WO2014057097A1 - Cellules souches mésenchymateuses modulées pour la thérapie cellulaire cardiaque - Google Patents

Cellules souches mésenchymateuses modulées pour la thérapie cellulaire cardiaque Download PDF

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WO2014057097A1
WO2014057097A1 PCT/EP2013/071308 EP2013071308W WO2014057097A1 WO 2014057097 A1 WO2014057097 A1 WO 2014057097A1 EP 2013071308 W EP2013071308 W EP 2013071308W WO 2014057097 A1 WO2014057097 A1 WO 2014057097A1
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stem cells
mesenchymal stem
adult human
human mesenchymal
adult
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PCT/EP2013/071308
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Anne-Marie Rodriguez
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1329—Cardiomyocytes

Definitions

  • Regenerative medicine involves transplanting cells of interest with the goal of repairing and regenerating a target tissue and/or target organ.
  • heart failure is among the main causes of death in Western countries. According to the World Health Organization, about 16.7 million people die globally each year from cardiovascular disease, accounting for 29% of all deaths in the world. As adult cardiomyocytes lose their proliferative potential, they fail to allow regeneration of myocardium damage occurring after myocardial infarction or other cardiac diseases, such as genetic disorders. Statistics show that about 22% of men and about 44% of women will develop heart failure within 6 years of a heart attack.
  • MSCs mesenchymal stem cells
  • the first strategy consists in genetically manipulating mesenchymal stem cells before implantation into the damaged heart. For example, it has been reported that the therapeutic potential of stem cells significantly improves after introduction of different genes that stimulate the synthesis, by the stem cells, of cardioprotective factors.
  • Such genes include the Akt (Gnecchi et al., FASEB J., 2006, 20: 661-669; Gnecchi et al., Nature Med., 2005, 11: 367-368), GSKSb (Cho et al, Cir. Res., 2011, 108: 478-489), Protaglandin I synthase (Lian et al, Life Sci., 2011, 88: 455- 464), VGEF and HGF (Deuse et al., Circulation, 2009, 120: S247-S254) and SDF-1 (Tang et al., Eur. J. Cardiothorac. Surg., 2009, 36: 644-650) genes.
  • the second approach used to optimize the cardiac regenerating potential of stem cells consists in pre-conditioning the stem cells under hypoxic conditions (Chacko et al., Am. J. Physiol. Cell Physiol., 2011, 299: C1562-C1570; Fang et al., J. Mol. Cell. Cardiol., 2011, 839-847), or with pharmacologic substances such as pioglitazone (Shinmura et al., Stem Cells, 2011, 29: 357-366), erythropoietin (Zhang et al., Cardiology, 2007, 108: 228-236), several chemokines or growth factors including the factors SDF-1 (Pasha et al, Cardiovasc.
  • the present invention encompasses the recognition by the applicants that the innate humoral regenerative function of mesenchymal stem cells can be improved through cell interaction and communication with distressed cardiomyocytes.
  • hMADS human multipotent adipose derived stem cells
  • mouse adult terminally-differentiated cardiomyocytes which can be considered to be in a distressed state to mimic in vivo microenvironment after the onset of myocardial infarction results in cell-to-cell communication processes between stem and cardiac cells which trigger changes in the hMADS secretome expression and consequently enhance the hMADS effectiveness in promoting angiogenesis and chemoattraction of bone marrow-derived mesenchymal progenitors, these two processes being of key importance for cardiac repair.
  • hMADS Compared to other pre-conditioning methods known in the art, co-culture of hMADS with distressed cardiomyocytes results in alteration of the release, by hMADS, of diverse group of diffusible molecules with known cardioprotective properties.
  • Some of these cardioprotective factors have been identified by the applicants, including VEGF, HGF, SDF-lcc, MCP-3, IL6, and GROcc.
  • the paracrine activation that is obtained by coculture is advantageously gradual and transient, a chemical gradient being required for cardiac regeneration.
  • cardiomyocytes have an effect that could be assimilated to that of a vaccine since mesenchymal stem cells co-cultured with cardiomyocytes retain the memory of signals from distressed cardiomyocytes and their paracrine activation is consequently significantly enhanced following a subsequent exposition to cardiomyocytes.
  • co-culturing does not exhibit the drawbacks inherent to genetic modifications and pharmacologic treatments or the immunologic tolerance associated with the use of biomaterials.
  • the present invention provides a method for modulating the secretome, in particular the cardioprotective secretome, of adult human mesenchymal stem cells, said method comprising a step of coculturing adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes in an appropriate culture medium.
  • the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are in physical contact during the coculture.
  • modulating the secretome results in an amount of at least one cardioprotective factor released by the cocultured adult human mesenchymal stem cells that is higher than the amount of the same at least one cardioprotective factor released by naive adult human mesenchymal stem cells.
  • the amount of the at least one cardioprotective factor released by cocultured adult human mesenchymal stem cells may be at least 1.25 times higher than the amount of the same at least one cardioprotective factor released by naive adult human mesenchymal stem cells.
  • the at least one cardioprotective factor is selected from the group consisting of VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), SDF-lcc (stromal-derived factor- 1 alpha), MCP-3 (monocyte chemotactic protein 3), IL6 (interleukin-6), GROcc (growth regulated oncogene alpha), and any combination thereof.
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • SDF-lcc stromal-derived factor- 1 alpha
  • MCP-3 monocyte chemotactic protein 3
  • IL6 interleukin-6
  • GROcc growth regulated oncogene alpha
  • the adult human mesenchymal stem cells are derived from a tissue selected from the group consisting of adipose tissue, skeletal muscle, bone marrow, dental pulp, blood, umbilical cord blood, and any combination thereof.
  • the adult human mesenchymal stem cells are derived from a tissue obtained from a healthy adult donor.
  • following co-culturing the preconditioned adult human mesenchymal stem cells are separated from the adult fully differentiated cardiomyocytes after coculture, and optionally stored prior to use.
  • the present invention provides preconditioned adult human mesenchymal stem cells obtainable, or obtained, by a method according to the invention.
  • the preconditioned adult human mesenchymal stem cells are characterized in that, in response to de novo contact with adult fully differentiated cardiomyocytes, the preconditioned adult human mesenchymal stem cells release at least one cardioprotective factor in a higher amount than naive adult human mesenchymal stem cells, all other things being equal.
  • the preconditioned adult human mesenchymal stem cells are characterized in that the release of the at least one cardioprotective factor released by preconditioned adult human mesenchymal stem cells is at least 1.25 times higher than the amount of the same at least one cardioprotective factor released by naive adult human mesenchymal stem cells.
  • the preconditioned adult human mesenchymal stem cells are characterized in that the at least one cardioprotective factor is selected from the group consisting of VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), SDF-lcc (stromal-derived factor- 1 alpha), MCP-3 (monocyte chemotactic protein 3), IL6 (interleukin-6), GROcc (growth regulated oncogene alpha), and any combination thereof.
