WO2015076717A2 - Mscs in the treatment of cardiac disorders - Google Patents

Abstract

The present invention relates to, inter alia, methods for culturing mesenchymal stem cells (MSCs) (without the use of non-human serum components), MSCs and extracellular vesicles obtained from such culturing methods, having advantageous polypeptide profiles which are key for immuno-modulatory efficacy. Further, the present invention pertains to pharmaceutical compositions comprising such MSCs and/or extracellular vesicles, and medical treatment and prophylactic methods and medical uses of MSCs and/or extracellular vesicles in a variety of diseases, notably myocarditis, pericarditis, cardiomyopathy and specifically dilated cardiomyopathy, and/or post-cardiotomy syndrome, and related diseases and ailments, such as organ failure.

Description

MSCs in the treatment of cardiac disorders

Technical Field

The present invention relates to, inter alia, methods for culturing mesenchymal stem cells (MSCs) (without the use of non-human serum components), MSCs obtained from such culturing methods, MSCs and/or extracellular vesicles derived from said MSCs having proteomic profiles which ensure immuno-modulatory capacity, pharmaceutical compositions comprising such MSCs, and medical treatment and prophylactic methods and medical uses of MSCs and/or extracellular vesicles in cardiac diseases and disorders, specifically myocarditis and other autoimmune and inflammatory disorders.

Background Art

Mesenchymal stem cells (MSCs) can be found in inter alia the bone marrow, blood, adipose tissue, dermis, periosteum and various other tissues and MSCs can differentiate into a variety of cell types, including adipose, areolar, osseous, cartilaginous, elastic, marrow stroma, muscle, fibrous connective tissue, and cardiac tissue, depending upon external milieu and stimulants. Due to their cellular origin and their phenotype, MSCs do normally not stimulate adverse immune responses, potentially enabling using unrelated human donors in clinical settings.

MSCs are conventionally isolated from the tissue, purified, and then expanded in an appropriate culture medium. Suitable culture medium for MSCs may comprise various components to promote MSC expansion, for instance growth factors, cytokines, and serum. After the isolation, purification, and culture expansion steps, the MSCs are washed and centrifuged, followed by freezing in a suitable cryopreservation medium. At the time of treatment, the MSCs are thawed just prior to administration to a patient. The current conventional MSC culturing process typically requires 2 to 10 weeks to isolate, expand, harvest and purify a suitable number of cells to constitute a pharmaceutical treatment. In some cases a pharmaceutical treatment consists of 1 dose, whereas in other cases a pharmaceutical treatment may consist of 2 or more doses. Unfortunately, MSC therapy is often urgently needed, i.e. in close connection with diagnosis or presentation of clinical symptoms, meaning that short manufacturing times and MSC populations with pre-validated therapeutic efficacy are crucial factors behind clinical success.

In vitro findings indicate that MSC are immunosuppressive, and rodent, baboon or human MSCs suppress lymphocyte proliferation in mixed lymphocyte cultures, as well as inhibiting the formation of cytotoxic T-cells and NK-cells. The unique immunoregulatory and regenerative properties of MSCs make them an attractive tool for cellular treatment of autoimmunity and inflammation and MSCs have been applied in a variety of clinical contexts, for instance in the treatment of severe graft- versus-host disease (Le Blanc, 2004, Lancet), but also for treating multiple sclerosis and Crohn's disease. However, there are numerous other severe indications for which there is currently a lack of efficient medical treatments, and the present invention thus aims to expand the usage of off-the-shelf MSCs that are rapidly available for administration to a patient, obtained from cell cultures devoid of non- human animal products, to additional indications where there is an unmet medical need. Furthermore, although MSCs are being investigated in several ongoing clinical trials very little is known about what makes MSCs clinically effective, i.e. which characteristics that are necessary for MSCs to be capable of exerting the desired therapeutic effects in a given disease.

Summary of the Invention

The present invention relates to, inter alia, methods for culturing mesenchymal stem cells (MSCs) (without the use of non-human serum components), MSCs obtained from such culturing methods, MSCs and/or extracellular vesicles having clinically efficacious proteomics profiles and immuno-modulatory capacity, pharmaceutical compositions comprising such MSCs, and medical treatment and prophylactic methods and medical uses of MSCs in myocarditis and/or other inflammatory and/or autoimmune cardiac disorders. In one aspect, the present invention relates to a method for culturing mesenchymal stem cells (MSCs), comprising the steps of (i) obtaining mesenchymal cells from a suitable source, and (ii) culturing the cells in a culture medium comprising lyzed human trombocytes, as well as, in additional aspects, selection methods and immuno-modulation criteria, and cell culture compositions for culturing MSCs and MSCs and/or extracellular vesicles obtainable by the methods of the present invention. Further, in yet other aspects, the instant invention pertains to pharmaceutical compositions, methods for preparing said pharmaceutical compositions, and medical uses involving MSCs and/or extracellular vesicles, as well as treatment methods involving MSCs and/or extracellular vesicles.

In another aspect, the present invention pertains to methods for selecting clinically effective immuno-modulatory MSCs and related paracrine factors (e.g. extracellular vesicles such as exosomes), by utilizing proteomics profiling and by assessing the functional characteristics of the MSCs. More specifically, the MSCs of the present invention display certain characteristics in terms of polypeptide expression as well as in terms of polypeptide expression in extracellular fractions (which may comprise extracellular vesicles such as exosomes and, additionally, extracellular polypeptides).

In a further aspect, the instant invention pertains to MSCs and/or extracellular vesicles obtainable by the methods according to the present invention.

In a further aspect, the present invention pertains to a method of preparing a pharmaceutical composition comprising MSCs, comprising the steps of culturing MSCs of crista iliaca and/or sternum origin in a cell culture composition comprising lyzed human trombocytes, passaging the MSCs in the culture not more than 15 times, and, re-suspending the MSCs in saline solution to a final concentration of between approximately 5x10⁵ and 5x10⁶ MSCs per ml.

In a further aspect, the instant invention pertains to pharmaceutical compositions in accordance with the present invention for use in medicine, specifically, in yet another aspect, for use in the treatment and/or prophylaxis of myocarditis and/or other inflammatory cardiomyopathies. In yet another aspect, the present invention relates to a mesenchymal stem cell ( SC), and populations comprising such MSCs and/or extracellular vesicles, with validated immuno-modulatory properties, wherein the MSCs may display a spindle- shape morphology and express CD73, CD90, and CD105, and wherein the MSCs are devoid of CD34, CD45, CD14, and CD3. The MSCs may be either positive or negative for H LA-ABC (MHC class I) and/or HLA-DR (MHC class II), for instance depending on the degree of priming/activation of the MSCs. Further, the MSCs may be negative for CD1 1 b, CD19, and/or CD31.

In a further aspect, the instant invention pertains to a method of treating and/or preventing diseases and/or disorders selected from the group comprising myocarditis and/or other inflammatory and/or autoimmune disorders, comprising the steps of providing a therapeutic dose of MSCs, and administering the therapeutic dose to a patient suffering from any one of the abovementioned diseases or disorders.

Thus the present invention provides a novel therapeutic modality, namely MSCs and/or extracellular vesicles obtainable from the MSCs displaying efficacious proteomics profiles, potency in in vitro immuno-modulatory assays, and expanded/cultured via surprisingly advantageous methods, for the treatment of various illnesses where there is currently a significant unmet medical need, notably myocarditis and/or similar inflammatory cardiomyopathies and related disorders and illnesses. The polypeptide profiling and the proven immuno-modulatory activity result in MSCs that are clinically effective, in stark contrast to many MSC preparations often described in the prior art. Thus, the absence of non-human components during the culturing processes, the advantageous components comprised in the cell culture compositions and in the pharmaceutical compositions, and the methods and protocols for ensuring immuno-modulatory capacity are all factors that contribute to rapid production of high-potency MSCs for highly efficacious clinical treatment of these severe illnesses. Brief Description of the Drawings

Figure 1. EF increased strongly after administration of the MSCs in a male human patient.

