WO2010141877A1

WO2010141877A1 - Method of treating stem cells

Info

Publication number: WO2010141877A1
Application number: PCT/US2010/037487
Authority: WO
Inventors: Tarun Chakravarty; Christian B. Allan
Original assignee: Cedars Sinai Medical Center; RegeneRx Biopharmaceuticals Inc
Current assignee: Cedars Sinai Medical Center; RegeneRx Biopharmaceuticals Inc
Priority date: 2009-06-04
Filing date: 2010-06-04
Publication date: 2010-12-09
Anticipated expiration: 2011-12-04

Abstract

A method of treatment for enhancing stem cells includes providing a population of stem cells in vitro and contacting the population of stem cells with a stem cell-enhancing amount of the polypeptide agent thymosin beta 4 (Tβ4).

Description

METHOD OF TREATING STEM CELLS

1. Background of the Invention

2. Cross-Reference to Related Applications

[001] This application claims benefit of U.S. provisional application No. 61/184,247 filed on June 4, 2009, all of which is incorporated herein by reference in its entirety for all purposes.

3. Field of the Invention

[002] The present invention relates to the field of treating stem cells.

4. Description of the Background Art

[003] Stem cell therapy is a promising therapeutic treatment which sometimes involves extracting stem cells from tissue of a subject, culturing the stem cells in vitro to form an enlarged population of stem cells, and then introducing the enlarged population of stem cells back into the subject's tissue. [004] Heart disease is a leading cause of death in newborns and in adults. [005] Coronary artery disease results in acute occlusion of cardiac vessels leading to loss of dependent myocardium. Such events are one of the leading causes of death in the Western world. Because the heart is incapable of sufficient muscle regeneration, survivors of myocardial infarctions typically develop chronic heart failure with over ten million cases in the United States alone. While more commonly affecting adults, heart disease in children is the leading non-infectious cause of death in the first year of life and often involves abnormalities in cardiac cell specification, migration or survival.

[006] There are many causes of myocardial and coronary vessel and tissue injuries, including but not limited to myocardial ischemia, clotting, vessel occlusion, infection, developmental defects or abnormalities and other such myocardial events. Myocardial infarction results from blood vessel disease in the heart. It occurs when the blood supply to part of the heart is reduced or stopped (caused by blockage of a coronary artery, as one example). The reduced blood supply causes injuries to the heart muscle cells and may even kill heart muscle cells. The reduction in blood supply to the heart is often caused by narrowing of the epicardial blood vessels due to plaque. These plaques may rupture causing hemorrhage, thrombus formation, fibrin and platelet accumulation and constriction of the blood vessels.

[007] Recent evidence suggests that a population of extracardiac or intracardiac stem cells may contribute to maintenance of the cardiomyocyte population under normal circumstances. Efforts to promote cardiac repair by introduction or recruitment of exogenous stem cells hold promise but typically involve isolation and introduction of autologous or donor progenitor cells. While the stem cell population may maintain a delicate balance between cell death and cell renewal, it is insufficient for myocardial repair after acute coronary occlusion. Introduction of isolated stem cells may improve myocardial function, but this approach has been controversial, and requires isolation of autologous stem cells or use of donor stem cells along with immunosuppression. Efforts to coax pluripotent embryonic stem cells into a cardiomyocyte lineage remain unsuccessful. Technical hurdles of stem cell delivery and differentiation have thus far prevented broad clinical application of cardiac regenerative therapies.

[008] There remains a need in the art for improved methods for enhancing stem cells.

