WO2014057097A1 - Modulated mesenchymal stem cells for cardiac cell therapy - Google Patents

Abstract

The present invention relates to methods for obtaining mesenchymal stem cells with improved cardiac regenerative potential. The methods comprise a step of co-culturing adult human mesenchymal stem cells with adult fully differentiated cardiomyocytes, which leads to a modulation of the cardioprotective secretome of the pre-conditioned mesenchymal stem cells. The invention also relates to pharmaceutical compositions comprising such pre-conditioned mesenchymal stem cells, and methods for using them for the treatment of cardiac pathologies and/or for cardiac tissue reconstructions or regeneration.

Description

MODULATED MESENCHYMAL STEM CELLS FOR CARDIAC CELL THERAPY

Related Application

The present patent application claims priority to European Patent Application No. EP 12 306 264 filed on October 12, 2012. The content of the European Patent Application is incorporated herein by reference in its entirety.

Background of the Invention

For several years, technologies in the field of regenerative medicine have focussed on stem cells, as these cells have the capacity to differentiate into specialized cell types. Indeed, certain tissues or organs, such as heart tissue and neural tissue cannot regenerate alone or, at least, cannot regenerate efficiently, due to their very limited capacity of self-renewal. Regenerative medicine involves transplanting cells of interest with the goal of repairing and regenerating a target tissue and/or target organ.

Thus, 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.

With regard to this serious public health problem, rebuilding the injured heart is a critical challenge. During the past decade, intensive efforts have been devoted to the development of methods for cardiac repair based on cell transplantation. Different cell types have been utilized in such methods, including skeletal myoblasts, neonatal myocytes, hematopoietic stem cells and mesenchymal and embryonic stem cells. But each of these approaches exhibits limitations (e.g., extensive death of transplanted cells, lack of electrochemical junctions with host cardiomyocytes, risk of immune rejection, and teratoma formation) (Rosenthal et al., N. Engl. J. Med., 2003, 349(3): 267-274; Torella et al, Circ. Res., 2004, 94(4): 514-524; Leri et al, Circ. Res., 2004, 94(2): 132-134). Even if some of these strategies provide beneficial effects, these are mainly due to neangiogenesis and arteriogenesis. However, none of these methods promote cardiac remuscularization, which is vital to improve heart function in the long term (Invernici et al., Exp. Cell Res., 2008, 314: 366-376).

Cell therapies based on mesenchymal stem cells (MSCs) are among the most promising approaches to rebuild damage heart due to the MSC plasticity, immune privilege and strong self -renewal ability (D'Ippolito et al, J. Cell Sci., 2004, 117: 2971-2981; Reyes al, Blood, 2001, 98: 2615-2625; Rodriguez al, J. Exp. Med., 2005, 201: 1397-1405). Studies performed during the past decade have established the proof of concept that intramyocardial delivery of MSC from various tissue origins improves hear function after infarction (Shake al, Ann. Thorac. Surg., 2002, 73: 1919-1925; Zeng al, Circulation, 2007, 115: 1866-1875; Zimmet al., Basic Res. Cardiol., 2005, 100: 471-481). Nevertheless, the mechanisms by which mesenchymal stem cells exert their therapeutic action remain so far unclear. Therefore, there needs to be a deeper understanding of mesenchymal stem cells biology to improve the modest efficiency of existing cardiac cell therapies.

Different strategies have been developed in order to optimize the cardiac regeneration potential of mesenchymal stem cells (Mohsin et al., Circ. Res., 2011, 109: 1415-1428; Ranganath et al., Cell Stem Cell, 2012, 10: 244-258). 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. Res., 2008, 77: 134-142), FGF, BMP-2 and IGF-1 (Hahn et ah, JACC, 2008, 51: 933-944). Still another approach consists in co-injecting the stem cells with biomaterials (Jin et ah, Eur. J. Heart Fail., 2009, 11: 147-153; Karam et ah, Biomaterials, 2012, 33: 1733-1743). However these approaches exhibit several limitations including side effects in the case of co-injection with biomaterials, constitutive expression of the genes of interest instead of the desired transient expression under the form of a gradient in the case of genetically modified stem cells, and a microenvironment that only partially mimics the microenvironment found in a damaged heart tissue in the case of pharmacologic pre-conditioning. Therefore, there still remains, in the art, an ongoing and undisputed need for a new strategy to improve the modest efficiency of existing cardiac cell therapies.

Summary of the Invention

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. In particular, the applicants have shown that coculturing human multipotent adipose derived stem cells (hMADS) used as a model for mesenchymal stem cells with 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. 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. In addition, the paracrine activation that is obtained by coculture is advantageously gradual and transient, a chemical gradient being required for cardiac regeneration. Furthermore, pre-conditioning with cardiomyocytes has 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. Finally, co-culturing does not exhibit the drawbacks inherent to genetic modifications and pharmacologic treatments or the immunologic tolerance associated with the use of biomaterials.

Accordingly, in one aspect, 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.

In certain embodiments, the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are in physical contact during the coculture.

In certain embodiments, 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. For example, 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.

In certain embodiments, 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.

In certain embodiments, 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. In certain embodiments, the adult human mesenchymal stem cells are derived from a tissue obtained from a healthy adult donor. In certain embodiments, 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.

In another aspect, the present invention provides preconditioned adult human mesenchymal stem cells obtainable, or obtained, by a method according to the invention.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In another aspect, the present invention provides 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.

In certain embodiments, the preconditioned adult human mesenchymal stem cells have been separated from the adult fully differentiated cardiomyocytes with which they have been cocultured. In a related aspect, 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.

In yet another aspect, 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.

In certain embodiments, 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.

In certain embodiments, 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.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments. Brief Description of the Figures

Figure 1 is a set of graphs showing that co-culture alters hMADS secretome. Histograms representing (A) relative secretion level and (B) relative mRNA abundance of factors found differently expressed in co-cultures determined by Luminex assays from conditioned media from 24 hour-single cultured mouse cardiomyocytes (CM, n=8), hMADS cells alone (HM, n=8) or in co-culture without or with transwell insert (CC, n=8 or CCTW, n=6, respectively). * p<0.05; ** p<0.01, *** p<0.001.

