WO2025117472A1 - Treatment of heart failure using pluripotent stem cell derived cardiomyocytes and derivatives thereof - Google Patents

Abstract

Compositions of matter, cell types, and protocols useful for treatment of myocardial infarction and heart failure. Methods for the generation of autologous cardiomyocyte progenitor cells and cardiomyocytes and methods of administration in a manner capable of stimulating regeneration. Induced pluripotent stem cells can be treated with modulators of the Wnt pathway followed by exposure to cardiogenic agents such as insulin and/or cell conditioned media. Administration of cardiomyocyte progenitors can be performed in the presence of angiogenic and/or anti-inflammatory cells to assist in viability and function of engrafted cells.

Description

IMMORTA-HEART-PCT TREATMENT OF HEART FAILURE USING PLURIPOTENT STEM CELL DERIVED CARDIOMYOCYTES AND DERIVATIVES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims benefit of U.S. Provisional Patent Application Serial No.63/604,867, filed on November 30, 2023, entitled “TREATMENT OF HEART FAILURE USING PLURIPOTENT STEM CELL DERIVED CARDIOMYOCYTES AND DERIVATIVES THEREOF”, the contents of which are incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The invention concerns the fields of cell biology, regenerative medicine and cardiology. Specifically, the invention relates to approaches for generating therapeutic cell populations and methods of use thereof for treating myocardial infarction. BACKGROUND OF THE INVENTION [0003] One type of regenerative cell which possesses significant clinical promise is the induced pluripotent stem cells (iPSC), which can be generated directly from somatic adult cells. This technology was pioneered over a decade ago by the introduction of four specific genes encoding transcription factors Oct3/4, Sox2, c-Myc and Klf4 into mouse adult fibroblasts under embryonic stem (ES) cell culture conditions. [0004] iPSCs can be differentiated into many different types of cells such as neurons, cardiomyocytes and hepatocytes for potential therapies. These cells can also be used for understanding underlying disease mechanisms and screening for therapeutics which may serve to alleviate disease conditions. One of the first cell types derived from iPSC technology was cardiomyocytes. These iPSC-derived cardiomyocytes appear to express the proper ion channel repertoire, as well as structural and other proteins found in normal cardiomyocytes. However, it is challenging to fully differentiate iPSCs into cardiomyocytes having an adult phenotype. Rather, development often stalls during the immature phase, where, compared to the mature or adult phenotype, the immature cardiomyocytes tend to have a rounded morphology, disorganized sarcomere, lack of T-tubule, differ in gene expression profile, and differ in action potential profile. At present, the potential for immature cardiomyocytes as a therapeutic approach is limited. IMMORTA-HEART-PCT [0005] A variety of different approaches have been employed to improve maturation status of iPSC-derived cardiomyocytes, including long-term cardiomyocyte culture, cultivation of cells on a substrate which has stiffness close to that of native myocardium, seeding cells to a patterned substrate providing topographical cues, applying mechanical loading to cells and directed electrical stimulation. All these methods can result in cardiomyocytes that structurally and functionally resemble an adult-like phenotype. However, each system has its weaknesses, such as difficulty to achieve high throughput screening, and the high level of technical knowledge required. [0006] Novel methods for generating cardiomyocytes represent promising approaches for treatment of cardiovascular diseases. Despite the broad use of cholesterol lowering agents and better control of co-morbid illnesses (i.e., diabetes, hypertension, etc.) that contribute to ischemic cardiovascular disease, acute myocardial infarction remains common with a reported annual incidence of 1.1 million cases in the United States alone. Acute myocardial infarction causes necrotic and apoptotic myocardial cell death resulting in ventricular remodeling, which is a precursor to subsequent cardiac dysfunction, congestive heart failure and resulting cardiac related adverse events including death in a significant percentage of patients. The extent of myocardial cell loss is dependent on the location and duration of coronary artery occlusion, existing collateral coronary circulation and the condition of the cardiac microvasculature. [0007] Myocardial cell loss along with evolving microvascular insufficiency and inadequate collateral circulation development can result in chronic hemodynamic overload and eventual congestive heart failure. Once developed the prognosis is poor with observed median survivals of 0.7 and 3.2 years in men and women, respectively. Thus, therapies are applied to limit or prevent ventricular remodeling in order to avoid left ventricular dilatation and late congestive heart failure after myocardial infarction. [0008] Newer modalities resulting in immediate re-perfusion of occluded coronary arteries have been successful in limiting the extent of myocardial infarcts, ameliorating angina pectoris, improving function of remaining viable myocardium and limiting subsequent infarcts. Unfortunately, these interventions have not significantly altered the resulting cardiac dysfunction caused by myocardial cell loss and peri-infarct zone micro-vascular insufficiency. Myocardial cells have virtually no ability to regenerate, thus myocardial infarction leads to permanent cardiac dysfunction due to contractile-muscle cell loss and replacement with non- functioning fibrotic scaring. Moreover, compensatory hypertrophy of viable cardiac muscle leads to micro-vascular insufficiency that results in further demise in cardiac function by causing myocardial muscle hibernation and apoptosis of hypertrophied myocytes in the peri-infarct IMMORTA-HEART-PCT zone. Consequently, new strategies are in development to limit or prevent cardiac dysfunction after acute injury and to restore cardiac function in the chronic dysfunction setting. [0009] In the setting of brief (3 to 5 minutes) coronary artery occlusion, energy metabolism is impaired leading to demonstrable cardiac muscle dysfunction that can persist for up to 48 hours despite immediate reperfusion. This so-called stunned myocardium phenomenon occurs subsequent to reperfusion and is thought to be a result of reactive oxygen molecules. The process is transient and not associated with an inflammatory response. Immediately following a myocardial infarction, transient generalized cardiac dysfunction (stunned myocardium) uniformly occurs. After successful revascularization significant recovery from stunning occurs within three to four days, although complete recovery may take much longer. [0010] Coronary artery occlusion of more significant duration leading to myocardial ischemia is associated with a significant inflammatory response that begins immediately after reperfusion and can last for up to several weeks. The inflammatory cascade following reperfusion is complex, initially contributing to myocardial damage but then later leading to healing and scar formation. This complex process appears to occur in two phases. In the first so called “hot” phase (within the first 5 days), reactive oxygen species in the ischemic myocardial tissue along with complement activation, generate a leukocyte chemotaxis signal and initiate a cytokine cascade. Mast cell degranulation, TNFalpha release and increased IL-6, ICAM-1, selectin (L, E and P) and integrin (CD11a, CD11b and CD18) expression all appear to contribute to neutrophil accumulation and degranulation in ischemic myocardium. Neutrophils contribute significantly to myocardial cell damage and death through micro-vascular obstruction, and activation of neutrophil respiratory burst pathways after ligand-specific adhesion to cardiac myocytes. During the “hot” phase, angiogenesis is inhibited due to the release of angiostatic substances including Interferon gamma inducible protein (IP 10). In the second phase, the cardiac repair process begins (days 7 to 14) that eventually lead to scar formation (days 14 to 21) and subsequent ventricular remodeling (days 21 to 90). Soon after reperfusion, monocytes infiltrate the infarcted myocardium. Attracted by complement (C 