SUMMARY OF THE INVENTION

[009] In accordance with one aspect of the present invention, a method of treatment for enhancing stem cells comprises providing a population of stem cells in vitro and contacting said population of stem cells in vitro with a stem cell- enhancing amount of a stem cell-enhancing polypeptide agent comprising, consisting essentially of or consisting of thymosin beta 4 (Tβ4), so as to form enhanced stem cells. In certain embodiments the stem cells are cardiac stem cells. [0010] In accordance with one aspect of the present invention, a method of treatment for enhancing cardiac stem cells comprises providing a population of cardiac stem cells in vitro and contacting said population of cardiac stem cells with a stem cell-enhancing amount of a stem cell-enhancing polypeptide agent comprising, consisting essentially of or consisting of thymosin beta 4 (Tβ4), so as to form enhanced cardiac stem cells. In certain embodiments the contacting can be in vitro, or in vivo in a subject to be treated. [0011] In accordance with another aspect, the invention is a method of treatment for treating, at least partly preventing, inhibiting, or reducing damage to a subject's tissue, or for promoting growth of a subject's tissue, comprising providing a population of stem cells in vitro and contacting the population of stem cells in vitro with a stem cell enhancing-amount of a stem cell-enhancing polypeptide agent comprising, consisting essentially of or consisting of thymosin beta 4 (Tβ4), so as to form enhanced stem cells, and then administering the enhanced stem cells to the subject's tissue to treat, prevent, inhibit or reduce damage to the subject's tissue, or to promote growth of the subject's tissue. In certain embodiments, an isolated population of stem cells can be administered to tissue of a subject along with a polypeptide agent as described herein, separately or concurrently therewith, so as to enhance the stem cells in vivo, and thereby affect the subject's tissue according to the invention.

[0012] In accordance with another aspect, the invention is a method of treatment for treating, at least partly preventing, inhibiting, or reducing damage to coronary tissue, or for promoting growth of cardiac tissue, comprising providing a population of cardiac stem cells in vitro and contacting the population of cardiac stem cells in vitro with a stem cell enhancing-amount of a stem cell-enhancing polypeptide agent comprising, consisting essentially of or consisting of thymosin beta 4 (Tβ4), so as to provide enhanced cardiac stem cells, and then administering the enhanced cardiac stem cells to a subject's coronary tissue to treat, prevent, inhibit or reduce damage to the coronary tissue, or to promote growth of the cardiac tissue. Alternatively, an isolated population of cardiac stem cells can be administered to cardiac tissue of a subject along with a polypeptide agent as described herein, separately or concurrently therewith, so as to enhance the stem cells in vivo, and thereby affect the coronary tissue according to the invention.

[0013] In certain embodiments, the stem cells are autologous to the subject's tissue. [0014] The invention also applies to enhanced stem cells as described herein.

DETAILED DESCRIPTION OF THE INVENTION

[0015] One embodiment of the present invention provides that damage to a subject's tissue (e.g., cardiac tissue) can be prevented, treated, inhibited or reduced, or growth of a subject's tissue can be promoted, by administering stem cells to the tissue after pretreating and enhancing the stem cells (herein sometimes referred to as "enhanced stem cells"), with a polypeptide agent as described herein, which agent has stem cell-enhancing activity of Tβφ

[0016] In certain procedures, enhancement of stem cells has involved contacting the stem cells with two or three different active agents, such as positive effectors, negative effectors and/or ancillary effectors. The term "positive effector" refers to a molecule capable of activating or otherwise enhancing or promoting cell proliferation, cell engraftment, cell migration, cell differentiation and/or cell cycle re-entry of differentiated cells. The term "negative effector" refers to a molecule which inhibits or otherwise reduces apoptotic cell death and inflammation related to tissue injury. The term "ancillary effector" refers to a molecule that can be used or administered in conjunction with a positive and/or a negative effector, which contributes to the beneficial treatment of injured tissue (e.g., cardiac tissue). Exemplary functions of an ancillary effector include, but are not limited to, facilitating the functions of positive and/or negative effectors or acting as an angiogenic agent, or facilitating cell-to-cell interaction or communication. Non- limiting examples of ancillary effectors include P38 MAP kinase inhibitors, phosphodiesterase inhibitors, stem cell factors and transforming growth factor (TGF) (e.g., TGFβ or TGFβ3). It surprisingly has been discovered that stem cells can be enhanced in vitro utilizing only a single active agent, a polypeptide agent in accordance with the present invention.

