefrom in a cell culture medium; and isolating the cell culture medium.
  • the mesenchymal stem cell may be produced by a process comprising obtaining a cell by dispersing a embryonic stem (ES) cell colony.
  • ES embryonic stem
  • the cell, or a descendent thereof may be propagated in the absence of co-culture in a serum free medium comprising FGF2.
  • the particle may be produced or isolated in a number of ways. Such a method may comprise isolating the particle from a mesenchymal stem cell (MSC). Such a method may comprise isolating the particle from an mesenchymal stem cell conditioned medium (MSC-CM).
  • MSC mesenchymal stem cell
  • MSC-CM mesenchymal stem cell conditioned medium
  • the particle may be isolated for example by being separated from non-associated components based on any property of the particle.
  • the particle may be isolated based on molecular weight, size, shape, composition or biological activity.
  • the conditioned medium may be filtered or concentrated or both during, prior to or subsequent to separation.
  • it may be filtered through a membrane, for example one with a size or molecular weight cut-off. It may be subject to tangential force filtration or ultrafiltration.
  • filtration with a membrane of a suitable molecular weight or size cutoff as described in the Assays for Molecular Weight elsewhere in this document, may be used.
  • the conditioned medium may be subject to further separation means, such as column chromatography.
  • column chromatography high performance liquid chromatography (HPLC) with various columns may be used.
  • HPLC high performance liquid chromatography
  • the columns may be size exclusion columns or binding columns.
  • One or more properties or biological activities of the particle may be used to track its activity during fractionation of the mesenchymal stem cell conditioned medium (MSC-CM).
  • MSC-CM mesenchymal stem cell conditioned medium
  • light scattering, refractive index, dynamic light scattering or UV-visible detectors may be used to follow the particles.
  • a therapeutic activity such as cardioprotective activity may be used to track the activity during fractionation.
  • a mesenchymal stem cell particle may be produced by culturing mesenchymal stem cells in a medium to condition it.
  • the mesenchymal stem cells may comprise HuES9.E1 cells.
  • the medium may comprise DMEM.
  • the DMEM may be such that it does not comprise phenol red.
  • the medium may be supplemented with insulin, transferrin, or selenoprotein (ITS), or any combination thereof.
  • It may comprise FGF2.
  • It may comprise PDGF AB.
  • the concentration of FGF2 may be about 5 ng/ml FGF2.
  • the concentration of PDGF AB may be about 5 ng/ml.
  • the medium may comprise glutamine-penicillin-streptomycin or b-mercaptoethanol, or any combination thereof.
  • the cells may be cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more, for example 3 days.
  • the conditioned medium may be obtained by separating the cells from the medium.
  • the conditioned medium may be centrifuged, for example at 500 g. it may be concentrated by filtration through a membrane.
  • the membrane may comprise a >1000 kDa membrame.
  • the conditioned medium may be concentrated about 50 times or more.
  • the conditioned medium may be subject to liquid chromatography such as HPLC.
  • the conditioned medium may be separated by size exclusion. Any size exclusion matrix such as Sepharose may be used.
  • a TSK Guard column SWXL, 6 ⁇ 40 mm or a TSK gel G4000 SWXL, 7.8 ⁇ 300 mm may be employed.
  • the eluent buffer may comprise any physiological medium such as saline. It may comprise 20 mM phosphate buffer with 150 mM of NaCl at pH 7.2.
  • the chromatography system may be equilibrated at a flow rate of 0.5 ml/min.
  • the elution mode may be isocratic. UV absorbance at 220 nm may be used to track the progress of elution. Fractions may be examined for dynamic light scattering (DLS) using a quasi-elastic light scattering (QELS) detector.
  • DLS dynamic light scattering
  • QELS quasi-elastic light scattering
  • Fractions which are found to exhibit dynamic light scattering may be retained. For example, a fraction which is produced by the general method as described above, and which elutes with a retention time of 11-13 minutes, such as 12 minutes, is found to exhibit dynamic light scattering. The r h of particles in this peak is about 45-55 nm. Such fractions comprise mesenchymal stem cell particles such as exosomes.
  • the property of a mesenchymal stem cell may comprise a property of a medium conditioned by a mesenchymal stem cell (MSC-CM). Methods of producing such a mesenchymal stem cell conditioned medium are described elsewhere in this document and are illustrated in for Example 1 below.
  • the property may comprise a biological property such as a biological activity.
  • biological activities include cardioprotection, reduction of oxidative stress and reduction of infarct size.
  • the particle may have a property of mesenchymal stem cells and/or mesenchymal stem cell conditioned medium (MSC-CM) comprising cardioprotection
  • the cardioprotection may comprise restoration or maintenance of cardiac function during ischemia and/or reperfusion.
  • Cardioprotection may for example be assayed using any one or more of the methods described in Examples 5, 10, 14 and 20.
  • the particle may have a property of mesenchymal stem cells and/or mesenchymal stem cell conditioned medium (MSC-CM) comprising the ability to reduce oxidative stress (or cytoprotection).
  • MSC-CM mesenchymal stem cell conditioned medium
  • the reduction of oxidative stress may for example be assayed using an in vitro assay of hydrogen peroxide (H 2 O 2 )-induced cell death.
  • hydrogen peroxide (H 2 O 2 )-mediated oxidative stress is induced in human leukemic CEM cells and cell viability is monitored by Trypan blue-exclusion.
  • Human leukemic CEM cells are incubated with particle, conditioned medium or mesenchymal stem cell (with saline as a control) and treated with 50 ⁇ M H 2 O 2 to induce oxidative stress.
  • Cell viability is assessed using Trypan Blue exclusion at 12, 24, 36 and 48 hours after H 2 O 2 treatment.
  • the reduction of oxidative stress may further be assayed using an in vivo assay of DNA oxidation.
  • In vivo oxidative stress may also be assayed as follows. Pigs are treated with the particle, conditioned medium or mesenchymal stem cell (with saline as a control). Tissue sections of pig heart are obtained. Nuclear oxidative stress in tissue sections of treated and untreated pigs is quantified by 8-OHdG immunostaining for oxidized DNA. The tissue sections are assayed for intense nuclear staining indicative of DNA oxidation and oxidative stress.
  • the particle may have a property of mesenchymal stem cells and/or mesenchymal stem cell conditioned medium (MSC-CM) comprising the ability to reduce infarct size.
  • MSC-CM mesenchymal stem cell conditioned medium
  • Infarct size may for example be assayed using any one or more of the methods described in Examples 6 and 13.
  • the particle may have a molecular weight of greater than 100 kDa. It may have a molecular weight of greater than 500 kDa. For example, it may have a molecular weight of greater than 1000 kDa.
  • the molecular weight may be determined by various means. In principle, the molecular weight may be determined by size fractionation and filtration through a membrane with the relevant molecular weight cut-off. The particle size may then be determined by tracking segregation of component proteins with SDS-PAGE or by a biological assay.
  • the particle may have a molecular weight of greater than 100 kDa.
  • the particle may be such that most proteins of the particle with less than 100 kDa molecular weight segregate into the greater than 100 kDa molecular weight retentate fraction, when subject to filtration.
  • most proteins of the particle with less than 500 kDa molecular weight may segregate into the greater than 500 kDa molecular weight retentate fraction. This indicates that the particle may have a molecular weight of more than 500 kDa.
  • the particle may have a molecular weight of more than 1000 kDa.
  • the particle may be such that when subject to filtration with a membrane with a molecular weight cutoff of 1000 kDa, the relevant biological activity substantially or predominantly remains in the retentate fraction.
  • biological activity may be absent in the filtrate fraction.
  • the biological activity may comprise any of the biological activities of the particle described elsewhere in this document.
  • the biological activity may comprise reduction of infarct size, as assayed in any suitable model of myocardia ischemia and reperfusion injury.
  • the biological activity may be assayed in a mouse or pig model, such as described in the Examples.
  • myocardial ischemia is induced by 30 minutes left coronary artery (LCA) occlusion by suture ligation and reperfusion is initiated by removal of suture.
  • Mice are treated with liquid containing the particles (such as unfractionated MSC-CM), filtrate (such as ⁇ 100 or 1,000 kD fraction), retentate (such as >1000 kD retentate) or saline intravenously via the tail vein, 5 minutes before reperfusion. 24 hours later, the hearts are excised. Before excision, the Area At Risk (AAR) is determined by religating the LCA and then perfusing Evans blue through the aorta.
  • AAR Area At Risk
  • AAR is defined as the area not stained by the dye and is expressed as a percentage of the left ventricular wall area. Infarct size is assessed 24 hours later using Evans blue and TTC. Where the relative infarct size is significantly reduced in animals treated with mesenchymal stem cell conditioned medium (MSC-CM) and the retentate (such as a >1000 kD) fraction when compared to saline, this indicates that the particle has a molecular weight which is higher than the relevant cutoff of the membrane (e.g., greater than 1000 kDa).
  • MSC-CM mesenchymal stem cell conditioned medium
  • retentate such as a >1000 kD
  • the particle may have a size of greater than 2 nm.
  • the particle may have a size of greater than 5 nm, 10 nm, 20 nm, 30 nm, 40 nm or 50 nm.
  • the particle may have a size of greater than 100 nm, such as greater than 150 nm.
  • the particle may have a size of substantially 200 nm or greater.
  • the particle or particles may have a range of sizes, such as between 2 nm to 20 nm, 2 nm to 50 nm, 2 nm to 100 nm, 2 nm to 150 nm or 2 nm to 200 nm.
  • the particle or particles may have a size between 20 nm to 50 nm, 20 nm to 100 nm, 20 nm to 150 nm or 20 nm to 200 nm.
  • the particle or particles may have a size between 50 nm to 100 ⁇ m, 50 ⁇ m to 150 nm or 50 nm to 200 nm.
  • the particle or particles may have a size between 100 nm to 150 nm or 100 nm to 200 nm.
  • the particle or particles may have a size between 150 nm to 200 nm.
  • the size may be determined by various means. In principle, the size may be determined by size fractionation and filtration through a membrane with the relevant size cut-off. The particle size may then be determined by tracking segregation of component proteins with SDS-PAGE or by a biological assay.
  • the size may also be determined by electron microscopy, as described in Example 21.
  • the size may comprise a hydrodynamic radius.
  • the hydrodynamic radius of the particle may be below 100 nm. It may be between about 30 nm and about 70 nm.
  • the hydrodynamic radius may be between about 40 nm and about 60 nm, such as between about 45 nm and about 55 nm.
  • the hydrodynamic radius may be about 50 nm.
  • the hydrodynamic radius of the particle may be determined by any suitable means, for example, laser diffraction or dynamic light scattering.
  • An example of a dynamic light scattering method to determine hydrodynamic radius is set out in Example 33 below.
  • the particle may comprise one or more proteins secreted by a mesenchymal stem cell.
  • the particle may comprise one or more proteins present in mesenchymal stem cell conditioned medium (MSC-CM).
  • the particle may comprise 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more or 70% or more of these proteins.
  • the particle may comprise substantially about 75% of these proteins.
  • the proteins may be defined by reference to a list of proteins or gene products of a list of genes.
  • the proteins may be selected from those set out in Table D1 below.