Figure 2. TAPSE measurements in a male human patient showed a substantial increase in response to MSC treatment.

Figure 3. ProBNP in a male human patient decreased sharply in response to MSC treatment.

Figure 4. MSC phenotype and responsiveness towards inflammatory stimuli.

Figure 5. Mice with virus-induced myocarditis exhibited a 50% reduction in mortality at two weeks after infection when treated with MSCs of placental and bone marrow origin.

Figure 6. Inflammatory cytokines can activate ("prime") MSCs, which is considered a key step (also known as "licensing") for MSCs to efficiently control inflammation. The characteristic up-regulation of ICAM-1 (CD54), VCAM-1 (CD106), H LA-ABC, and HLA-DR upon 48 hours of cell exposure towards the pro-inflammatory IFN-γ and TNF-a was confirmed by FACS on the MSCs.

Figure 7. MSCs promote in vitro generation of regulatory T-cells. MSCs are capable of promoting the induction and expansion of immune suppressive regulatory T-cells (TRegs)- Co-culturing MSCs in different ratios with purified healthy control T-cells led to an increase of the CD25⁺CD127^dim T_Reg-fraction among the CD4⁺ T-cells. Control T-cells were stimulated using activating anti-CD3 and anti-CD28 microbeads. The left panel shows a representative flow cytometry (FACS) dot plot analysis and the right panel the mean values (columns) of two independent experiments performed in duplicates. Detailed Description of the Invention

The present invention relates to, inter alia, methods for culturing mesenchymal stem cells (MSCs) (without the use of non-human serum components), methods for selecting clinically potent immuno-modulatory MSCs and/or extracellular components (primarily extracellular vesicles such as exosomes) for the treatment of myocarditis, pericarditis, post-cardiotomy syndrome, cardiomyopathy and specifically dilated cardiomyopathy, and associated conditions. Furthermore, the invention also pertains to MSCs and/or extracellular vesicles having clinically efficacious proteomics profiles, pharmaceutical compositions comprising such MSCs and/or vesicles, and medical treatment and prophylactic methods and medical uses of MSCs in a variety of diseases, notably inflammatory and/or autoimmune cardiac diseases and disorders .

Myocarditis is an inflammatory disease of the heart that can be acute, subacute, or chronic, and there may be either focal or diffuse involvement of the myocardium. Both systemic and cardiac symptoms may be seen and in the early stages of e.g. viral myocarditis the patient may have fever, myalgias, and muscle tenderness. The muscle symptoms are attributable to myositis induced by a myotrophic virus. In symptomatic patients, the cardiac presentation may be one of an acute cardiomyopathy.

Myocarditis may be caused by infectious organisms such as viruses, bacteria, fungi, protozoa, and helminths, or by various toxins. The disease can also be associated with systemic illness including granulomatous, collagen- vascular, and autoimmune diseases. However, viral infection is the most common cause of myocarditis and the most frequently implicated viruses are Coxsackievirus B, echovirus, influenza virus, Epstein-Barr virus, and the viruses of childhood exanthematous diseases. However, most pathogenic viruses may replicate in the heart and induce myocarditis.

Myocarditis may be focal or diffuse, involving any or all cardiac chambers. Severe diffuse myocarditis can result in dilatation of all cardiac chambers and there may be mural thrombus formation in any chamber. Histological examination reveals cellular infiltrates, which are usually mononuclear, but may be neutrophilic or occasionally eosinophilic. The infiltrates are of varying severity; and are often associated with myocyte necrosis and disorganization of the myocardial cytoskeleton. With subacute and chronic myocarditis, interstitial fibrosis may replace fiber loss, and myofiber hypertrophy may be seen.

The so called "Dallas criteria" are frequently used to define the disease as acute or borderline myocarditis. Active myocarditis is typically defined as "an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes not typical of the ischemic damage associated with coronary heart disease". Borderline myocarditis is the term used when the inflammatory infiltrate is too sparse, or myocyte injury is not demonstrated. The myocarditis process is in the lion's share of cases thought to be initiated by an infectious agent (usually viral). Both direct viral-induced myocyte damage and post-viral immune inflammatory reactions may contribute to myocyte damage and necrosis. Inflammatory lesions and the necrotic process may persist for months, although viruses normally only replicate in the heart for at most 2 or 3 weeks after infection. The MSCs and/or the extracellular vesicles and/or other paracrine factors obtainable from the MSCs of the present invention are suitable for treating and alleviating myocarditis (and other related disorders and diseases) in all Dallas criteria disease states (e.g. active myocarditis, borderline myocarditis, subacute myocarditis, chronic myocarditis), and additionally the MSCs according to the present invention are suitable for treatment of virus-induced, bacteria-induced, parasite-induced and/or autoimmunity-induced myocarditis.

A variety of findings indicates a pathogenic role for immune and/or autoimmune processes. These findings include infiltration with predominately T-lymphocytes, the presence of activated macrophages, B-cells, cytokines, and adhesion molecules, and the expression of major histocompatibility complex antigens in the myocardium.

Circulating autoantibodies have been demonstrated in myocarditis patients and may persist for a prolonged period. The antibodies are typically directed against mitochondria and contractile proteins and the beta-adrenergic receptors. Experimental models have also implicated cytokines (such as interleukin-1 and tumor necrosis factor), oxygen-based free radicals, and microvascular changes as contributory pathogenic factors. Myocarditis of any origin may be suitable for prohylaxis and/or treatment using the MSCs of the present invention: the myocarditis may thus be associated with inflammation, infection (e.g. viral and/or bacterial and/or fungal and/or parasite infection), autoimmunity, injury, trauma, etc.

Where features, embodiments, or aspects of the present invention are described in terms of Markush groups, a person skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. The person skilled in the art will further recognize that the invention is also thereby described in terms of any combination of individual members or subgroups of members of Markush groups. Additionally, it should be noted that embodiments and features described in connection with one of the aspects and/or embodiments of the present invention also apply mutatis mutandis to all the other aspects, alternatives, and/or embodiments of the invention. For instance, aspects, alternatives, and/or embodiments described in connection with the methods for obtaining clinically effective immuno-modulatory MSCs (e.g. the immuno-modulation criteria) may also be applicable, relevant, and/or combined with teachings pertaining to, for instance, embodiments relating to the MSCs and/or extracellular vesicles obtainable by said methods, and/or the pharmaceutical compositions comprising the MSCs and/or extracellular vesicles. By way of another non-limiting example, aspects, alternatives, and/or embodiments described in connection with the method for culturing MSCs in the absence of non-human serum components may also be applicable to other aspects and/or embodiments as per the present invention, for instance the immuno-modulation selection criteria, meaning that the present invention, although it may not be explicitly mentioned herein, also encompasses combining the teachings relating to, for instance, the culturing methods with the teachings of the immuno-modulation selection methods. Similarly, aspects, alternatives, and/or embodiments described in connection with the MSCs and/or the extracellular vesicles may naturally also be applicable, relevant, and/or combined with teachings pertaining to the pharmaceutical compositions per se, which comprises said MSCs and/or extracellular vesicles.

For convenience and clarity, certain terms employed herein are collected below.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Generally, all polypeptides and/or nucleotides disclosed in the present application naturally encompass polypeptide and/or nucleotide sequences that have at least a reasonable resemblance to the polypeptide and/or polynucleotide in question, for instance a 50% sequence identity to the polypeptide and/or polynucleotide in question, preferably 70% sequence identity to the polypeptide and/or polynucleotide in question, more preferably a sequence identity of at least 80%, and even more preferably a sequence identify of at least 90% to the polypeptide and/or polynucleotide in question.