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • SDF-lcc stromal-derived factor- 1 alpha
  • MCP-3 monocyte chemotactic protein 3
  • IL6 interleukin-6
  • GROcc growth regulated oncogene alpha
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of preconditioned adult human mesenchymal stem cells of the invention, and at least one pharmaceutically acceptable carrier or excipient.
  • the preconditioned adult human mesenchymal stem cells have been separated from the adult fully differentiated cardiomyocytes with which they have been cocultured.
  • the present invention relates to the use of preconditioned adult human mesenchymal stem cells of the invention for the manufacture of a medicament or pharmaceutical composition.
  • the present invention provides preconditioned adult human mesenchymal stem cells or pharmaceutical composition thereof according to the invention for use in the treatment of a cardiac pathology and/or in cardiac tissue reconstruction or regeneration.
  • the preconditioned adult human mesenchymal stem cells used in the manufacture of a medicament or pharmaceutical composition or use in the treatment of a cardiac pathology and/or in cardiac tissue reconstruction or regeneration have been separated from the adult fully differentiated cardiomyocytes with which they have been cocultured.
  • the cardiac pathology is a member of the group consisting of heart failure, myocardial infarction, cardiac ischemia, and inherited genetic cardiomyopathies such as Duchenne muscular dystrophy and Emery Dreiffuss.
  • FIG. 2 is a set of graphs showing that co-culture improves hMADS paracrine functions.
  • A Representative photographs of an HUVEC spheroid and
  • (C) Upper panel: co-immunostaining for GATA-4 (red) and PH3 (green) of human BM-MSC revealing presence of cardiac progenitor GATA-4+/PH3+ like cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 ⁇ . Lower panel: migration of BMMSC after 24 hours exposure to supernatants (mean + SD of n 8 independent experiments).
  • FIG. 3 is a set of showing that co-culture improves hMADS paracrine functions.
  • C Upper panel: a representative flow cytometry dot blot of mouse neonatal CM stained with annexin V/ IP.
  • # p ⁇ 0.05 versus basal medium; * p ⁇ 0.05; ** p ⁇ 0.01, ns: no significant.
  • Figure 4 is a set of showing that latruculin A or nocodazole specifically inhibit TNT- like channels.
  • (B) Relative calcein and mitrotracker fluorescence mean changes in 24hour-cocultures treated with latrunculin A (LAT-A), nocodazole (NOCO), 18a-glycyrrhetinic acid (18a-GA) in presence or not of 0.4 or 1 ⁇ pore size transwell insert. Data represent the mean +SEM of at least n 4 independent experiments. *p ⁇ 0.05; **p ⁇ 0.01.
  • Figure 5 is a set of microscope pictures.
  • First line TNT channels interconnecting stem (arrowhead) to cardiac (asterix) cells composed of both f-actin (rhodamine-phalloidin staining, red) and microtubules (FITC conjugated a-tubulin, green) at 24 hour-coculture. Scale bar, 20 ⁇ .
  • Second line Transfer of calcein ⁇ Second line) (arrowhead, green) and mitotracker ⁇ Third line) (arrowhead, red) from the CM to hMADS along TNT like structures. HMADS were labelled with WGA (white) prior coculturing. Scale bar, 20 ⁇ .
  • Figure 7 is a set of graphs showing that disruption of heterologous TNT channels abrogates coculture-induced hMADS paracrine stimulation.
  • A-B Impact of latrunculin A and nocodazole treatments on coculture induced
  • A -angiogenesis
  • B -chemotaxis of human BM-MSC.
  • Figure 8 shows that coculture improves hMADS cell therapy efficacy.
  • LVEF Left ventricular ejection fraction
  • Figure 9 shows that coculture improves hMADS cell therapy efficacy.
  • B C) Immunostaining were performed at day-3. Nuclei were counterstained with DAPI. #, p ⁇ 0.05 versus basal medium; * p ⁇ 0.05; ** p ⁇ 0.01.
  • Figure 10 shows that second CM exposure reinforces paracrine stimulation of the first primed hMADS.
  • B Confocal microscopy showing CM with human stem cell mitochondria (red) (white arrows) in mouse hearts injected with hMADS alone or in coculture at day 3 post infarction. CMs are stained with cTnT (green). Stars design hMADS. Scale bar, 20 ⁇ .
  • the present applicants relates to a method for modulating the secretome of mesenchymal stem cells by coculturing adult mesenchymal human stem cells with adult fully differentiated cardiomyocytes, thereby enhancing or improving the cardiac regenerative potential of the mesenchymal stem cells.
  • the method disclosed herein for modulating the cardioprotective secretome of mesenchymal stem cells or for improving the cardiac therapy efficacy of mesenchymal stem cells comprises a step of coculturing adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes in an appropriate culture medium.
  • the term “coculturing” refers to a process in which at least two different types of cells are cultured together in an appropriate culture medium.
  • adult mesenchymal human stem cells and adult fully differentiated cardiomyocytes are cocultured.
  • an appropriate culture medium refers to a culture medium that contains nutrients necessary to support the growth and/or survival of the cocultured cells, but that does not contain any chemical reagent generally used for the cell fusion of stem cells with somatic cells, such as polyethylene glycol (PEG).
  • An appropriate culture medium may or may not further comprise growth factors.
  • growth factors of interest may be bFGF (also known as FGF-2), BMP2, IGF1, TNF-cc, TGF -l, BMP-2, BMP-4, Activin-A, FGF-2, FGF-4, IL-6, IGF-1, IGF-2, VEGF-A, EGF, and any combination of these or other growth factors.
  • an appropriate culture medium according to the invention may consist in a minimal medium in which cells can be alive or grow, such as for example Dulbecco modified Eagle's minimal essential medium (DMEM) supplemented or not with decomplemented fetal calf serum (FCS).
  • DMEM Dulbecco modified Eagle's minimal essential medium
  • FCS decomplemented fetal calf serum
  • Adult human mesenchymal stem cells and fully differentiated cardiomyocytes may be plated in coated (e.g. , gelatine - coated) or uncoated plates.
  • adult human mesenchymal stem cells generally refers to undifferentiated cells found in a differentiated (specialized) tissue and that are capable of making identical copies of themselves (self-renewal) for the lifetime of the organism.
  • Adult human mesenchymal stem cells that can be used in the context of the present invention thus include any suitable adult human stem cells (i.e., cells with an ability for self-renewal) derived from any suitable tissue using any appropriate isolation method.
  • adult human mesenchymal stem cells that can be used in the methods of the present invention include cells previously described in international patent application PCT/FR2003/002439 (the content of which is incorporated herein by reference in its entirety) and in Rodriguez et al, J. Exp.
  • human Multipotent Adipose tissue Derived Stem cells or hMADS.
  • Other adult human mesenchymal stem cells that can be used in the methods of the present invention are cells derived from adipose tissue and skeletal muscle of an adult person and obtained using a method disclosed in international patent application PCT/FR2003/002439 or a variation of that method developed by the present Applicants and described herein.
  • Such mesenchymal stem cells exhibit a very important ability for self -renewal and, in particular, are capable of sustained self-renewal during at least 130 doublings of the population.