  • Table D1 comprises the following proteins numbered 1 to 250, as well as the “proteins with unidentified functions”, in the paragraphs below:
  • IPI00021428 Actin, alpha skeletal muscle; 2. IPI00414057 Actin alpha 1 skeletal muscle protein; 3. IPI00008603 Actin, aortic smooth muscle; 4. IPI00021439 Actin, cytoplasmic 1; 5. IPI00023006 Actin, alpha cardiac; 6. IPI00021440 Actin, cytoplasmic 2; 7. IPI00025416 Actin, gamma-enteric smooth muscle; 8. IPI00479925 agrin; 9. IPI00015102 CD166 antigen precursor; 10. IPI00007423 Acidic leucine-rich nuclear phosphoprotein 32 family member B; 11. IPI00413331 36 kDa protein; 12. IPI00412577 34 kDa protein; 13.
  • IPI00413506 33 kDa protein; 14. IPI00418169 Hypothetical protein DKFZp686P03159; 15. IPI00003815 Rho GDP-dissociation inhibitor 1; 16. IPI00004656 Beta-2-microglobulin precursor; 17. IPI00218042 Splice Isoform BMP1-5 of Bone morphogenetic protein 1 precursor; 18. IPI00009054 Splice Isoform BMP1-3 of Bone morphogenetic protein 1 precursor; 19. IPI00014021 Splice Isoform BMP1-1 of Bone morphogenetic protein 1 precursor; 20. IPI00218040 Splice Isoform BMP1-4 of Bone morphogenetic protein 1 precursor; 21.
  • IPI00290085 Neural-cadherin precursor 31. IPI00029739 Splice Isoform 1 of Complement factor H precursor; 32. IPI00012011 Cofilin, non-muscle isoform; 33. IPI00007257 calsyntenin 1 isoform 2; 34. IPI00218539 Splice Isoform B of Collagen alpha-1(XI) chain precursor; 35. IPI00477350 Collagen, type XI, alpha 1; 36. IPI00329573 Splice Isoform Long of Collagen alpha-1(XII) chain precursor; 37. IPI00221384 Splice Isoform Short of Collagen alpha-1(XII) chain precursor; 38.
  • IPI00400935 Collagen alpha-1(XVI) chain precursor; 39. IPI00297646 Collagen alpha-1(I) chain precursor; 40. IPI00164755 Prepro-alpha2(I) collagen precursor; 41. IPI00304962 Collagen alpha-2(I) chain precursor; 42. IPI00021033 Collagen alpha-1(III) chain precursor; 43. IPI00167087 COL3A1 protein; 44. IPI00021034 Collagen alpha-1(IV) chain precursor; 45. IPI00479324 alpha 2 type IV collagen preproprotein; 46. IPI00306322 Collagen alpha-2(IV) chain precursor; 47. IPI00303313 Collagen alpha-1(V) chain precursor; 48.
  • IPI00220701 Splice Isoform 2 of Collagen alpha-3(VI) chain precursor; 57. IPI00072917 alpha 3 type VI collagen isoform 3 precursor; 58. IPI00021828 Cystatin B; 59. IPI00007778 Di-N-acetylchitobiase precursor; 60. IPI00295741 Cathepsin B precursor;
  • Fibronectin precursor (FN) (Cold-insoluble globulin) (CIG). Splice isoform 3; 81. IPI00339225 Splice Isoform 5 of Fibronectin precursor; 82. IPI00339319 Splice Isoform 11 of Fibronectin precursor; 83. IPI00556632 Splice Isoform 12 of Fibronectin precursor; 84. IPI00411462 Hypothetical protein DKFZp686B18150; 85. IPI00029723 Follistatin-related protein 1 precursor; 86. IPI00005401 Polypeptide N-acetylgalactosaminyltransferase 5; 87. IPI00219025 Glutaredoxin-1; 88. IPI00171411 Golgi phosphoprotein 2; 89. IPI00026314 Gelsolin precursor;
  • IPI100024284 Basement membrane-specific heparan sulfate proteoglycan core protein precursor; 98. IPI00297284 Insulin-like growth factor binding protein 2 precursor; 99. IPI00297284 Insulin-like growth factor binding protein 2 precursor; 100. IPI00029236 Insulin-like growth factor binding protein 5 precursor; 101. IPI00029236 Insulin-like growth factor binding protein 5 precursor; 102. IPI00029235 Insulin-like growth factor binding protein 6 precursor; 103. IPI00029235 Insulin-like growth factor binding protein 6 precursor; 104. IPI00016915 Insulin-like growth factor binding protein 7 precursor; 105. IPI00016915 Insulin-like growth factor binding protein 7 precursor; 106.
  • IPI00374397 PREDICTED similar to tropomyosin 4; 116. IPI00374732 PREDICTED: similar to PPIA protein; 117. IPI00402104 PREDICTED: similar to peptidylprolyl isomerase A isoform 1; cyclophilin A; peptidyl-pro; 118. IPI00455415 PREDICTED: similar to Heterogeneous nuclear ribonucleoprotein C-like dJ845O24.4; 119. IPI00454722 PREDICTED: similar to Phosphatidylethanolamine-binding protein; 120. IPI00454852 PREDICTED: similar to Teratocarcinoma-derived growth factor 1;
  • IPI00002802 Protein-lysine 6-oxidase precursor; 122. IPI00410152 latent transforming growth factor beta binding protein 1 isoform LTBP-1L; 123. IPI00220249 Latent transforming growth factor beta-binding protein, isoform 1L precursor; 124. IPI00220249 Latent transforming growth factor beta-binding protein, isoform 1L precursor”; 125. IPI00410152 latent transforming growth factor beta binding protein 1 isoform LTBP-1 L; 126. IPI00020986 Lumican precursor; 127. IPI00291006 Malate dehydrogenase, mitochondrial precursor; 128. IPI00005707 Macrophage mannose receptor 2 precursor; 129.
  • IPI00472718 peptidylprolyl isomerase A isoform 2; 152. IPI00000874 Peroxiredoxin-1; 153. IPI00024915 Peroxiredoxin-5, mitochondrial precursor; 154. IPI00375306 peroxiredoxin 5 precursor, isoform b; 155. IPI00012503 Splice Isoform Sap-mu-0 of Proactivator polypeptide precursor; 156. IPI00374179 proteasome activator subunit 1 isoform 2; 157. IPI00030154 Proteasome activator complex subunit 1; 158. IPI00168812 PTK7 protein tyrosine kinase 7 isoform d precursor; 159.
  • IPI00419941 PTK7 protein tyrosine kinase 7 isoform a precursor; 160. IPI00003590 Quiescin Q6, isoform a; 161. IPI00015916 Bone-derived growth factor (Fragment); 162. IPI00015916 Bone-derived growth factor; 163. IPI00298289 Splice Isoform 2 of Reticulon-4; 164. IPI00021766 Splice Isoform 1 of Reticulon-4; 165. IPI00013895 Calgizzarin; 166. IPI00010402 Hypothetical protein; 167. IPI00218733 Superoxide dismutase; 168. IPI00014572 SPARC precursor; 169.
  • IPI00005614 Splice Isoform Long of Spectrin beta chain, brain 1; 170. IPI00008780 Stanniocalcin-2 precursor; 171. IPI00301288 SEL-OB protein; 172. IPI00216138 Transgelin; 173. IPI00018219 Transforming growth factor-beta-induced protein ig-h3 precursor; 174. IPI00304865 transforming growth factor, beta receptor III”; 175. IPI00296099 Thrombospondin-1 precursor; 176. IPI00032292 Metalloproteinase inhibitor 1 precursor; 177. IPI00027166 Metalloproteinase inhibitor 2 precursor; 178. IPI00220828 Thymosin beta-4; 179. IPI00180240 thymosin-like 3;
  • IPI00552815 Collagen, type V, alpha 1; 207. IPI00552981CDNA PSEC0266 fis, clone NT2RP3003649, highly similar to Homo sapiens fibulin-1D mRNA; 208. IPI00180776 29 kDa protein; 209. IPI00552416 Filamin A, alpha;
  • IPI00640698 Actin, gamma-enteric smooth muscle; 211. IPI00514530 Actin, alpha 1, skeletal muscle; 212. IPI00556442 Insulin-like growth factor binding protein 2 variant (Fragment); 213. IPI00513782 Gelsolin; 214. IPI00478731 29 kDa protein; 215. IPI00396479 24 kDa protein; 216. IPI00334627 39 kDa protein; 217. IPI00555762 PTK7 protein tyrosine kinase 7 isoform a variant (Fragment); 218. IPI00658202 97 kDa protein; 219. IPI00006273 CYR61 protein; 220.
  • IPI00414489 Protein 231.
  • IPI00411463 Protein 232.
  • IPI00556415 Transgelin variant (Fragment);
  • IPI00718825 Calmodulin;
  • IPI00478156 17 kDa protein 235.
  • IPI00386621 CALM3 protein;
  • IPI00647001 Acidic 237.
  • IPI00642650 Similar to Stanniocalcin 2 precursor;
  • IPI00641471 Collagen-like protein; 239.
  • IPI00514669 SH3 domain binding glutamic acid-rich protein like 3; 240.
  • IPI00719422 Triosephosphate isomerase 241.
  • IPI00003734 Putative S100 calcium-binding protein H_NH0456N16.1; 242. IPI00029574 11 kDa protein; 243. IPI00641047 Gelsolin; 244. IPI00647556 Gelsolin; 245. IPI00654821 hypothetical protein LOC54845 isoform 1; 246. IPI00647572 Dickkopf related protein-3 precursor; 247. IPI00639879 Similar to Cytokinesis protein sepA; 248. IPI00657746 Similar to Dedicator of cytokinesis protein 8; 249. IPI00555993 Vascular endothelial growth factor receptor 3 variant; 250. IPI00552545 Dedicator of cytokinesis protein 8.
  • the proteins may be selected from the gene products of the genes set out in Table D2 below.
  • Table D2 comprises the following genes in the paragraphs below:
  • the particle may additionally, or alternatively, comprise any of the gene products of the 201 genes listed below in Table D3. These genes are characterised according to a biological process the gene is involved in.
  • the particle may be employed to affect, control or regulate any of these 58 biological processes.
  • the particle may additionally, or alternatively, comprise any of the gene products of the 201 genes listed below in Table D4. These genes are characterised according to a pathway the gene is involved in.
  • the particle may be employed to affect, control or regulate any of these 30 pathways.
  • the particle may additionally, or alternatively, comprise any of the gene products of the 593 genes and/or 794 gene products listed below in Table D5.
  • the particle may be employed to affect, control or regulate any of the biological processes or pathways the genes or gene products are involved in.
  • the particle may in particular comprise a vesicle.
  • the particle may comprise an exosome.
  • the Examples describe the isolation of the active component in the secretion that confers cardioprotection against the reperfusion injury.
  • the active component may comprise an exosome secreted by the mesenchymal stem cells (MSCs).
  • Exosomes are small membrane vesicles formed in late endocytic compartments (multivesicular bodies) first described to be secreted by reticulocytes in 1983 21 and subsequently found to be secreted by many cells types including various haematopoietic cells, tumours of haematopoietic or non-haematopoietic origin and epithelial cells 22 . They are distinct entities from the more recently described ‘ribonuclease complex’ also named exosome 23 .
  • Exosomes may be defined by morphological and biochemical parameters (see reviews 22,24-35 ). Accordingly, the particles described here may comprise one or more of these morphological or biochemical parameters.