The terms "positive for" and "negative for" in the context of the present invention (e.g. an MSC which is "positive for" a polypeptide and/or polynucleotide in question) shall be understood in accordance with the meaning normally given to the term within the biological and medical sciences, in essence a cell that is positive for a certain polypeptide and/or polynucleotide expresses said polypeptide and/or polynucleotide. The polypeptide and/or polynucleotide in question may be identified via various means, for instance using fluorescence-activated cell sorting (FACS) and/or immunohistochemical techniques and/or proteomics techniques such as LC-MS and/or 2D-PAGE. The term "positive for" may in certain instances be understood to comprise cell populations where at least 50% of the cells express the polypeptide (or polynucleotide or any other marker) in question, but preferably at least 70% or even more preferably at least 90% of the population expresses the polypeptide in question. The term "negative for" may, in the same vein, naturally be understood to be the opposite of the term "positive for", i.e. at least 50% - but preferably at least 70% or even more preferably at least 90% - of the cells of the population shall not express the polypeptide (or other suitable marker) in question.

The term "population", which may relate to MSCs or to extracellular vesicles such as exosomes, shall be understood to encompass a plurality of entities constituting a given population, for instance the individual MSCs which when present in a plurality constitute an MSC population. Thus, naturally, the present invention pertains also to the individual cells and vesicles of e.g. an MSC population or a population of extracellular vesicles, respectively.

The terms "subject" and/or "individual" and/or "patient" may be used interchangeably herein and are to be understood to refer broadly to an animal, for instance a human being, from whom cells can be obtained and/or to whom treatment, including prophylaxis or preventative treatment (for instance using the cells as per the present invention) is provided. Advantageously, the subject of the treatments as described in the context of the present invention is a mammal, preferably a human, or other mammals, preferably domesticated or production mammals.

The term "therapeutically effective amount" is to be understood to refer to an amount which results in an improvement, allevation, or remediation of the disease, disorder, or symptoms of the disease or condition.

The terms "administering," "introducing" and "transplanting" are used interchangeably for the purposes of the present invention, for instance in the context of the administration of the MSCs to a patient suffering from any of the diseases and/or disorders mentioned in the context of the present invention. A suitable method or route is one which leads to at least partial localization of the MSCs at a desired site. The cells may be administered (delivered) by any appropriate route which results in delivery of the cells and/or paracrine factors excreted from the cells to a desired location/tissue/site in the subject. The modes of administration suitable for the purposes of the present invention comprise for instance (without limitation) intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. The most advantageous modes of administration for most patients having the diseases and disorders described herein are probably intravenous injection, peripheral intravenous injection, central venous injection into the right atrium, injection into the right ventricle of the heart, and/or injection into the pulmonary trunk/artery. The phrase "pharmaceutically acceptable excipient" as used herein is to be understood to relate to a pharmaceutically acceptable material, composition or vehicle, for instance a solid or liquid filler, a diluent, an excipient, a carrier, a solvent or an encapsulating material, involved in suspending, maintaining the activity of or carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.

In one aspect, the present invention relates to a method for culturing mesenchymal stem cells (MSCs). The culturing method does not utilize any non-human components and therefore greatly reduces the risk of eliciting immune responses, thereby possibly improving immuno-modulation, homing, engraftment and/or therapeutic outcomes. In a first step, mesenchymal stem cells are obtained from a suitable source, followed by culturing the cells in a culture medium comprising lyzed human trombocytes (platelets), preferably at least 10⁷ (or even higher numbers, such as 10⁸ or 10⁹) lyzed human trombocytes per ml of culture medium.

MSCs may be obtained from various tissues, for instance bone marrow, blood, cord blood, placenta, Wharton's jelly, dermis, amniotic fluid, adipose tissue, and/or the periosteum, using either allogeneic or autologous sources. When using an allogeneic source of MSCs it is of utmost importance to obtain cells from a young, healthy donor, who is preferably 35 years of age, preferably under 30 years of age, even more preferably under 25 years of age, and most preferably under 20 years of age. MSCs may also be termed multipotent stromal cells, mesenchymal stromal cells, mesenchymal cells, fibrocytes, etc.

In one aspect, the present invention pertains to MSCs and populations of such MSCs having strong immuno-modulatory capacity and with effectiveness in treating myocarditis, pericarditis, post-cardiotomy syndrome, cardiomyopathies such as dilated cardiomyopathy, and various other similar inflammatory and/or autoimmune conditions. Ensuring that MSCs have clinically effective immuno-modulatory capacity is a crucial but in the prior art frequently overlooked aspect. The present invention comprises various criteria for assessing and validating immuno-modulatory properties of MSCs, e.g. by establishing that the MSCs and/or the MSC population may fulfil at least one of the following immuno-modulation criteria: (a) the MSC population is positive for at least one of the following polypeptides: vimentin (SEQ ID No 1), caldesmon (SEQ ID No 2), annexin A1 (SEQ ID No 3), 14-3-3 protein epsilon (SEQ ID No 4), ADP ribosylation factor 1 (SEQ ID No 5), calnexin (SEQ ID No 6), ADP ribosylation factor 5 (SEQ ID No 7), transforming protein RhoA (SEQ ID No 8), CD44 (SEQ ID No 9), coactosin-like protein (SEQ ID No 10), mitogen-activated protein kinase 3 (SEQ ID No 11), insulin-like growth factor- binding protein 7 (SEQ ID No 12), N-acetyl-glucosamine-6- sulfatase (SEQ ID No 13), cellular retinoic acid-binding protein 2 (SEQ ID No 14), transcription elongation factor B polypeptide 1 (SEQ ID No 15), NEDD8 (SEQ ID No 16), fatty acid-binding protein, heart (SEQ ID No 17). Preferably, the MSC population is positive for at least one of the following polypeptides: vimentin (SEQ ID No 1), annexin A1 (SEQ ID No 3) and/or insulin-like growth factor-binding protein 7 (SEQ ID No 12);

(b) a population of extracellular vesicles derived from the MSC population is positive for at least one of the following polypeptides: serotransferrin (SEQ ID No 18), versican core protein (SEQ ID No 19), annexin A2 (SEQ ID No 20), serine protease HTRA1 (SEQ ID No 21), insulin-like growth factor- binding protein 3 (SEQ ID No 22), connective tissue growth factor (SEQ ID No 23), vinculin (SEQ ID No 24), neuroblast differentiation associated protein AHNAK (SEQ ID No 25), microtubule-associated protein 1 B (SEQ ID No 26), fatty acid- synthase (SEQ ID No 27), triosephosphate isomerase (SEQ ID No 28), ATP-citrate synthase (SEQ ID No 29), calreticulin (SEQ ID No 30), vigilin (SEQ ID No 31), DNA-dependent protein kinase catalytic subunit (SEQ ID No 32), Rab GDP dissociation inhibitor beta (SEQ ID No 33), ATP synthase subunit beta, mitochondrial (SEQ ID No 34). Preferably, the population of extracellular vesicles derived from the MSC population is positive for at least one of the following polypeptides: serotransferrin (SEQ ID No 18), annexin A2 (SEQ ID No 20), and/or insulin-like growth factor-binding protein 3 (SEQ ID No 22);

(c) the MSC population in (a) displays the following order of polypeptide abundance: vimentin > Annexin A . Preferably, the MSC population exhibits the following order of polypeptide abundance: vimentin > Annexin A1 > CD44 > insulin-like growth factor binding protein 7 > fatty-acid binding protein 3;

(d) the extracellular vesicle population in (b) displays the following order of polypeptide abundance: serotransferrin > annexin A2. Preferably, the extracellular vesicle population displays the following polypeptide abundance: serotransferrin > annexin A2 > connective tissue growth factor

(e) the fold-increase expression of indoleamine 2,3- dioxygenase (IDO) in the MSC population is <10 when the MSCs are primed with 15 ng/mL TNF-alpha;

(f) the fold-increase expression of indoleamine 2,3- dioxygenase (IDO) in the MSC population is >100 when the MSCs are primed with 10 ng/mL IFN-gamma;

(g) the viability of polymorphonuclear neutrophils (PMNs) is increased by at least 20% (preferably at least 30% or even more preferably at least 50%) when co-cultured with MSCs from the MSC population primed with IFN-gamma or TNF-alpha in accordance with (e) or (f);

(h) the number of CD14⁺HLA-DR^low monocytes is increased at least 1.5-fold (more preferably at least 2-fold, or even more preferably at least 3-fold) when healthy control human peripheral blood mononuclear cells (PBMCs) are co-cultured with the MSC population primed with IFN-gamma or TNF-alpha in accordance with (e) or (f);

(i) the number of CD4⁺CD25^highCD127^low regulatory T-cells 0~Regs) is increased at least 1.5-fold (more preferably at least 2- fold, or even more preferably at least 3-fold) when healthy control human peripheral blood mononuclear cells (PBMCs) are co-cultured with the MSC population primed with IFN-gamma or TNF-alpha in accordance with (e) or (f).