Figure 2 is a set of graphs showing that co-culture improves hMADS paracrine functions. (A) Representative photographs of an HUVEC spheroid and (B) HUVEC spheroid sprout length and sprout number after 48 hours exposure to basal medium (0.8% FBS), or conditioned media from CM (CM), hMADS (HM), coculture without (CC), with transwell insert (TWCC) and, mix of hMADS and cardiomyocytes independent cultures (HM+CM) or VEGF (20ng/ml) (mean + SD of independent experiments repeated n= 8 for 0.8% FBS, n=4 for CM, n=8 for HM, n=8 for CC, n=4 for CCTW, n=6 and n=5 for VEGF). (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, 20μιη. Lower panel: migration of BMMSC after 24 hours exposure to supernatants (mean + SD of n=8 independent experiments). (D) Capillary relative- length and number after 24 hour exposure to basal medium (0.8% FBS) or conditioned media froHM, CC and CC treated or not with hHGF and/or hVEGF blocking antibodies. (E) Migration of BM-MSC after 24 hour-exposure to basal medium (0.8% FBS) or conditioned media from HM, CC or CC treated with neutralizing antibodies against hHGF, hVEGF, hSDF-l and hMCP3 (mean + SD of n=6 independent experiments). (F) Flow cytometry-estimated rate of annexin V+ apoptotic CM after 48-hour exposure to conditioned media (mean + SD of n=6 independent experiments). (G) Proliferation rate (MTT) of human cardiac fibroblasts after 48h-exposure to conditioned media (mean + SD of n=4 and n=8 independent experiments, respectively). (B-F) #, p<0.05 versus basal medium; * p<0.05; **p<0.01, ns: no significant. Figure 3 is a set of showing that co-culture improves hMADS paracrine functions. (A) Migration of BM-MSC after 24-hour exposure to different kinds of supernatants (mean + SD of n=8 independent experiments). (B) MTT assays revealing proliferation rate of human primary cardiac progenitor cells exposed during 48 hours to the different culture supernatants (mean + SEM of n=8 independent experiments). (C) Upper panel: a representative flow cytometry dot blot of mouse neonatal CM stained with annexin V/ IP. Lower panel: flow-cytometry-estimated rate of annexin V+ apoptotic CM after 48-hour exposure to conditioned media (mean + SD of n=6 independent experiments). (D) Upper panel: collagen synthesis (sirius red staining quantification) and proliferation rate (MTT assays) of human cardiac fibroblasts after 48h-exposure to conditioned media (mean + SEM of n=4 and n=8 independent experiments, respectively). #, 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. (A) Relative mean fluorescence fold increase for calcein and mitotracker in 24-hour co-cultivated hMADS without or with 0.4 or 1 μιη pore size transwell insert by reference to single hMADS. (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 μιη 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, 20μιη. 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, 20μιη.

Figure 6 is a set of graphs showing that disruption of heterologous TNT channels abrogates coculture-induced hMADS paracrine stimulation. Relative paracrine secretion of 24 hour co-cultivated hMADS compared to hMADS alone in absence (CTL) or presence of latrunculin A (LAT-A) or nocodazole (NOCO). Data represent the means+ SEM of at least n=6 independent experiments. * p<0.05; ** p<0.01.

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 and (B) -chemotaxis of human BM-MSC. Data represent the means+ SEM of at least n=6 independent experiments. * p<0.05; ** p<0.01.

Figure 8 shows that coculture improves hMADS cell therapy efficacy. (A) Left ventricular ejection fraction (LVEF) at day 5 and day 21 after experimental myocardial infarction and cell injection. (HBSS, n=7; CM, n=6; HM, n=8; CC, n=l l). * p<0.05 between CC and HM;† p<0.05 between CC and HBSS; # p<0.01 between CC and CM. (B) Trichrome Masson's staining (blue) and (C) quantification of cardiac fibrosis at day 21 post infarction from heart sections of mice treated with HBSS (n=7); CM (n=6); HM (n=8); CC (n=l l). (D) Quantification of peri-infarct capillary density at day-3 and -7 post-infarction (n=4 per group of mice and time). #, p<0.05 versus basal medium; * p<0.05; ** p<0.01.

Figure 9 shows that coculture improves hMADS cell therapy efficacy. (A) Quantification of GATA4+ cells in the infarct area at day-3 and -7 postinfarction (mean+ SEM of n=4 mice per group and time). (B) Quantification of double GATA4/GFP positive cells in the infarct area at day-3 post-infarction (mean+ SEM of n=4 mice per group and time). (C) Number of caspase 3 positive apoptotic CM in the peri-infarct area at day-3 and -7 post-infarction (mean +SEM of n=4 mice/per group and time). (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. (A) Luminex assays revealing secretome changes of day 4 co-cultivated hMADS (d4CC) prior and after a second 24hour-exposure to CM (d4 CC+CM). Results are expressed in mean + SEM of at least n=5 independent experiments for each condition, * p<0.05. (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, 20μιη.

Figure 11. Relative transcriptional gene expression changes (qPCR) of hMADS after 24 hours of coculture with CM (1HM+1CM: 1HM) compared to that of hMADS seeded in double number (2HM: 1HM). Results are expressed in relative means +SD of 3 independent experiments. *p<0.05, **p<0.01 and ***p<0.001. Detailed Description of the Invention

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. I - Methods for Modulating the Cardioprotective Secretome of 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.

As intended herein, the term "coculturing" refers to a process in which at least two different types of cells are cultured together in an appropriate culture medium. In the methods of the present invention, adult mesenchymal human stem cells and adult fully differentiated cardiomyocytes are cocultured.

As used herein, the term "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. By way of examples, 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. Thus, 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). Adult human mesenchymal stem cells and fully differentiated cardiomyocytes may be plated in coated (e.g. , gelatine - coated) or uncoated plates.

As used herein, the term "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. For example, 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. Med., 2005, 201(9): 1397-1405. These cells are called 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. Other 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. Med., 2004, 200(2): 123-135), and mesoangioblasts (Sampaolesi M et al, Nature, 2006, 444(7119): 574-579; Dellavalle A et al, Nat. Cell Biol., 2007, 9: 255-267). In particular, 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. Neuroimmunol., 2008, 197(2): 99-109) and have been shown to fail to elicit an immune response in allogeneic hosts after engraftment into the diseased heart (Henning et al, Cell Transplant, 2007, 16(9): 907-917). Furthermore, umbilical cord blood banks {e.g., Etablissement Francais du Sang, in France) provide secure and easily available sources of such cells for transplantation.

Other suitable mesenchymal stem cells include mesenchymal stem cells isolated from bone marrow or obtained by liposuction.

As used herein, the term "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. In the context of the present invention, adult fully differentiated cardiomyocytes may be from any appropriate mammal origin (e.g. , mouse, rat, rabbit, pig, dog or human origin).

In a particular embodiment, the method comprises steps of:

a) providing adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes, and

b) coculturing said cells in an appropriate culture medium.

In and during the coculture, 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

In this particular context, the term "providing" herein refers to a process in which cells are isolated and provided in a state suitable for in vitro culture. As used herein, the term "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). Preferably, "isolated" includes alteration of the cell' s relationship with the neighboring cells with which it is in direct contact, for example, by dissociation.

Within the context of the present invention, human stem cells are preferably adult human mesenchymal stem cells, which may be derived from a large variety of tissues. In certain embodiments, human mesenchymal stem cells are derived from adipose tissue. In other embodiments, human mesenchymal stem cells are derived from skeletal muscle. In yet other embodiments, human mesenchymal stem cells are derived from adipose tissue and skeletal tissue. In still other embodiments, human mesenchymal stem cells are derived from bone marrow, dental pulp, blood, and/or umbilical cord blood.

In most of the present document, 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. However, it is to be understood that 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. Examples of 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.

Similarly, it is to be understood that 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.

As already mentioned above, adult fully different cardiomyocytes may be from any appropriate mammal origin (e.g. , mouse, rat, rabbit, pig, dog or human origin). In the context of the present invention, 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. Thus, adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes may be independently isolated from any suitable tissue sample. The term "tissue sample", as used herein, refers to any sample of tissue harvested from a suitable mammal, as already mentioned above. In the context of the present invention, 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.

Methods of harvesting samples from tissues and organs are known in the art and can be used in the practice of the present invention. Preferably, methods of harvesting are not excessively destructive for the tissue being harvested. Thus, for example, human 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. For example, the tissue sample may be washed with a buffer solution (e.g. , buffered saline) optionally comprising antimytotic and/or antibiotic agents.