  • Exosomes are classically defined as “saucer-like” vesicles or a flattened sphere limited by a lipid bilayer with diameters of 40-100 nm and are formed by inward budding of the endosomal membrane. Like all lipid vesicles and unlike protein aggregates or nucleosomal fragments that are released by apoptotic cells, exosomes have a density of ⁇ 1.13-1.19 g/ml and float on sucrose gradients. Exosomes are enriched in cholesterol and sphingomyelin, and lipid raft markers such as GM1, GM3, flotillin and the src protein kinase Lyn suggesting that their membranes are enriched in lipid rafts.
  • exosomes contain ubiquitous proteins that appear to be common to all exosomes and proteins that are cell-type specific. Also, proteins in exosomes from the same cell-type but of different species are highly conserved.
  • the ubiquitous exosome-associated proteins include cytosolic proteins found in cytoskeleton e.g. tubulin, actin and actin-binding proteins, intracellular membrane fusions and transport e.g. annexins and rab proteins, signal transduction proteins e.g. protein kinases, 14-3-3 and heterotrimeric G proteins, metabolic enzymes e.g.
  • tetraspanins e.g. CD9, CD63, CD81 and CD82.
  • the tetraspannins are highly enriched in exosomes and are known to be involved in the organization of large molecular complexes and membrane subdomains.
  • exosomes examples include MHC class II molecules in exosomes from MHC class II-expressing cells, CD86 in dendritic cell-derived exosomes, T-cell receptors on T-cell-derived exosomes etc.
  • exosomes do not contain proteins of nuclear, mitochondrial, endoplasmic-reticulum or Golgi-apparatus origin.
  • highly abundant plasma membrane proteins are absent in exosomes suggesting that they are not simply fragments of the plasma membrane.
  • Many of the reported ubiquitous exosome-associated proteins are also present in the proteomic profile of the hESC-MSC secretion.
  • Exosomes are also known to contain mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location 36 .
  • the physiological functions of exosome remain poorly defined. It is thought to help eradicate obsolete proteins, recycle proteins, mediate tramission of infectious particles such as prions and viruses, induce complement resistance, facilitate immune cell-cell communication and transmit cell signaling 1,22,25-28,37-40 .
  • Exosomes have been used in immunotherapy for treatment of cancer 34
  • the particle may be used as a substitute for an MSC or MSC-CM, as described above
  • the particle may be used for any of the therapeutic purposes that MSCs or MSC-CMs are currently being used, or in the future may be used.
  • Mesenchymal stem cells and differentiated cells produced by the methods and compositions described here may be used for, or for the preparation of a pharmaceutical composition for, the treatment of a disease.
  • a disease may comprise a disease treatable by regenerative therapy, including cardiac failure, bone marrow disease, skin disease, burns, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease, etc and cancer. Accordingly, particles from MSCs may be used to treat such diseases.
  • Particles from mesenchymal stem cells such as those made according to the methods and compositions described here may be used for a variety of commercially important research, diagnostic, and therapeutic purposes.
  • the particles from mesenchymal stem cells may in particular be used for the preparation of a pharmaceutical composition for the treatment of disease.
  • disease may comprise a disease treatable by regenerative therapy, including cardiac failure, bone marrow disease, skin disease, burns, degenerative disease such as diabetes, Alzheimer's disease, Parkinson's disease, etc and cancer.
  • BM-MSCs bone marrow derived mesenchymal stem cells
  • the particles from MSCs described here may be used to treat diseases which these functions may have a role in, or whose repair or treatment involves any one or more of these biological processes.
  • the proteins expressed by the MSCs singly or in combination, preferably in the form of particles as described here, may be used to supplement the activity of, or in place of, the MSCs, or media conditioned by the MSCs, for the purpose of for example treating or preventing such diseases.
  • the 201 gene products expressed by the MSCs are shown to activate important signalling pathways in cardiovascular biology, bone development and hematopoiesis such as Jak-STAT, MAPK, Toll-like receptor, TGF-beta signalling and mTOR signaling pathways. Accordingly, the particles from the MSCs, etc, may be used to prevent or treat a disease in which any of these signalling pathways is involved, or whose etiology involves one or more defects in any one or more of these signalling pathways.
  • such particles may be used to treat cardiac failure, bone marrow disease, skin disease, burns and degenerative diseases such as diabetes, Alzheimer's disease, Parkinson's disease and cancer.
  • Such particles may also be used to treat myocardial infarction, a cutaneous wound, a dermatologic disorder, a dermatological lesion, dermatitis, psoriasis, condyloma, verruca, hemangioma, keloid, skin cancer, atopic dermatitis, Behcet disease, chronic granulomatous disease, cutaneous T cell lymphoma, ulceration, a pathological condition characterised by initial injury inducing inflammation and immune dysregulation leading to chronic tissue remodeling including fibrosis and loss of function, renal ischemic injury, cystic fibrosis, sinusitis and rhinitis or an orthopedic disease.
  • the particles may be used to aid wound healing, scar reduction, bone formation, a bone graft or bone marrow transplantation in an individual.
  • conditioned medium should be taken to include not only cell culture medium exposed to MSCs as well as such a composition comprising one or more, preferably substantially all, the polypeptides which are present in the conditioned medium.
  • the particles may also be used as sources for any of the proteins secreted or expressed by the MSCs.
  • the mesenchymal stem cell particle methods and compositions described here may be used for treatment or prevention of heart disease.
  • Heart disease is an umbrella term for a variety for different diseases affecting the heart. As of 2007, it is the leading cause of death in the United States, England, Canada and Wales, killing one person every 34 seconds in the United States alone. Heart disease includes any of the following.
  • Coronary artery disease is a disease of the artery caused by the accumulation of atheromatous plaques within the walls of the arteries that supply the myocardium.
  • Angina pectoris chest pain
  • myocardial infarction myocardial infarction
  • Cardiomyopathy is the deterioration of the function of the myocardium (i.e., the actual heart muscle) for any reason. People with cardiomyopathy are often at risk of arrhythmia and/or sudden cardiac death. Extrinsic cardiomyopathies—cardiomyopathies where the primary pathology is outside the myocardium itself comprise the majority of cardiomyopathies. By far the most common cause of a cardiomyopathy is ischemia.
  • the World Health Organization includes as specific cardiomyopathies: Alcoholic cardiomyopathy, coronary artery disease, congenital heart disease, nutritional diseases affecting the heart, ischemic (or ischaemic) cardiomyopathy, hypertensive cardiomyopathy, valvular cardiomyopathy, inflammatory cardiomyopathy.
  • Intrinsic cardiomyopathies weakness in the muscle of the heart that is not due to an identifiable external cause
  • DCM Dilated cardiomyopathy
  • HCM Hypertrophic cardiomyopathy
  • ARVC Arrhythmogenic right ventricular cardiomyopathy
  • RCM Restrictive cardiomyopathy
  • Noncompaction Cardiomyopathy the left ventricle wall has failed to properly grow from birth and such has a spongy appearance when viewed during an echocardiogram.
  • Cardiovascular disease is any of a number of specific diseases that affect the heart itself and/or the blood vessel system, especially the veins and arteries leading to and from the heart. Research on disease dimorphism suggests that women who suffer with cardiovascular disease usually suffer from forms that affect the blood vessels while men usually suffer from forms that affect the heart muscle itself. Known or associated causes of cardiovascular disease include diabetes mellitus, hypertension, hyperhomocysteinemia and hypercholesterolemia.
  • Types of cardiovascular disease include atherosclerosis
  • Ischaemic heart disease is disease of the heart itself, characterized by reduced blood supply to the organs. This occurs when the arteries that supply the oxygen and the nutrients gets stopped and the heart will not get enough of the oxygen and the nutrients and will eventually stop beating.
  • Heart failure also called congestive heart failure (or CHF), and congestive cardiac failure (CCF) is a condition that can result from any structural or functional cardiac disorder that impairs the ability of the heart to fill with or pump a sufficient amount of blood throughout the body.
  • Cor pulmonale is a failure of the right side of the heart.
  • Hypertensive heart disease is heart disease caused by high blood pressure, especially localised high blood pressure. Conditions that can be caused by hypertensive heart disease include: left ventricular hypertrophy, coronary heart disease, (Congestive) heart failure, hypertensive cardiomyopathy, cardiac arrhythmias, inflammatory heart disease, etc.
  • Inflammatory heart disease involves inflammation of the heart muscle and/or the tissue surrounding it.
  • Endocarditis comprises inflammation of the inner layer of the heart, the endocardium. The most common structures involved are the heart valves. Inflammatory cardiomegaly.
  • Myocarditis comprises inflammation of the myocardium, the muscular part of the heart.
  • Valvular heart disease is disease process that affects one or more valves of the heart.
  • the valves in the right side of the heart are the tricuspid valve and the pulmonic valve.
  • the valves in the left side of the heart are the mitral valve and the aortic valve. Included are aortic valve stenosis, mitral valve prolapse and valvular cardiomyopathy.
  • the particles as described in this document may be delivered to the human or animal body by any suitable means.
  • the delivery system may comprise a source of particles, such as a container containing the particles.
  • the delivery system may comprise a dispenser for dispensing the particles to a target.
  • a delivery system for delivering a particles comprising a source of particles as described in this document together with a dispenser operable to deliver the particles to a target.
  • Delivery systems for delivering fluid into the body include injection, surgical drips, catheters (including perfusion catheters) such as those described in U.S. Pat. No. 6,139,524, for example, drug delivery catheters such as those described in U.S. Pat. No. 7,122,019.
  • Delivery to the lungs or nasal passages may be achieved using for example a nasal spray, puffer, inhaler, etc as known in the art (for example as shown in U.S. Design Pat. D544,957.
  • Delivery to the kidneys may be achieved using an intra-aortic renal delivery catheter, such as that described in U.S. Pat. No. 7,241,273.
  • the particles may for example be used for the treatment or prevention of atherosclerosis.
  • perfusion of particles may be done intravenously to stabilize atherosclerotic plaques or reduce inflammation in the plaques.
  • the particles may be used for the treatment or prevention of septic shock by intravenous perfusion.
  • the particles may be used for the treatment or prevention of heart failure. This may be achieved by chronic intracoronary or intramyocardially perfusion of particles to retard remodeling or retard heart failure.
  • the particles may be used for the treatment or prevention of lung inflammation by intranasal delivery.
  • the particles may be used for the treatment or prevention of dermatological conditions e.g. psoriasis. Long term delivery of particles may be employed using transdermal microinjection needles until the condition is resolved.
  • the particles may be delivered for example to the cardiac tissue (i.e., myocardium, pericardium, or endocardium) by direct intracoronary injection through the chest wall or using standard percutaneous catheter based methods under fluoroscopic guidance for direct injection into tissue such as the myocardium or infusion of an inhibitor from a stent or catheter which is inserted into a bodily lumen.
  • cardiac tissue i.e., myocardium, pericardium, or endocardium
  • the particles may be delivered for example to the cardiac tissue (i.e., myocardium, pericardium, or endocardium) by direct intracoronary injection through the chest wall or using standard percutaneous catheter based methods under fluoroscopic guidance for direct injection into tissue such as the myocardium or infusion of an inhibitor from a stent or catheter which is inserted into a bodily lumen.
  • any variety of coronary catheter, or a perfusion catheter, may be used to administer the compound.
  • the particles may be coated or impregnated on a stent that is placed in a coronary vessel.
  • Mesenchymal stem cells and differentiated cells made according to the methods and compositions described here, and particles derived therefrom, may also be used for tissue reconstitution or regeneration in a human patient in need thereof.
  • the cells are administered in a manner that permits them to graft to the intended tissue site and reconstitute or regenerate the functionally deficient area.