Furthermore, the extracellular vesicle population obtainable from the MSC population may preferably be negative for (i.e. devoid of) at least one of the following polypeptides: LIM domain only protein7 (SEQ ID No 35), LIM domain and actin- binding protein 1 (SEQ ID No 36), coatomer protein complex, subunit beta 2 (Beta prime), isoform CRA b (SEQ ID No 37), ribonuclease inhibitor (SEQ ID No 38), PDZ and LIM domain protein 5 (SEQ ID No 39), reticulocalbin-1 (SEQ ID No 40), early endosome antigen 1 (SEQ ID No 41), septin-2 (SEQ ID No 42), actin-related protein 2/3 complex subunit 2 (SEQ ID No 43), septin 11 (SEQ ID No 44).

Naturally, the MSC culture (i.e. the MSC population, the population of extracellular vesicles derived from the MSCs, and both the MSC population and the population of extracellular vesicles derived from the MSCs) may fulfil only one (1) of the above criteria, but preferably several criteria are met. For instance, the culture may meet criteria (a) and (b), (a) and (c), (b) and (c), (a) and (d), (a) and (e), (b) and (d), (a) and (e), (a), (b), and (c), (a), (b), and (d), (b), (c), and (d), (c), (d), and (e), (d) and (e), (a), (f), and (g), (e), (f), and (g), (a), (b), and (g), (a), (c), and (f), etc., etc. Thus, the MSC culture may meet all possible combinations and permutations of the above criteria (a)-(i) without departing from the scope of the present invention.

In essence, based on screening of a large number of MSC cultures the inventors have realized that certain polypeptide profiles of both cells and extracellular components (notably extracellular vesicles such as exosomes but also more generally the paracrine factors, primarily polypeptides, existing outside the MSCs) contribute strongly to the therapeutic immuno-modulatory efficacy. Some of the key polypeptide expression features are summarized in the tables below but other polypeptide expression patterns in addition to the ones explicitly mentioned have also been linked by the inventors to immuno-modulatory properties.

Polypeptide Expression - Active MSC-derived Extracellular Vesicles

Preferably positive for at SEQ Preferably negative for at least SEQ least one of: ID No one of: ID No

Serotransferrin 18 LIM domain only protein 7 35

Versican core protein 19 LIM domain and actin-binding 36 protein 1

Annexin A2 20 Coatomer protein complex, subunit 37 beta 2 (Beta prime), isoform

CRA b

Serine protease HTRA1 21 Ribonuclease inhibitor 38

IGFBP3 22 PDZ and LIM domain protein 5 39

Connective tissue GF 23 Reticulocalbin-1 40

Vinculin 24 Early endosome antigen 1 41

Neuroblast differentiation- 25 Septin-2 42 associated protein AHNAK

Microtubule-associated 26 Actin-related protein 2/3 complex 43 protein subunit 2

Fatty acid-synthase 27 Septin 1 44

Triosephosphate isomerise 28

ATP-citrate synthase 29

Calreticulin 30 Vigilin 31

DNA-dependent protein 32

kinase catalytic subunit

Rab GDP dissociation 33

inhibitor beta

ATP synthase subunit beta, 34

mitochondrial

The sources of the MSCs as per the present invention may be selected from the group comprising bone marrow, cord blood, amniotic tissue, Wharton's jelly, tooth bud, adipose tissue, embryonic or fetal material, but various other sources of MSCs could also be applicable. MSCs of bone marrow origin are normally of crista iliaca origin and/or of sternum origin.

Importantly, in order to retain the immuno-modulatory potential of the MSCs (for instance the order of abundance between vimentin and annexin A1 , or any of the other abundance patterns of the MSCs and/or the extracellular vesicles referred to above), the MSCs shall preferably not be passaged more than 5 times before clinical use.

In yet another aspect, the present invention pertains to a population of immunomodulatory MSCs fulfilling at least one of the above-mentnioned criteria, which may be obtainable by the methods of the present invention.

In a further aspect, the present invention relates to a population of immunomodulatory MSCs having the following antigen profile: CD73+, CD90+, CD105+, CD34-, CD45-, CD14-, and CD3-. In a further embodiment, the MSC population may be positive for vimentin and/or Annexin A1 , and further positive for insulin-like growth factor binding protein 7 and/or fatty-acid binding protein 3 and/or Annexin A1.

MSCs are normally defined as functional and biologically active through a colony unit forming (CFU) test and differentiation into adipocytes (fat cells), osteoblasts (bone cells), and chondrocytes (cartilage cells). Madeira and co-authors (PLOS One, 2012) have compared biologically active and inactive MSCs and found that in inactive cells, expression of annexin A1 is upregulated 1.5 fold. Annexin A1 is a known apoptosis- related protein, which impacts adaptive and innate immunity. In contrast, expression of vimentin, which is a cellular cytoskeleton component, is downregulated 2.5 fold in biologically inactive MSCs in comparison with that in the active ones. This probably reflects downregulation in proliferation capacity of the inactive MCSs. However, what the inventors of the present inventions have unexpectedly found is that it is clearly preferential with a larger abundance of vimentin (and related polypeptides) than of annexin A1 (and related polypeptides). Additionally, the present inventors have found that the abundance of both Annexin A1 and Annexin A2 are higher in the extracellular fraction (e.g. in extracellular vesicles such as exosomes) of immunomodulatory MSCs than of non-immunomodulatory MSCs and that this is an important factor for immuno-modulatory capacity in e.g. cardiac inflammation disorders.

Similarly, other abundance patterns have been shown in accordance with the present invention to result in enhanced immuno-modulatory potency, for instance in the context of the whole cells lysates: Vimentin > Caldesmon, Vimentin > CD44, and Vimentin > Annexin A1 > CD44. And, in the context of the fraction containing extracellular vesicles: Serotransferrin > Versican core protein, Serotransferrin > Annexin A2 > Connective tissue growth factor, and Serotransferrin > Annexin A2 > Vinculin. Importantly, the different polypeptide profiles of the MSCs and the extracellular vesicles may co-exist, for instance in that the whole cell lysate (i.e. the MSC polypeptide pattern) may display a greater abundance of vimentin than of Annexin A1 , whereas the extracellular vesicle fraction derived from the MSC population exhibits a greater abundance of serotransferrin than of Annexin A2.

The present inventors have also realized that certain polypeptides play an extraordinarily important role in the modulation of the immune system. Another immuno-modulation criteria as per the present invention relates to insulin-like growth factor binding protein 7 (IGFBP7), which is key to inducing T cells to switch to regulatory T cells, an important mechanism behind the immuno-modulatory capacity of MSCs. Thus, in a preferred embodiment the immuno-modulatory MSCs (and naturally the MSC populations thereof) effective in treating myocarditis of the present invention may also meet the criteria of being positive for IGFBP7, and further the cell fraction and the extracellular fraction (e.g. the extracellular vesicles) may express approximately equal amounts (+/- 30%, but preferably +/- 20%) of IGFBP7 (i.e have approximately equal abundance of the polypeptide in question). In contrast, the extracellular fraction from non-immunomodulatory MSCs (i.e. MSCs not meeting the immuno-modulation criteria as per the present invention) may be distinguished from immuno-modulatory MSCs by having a significantly lower IGFBP7 abundance.