In certain embodiments, 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. , using one or more proteolytic enzymes or combination of 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.

Any suitable clonal selection and cell separation techniques may be used in the practice of the present invention. 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. In certain embodiments, 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).

In certain embodiments, adult human mesenchymal stem cells may be obtained using a method comprising one or more of the following steps:

a) enzymatic digestion of a tissue sample (e.g. , in the presence of collagenase for about 10 minutes or less) to obtain a cellular fraction;

b) in vitro culture of the cellular fraction; and c) selection, from the cellular fraction, of a cell sub-population of cells called "CA" and exhibiting an adhesion rate lower than about 12 hours to obtain adult human mesenchymal stem cells.

If the tissue sample is an adipose tissue, 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.

Before coculture, 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% C0₂ 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.).

If desired, 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).

If desired, adult human mesenchymal stem cells and/or adult fully differentiated cardiomyocytes, either freshly isolated from a tissue sample or following expansion in culture, can be independently cryopreserved for future use in a coculture according to the present invention. In such a case, the cells are preferably cryopreserved under such conditions that most of the cells are viable upon recovery (i.e., thawing). Preferably, 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. Preferably, 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. Typically, the cryopreservation medium contains dimethyl sulfoxide (DMSO). The cryopreservation medium may further comprise cryopreservation agents such as, methylcellulose. Once frozen, the adult mesenchymal stem cells and the adult fully differentiated cardiomyocytes may be independently stored indefinitely under liquid nitrogen until needed, as long as care is taken to prevent the possibility of accidental thawing or warming of the frozen cells at any time during their storage period. 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.

Coculturing of Adult Human Mesenchymal Stem Cells and Adult Fully Differentiated Cardiomyocytes

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. As used herein, 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.

In certain embodiments, 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. In other embodiments, 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. In certain embodiments, the cells are cocultured in a culture medium that does not comprise any growth factors.

In other particular embodiments, 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. One skilled in the art will know how to determine such an amount of time. In certain embodiments, 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.

Moreover, 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. 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. For example, in certain embodiments, 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. In certain embodiments, 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.

However, in other embodiments, after coculture, 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

The term "secretome", as used herein, 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".

Thus, in certain embodiments, a method for modulating the secretome of adult human mesenchymal stem cells according to the present invention advantageously results in an increase in the release of at least one cardioprotective factor by the cocultured mesenchymal stem cells. The term "an increase in the release of at least one cardioprotective factor", as used herein, 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.

In certain embodiments, 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. For example, 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. The general functions of such 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.

Among the cardioprotective factors released by co-cultured mesenchymal stem cells, 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), thrombospondin-1 (THBS1), thymosin- 4 (TMSB4), transforming growth factor-β (TGF-β), tumor necrosis factor-cc (TNF-cc), and any combination thereof.

The applicants have observed that co-culturing led to a decrease in the release of soluble factors reported to be deleterious for heart repair, such as matrix metalloproteinase-3 (MMP3) and monocyte chemoattractant protein-1 (MCP-1). However, the decreased release of these deleterious factors did not require a physical contact between the co-culture mesenchymal stem cells and cardiomyocytes.

The applicants have shown that in vitro preconditioning of mesenchymal stem cells with cardiomyocytes through co-culture improves the effectiveness of mesenchymal stem cells in promoting angiogenesis and chemoattraction of bone- marrow-derived mesenchymal progenitors. Superior beneficial effects of co- cultivated versus naive mesenchymal stem cells are explained by a stronger paracrine stimulation of pre-conditioned cells compared to naive ones in response to in vivo cellular interaction with resident cardiomyocytes.

Accordingly, in certain embodiments, 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.

In certain embodiments, 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.

II - Preconditioned Mesenchymal Stem Cells

In another aspect, 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. In certain embodiments, 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.

The term "substantially homogeneous population", as used herein in relation to a population of preconditioned adult human mesenchymal stem cells, 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. The term "heterogeneous population", as used herein in relation to a population of preconditioned adult human mesenchymal stem cells, refers to a population of cells comprising preconditioned adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes. Generally, 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. In certain embodiments, 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.

As mentioned above, the Applicants have found that the paracrine stimulation of mesenchymal stem cells could be re-induced by subsequent contact with cardiomyocytes. In other words, 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.

Accordingly, in certain embodiments, in response to subsequent contact with adult fully differentiated 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. In certain embodiments, 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. For example, 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 Applicants have also found that 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. In certain embodiments, the cardioprotective factor is VEGF, SDF-lcc, MCP-3, IL6, GROcc, and any combination thereof.

In certain embodiments, 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. For example, the increase in the amount may be by a factor of about 2, about 3, about 4, about 5, or more than 5. In certain embodiments, preconditioned adult human mesenchymal stem cells according to the invention are characterized by a release of VEGF that is equal to at least 500 pg/ml/10⁵ cells or to at least 600 pg/ml/10⁵ cells; and/or by a release of HGF that is equal to at least 300 pg/ml/10⁵ cells or to at least 400 pg/ml/10⁵ cells; and/or by a release of GROa that is equal to at least 1200 pg/ml/10⁵ cells or to at least 1300 pg/ml/10⁵ cells or to at least 1400 pg/ml/10⁵ cells or to at least 1500 pg/ml/10⁵ cells; and/or by a release of IL-6 that is equal to at least 2000 pg/ml/10⁵ cells or to at least 2100 pg/ml/10⁵ cells or to at least 2200 pg/ml/10⁵ cells or to at least 2300 pg/ml/10⁵ cells or to at least 2400 pg/ml/10⁵ cells; and/or by a release of MCP-3 is equal to at least 35 pg/ml/10⁵ cells or to at least 45 pg/ml/10⁵ cells; and/or by a release of SDF-1 is equal to at least 80 pg/ml/10⁵ cells or to at least 90 pg/ml/10⁵ cells; and/or by a release of MCP-1 is equal to at most 600 pg/ml/10⁵ cells or to at most 90 pg/ml/10⁵ cells; and/or by a release of MMP-3 that is equal to at most 20,000 pg/ml/10⁵ cells or at most 15,000 pg/ml/10⁵ cells or at most 10,000 pg/ml/10⁵ cells. In certain embodiments, 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/10⁵ cells; and/or by a release of FGF-2 that is of between 12 and 30 pg/ml/10⁵ cells; and/or by a release of G-CSF that is of between 12 and 30 pg/ml/10⁵ cells; and/or by a release of SCF that is of between 2.5 and 5 pg/ml/10⁵ cells. In certain embodiments, 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.

In yet other embodiments, 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/10⁵ cells {e.g. , at least 1500, at least 2000 or at least 2500 pg/ml/10⁵ cells); and/or by a release of HGF that is equal to at least 600 pg/ml/10⁵ cells (e.g. , at least 1000, at least 2000, at least 3000 or at least 3500 pg/ml/10⁵ cells); and/or by a release of GROa that is equal to at least 2000 pg/ml/10⁵ cells (e.g. , at least 3000, at least 4000 or at least 5000 pg/ml/10⁵ cells); and/or by a release of IL-6 that is equal to at least 4000 pg/ml/10⁵ cells (e.g. , at least 6000, at least 8000, at least 10000, or at least 15000 pg/ml/10⁵ cells); and/or by a release of MCP-3 is equal to at least 100 pg/ml/10⁵ cells (e.g. , at least 200, at least 300, at least 400 or at least 500 pg/ml/10⁵ cells); and/or by a release of SDF-1 is equal to at least 200 pg/ml/10⁵ cells (e.g. , to at least 300, at least 400, or at least 450 pg/ml/10⁵ cells). In certain embodiments, 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. In certain embodiments, 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.