  • mesenchymal stem cells also exhibiting an important capacity for self-renewal and that may be used in the practice of the methods of the present invention include, but are not limited to, adult multilineage inducible (MIAMI) cells (D'Ippolito et al, J. Cell Sci., 2004, 117: 2971-2981), MAPC (also known as MPC) (Reyes al, Blood, 2001, 98: 2615-2625), cord blood derived stem cells (Kogler G et al, J. Exp.
  • MIAMI adult multilineage inducible
  • umbilical cord blood stem cells are easy to expand in vitro, are multipotent, have been reported to be non-immunogenic (Wang et al, Immunology, 2009, 126(2): 220-232; Ji et al, ", J.
  • mesenchymal stem cells include mesenchymal stem cells isolated from bone marrow or obtained by liposuction.
  • adult fully differentiated cardiomyocytes refers to the cells specialized for a particular function and composing the cardiac muscle, and that do not have the ability to generate other kinds of cells.
  • adult fully differentiated cardiomyocytes may be from any appropriate mammal origin (e.g. , mouse, rat, rabbit, pig, dog or human origin).
  • the method comprises steps of:
  • the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are in physical contact. Providing Adult Human Mesenchymal Stem Cells and Adult Differentiated Cardiomyocytes
  • the term “providing” herein refers to a process in which cells are isolated and provided in a state suitable for in vitro culture.
  • isolated refers to a cell which has been separated from at least some components of its natural environment. This term includes gross physical separation of the cell from natural environment (e.g. , removal from the donor).
  • isolated includes alteration of the cell' s relationship with the neighboring cells with which it is in direct contact, for example, by dissociation.
  • human stem cells are preferably adult human mesenchymal stem cells, which may be derived from a large variety of tissues.
  • human mesenchymal stem cells are derived from adipose tissue.
  • human mesenchymal stem cells are derived from skeletal muscle.
  • human mesenchymal stem cells are derived from adipose tissue and skeletal tissue.
  • human mesenchymal stem cells are derived from bone marrow, dental pulp, blood, and/or umbilical cord blood.
  • the method for modulating mesenchymal stem cells secretome is described as involving the coculture of adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes in an appropriate culture medium.
  • the present invention encompasses methods for modulating mesenchymal stem cells secretome wherein adult human stem cells (i.e. , adult human non-mesenchymal stem cells) are used in place of adult human mesenchymal stem cells.
  • tissues from which adult human (non-mesenchymal) stem cells may be obtained include, but are not limited to, tissues of endothermal origin such as the liver and pancreas, and tissues of ectodermal origin such as the cornea and/or the retina of the eye, the brain and the skin.
  • the present invention encompasses methods for modulating the secretome of mesenchymal stem cells, wherein non- human mammalian stem cells are used in place of adult human stem cells.
  • Adult non-human mammalian stem cells that can be used in the practice of the present invention include any adult stem cells of non-human mammalian origin, such as, for example, of mouse, rat, dog, cat, pig, guinea pig, hamster, or non-human primates, and the like.
  • adult fully different cardiomyocytes may be from any appropriate mammal origin (e.g. , mouse, rat, rabbit, pig, dog or human origin).
  • a cell is "derived from" a subject or a sample (e.g. , a biological sample) if the cell is obtained (e.g. , isolated, extracted, or purified) from the subject or sample.
  • a cell derived from an organ, tissue, cell line, etc. may be modified in vitro after it is obtained. Such a modified cell is still considered to be derived from the original source.
  • adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes may be independently isolated from any suitable tissue sample.
  • tissue sample refers to any sample of tissue harvested from a suitable mammal, as already mentioned above.
  • tissue samples are preferably obtained from healthy donors.
  • the donor may be of any age. However, in certain embodiments, the donor is a healthy adult.
  • tissue samples are preferably not obtained by liposuction. Isolation of cells of interest from a tissue sample preferably occurs in an aseptic environment. In embodiments where the tissue sample is solid or semi-solid, blood and debris are removed from the tissue sample prior to isolation of the cells.
  • the tissue sample may be washed with a buffer solution (e.g. , buffered saline) optionally comprising antimytotic and/or antibiotic agents.
  • the different cell types present in the tissue sample are fractionated into subpopulations from which the cells of interest can be isolated. This may be accomplished using techniques for cell separation including but not limited to, mechanical treatment (e.g. , mincing or shear forces) and/or enzymatic digestions (e.g.
  • proteolytic enzymes such as neutral proteases, metallopro teases, serine proteases, deoxyribonucleases, for example, collagenase, trypsin, chymotrypsin, thermolysin, dispase, elastase, hyaluronidase, pepsin, and the like to dissociate the tissue sample into its component cells, followed by cloning and selection of specific cell types.
  • proteolytic enzymes such as neutral proteases, metallopro teases, serine proteases, deoxyribonucleases, for example, collagenase, trypsin, chymotrypsin, thermolysin, dispase, elastase, hyaluronidase, pepsin, and the like to dissociate the tissue sample into its component cells, followed by cloning and selection of specific cell types.
  • Suitable methods of cell selection and/or separation include, but are not limited to, selection based on morphologic and/or biochemical markers, selective growth of desired cells (positive selection), selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation, and the like.
  • adult human mesenchymal stem cells are isolated and obtained as described in international patent application PCT/FR2003/002439 (WO/2004/013275) with the difference that any suitable tissue sample may be used (including those described above) and that the donor may be an adult donor and not just a child of less than 10 years of age. Other differences include the fact that the stem cells are not necessarily quiescent or do not necessarily have the ability to become quiescent (in contrast to the method disclosed in PCT/FR2003/002439).
  • adult human mesenchymal stem cells may be obtained using a method comprising one or more of the following steps:
  • the method further comprises, prior to step (b), a step of elimination of adipocytes from the digested tissue sample obtained in step (a) (e.g., by filtration), which leads to a cellular fraction essentially free of adipocytes.
  • adult human mesenchymal stem cells may be cultured according to standard cell culture techniques. For example, cells are often grown in a suitable vessel in a sterile environment at 37°C in an incubator containing a humidified 95% air - 5% C0 2 atmosphere. Vessels may contain stirred or stationary cultures. Cell culture techniques are well known in the art and established protocols are available for the culture of diverse cell types (see, for example, R.I. Freshney, "Culture of Animal Cells: A Manual of Basic Technique", 2 nd Edition, 1987, Alan R. Liss, Inc.).
  • cell viability can be determined, prior to coculture, for example, using standard techniques including histology, quantitative assessment with radioisotopes, visual observation using a light or scanning electron microscope or a fluorescent microscope. Alternatively, cell viability may be assessed by Fluorescence-Activated Cell Sorting (FACS).
  • FACS Fluorescence-Activated Cell Sorting
  • adult human mesenchymal stem cells and/or adult fully differentiated cardiomyocytes can be independently cryopreserved for future use in a coculture according to the present invention.
  • the cells are preferably cryopreserved under such conditions that most of the cells are viable upon recovery (i.e., thawing).