Conversely, immuno-modulatory MSCs with therapeutic activity in e.g. myocarditis may be essentially devoid (or at least have a very low expression/abundance) of IGFBP2 both in the whole cell fraction and in the extracellular fraction (e.g. in the extracellular vesicles), whereas MSCs without immuno-modulatory capacity may have a significantly higher IGFBP2 expression.

Naturally, the abovementioned profiles may be detected/assessed either at the point of obtaining the material from a donor, at various time points during the expansion/culturing of the MSCs prior to clinical application, at various time points after the cells have been cultured in vitro, and/or at the point when it is time to administer the MSCs and/or extracellular vesicles to a patient to be treated. In a further embodiment, the MSC population may be positive for the CD44 antigen, and CD44 may advantageously be present in a lower abundance than Annexin A1.

In a further embodiment, when the cells are to be utilized for medical treatment of a patient suffering from any of the diseases and disorders that may be prevented, treated, cured or alleviated by MSCs, the cells may be harvested (and subsequently used for therapeutic treatment) when the population of MSCs have reached at most 750x10⁶ cells, preferably at most 500x10⁶ cells, starting from at most 60 ml (cm³) of bone marrow aspirate. Thus, the entire procedure from obtaining the cells to the preparing a pharmaceutical composition entails aspirating not more than 60 ml (preferably not more than 40 ml and even more preferably not more than 30 ml) from the bone marrow of a healthy donor (preferably from crista iliaca and/or sternum), expanding the MSCs obtained from the aspirate to not more than 750*10⁶ cells, and, finally, harvesting the cells to prepare a pharmaceutical composition to be administered to a patient. The MSCs (which may preferably be obtained from bone marrow or from cord blood or from amnion or from Wharton's jelly, etc.) obtained by the methods of the present invention display a high therapeutic potency and a significantly smaller number of cells may hence be administered to a patient without reducing the therapeutic efficacy, thereby simplifying and speeding up the entire procedure. One key aspect behind the enhanced therapeutic potency lies in the lower number of cell divisions the MSCs in accordance with the present invention have undergone, meaning that their beneficial immuno-modulating characteristics are conserved. When MSCs are obtained from the donor they may be frozen immediately, followed by thawing and starting of the cell culture upon the need for therapeutic intervention (alternatively, cells may be obtained (and cultivated directly) from a donor when a clinical need for MSCs arises). Surprisingly, it is crucial to not cultivate the MSCs for any longer period of time, preferably less than 15 passages (after obtaining the cells from the donor or after thawing), more preferably less than 5 passages, and even more preferably not more than 2 passages, in order to conserve the potency of the MSCs. The limited expansion of the bone marrow aspirate to preferably not more than 750x10⁶ cells from not more than 60 ml of aspirate (preferably not more than 50 ml, preferably not more than 40 ml, or even more preferably not more than 40 ml), and the limited number of passages, represent a completely novel approach in comparison to the existing art, with clearly beneficial therapeutic effects. Also, the application of the above-mentioned immuno-modulation selection criteria means that the MSCs according to the present invention has a completely different potency and molecular profile than MSC populations conventionally used in the art.

According to one method, the MSC population is cultured (in vitro or ex vivo) on cell plastic surfaces (e.g. in a flask) so as to enrich for mesenchymal stem cells by removing non-adherent cells (i.e. non-mesenchymal stem cells). Thus, according to some aspects of some embodiments of the invention, the mesenchymal stem cells are "adherent" or "plastic- adherent" cells (e.g. cells remaining adhered to the plastic surface after removal of non- adherent, non-mesenchymal cells).

In yet another aspect, the present invention pertains to a cell culture composition for culturing MSCs. The cell culture composition comprises a cell culture medium (for instance Dulbecco's Modified Eagles Medium (DMEM), having a glucose concentration of between 0.5 and 2 g/l (preferably 1 g/l) (e.g. Gibco, InVitrogen Corp, # 31885-023)), and lyzed human trombocytes (platelets), preferably at least 10⁷ lyzed platelets per ml of cell culture composition. The inclusion of lyzed human trombocytes is highly advantageous as it reduces the risk of immune responses to foreign antigens introduced when using conventional non-human serum (e.g. fetal calf serum). The platelet-containing culture medium/composition may be obtained using the following method: (1) isolating platelets from human blood (using conventional isolation techniques), (2) lyzing the platelets (preferably using radiation, e.g. ionizing radiation at around 24 Gy), (3) mixing the platelet lyzate and heparin, (4) mixing the platelet-heparin mixture with DMEM or any other suitable cell culture medium (the medium preferably containing suitable additives, such as antibiotics and/or antimycotics), and (5) centrifuging to eliminate any aggregates (normally at approximately 900 g for approximately 10 minutes).

The cell culture composition may comprise additional components, such as conventional antibiotic and antimycotic drugs (e.g. antibiotic/antimycotic, 100x Gibco, #15240-062), nutrients, or other additives. The DMEM normally comprises the standard ingredients of low glucose DMEM, namely amino acids, salts (normally calcium chloride, potassium chloride, magnesium sulfate, sodium chloride, and monosodium phosphate), glucose, iron, and vitamins (typically folic acid, nicotinamide, riboflavin, and vitamin B12).

In a further aspect, the present invention relates to a pharmaceutical composition comprising between 1 *10⁵ and (100,000) and 5*10⁶ (5,000,000) MSCs per ml in a pharmaceutically acceptable carrier. The MSCs in the pharmaceutical composition may advantageously be obtained by the methods according to the present invention but the MSCs may alternatively be obtained using other suitable methods.

In one embodiment, the pharmaceutical composition may further comprise plasma of blood type AB. Surprisingly, plasma of blood type AB may advantageously be used irrespective of the blood type of the patient to which the pharmaceutical composition is to be administered. Without wishing to be bound by any theory, it is surmised that the absence of antibodies against either A or B antigens is advantageous when administering the pharmaceutical composition to a patient.

The plasma (preferably of blood type AB) may be present at any concentration above 1%, preferably around 10%. The plasma is obtained fresh, normally cryo- reduced, and subsequently stored at -20°C until use. In one embodiment, the pharmaceutically acceptable carrier may be an aqueous solution comprising at least 5% w/v sodium chloride, but other pharmaceutically and physiologically acceptable carriers may also be employed. The concentration of sodium chloride in the aqueous solution is preferably around 9% w/v.

In further embodiments, the MSCs of the pharmaceutical composition may be from an allogeneic and/or an autologous cell source. If the cells of the pharmaceutical composition are obtained from an allogeneic donor it is crucial that the donor is a young, healthy individual under 30 years of age, preferably under 25 years of age, and even more preferably under 20 years of age.

In a further aspect, the present invention relates to a method for preparing a pharmaceutical composition comprising MSCs, comprising the following steps of culturing MSCs of crista iliaca and/or sternum origin in a cell culture composition comprising lyzed human trombocytes, passaging said MSCs not more than 5 times, and, re-suspending the MSCs so obtained in saline solution to a final concentration of between approximately 1 *10⁵ and 5x10⁶ MSCs per ml, preferably 5x10⁵ and 3x10⁶ MSCs per ml.

The pharmaceutical composition may be obtained by aspirating between 20-60 ml (e.g. 20, 25, 30, 35, 40, 45, 50, 55, or 60 ml) of bone marrow from either sternum or Crista iliaca using an aspirate needle that is inserted through the skin using manual pressure until the needle hits the bone. When the needle hits the bone, with a twisting motion of the hand the needle is advanced through the bone cortex and into the marrow cavity. Once the needle is in the marrow cavity, a syringe is attached and used to aspirate liquid bone marrow. A twisting motion is performed during the aspiration to avoid excess content of blood in the sample, which might be the case if an excessively large sample from one single point is taken.