  • the methods and compositions described here may be used to modulate the differentiation of stem cells.
  • Mesenchymal stem cells and differentiated cells and particles derived therefrom may be used for tissue engineering, such as for the growing of skin grafts.
  • Modulation of stem cell differentiation may be used for the bioengineering of artificial organs or tissues, or for prosthetics, such as stents.
  • Mesenchymal stem cells and differentiated cells made by the methods and compositions described here and particles derived therefrom may be used for the treatment of cancer.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • glial cell tumors such as glioblastoma and neurofibromatosis
  • cervical cancer ovarian cancer
  • liver cancer bladder cancer
  • hepatoma hepatoma
  • breast cancer colon cancer
  • colorectal cancer endometrial carcinoma
  • salivary gland carcinoma salivary gland carcinoma
  • kidney cancer renal cancer
  • prostate cancer prostate cancer
  • vulval cancer thyroid cancer
  • hepatic carcinoma various types of head and neck cancer.
  • solid tumor cancer including colon cancer, breast cancer, lung cancer and prostrate cancer
  • hematopoietic malignancies including leukemias and lymphomas
  • Hodgkin's disease aplastic anemia
  • skin cancer and familiar adenomatous polyposis.
  • Further examples include brain neoplasms, colorectal neoplasms, breast neoplasms, cervix neoplasms, eye neoplasms, liver neoplasms, lung neoplasms, pancreatic neoplasms, ovarian neoplasms, prostatic neoplasms, skin neoplasms, testicular neoplasms, neoplasms, bone neoplasms, trophoblastic neoplasms, fallopian tube neoplasms, rectal neoplasms, colonic neoplasms, kidney neoplasms, stomach neoplasms, and parathyroid neoplasms.
  • Breast cancer, prostate cancer, pancreatic cancer, colorectal cancer, lung cancer, malignant melanoma, leukaemia, lympyhoma, ovarian cancer, cervical cancer and biliary tract carcinoma are also included
  • the mesenchymal stem cells and differentiated cells made according to the methods and compositions described here may also be used in combination with anticancer agents such as endostatin and angiostatin or cytotoxic agents or chemotherapeutic agent.
  • anticancer agents such as endostatin and angiostatin or cytotoxic agents or chemotherapeutic agent.
  • drugs such as such as adriamycin, daunomycin, cis-platinum, etoposide, taxol, taxotere and alkaloids, such as vincristine, and antimetabolites such as methotrexate.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. I, Y, Pr), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • the term includes oncogene product/tyrosine kinase inhibitors, such as the bicyclic ansamycins disclosed in WO 94/22867; 1,2-bis(arylamino) benzoic acid derivatives disclosed in EP 600832; 6,7-diamino-phthalazin-1-one derivatives disclosed in EP 600831; 4,5-bis(arylamino)-phthalimide derivatives as disclosed in EP 516598; or peptides which inhibit binding of a tyrosine kinase to a SH2-containing substrate protein (see WO 94/07913, for example).
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include Adriamycin, Doxorubicin, 5-Fluorouracil (5-FU), Cytosine arabinoside (Ara-C), Cyclophosphamide, Thiotepa, Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincristine, VP-16, Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin, Aminopterin, Dactinomycin, Mitomycins, Nicotinamide, Esperamicins (see U.S. Pat.
  • the particles described here may be isolated or produced from mesenchymal stem cell conditioned medium (MSC-CM).
  • MSCs suitable for use in the production of conditioned media and particles may be made by any method known in the art.
  • MSCs may be made by propagating a cell obtained by dispersing a embryonic stem (ES) cell colony, or a descendent thereof, in the absence of co-culture in a serum free medium comprising FGF2. This is described in detail in the sections below.
  • ES embryonic stem
  • MSC mesenchymal stem cells
  • MSC-like cells from hESCs
  • hTERT human telomerase reverse transcriptase
  • the particles may therefore be made from MSCs derived by the use of a clinically relevant and reproducible protocol for isolating similar or identical (such as homogenous) MSC populations from differentiating hESCs.
  • the method comprises dispersing a embryonic stem (ES) cell colony into cells. The cells are then plated out and propagated. The cells are propagated in the absence of co-culture in a serum free medium comprising fibroblast growth factor 2 (FGF2), in order to obtain mesenchymal stem cells (MSCs).
  • FGF2 fibroblast growth factor 2
  • the protocol does not require serum, use of mouse cells or genetic manipulations and requires less manipulations and time, and is therefore highly scalable.
  • the protocol may be used for the isolation of MSCs from two different hESC lines, HuES9 and H-1 and also a third one, Hes-3.
  • Human ES cell derived MSCs (hESC-MSCs) obtained by the methods and compositions described here are remarkably similar to bone-marrow derived MSCs (BM-MSCs).
  • the embryonic stem cell culture may comprise a human embryonic stem cell (hESC) culture.
  • hESC human embryonic stem cell
  • a method of generating mesenchymal stem cells comprises trypsinizing and propagating hESCs without feeder support in media supplemented with FGF2 and optionally PDGF AB before sorting for CD105+CD24-cells.
  • the method may comprise sorting for CD105+, CD24 ⁇ cells from trypsinized hESCs one week after feeder-free propagation in a media supplemented with FGF2 and optionally PDGF AB will generate to generate a hESC-MSC cell culture in which at least some, such as substantially all, or all cells are similar or identical (such as homogenous) to each other.
  • the MSCs produced by this method may be used to produce mesenchymal stem cell conditioned medium (MSC-CM), from which the particles may be isolated.
  • MSC-CM mesenchymal stem cell conditioned medium
  • One method of producing mesenchymal stem cells may comprise dispersing or disaggregating an embryonic stem cell colony into cells.
  • the embryonic stem cell colony may comprise a huES9 colony (Cowan C A, Klimanskaya I, McMahon J, Atienza J, Witmyer J, et al. (2004) Derivation of embryonic stem - cell lines from human blastocysts . N Engl J Med 350: 1353-1356) or a H1 ESC colony (Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A, Swiergiel J J, et al. (1998) Embryonic Stem Cell Lines Derived from Human Blastocysts . Science 282: 1145-1147).
  • the cells in the colony may be disaggregated or dispersed to a substantial extent, i.e., at least into clumps.
  • the colony may be disaggregated or dispersed to the extent that all the cells in the colony are single, i.e., the colony is completely disaggregated.
  • the disaggregation may be achieved with a dispersing agent.
  • the dispersing agent may be anything that is capable of detaching at least some embryonic stem cells in a colony from each other.
  • the dispersing agent may comprise a reagent which disrupts the adhesion between cells in a colony, or between cells and a substrate, or both.
  • the dispersing agent may comprise a protease.
  • the dispersing agent may comprise trypsin.
  • the treatment with trypsin may last for example for 3 minutes or thereabouts at 37 degrees C.
  • the cells may then be neutralised, centrifuged and resuspended in medium before plating out.
  • the method may comprise dispersing a confluent plate of human embryonic stem cells with trypsin and plating the cells out.
  • the disaggregation may comprise at least some of the following sequence of steps: aspiration, rinsing, trypsinization, incubation, dislodging, quenching, re-seeding and aliquoting.
  • aspiration rinsing
  • trypsinization trypsinization
  • incubation dislodging
  • quenching quenching
  • re-seeding aliquoting.
  • the following protocol is adapted from the Hedrick Lab, UC San Diego (http://hedricklab.ucsd.edu/Protocol/COSCell.html).
  • the media is aspirated or generally removed from the vessel, such as a flask.
  • the cells are rinsed with a volume, for example 5-10 mls, of a buffered medium, which is may be free from Ca 2+ and Mg 2+ .
  • the cells may be rinsed with calcium and magnesium free PBS.
  • an amount of dispersing agent in buffer is added to the vessel, and the vessel rolled to coat the growing surface with the dispersing agent solution. For example, 1 ml of trypsin in Hank's BSS may be added to a flask.
  • the cells are left for some time at a maintained temperature.
  • the cells may be left at 37° C. for a few minutes (e.g., 2 to 5 minutes).
  • the cells may be dislodged by mechanical action, for example by scraping or by whacking the side of the vessel with a hand. The cells should come off in sheets and slide down the surface.
  • a volume of medium is added to the flask.
  • the medium may comprise a neutralising agent to stop the action of the dispersing agent.
  • the dispersing agent is a protease such as trypsin
  • the medium may contain a protein, such as a serum protein, which will mop up the activity of the protease.
  • 3 ml of serum containing cell culture medium is added to the flask to make up a total of 4 mls.
  • the cells may be pipetted to dislodge or disperse the cells.
  • the cells are re-seeded into fresh culture vessels and fresh medium added.
  • a number of re-seedings may be made at different split ratios.
  • the cells may be reseeded at 1/15 dilution and 1/5 dilution.
  • the cells may be re-seeded by adding 1 drop of cells into a 25 cm 2 flask and 3 drops into another to re-seed the culture, and 7-8 mls media is then added to each to provide for 1/15 dilution and 1/5 dilution from for example a 75 cm 2 flask.
  • the cells may be aliquoted into new dishes or whatever split ratio is desired, and media added.
  • the method includes the following steps: human ES cells are first grown suspended in non-adherent manner to form embryoid bodies (EBs). 5-10 day old EBs are then trypsinized before plating as adherent cells on gelatine coated tissue culture plates.
  • EBs embryoid bodies
  • the disaggregated cells may be plated and maintained as a cell culture.
  • the cells may be plated onto a culture vessel or substrate such as a gelatinized plate.
  • a culture vessel or substrate such as a gelatinized plate.
  • the cells are grown and propagated without the presence of co-culture, e.g., in the absence of feeder cells.
  • the cells in the cell culture may be grown in a serum-free medium which is supplemented by one or more growth factors such as fibroblast growth factor 2 (FGF2) and optionally platelet-derived growth factor AB (PDGF AB), at for example 5 ng/ml.
  • FGF2 fibroblast growth factor 2
  • PDGF AB platelet-derived growth factor AB
  • the cells in the cell culture may be split or subcultured 1:4 when confluent, by treatment with trypsin, washing and replating.
  • the cells may be cultured in the absence of co-culture.
  • co-culture refers to a mixture of two or more different kinds of cells that are grown together, for example, stromal feeder cells.
  • the inner surface of the culture dish is usually coated with a feeder layer of mouse embryonic skin cells that have been treated so they will not divide.
  • the feeder layer provides an adherent surface to enable the ES cells to attach and grow.
  • the feeder cells release nutrients into the culture medium which are required for ES cell growth.
  • the ES and MSC cells may be cultured in the absence of such co-culture.
  • the cells may be cultured as a monolayer or in the absence of feeder cells.
  • the embryonic stem cells may be cultured in the absence of feeder cells to establish mesenchymal stem cells (MSC).
  • the dissociated or disaggregated embryonic stem cells may be plated directly onto a culture substrate.
  • the culture substrate may comprise a tissue culture vessel, such as a Petri dish.
  • the vessel may be pre-treated.
  • the cells may be plated onto, and grow on, a gelatinised tissue culture plate.
  • An example protocol for the gelatin coating of dishes follows. A solution of 0.1% gelatin in distilled water is made and autoclaved. This may be stored at room temp. The bottom of a tissue culture dish is covered with the gelatin solution and incubated for 5-15 min. Remove gelatin and plates are ready to use. Medium should be added before adding cells to prevent hypotonic lysis.
  • the dissociated or disaggregated embryonic stem cells may be cultured in a medium which may comprise a serum-free medium.