  • more than about 50%, 75%, 80%, or 85% of the cryopreserved cells are viable after recovery. More preferably, more than about 90% of the cryopreserved cells are viable after recovery. Even more preferably, more than about 95% or about 99% of the cryopreserved cells are viable after recovery.
  • the cryopreservation conditions are such that viable cells have identical morphologic and functional characteristics as the cells prior to cryopreservation.
  • Methods for the cryopreservation of different types of cells are known in the art. Any suitable method of cryopreservation may be used in the practice of the present invention.
  • the cryopreservation medium contains dimethyl sulfoxide (DMSO).
  • the cryopreservation medium may further comprise cryopreservation agents such as, methylcellulose.
  • the cells When the cells are to be used in a method of the present invention, they can be thawed under controlled conditions, for example by transferring the vial(s) containing frozen cells to a water bath set at 37°C. The thawed contents of the vial(s) may then be rapidly transferred under sterile conditions to a culture vessel containing an appropriate medium. The thawed cells can then be tested for viability, growth properties, etc.
  • Coculture of adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes may be carried out using any suitable method.
  • the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are cocultured under conditions where they are in physical contact.
  • the applicants have found that during the coculture, it is crucial that the stem cells and cardiomyocytes be in physical contact to allow cell-to-cell communications.
  • the term "physical contact” has its general meaning. For example, cells are in physical contact with each other when they are in a conformation or arrangement that allows for intercellular exchange of materials and/or information to take place without the involvement of a soluble factor.
  • Such conformations or arrangements include, but are not limited to, configurations comprising junction gaps, intercellular nanotubes, interactions between membrane receptors and membrane ligands, and the like.
  • the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are first put in suspension together in an appropriate culture medium before being plated.
  • the adult human mesenchymal stem cells are plated in an appropriate culture medium in order to obtain a cell lawn of mesenchymal stem cells, and then adult fully differentiated cardiomyocytes are added onto the plate of mesenchymal stem cells.
  • the cells are cocultured in a culture medium that does not comprise any growth factors.
  • adult human mesenchymal stems cells are plated on coated plates, e.g., gelatine-coated plates.
  • adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes may be cocultured for any efficient amount of time, i.e. any amount of time that is necessary to allow stimulation of the paracrine activity of adult human mesenchymal stem cells.
  • adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are cocultured for at least about 12 hours and preferably for at least about 24 hours in an appropriate culture medium, as described herein.
  • adult human stem cells and adult fully differentiated cardiomyocytes may be cocultured in any efficient ratio, i.e., in any ratio that leads to the stimulation of the paracrine activity of adult human mesenchymal stem cells.
  • any efficient ratio i.e., in any ratio that leads to the stimulation of the paracrine activity of adult human mesenchymal stem cells.
  • One skilled in the art will know how to determine such a ratio, and will also know how to identify optimal ratio conditions for the most efficient stimulation of the paracrine activity of adult human mesenchymal stem cells.
  • adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are coculture in a ratio of about 1:2, about 1: 1, or about 2: 1.
  • the coculture containing the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes is used in a cell-based therapeutic method as described herein.
  • the preconditioned adult human mesenchymal stem cells are separated from the adult fully differentiated cardiomyocytes. Separation may be performed using any suitable method, for example they may be separated by cell sorting flow, cytometry or by immunomagnetic beads coated with an antibody allowing discrimination between stem and cardiac cells. After separation, the preconditioned adult human mesenchymal stem cells may be used in a cell-based therapeutic method as described herein. Optionally, prior to being used, the separated preconditioned adult human mesenchymal stem cells may be stored under suitable conditions. Modulated Secretome and Improved Cardiac Cell Therapy Efficacy of Cocultured Mesenchymal Stem Cells
  • secretome has its art understood meaning, and refers to the set of proteins secreted by a cell.
  • the applicants have found that coculture of adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes led to an increase in the release, by the adult human mesenchymal stem cells, of soluble molecules that can be involved in or beneficial to cardiac repair. These molecules are herein called "cardioprotective factors”.
  • a method for modulating the secretome of adult human mesenchymal stem cells advantageously results in an increase in the release of at least one cardioprotective factor by the cocultured mesenchymal stem cells.
  • an increase in the release of at least one cardioprotective factor refers to an amount of a cardioprotective factor released by cocultured adult human mesenchymal stem cells that is higher than the amount of the same cardioprotective factor released by naive adult human mesenchymal stem cells, all other things being equal.
  • Naive adult human mesenchymal stem cells are adult human mesenchymal stem cells that have not been submitted to any co-culture, pre-conditioning, genetic modification or other type of treatment.
  • the amount of a cardioprotective factor released by cocultured adult human mesenchymal stem cells is at least 1.25 times higher than the amount of the same cardioprotective factor released by naive adult human mesenchymal stem cells, all other things being equal.
  • the increase in the amount may be by a factor of about 1.5, about 1.75, about 2, about 2.5; about 3, about 4, about 5 or more than 5.
  • the at least one cardioprotective factor may be any soluble molecule known in the art to be secreted by adult human mesenchymal stem cells and to be involved in or beneficial to cardiac repair.
  • soluble molecules may be for example: cytoprotection, angiogenesis, cell proliferation, cell migration, vessel stabilization, development, cell differentiation, cell growth, cell stabilization, cell contractility, inflammatory response, tubule formation, monocyte migration, monocyte proliferation, progenitor cell homing, and the like.
  • the applicants have identified VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), SDF- l cc (stromal-derived factor- 1 alpha), MCP-3 (monocyte chemotactic protein 3), IL6 (interleukin-6), and GROcc (growth regulated oncogene alpha). Therefore, in certain embodiments, the at least one cardioprotective factor is selected from the group consisting of VEGF, HGF, SDF- l cc, MCP-3, IL6, GROcc, and any combination thereof.
  • the at least one cardioprotective factor may also be any of adrenomedullin (ADM), angio-associated migratory protein (AAMP), angiogenin (ANG), angiopoetin- 1 (AGPT1), bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-6 (BMP6), connective tissue growth factor (CTGF), endothelin-1 (EDN1), fibroblast growth factor-7 (FGF7), insulin-like growth factor-1 (IGF-1), interleukin-11 (IL-11), kit ligand/stem cell factor (KITLG (SCF)), macrophage migration inhibitory factor (MIF), matrix metalloproteinase-9 (MMP9), macrophage- specific colony- stimulating factor (M-CSF), placental growth factor (PGF), plasminogen activator (PA), pleiotrophin (PTN), secreted frizzled-related protein-1 (SFRP1), secreted frizzled-related protein-2 (SFRP2), thrombo
  • MMP3 matrix metalloproteinase-3
  • MCP-1 monocyte chemoattractant protein-1
  • a method for modulating the secretome of adult human mesenchymal stem cells, and in particular for increasing the release of at least one cardioprotective factor by adult human mesenchymal stem cells, according to the invention results in adult human mesenchymal stem cells with improved pro-angiogenic properties and/or improved pro-chemotactic properties.