5a), TGF-B1 and MCP-1, monocytes differentiate into macrophages that initiate the healing process through scavenging dead tissue, regulating extracellular matrix metabolism and inducing fibroblast proliferation. Secretion of IL-10 by infiltrating lymphocytes also promotes healing by down-regulating inflammatory cytokines and influencing tissue remodeling. Mast cells appear to also be involved in the later stages of myocardial repair by participating in the formation of fibrotic scar tissue. Stem Cell Factor (SCF) is a potent attractor of mast cells. SCF mRNA has been shown to be upregulated in ischemic myocardial segments in a canine model of myocardial infarction and IMMORTA-HEART-PCT thus may contribute to mast cell accumulation at ischemic myocardial sites. Excreted mast cell products (TGF-beta, bFGF, VEGF and gelatinases A and B) induce fibroblast proliferation, influence extracellular matrix metabolism and induce angiogenesis. [0011] Thus, it would appear that the ideal opportunity to intervene with novel approaches intended to reduce or prevent ventricular remodeling would be between 5 to 10 days after revascularization to avoid the hot phase but before the initiation of permanent scar formation. [0012] Among survivors of myocardial infarction, residual cardiac function is influenced most by the extent of ventricular remodeling. Alterations in ventricular topography occur in both infarcted and healthy cardiac tissue after myocardial infarction. Specifically, ventricular dilatation causes a decrement in global cardiac function and is affected most by the infarct size, infarct healing and ventricular wall stresses. Recent efforts to minimize remodeling have been successful by limiting infarct size through rapid reperfusion using thrombolytic agents and mechanical interventions, along with reducing ventricular wall stresses by judicious use of pre- load therapies and proper after load management. Regardless of these interventions, a substantial percentage of patients experience clinically relevant and long-term cardiac dysfunction after myocardial infarction. [0013] Accordingly, there remains a need for improved systems and methods for further maturation of cardiomyocyte precursor cells that can be useful for treatment of disease conditions such as myocardial infarction. SUMMARY OF THE INVENTION [0014] The invention provides compositions of cardiomyocytes for use in regenerative medicine applications and methods of use thereof. In one embodiment, compositions of a cell culture media capable of producing a cardiomyocyte are provided. The invention discloses methods of differentiation of pluripotent stem cells such as iPSCs to provide a source of cardiomyocytes or cardiomyocyte progenitors and methods of use thereof for treating a subject with a cardiovascular disorder such as myocardial infarction or heart failure. [0015] In one embodiment, compositions and methods are provided for culturing iPSCs and cell populations derived thereof, wherein the cultures comprise one or a plurality of factors selected from the group consisting of growth factors, anti-inflammatory factors, and angiogenic factors, and wherein the factors promote viability and function of cardiomyocytes produced using the culture methods of the invention. Specific embodiments of the invention provide IMMORTA-HEART-PCT compositions of cells and cell culture media for producing cardiomyocytes having a mature phenotype, and methods of generating the same. [0016] In one embodiment, the invention provides compositions of support cells and factors for inducing differentiation of a cardiomyocyte from a pluripotent stem cell in a cell culture system. In another embodiment, compositions of exosomes are provided to the culture for enhancing the functions or activity of a cardiomyocyte. In another embodiment, compositions of exosomes are provided in a cell culture system of the invention. [0017] Embodiments of the invention provide compositions of cell culture media for differentiating a pluripotent stem cell into a cardiomyocyte. In certain embodiments, one or a plurality of support cells or scaffolds are provided for culturing an iPSC and for its stepwise differentiation into a cardiomyocyte. In one embodiment, the one or plurality of support cells or scaffolds comprise a modified cell culture substrate or surface, wherein the modified cell culture substrate comprises one or a plurality of factors selected from the group consisting of: growth factors, cytokines, anti-inflammatory molecules, and/or exosomes. In one embodiment, pluripotent stem cells such as iPSCs are plated on the modified cell culture substrate in culture medium until reaching confluency of at least 75%, wherein the modified cell culture substrate comprises one or a plurality of the following components: decellularized cardiac tissue, hyaluronic acid, fibronectin, vitronectin, collagen II, and collagen IV. In one embodiment, decellularized cardiac tissue comprises an extracellular matrix, wherein the extracellular matrix comprises structural and functional molecules from cardiac tissue, and wherein the molecules comprise proteins, glycosaminoglycans, proteoglycans, and other components. In one embodiment, the decellularized cardiac tissue comprises one or a plurality of the following molecules or factors: thrombospondin, tenascin, versican, syndecan, biglycan, hyaluronan, heparan sulfate, collagen, fibronectin, elastin, and laminin. These embodiments provide an iPSC population that is suitable for subsequent differentiation steps to produce a population of cardiomyocytes. [0018] In one embodiment, a method for generating a cardiomyocyte from a cultured iPSC is provided, the method comprising: a) exposing the iPSC to a first growth medium comprising a Wnt activator, wherein the iPSC is induced to differentiate into a mesoderm cell; b) exposing the mesoderm cell from (a) to a second growth medium comprising a Wnt inhibitor, wherein the mesoderm cell is induced to differentiate into a cardiac mesoderm cell; and c) exposing the cardiac mesoderm cells from (b) to a third growth medium comprising insulin, wherein the cardiac mesoderm cell is induced to differentiate into a cardiomyocyte. IMMORTA-HEART-PCT [0019] In yet another embodiment, a cell culture system for differentiation of iPSCs into cardiomyocytes comprises one or a plurality of additional cell types for enhancing the differentiation process, wherein the cell types may include lymphocytes, connective tissue cells, multipotent stem cells, or combinations thereof that may be selected based on their capacity to produce growth factors and/or anti-inflammatory factors. In a specific embodiment, a cell type for inclusion in a cell culture system of the invention is selected from the group consisting of a monocyte, a fibroblast, a macrophage, a mesenchymal stem cell, or combinations thereof. In one embodiment, various concentrations of prostaglandin E2 (PGE2) are provided in a cell culture system, preferably with at least one cell type. [0020] In one embodiment, a kit for practicing the methods of the invention is disclosed, wherein the kit comprises an animal-component-free cell culture medium, which may comprise at least one or more of the following components: L-ascorbic acid-2-phosphate magnesium, sodium selenium, fibroblast growth factor 2 (FGF2), sodium bicarbonate (NaHCO3), insulin, transferrin, and transforming growth factor beta 1 (TGFβ1). In certain embodiments, the kit comprises one or a plurality of the following proteins or molecules: a Wnt activator (e.g., CHIR99021), a Wnt inhibitor (e.g., XAV939), insulin, a ROCK inhibitor, and a histone deacetylase inhibitor. [0021] In one embodiment, a method for treating a subject with myocardial infarction is provided using cardiomyocytes generated using the methods of the invention. In other embodiments, a method of treatment comprises administering a cardiomyocyte comprising an exosome to a subject with myocardial infarction. In yet another embodiment, a method of treatment comprises administering an exosome derived from a cardiomyocyte or from another cell source to a subject in need thereof. [0022] A summary is provided below based on numbered aspects of the invention. [0023] 1. A method of treating heart failure comprising the steps of: a) obtaining a pluripotent stem cell population; b) differentiating said pluripotent stem cell into a cardiomyocyte progenitor cell; c) implanting said