After aspiration, the cells are normally washed in a suitable liquid, for instance phosphate-buffered saline (PBS), and the washed cells may then be re-suspended in Dulbecco's modified Eagle's medium-low glucose supplemented with trombocyte (platelet) lysate and plated at a density of e.g. 160D000 cells per cm². Cultures are maintained at 37°C in a humidified atmosphere containing 5% CO₂, preferably in 175 cm² flasks. When the cultures are near confluence (>80%), the cells may be detached by treatment with trypsin and EDTA and replated at a density of 4000 cells per cm². When approximately 2x10⁶ cells or more are obtained, the cells are harvested and either cryopreserved in 10% dimethyl sulphoxide (DMSO) or washed repeatedly with PBS and re-suspended to a final concentration of between approximately 5x10⁵ and 4x10⁶ cells per ml in saline solution. The saline solution into which the MSCs are re-suspended may preferably comprise blood plasma, preferably plasma of blood type AB.

After aspiration of the MSCs from the bone marrow of a donor, it is crucial to not cultivate the MSCs for any longer period of time, preferably less than 5 passages (after obtaining the cells from the donor or after thawing), and even more preferably not more than 2 passages, in order to conserve the potency of the MSCs Various criteria for release of mesenchymal stem cells for clinical use need to be met, including the absence of visible clumps and the absence of contamination by pathogens (as documented by aerobic and anaerobic cultures before release), the MSCs shall preferably display a spindle-shape morphology, have a viability that preferably is greater than 95%, and immune phenotyping shall preferably show expression of CD73, CD90, and CD105 surface molecules (>90%) and absence of CD34, CD45, CD14, and CD31. Additionally, the immuno-modulation criteria as per the present invention may be applied at least once during the preparation of the MSC-containing pharmaceutical composition, in order to ensure reliability and repeatability.

Administration of the MSCs of the present invention may increase the levels of circulating CD4⁺CD25^highCD127^low TRegs, modulating the immune system. Furthermore, the MSCs in accordance with the present invention may be negative for the endothelial marker CD31. Inflammatory cytokines can prime MSCs, which is considered a key step for MSCs to efficiently control inflammation. The characteristic up-regulation of ICAM-1 (CD54), VCAM-1 (CD106), H LA-ABC, and HLA-DR upon 48 hours of cell exposure towards the pro-inflammatory IFN-γ and TNF-a was confirmed by FACS on the MSCs. lndoleamine-2,3-dioxygenase (IDO) holds an important role for MSC-mediated immune regulatory functions. As abovementioned, the MSCs as per the present invention may exhibit a substantial up-regulation of IDO expression when "primed" with TNF-a or IFN-γ for 48 hours, as assessed for instance by quantitative PCR.

Levels of several pro-inflammatory miRNAs, including miR-155 and miR-125a, that may be elevated prior to MSC administration may demonstrate a significant decline 24 hours after treatment. In contrast several anti-inflammatory miRNAs (such as miR-328, miR-26b, miR-30d, miR-29b) that may be suppressed prior to treatment may demonstrate a significant increase already by day 1 after administration of the MSC in accordance with the present invention.

In yet additional embodiments, the pharmaceutical composition as per the present invention may further comprise exosomes and/or other extracellular vesicles or components obtainable from the MSCs. Extracellular vesicles (such as exosomes) may be added to the pharmaceutical composition from a separate culture of MSCs or they may be derived from the MSCs that are themselves to be included in the pharmaceutical composition.

To optimize the properties of the pharmaceutical composition and to avoid coagulation upon injection, the pharmaceutical composition may comprise heparin or fragmin or any other anticoagulation factor. Alternatively, heparin or fragmin or any other anticoagulation factor may be administered separately to the patient. Additionally, any medical devices used to deliver the MSCs population to the patient may be heparinzed prior to use, again to avoid coagulation.

The present invention, in a further aspect, thus relates to the use of the pharmaceutical compositions according to the present invention for use in medicine, and specifically in the treatment of diseases and disorders such as myocarditis and/or other inflammatory and/or autoimmune conditions of the heart. Myocarditis (inflammatory cardiomyopathy) is an inflammation of the heart muscle, believed to be caused by an autoimmune response. Myocarditis is most often due to infection of the heart muscle by common viruses or bacteria with an inflammatory infiltrate, causing damage to the heart muscle, without the occlusion of the coronary arteries. In some many cases the inflammatory condition results in dilation of the ventricle/ventricles. Post partum myocarditis is a related disorder that would benefit from treatment with MSCs according to the present invention. The MSCs of the present invention are basically applicable in all types of immune-mediated cardiac disorders and/or diseases, for instance post-card iotomy syndrome (an ischemia- reperfusion and cardiomyocyte injury of inflammatory nature occurring after cardiotomy), pericarditis, dilated cardiomyopathy, etc.

The pharmaceutical composition comprising the MSCs may advantageously be administered to a patient suffering from e.g. myocarditis more than once within a certain time period, for instance the pharmaceutical composition may be administered within 1 week of the first dose, within 2 weeks of the first dose, within 3 weeks of the first dose, within 1 month of the first dose, within 2 months of the first dose, within 6 months of the first dose, and even within 1 year of the first dose, in order to enhance the therapeutic effect. Additionally, the pharmaceutical composition comprising the MSCs may be administered with longer intervals as well, either in response to disease recidivism or as a part of the regular treatment.

Prior to administering the MSC composition the patient may be treated with fragmin and/or heparin to reduce cell trapping in the lungs and to prevent the MSCs to cause pulmonary clotting. Furthermore, the patient may also be pre-treated with steroid such as prednisolone, antihistamines, and antibiotics, in a conventional manner.

The pharmaceutical composition comprising the cell dose may advantageously be administered to the patient via infusion through a central venous catheter.

The administration route of the pharmaceutical composition comprising the MSCs may be important to achieve therapeutic efficacy. For instance, in the treatment of myocarditis and/or other inflammatory/autoimmune cardiac disorders the pharmaceutical composition is preferably administered via peripheral intravenous injection, central venous injection into the right atrium, injection into the right ventricle of the heart, and/or injection into the pulmonary trunk/artery.

In a further aspect, the present invention relates to a method of preventing, treating, and/or alleviating diseases and/or disorders selected from the group comprising myocarditis and/or similar inflammatory cardiac disorders and diseases. The treatment methods may comprise the steps of providing a therapeutic dose of MSCs and/or exosomes obtainable from said MSCs, and administering said therapeutic dose to a patient in need thereof. In one embodiment, the therapeutic dose of MSCs and/or exosomes obtainable from said MSCs comprises at least 100,000 MSCs per kg of body weight, preferably at least 500,000 MSCs per kg of body weight, more preferably at least 1 ,000,000 MSCs per kg of body weight, or even more preferably at least 2,000,000 MSCs per kg of body weight.

The present invention will now be described by non-limiting examples and experiments, which in no way limits and/or restricts the scope of the invention but merely serves to exemplify aspects, embodiments, and alternatives of the present invention. Examples

Expansion of mesenchymal stem cells

Twenty-eight ml of bone marrow was aspirated from a HLA-mismatched third party healthy male volunteer. Clinical-grade MSCs were generated under good manufacturing practice (GMP) conditions. Bone marrow mononuclear cells (146x10⁶) were seeded into 175 cm² flasks (Falcon, Franklin Lakes, New Jersey, USA) in Dulbecco's modified Eagles Medium-Low Glucose (DMEM-LG, Life Technologies, Gaithersburg, MD, USA) supplemented with lysed human platelets (final concentration between 10⁷ to 10⁹ cells/mL, preferably 10⁸/mL). When the cultures were near confluence (>80%), the cells were detached by treatment with trypsin and EDTA (Invitrogen, Grand Island, NY, USA) and re-plated once at a density of 4,000 cells/cm². MSCs (adherent cells) were harvested and cryopreserved in 10% Dimethyl Sulfoxide (WAK-Chemie Medical GmbH, Germany). After thawing, the cells were washed three times in PBS and re-suspended in 0.9 % saline solution with the addition of 10% AB plasma, to a final concentration of 2x10⁶ cells/ml. MSC release criteria for clinical use included: absence of visible clumps, spindle shape morphology, absence of contamination by pathogens (bacteria and mycoplasma) and viability >95%. MSCs expressed CD73, CD90, CD105, H LA-ABC and were negative for CD14, CD31 , CD34, CD45 and HLA-DR (Figure 4).