  • a method for modulating the secretome of adult human mesenchymal stem cells, and in particular for increasing the release of at least one cardioprotective factor by adult human mesenchymal stem cells, according to the invention results in adult human mesenchymal stem cells with an improved cardiac cell therapy efficacy.
  • the present invention relates to a population of preconditioned adult human mesenchymal stem cells, obtainable or obtained according to a method of the invention or an obvious variation thereof.
  • the population of preconditioned adult human mesenchymal stem cells is substantially homogeneous. In other embodiments, the population of preconditioned adult human mesenchymal stem cells is heterogeneous.
  • substantially homogeneous population refers to a population of adult human mesenchymal stem cells wherein the majority (e.g., at least about 80%, preferably at least about 90%, more preferably at least about 95%) of the total number of cells are preconditioned adult human mesenchymal stem cells.
  • heterogeneous population refers to a population of cells comprising preconditioned adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes.
  • a heterogeneous population of preconditioned adult human mesenchymal stem cells comprises at least about 40%, preferably at least about 50%, more preferably at least about 60% of preconditioned adult human mesenchymal stem cells.
  • the present invention relates to preconditioned adult human mesenchymal stem cells that have been separated from the adult fully differentiated cardiomyocytes with which they have been cultured.
  • preconditioned mesenchymal stem cells are characterized by their ability to undergo stronger paracrine stimulation than naive ones in response to de novo contact with cardiomyocytes.
  • the pre-conditioned adult human mesenchymal stem cells of the invention release at least one cardioprotective factor in a higher amount than naive adult human mesenchymal stem cells, all other things being equal.
  • the at least one cardioprotective factor may be any of the cardioprotective factor mentioned above.
  • the amount of a cardioprotective factor released by preconditioned adult human mesenchymal stem cells is at least 1.25 times higher than the amount of the same cardioprotective factor released by naive adult human mesenchymal stem cells, all other things being equal.
  • the increase in the amount may be by a factor of about 1.5, about 1.75, about 2, about 2.5; about 3, about 4, about 5, about 6, about 7, about 8, about 9 or more than 9.
  • the magnitude of the paracrine response of preconditioned mesenchymal stem cells to a second exposure to cardiomyocytes is equal or higher than that obtained by co-culture of mesenchymal stem cells with cardiomyocytes ⁇ i.e., in response to the first contact with cardiomyocytes). Accordingly, in certain embodiments, the amount of at least one cardioprotective factor released by preconditioned adult human mesenchymal stem cells in response to de novo contact with adult fully differentiated cardiomyocytes is equal or higher than the amount of the same cardioprotective factor released by adult human mesenchymal stem cells co-cultured with adult fully differentiated cardiomyocytes.
  • the at least one cardioprotective factor may be any of the cardioprotective factor mentioned above.
  • the cardioprotective factor is VEGF, SDF-lcc, MCP-3, IL6, GROcc, and any combination thereof.
  • the amount of a cardioprotective factor released by preconditioned adult human mesenchymal stem cells in response to de novo contact with adult fully differentiated cardiomyocytes is equal or at least 1.5 times higher than the amount of the same cardioprotective factor released by adult human mesenchymal stem cells co-cultured with adult fully differentiated cardiomyocytes, all other things being equal.
  • the increase in the amount may be by a factor of about 2, about 3, about 4, about 5, or more than 5.
  • preconditioned adult human mesenchymal stem cells are characterized by a release of VEGF that is equal to at least 500 pg/ml/10 5 cells or to at least 600 pg/ml/10 5 cells; and/or by a release of HGF that is equal to at least 300 pg/ml/10 5 cells or to at least 400 pg/ml/10 5 cells; and/or by a release of GROa that is equal to at least 1200 pg/ml/10 5 cells or to at least 1300 pg/ml/10 5 cells or to at least 1400 pg/ml/10 5 cells or to at least 1500 pg/ml/10 5 cells; and/or by a release of IL-6 that is equal to at least 2000 pg/ml/10 5 cells or to at least 2100 pg/ml/10 5 cells or to at least 2200 pg/ml/10 5 cells or to at least 2300 pg/ml/10 5 cells or to at least 2400
  • preconditioned adult human mesenchymal stem cells according to the invention are further characterized by a release of PDGF- BB that is of between 10 and 20 pg/ml/10 5 cells; and/or by a release of FGF-2 that is of between 12 and 30 pg/ml/10 5 cells; and/or by a release of G-CSF that is of between 12 and 30 pg/ml/10 5 cells; and/or by a release of SCF that is of between 2.5 and 5 pg/ml/10 5 cells.
  • preconditioned adult human mesenchymal stem cells according to the invention are further characterized by a release of LIF and/or IL-1 and/or IL-10 and/or TARC that is not detectable.
  • preconditioned adult human mesenchymal stem cells are characterized in that, in response to de novo contact with adult fully differentiated cardiomyocytes, they exhibit a release of VEGF that is equal to at least 1200 pg/ml/10 5 cells ⁇ e.g. , at least 1500, at least 2000 or at least 2500 pg/ml/10 5 cells); and/or by a release of HGF that is equal to at least 600 pg/ml/10 5 cells (e.g. , at least 1000, at least 2000, at least 3000 or at least 3500 pg/ml/10 5 cells); and/or by a release of GROa that is equal to at least 2000 pg/ml/10 5 cells (e.g.
  • IL-6 that is equal to at least 4000 pg/ml/10 5 cells (e.g. , at least 6000, at least 8000, at least 10000, or at least 15000 pg/ml/10 5 cells); and/or by a release of MCP-3 is equal to at least 100 pg/ml/10 5 cells (e.g. , at least 200, at least 300, at least 400 or at least 500 pg/ml/10 5 cells); and/or by a release of SDF-1 is equal to at least 200 pg/ml/10 5 cells (e.g.
  • these preconditioned adult human mesenchymal stem cells are further characterized by a release of LIF and/or IL- 1 and/or IL- 10 and/or TARC, in response to de novo contact with adult fully differentiated cardiomyocytes, that is not detectable.
  • preconditioned adult human mesenchymal stem cells according to the invention are characterized by improved pro-angiogenesis properties and/or pro-chemotactic properties compared to naive adult human mesenchymal stem cells.
  • preconditioned adult human mesenchymal stem cells according to the invention are characterized by an improved cardiac cell therapy efficacy compared to naive adult human mesenchymal stem cells.
  • a further aspect of the invention relates to the use of preconditioned adult human mesenchymal stem cells obtained using a method of the invention for the manufacture of a medicament or pharmaceutical composition for the treatment of a cardiac pathology.
  • the invention also relates to a pharmaceutical composition comprising adult human mesenchymal stem cells preconditioned by coculture with adult fully differentiated cardiomyocytes and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutical composition according to the present invention may further comprise at least one biologically active substance or bioactive factor.
  • adult human mesenchymal stem cells preconditioned by coculture with adult fully differentiated cardiomyocytes may only contain preconditioned mesenchymal stem cells (i.e. , preconditioned adult human mesenchymal stem cells after separation from adult fully differentiated cardiomyocytes with which they have been cultured) or alternatively may contain preconditioned mesenchymal stem cells and adult fully differentiated cardiomyocytes.