cardiomyocyte progenitor cell into cardiac tissue; and optionally d) providing adjuvant cells and/or chemicals to enhance viability and function of said engrafted cardiomyocyte progenitor. [0024] 2. The method of aspect 1, wherein said heart failure is associated with loss of cardiomyocytes. [0025] 3. The method of aspect 1, wherein said loss of cardiomyocytes is caused by apoptosis. IMMORTA-HEART-PCT [0026] 4. The method of aspect 3, wherein said apoptosis is mediated by a death receptor. [0027] 5. The method of aspect 4, wherein said death receptor is a member of the TNF- alpha receptor superfamily. [0028] 6. The method of aspect 4, wherein said death receptor is TNF-alpha receptor p55. [0029] 7. The method of aspect 4, wherein said death receptor is TNF-alpha receptor p75. [0030] 8. The method of aspect 4, wherein said death receptor is NGF receptor. [0031] 9. The method of aspect 4, wherein said death receptor is TWEAK receptor. [0032] 10. The method of aspect 4, wherein said death receptor is TRAIL receptor. [0033] 11. The method of aspect 4, wherein said death receptor is Fas receptor. [0034] 12. The method of aspect 4, wherein said death receptor is Apo2. [0035] 13. The method of aspect 3, wherein said apoptosis is mediated by caspase 3. [0036] 14. The method of aspect 3, wherein said apoptosis is mediated by mitochondria initiated signals. [0037] 15. The method of aspect 3, wherein said apoptosis is mediated by caspase 8. [0038] 16. The method of aspect 3, wherein said apoptosis is mediated by caspase 9. [0039] 17. The method of aspect 1, wherein said loss of cardiomyocytes is caused by necrosis. [0040] 18. The method of aspect 1, wherein said loss of cardiomyocytes is caused by ferroptosis [0041] 19. The method of aspect 1, wherein said loss of cardiomyocytes is caused by pyroptosis. [0042] 20. The method of aspect 1, wherein said loss of cardiomyocytes is caused by NLRP3 activation. [0043] 21. The method of aspect 1, wherein said loss of cardiomyocytes is caused by activation of an innate immune receptor. [0044] 22. The method of aspect 21, wherein said innate immune receptor is RIG1. [0045] 23. The method of aspect 21, wherein said innate immune receptor is STING. [0046] 24. The method of aspect 21, wherein said innate immune receptor is toll like receptor 2. [0047] 25. The method of aspect 21, wherein said innate immune receptor is toll like receptor 3. [0048] 26. The method of aspect 21, wherein said innate immune receptor is toll like receptor 5. IMMORTA-HEART-PCT [0049] 27. The method of aspect 21, wherein said innate immune receptor is toll like receptor 7. [0050] 28. The method of aspect 21, wherein said innate immune receptor is toll like receptor 8. [0051] 29. The method of aspect 21, wherein said innate immune receptor is toll like receptor 9. [0052] 30. The method of aspect 1, wherein said pluripotent stem cell is an induced pluripotent stem cell. [0053] 31. The method of aspect 1, wherein said pluripotent stem cell is differentiated into a cardiomyocyte progenitor by obtaining an embryoid body, dissociating said embryoid body, and exposing cells from the cardiogenic region of said embryoid body to inductive factors. [0054] 32. The method of aspect 31, wherein said cells from said embryoid body are cultured on hyaluronic acid. [0055] 33. The method of aspect 31, wherein said cells from said embryoid body are cultured on fibronectin. [0056] 34. The method of aspect 31, wherein said cells from said embryoid body are cultured on collagen II. [0057] 35. The method of aspect 31, wherein said cells from said embryoid body are cultured on collagen IV. [0058] 36. The method of aspect 31, wherein said cells from said embryoid body are cultured on decellularized cardiac tissue. [0059] 37. The method of aspect 31, wherein said cells from said embryoid body are cultured in a liquid media containing one or more of the following: a) L-ascorbic acid-2- phosphate; b) magnesium; c) sodium; d) selenium; e) fibroblast growth factor 2 (FGF2); f) Insulin; g) NaHCO; ; h) transferrin and; i) transforming growth factor beta 1 (TGFβ1). [0060] 38. The method of aspect 37, wherein a Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor is added to the culture. [0061] 39. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-1 receptor in said cells by more than 25%. [0062] 40. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-1 receptor in said cells by more than 50%. IMMORTA-HEART-PCT [0063] 41. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-1 receptor in said cells by more than 100%. [0064] 42. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-2 receptor in said cells by more than 25%. [0065] 43. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-2 receptor in said cells by more than 50%. [0066] 44. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-2 receptor in said cells by more than 100%. [0067] 45. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-5 receptor in said cells by more than 25%. [0068] 46. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-5 receptor in said cells by more than 50%. [0069] 47. The method of aspect 38, wherein said ROCK inhibitor is added at a concentration and duration to induce upregulation of FGF-5 receptor in said cells by more than 100%. [0070] 49. The method of aspect 38, wherein said ROCK inhibitor is administered at a concentration between 1 μM and 120 μM. [0071] 50. The method of aspect 38, wherein said ROCK inhibitor is administered at a concentration between 2 μM and 50 μM. [0072] 51. The method of aspect 38, wherein said ROCK inhibitor is administered at a concentration between 4 μM and 40 μM. [0073] 52. The method of aspect 38, wherein said ROCK inhibitor is administered at a concentration between 8 μM and 12 μM. [0074] 53. The method of aspect 38, wherein a histone deacetylase inhibitor is added to the culture. [0075] 54. The method of aspect 53, wherein said histone deacetylase inhibitor is sulforaphane. IMMORTA-HEART-PCT [0076] 55. The method of aspect 53, wherein said histone deacetylase inhibitor is valproic acid. [0077] 56. The method of aspect 53, wherein said histone deacetylase inhibitor is phenylbutyrate. [0078] 57. The method of aspect 53, wherein said histone deacetylase inhibitor is sodium phenylbutyrate. [0079] 58. The method of aspect 53, wherein said histone deacetylase inhibitor is trichostatin A. DETAILED DESCRIPTION OF THE INVENTION [0080] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. [0081] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. [0082] Unless defined otherwise, the technical terms used herein have the same meaning as is commonly understood by one of skill in the art. [0083] As used herein, the term “subject” means any vertebrate organism having a circulatory system. In some embodiments, a subject is a human or an animal. [0084] As used herein, the terms “treatment”, “therapy”, or “therapeutic” refer to an approach for obtaining beneficial results in the disease condition or symptoms of a subject, for example, using a drug or a medical device. Beneficial results may be obtained in clinical trials or from other clinical experiences and may include alleviation of symptoms, diminishing disease, stabilizing disease, preventing a disease from spreading, delaying disease progression, inducing remission, diminishing disease severity, or slowing its recurrence, and/or affording improvements in a subject’s quality of life. IMMORTA-HEART-PCT [0085] As used herein, “exosome” refers to a bi-lipid membrane extracellular vesicle of approximately 30-150 nm in diameter (average size of approximately 100 nm) that is secreted by a cell. In the context of the invention, an exosome is a type of extracellular vesicle. Exosomes may carry components from cell membranes or from a cell’s components including proteins and nucleic acids including but not limited to messenger RNA (mRNA), microRNA (miRNA), long-non- coding RNA (lncRNA), and circular RNA (circRNA) that can be transmitted to recipient cells. In certain embodiments, exosomes are identified by the expression of surface proteins unique to the endosomal pathway or fragments thereof including but not limited to tetraspanin molecules such as CD9, CD63, CD81, heat shock proteins (e.g., HSP70), lysosomal proteins (e.g., Lamp2b), tumor-sensitive gene 101 (Tsg101), and fusion proteins (e.g., flotillin and annexin). [0086] As used herein, “induced pluripotent stem cell” (iPSC) refers to a