  • serum-free media may comprise cell culture media which is free of serum proteins, e.g., fetal calf serum.
  • Serum-free media are known in the art, and are described for example in U.S. Pat. Nos. 5,631,159 and 5,661,034. Serum-free media are commercially available from, for example, Gibco-BRL (Invitrogen).
  • the serum-free media may be protein free, in that it may lack proteins, hydrolysates, and components of unknown composition.
  • the serum-free media may comprise chemically-defined media in which all components have a known chemical structure. Chemically-defined serum-free media is advantageous as it provides a completely defined system which eliminates variability allows for improved reproducibility and more consistent performance, and decreases possibility of contamination by adventitious agents.
  • the serum-free media may comprise Knockout DMEM media (Invitrogen-Gibco, Grand Island, N.Y.).
  • the serum-free media may be supplemented with one or more components, such as serum replacement media, at a concentration of for example, 5%, 10%, 15%, etc.
  • the serum-free media may comprise or be supplemented with 10% serum replacement media from Invitrogen—Gibco (Grand Island, N.Y.).
  • the serum-free medium in which the dissociated or disaggregated embryonic stem cells are cultured may comprise one or more growth factors.
  • growth factors include PDGF, EGF, TGF-a, FGF, NGF, Erythropoietin, TGF-b, IGF-I and IGF-II.
  • the growth factor may comprise fibroblast growth factor 2 (FGF2).
  • the medium may also contain other growth factors such as platelet-derived growth factor AB (PDGF AB). Both of these growth factors are known in the art.
  • the method may comprise culturing cells in a medium comprising both FGF2 and PDGF AB.
  • the medium may comprise or further comprise epidermal growth factor (EGF).
  • EGF epidermal growth factor
  • Use of EGF may enhance growth of MSCs.
  • EGF may be used at any suitable concentration, for example 5-10 ng/ml EGF.
  • EGF may be used in place of PDGF.
  • EGF is a protein well known in the art, and is referred to as symbol EGF, Alt. Symbols URG, Entrez 1950, HUGO 3229, OMIM 131530, RefSeq NM — 001963, UniProt P01133.
  • FGF2 is a wide-spectrum mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels in many tissues and cell types and reaches high concentrations in brain and pituitary. FGF2 has been implicated in a multitude of physiologic and pathologic processes, including limb development, angiogenesis, wound healing, and tumor growth. FGF2 may be obtained commercially, for example from Invitrogen-Gibco (Grand Island, N.Y.).
  • Platelet Derived Growth Factor is a potent mitogen for a wide range of cell types including fibroblasts, smooth muscle and connective tissue.
  • PDGF which is composed of a dimer of two chains termed the A chain and B chain, can be present as AA or BB homodimers or as an AB heterodimer.
  • Human PDGF-AB is a 25.5 kDa homodimer protein consisting of 13.3 kDa A chain and 12.2 B chain.
  • PDGF AB may be obtained commercially, for example from Peprotech (Rocky Hill, N.J.).
  • the growth factor(s), such as FGF2 and optionally PDGF AB, may be present in the medium at concentrations of about 100 pg/ml, such as about 500 pg/ml, such as about 1 ng/ml, such as about 2 ng/ml, such as about 3 ng/ml, such as about 4 ng/ml, such as about 5 ng/ml.
  • the medium contains FGF2 at about 5 ng/ml.
  • the medium may also contain PDGF AB, such as at about 5 ng/ml.
  • Cells in culture will generally continue growing until confluence, when contact inhibition causes cessation of cell division and growth. Such cells may then be dissociated from the substrate or flask, and “split”, subcultured or passaged, by dilution into tissue culture medium and replating.
  • the methods and compositions described here may therefore comprise passaging, or splitting during culture.
  • the cells in the cell culture may be split at a ratio of 1:2 or more, such as 1:3, such as 1:4, 1:5 or more.
  • the term “passage” designates the process consisting in taking an aliquot of a confluent culture of a cell line, in inoculating into fresh medium, and in culturing the line until confluence or saturation is obtained.
  • the method may further comprise a selection or sorting step, to further isolate or select for mesenchymal stem cells.
  • the selection or sorting step may comprise selecting mesenchymal stem cells (MSC) from the cell culture by means of one or more surface antigen markers.
  • MSC mesenchymal stem cells
  • the use of a selection or sorting step further enhances the stringency of sorting and selection specificity for MSCs and furthermore potentially reduces possible contamination from embryonic stem cells such as hESCs and other hESC-derivatives from the starting material. This would then further reduce the risk of teratoma formation and further increase the clinical relevance of the protocol we describe.
  • FACS fluorescence activated cell sorting
  • MSCs a number of candidate surface antigens known to be associated with MSCs e.g. CD105, CD73, ANPEP, ITGA4 (CD49d), PDGFRA
  • some of the MSC associated surface antigens e.g. CD29 and CD49e are also highly expressed in ES cells such as hESCs and their expression are verified by FACS analysis.
  • the association of a surface antigen with MSCs may not be sufficient to qualify the antigen as a selectable marker for isolating MSCs from ES cells such as hESC. Accordingly, the selection or sorting step may employ antigens which are differentially expressed between MSCs and ES cells.
  • the selection or sorting step of our method may positively select for mesenchymal stem cells based on the expression of antigens.
  • antigens may be identified by, for example, comparing the gene expression profiles of hESCs and hESCMSCs.
  • the selection or sorting may specifically make use of any of the antigens shown in Table E1A and E1B below.
  • the selection or sorting step of our method may positively select for mesenchymal stem cells based on the expression of antigens which are identified as expressed on MSCs, but not expressed on ES cells such as hESCs.
  • CD73 is highly expressed on MSCs, while being not highly expressed on hESCs. Both CD73 and CD105 are highly expressed surface antigens in MSCs and are among the top 20 highly expressed surface antigens in hESC-MSCs relative to hESC, the use of either CD73 or CD105 (or both) as selectable marker for putative MSCs will be equally effective in sorting for putative MSCs generated by differentiating hESCs.
  • the selection or sorting step may negatively select against antigens based on surface antigens that are highly expressed as surface antigen on embryonic stem cells (ES cells) such as hESCs, and not mesenchymal stem cells e.g., hESC-MSC. Selection or sorting may be based on known or previously identified hESC-specific surface antigens such as MIBP, ITGB1BP3 and PODXL, and CD24.
  • ES cells embryonic stem cells
  • hESC-MSC mesenchymal stem cells
  • CD24 may be used as a negative selection or sorting marker either on its own, or in conjunction with CD105 as a positive selectable marker for isolating putative MSCs from differentiating hESC cultures.
  • MSCs Mesenchymal stem cells derived from adult bone marrow have emerged as one of the most promising stem cell types for treating cardiovascular disease (Pittenger and Martin, 2004). Although the therapeutic effects of autologous MSCs have been attributed to their potential to differentiate into many different reparative or replacement cell types such as cardiomyocytes, endothelial cells and vascular smooth cells (Minguell and Erices, 2006; Zimmet and Hare, 2005), the differentiation efficiency of transplanted MSCs into therapeutically relevant numbers of functional reparative cells in injured tissues remains to be established.
  • a chemically defined serum free culture medium is conditioned by MSCs derived from human embryonic stem cells (hESCs), using a clinically compliant protocol.
  • MSCs derived from human embryonic stem cells (hESCs)
  • hESCs human embryonic stem cells
  • Three polyclonal, karyotypically stable, and phenotypically MSC-like cultures, that do not express pluripotency-associated markers but displayed MSC-like surface antigens (CD29+, CD44+, CD49a+/e+, CD105+, CD166+, CD34 ⁇ , CD45 ⁇ ) and gene expression profile, are generated by trypsinization and propagation of hESCs from either HuES9 hESC line or H1 hESC line in feeder- and serum-free selection media 15 .
  • HuES9.E1 can be stably expanded for at least 80 population doublings.
  • hESC-derived MSC cultures are transferred to a chemically defined, serum free culture medium to condition the medium for three days before the media containing MSC secretions are collected, clarified by centrifugation, concentrated 25 times using 10 kDa MW cut-off ultrafiltration membranes and sterilized by filtration through a 220 nm filter.
  • the secretory proteome is analyzed by multidimensional protein identification technology (MuDPIT) and cytokine antibody array analysis, and revealed the presence of 201 unique gene products. Computational analyses disclosed that this CM holds potential cytoprotective properties 16 .
  • the saline group is added to assess a potential effect of fresh, non-conditioned culture medium.
  • MI is induced by 75 minutes of proximal left circumflex coronary artery (LCxCA) ligation and 4 hours of subsequent reperfusion.
  • An ischemic period of 75 minutes is selected to inflict severe myocardial injury without inducing completely transmural myocardial infarction.
  • the 4 hour reperfusion period is used, because infarct size measurement using TTC staining is most reliable after 3 hours of reperfusion 17 . After longer periods of reperfusion, it becomes more difficult to assess oxidative stress status and apoptotic mechanisms.
  • Transonic flow probes Transonic Systems Inc, Ithaca, N.Y.
  • a wire is placed around the inferior caval vein to enable functional measurements under varying loading conditions for PV loops.
  • heparin After functional measurements, 10.000 IU of heparin are administered intravenously and sutures are tightened to occlude the proximal LCxCA. Internal defibrillation with 50 J is used when ventricular fibrillation occurred. After 75 minutes of ischemia, the LCxCA is reopened by release of the suture. Immediately following reperfusion, Nitroglycerine (0.1 mg to prevent no-reflow) is infused through the LCxCA via the guiding catheter, followed by intracoronary treatment with MSC-CM, non-CM or saline. After 4 hours of reperfusion, the final functional measurements are performed and the heart is explanted for infarct size analysis.
  • LV pressure and volume are measured using the conductance catheter method, as described previouslyl 18 .
  • LV pressure and volume signals derived from the conductance catheter are displayed and acquired at a 250-Hz sampling rate with a Leycom CFL-512 (CD Leycom).
  • SWT Systolic wall thickening
  • FAS fractional area shortening
  • LVEF left ventricular ejection fraction
  • the end-diastolic chamber stiffness is quantified by means of linear regression of the end-diastolic pressure-volume relationship. Echocardiography and PV loops are measured before MI, 1 hour after ischemia and 4 hours after reperfusion. To challenge stunned myocardium, additional measurements are performed during pharmaceutically induced stress by intravenous dobutamine infusion (2.5 ⁇ g/kg/min and 5.0 ⁇ g/kg/min).
  • the LCxCA pigs
  • LCA gallate
  • AAR area at risk
  • the heart is then excised, the LV is isolated and cut into 5 slices from apex to base.
  • the slices are incubated in 1% triphenyltetrazolium chloride (TTC, Sigma-Aldrich Chemicals, Zwijndrecht, the Netherlands) in 37° C.
  • Sörensen buffer (13.6 g/L KH 2 PO 4 +17.8 g/L Na 2 H PO 4 .2H 2 O, pH 7.4) for 15 minutes to discriminate infarct tissue from viable myocardium.
  • infarct size is calculated as a percentage of the AAR and of the LV.
  • Human leukemic CEM cells are incubated in either CM or non-CM, and treated with 50 ⁇ M H 2 O 2 to induce oxidative stress. Cell viability is assessed using Trypan Blue exclusion at 12, 24, 36 and 48 hours after H 2 O 2 treatment.
  • Nuclear oxidative stress in the ischemia and reperfusion area is assessed by immunostaining for 8-hydroxy-2′-deoxyguanosine (8-OHdG), a product of oxidative stress to DNA.