In certain embodiments, 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.

Ill - Pharmaceutical Compositions and Use Thereof

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. In certain embodiments, a pharmaceutical composition according to the present invention may further comprise at least one biologically active substance or bioactive factor. As indicated above, 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.

As used herein, 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. Examples of 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.

As used herein, the term "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. As will be acknowledged by one skilled in the art, 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. For example, 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.

Another aspect of the invention pertains to such pharmaceutical compositions for use in the treatment of pathologies, and/or for use in tissue reconstruction regeneration. 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.

In the context of the invention, the term "treating" or "treatment", as used herein, 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.

As used herein, 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.

As used herein, 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.

As used herein, the term "cardiac pathology" refers to any disease or condition affecting the heart, in particular to any disease or condition associated with cardiac tissue damage. The term "pathology associated with cardiac tissue damage" refers to any disease or clinical condition characterized by cardiac tissue injury, dysfunction, defect or abnormality. Thus, 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. Examples of 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.

In most methods of treatment of the present invention, the preconditioned adult human mesenchymal stem cells are allogenic to the subject being treated. As used herein, 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. Generally, 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. Alternatively, 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. In certain embodiments, 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) and 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).

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data are actually obtained.

Some of the results presented below have been described in a manuscript (Figeac et ah, "Nanotubular crosstalk with distressed cardiomyocytes stimulates the paracrine repair function of mesenchymal stem cells") submitted for publication to Stem Cells. Materials and Methods

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 (Etablissement 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 μg/ml streptomycin, and 10 mM HEPES (Invitrogen), in a 5% C0₂ atmosphere at 37 °C whereas human cardiac primary cells were expanded as specifically recommended by manufacturers.

Co-culture between hMADS and mouse adult Cardiomyocytes (CM). Adult ventricular CM were isolated from hearts of 2 to 5 months-old male mice from C57BL/6J (Janvier) GCAG-GFP transgenic strains (Okabe et al, FEBS Lett., 1997, 407: 313-319) as previously described (Mitra and Morad, Am. J. Physiol., 1985, 249: H1056-1060). Immediately after their isolation, cardiac myocytes were co-cultured with hMADS in a 1: 1 ratio in DMEM supplemented with 10% FBS (Gibco BRL). Each of the two co-cultivated cell types was seeded at a density of 3500 cells/cm . For co-culture without physical contact between the 2 cell types, cardiomyocytes were seeded on cell culture inserts containing polycarbonate membrane (0.4-μιη or Ιμιη 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 10⁵ 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. 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.

Chemotactic activities of culture supernatants were assessed by using μ-slide chemotaxis (Ibidi) seeded with 18x103 hBMDC per channel. Neutralizing antibodies against human VEGF165 (0.08μg/ml), human HGF (0Λμg/m\), human MCP-3 (20μg/ml) and human SDF-1 (3μg/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).

Real-time PCR Assays. Total 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).

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 μ-slide chemotaxis (Ibidi) seeded with 18.10 hBMDC per channel. Cells were exposed during 24 hours to a chemical gradient between basal medium (DMEM + 0.8% FBS) and the different kind of conditioned supernatants and then fixed and counted. Immunocytochemistry. The cells were fixed with 4% PFA and stained with antibodies (Ab) against GATA-4 (goat polyclonal Ab (pAb), 1:20, R&D Systems) and phospho Histone H3 (pH3, rabbit pAb, 1: 100, Abeam). Donkey secondary anti- goat or -rabbit antibodies (FITC- or Cy3- conjugated, 1: 100) were purchased from Jackson ImmunoResearch Laboratories Inc and Wheat Germ Agglutinin conjugated to Alexa 647 (10μg/ml) was from Invitrogen. For co-staining with phalloidin- rhodamine (5 μg/ml, Sigma-Aldrich) and FITC conjugated cc-tubulin (mouse mAb, 1:100, Abeam), 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 (ΙμΜ) or calcein AM (ΙμΜ) (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 μΜ 18α- 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. All animal procedures were in accordance with the regulations issued by the French Ministry of Agriculture and were approved by the prefecture of our administrative district (Prefecture du Val de Marne, France; Licenses to conduct animal research: B 94-028-7 and A94-028- 245). Myocardial infarction was induced in 8 to 10 week-old male C57BL6/J mice by occluding the left coronary artery for 90 minutes and reperfusing the artery for 10 minutes. Just after surgery, approximately 2.0x10⁵ co-cultured cells in a total volume of 20 μΐ were injected into the myocardium surrounding the infarcted site. Control mice received HBSS or similar amount of hMADS or CM. Bone marrow transplantation experiments. 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 (3xl0⁶ 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).

LVEF was computed as (LVEDV-LVESV)*100/LVEDV. All measurements were averaged over three consecutive cardiac cycles, and the images were analyzed by a blinder reviewer.

Heart histology. Mice were sacrificed at day-3, -7, -21 post-injection. Hearts were either directly snap-frozen in isopentane precooled with liquid nitrogen, or for GFP detection, previously perfusion-fixed with 4% paraformaldehyde and saturated in 20% sucrose overnight. Cryostat serial sections of either 5 or 8 μιη in thickness were prepared from frozen hearts for FISH or histology/immunohistochemistry respectively. Fibrosis extent was determined from tissue sections separated by 300 μιη from the apex to the base after Mas son's trichrome staining (Sigma-Aldrich) and quantification using image analysis software (ImageJ 1.42q, NIH). Infarct size area was calculated by the sum of partial scar areas between sections and expressed as a percentage of total LV endocardial wall area.

Capillary density of peri-infarct area was determined after micro vessel staining with isolectin B4 (40μg/ml, Sigma-Aldrich) and counting from at least 20 randomly selected fields in border areas by a blinded investigator.

Immunohistochemistry. Frozen heart sections were fixed with 4% paraformaldehyde and incubated with antibodies against human GATA-4 (goat pAb, 1:50, R&D systems), Phospho Histone H3 (rabbit pAb, 1: 100, Abeam), hLamin AC (mouse mAb, 1: 100, Novocastra), CD31 (rabbit pAb, 1: 100, Abeam), cardiac Troponin I (rabbit pAb, 1: 100; Abeam), cardiac Troponin T (mouse mAb, 1: 100, Abeam), GFP (rabbit pAb, 1: 100, Gene Tex), Caspase-3 (rabbit pAb, 1:250, Cell Signaling), human mitochondria (mouse mAb, 1: 100, Abeam). Secondary 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

(GraphPad Software, USA) and results are reported as mean +SD. Levels of soluble factor secretion between the different kinds of culture were compared using a two- tailed paired test. For cardiac function measurements, LVEF differences between groups and time (day 5 to 21) were evaluated by analysis of repeated measures followed by post-hoc Student's test for each treatment group. Other between-group comparisons of mean values were performed using a two-tailed Student's t-test. P values smaller than 0.05 were considered significant. Results

Cell-to-cell communications with cardiomyocytes (CM) alter the mesenchymal stem cells (MSC) secretome. To determine whether early processes of cell-to-cell communication between hMADS and distressed adult CM could alter hMADS secretome, the Applicants measured by luminex assays the concentration of 20 soluble human factors selected according to their cardiac repair properties (Vandervelde et al, J. Mol. Cell Cardiol, 2005, 39: 363-376; Williams et al, Circ. Res., 2011, 109: 923-940). The levels of these factors were determined in supernatants from either hMADS culture alone or co-cultured with CM for 24 hours, at which time most of the primary CM were dead. Changes in secretion levels were found for 8 of these factors. Compared to conditioned media from hMADS alone, those from co-cultures contained higher amounts of VEGF (x 1.61+0.28), HGF (x5.3+0.94), SDF1 (xl.31+0.1), MCP-3 (xl.58+0.13), IL6 (xl.44+0.18) and GROa (x2.40+0.31) and decreased concentration of MCP-1 (xO.74+0.07) and MMP-3 (xO.54+0.07) (Table 1 and Figure 1A, B).