type of pluripotent stem cell that can be reprogrammed from adult somatic cells (e.g., from skin or blood cells, or from another tissue source). A distinct type of pluripotent stem cell is the embryonic stem cell that is derived from an embryoid body. [0087] As used herein, “mesenchymal stem cell” (MSC) refers to a cell having the following characteristics: a) adherence to plastic; b) expression of CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative; and c) possessing the ability to differentiate to osteogenic, chondrogenic and adipogenic lineage. Other cells possessing mesenchymal-like properties are included within the definition of “mesenchymal stem cell”, with the condition that said cells possess at least one of the following characteristics: a) regenerative activity; b) production of growth factors; c) ability to induce a healing response, either directly, or through elicitation of endogenous host repair mechanisms. As used herein, “mesenchymal stromal cell” or “mesenchymal stem cell” can be used interchangeably. Said MSC can be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, placenta, amniotic tissue, derived from pluripotent stem cells, and tooth. In some definitions of “MSC”, said cells include cells that are CD34 positive upon initial isolation from tissue but are similar to cells described about phenotypically and functionally. Mesenchymal stem cells include cells that are isolated from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof and satisfy the International Society for Cellular Therapy (ISCT) criteria either before or after expansion. In some contexts, mesenchymal stem cells include cells described in the literature as bone marrow stromal stem cells (BMSSC), marrow-isolated adult multipotent inducible cells (MIAMI) cells, multipotent adult IMMORTA-HEART-PCT progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), MultiStem®, Prochymal®, remestemcel-L, Mesenchymal Precursor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX-PAD, AlloStem®, Astrostem®, Ixmyelocel-T, MSC-NTF, NurOwn™, Stemedyne™-MSC, Stempeucel®, StempeucelCLI, StempeucelOA, HiQCell, Hearticellgram-AMI, Revascor®, Cardiorel®, Cartistem®, Pneumostem®, Promostem®, Homeo-GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Regenerative Cells (ERC), adipose-derived stem and regenerative cells (ADRCs). In one embodiment MSC donor lots are generated from umbilical cord tissue. Means of generating umbilical cord tissue MSC have been disclosed in the prior art. [0088] As used herein, “cardiac mesoderm cell” refers to a cardiac-lineage cell of mesodermal origin that has the capacity to give rise to a cardiac cell under the appropriate conditions, comprising a progenitor cell population for the heart. In certain embodiments, a cardiac mesoderm cell is characterized by expression of specific genes, e.g., Mesp1 (mesoderm posterior 1), and/or specific cardiac-specific transcription factors, e.g., Gata4, Hand1, Hand2, Nkx2-5, and Wt1. In certain embodiments, a cardiac mesoderm cell may have the ability to differentiate into one or a plurality of cardiac cell lineages, including cardiomyocytes, endothelial cells, and epicardial cells. In certain aspects, the terms “cardiac mesoderm cell” and “cardiac progenitor cell” may be used interchangeably. [0089] As used herein, “Wnt inhibitor” refers to a protein or small molecule that antagonizes or blocks signaling through one or a plurality of members of the Wnt/beta-catenin family of glycoproteins that pass signals from outside of a cell through cell surface receptors to inside of the cell. To inhibit Wnt signaling in a cell culture system, small molecule inhibitors, biological molecules, natural compounds, recombinant proteins, or other agents may be utilized. In some embodiments, a Wnt inhibitor is a compound selected from the group consisting of: C59, IWR-1, IWP-2, IWP-4, XAV939, and combinations thereof. [0090] As used herein, “Wnt activator” refers to a protein or small molecule that acts as an agonist or stimulator of Wnt signaling. Examples of Wnt activators include 6-bromoindirubin- 3-oxime (BIO), CHIR99021, BML-284, recombinant Wnt proteins, and R-spondin family members. Certain embodiments may utilize Wnt mimics or Wnt surrogates to stimulate the Wnt pathway. [0091] As used herein, “cardiomyocyte” refers to a muscle cell in the heart that is responsible for the contractions of the heart, which may comprise an atrial or ventricular cardiomyocyte, and which may be defined based on expression of one or a plurality of surface markers including but not limited to sarcomeric markers such as alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin. IMMORTA-HEART-PCT [0092] As used herein, “cardiomyocyte maturation” refers to a constellation of changes to cell structure, metabolism, function, and gene expression that convert an immature or fetal cardiomyocyte to a mature or adult cardiomyocyte. Cardiomyocyte maturation from an immature/fetal cell may comprise one or a plurality gene expression changes and corresponding functional changes selected from the group consisting of: a) an change in myofibril/sarcomere components to a mature form due to transcriptional changes or alternative splicing; b) an increase in ventricular ion channels, e.g., an increase in KCNJ2; c) a decrease of automaticity ion channels, e.g., HCN4; d) an increase of Ca2+ handling molecules, e.g., Cav1.2, RYR2, and SERCA2; e) a decrease in glycolysis, e.g., HK1, PKM activation of mitochondrial biogenesis, fatty acid oxidation, and oxidative phosphorylation (e.g., PPARGC1A, PPARA, ESRRA); f) silencing of cell cycle genes, e.g., CDK1, CCNB1; g) Changes in expression of cell adhesion genes, e.g., ICD and costamere components. [0093] Although differentiation of human pluripotent stem cells into cardiomyocytes has been described in the prior art, the cells described thus far exhibit immature phenotypes that resemble fetal cardiomyocytes and therefore exhibit functional deficiencies such as electrophysiology defects. Despite tremendous progress in promoting iPSC-derived cardiomyocyte maturation by tissue engineering-based methods, there remains a bottleneck that impedes the use of iPSC-derived cardiomyocytes for therapeutic purposes. Myocardial infarction, marked by the loss of cardiomyocytes is mediated by diverse mechanisms and molecules including TNF receptors (p55 and p75), NGF receptor, TWEAK receptor, TRAIL receptor, Fas receptor, Apo2, caspase 3, mitochondria-initiated signals, caspase 8, caspase 9, apoptosis, necrosis, ferroptosis, pyroptosis, NLRP3 activation, and signals through innate immune receptors including RIG1, STING, and toll-like receptors (e.g., TLR2, TLR3, TLR5, TLR7, TLR8 and TLR9). To overcome the limitations of the prior art, there is a need for novel methods for generating functional cardiomyocytes from pluripotent stem cells to replace or regenerate cardiomyocytes that have succumbed to these cellular damage or death mechanisms. [0094] The present invention teaches methods differentiation of pluripotent stem cells such as iPSCs into cardiomyocyte progenitor cells and/or cardiomyocytes that are useful for treating a cardiovascular disease such as myocardial infarction or heart failure in a subject in need thereof. The invention provides methods for producing cardiomyocyte progenitors and/or cardiomyocytes that are viable and functional upon administration to a subject. Disclosed herein are cell compositions and culture systems comprising support cells that provide one or a plurality of growth factors, angiogenic factors, and antiapoptotic factors that contribute to differentiation of functional therapeutic cell populations. IMMORTA-HEART-PCT [0095] Embodiments of the invention provide compositions of cell culture media for differentiating a pluripotent stem cell into a cardiomyocyte. In certain embodiments, one or a plurality of support cells or scaffolds are provided for culturing an iPSC and for its stepwise differentiation into a cardiomyocyte. In one embodiment, the one or plurality of support cells or scaffolds comprise a modified cell culture substrate or surface, wherein the modified cell culture substrate comprises one or a plurality of factors selected from the group consisting of: growth factors, cytokines, anti-inflammatory molecules, and/or exosomes. In one embodiment, pluripotent stem cells such as iPSCs are plated on the modified cell culture substrate in culture medium