[0017] The invention also includes stem cells which have been enhanced by the methods described herein, and compositions containing same. [0018] In certain embodiments, the stem cells which are enhanced according to the present invention are autologous to the tissue of the subject being treated. [0019] According to certain embodiments of the invention, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% (or 1% or less) of a given value or range.

[0020] According to certain embodiments of the invention, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by, but not limited to, intramyocardial, pulmonary (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptom thereof, is being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0021] According to certain embodiments of the invention, the term "autologous" refers to organs, tissues, cells, fluids or other bioactive molecules that are reimplanted in the same individual that they originated from. Non-limiting examples of autologous transplants or grafts include bone, bone marrow, skin biopsy, heart biopsy, cartilage and blood and stem cells, e.g., CDCs. [0022] According to certain embodiments of the invention, the term "cardiac cells" refers to any cells present in the heart that provide a cardiac function, such as heart contraction or blood supply, or otherwise serve to maintain the structure of the heart. Cardiac cells as used herein encompass cells that exist in the epicardium, myocardium or endocardium of the heart. Cardiac cells also include, for example, cardiac muscle cells or cardiomyocytes; cells of the cardiac vasculatures, such as cells of a coronary artery or vein. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac conducting cells and cardiac pacemaking cells that constitute the cardiac muscle, blood vessels and cardiac cell supporting structure.

[0023] According to certain embodiments of the invention, the term "cardiac function" refers to the function of the heart, including global and regional functions of the heart. The term "global" cardiac function as used herein refers to function of the heart as a whole. Such function can be measured by, for example, stroke volume, ejection fraction, cardiac output, cardiac contractility, etc. The term "regional cardiac function" refers to the function of a portion or region of the heart. Such regional function can be measured, for example, by wall thickening, wall motion, myocardial mass, segmental shortening, ventricular remodeling, new muscle formation, the percentage of cardiac cell proliferation and programmed cell death, angiogenesis and the size of fibrous and infarct tissue. Cardiac cell proliferation may be assessed by the increase in the nuclei or DNA synthesis of cardiac cells, cell cycle activities or cytokinesis. Programmed cell death may be measured by TUNEL assay that detects DNA fragmentation. Angiogenesis may be detected by the increase in arteriolar and/or capillary densities. Techniques for assessing global and regional cardiac function are known in the art. For example, techniques that can be used to measure regional and global cardiac function include, but are not limited to, echocardiography (e.g., transthoracic echocardiogram, transesophageal echocardiogram or 3D echocardiography), cardiac angiography and hemodynamics, radionuclide imaging, magnetic resonance imaging (MRI), sonomicrometry and histological techniques. [0024] According to certain embodiments of the invention, the term "cardiac tissue" refers to tissue of the heart, for example, the epicardium, myocardium or endocardium, or portion thereof, of the heart. According to certain embodiments of the invention the term "injured" cardiac tissue refers to a cardiac tissue that is, for example, ischemic, infarcted, reperfused, or otherwise focally or diffusely injured or diseased. Injuries associated with a cardiac tissue include any areas of abnormal tissue in the heart, including any areas caused by a disease, disorder or injury and includes damage to the epicardiurn, endocardium and/or myocardium. Non- limiting examples of causes of cardiac tissue injuries include acute or chronic stress (e.g., systemic hypertension, pulmonary hypertension or valve dysfunction), atheromatous disorders of blood vessels (e.g., coronary artery disease), ischemia, infarction, inflammatory disease and cardiomyopathies or myocarditis. [0025] According to certain embodiments of the invention "engraftment" refers to a process by which transplanted stem cells (e.g., autologous stem cells) are accepted by a host tissue, survive and persist in that environment. In certain embodiments, the transplanted stem cells further reproduce.