  • the term "pharmaceutically acceptable carrier or excipient” refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the preconditioned mesenchymal stem cells, and which is not excessively toxic to the host at the concentrations at which it is administered.
  • suitable pharmaceutically acceptable carriers or excipients include, but are not limited to, water, salt solution (e.g. , Ringer's solution), alcohols, oils, gelatins, carbohydrates (e.g. , lactose, amylase or starch), fatty acid esters, hydroxymethylcellulose, and polyvinyl pyroline.
  • Pharmaceutical compositions may be formulated as liquids, semi-liquids (e.g. , gels) or solids (e.g. , matrix, lattices, scaffolds, and the like). If desired, the pharmaceutical composition may be sterilized.
  • biologically active substance or bioactive factor refers to any molecule or compound whose presence in a pharmaceutical composition of the invention is beneficial to the subject receiving the composition.
  • biologically active substances or bioactive factors suitable for use in the practice of the present invention may be found in a wide variety of families of bioactive molecules and compounds.
  • a biologically active substance or bioactive factor useful in the context of the present invention may be selected from anti-inflammatory agents, anti-apoptotic agents, immunosuppressive or immunomodulatory agents, antioxidants, growth factors, and drugs.
  • a related aspect of the invention concerns a method for treating a subject suffering from a pathology associated with cardiac tissue damage, said method comprising a step of administering to the subject an efficient amount of adult human mesenchymal stem cells preconditioned by coculture with adult fully differentiated cardiomyocytes, or a pharmaceutical composition thereof.
  • treating refers to a method that is aimed at delaying or preventing the onset of a pathology, at reversing, alleviating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the symptoms of the pathology, at bringing about ameliorations of the symptoms of the pathology, and/or at curing the pathology.
  • the term “subject” refers to mammal, preferably a human being, that can suffer from a pathology associated with cardiac tissue damage, but may or may not have the pathology.
  • the term “subject” does not denote a particular age, and thus encompasses adults, children, and newborns.
  • the term "efficient amount” refers to any amount of a population of pre-conditioned mesenchymal stem cells (or a pharmaceutical composition thereof) that is sufficient to achieve the intended purpose.
  • cardiac pathology refers to any disease or condition affecting the heart, in particular to any disease or condition associated with cardiac tissue damage.
  • pathology associated with cardiac tissue damage refers to any disease or clinical condition characterized by cardiac tissue injury, dysfunction, defect or abnormality.
  • the term encompasses, for example, injuries, degenerative diseases and genetic diseases. Examples of cardiac degenerative diseases include, but are not limited to, heart failure, myocardial infarction, cardiac ischemia, myocarditis, arrhythmia, and the like.
  • cardiac genetic diseases include, but are not limited to, Duchenne muscular dystrophy, Emery Dreiffuss dilated cardiomyopathy, mental retardation caused by genetic abnormality such as fragile X chromosome and other inborn errors of metabolism such as phenylketonura gene defect, and the like.
  • the preconditioned adult human mesenchymal stem cells are allogenic to the subject being treated.
  • the term “allogenic” has its art understood meaning. More specifically, the term “allogenic”, when used herein in relation to the preconditioned adult human mesenchymal stem cells, means (1) that neither the adult human mesenchymal stem cells nor the adult fully differentiated cardiomyocytes used in the coculture were obtained from the subject to be treated, and (2) that the adult fully differentiated cardiomyocytes were obtained from a donor of the same species as the subject to be treated.
  • Preconditioned adult human mesenchymal stem cells (or a pharmaceutical composition thereof) according to the present invention may be administered to a subject using any suitable method.
  • the method of administration will be selected based on the site of tissue damage to be treated. Suitable methods of administration include, but are not limited to, parenteral methods such as intravenous, intra-arterial, intracardial (e.g., epicardial, intramyocardial), and percutaneous administration.
  • the administration method is preferably an intracardial administration.
  • Preconditioned adult human mesenchymal stem cells (or a pharmaceutical composition thereof) according to the present invention may be delivered at or near the site of tissue damage or degeneration of the deficient heart of the subject to be treated.
  • Patients may receive a single administration of preconditioned adult human mesenchymal stem cells (or a pharmaceutical composition thereof). Alternatively, they may receive at least two administrations of the preconditioned adult human mesenchymal stem cells.
  • Preconditioned adult human mesenchymal stem cells (or a pharmaceutical composition thereof) according to the present invention may be implanted in as subject alone or in combination with other cells, and/or in combination with other biologically active factors, reagents or drugs. As will be appreciated by those skilled in the art, these other cells, biologically active factors, reagents and drugs may be administered simultaneously (i.e. , substantially at the same time) or sequentially with (e.g. , prior to and/or following administration of) the preconditioned stem cells of the invention.
  • preconditioned adult human mesenchymal stem cells of the invention may be seeded and grown on a scaffold or any other three-dimensional tissue engineered construct support, either alone or in combinations with other cells, and /or in combination with biologically active factors or reagents.
  • the scaffold or construct which may be configured to replace a portion of the heart, can then be implanted into a subject.
  • a treatment according to the present invention further comprises pharmacologically immunosuppressing the subject prior to initiating the cell-based treatment. Methods for the systemic or local immunosuppression of a subject are well known in the art. However, in other embodiments, a treatment according to the present invention will not require to pharmacologically immunosuppression the subject prior to administration of preconditioned adult human mesenchymal stem cells (or a pharmaceutical composition thereof) according to the present invention.
  • Administration regimens including the optimal time of administration, e.g., following a heart attack
  • effective dosages to be used in the methods of treatment of the present invention can be readily determined by good medical practice based on the nature of the pathology of the subject, and will depend on a number of factors including, but not limited to, the extent of the symptoms of the pathology and extent of damage, degeneration and/or dysfunction of the cardiac tissue of interest, and characteristics of the subject (e.g., age, body weight, gender, general health, and the like).
  • Human primary cells, cell lines and culture conditions Human Multipotent Adipose Derived stem cells (hMADS) were isolated using previously described procedure (Rodriguez et ah, J. Exp. Med., 205, 201: 1397-1405).
  • Human bone marrow derived stem cells (hBMSC) were generously given by Dr. Helene Rouard (Etablatorium Francais du Sang (EFS), Creteil, France).
  • Human primary adult heart fibroblasts and progenitors were purchased from PromoCell (Heidelberg, Germany) or Innoprot (Bizkaia, Spain), respectively.