until reaching confluency of at least 75%, wherein the modified cell culture substrate comprises one or a plurality of the following components: decellularized cardiac tissue, hyaluronic acid, fibronectin, vitronectin, collagen II, and collagen IV. In one embodiment, decellularized cardiac tissue comprises an extracellular matrix, wherein the extracellular matrix comprises structural and functional molecules from cardiac tissue, and wherein the molecules comprise proteins, glycosaminoglycans, proteoglycans, and other components. In one embodiment, the decellularized cardiac tissue comprises one or a plurality of the following molecules or factors: thrombospondin, tenascin, versican, syndecan, biglycan, hyaluronan, heparan sulfate, collagen, fibronectin, elastin, and laminin. These embodiments provide an iPSC population that is suitable for subsequent differentiation steps to produce a population of cardiomyocytes. [0096] Embodiments of the invention provide cell culture systems comprising a cell culture medium that is suitable for supporting differentiation of a pluripotent stem cell into a cardiomyocyte. In one embodiment, a method for differentiating cultured iPSCs into cardiomyocytes is provided, the method comprising: a) exposing the iPSCs to a first growth medium comprising a Wnt activator, wherein the iPSCs are induced to differentiate into mesoderm cells; b) exposing the population of mesoderm cells from (a) to a second growth medium comprising a Wnt inhibitor, wherein the mesoderm cells are induced to differentiate into cardiac mesoderm cells; and c) exposing the population of cardiac mesoderm cells from (b) to a third growth medium comprising insulin, wherein the cardiac mesoderm cells are induced to differentiate into cardiomyocytes. In certain embodiments, steps (a) through (c) may be performed using various types of culture media to ensure the viability and function of differentiated cell types. [0097] In certain embodiments, the differentiation of cardiomyocytes in a cell culture system may be confirmed by various methods known in the art, for example, by determining a cardiomyocyte electrophysiological profile, by determining responsiveness to known IMMORTA-HEART-PCT cardioactive drugs, by contacting the cell population with an antibody specific for a cardiomyocyte-specific molecule, and determining the percentage of cells positive for expression or the levels of expression of the molecule, or by other methods known in the art. [0098] In one embodiment, a cell culture medium for differentiation of iPSCs into cardiomyocytes comprises an animal component-free culture medium is utilized which consists of Dulbecco's Modified Eagle Medium F-12 comprising one or a plurality of the following factors: L-ascorbic acid-2-phosphate magnesium, sodium, selenium, fibroblast growth factor 2 (FGF2), insulin, sodium bicarbonate, transferrin, and transforming growth factor beta 1 (TGFβ1). In one embodiment, the animal component-free culture medium comprises an inhibitor of Rho- associated, coiled-coil containing protein kinase (ROCK). In certain embodiments, an inhibitor of ROCK is provided at a concentration between 1 μM and 120 μM, between 2 μM and 50 μM, between 4 μM and 40 μM, between 8 μM and 12 μM, or a concentration or range defined by any two of these values. In certain embodiments, an inhibitor of ROCK is provided at a concentration that is sufficient to induce the upregulation of one or a plurality of the following growth factors in a cultured cell: FGF-1, FGF-2, and FGF-5, and wherein the expression levels of the one or plurality of growth factors in a cultured cell is increased by at least 25%, at least 50%, or at least 100% as measured by methods known to one of ordinary skill in the art. [0099] In another embodiment, a cell culture medium for differentiation of iPSCs into cardiomyocytes comprises a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is selected from the group consisting of: sulforaphane, valproic acid, phenylbutyrate, sodium phenylbutyrate, trichostatin A, or a combination thereof. [0100] In a specific embodiment, a method for differentiating iPSCs into mesoderm cells is provided, wherein the method comprises culturing the iPSCs in a cell culture medium comprising a Wnt activator, wherein the Wnt activator comprises CHIR99021, and wherein CHIR99021 is provided at a concentration between 10 μM and 14 μM. In certain embodiments, the duration of cell culture is between 18 and 30 hours for differentiation of iPSCs into mesoderm cells. [0101] In another specific embodiment, a method for differentiating mesoderm cells into cardiac mesoderm cells is provided, wherein the method comprises culturing the mesoderm cells in a cell culture medium comprising a Wnt inhibitor, wherein the Wnt inhibitor comprises XAV939, and wherein XAV939 is provided at a concentration between 1.5 μM and 2.5 μM. In certain embodiments, the duration of cell culture is between 48 and 100 hours for differentiation of mesoderm cells into cardiac mesoderm cells. [0102] In yet another embodiment, a method for differentiating cardiac mesoderm cells into cardiomyocytes is provided, wherein the method comprises culturing the cardiac mesoderm IMMORTA-HEART-PCT cells in a cell culture medium comprising insulin. In certain embodiments, the duration of cell culture is between 5 and 15 days for differentiation of cardiac mesoderm cells into cardiomyocytes. [0103] In certain embodiments, a method of the invention employs embryoid body formation as a platform for differentiation of a pluripotent stem cell into a cardiac lineage cell such as a cardiac mesoderm cell or a cardiomyocyte. In certain embodiments, iPSCs are cultured in suspension, allowing the iPSCs to form spherical embryoid bodies, and a culture medium is provided that is suitable for inducing differentiation into cardiac progenitor cells or cardiomyocytes. Considerations in embryoid body formation include the cell density and the growth medium that is selected, as can be determined by one of ordinary skill in the art. In certain embodiments, an embryoid body comprises iPSCs that undergo differentiation into cardiac lineage cells. In one embodiment, an embryoid body is dissociated to obtain an iPSC population. In one embodiment, iPSCs derived from an embryoid body are cultured in a culture medium comprising one or a plurality of the following factors: decellularized cardiac tissue, hyaluronic acid, fibronectin, vitronectin, collagen II, and collagen IV. In one embodiment, iPSCs from an embryoid body are cultured in a culture medium comprising one or a plurality of the following factors: ascorbic acid-2-phosphate magnesium, sodium, selenium, fibroblast growth factor 2 (FGF2), insulin, sodium bicarbonate, transferrin, and transforming growth factor beta 1 (TGFβ1), an inhibitor of ROCK, a Wnt inhibitor, a Wnt activator, and a histone deacetylase inhibitor. [0104] In yet another embodiment, a cell culture system for differentiation of iPSCs into cardiomyocytes comprises one or a plurality of cell types for enhancing the differentiation process, wherein the cell types may include lymphocytes, connective tissue cells, multipotent stem cells, or combinations thereof that may be selected based on their capacity to produce growth factors and/or anti-inflammatory factors. In a specific embodiment, a cell type for inclusion in a cell culture system of the invention is selected from the group consisting of a monocyte, a fibroblast, a macrophage, a mesenchymal stem cell, or combinations thereof. In one embodiment, various concentrations of prostaglandin E2 (PGE2) are provided in a cell culture system, preferably with at least one cell type. In one embodiment, PGE2 is added to a cell culture system to induce the production of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), and epidermal growth factor (EGF) by cell types such as monocytes, fibroblasts, or co-cultured cells. In a specific embodiment, the invention provides cell cultures comprising macrophages having an “M2 phenotype” cells that synergize with fibroblasts and endow said fibroblasts with the ability to induce cardiac regeneration, wherein IMMORTA-HEART-PCT the M2 phenotype classically refers to a macrophage that is CD163+ and CD206+, produces IL- 10, TFG-β, and low levels of pro-inflammatory cytokines, and promotes angiogenesis and tissue remodeling. [0105] In one embodiment, a kit for practicing the methods of the invention