[0026] According to certain embodiments of the invention, the terms "generate," "generation" and "generating" refer to the production of new tissue cells in a subject and optionally the further differentiation into mature, functioning tissue cells. In some embodiments, generation of tissue cells comprises regeneration of the tissue cells. In certain embodiments, generation of tissue cells comprises improving survival, engraftment and /or proliferation of the tissue cells. [0027] The term "peri-infarct zone" refers to an area at the junction between the normal tissue and the infarcted tissue, i.e., an area of a dying or dead heart tissue resulting from obstruction of blood flow to the heart muscle that results from a relative or absolute insufficiency of blood supply. In certain embodiments of the methods provided herein, the stem cells are administered into the peri-infarct zone of the cardiac tissue.

[0028] According to certain embodiments of the invention, the terms "preserve," "preservation of and "preserving" in the context of injured tissue refer to protection and/or maintenance of the subject's tissue, or the functions thereof, such that the tissue is not further injured or compromised, or that the rate of further injury or compromise is slowed relative to the rate in the absence of the intervention at issue. In certain embodiments, preserving injured subject tissue comprises prevention or reduction of apoptosis of cells (e.g., cardiomyocytes or stem cells). In certain embodiments, preserving injured tissue comprises prevention or reduction of cell inflammation.

[0029] According to certain embodiments of the invention the terms "regenerate," "regeneration" and "regenerating" in the context of injured tissue refer to the process of growing and/or developing new tissue in a subject (e.g., in a heart or cardiac tissue that has been injured, for example, injured due to ischemia, infarction, reperfusion, or other disease). In certain embodiments, cardiac tissue regeneration comprises activation and/or enhancement of cell proliferation. In certain embodiments, cardiac tissue regeneration comprises activation and/or enhancement of cell migration.

[0030] The term "stem cells" refers to cells that have the capacity to self-renew and to generate differentiated progeny. The term "pluripotent stem cells" refers to stem cells that has complete differentiation versatility, i.e., the capacity to grow into any of the fetal or adult mammalian body's approximately 260 cell types. For example, pluripotent stem cells have the potential to differentiate into three germ layers: endoderm (e.g., blood vessels), mesoderm (e.g., muscle, bone and blood) and ectoderm (e.g., epidermal tissues and nervous system), and therefore, can give rise to any fetal or adult cell type. The term "induced pluripotent stem cells" refers to differentiated mammalian somatic cells (e.g., adult somatic cells, such as skin) that have been reprogrammed to exhibit at least one characteristic of pluripotency. The term "multipotent stem cells" refers to a stem cell that has the capacity to grow into any subset of the fetal or adult mammalian body's approximately 260 cell types. For example, certain multipotent stem cells can differentiate into at least one cell type of ectoderm, mesoderm and endoderm gem layers. The term "embryonic stem cells" refers to stem cells derived from the inner cell mass of an early stage embryo, e.g., human, that can proliferate in vitro in an undifferentiated state and are pluripotent. The term "cardiac stem cells" refers to stem cells obtained from or derived from cardiac tissue. The term "cardiosphere-derived cells (CDCs)" as used herein refers to undifferentiated cells that grow as self-adherent clusters from subcultures of postnatal cardiac surgical biopsy specimens. CDCs can express stem cell as well as endothelial progenitor cell markers, and typically possess properties of adult cardiac stem cells. For example, human CDCs can be distinguished from human cardiac stem cells in that human CDCs typically do not express multidrug resistance protein i (MDRI; also known as ABCBi), CD45 and CD133 (also known as PROMi). See, e.g., Passier et al. (2008) Nature 453:322. CDCs are capable of long-term self-renewal, and can differentiate in vitro to yield cardiomyocytes or vascular cells after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse. See also U.S. Pub. No. 2008/0267921, which is herein incorporated by reference in its entirety. The term "bone marrow stem cells" refers to stem cells obtained from or derived from bone marrow. The term "placenta-derived stem cells" or "placental stem cells" refers to stem cells obtained from or derived from a mammalian placenta, or a portion thereof (e.g., amnion or chorion). The term "amniotic stem cells" refers to stem cells collected from amniotic fluid or amniotic membrane. The term "embryonic germ cells" refers to cells derived from primordial germ cells, which exhibit an embryonic pluripotent cell phenotype. The term "spermatocytes" refers to male gametocytes derived from a spermatogonium. [0031] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, rabbits, etc.) or a primate (e.g., monkey and human) having an injured tissue. In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal with acute heart failure. In another embodiment, the subject is a mammal with chronic heart failure. [0032] As used herein, the terms "treat," "treatment" and "treating" refer to the reduction or amelioration of the progression, severity, and/or duration of a tissue injury or a symptom thereof. Treatment as used herein includes, but are not limited to, preserving injured tissue, regenerating new tissue, increasing blood flow to the injured tissue, and may include at least one of increasing myocardial perfusion, improving global cardiac function (e.g., stroke volume, ejection fraction, and cardiac output) and regional cardiac function (e.g., ventricular wall thickening, segmental shortening and heart pumping).