  • hMADS and hBMSC were cultured in Dulbecco's Modified Eagle Medium (DMEM) 1 g/1 glucose containing 10% heat inactivated fetal bovine serum (FBS) (Dominique Dutscher), 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 10 mM HEPES (Invitrogen), in a 5% C0 2 atmosphere at 37 °C whereas human cardiac primary cells were expanded as specifically recommended by manufacturers.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS heat inactivated fetal bovine serum
  • HEPES Invitrogen
  • CM mouse adult Cardiomyocytes
  • cardiomyocytes were seeded on cell culture inserts containing polycarbonate membrane (0.4- ⁇ or ⁇ size pore, Millicell, Millipore), which were placed in 35-mm dishes plated with hMADS. Collection and Biological Activities of culture conditioned media. For collecting conditioned media from single or cocultures, hMADS, adult cardiomyocytes or co-cultures were seeded at 10 5 cells/mL in DMEM supplemented with 0.8% FBS (to avoid artefactual contamination by the soluble factors which are contained in large amounts in the bovine serum) during 24 hours. Twenty four hours later, supernatants were collected, centrifuged at 4300 rpm for 5 minutes to remove cells debris and were then frozen.
  • polycarbonate membrane 0.4- ⁇ or ⁇ size pore, Millicell, Millipore
  • Cytokines from culture supernatants were measured by luminex using MILLIPLEX MAP kits (Millipore) and the Bioplex 200 system (Bio-Rad). Supernatant angiogenic activities were evaluated on human umbilical vein endothelial cells (HUVEC) by 2D and 3D-angiogenesis (Promocell, GmbH) assays.
  • MILLIPLEX MAP kits Millipore
  • Bioplex 200 system Bio-Rad
  • Chemotactic activities of culture supernatants were assessed by using ⁇ -slide chemotaxis (Ibidi) seeded with 18x103 hBMDC per channel.
  • Neutralizing antibodies against human VEGF165 (0.08 ⁇ g/ml), human HGF (0 ⁇ g/m ⁇ ), human MCP-3 (20 ⁇ g/ml) and human SDF-1 (3 ⁇ g/ml) were from R&D systems.
  • Anti- apoptotic effects of supernatants were evaluated on mouse neonatal cardiomyocytes isolated from 1 to 3 days old C57BL/6J mice (Burger et ah, Cardiovasc. Res., 2006, 72: 51-59) by PE-Annexin V staining (BD Pharmingen) and flow cytometry analysis.
  • MTT assays (Sigma Aldrich) were performed on cardiac fibroblasts or progenitors initially seeded at 10 4 cells/cm 2 as previously reported (Mosmann et al, J. Immunol. Methods, 1983, 65: 55-63).
  • RNA were extracted using the Qiagen RNeasy Mini Kit (Qiagen) and then reversetranscribed using the Superscript First-Strand Synthesis System (Invitrogen) and Oligo(dT)20. Quantitative RT-PCR reactions were performed in triplicate on a 7900 real-time PCR detection system (Applied Biosystems) using Platinium SYBR Green qPCR SuperMix (Invitrogen). PCR conditions were 50°C for 2 minutes, 95°C for 2 minutes, 45 cycles at 95°C for 15 seconds, and 60°C for 45 seconds, using GAPDH as the reference gene. Results are reported as mean +SD.
  • Angiogenesis assays Angiogenic effects of culture conditioned media were evaluated with a 3D-angiogenesis assay containing human umbilical vein endothelial cells (HUVEC) embedded in a collagen matrix (Promocell GmbH). After 48 hour- exposure, HUVEC sprout number and sprout length were quantified through 10 randomly photographed spheroids using image J 1.42q software (National Institutes of Health).
  • HUVEC human umbilical vein endothelial cells
  • MTT assays and Sirius red quantification After 48h exposure with the different kind of conditioned culture media, proliferation rate of human primary cardiac fibroblasts or progenitors initially seeded at 10 4 cells/cm 2 were determined by MTT assays (Sigma Aldrich) as previously reported (Mosmann et ah, J Immunol Methods, 1983, 65: 55-63). After 48 hours exposure with conditioned culture media, collagen synthesis by cardiac fibroblasts seeded at confluence (1.5 10 5 cells/cm 2 ) was estimated by Sirius red staining (vWR) and spectrophotometer reading at OD 540nm.
  • Cardiomyocyte apoptosis Anti-apoptotic effects of supernatants were evaluated after 48 hours exposure of mouse neonatal cardiomyocytes because they survive better in vitro than their adult counterparts. These cells were isolated from 1- to-3 day old C57BL/6J mice as earlier described (Burger et ah, Cardiovasc Res. 2006, 72: 51-59). Apoptotic cell rate was evaluated following PE-Annexin V staining (BD Pharmingen) and flow cytometry analysis. Chemotaxis assays. Chemo tactic activities of the different kinds of culture supernatants were assessed by using ⁇ -slide chemotaxis (Ibidi) seeded with 18.10 hBMDC per channel.
  • Ibidi ⁇ -slide chemotaxis
  • hMADS cells were fixed with 4% PFA then with cold acetone. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich). Fluorescence was analysed with a Zeiss Axioplan 2 Imaging microscope. Intercellular dye exchanges and inhibition of cell-to-cell communication pathways. Prior to co-culturing, the cardiomyocytes were labelled with MitoTracker Red FM ( ⁇ ) or calcein AM ( ⁇ ) (Molecular Probes).
  • Intercellular exchanges were examined by flow-cytometry or conventional microscopy (Zeiss Axioplan 2 Imaging microscope). To inhibit gap junctions as well as f-actin or a-tubulin polymerization, fresh cocultures were treated during 24 hours with 100 ⁇ 18 ⁇ - glycyrrhetinic acid (18 a-GA, Sigma-Aldrich), 2.5x10 - " 8 M latrunculin A (Invitrogen) or 5x10 - " 8 M nocodazole (Sigma-Aldrich) respectively. Mouse myocardial infarction and cell injections.
  • Bone marrow cells were obtained from GCAG-GFP transgenic mice by flushing the femurs and injected retro-orbitally into 8 week-old C57BL/6 mice (3xl0 6 cells per mouse) previously irradiated at 9 Gy. Recipient mice were treated with lOmg/kg/day ciprofloxacin for 14 days. Blood chimerism of >90 was controlled at 8 weeks post- transplantation. Echocardiography. Echocardiography was performed before MI, 5 and 20 days post infarction using a 13-MHz linear transducer (VIVID 7 Echocardiogram, GE Medical System).
  • LV areas (A) and lengths (L) were measured at end-systole (ES) and end-diastole (ED) according to the American Society of Echocardiography leading-edge method. End-diastolic volume (LVEDV) and end- systolic volume (LVESV) were calculated using the single-plane area-length method as previously described (Scorsin et ah, J Thorac Cardiovasc Surg. 2000, 119: 1169-1175).
  • Capillary density of peri-infarct area was determined after micro vessel staining with isolectin B4 (40 ⁇ g/ml, Sigma-Aldrich) and counting from at least 20 randomly selected fields in border areas by a blinded investigator.