is disclosed, wherein the kit comprises an animal-component-free cell culture medium (e.g., Dulbecco's Modified Eagle Medium F-12,) wherein the cell culture medium comprises one or a plurality of the following components: L-ascorbic acid-2-phosphate magnesium, sodium selenium, fibroblast growth factor 2 (FGF2), sodium bicarbonate, insulin, transferrin, and transforming growth factor beta 1 (TGFβ1). In certain embodiments, the kit comprises one or a plurality of the following proteins or compounds or molecules: a Wnt activator (e.g., CHIR99021), a Wnt inhibitor (e.g., XAV939), insulin, a ROCK inhibitor, and a histone deacetylase inhibitor. [0106] The invention also provides methods for treating a subject with myocardial infarction by administration of cardiomyocytes generated using the methods of the invention to the subject. In certain embodiments, a method of treatment comprises administering a cardiomyocyte comprising an exosome to a subject with myocardial infarction. In yet another embodiment, a method of treatment comprises administering an exosome derived from a cell culture medium described herein to a subject in need thereof. In specific embodiments, exosomes are derived and isolated from one or a plurality of following cultured cell types: a) cardiac progenitor cells (i.e., cardiac mesoderm cells); b) cardiomyocytes; c) fibroblasts; d) monocytes; e) macrophages; and f) mesenchymal stem cells. In certain embodiments, exosomes are isolated from a cell type that has been cultured with PGE2 in combination with one or a plurality of other growth factors. In certain embodiments, an exosome is administered to subject to enhance viabilty and function of an engrafted cardiomyocyte. [0107] With respect to the methods of treatment of a subject, administration of cardiomyocytes is not intended to be limited to a particular mode of administration, dose, or frequency of administration. In certain embodiments, a cardiomyocyte or another cell type is adminisered to a subject by a subcutaneous, intramuscular, intravenous, or intramyocardial route (i.e., adminsitration into the cardiac tissue), or any other route sufficient to provide appropriate dosing to prevent or treat the disease. Cardiomyocytes can be administered to a subject in a single dose or multiple doses. When multiple administrations of cardiomyocytes are performed, the dosages can be spaced or performed at specific intervals, e.g., at 1 week, 1 month, and 2 months, or at daily, weekly, or monthly intervals. One or more growth factors, cytokines, small molecules, or other cells are also administered before, during, or after administration of the cells, further biasing those cells to a specific cell type toward a specific IMMORTA-HEART-PCT function or phenotype or to enhance the viability of the engrafted cells. In embodiments involving administrations of other cell types, similar flexibility in dosing regimens and routes of administration is contemplated. [0108] In one embodiment, the invention teaches the extraction of exosomes from cultures of cardiomyocytes generated using the methods of the invention, concentration of said exosomes, and administration of said exosomes to a subject for the purpose of stimulating angiogenesis. Without being restricted to mechanism, exosomes produced in the cell culture systems described herein may stimulate angiogenesis by acting as mitogens for endothelial cells, and/or by inducing production of angiogenic cytokines in cells of the body upon administration to the subject. In certain embodiments, a method of treatment of myocardial infarction comprises administration of cardiomyocytes and exosomes derived from one or a combination of cell types to a subject in need thereof. In certain embodiments, the cardiomyocytes and/or exosomes derived from a cell culture system of the invention and administered to a subject are autologous. Specific embodiments of the invention provide autologous cardiomyocytes that induce myocardial regeneration in an animal model or in a human subject post-myocardial infarction. Other embodiments provide autologous cardiomyocytes that can be administered to a subject preserve cardiac performance post-myocardial infarction in a subject. [0109] In one embodiment, a subject with myocardial infarction is provided with a therapeutic cell population comprising cardiomyocytes or cardiac progenitor cells alone or in combination with one or a plurality of other cell types selected from the group consisting of: a) fibroblasts; b) monocytes; c) macrophages (e.g., M2-type macrophages); and d) mesenchymal stem cells. In a specific embodiment, iPSC-derived progenitor cells are administered alone, or in combination with mesenchymal stem cells to augment regeneration of myocardium in a subject in need thereof. In one embodiment, prior to, concurrent with, and/or subsequent to administration of cells, extracorporeal shock wave therapy is utilized to augment regenerative activity of the cells prior to, concurrent with, and/or subsequent to administration of iPSC- derived progenitor cells to the subject. [0110] In one embodiment, a method of treating myocardial infarction is provided, the method comprising: a) obtaining a pluripotent stem cell population; b) differentiating said pluripotent stem cell into a cardiac-lineage cell such as a cardiomocyte or a cardiomyocyte progenitor cell using a method of the invention; c) implanting said cardiac-lineage cell into cardiac tissue; and optionally, d) providing adjuvant cells and/or chemicals to enhance viability and function of said engrafted cardiac-lineage cell. IMMORTA-HEART-PCT [0111] Embodiments of the invention involve monitoring a subject for parameters of cardiac function prior to and following application of the methods of the invention to the subject. By way of example, quantification of infarct size can be performed on the subject using methods known in the art (e.g., infarct size volume by MRI). In certain embodiments, selection of a subject for application of the compositions and methods of the invention may be performed my measuring myocardial infarction by one or a plurality of the following tests or methods: a) Biomarkers such as Troponin tests; b) 2-D and 4-D echocardiography; c) Cardiovascular magnetic resonance; d) EGC; and e) Myocardial Infarction Dimensional Assessment Scale (MIDAS). One or more of these tests are applicable for monitoring the response to treatment of a subject with a method of the invention. [0112] Methods are provided for isolating exosomes that are useful for treating a subject with myocardial infarction. In one embodiment, a strong or weak, preferably strong, anion exchange may be performed for isolation of exosomes from a cell culture system. In a specific embodiment, the chromatography is performed under pressure. Thus, more specifically, it may consist of high-performance liquid chromatography (HPLC). Different types of supports may be used to perform the anion exchange chromatography. More preferably, these may include cellulose, poly(styrene-divinylbenzene), agarose, dextran, acrylamide, silica, ethylene glycol- methacrylate co-polymer, or mixtures thereof, e.g., agarose-dextran mixtures. In a specific embodiment, this invention relates to a method of preparing exosomes from a culture of cells containing cardiomyocytes, comprising at least one step during which the biological sample is treated by anion exchange chromatography on a support selected from cellulose, poly(styrene- divinylbenzene), silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer, alone or in mixtures, optionally functionalized. In addition, to improve the chromatographic resolution, within the scope of the invention, it is preferable to use supports in bead form. Ideally, these beads have a homogeneous and calibrated diameter, with a sufficiently high porosity to enable the penetration of the objects under chromatography (i.e., the exosomes). In this way, given the diameter of exosomes (generally between 50 and 100 nm), to apply the invention, it is preferable to use high porosity gels, particularly between 10 nm and 5 microns, more preferably between approximately 20 nm and approximately 2 microns, even more preferably between about 100 nm and about 1 micron. For the anion exchange chromatography, the support used must be functionalized using a group capable of interacting with an anionic molecule. Generally, this group is composed of an amine which