Further, various inflammatory cytokines can activate ("prime") MSCs, which is considered a key step (also known as "licensing") for MSCs to efficiently control inflammation. The characteristic up-regulation of ICAM-1 (CD54), VCAM-1 (CD106), H LA-ABC, and HLA-DR upon 48 hours of cell exposure towards the proinflammatory IFN-Y and TNF-a was confirmed by FACS for the MSCs of the present invention. Additionally, indoleamine-2,3-dioxygenase (IDO) holds an important role for MSC-mediated immune regulatory functions and the MSCs of the present invention exhibited a substantial up-regulation of IDO expression when "primed" with TNF-a or IFN-γ for 48 hours as assessed by quantitative PCR. Cell Purification and Co-cultures

Peripheral blood mononuclear cells (PBMCs) retrieved from the patient and healthy donors were isolated by density gradient-based centrifugation and stored in 10% dimethyl sulfoxide (DMSO) in liquid nitrogen until further analysis.

For further purification of T-cells, a paramagnetic bead-based selection was utilized (Miltenyi Biotec, Bergisch Gladbach Germany). The same BM-MSCs as utilized in the patient, were co-cultured with allogeneic T-cells (MSC:T-cell ratio 1 :5, 1 :10) for 5 days. T-cells were stimulated with activating anti-CD2/CD3/CD28 antibodies (Miltenyi Biotec) in a 0.5 bead per cell ratio. PBMCs were cultured for five days in the presence of MSCs (MSCPBMC ratio 1 :5 and 1 :10) and the monocytic compartment was subsequently analyzed.

In order to assess the capability of the utilized BM-MSCs to respond towards inflammatory licensing, MSCs were cultured in the presence of TNF-a (15 ng/mL) or IFN-y (10ng/mL) for 48h. IDO relative gene expression was measured and compared to untreated cells by quantitative PCR.

Polymorphonuclear leukocytes (PMNs) from buffy coats of healthy donors were ultrapurified under endotoxin-free conditions. PMNs (> 95% purity) and MSCs were co-cultured for up to 40h in presence or absence of 100 ng/ml of LPS. In all cases, MSC were plated 72 hours before the start of co-cultures. In selected experiments, MSCs were previously exposed for 48h to recombinant human IFN-γ (10 ng/ml) and TNF-a (15 ng/ml). Then MSCs were gently washed twice with fresh medium to remove any residual cytokine, and finally co-cultured with freshly isolated PMNs.

Reagents

DMEM-LG, trypan blue (0.4%) and LPS (Ultra-Pure E.coli LPS) were purchased from Invitrogen (Carlsbad, CA, USA); RPMI 1640 medium, dimethyl sulfoxide, penicillin-streptomycin from Sigma-Aldrich (St. Louis, MO USA). Recombinant human IFN-γ was obtained from Peprotech (Rocky Hill, NJ USA) and R&D Systems (Minneapolis, MN, USA) and TNF-a from R&D Systems. Antibodies and flow cytometry

Cells were stained according to the manufacturer's instruction with fluorochrome- coupled antibodies as detailed in Supplemental Table 1. The LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit (Life technologies, Carlsbad, CA, USA) was used for the exclusion of dead cells for PBMC analysis, Propidium Iodide (PI) (Invitrogen) staining for testing the viability of MSCs. For performing intracellular stainings, cells were treated with the BD Cytofix/Cytoperm Fixation/Permeabilisation Kit (BD Biosciences, San Jose, CA USA). After co-culture with MSCs, PMNs were identified on the basis of their typical morphological parameters (forward scatter/side scatter) and their CD45 expression. Levels of PMN apoptosis were assessed by using a Annexin-V- FITC staining kit (Miltenyi Biotec).

Cells were analyzed using a FACS Canto II cytometer (BD Biosciences, San Jose, CA USA) and FlowJo Version 9.5 software (TreeStar, Ashland, OR, USA) software.

Table. Antibody list.

Antibody Fluorochrome Clone Isotype Company

ARG1 APC Sheep R&D Systems

CCR2 AF 647 TG5/CCR2 Mouse Biolegend

CD105 FITC SN6h Ms Ancell

CD106 PE 51-10C9 Ms BD Biosciences

CD11b PE Cy7 ICRF44 Mouse Biolegend

Rat

CD11b Vio Green M1/70.15.11.5 Miltenyi Biotec

lgG2b

CD11c PE Cy7 3.9 Mouse Biolegend

CD123 APC 6H6 Mouse Biolegend

CD127 PerCP Cy5.5 A019D5 Mouse Biolegend

CD 137 PE Cy7 4B4-1 Mouse Biolegend

CD14 PerCP Cy5.5 M5E2 Mouse Biolegend

CD16 PB 3G8 Mouse Biolegend

CD16 FITC 3G8 Mouse Biolegend

CD16 APC VEP13 Mouse Miltenyi Biotec

CD206 APC Cy7 15.2 Mouse Biolegend

CD226 PE 11A8 Mouse Biolegend

CD25 APC M-A251 Mouse BD Biosciences

CD279 PE EH12.2H7 Mouse Biolegend

CD3 FITC SK7 Ms BD Biosciences CD3 PB UCHT1 Mouse Biolegend

CD3 PE Cy7 HIT3a Mouse Biolegend

CD31 FITC WM59 Ms BD Biosciences

CD31 PE WM59 Mouse Biolegend

CD314 PE Cy7 1 D11 Mouse Biolegend

CD33 APC WM53 Mouse Biolegend

CD33 PE WM53 Mouse BD Biosciences

CD34 PE 8G12 Mouse BD Biosciences

CD4 FITC RPA-T4 Mouse Biolegend

CD4 PE Cy7 Sk3 Mouse Biolegend

CD4 APC Cy7 OKT4 Mouse Biolegend

Mouse

CD45 APC Vio770 5B1 Miltenyi Biotec lgG2a

Mouse

igG_2b,

CD45/CD14 FITC/PE ΜφΡ9, 2D1 K, BD Biosciences

Mouse

lgG1 , K

CD45RA PE Cy7 HI100 Mouse BD Biosciences

CD54 PE HA58 Mouse BD Biosciences

CD56 APC Cy7 HCD56 Mouse Biolegend

Mouse

CD62L Vio Blue 145/15 Miltenyi Biotec lgG1

Mouse

CD64 Vio Blue 10.1.1 Miltenyi Biotec lgG1

CD69 AF 647 FN50 Mouse Biolegend

CD73 PE AD2 Mouse BD Biosciences

CD8 PerCP Cy5.5 Sk1 Mouse Biolegend

CD8 FITC HIT8a Mouse Biolegend

CD80 PE L307.4 Ms BD Biosciences

CD83 PE HB15e Mouse Biolegend

CD86 PB IT2.2 Mouse Biolegend

CD90 FITC 5.00E+10 Ms BD Biosciences

HLA class I PE G46-2.6 Mouse BD Biosciences

HLA class I PE W6/32 Ms DAKO

HLA class II FITC CR3/43 Ms DAKO

HLA-DR APC Cy7 L243 Mouse Biolegend HLA class II Mouse

FITC 679.1 Mc7 Beckham Coulter DR, DQ, IgGi, K

HLA-DR APC Cy7 L243 Mouse Biolegend

HLA-DR FITC G46-6 Mouse BD Biosciences

IDO AF 488 700838 Mouse R&D Systems

UCHT1 , HCD14, Mouse

Lineage FITC Biolegend

HIB19, 2H7, lgG1 , K;

RNA preparation and quantitative RT-PCR

Total RNA was extracted (RNeasy mini kit; Qiagen, Hilden, Germany) and cDNA prepared (Superscript First Strand Synthesis System, Life Technologies) using a Mastercycler nexus (Eppendorf, Hamburg, Germany). Messenger RNA levels were quantified by qPCR (Quantitect SYBR Green PCR Kit; Qiagen) on a Rotor Gene Q (Qiagen). Relative gene expression was determined by normalizing the expression to beta2-microglobulin. The following gene-specific primers were used (forward and reverse sequence): ido, 5'-GCATTTTTCAGTGTTCTTCGCATA-3' and 5'- TCATACACCA- GACCGTCTGATAGC-3' ; beta2-microglobulin 5'-TGC-TGT-CTC- CAT-GTT-TGA-TGT-ATC-T-3' and 5'-TCT-CTG-CTC-CCC-ACC-TCT-AAG-T-3\