  • FITC-, CY3- or Cy5- conjugated antibodies were from Jackson Immunoresearch laboratories (1: 100,). Sections were counterstained with dapi (Sigma-Aldrich) and fluorescence was analysed by conventional (Zeiss Axioplan 2 Imaging microscope) or confocal microscopy (Zeiss LSM 510 Meta). Fluorescence in situ hybridization (FISH). FISH experiments were performed on heart sections as described by Matsuura (Matsuura et ah, J Clin Invest. 2009, 119: 2204-2217), using NICK-translated human Cy-3 COT-1 and mouse biotinylated COT-1 DNA probes (Roche Diagnostic). Mouse biotin-labeled DNA was detected with streptavidin fluorescein conjugate (Sigma-Aldrich). Statistical Analysis. Statistical analysis was done using Prism 5.04 Software
  • CM mesenchymal stem cells
  • MSC mesenchymal stem cells
  • CM mesenchymal stem cells
  • endothelial HUVEC cells exhibited a significantly higher relative sprout length and sprout number when exposed to co-culture compared to control hMADS or CM conditioned media or basal medium ( Figure 2A- B).
  • co-culture-conditioned media induced a significant faster migration of bone marrow derived stem cells, some of which exhibited a GATA-4+/PH3+ cardiac progenitor phenotype (168+28% compared to basal medium) than naive hMADS (128+22%) or CM (112+19%) (Figure 3A).
  • Tunnelling nanotubes (TNT) mediated cell-to-cell communication can be selectively inhibited by latrunculin A or nocodazole treatments.
  • TNT Tunnelling nanotubes
  • the Applicants then developed a drug strategy approach to specifically disrupt TNT by using 2.5 x 10 - " 8 M latrunculin A or 5.0 x 10 - " 8 M nanodazole, which inhibit polymerization of f-actin and microtubule TNT components, respectively. Consequences of these pharmacological treatments were evaluated in mixed cultures of hMADS with mouse CM preloaded both the small gap junction diffusible molecule, calcein and the mitochondria-label mitotracker.
  • TNT Heterologous tunnelling nanotubes
  • MSC mesenchymal stem cells
  • TNT TNF-cc or IFN- ⁇ prior to coculture with cardiomyocytes.
  • the number of TNT connecting stem to cardiac cells was increased and the transcription of TNT-dependent cytokines was significantly activated (data not shown), suggesting that TNT cell-to-cell communication is sensitive to inflammatory stimuli.
  • human cells exhibiting endothelial or cardiac phenotype were never detected (not shown) while hybrid cells were difficult to observe (an average of 1 synkaryon detected per 20 heart sections) (not shown).
  • mice infracted hearts were injected with co-cultures made with GFP+ CM to easily identify cells derived from CM somatic reprogramming.
  • some rare GFP-positive CM were detectable suggesting that in vitro reprogrammed cardiac cells could have the potency to terminally differentiate into mature CM.
  • Luminex assays showed that at this time point, secretome differences between hMADS grown alone or in co- culture were flattened for most of the soluble factors except to MCP-1 and MMP-3 ( Figure 10A). This suggests that the higher regenerative capacity of co-cultivated hMADS was unlikely due to a higher production of cardioprotective factors at the time of cell injection.
  • VEGF and HGF in co-cultivated hMADS accounts for the enhanced pro-angiogenic activity (Jayasankar et al, Circulation, 2003, 108(Suppl. 1): 11230-236; Tao et al, Proc. Natl. Acad. Sci. USA, 2011, 108: 2064-2069) while over- secretion of VEGF, HGF, SDF-la and MCP-3 by means of coculture participates in the heightened bone marrow-derived progenitor recruitment potential of hMADS (Kucia et al, Circ.
  • stimulated release of other factors such as IL-6 and GRO-a by co-cultivated hMADS may indirectly enhance their pro-angiogenic effects by respectively activating either the secretion of VEGF or the myocardial homing of bone marrow-derived endothelial progenitors (Kinnaird et al, Circ. Res., 2004, 94: 678-685; Kocher et al, J. Mol. Cell. Cardiol., 2006, 40: 455-464).
  • the enhanced salutary effects of co-cultivated hMADS may also be due to down -regulation of "deleterious" diffusible molecules such as MCP-1, previously reported to exacerbate myocardial inflammation (Niu et al, Clin. Sci., 2009, 117: 95-109) and MMP-3, incriminated in extracellular matrix degradation resulting in adverse left ventricular remodeling post-acute myocardial infarction (Kelly et al, Eur. J. Heart Fail., 2008, 10: 133-139).
  • CM stress signal spreading along these structures trigger secretome alteration of hMADS with the aim to enhance broken heart repair.
  • the present results strongly indicate that membrane tunneling bridges enriched of f-actin and microtubules, already found to connect MSC and CM (Acquistapace et al, Stem Cells, 2011, 29: 812-824; He et al, Cardiovasc. Res., 2011, 92: 39-47), are clearly involved in the hMADS paracrine switch of key mediators of heart repair encompassing VEGF, HGF, SDF-1 and MCP-3 and are mainly responsible of the heightened angiogenic and chemotactic effects of hMADS following co-culture.
  • hMADS paracrine changes is mediated by other pathways of cell-to-cell communications, as illustrated by the secretion of IL-6, GRO-a, MMP-3 and MCP-1.
  • uptake by hMADS of large microparticles >0.4 ⁇
  • apoptotic bodies from cardiac origin Burghoff et al, Cardiovasc.
  • TNT effects is extremely difficult to assess in vivo, this issue could be addressed indirectly by comparing the efficacy of cell therapy as a function of increasing doses of administered cells and/or by using different routes of cell delivery.
  • cells may be unable to interact with CM because they may clump when delivered at high concentrations or be retained in interstitial spaces when intravenously injected, the repairing processes involving TNT should be minimized.
  • hMADS should induce an immune rejection earlier after their transplantation, thus compromising heart repair, as reported for human engrafted MSC in rat infarcted myocardium (Lai et al, J. Mol. Cell Cardiol., 2010, 48: 1215-1224; Grinnemo et al, Ann. Med., 2006, 38: 144-153; Grinnemo et al, J. Thorac. Cardiovasc. Surg., 2004, 127: 1293-1300).
  • the improved regenerative properties of cocultured cells are unlikely to result from an immune survival advantage over their naive counterparts as both human cells were similarly cleared in immunocompetent mouse hearts.

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Abstract

La présente invention concerne des procédés pour obtenir des cellules souches mésenchymateuses ayant un potentiel de régénération cardiaque amélioré. Les procédés comprennent une étape de co-culture de cellules souches mésenchymateuses humaines adultes avec des cardiomyocytes complètement différenciés adultes, ce qui entraîne une modulation du secrétome cardioprotecteur des cellules souches mésenchymateuses pré-conditionnées. L'invention concerne également des compositions pharmaceutiques comprenant de telles cellules souches mésenchymateuses pré-conditionnées, et leurs procédés d'utilisation pour le traitement de pathologies cardiaques et/ou pour les reconstructions ou la régénération de tissu cardiaque.
PCT/EP2013/071308 2012-10-12 2013-10-11 Cellules souches mésenchymateuses modulées pour la thérapie cellulaire cardiaque WO2014057097A1 (fr)

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WO2016102597A1 (fr) * 2014-12-23 2016-06-30 Mesoblast International Sàrl Procédé pour traiter l'insuffisance cardiaque
CN107257687A (zh) * 2014-12-23 2017-10-17 迈索布拉斯特国际有限公司 用于治疗心力衰竭的方法
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