Similar changes were found at the transcriptional level in the human genome strengthening the stem cell origin of paracrine activation (Figure IB). In addition, CFSE flow cytometry analysis and qRT-PCR experiments demonstrated that changes in secretome cannot be ascribed either to hMADS proliferative changes in the presence of CM or to differences in cell density between coculture and monoculture (Figure 11).

Paracrine differences existing between co-cultured and control hMADS, except for MCP-1 and MMP-3, were abrogated in co-cultures performed with a cell culture insert impeding physical interaction between hMADS and CM (Table 1 and Figure 1). These data indicate that preconditioning with CM alters the secretome of hMADS through several cell-cell communication routes including paracrine signaling and heterologous direct cell-cell contact. Table 1. Luminex assays from conditioned media from 24 hour-single cultured mouse cardiomyocytes (CM, n=7), hMADS cells alone (HM, n=14) or in co-culture without or with transwell insert (CC, n=14 or CCTW n=7 respectively).

Cell-to-cell communications with cardiomyocytes (CM) enhance the paracrine regenerative effects of mesenchymal stem cells (MSC). To assess the physiological impact of co-culture-induced hMADS secretome changes, conditioned media from either co-cultured or naive hMADS were analyzed relative to their effects on angiogenesis, bone marrow (BM) cell chemo taxis, cardiac progenitor cell activation, CM survival and cardiac fibroblast activities.

The Applicants found that 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). Similarly, 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). Importantly, supernatants collected from co-cultures wherein hMADS and CM were physically separated failed to stimulate hMADS pro-angiogenic and pro-chemotactic properties suggesting that these phenomena were triggered by direct contact between stem and cardiac cells (Figure 2, and Figure 3A). In contrast, co-culture preconditioning was found not to alter the paracrine ability of naive hMADS to stimulate proliferation of human primary cardiac progenitor cells (which was basically inexistent) (Figure 3B), to protect CM against serum withdrawal-induced apoptosis (Figure 3C) and to inhibit cardiac fibroblast proliferation and collagen synthesis (Figure 3D).

In addition, assays performed with neutralizing antibodies against some human- specific growth factors upregulated in cocultures, revealed that hVEGF and hHGF triggered the pro-angiogenic activity of cocultured hMADS whereas hVEGF, - hHGF, -hMCP-3 and -hSDF-la participated in their pro-chemotactic effects (Figure 2D, E).

Tunnelling nanotubes (TNT) mediated cell-to-cell communication can be selectively inhibited by latrunculin A or nocodazole treatments. To assess the involvement of TNT highway in the alteration of hMADS secretome, 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. Flow-cytometry analysis showed that consistent with a TNT transport, mitochondria and at a significantly lower efficiency, calcein were transferred from the CM to the hMADS within the first 24 hours of co-culture (mean fluorescence increase versus hMADS of 229.6+62.78 and 6.98+2.74, respectively) (Figure 4A). Rate of conveyed mitochondria was significantly decreased after co-culture-treatment with either latrunculin A or nocodazole (approximately 46% and 20% of inhibition, respectively) whereas flow of calcein was sensitive to only latrunculin A (around 54% of inhibition) (Figure 4B). Additionally, these intercellular exchanges were not altered by gap junction inhibitor CC-18GA indicating that inhibitory action of latrunculin A or nocodazole was not attributable to blockade of GAP channel activity (Figure 4A).

To exclude a potential interference of these drugs with cell signalling processes mediated by either exosomes/microvesicules or larger microparticules like apoptotic bodies, co-cultures were performed with TW insert of 0.4 or 1 μιη pore size, respectively. Under these conditions, intercellular traffics of calcein and organelles were dramatically decreased (mean fluorescence fold increase compared to control hMADS for 0.4 and 1 μιη TW co-cultures, respectively of 1.1+0.77 and 1.2+0.23 for calcein and 5.2+1.54 and 4.6+1.38 for mitotracker) but nevertheless remained unchanged by addition of latrunculin A or nocodazole (Figure 4A,B=Figure 3 AC). In agreement with these results, the Applicants showed that these drugs do not affect phagocytosis or endocytosis processes in hMADS through exposure with pHrodo red S. aureus bioparticles or pHrodo green dextran, respectively (data not shown).

Together with microscopic observations showing calcein and mitrotracker molecules inside heterologous TNT channels mainly composed of f-actin and microtubules (Acquistapace et ah, Stem Cells, 2011, 29: 812-824) (Figure 5), these results provided evidences that in early co-cultures, intercellular exchanges were primarily mediated by the means of TNT network which can be specifically disrupted by latrunculin A and nocodazole at the concentration of 2.5x10 -^"8 M and 5.0x10 -^"8 M, respectively, without affecting functionality of gap junction or membrane vesicle communication pathways.

Heterologous tunnelling nanotubes (TNT) connection mediate mesenchymal stem cells (MSC) secretome changes improving angiogenesis and chemotaxis. To assess the role of TNT in hMADS secretome changes during co- culturen, luminex assays were performed on supernatants from latrunculin A- or nocodazole-treated co-cultures. Both drug-treatments abrogated the secretory stimulation of VEGF, HGF, MCP-3 and SDF-1 supporting a role of nanotubular connections made of f-actin and microtubule in this phenomenon. In contrast, these drugs unaffected the co-culture-induced secretion of IL-6, GRO-CC, MMP3 and MCP- 1 suggesting that production of these factors by hMADS was triggered by other cell- to-cell communication mechanisms (Figure 6). In agreement with these results, the Applicants found that the paracrine ability of hMADS to promote HUVEC angiogenesis and BM-MSC chemotaxis failed to be induced in cocultures exposed to drug (Figure 7A,B), indicating that TNT composed of both f-actin and microtubules play a critical role in the modulation of MSC paracrine regenerative responses to CM-emitted cues.

Finally, the Applicants investigated whether TNT might be affected by inflammation, a key component of the infarcted microenvironment, by priming hMADS with TNF-cc or IFN-γ prior to coculture with cardiomyocytes. Under these conditions, 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.