[0033] In certain embodiments, a population of stem cells are pretreated (contacted) in vitro with a polypeptide agent as described herein for a time period of from about i hour to about 7 days, e.g., about 1-100 hours, about 10-80 hours, or about 40-80 hours. Alternatively, the contacting can be about 14-16 hours. The concentration of said polypeptide during said contacting may be about i-2oug/ml, e.g., about ioug/ml.

[0034] Certain embodiments of the invention are directed to increasing survival of, engraftment of or proliferation of stem cells in tissue of a subject. [0035] Certain embodiments include the steps of extracting stem cells from a subject's tissue, cultivating said stem cells as is known in the art so as to increase their number and form a population of stem cells, and then enhancing the stem cells in accordance with the invention. Alternatively, the stem cells can be cultivated and enhanced simultaneously. An enhanced population of stem cells then can be introduced into tissue of a subject.

[0036] In accordance with one aspect of the invention, a population of cells is made up of a substantial portion of stem cells, e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of all cells of a population being stem cells. [0037] Enhanced cardiac stem cells according to the invention may include cardiac stem cells with increased migration capabilities as compared to untreated stem cells, as well as an increase in a number of migrating cardiac stem cells in a population of cardiac stem cells as compared to an untreated population. [0038] Damage to coronary tissue may be treated, at least partly prevented, inhibited or reduced, or growth of cardiac tissue can be promoted by up-regulation of or increasing integrin linked kinase (ILK), up-regulation of or increasing protein kinase B (Akt), up-regulation of or increasing phosphatidylinositol 3-kinase (PI3K) activity, down-regulation of or reducing cardiomyocyte cell death and hibernation of cardiomyocytes, or a combination thereof. [0039] In accordance with one embodiment, stem cells which have been enhanced utilizing a polypeptide agent as described herein are administered to a subject in need of treatment. The subject may be a mammal, preferably human. [0040] In accordance with one embodiment, the polypeptide agent is thymosin β4 (Tβ4 or TB4). Thymosin β4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin β4 was originally isolated from the thymus and is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Several roles have been ascribed to this protein including a role in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration and vascularization. [0041] In certain embodiments, the invention is applicable to stem cell- enhancing polypeptides that are functional equivalents of Tβ4 which may include Tβ4 isoforms, analogues or derivatives having stem cell-enhancing activity of Tβ4, oxidized Tβ4, an N-terminal variant of Tβ4 having stem cell-enhancing activity of Tβ4, a C-terminal variant of Tβ4 having stem cell-enhancing activity of Tβ4, Tβ^ala, Tβ9, Tβio, Tβn, Tβi2, Tβi3, Tβi4, Tβi5, gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adservertin, propomyosin, fincilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, β-actinin and acumentin having stem cell enhancing activity of Tβ4, and which perform substantially the same function as Tβ4, in substantially the same way, to achieve substantially the same result. [0042] Many Tβ4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of Tβ4. Such isoforms include, for example, Tβ4^ala, Tβ9, Tβio, Tβn, Tβi2, Tβiβ, Tβi4 and Tβi5. These isoforms, along with Tβ4, share an amino acid sequence, LKKTET, or LKKTNT that may be utilized in enhancing stem cells.