may be ternary or quaternary, which defines a weak or strong anion exchanger, respectively. Within the scope of this invention, it is particularly advantageous to use a strong anion exchanger. In this way, according to the IMMORTA-HEART-PCT invention, a chromatography support as described above, functionalized with quaternary amines, is used. Therefore, according to a more specific embodiment of the invention, the anion exchange chromatography is performed on a support functionalized with a quaternary amine. Even more preferably, this support should be selected from poly(styrene-divinylbenzene), acrylamide, agarose, dextran and silica, alone or in mixtures, and functionalized with a quaternary amine. Examples of supports functionalized with a quaternary amine include the gels SOURCEQ. MONO Q, Q SEPHAROSE.RTM., POROS.RTM. HQ and POROS.RTM. QE, FRACTOGEL.RTM.TMAE type gels and TOYOPEARL SUPER.RTM.Q gels. [0113] A particularly preferred support to perform the anion exchange chromatography comprises poly(styrene-divinylbenzene). An example of this type of gel which may be used within the scope of this invention is SOURCE Q gel, particularly SOURCE 15 Q (Pharmacia). This support offers the advantage of very large internal pores, thus offering low resistance to the circulation of liquid through the gel, while enabling rapid diffusion of the exosomes to the functional groups, which are particularly important parameters for exosomes given their size. The biological compounds retained on the column may be eluted in different ways, particularly using the passage of a saline solution gradient of increasing concentration, e.g., from 0 to 2 M. A sodium chloride solution may particularly be used, in concentrations varying from 0 to 2 M, for example. The different fractions purified in this way are detected by measuring their optical density (OD) at the column outlet using a continuous spectro-photometric reading. As an indication, under the conditions used in the examples, the fractions comprising the exosomes are eluted at an ionic strength comprised between approximately 350 and 700 mM, depending on the type of vesicles. Different types of columns may be used to perform this chromatographic step, according to requirements and the volumes to be treated. It is understood that higher volumes may also be treated, by increasing the volume of the column, for example. In addition, to perform this invention, it is also possible to combine the anion exchange chromatography step with a gel permeation chromatography step. In this way, according to a specific embodiment of the invention, a gel permeation chromatography step is added to the anion exchange step, either before or after the anion exchange chromatography step. Preferably, in this embodiment, the permeation chromatography step takes place after the anion exchange step. In addition, in a specific variant, the anion exchange chromatography step is replaced by the gel permeation chromatography step. The exosomes may also be purified using gel permeation liquid chromatography, particularly when this step is combined with an anion exchange chromatography or other treatment steps of the biological sample, as described in detail below. To perform the gel permeation chromatography step, a support selected from IMMORTA-HEART-PCT silica, acrylamide, agarose, dextran, ethylene glycol-methacrylate co-polymer or mixtures thereof, e.g., agarose-dextran mixtures, are preferably used. [0114] In addition, according to a preferred embodiment of the invention, the biological sample is treated, prior to the chromatography step, to be enriched with exosomes (enrichment stage). In this way, in a specific embodiment, this invention relates to a method of preparing exosomes from a biological sample, characterized in that it comprises at least: b) an enrichment step, to prepare a sample enriched with exosomes, and c) a step during which the sample is treated by anion exchange chromatography and/or gel permeation chromatography. [0115] In one embodiment, the cell culture supernatant treated so as to be enriched with exosomes. In particular, the cell culture supernatant may comprise a population of exsosome- producing cells that can be enriched or purified using techniques such as centrifugation, clarification, ultrafiltration, nanofiltration and/or affinity chromatography, particularly with clarification and/or ultrafiltration and/or affinity chromatography. Therefore, a preferred method of preparing exosomes according to this invention more particularly comprises the following steps: a) culturing a population of exosome-producing cells under conditions enabling the release of vesicles, b) a step of enrichment of the sample in exosomes, and c) an anion exchange chromatography and/or gel permeation chromatography treatment of the sample. [0116] As indicated above, the supernatant enrichment step may comprise one or more centrifugation, clarification, ultrafiltration, nanofiltration and/or affinity chromatography steps on the supernatant. In one embodiment, the enrichment comprises the elimination of cells and/or cell debris (clarification), possibly followed by a concentration and/or affinity chromatography step. In another specific embodiment, the enrichment step comprises an affinity chromatography step, optionally preceded by a step of elimination of cells and/or cell debris (clarification). A preferred enrichment step comprises elimination of cells and/or cell debris (clarification), a concentration step, followed by affinity chromatography. The cells and/or cell debris may be eliminated by centrifugation of the sample, for example, at a low speed, preferably below 1000 g, between 100 and 700 g, for example. Preferred centrifugation conditions during this step are approximately 300 g or 600 g for a period between 1 and 15 minutes, for example. The cells and/or cell debris may also be eliminated by filtration of the sample, possibly combined with the centrifugation described above. The filtration may particularly be performed with successive filtrations using filters with a decreasing porosity. For this purpose, filters with a porosity above 0.2 microns, e.g., between 0.2 and 10 microns, are preferentially used. It is particularly possible to use a succession of filters with porosities of 10 microns, 1 micron, 0.5 microns and 0.22 microns. A concentration step may also be performed, IMMORTA-HEART-PCT in order to reduce the volumes of sample to be treated during the chromatography stages. In this way, the concentration may be obtained by centrifugation of the sample at high speeds, e.g., between 10,000 and 100,000 g, to cause the sedimentation of the exosomes. This may consist of a series of differential centrifugations, with the last centrifugation performed at approximately 70,000 g. The exosomes in the pellet obtained may be taken up with a smaller volume and in a suitable buffer for the subsequent steps of the process. The concentration step may also be performed by ultrafiltration. In fact, this ultrafiltration allows both to concentrate the supernatant and perform an initial purification of the vesicles. According to one embodiment, the biological sample (e.g., the supernatant) is subjected to an ultrafiltration, preferably a tangential ultrafiltration. Tangential ultrafiltration consists of concentrating and fractionating a solution between two compartments (filtrate and retentate), separated by membranes of determined cut-off thresholds. The separation is carried out by applying a flow in the retentate compartment and a transmembrane pressure between this compartment and the filtrate compartment. Different systems may be used to perform the ultrafiltration, such as spiral membranes, flat membranes or hollow fibers. Within the scope of the invention, the use of membranes with a cut-off threshold below 1000 kDa, preferably between 300 kDa and 1000 kDa, or even more preferably between 300 kDa and 500 kDa, is advantageous. The affinity chromatography step can be performed in various ways, using different chromatographic support and material. It is advantageously a non-specific affinity chromatography, aimed at retaining (i.e., binding) certain contaminants present within the solution, without retaining the objects of interest (i.e., the exosomes). Preferably, an affinity chromatography on a dye is used, allowing the elimination (i.e., the retention) of contaminants such as proteins and enzymes, for