Co-culture with cardiomyocytes (CM) improves mesenchymal stem cell (MSC) cardiac cell therapy efficacy. Finally, the Applicants investigated whether in vitro preconditioning of hMADS cells with CM might impact their cardiac cell therapy efficacy. Mouse hearts previously subjected to experimental myocardial ischemia were injected with human cells grown alone or in co-culture, mouse CM or HBSS saline solution. From day 5 to day 21 post-injection, mice treated with either hMADS, HBSS or mouse CM exhibited no significant changes in their left ventricular ejection fraction (LVEF) whereas LVEF in mice treated with co-cultured cells was improved by 35% (Figure 8A). In addition, quantification of Masson's Trichrome staining of heart sections indicated that infarct size was significantly reduced in myocardium injected with co-cultured cells compared to the other control conditions (Figure 8B,C). These results underline that hMADS notably improved their heart regenerative capacity following a prior step of co-culturing with mouse adult CM. In addition, the Applicants found that a similar number of cocultured or naive hMADS cells were present in mouse hearts at Day 3 following infarction and cell delivery, while at Day 7, both kinds of human cells were hardly detectable (data not shown). These results indicate that the regenerative capacity of cocultured cells cannot be attributed to a survival advantage over their non-cocultured counterparts.

To assess further knowledge on why cocultures were superior in restoring hear function than naive hMADS, the Applicants examined angiogenesis, CM apoptosis and cardiac progenitor mobilization in mouse hearts at day 3 and day 7 post- infarction injected with HBSS, CM or hMADS grown alone or in co-culture. Higher capillary density assessed by isolectin B4 staining was found in the border zone of co-culture-treated hearts compared to the other animal groups at day 3 and day 7 (capillary density at day 3 and 7 respectively: 1644+97/mm2 and 1700+83/mm2 for coculture (CC), 1231+43/mm² and 1212+114/mm² for hMADS (HM), 1154+159/mm² and 1263+132/mm² for CM, and 1083+191/mm2 and 1210+64mm for HBSS group) (Figure 8D). Additionally, the proportion of cardiac progenitor like cells present in the peri-infarcted myocardium and evaluated by counting nuclei expressing the early cardiac commitment GATA-4 marker was found significantly higher in mice treated with co-cultured MSC than in those treated with MSC alone, mouse CM or HBSS at day 3 and day 7 post-ischemia and cell transplantation (Figure 9A). To determine whether these cells originated in bone marrow, the Applicants quantified the proportion of GATA- 4+/GFP+ in chimeric mice reconstituted with GFP BM cells at day 3 post-infarction chosen as optimal time point to really discriminate homing to self -renewal of circulating progenitor cells at the infarction site. They found that GATA-4+/GFP+ cells were more abundant in co-culture treated mice than in hMADS-, CM- or HBSS- injected mice (Figure 9B) indicating the increased concentration of GATA-4+ cells at the myocardial injury site of co-culture-treated hearts was due at least in part to a greater mobilization of BM derived cells having a cardiac progenitor-like phenotype.

Finally, the rate of apoptotic CM after caspase-3 immuno staining was found similar in mice peri-infarct area treated with hMADS and cocultures at day 3 and day 7 post-injection, with a tendency although not statistically significant to be inferior compared to CM- or HBSSinjected mouse infarcts at day 3 (Figure 9C). Consistent with in vitro observations, our in vivo results indicate that improved cardiac cell therapy efficacy of hMADS through co-culture is associated with an enhancement of the angiogenesis and myocardial homing of BM-derived cells having a cardiac progenitor-like phenotype. The fact that neither endothelial or cardiac trandifferentiation nor permanent cell fusion were observed in infarcted hearts treated with naive- or co-cultivated-hMADS (data not shown), suggests that the repairing properties of engrafted cocultured cells are likely to be mediated by a paracrine process. Transdifferentation, permanent cell fusion and cardiomyocyte (CM) somatic reprogramming cannot explain improved cardiac cell therapy of co- cultured mesenchymal stem cell (MSC). The Applicants next investigated the role of transdifferentiation and permanent cell fusion in the cardiac benefit of co-cultured cells, by performing immunohistochemistry combining hLamin A/C with endothelial CD31 or cardiac early GATA-4 or late cTnT commitment markers or FISH experiments in mouse infracted hearts at day 3 and 7 following naive- or co- cultivatedhMADS cell injection. In all the conditions, 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).

Finally, the Applicants analyzed whether in vitro mouse CM reprogramming- derived cells (Acquistapace et ah, Stem Cells, 2011, 29: 812-824), which were present in a small number in the co-cultures (less than 1% of the total of transplanted cells), could have a role in myocardial repair. For this, mice infracted hearts were injected with co-cultures made with GFP+ CM to easily identify cells derived from CM somatic reprogramming. At day 21 post-injection, some rare GFP-positive CM were detectable suggesting that in vitro reprogrammed cardiac cells could have the potency to terminally differentiate into mature CM. Nevertheless, according to the scarcity of these events, this phenomenon seems unlikely to be effective in the cardiac regeneration promoted by co-culture engraftment (not shown). Taken together, these results exclude transdifferentiation, permanent cell fusion and in vitro somatic CM reprogramming as mechanisms accounting for heart repair benefits of co-cultured cells and thus indirectly support a paracrine regenerative mechanism. Co-culture-mediated mesenchymal stem cell (MSC) paracrine changes are transient but re-inducible by new exposure to cardiomyocytes (CM). To gain further insights on whether the paracrine regenerative effects of hMADS was improved by the co-culture, secretome of hMADS was examined at day 4-coculture corresponding at the stage in which cells were injected. 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. Nevertheless, when these first primed hMADS were exposed de novo to CM to mimic what happens after intramyocardial delivery of co- cultivated cells, an increased release of VEGF (x5.3+0.38), MCP-3 (x8.11+1.83), HGF (x3.6+1.18) and SDF-1 (xl.30+0.28), IL-6 (x2.27+0.33) and GROa (x9.5+1.8) was observed (Figure 10A). Of particular interest was the finding that the magnitude of the paracrine response of hMADS to the second CM exposure is equal or higher (depending on the cytokines) than that obtained after the first CM preconditioning (Figure 10A). These results reveal that the paracrine stimulation of hMADS through co-culture is transient but can be re-induced by de novo contact with CM. Finally, in the attempt to correlate in vitro observations with in vivo stronger cardiac regenerative potential of co-cultivated cells, the Applicants examined whether these cells could directly communicate with resident CM by examining heterocellular exchanges of human stem cell mitochondria at the site of infarction. Immunohistochemistry analysis with anti- human mitochondria and cardiac troponin T antibodies revealed the presence of some CM containing human mitochondria at the peri-infarct borders in day 3-treated hearts with hMADS grown alone or in co- culture (Figure 10B). These observations reinforce the hypothesis that stem cells and resident CM physically interacted with each other in vivo and that such interaction might boost paracrine function of co-cultivated hMADS. Discussion