[0043] International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses isoforms of Tβ4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET and conservative variants thereof, which may be utilized with the present invention. International Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Thymosin β4 which may be utilized in accordance with the present invention. [0044] Thus, it is specifically contemplated that polypeptide agents such as known Tβ4 isoforms, such as those listed above, as well as Tβ4 isoforms not yet identified, will be useful in the invention.

[0045] In certain embodiments, the stem cells which are enhanced according to the present invention are cardiosphere-derived stem cells (CDC). [0046] As sometimes used herein, the terms "stem cell-enhancing" and "stem cell-enhanced" mean stem cells in which migration has been increased or promoted, or a number of migrating stem cells in a population of stem cells has been increased, as compared to an untreated population. In certain embodiments, the number of migrating stem cells in a population is increased by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, in a population of stem cells, as compared to an untreated population of stem cells. In certain embodiments, the number of migrating stem cells is increased by about 1.5-1.8 times in a population of stem cells treated in accordance with the present invention, as compared to an untreated population of stem cells in vitro.

[0047] In certain embodiments, stem cells are enhanced in accordance with the present invention with a polypeptide in accordance with the present invention at a concentration of about i-2oug/ml of the polypeptide, e.g., about ioug/ml. After stem cells are enhanced in accordance with the present invention, they can be administered to tissue of a subject in vivo according to any suitable procedure. Additionally, a polypeptide agent as described herein can be administered to tissue of a subject together with the stem cells, separately or concurrently therewith, so as to enhance the stem cells in vivo.

[0048] The beneficial effects of previously attempted cardiac stem cell therapy appear to be limited in part by poor cell survival in the post-ischemic environment. Polypeptide agents such as Tβ4 promote cardioprotection by cardiomyocyte survival, limiting inflammation and increasing angiogenesis. Coadministration of TB4 with enhanced cardiac stem cells, e.g., cardiosphere-derived stem cells (CDC), can improve functional outcomes by increased cardio-protection and promotion of angiogenesis.

[0049] In addition, other polypeptide agent molecules related to Tβ4 can similarly be employed in the methods of the invention. Such molecules may include gelsolin, vitamin D binding protein (DBP), profilin, cofilin, adsevertin, propomyosin, fincilin, depactin, Dnasel, vilin, fragmin, severin, capping protein, β- actinin and acumentin, for example , which agents have stem cell enhancing activity of Tβφ

[0050] Thus, in accordance with one aspect, the present invention may utilize polypeptide agents such as peptides or peptide fragments comprising or consisting essentially of amino acid sequence LKKTET or LKKTNT, or conservative variants thereof, including amino acid sequences KLKKTET and/or LKKTETQ (collectively sometimes referred to as LKKTET or LKKTNT peptides).

[0051] As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. [0052] The phosphatidylinositol 3-kinase (PI3K) and the integrin-linked kinase (ILK) and AkT signaling pathways may mediate survival signals and thus play an important role in preventing damage to cardiac tissue after an ischemic insult. AkT is a serine-threonine kinase which may play a role in cell and tissue survival by influencing a number of downstreaming pathways which may inhibit apoptosis. The PI3K and ILK kinases also may activate AkT following stimulation with a variety of membrane receptors, hormones, cytokines, chemokines, and other cellular molecules.

[0053] In one embodiment, the invention provides a method for treating, preventing, inhibiting or reducing tissue damage in a subject by contacting the damaged site with stem cells which have been enhanced utilizing a polypeptide agent as described herein. The contacting may be direct or systemically. Examples of contacting the damaged site include contacting the site with a composition comprising stem cells which have been enhanced utilizing a polypeptide agent as described herein.

[0054] Administration may include, for example, injection of enhanced stem cells directly into a subject's tissue such as heart muscle tissue or myocardium, alone or in combination with a polypeptide agent as described herein, which polypeptide agent can also separately be administered by intravenous, intraperitoneal, intramuscular or subcutaneous injections, or inhalation, transdermal or oral administration.