  • 8-OHdG 8-hydroxy-2′-deoxyguanosine
  • tissue sections are incubated with 10% normal horse serum for 30 minutes, mouse-anti-8-OHdG (OXIS international, Foster City, Calif., USA) 1:20 in 0.1% PBSA over night at 4° C., biotin labeled horse-anti-mouse (Vector laboratories, Burlingame, Calif., USA) 1:500 for 1 hour and with streptavidin-HRPO 1:1000 for 1 hour.
  • mouse-anti-8-OHdG OXIS international, Foster City, Calif., USA
  • biotin labeled horse-anti-mouse Vector laboratories, Burlingame, Calif., USA
  • the sections are incubated with H 2 O 2 -diaminobenzidine for 10 minutes.
  • the amount of 8-OHdG positive nuclei is quantified in 4 randomly picked fields per section with digital image microscopy software Analysis (Olympus, Weg, Germany) at 200 ⁇ magnification.
  • Protein is isolated from frozen tissue samples collected from the ischemia/reperfusion area of pigs using 1 ml Tripure Isolation Reagent (Boehringer, Mannheim, Germany) according to the manufacturer's protocol. For western blotting, 8 ⁇ g total protein is separated on a 10% SDS-PAGE gel, transferred onto a Nitrocellulose C membrane (Amersham, Buckinghamshire, UK) and blocked using Phosphate Buffered Saline (PBS)—0.1% Tween—5% Protifar (Nutricia, Netherlands).
  • PBS Phosphate Buffered Saline
  • the membrane is incubated with a rabbit antibody for phosphoSMAD2 1:1000 (Cell Signalling Technology), for active caspase 3 1:100 (Chemicon, Germany), or for beta-tubulin 1:5000 (Abcam, Cambridge, UK), and subsequently with goat-anti-rabbit HRP 1:2000 (DAKO, Glostrup, Denmark).
  • Chemiluminescence substrate NNk Life Science Products is used for detection; the bands are analyzed using the Gel Doc 1000 system (Biorad, Veenendaal, Netherlands).
  • the MSC-CM is prepared by sterile filtration through a 220 nm filter and concentrated through a 10 nm filter and therefore contains components between 10 and 220 nm. Subsequently, a ⁇ 1000 kDa fraction is prepared by filtering the MSC-CM through a 1000 kDa MW cut-off membrane with a 100 nm nominal pore size (Pall Corporation, Singapore), generating a fraction containing products between 10 and 100 nm.
  • a mouse or pig model of ischemia and reperfusion injury is used. MI is induced by 30 minutes left coronary artery (LCA) occlusion and subsequent reperfusion. Mice are treated with 20 ⁇ l unfractionated MSC-CM (10-220 nm), the ⁇ 1000 kDa fraction (10-100 nm), or saline intravenously via the tail vein, 5 minutes before reperfusion. Infarct size is assessed 24 hours later using Evans blue and TTC as described previously.
  • Infarct size compared to the area at risk (AAR) as well as compared to the LV, is markedly reduced in pigs treated with MSC-CM compared to those treated with non-CM and saline ( FIG. 1 ).
  • MSC-CM treatment resulted in approximately 60% reduction of infarct size.
  • the AAR is similar in all pigs, which indicates that the initial ischemic injury is similar in all pigs (Table E1 below).
  • Intravenous infusion of the ⁇ 1 -adrenergic receptor agonist dobutamine further increased systolic wall thickening in the MCS-CM treated pigs, whereas no improvement is seen in the control groups. Also global left ventricular systolic function decreased due to the ischemia ( FIG. 2B ). In the pigs treated with CM, the fractional area shortening increased after reperfusion, almost back to the baseline level, and increased above baseline level during dobutamine infusion.
  • CM nuclear oxidative stress in tissue sections of pigs treated with CM, non-CM or saline is quantified by 8-OHdG immunostaining for oxidized DNA. Intense nuclear staining indicative of DNA oxidation is observed in sections of non-CM or saline-treated pigs compared to CM-treated pigs ( FIGS. 3B-D ). In addition, there are also significantly more positive nuclei in non-CM or saline-treated pigs ( FIG. 3E ).
  • CM can confer cytoprotection against oxidative stress in vitro and in vivo.
  • CM treatment resulted in reduced pSMAD2 expression, indicating that TGF- ⁇ signaling via ALK-5 is reduced ( FIGS. 4A , B).
  • Reperfusion injury causes cell death through apoptosis rather than necrosis 20-22 .
  • CM treatment reduces apoptosis during reperfusion
  • active caspase-3 levels are lower compared to both non-CM control and saline control suggesting that CM inhibits apoptosis in vivo ( FIGS. 4C , D).
  • Unfractionated MSC-CM contains products between 10 and 220 nm. In order to come closer to identifying the cardioprotective factor(s) within the MSC-CM, a ⁇ 1000 kDa fraction is generated containing products ranging in size from 10-100 nm. Unfractionated MSC-CM confers cardioprotection, whereas the ⁇ 1000 kDa fraction does not ( FIG. 5 ), indicating that the cardioprotective factor(s) are in the >1000 kDa fraction with size ranging from 100 to 220 nm.
  • NCM non-conditioned medium
  • ITS insulin-transferrin-selenoprotein supplement
  • FGF2 FGF2, EGF and PDGF AB
  • proteins secreted by the cells are in complexes, and these secretion complexes are larger than 100 kD Proteins that are added exogenously to the culture medium as supplements, and that are less than 100 kD are readily filtered through membranes with MW cut off of 100 kD.
  • MI is induced by 30 minutes left coronary artery (LCA) occlusion by suture ligation and reperfusion is initiated by removal of suture.
  • Mice are treated with 20 ⁇ l unfractionated MSC-CM (10-220 nm), 20 ⁇ l of ⁇ 100 or 1,000 kD fraction, 4 ⁇ l of >1000 kD retentate or saline intravenously via the tail vein, 5 minutes before reperfusion. 24 hours later, the hearts are excised. Before excision, the Area At Risk (AAR) is determined by relegating the LCA and then perfusing Evans blue through the aorta. AAR is defined as the area not stained by the dye and is expressed as a percentage of the left ventricular wall area. Infarct size is assessed 24 hours later using Evans blue and TTC as described previously.
  • AAR Area At Risk
  • Electron microscopy analysis of the Conditioned Medium is performed using standard methodology. Briefly, the Conditioned Medium in PBS is loaded onto formwar carbon coated grids (Ted Pella Inc, Redding, Calif., USA cat no 01800N-F), fixed in 2.5% glutaraldehyde, washed, contrasted in 2% uranyl acetate, embedded in a mixture of uranyl acetate (0.8%) and methyl cellulose (0.13%), and examined under an electron microscope. Consistent with the size fractionation studies above, we observed the presence of numerous vesicles of ⁇ 50-150 nm, suggesting that these vesicles are the putative active complexes in the secretion ( FIG. 11 ).
  • the hydrophobic lipid/steroid components of Conditioned Medium and NCM are extracted by Folch procedure. Briefly, 50 ml of Conditioned Medium or NCM is vigorously mixed with 5 ml of chloroform and 2 ml of methanol. The organic and aqueous phases are allowed to separate. The bottom chloroform layer is removed and evaporated to dryness by speedvac.
  • the residue is reconstituted in methanol for LC-MS/MS analysis.
  • the sample is then injected to a normal-phase (silica phase) HPLC column with dichloromethane/methanol/water/ethylamine mobile phase.
  • the eluate is then ionized online by nanospray to LTQ-FTMS/Orbitrap.
  • the LTQ-FTMS/Orbitrap is run at alternate positive and negative modes for detection of lipid/steroid with different chemical properties.
  • the top 5 precursor ions of each MS scan are further analyzed by MS/MS scan
  • the molecules are therefore characterized by a combination of FTMS and LTQ.
  • the precursor mass of each tandem mass spectrum is first matched to a candidate in a lipid and metabolic database.
  • MS/MS spectra of ions that have a ⁇ 5 pmm mass error to any molecule in the database are then compared to known standard spectra or spectra predicted by Mass Frontier program
  • Mass spectrometry analysis of chloroform extract from the Conditioned Medium revealed the presence of lipids commonly found in plasma membrane, namely phospholipids, glycolipids, and steroids and also in exosomes 35 .
  • the phospholipids include phosphatidyl serine and phosphatidyl inositol, phosphatidyl choline, shingomyelin, ceramides; glycolipid such as cerebroside and steroids such as cholesterol.
  • exosomes has microdomains known as lipid rafts in their lipid membranes 22,24-35 41,42 .
  • Exosomes are cholesterol-rich and their cholesterol-phospholipid ratio generally exceed the ratio of 0.3-0.4 (mol/mol) ratio found in plasma membrane 35 .
  • These rafts are characterized by their resistance to dissolution by non-ionic detergents such as Triton X-100 or Brij-98 at low temperatures, and their sensitivity to cyclodextrin that binds cholesterol.
  • non-ionic detergents such as Triton X-100 or Brij-98 at low temperatures
  • Generally insoluble in detergents such as triton X-100 and detergent insolubility is often used to identify the presence of lipid rafts.
  • Trizol extraction of the Conditioned Medium followed by isopropanol precipitation as commonly used in the extraction of RNA from cells produces a pellet that in water has an 260:280 nm absorbance ratio of 1.9, suggesting that it may be RNA.
  • exosomes contain mRNAs and microRNAs 36 .
  • This pellet is assayed for sensitivity to RNase activity.
  • the Conditioned Medium will also treated with RNases before extraction with trizol. These assays determine if the pellet is RNA, and if the RNA is sequestered in lipid vesicles such as exosomes.
  • RNA is assayed by generic gene expression assays such as microarrary, sequencing, RT-PCR and in vitro translation assays to determine the composition and functions of RNA.
  • the RNAs are translated in vitro using standard commercially available reticulocyte lysate system with and without 15 N-leucine.
  • the translated protein products are identified by mass spectrometry.
  • the proteomic profile of the Conditioned Medium also describes the presence of proteins that are known to be membrane-bound. Some notable but not exhaustive examples include CD9, CD109, thy-1 20 . Other known surface antigen of exosome such as CD24 that is found on the surface of exosomes secreted in the urine 45 is not expressed in MSCs 19 or its secretion 20 .
  • the Conditioned Medium is biotinylated using standard commercially available biotinylation kits.
  • the proteins is separated on standard SDS-PAGE, transferred on nylon or nitrocellulose, and probed with avidin-peroxidase using standard protocols in western blot analysis. In this protocol, only proteins that are on the surfaces on the complexes and are physically accessible to biotin are biotinlyated. All the proteins that are within the complex and are therefore not physically accessible are be biotinylated.
  • the biotinylated proteins are also isolated using avidin-affinity chromatography and identify using LC/MS. The identity of these proteins is confirmed by western blot analysis, immunoelectron microscopy and gene expression of MSCs.
  • the suspension is assayed for the biological activities that are computationally predicted for the Conditioned Medium 20 , and is tested for cardioprotective effects in the mouse and pig models as described above or below, respectively.
  • the saline group is added to assess a potential effect of fresh, non-conditioned culture medium.
  • MI is induced by 75 minutes of proximal left circumflex coronary artery (LCxCA) ligation and 4 hours of subsequent reperfusion.
  • An ischemic period of 75 minutes is selected to inflict severe myocardial injury without inducing completely transmural myocardial infarction.