Intensive efforts are currently devoted to characterize the mechanisms whereby MSC can repair defective hearts, such basic knowledge being of critical importance for rational design of MSC based-cell therapies and optimization of existing cell therapies. Several studies, including the ones based on clinical trials, tend to attribute cardiac benefic of transplanted MSC to paracrine effects (Wollert et al. , Nat. Rev. Cardiol., 2010, 7: 204-215). However, it remains so far unclear if the diffusible mediators underlying cardiovascular regeneration are produced by intramyocardiacally engrafted MSC in a constitutive fashion or rather in response to entry contact with injured heart microenvironment. The present study enlightens this fundamental but largely unexplored question through co-culture assays of hMADS used herein as MSC model with mouse adult CM in a suffering state to mimic in vivo microenvironment after the onset of myocardial infarction (Acquistapace et al., Stem Cells, 2011, 29: 812-824; Sambrano et al, Stem Cells, 2002, 25: 245-251). Besides confirming that hMADS, as similarly reported for other types of MSC (Caplan et al, J. Cell Biochem., 2006, 98: 1076-1084; Liu et al, Cytokine, 2005, 32: 270-279; Sadat et al, Biochem. Biophys. Res. Commun., 2007, 363: 674-679) constitutively secrete chemical factors limiting myocardial loss and scarring or stimulating angiogenesis, the present study importantly uncovers a novel mechanism by which the "innate" humoral regenerative function of MSC can be improved through cell interaction and communication with distressed CM. More specifically, in vitro evidence is provided that cell-to-cell communication processes between stem and cardiac cells trigger changes in the hMADS secretome expression and consequently enhance the hMADS effectiveness in promoting angiogenesis and chemoattraction of BM-derived mesenchymal progenitors, these two latter processes being of key importance for cardiac repair (Gnecchi et al, Nat. Med., 2005, 11: 367- 368; Huang et al, Circulation, 2011, 124: S46-54; Uemura et al, Cir. Res., 2006, 98: 1414-1421; Vrijsen et al, Curr. Opin. Organ. Transplant, 2009, 14: 560-565). Even though the present report does not provide exhaustive identification of the hMADS-released factors responsible of the above-stated effects, it clearly shows that the production of some diffusible molecules with known cytoprotective properties is markedly increased by the means of co-cultures. These factors are therefore strong candidates as mediators of hMADS phenotypic switch. In particular, the Applicants report that the upregulated secretion of 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. Res., 2004, 95: 1191-1199; Schenk et al, Stem Cells, 2007, 25: 245-251; Son et al, Stem Cells, 2006, 24: 1254-1264; Tang et al, Mol. Cells, 2010, 29: 9-19).

Additionally, 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). Concomitantly with the increased production of soluble factors considered as beneficial for cardiovascular repair, 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).

In addition to the speculative role of the above- stated soluble factors, it is possible that other candidates not selected in the present study might contribute to the paracrine functional modifications of hMADS. Identification of these factors by transcriptomic/proteomic approaches combined with functional assays might surely help in understanding how cell-to-cell communications with CM modulate MSC secretome expression and thus repair potential. Finally, in regards with the present findings, it is worth noting that early cell-to-cell communication events between MSC and CM seem to affect exclusively the paracrine action of hMADS on angiogenesis and BM chemo taxis but not that exerted on heart-derived progenitor cells, cardiac fibroblasts and apoptotic CM. Nevertheless, these observations do not in any way preclude that such mechanisms could take place later in the coculture or that hMADS could act on these cell types by a paracrine independent pathway.

Another important finding unveiled by the present work concerns the characterization of the cell- to cell communication routes involved in the hMADS secretome alteration and more precisely reveals a novel functional facet of nanotubular connections between stem and cardiac cells consisting in directly regulating the regenerative paracrine properties of MSC. So far, previous in vitro studies suggested that this way of cell to cell communication was a common salvage process by which MSC could directly rescue various kinds of somatic cells exposed to stress signals such as CM (Acquistapace et al, Stem Cells, 2011, 29: 812-824; Cselenyak et al, BMC Cell Biol, 2010, 11: 29), astrocytes (Wang et al, Cell Death Differ., 2011, 18: 732-742) and endothelial cells (Yasuda et al, Aging, 2011, 3: 597- 608). Nevertheless, until now, very little was documented on whether this process allowing bidirectional intercellular exchanges impacts MSC cell fate. Despite the existence of several studies sustaining that transfer of cardiac cellular compounds through MSC by the means of tunneling conduits might favor MSC transdifferentiation into cardiac lineage, others works fail to confirm this mechanism. This apparent discrepancy is likely to be ascribed to differences in developmental stage of CM used for co-culture. In fact, studies reporting cardiac MSC transdifferentiation were generally conducted with healthy neonatal cardiac cells capable of division (Nishiyama et al, Stem Cells, 2007, 25: 2017-2024; Rose et al, Stem Cells, 2008, 26: 2884-2892; Yoon et al, Ann. Hematol., 2005, 84: 715- 721). In contrast, such a phenomenon is not observed in non-replicative adult CM characterized by a suffering state (Acquistapace et al, Stem Cells, 2011, 29: 812- 824; Gallo et al, J. Cell Biochem., 2007, 100: 86-99).

Here, is provided the first evidence that CM stress signal spreading along these structures trigger secretome alteration of hMADS with the aim to enhance broken heart repair. In fact, 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. Even occurrence of such a phenomenon is extremely difficult to assess in vivo, its physiological relevance is supported by the earlier detection of connections made of f-actin and microtubules in mouse heart tissue (He et al, Cardiovasc. Res., 2011, 92: 39-47). The Applicants postulate that the paracrine activation of hMADS might be triggered by stress signals sent by CM along TNT. Although the nature of these signals remains to be elucidated, miRNA, proteins or organelles such as lysosomes or mitochondria might contribute to different extents to the phenomenon observed. Besides the role of TNT cell-to cell crosstalk, the present study also shows that part of 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. For instance, uptake by hMADS of large microparticles (>0.4μιη) (Lai et al, J. Mol. Cell Cardiol., 2010, 48: 1215-1224) or apoptotic bodies from cardiac origin (Burghoff et al, Cardiovasc. Res., 2008, 77: 534-543; Hristov et al, Blood, 2004, 104: 2761-2766) might be the mechanism stimulating the release of IL6 and GRO-a as it didn't occur in cocultures with 0.4 μιη pore size transwell insert. On the other hand, soluble factors or exosomes/ small microvesicles (Camussi et al, Kidney Int., 2010, 78: 838-848) might be likely involved in the decreased production of MMP-3 and MCP-1 since this decrease persisted in indirect co-cultures with 0.4μιη pore size transwell insert. Nevertheless, according to the present data, these latter cell-to-cell communication pathways do not seem responsible of the changes observed in hMADS cells during co-culture and therefore their biological significance is questionable.

Beyond the scope of the present study, the data obtained here raise a series of fundamental questions as to what is the nature of the cues emitted by the distressed CM and transported along the nanotubular highway which activate MSC secretome and what are the molecular mechanism/signaling pathways underlying the hMADS phenotypic changes? Although to be formally proven, the Applicants hypothesize that hypoxia, which naturally occurs during a heart attack, might be a key inducer of the CM-stress factors triggering MSC paracrine alteration. This hypothesis is strengthened by a series of studies showing that MSC enhanced their paracrine regenerative properties through release of pro-survival and angiogenic factors after either hypoxic preconditioning (Chacko et al., Am. J. Physiol. Cell Physiol., 2010; 229: C1562-1571; Fang et al, J. Mol. Cell Cardiol., 2011, 51: 839-847; Hu et al, J. Thorac. Cardiovasc. Surg., 2008, 135: 799-808) or after stimulation of Akt whose expression is inducible by hypoxia (Gnecchi et al, FASEB J., 2006, 20: 661-669). Future prospects in this field will highlight the role of hypoxia but also will lead to determine if among the stress signalling mediators produced by the CM are microRNA, DNA, proteins, organelles or another kind of cellular compounds.