[0055] Stem cells which have been enhanced utilizing a polypeptide agent as described herein may be administered in any suitable tissue damage-treating, - preventing, -inhibiting or -reducing amount.

[0056] Stem cells which have been enhanced utilizing a polypeptide agent as described herein can be administered as a single administration, daily, every other day, etc., for multiple days, weeks or months, etc., with a single administration or multiple administrations per day of administration, such as applications 2, 3, 4 or more times per day of administration.

[0057] In certain embodiments, stem cells are administered to tissue of the subject in amounts of about lc^-io¹⁰ cells (e.g., about 10⁵ cells), together with, or without, a polypeptide agent in accordance with the present invention. The enhanced stem cells maybe administered intravenously (by IV) and/or directly to tissue (e.g., intramyocardially) to a subject, e.g., after injury caused by, for example, myocardial infarction (MI). In certain embodiments, enhanced stem cells and/or polypeptide agent are administered a plurality of times post-injury within the first forty-eight hours of the injury (e.g., hourly, every two, three, four... hours, etc.). Alternatively, after enhanced stem cells are initially administered, a polypeptide agent in accordance with the present invention maybe administered once or a plurality of times before and/or after administration of the enhanced stem cells. [0058] Additionally, other agents that assist in treating, preventing, inhibiting or reducing damage to a subject's tissue, or for promoting growth of a subject's tissue, may be added to a composition along with stem cells which have been enhanced utilizing a polypeptide agent as described herein. Such agents may include a polypeptide agent as described herein (e.g., Tβ4), angiogenic agents, growth factors, and/or agents that direct differentiation of cells.

[0059] The invention also includes a pharmaceutical composition comprising a therapeutically effective amount of stem cells which have been enhanced utilizing a polypeptide agent as described herein, in a pharmaceutically acceptable carrier, such as water for injection, and optionally further comprising a polypeptide agent as described herein (e.g., Tβ4) and/or other agents. [0060] The actual dosage, formulation or composition that treats or prevents damage to cardiac tissue may depend on many factors, including the size and health of a subject. Persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, supra, and the references cited therein, to determine the appropriate dosage to use to determine the amount of polypeptide agent as described herein to include in an inventive composition, or to administer separately.

[0061] Suitable formulations for administration may include a polypeptide agent as described herein at a concentration within the range of, e.g., about 0.001 - 30% by weight, about 0.01 - 10% by weight, about 0.01 - 0.1% by weight, or at a concentration of about 0.05% by weight.

[0062] The therapeutic approaches described herein involve various routes of administration or delivery of reagents or compositions comprising a polypeptide agent as described herein, including any conventional administration techniques to a subject. The methods and compositions using or containing a polypeptide agent as described herein, and/or other agents utilized with the invention may be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable non-toxic excipients or carriers.

[0063] The invention is further illustrated by the following example, which is not to be construed as limiting.

Example

Methods

[0064] CDCs were cultured from endomyocardial biopsies and pretreated in TB4 (loug/ml) for 14-16 hours. Akt phosphorylation was assessed by western blot, cell migration by Boyden chamber assay and angiogenesis by matrigel assay. SCID mice were divided into 4 groups: CDCs, TB4-pretreated CDCs, TB4 and PBS. Depending on the treatment group, 10⁵ CDCs (in TB4 or PBS) or TB4 or PBS were injected intramyocardially immediately post-MI. Cardiac function was assessed at day-2 and week-3 post-MI by echocardiography and scar area was assessed by Masson's trichrome staining. Results

[0065] TB4 increased the number of migrating CDCs by 1.5-.1.8 times (p < 0.05) in a concentration-dependent manner, with maximum effect occurring at 10 ug/ml. At 3.5 hours, the total length of capillary-like tubules formed in matrigel was significantly greater in TB4 treated cells (p < 0.05). TB4 upregulated Akt phosphorylation in CDCs without altering the total Akt level. Although ejection fraction (EF) and fractional area change (FAC) was similar in all groups on Day 2 post-MI; at 3 weeks, EF stabilized in the TB4-pretreated CDC group, whereas in all 3 control groups, it decreased significantly (p < 0.05). These changes in global function were paralleled by similar significant differences in function of the periinfarct segment, as assessed by fractional area change.