Exosome purification and miRNA isolation

Briefly, 1 ,5-2 ml of serum from each time-point was purified using the EXO50 (Exosome Diagnostics, Martinsried, Germany). The eluted RNA was processed for microRNA analysis using the Low Sample Input protocol for the TaqMan® OpenArray® Human MicroRNA Panel (Life Technologies, US). Megaplex™ RT Primers, Human Pool A and Human Pool B were used for reverse transcription followed by a pre-amplification step according to manufacturer's protocol. The pre- amplified material was diluted 1 :20 in TE (pH8.0) and analyzed on the TaqMan OpenArray Human MicroRNA Panel for final qPCR detection. A global normalization was applied and data was presented in comparison to a healthy control sample. Assessment of immuno-modulatory capacity

Ex- Vo expanded MSCs were pre-treated (primed MSCs, pMSCs) or not (MSCs) with 10 ng/ml of IFN-γ and 15 ng/ml of TNF-a for 48 hours before used in co-cultures with PMNs with or without activation by endotoxin (100 ng/ml of lipopolysaccharides (LPS)). Following inflammatory priming, the MSCs up-regulated cell surface expression of CD54 (ICAM-1), CD106 (VCAM-1) and H LA-ABC and -DR, as well as the expression of indoleamine 2,3-dioxygenase (IDO), a potent mediator of many MSC immune regulatory functions (Figures 6 and 7).

Furthermore, active (i.e. immuno-modulatory) MSC populations were compared to non-active populations on a proteomics level, analyzing both whole cell fractions and extracellular fractions comprising e.g. extracellular vesicles such as exosomes. To assess the profiles of the MSCs and extracellular vesicles, MSCs (active and non- active) were grown in serum free media for 48 hours. Conditioned media was collected and the extracellular vesciels were isolated using ultracentrifugation in accordance with the protocol of Thiery and Sleeman (2006). Total protein lysates were generated on MSCs and extracellular fractions followed by tryptic digestion. Digestion was analyzed using 4 hours nano reverse phase chromatography gradient prior to mass spectrometry (nanoLC-MS/MS) analyses. Various patterns, comparisons, and rankings were made to identify the proteomics parameters determining immuno-modulatory capacity and to define immuno-modulation criteria.

Statistical Analysis

Wilcoxon paired test was used to compare the differences between two different groups. One-way ANOVA analysis was used to statistically evaluate the difference of sample means among multiple groups. Significant level was set a p-value <0.05.

MSC treatment of virus-induced myocarditis in mice

Specific pathogen free in-bred, 4 week old, male balb/c mice were inoculated with 10x6 plaque forming units of CVB3(strain Nancy) diluted in PBS to a final volume of 0.1 ml. Virus-inoculated mice were randomised to 10x6 MSC iv when myocarditis had been confirmed.

Mice treated with between 5^*10⁵ and 2^*10⁶ MSC of different origin (bone marrow and placenta) had a 50 % reduced mortality at two weeks after infection.

Male patient with myocarditis

A 48-year male presents with acute heart failure due to myocarditis. Left ventricular ejection fraction is measured to 12%. The patient has TAPSE 8 mm and ventricular arrhythmias. On the basis of acute heart failure the patient receives 2,000,000 MSCs/kg of body weight through a central venous catheter.

Prior to MSC infusion the patient is treated with fragmin/heparin to reduce cell trapping in the lungs and to prevent the cells to cause pulmonary clothing.

Within 7 days post treatment the patient's ejection fraction has more than doubled to 25%, with further improvements measured over the coming 3 months.

During the same time span TAPSE increased from 8 mm to 12 mm, and ProBNP was decreased from 19,000 to 5,000.

Claims

Claims

1. A mesenchymal stem cell (MSC) population, wherein the MSC population fulfils at least one of the following immuno-modulation criteria:

(a) the MSC population is positive for at least one of the following polypeptides: vimentin (SEQ ID No 1), annexin A1 (SEQ ID No 3) and/or insulin-like growth factor-binding protein 7 (SEQ ID No 12);

(b) a population of extracellular vesicles derived from the MSC population is positive for at least one of the following polypeptides: serotransferrin (SEQ ID No 18), annexin A2 (SEQ ID No 20), and/or insulin-like growth factor-binding protein 3 (SEQ ID No 22);

(c) the MSC population in displays the following order of polypeptide abundance: vimentin > Annexin A1 ;

(d) the MSC population and the population of extracellular vesicles derived from the MSC population expresses approximately equal amounts (+/- 30%) of IGFBP7;

(e) the extracellular vesicle population displays the following order of polypeptide abundance: serotransferrin > annexin A2;

(f) the fold-increase expression of indoleamine 2,3- dioxygenase (IDO) in the MSC population is >100 when the MSCs are primed with 10 ng/mL IFN-gamma;

2. The MSC population according to any claim 1 , wherein the MSC population is passaged at most 5 times before clinical use.

3. The MSC population according to any one of claims 1 to 2, wherein the MSC population expresses a larger abundance of vimentin than of CD44.

4. A vesicle population comprising extracellular vesicles derived from the MSC population of any one of claims 1 to 3.

5. The vesicle population according to claim 4, wherein the extracellular vesicles are exosomes.

6. A pharmaceutical composition comprising the MSC population of any one of claims 1 to 3 and/or the vesicle population according to any one of claims 4 to 5.

7. The pharmaceutical composition according to claim 6, further comprising blood plasma.

8. The MSC population according to any one of claims 1 to 3 and/or the vesicle population according to any one of claims 4 to 5 and/or the pharmaceutical composition according to any one of claims 6 to 7 for use in medicine.

9. The MSC population according to any one of claims 1 to 3 and/or the vesicle population according to any one of claims 4 to 5 and/or the pharmaceutical composition according to any one of claims 6 to 7 for use in the treatment of myocarditis, pericarditis, cardiomyopathy and specifically dilated cardiomyopathy, and/or post-cardiotomy syndrome.

10. The MSC population according to any one of claims 1 to 3 and/or the vesicle population according to any one of claims 4 to 5 and/or the pharmaceutical composition according to any one of claims 6 to 7 for use in the treatment of of myocarditis, pericarditis, cardiomyopathy and specifically dilated cardiomyopathy, and/or post-cardiotomy syndrome, wherein the composition is administered via intravenous injection, peripheral intravenous injection, central venous injection into the right atrium, injection into the right ventricle of the heart, and/or injection into the pulmonary trunk/artery.

11. The MSC population according to any one of claims 1 to 3 and/or the vesicle population according to any one of claims 4 to 5 and/or the pharmaceutical composition according to any one of claims 6 to 7 for use in the treatment of of myocarditis, pericarditis, cardiomyopathy and specifically dilated cardiomyopathy, and/or post-cardiotomy syndrome, wherein the patient suffering from myocarditis, pericarditis, cardiomyopathy and specifically dilated cardiomyopathy, and/or post-cardiotomy syndrome is eligible for and/or is undergoing extra-corporal membranous oxygenation (ECMO) treatment.

12. The MSC population according to any one of claims 1 to 3 and/or the vesicle population according to any one of claims 4 to 5 and/or the pharmaceutical composition according to any one of claims 6 to 7 for use in the treatment of of myocarditis, pericarditis, cardiomyopathy and specifically dilated cardiomyopathy, and/or post-cardiotomy syndrome, wherein the patient to be treated has elevated levels of at least one of the following microRNAs (miRs): miR-409-3P, miR-886-5P, miR-324-3P, miR-222, miR-125A-5P, miR-339-3P, and/or miR-155.