  • the 4 hour reperfusion period is used, because infarct size measurement using TTC staining is most reliable after 3 hours of reperfusion 46 .
  • MSC-CM 1.0 ml, 2.0 mg protein
  • non-CM 1.0 mg protein
  • saline an additional intracoronary bolus MSC-CM (4.0 ml, 8.0 mg protein), non-CM or saline is given.
  • Myocardial infarct size and function are assessed 4 hours after reperfusion.
  • MI left coronary artery
  • Mice are treated with unfractionated conditioned medium, ⁇ 1000 kD fraction, ⁇ 500 kD fraction, ⁇ 300 fraction kD, ⁇ 100 kD fraction or saline intravenously via the tail vein, 5 minutes before reperfusion. Infarct size is assessed the following day (24 hours after reperfusion).
  • the distal tip of a Swan Ganz catheter is placed into the pulmonary artery via the internal jugular vein.
  • Transonic flow probes Transonic Systems Inc, Ithaca, N.Y.
  • a wire is placed around the inferior caval vein to enable functional measurements under varying loading conditions for PV loops.
  • 10.000 IU of heparin are administered intravenously and sutures are tightened to occlude the proximal LCxCA. Internal defibrillation with 50 J is used when ventricular fibrillation occurred. After 75 minutes of ischemia, the LCxCA is reopened by release of the suture.
  • Nitroglycerine (0.1 mg to prevent no-reflow) is infused through the LCxCA via the guiding catheter, followed by intracoronary treatment with MSC-CM, non-CM or saline. After 4 hours of reperfusion, the final functional measurements are performed and the heart is explanted for infarct size analysis.
  • Mice are anesthetized with Fentanyl (0.05 mg/kg), Dormicum (5 mg/kg) and Domitor (0.5 mg/kg) and intubated using a 24-gauge intravenous catheter with a blunt end.
  • Mice are artificially ventilated at a rate of 105 strokes/min using a rodent ventilator with a mixture of O 2 and N 2 O (1:2 vol/vol) to which isoflurane (2.5-3.0% vol/vol) is added.
  • the mouse is placed on a heating pad to maintain the body temperature at 37° C.
  • the chest is opened in the third intercostal space and an 8-0 prolene suture is used to occlude the left coronary artery (LCA) for 30 minutes.
  • the chest is closed and the following day (24 hours later), the hearts are explanted for infarct size analysis.
  • LCA left coronary artery
  • the ECG, arterial pressure and cardiac output, are digitized at a sampling rate of 250 Hz and stored for offline analysis (Leycom CFL-512, CD Leycom).
  • Left ventricular (LV) pressure and volume are measured using the conductance catheter method, as described previously 47 .
  • LV pressure and volume signals derived from the conductance catheter are displayed and acquired at a 250-Hz sampling rate with a Leycom CFL-512 (CD Leycom).
  • Systolic wall thickening is calculated as [(WT(ES) ⁇ WT(ED))/WT(ED)]*100%, fractional area shortening (FAS) as [(LVia(ES) ⁇ LVia (ED))/LVia (ED)]*100%, and left ventricular ejection fraction (LVEF) as [(EDV ⁇ ESV)/EDV]*100%.
  • the end-diastolic chamber stiffness is quantified by means of linear regression of the end-diastolic pressure-volume relationship. Echocardiography and PV loops are measured before MI, 1 hour after ischemia and 4 hours after reperfusion. To challenge stunned myocardium, additional measurements are performed during pharmaceutically induced stress by intravenous dobutamine infusion (2.5 ⁇ g/kg/min and 5.0 ⁇ g/kg/min).
  • the LCxCA pigs
  • LCA gallate
  • Evans blue dye is infused through the coronary system to delineate the area at risk (AAR).
  • AAR area at risk
  • the slices are incubated in 1% triphenyltetrazolium chloride (TTC, Sigma-Aldrich Chemicals, Zwijndrecht, the Netherlands) in 37° C. Sörensen buffer (13.6 g/L KH 2 PO 4 +17.8 g/L Na 2 H PO 4 .2H 2 O, pH 7.4) for 15 minutes to discriminate infarct tissue from viable myocardium.
  • TTC triphenyltetrazolium chloride
  • infarct size is calculated as a percentage of the AAR and of the LV.
  • HuES9.E1 cells are cultured as described previously (Lian et al., 2007; Sze et al., 2007).
  • HuES9.E1 cell cultures are washed three times with PBS and cultured overnight in a chemically defined medium consisting of DMEM without phenol red (catalog number 31053, Invitrogen) and supplemented with insulin, transferrin, and selenoprotein (ITS) (Invitrogen), 5 ng/ml FGF2 (Invitrogen), 5 ng/ml PDGF AB (Peprotech, Rocky Hill, N.J.), glutamine-penicillin-streptomycin, and b-mercaptoethanol. The cultures are then rinsed three times with PBS, and then fresh defined medium is added.
  • DMEM without phenol red
  • ITS selenoprotein
  • CM sample is prepared by concentrating CM 50 ⁇ using 100 kDa MWCO tangential force filtration (TFF). All other concentrations are performed using ultrafiltration membrane. All CM and other differently processed CMs are 0.2 micron filtered after all procedures and before being stored or used.
  • CM dialyzed conditioned
  • NCM non-conditioned media
  • the samples are then desalted by passing the digested mixture through a conditioned Sep-Pak C-18 SPE cartridge (Waters, Milford, Mass., USA), washed twice with a 3% acetonitrile (ACN) (JT Baker, Phillipsburg, N.J.) and 0.1% formic acid (FA) buffer, and eluted with a 70% ACN and 0.1% FA buffer.
  • ACN 3% acetonitrile
  • F formic acid
  • the eluted samples are then dried to about 10% of their initial volumes by removing organic solvent in a vacuum centrifuge.
  • offline peptide fractionation is carried out with a HPLC system (Shimadzu, Japan) through a Polysulfoethyl SCX column (200 mm ⁇ 4.6 mm) (PolyLC, USA).
  • Mobile phase A (5 mM KH4PO4+30% acetonitrile)
  • mobile phase B (5 mM KH4PO4+30% acetonitrile+350 mM KCl) at 1 ml/min.
  • Eight fractions are collected and dried with a vacuum centrifuge.
  • Fractionated samples are loaded into the autosampler of a Shimadzu DGU-20A3 C18 reverse phase HPLC system coupled online to a LTQ-FT ultra linear ion trap mass spectrometer (Thermo Electron, San Jose, Calif.) fitted with a nano-spray source.
  • Injected peptides are trapped in a Zorvax 300SB-C18 enrichment column (5 mm ⁇ 03 mm, Agilent Technologies, Germany) and eluted into a nano-bored C18 packed column (75 ⁇ m ⁇ 100A, Michrom Bioresources, Auburn, Calif.).
  • a 90 minute gradient at 200nl/min flow rate is used to elute the peptides into the mass spectrometer.
  • the LTQ is operated in a data-dependent mode by performing MS/MS scans for the 8 of the most intense peaks from each MS scan in the FTMS.
  • MS/MS (dta) spectra of the eight SCX fractions are combined into a single mascot generic file by a home-written program.
  • Protein identification is achieved by searching the combined data against the IPI human protein database (version 3.34; 67,758 sequences) via an in-house Mascot server (Version 2.2, Matrix Science, UK).
  • the search parameters are: a maximum of 2 missed cleavages using trypsin; fixed modification is carbaminomethylation of cysteine and variable modifications is oxidation of methionine.
  • the mass tolerances are set to 20 ppm and 0.8 Da for peptide precursor and fragment ions respectively. Protein identifications are accepted as true positive if two different peptides are found to be with scores greater than the homology scores.
  • the instrument setup consisted of a liquid chromatography system with a binary pump, an auto injector, a thermostated column oven and a UV-visible detector operated by the Class VP software from Shimadzu Corporation (Kyoto, Japan).
  • the Chromatography columns used are TSK Guard column SWXL, 6 ⁇ 40 mm and TSK gel G4000 SWXL, 7.8 ⁇ 300 mm from Tosoh Corporation (Tokyo, Japan).
  • the following detectors, Dawn 8 (light scattering), Optilab (refractive index) and QELS (dynamic light scattering) are connected in series following the UV-visible detector.
  • the last three detectors are from Wyatt Technology Corporation (California, USA) and are operated by the ASTRA software.
  • the components of the sample are separated by size exclusion i.e. the larger molecules will elute before the smaller molecules.
  • the eluent buffer used is 20 mM phosphate buffer with 150 mM of NaCl at pH 7.2. This buffer is filtered through a pore size of 0.1 ⁇ m and degassed for 15 minutes before use.
  • the chromatography system is equilibrated at a flow rate of 0.5 ml/min until the signal in Dawn 8 stabilized at around 0.3 detector voltage units.
  • the UV-visible detector is set at 220 nm and the column is oven equilibrated to 25° C.
  • the elution mode is isocratic and the run time is 40 minutes.
  • the volume of sample injected ranged from 50 to 100 ⁇ l.
  • the % area of the exosome peak vs. all other peaks is integrated from the UV-visible detector.
  • the hydrodynamic radius, R h is computed by the QELS and Dawn 8 detectors.
  • the highest count rate (Hz) at the peak apex is taken as the R h .
  • Peaks of the separated components visualized at 220 nm are collected as fractions for further characterization studies.
  • sucrose gradient density equilibrium centrifugation 14 sucrose solutions with concentrations from 22.8-60% (w/v) are prepared. The most concentrated solution is layered at the bottom of SW60Ti ultracentrifuge tube (Beckman Coulter Inc., Fullerton Calif., USA), followed by the next highest sucrose concentration. CM is carefully loaded on top before ultracentrifuged for 16.5 hours at 200,000 ⁇ g, 4° C. in a SW60Ti rotor (Beckman Coulter Inc.). 16 fractions are collected from top to the bottom of the sucrose gradient. The densities of all the sucrose fractions are calculated using a microbalance, and consolidated into to 13 fractions.
  • the CM is pretreated with a cell lysis buffer (Cell Extraction Buffer, Biovision, www.BioVision.com) before being loaded on sucrose gradient density equilibrium centrifugation.
  • the lysis buffer is added to CM in a 1:1 volume ratio with cocktail of protease inhibitors (Halt Protease Inhibitor Cocktail, EDTA-Free, Thermo Scientific, www.thermofisher.com).
  • the mixture is incubated for 30 min at room temperature with gentle shaking.
  • Protein concentration of CM is quantified using NanoOrange Protein Quantification kit (Invitrogen) according to the manufacturer's instructions.
  • CM Total proteins of CM are separated on polyacrylamide gels, before transfer to a nitrocellulose membrane (Amersham Biosciences, Uppsala, Sweden).
  • the membrane is blocked, incubated with mouse antibodies against human CD9, CD81, SOD-1, pyruvate kinase, Alix, Tsp-1 followed by horseradish peroxidase-coupled secondary antibodies against the mouse primary antibody.
  • the blot is then incubated with a chemiluminescent HRP substrate to detect bound primary antibody, and therefore the presence of the antigen.
  • Cholesterol, sphingomyelin and phosphatidylcholine concentrations in two independent preparations of CM and pellet from the ultracentrifugation of CM at 100,000 ⁇ g for 2 hours at 4° C. are measured using commercially available assay kits. Cholesterol is measured using Amplex® Red Cholesterol Assay kit (Molecular Probes, USA), sphingomyelin by the Sphingomyelin Assay Kit (Cayman Chemical Company, Ann Arbor, Mich., USA) and phosphatidylcholine is measured using the Phosphatidylcholine Assay Kit (Cayman Chemical Company, Ann Arbor, Mich., USA).