Conclusion

[0066] Modification of the cellular environment by pretreatment of CDCs with TB4 results in an improvement in global and regional cardiac function, possibly through upregulation of prosurvival and proangiogenic pathways, providing an avenue for improving the outcomes of cardiac stem cell therapy.

Claims

CLAIMS:

1. A method of treatment for enhancing stem cells comprising providing a population of stem cells in vitro and contacting said population of stem cells with a stem cell-enhancing amount of a polypeptide agent comprising thymosin beta 4 (Tβ4), so as to form an in vitro population of enhanced stem cells.

2. The method of claim 1 wherein said population is made up of greater than 50% said stem cells.

3. The method of claim 1 wherein said contacting is at a concentration of said polypeptide of about 1 - 20 ug/ml said polypeptide.

4. An enhanced in vitro population of stem cells formed in accordance with the method of claim 1.

5. A pharmaceutical combination comprising enhanced stem cells formed in accordance with the method of claim 1, the combination further comprising said polypeptide agent.

6. The combination of claim 5 comprising about 10¹ - 10 ¹⁰ said enhanced stem cells and said polypeptide agent at a concentration of about 1 - 20 ug/ml.

7. A method of treatment for treating, preventing, inhibiting or reducing damage to a subject's tissue, or for promoting growth of a subject's tissue, comprising administering enhanced stem cells to said subject's tissue, formed according to claim 1.

8. The method of claim 7 wherein said stem cells are autologous to the subject.

9. The method of claim 7 further comprising administering said polypeptide agent to said subject in a formulation wherein said polypeptide agent is at a concentration of about 0.001 - 30% by weight.

10. The method of claim 7 wherein about 10¹ - 10¹⁰ said enhanced stem cells are administered to said subject.

11. A method of treatment for enhancing cardiac stem cells comprising providing a population of stem cells in vitro and contacting said population of cardiac stem cells with a stem cell-enhancing amount of a polypeptide agent comprising thymosin beta 4 (Tβ4), so as to form enhanced stem cells.

12. The method of claim 11 wherein said contacting is in vitro.

13. The method of claim 11 wherein said contacting is in vivo.

14. The method of claim 11 wherein said contacting is at a concentration of said polypeptide of about 1 - 20 ug/ml said polypeptide.

15. The method of claim 11 wherein said stem cells are cardiosphere-derived stem cells (CDCs).

16. Enhanced cardiac stem cells formed in accordance with the method of claim 11.

17. The enhanced stem cells of claim 16 which are CDC stem cells.

18. A pharmaceutical combination comprising enhanced cardiac stem cells formed in accordance with the method of claim 11, the combination further comprising said polypeptide agent.

19. The combination of claim 18 wherein said stem cells are CDC stem cells.

20. The combination of claim 18 comprising about 10¹ - 10¹⁰ said enhanced cardiac stem cells and said polypeptide agent at a concentration of about 1 - 20 ug/ml.

21. A method of treatment for treating, preventing, inhibiting or reducing damage to coronary tissue, or for promoting growth of coronary tissue, comprising administering enhanced cardiac stem cells formed according to claim 11 to cardiac tissue of a subject.

22. The method of claim 21 wherein said stem cells are CDC stem cells.

23- The method of claim 21 further comprising administering said polypeptide agent to said subject in a formulation wherein said polypeptide agent is at a concentration of about 0.001 - 30% by weight.

24. The method of claim 21 wherein about 10¹ - 10¹⁰ said enhanced cardiac stem cells are administered to said subject.

25. The method of claim 23 wherein said polypeptide agent is administered to said subject a plurality of times within 48 hours of myocardial infarction of said subject.