Finally, one of the most exciting outcomes of the present work is that in vitro preconditioning of stem cells with CM through co-culture improves their cell therapy efficacy. Higher functional recovery of hearts engrafted with co-cultured hMADS results, at least in part, from more efficient paracrine mechanisms in promoting angiogenesis and BM cell myocardial homing. Superior beneficial effects of co- cultivated versus naive hMADS cannot seemingly be explained by differences in survival or secretome expression at the time of intramyocardial cell injection but rather by a stronger paracrine stimulation of preconditioned cells compared to naive ones in response to in vivo cellular interaction with resident CM. This phenomenon probably encompasses TNT-mediated heterologous communications between MSC and CM as supported by in vivo evidence of transfer of stem mitochondria inside myocardial cells. The priming of MSC with CM reported here has analogies with that earlier reported in vitro for hematopoietic cells with BM environment, such priming leading to a better BM engraftment (Massollo et ah, Exp. Hematol., 2010, 38: 968-977). Although the present study does not formally demonstrate that the improved regenerative properties of cocultured cells in vivo is due to TNT interactions with dying CM, this hypothesis is supported by the fact that (i) TNT communication between stem and cardiac cells probably occur in vivo , as suggested by mitochondria exchanges from stem to myocardial cells preferentially ensured by these structures and (ii) in vitro, MSC subjected to a second CM exposure activated their secretome more efficiently than during the first one and as a function of the CM ratio. Based on these observations, the activation of hMADS secretome following engraftment might be expected to be greater than in vitro since MSC in vivo should encounter a broader range of CM. Albeit a direct implication 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. In this context, as 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.

Besides a role for TNT, other mechanisms cannot be excluded that could account for the heart repair properties of co-cultivated MSC. These might involve such as those promoted by direct cell-to-cell contact with CM or cell-to-cell communications with neighbouring endothelial, fibroblast and/or resident cardiac stem cells. Furthermore, the present study raises questions about the in vivo biological significance of "stem" mitochondrial transfer into CM. D espite the fact that such a process was found to protect CM against apoptosis in vitro (data not shown), this role was not confirmed by the present in vivo experiments since stem cell administration into infarcted myocardium did not significantly decrease cardiac apoptosis. Although the implications of this phenomenon in vivo remains to be confirmed, the Applicants speculate that a transfer of human stem cell mitochondria should dramatically improve the bioenergetic status of the cardiac cells and perhaps also the overall metabolism to combat injury.

The present study, addressing for the first time the cardiac regenerative potential of hMADS in vivo, argues against the commonly accepted concept that MSC infusion affords functional benefit in the infarcted myocardium (Shake et al, Ann. Thorac. Surg., 2002, 73: 1919-1926; Zeng et al, Circulation, 2007, 115: 1866- 1875; Zimmet et al, Basic Res. Cardiol., 2005, 100 : 471-481; Tomita et al, J. Thorac. Cardiovasc. Surg., 2002, 123: 1132-1140; Orlic et al, Nature, 2001, 410: 701-705), since hMADS fail to promote significant functional recovery of injured heart function. Nevertheless, similar negative findings about the therapeutic effectiveness of MSC have been also previously documented by others (Tatsumi et al, Circ J.2008, 72: 1351-1358; Schuleri et al, Eur Heart J., 2009, 30: 2722-2732; Grinnemo et al, Ann. Med., 2006, 38: 144-153; Flynn et al, Stem Cell. Res. Ther. 2012, 3: 36; Dayan et al, Interact. Cardiovasc. Thorac. Surg., 2012, 14: 516-520; Bartunek et al, Am. J; Physiol. Heart. Circ. Physiol, 2007, 292: H1095-1104). This apparent discrepancy might be explained by several factors including the source and species of MSC, number and mode of cell delivery, animal model and extent of left ventricular dysfunction (Gaebel et al, PLoS One, 2011, 6: el5652). Nevertheless, of important note is that the present model significantly differs from previously described studies regarding the immunological context in which the Applicants experiments were performed. Indeed, most of the positive studies on human MSC have used immunodeficient animals (Gaebel et al, PLoS One, 2011, 6: el5652; Toma et al, Circulation, 2002, 105: 93-98; Shinmura et al, Stem Cells, 2012, 29: 357-366; Otto Beitnes et al, Cell Transplant, 2012, 21: 1697-1709; Kim et al, Cardiovasc. Res., 2012,95: 495-506; Kim et al, Cardiovasc. Res. 2012, 95: 495-506) while, in sharp contrast, our experiments were carried out on immunocompetent mice. In this xenogeneic context, 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). Importantly, 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. Nevertheless, it cannot be excluded that the enhanced benefit of cocultured cells may be partially explained by a differential response of cocultured and naive hMADS towards the inflammation associated to myocardial infarction. Although the role of inflammation requires further investigations, the present in vitro observations support the idea that TNT cell-to-cell communication can be improved when hMADS are primed with inflammatory cytokines prior to coculture.

To conclude, the present study provides new insights into the repair mechanisms underlying paracrine therapeutic benefits of MSC and supports the intriguing possibility that reparative capacities of stem cell can be enhanced by exogenously manipulating these mechanisms.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

Claims

Claims

A method for modulating the 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 to obtain preconditioned adult human mesenchymal stem cells.

The method according to claim 1, wherein the adult human mesenchymal stem cells and adult fully differentiated cardiomyocytes are in physical contact during the coculture.

The method according to claim 1 or claim 2 further comprising a step of separating the preconditioned adult human mesenchymal stem cells from the adult fully differentiated cardiomyocytes after coculture, and optionally storing the separated preconditioned adult human mesenchymal stem cells.

The method according to any one of claim 1 to 3, wherein 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 method according to claim 4, wherein the amount of the at least one cardioprotective factor released by cocultured 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 method according to claim 4 or claim 5, wherein 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.

The method according to any one of claims 1 to 6, wherein 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 method according to any one of claims 1 to 7, wherein the adult human mesenchymal stem cells are derived from a tissue obtained from a healthy adult donor.

Preconditioned adult human mesenchymal stem cells obtainable by a method according to any one of claims 1 to 8.

Preconditioned adult human mesenchymal stem cells according to claim 9, wherein 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.

Preconditioned adult human mesenchymal stem cells according to claim 10, wherein the amount 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.

Preconditioned adult human mesenchymal stem cells according to claim 10 or claim 11, wherein 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.

A pharmaceutical composition comprising an effective amount of preconditioned adult human mesenchymal stem cells according to any one of claims 9 to 12, and at least one pharmaceutically acceptable carrier or excipient.

14. Preconditioned adult human mesenchymal stem cells according to any one of claims 9 to 12 or the pharmaceutical composition according to claim 13 for use in the treatment of a cardiac pathology and/or in cardiac tissue reconstruction or regeneration.

15. Preconditioned adult human mesenchymal stem cells or the pharmaceutical composition for use according to claim 14, wherein 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.

16. Method for treating a cardiac pathology or for reconstructing cardiac tissue in a subject, comprising a step of administering to the subject in need thereof an efficient amount of preconditioned adult mesenchymal stem cells obtained using a method according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 13.

17. The method according to claim 16, wherein the preconditioned adult mesenchymal stem cells are administered in combination with one or more of: other cells, biologically active factors, and drugs.

18. The method according to claim 16 or 17, wherein the preconditioned adult mesenchymal stem cells are administered seeded and grown on a scaffold.

19. The method according to any one of claims 16 to 18, wherein the wherein 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.