  • CM is treated with/without triton x or lysis buffer for 30 min at 4° C. with gentle shaking.
  • Proteolytic digestion is allowed to carry out by adding trypsin to the treated CM for 3 seconds to 20 minutes at room temperature with gentle shaking. The digestion is stopped using a trypsin inhibitor, PMSF.
  • RNA microarray Two biological replicates of total cellular RNA from MSCs and two biological replicates of secreted RNA from CM are analysed by miRNA microarray.
  • the hybridization and data analysis is outsourced to LC Sciences, LLC (www.LCsciences.com).
  • the chip contained probes for miRNA transcripts listed in Sanger miRBase Release 10.1 (http://www.sanger.ac.uk/Software/Rfam/mirna/).
  • CM that is concentrated by ⁇ 125 times against a similar membrane is cardioprotective in a mouse model of ischemia/reperfusion injury.
  • filteration through filters with a MWCO smaller than 0.2 ⁇ m such as 100 kDa, 300 kDa, 500 kDa or 1000 kDa are not cardioprotective ( FIG.
  • CM that is concentrated against a 1000 kDa membrane (Timmers et al., 2008) or a 100 kDa membrane is cardioprotective ( FIG. 14 ).
  • the active fraction consisted of large complexes of >1000 kDa or having a diameter of 50-100 nm.
  • the particles in the CM are exosomes. Exosomes are formed from multivesicular bodies (Fevrier and Raposo, 2004; Keller et al., 2006) with a bilipid membrane that has the same orientation as plasma membrane. They are known to be produced by many cell types and are thought to be important in intercellular communications.
  • Exosomes have diameters of 40-100 nm. Exosomes have been shown to be secreted by many cell types and the protein composition of these exosomes appeared to be cell specific. However, some proteins such as CD9, pyruvate kinase and alix appear to be commonly expressed in the exosomes (Sze et al., 2007). We have previously identified about 201 proteins in the secretion (Sze et al., 2007).
  • TSP-1, SOD-1 and pyruvate kinase did not co-immunoprecipitated with CD81, suggesting that these proteins are not present in CD81+ exosomes or not present in exosomes at all. ( FIG. 15 ).
  • TIMP1, TIMP2, TNFRSF11B, LGALS3, ALCAM, DCN, SFRP1, GDF15, PDGFC, PTX3, LTBP1, IGFBP2, GREM1, IGFBP7, MIF, MMP1, PLAU, INHBA and THBS1 were identified by LC MS/MS and antibody array, PPIA, HIST1H4, PPIB, HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, HIST2H2AA3, HIST2H2AA4, HIST2H4A, HIST2H 4 B, HIST4H4, HLA-A, HLA-B, SDCBP, TUBA1A, TUBA6, TUBA8, GAPDH, TUBB, TUBB2C
  • GPC5, IGF2R, CHRDL1, GRN, VEGFC, IL13, IL15, EML2, IL15RA, IL1RAP, MMP10, IL2, GZMA, IL21R, IL3, IL6, IL6ST, IL8, HGF and THBS are identified by antibody array.
  • the remaining proteins are identified by LC MS/MS.
  • the CM is ultracentrifuged at 200,000 g for two hours. There is a >200 fold enrichment of CD9 an exosome-associated protein in the pellet with no detectable level of CD9 in the supernatant ( FIG. 16 ). Ultracentrifugation at 100,000 g for one hour is not sufficient to sediment all the CD9 ( FIG. 16 ). Filtration of the CM through a filter with a MWCO of 500 kDa followed by centrifugation of either the filtrate or retentate at 200,000 g for 2 hours generated a pellet in the retentate fraction ( FIG. 16 ). CD9 which had MW of 19 kDa respectively are highly enriched in this pellet.
  • the CM is fractionated on a sucrose density gradient by equilibrium ultracentrifugation.
  • the density of exosomes ranges from 1.13 g ml ⁇ 1 to 1.19 g ml-1 and float on sucrose gradients. Flotation on sucrose gradients readily separate exosomes from contaminating material such as protein aggregates or nucleosomal fragmentst (Thery et al., 2002). Fractions from the sucrose gradient are then analysed for the presence CD9, CD81, Tsp1, SOD-1 and pyruvate kinase along the gradient ( FIG.
  • CM is treated with a cell lysis buffer ( FIG. 17B ) before being fractionated on a sucrose density gradient.
  • This pre-treatment with a plasma membrane solubilization reagent restored each of the apparent densities to the expected density of proteins that correlated to their molecular weight. Therefore, the exosome-associated proteins are localized in lipid vesicles, consistent with our exosome hypothesis.
  • the concentration of sphingomyelin and phosphatidylcholine, the major phospholipids of the plasma membrane, and cholesterol is determined ( FIG. 17C ).
  • the relative concentration of these lipids per ⁇ g protein is higher in the CM relative to the non-conditioned medium.
  • ultracentrifugation of the CM at 200,000 g for 2 hours significantly increased the concentration of the lipids ( FIG. 17C ).
  • exosome-associated proteins include many known membrane proteins such as CD9 and cytosolic proteins such as SOD1, we therefore determined if these proteins are similarly localized on the lipid membrane and lumen of the vesicles.
  • CM is subjected to limited trypsinization over time ( FIG. 18A ).
  • CD9 which has a similar MW as SOD1 is relatively more susceptible to trypsin digest than SOD1.
  • Digestion of SOD1 is observed only after more than 50% of CD9 had been digested ( FIG. 18A ).
  • tryptic digestion of CD9 generated three tryptic peptide intermediates, suggesting that CD9 has domains with different trypsin-sensitivity.
  • the three susceptible tryptic peptide intermediates are mapped to the transmembrane or cytoplasmic domains.
  • CD9 a known membrane protein is also membrane-bound in the exosome and is oriented in the same direction as CD9 in the plasma membrane while cytosolic SOD-1 is localized in the lumen and could be digested only when the integrity of the membrane is compromised by the digestion of membrane proteins.
  • RNAs are secreted by cells in exosomes (Smalheiser, 2007; Taylor and Gercel-Taylor, 2008; Valadi et al., 2007).
  • CM is extracted for RNA by Trizol to yield 5-6 ug RNA per mg protein.
  • FIG. 19A When separated on a glyoxal-agarose gel ( FIG. 19A ) or an urea-PAGE ( FIG. 19B ), the RNA contained undetectable level of 18S and 28S ribosomal RNA with most RNAs being ⁇ 300 nt.
  • RNAse are protected by a lipid membrane, analogous to a cell membrane by treating the CM with a SDS-based cell lysis buffer, cylcodextrin or phospholipase A2. After treatment with one of the four reagents, the CM is exposed to RNAse and then extracted for RNA.
  • RNAs of ⁇ 70-100 nt are more sensitive to RNAse III activity than those of smaller MW, suggesting that the larger RNAs are double stranded ( FIG. 19D ).
  • RNAs are shown to be in lipid vesicles, we next determined the buoyant density of these vesicles using sucrose gradient equilibrium ultracentrifugation.
  • CM CM pretreated with lysis buffer or a set of RNA MW markers is loaded onto a sucrose density gradient and ultracentrifuged as described in FIGS. 4 a, b .
  • the gradients are then removed in thirteen fractions and each fraction is then extracted for RNA.
  • the secreted RNAs equilibrated at a density of 1.074-1.1170 g/ml ( FIG. 20 ).
  • RNA MW markers exhibited a buoyant density of 1.115-1.1170 g/ml and pretreatment of the CM with lysis buffer before centrifugation caused an increase in density of the secreted RNA to that of RNA MW markers i.e. 1.115-1.145 g/ml ( FIG. 20C ). These observations are therefore consistent with the RNAs being encapsulated in a lipid vesicle and thus had an apparent density that is much lower than soluble RNA. Pretreatment of the CM with lysis buffer released the RNA and resulted in the RNA sedimenting at the density of RNA markers.
  • RNA-containing Vesicles are not in CD81 Containing Exosomes
  • CD9, CD81 and Alix are co-immunoprecipitated by anti CD81 antibodies.
  • RNA also immunoprecipitate with CD81. After immunoprecipitation, the RNA is not present in the precipitate but remained in the supernatant ( FIG. 21 ). Therefore, the secreted RNAs are not sequestered in CD81+, CD81+CD9+, or CD81+CD9+Alix+ vesicles.
  • miRNAs are present in both MSC and CM. These are: hsa-let-7a, hsa-miR-149*, hsa-miR-214, hsa-let-7b, hsa-miR-221, hsa-let-7c, hsa-miR-26a, hsa-miR-151-5p, hsa-miR-222, hsa-let-7d, hsa-miR-100, hsa-miR-320, hsa-let-7e, hsa-miR-103, hsa-let-7f, hsa-miR-181a, hsa-miR-574-3p, hsa-miR-574-5p, hsa-let-71, hsa-miR-107, hsa-miR-575, hsa-miR-125a-5p
  • 16 miRNAs are detectable in the CM but are present at below detection level in MSCs. These are: hsa-let-7b*, hsa-miR-124, hsa-miR-296-5p, hsa-miR-765, hsa-miR-1228, hsa-miR-1238, hsa-let-7d*, hsa-miR-150*, hsa-miR-493*, hsa-miR-933, hsa-miR-1234, hsa-miR-122, hsa-miR-198, hsa-miR-572, hsa-miR-1224-5p an dhsa-miR-1237.
  • miRNAs are present in MSC only: hsa-miR-24-2*, hsa-miR-98, hsa-miR-484, hsa-miR-25, hsa-miR-99a, hsa-miR-151-3p, hsa-miR-491-5p, hsa-miR-99b, hsa-miR-503, hsa-miR-26b, hsa-miR-152, hsa-miR-505*, hsa-miR-27a, hsa-miR-155, hsa-miR-324-5p, hsa-miR-532-5p, hsa-miR-27b, hsa-miR-106a, hsa-miR-328, hsa-let-7g, hsa-miR-27b*
  • CM contained significant levels of anti-guide miRNA (denoted with an asterisk).
  • the relative ratios of let7b to let7b*, let7d to let7d* and miR-191 to miR-191* in CM are much reduced compared to that in the MSCs ( FIG. 22B ).
  • cleavage of the stem-loop pre-miRNA generates the mature guide miRNA and the anti-guide miRNA*. The latter is usually degraded in the cell.
  • One possible explanation for the low ratio of guide miRNA to anti-guide miRNA is that microRNAs in the secretion are pre-miRNAs, and not mature RNAs.
  • RNAse III treatment will confirm the presence of pre-miRNA by degrading the pre-miRNA and render it undetectable by RT-PCR.
  • Cardioprotective Secretion Contains only Particles with Hydrodynamic Radius of 45-55 nm in the Detectable Range of 1-1000 nm
  • CM and NCM is first separated by size exclusion on a HPLC ( FIG. 13 ) and each eluting peak as measured by absorbance at 220 nm is then examined for dynamic light scattering (DLS) using a quasi-elastic light scattering (QELS) detector.
  • DLS dynamic light scattering
  • QELS quasi-elastic light scattering
  • this eluting peak are harvested and tested in a mouse model of myocardial ischemia/reperfusion injury as previously described above and in (Timmers et al., 2008).

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CN102014934B (zh) 2014-08-20
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