instance albumin, kinases, dehydrogenases, clotting factors, interferons, lipoproteins, or also co- factors, etc. More preferably, the support used for this chromatography step is a support as used for the ion exchange chromatography, functionalized with a dye. The support is more preferably agarose. It should be understood that any other support and/or dye or reactive group allowing the retention (binding) of contaminants from the treated biological sample can be used in the instant invention. [0117] In one embodiment, an exosome preparation process within the scope of this invention comprises the following steps: a) the culture of a population of exosome-producing cells under conditions enabling the release of vesicles; b) the treatment of the culture supernatant with at least one ultrafiltration or affinity chromatography step, to produce a biological sample enriched with exosomes; and c) an anion exchange chromatography and/or gel permeation chromatography treatment of the biological sample. In a preferred embodiment, IMMORTA-HEART-PCT step b) above comprises a filtration of the culture supernatant, followed by an ultrafiltration, preferably tangential. In another preferred embodiment, step b) above comprises a clarification of the culture supernatant, followed by an affinity chromatography on dye. In addition, after step c), the material harvested may, if applicable, be subjected to one or more additional treatment and/or filtration stages d), particularly for sterilization purposes. For this filtration treatment stage, filters with a diameter less than or equal to 0.3 microns are preferentially used, or even more preferentially, less than or equal to 0.25 microns. After step d), the material obtained is, for example, distributed into suitable devices such as bottles, tubes, bags, syringes, etc., in a suitable storage medium. The purified vesicles obtained in this way may be stored cold, frozen or used extemporaneously. Therefore, a specific preparation process within the scope of the invention comprises at least the following steps: c) an anion exchange chromatography and/or gel permeation chromatography treatment of the biological sample, and d) a filtration step, particularly sterilizing filtration, of the material harvested after stage c). In a first variant, the process according to the invention comprises: c) an anion exchange chromatography treatment of the biological sample, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). In another embodiment, the process according to the invention comprises: c) a gel permeation chromatography treatment of the biological sample, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). According to a third variant, the process according to the invention comprises: c) an anionic exchange treatment of the biological sample followed or preceded by gel permeation chromatography, and d) a filtration step, particularly sterilizing filtration, on the material harvested after step c). [0118] In one embodiment, therapeutic factors for stimulating angiogenesis are derived from tissue culture that may contain exosomes or may not contain exosomes but contain factors capable of stimulating angiogenesis. Culture conditioned media may be concentrated by filtering/desalting means known in the art including use of Amicon filters with specific molecular weight cut-offs, said cut-offs may select for molecular weights higher than 1 kDa to 50 kDa. Supernatant may alternatively be concentrated using means known in the art such as solid phase extraction using C18 cartridges that are used to adsorb small hydrophobic molecules from cell culture supernatant and allows for the elimination of salts and other polar contaminants. It may be desirable to use other adsorption means in order to purify certain compounds from said supernatant. Said concentrated supernatant may be assessed directly for biological activities useful for the practice of this invention or may be further purified. Further purification may be performed using, for example, gel filtration using a Bio-Gel P-2 column. Cell supernatant IMMORTA-HEART-PCT concentrates extracted by C18 cartridge may be dissolved in 0.5 ml of 20 mM Tris buffer, pH 7.2 and run through the column. Fractions may be collected from the column and analyzed for biological activity. Other purification, fractionation, and identification means are known to one skilled in the art and include anionic exchange chromatography, gas chromatography, high performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. Administration of supernatant active fractions may be performed locally or systemically for treatment of a subject in need thereof.

Claims

IMMORTA-HEART-PCT Claims 1. A method for treating myocardial infarction in a subject, the method comprising: a) obtaining a pluripotent stem cell population; b) differentiating the pluripotent stem cell population into cardiac-lineage cells in cell culture; and c) implanting the cardiac-lineage cells into cardiac tissue of the subject. 2. The method of Claim 1, wherein the cardiac-lineage cell is a cardiomyocyte. 3. The method of Claim 1, wherein the cardiac-lineage cell is a cardiac progenitor cell. 4. The method of Claim 1, wherein the pluripotent stem cell is an induced pluripotent stem cell. 5. The method of Claim 1, wherein the pluripotent stem cell is obtained from an embryoid body. 6. The method of Claim 5, wherein the pluripotent stem cell from an embryoid body is cultured in a culture medium comprising one or a plurality of the following factors: L-ascorbic acid-2- phosphate; b) magnesium; c) sodium; d) selenium; e) fibroblast growth factor 2 (FGF); f) insulin; g) sodium bicarbonate; h) transferrin; and i) transforming growth factor beta 1 (TGFβ1). 7. The method of Claim 1, wherein the pluripotent stem cell is cultured on a modified cell culture substrate comprising one or a plurality of the following factors: a) decellularized cardiac tissue; b) hyaluronic acid; c) fibronectin; d) vitronectin; e) collagen II; and f) collagen IV. 8. The method of Claim 7, wherein the culture comprises an inhibitor of Rho-associated, coiled- coil containing protein kinase (ROCK). 9. The method of Claim 8, wherein the inhibitor of ROCK is added to the culture at a concentration between 1 μM and 120 μM. IMMORTA-HEART-PCT 10. The method of Claim 7, wherein the culture comprises a histone deacetylase inhibitor. 11. The method of Claim 10, wherein the histone deacetylase inhibitor comprises one or a plurality of the following compounds: a) sulforaphane; b) valproic acid; c) phenylbutyrate; d) sodium phenylbutyrate; and e) trichostatin A. 12. The method of Claim 1 further comprising providing an exosome population to the subject to enhance viability and function of the cardiac-lineage cell, wherein the exosome population is derived form one or a plurality of the following cell types: a) cardiac progenitor cells, (e.g., cardiac mesoderm cells); b) cardiomyocytes; c) fibroblasts; d) macrophages; and e) mesenchymal stem cells. 13. The method of Claim 1, wherein the differentiation of the pluripotent stem cell into a cardiomyocyte comprises the follow steps: a) exposing the iPSC to a first growth medium comprising a Wnt activator, wherein the iPSC is induced to differentiate into a mesoderm cell; b) exposing the mesoderm cell from (a) to a second growth medium comprising a Wnt inhibitor, wherein the mesoderm cell is induced to differentiate into a cardiac mesoderm cell; and c) exposing the cardiac mesoderm cells from (b) to a third growth medium comprising insulin, wherein the cardiac mesoderm cell is induced to differentiate into a cardiomyocyte. 14. The method of Claim 13, wherein the Wnt activator comprises CHIR99021 15. The method of Claim 14, wherein CHIR99021 is provided at a concentration between 10 μM and 14 μM . 16. The method of Claim 13, wherein the Wnt inhibitor comprises XAV939. 17. The method of Claim 16, wherein XAV939 is provided at a concentration between 1.5 μM and 2.5 μM. 18. The method of Claim 1, wherein the cell culture comprises one or a plurality of additional cell types for enhancing the differentiation into a cardiomyocyte. 19. The method of Claim 18, wherein the one or plurality of additional cell types are selected from the group consisting of: a) monocytes; b) fibroblasts c) macrophages; and d) mesenchymal stem cells. IMMORTA-HEART-PCT 20. The method of Claim 19, wherein the macrophages are M2 macrophages.