Immortalized cardiac stem cells for cardiac repair

Abstract

Embodiments of the present disclosure relate to compositions and methods of use related to certain immortalized cells, including cardiac muscle stem cells, obtained from pediatric or neonatal individuals. In certain embodiments, the immortalized cells, or conditioned medium derived from the cells, or a partial or entire secretome thereof, are provided to an individual in need thereof in an effective amount, either alone or in combination with cardiac muscle stem cells.

Description

[Technical Field]

[0001] The field of the present disclosure relates to at least the fields of cell biology, molecular biology, and medicine, including cardiology. [Background technology]

[0002] Heart disease is a leading cause of death in adults, and advances in surgical techniques for congenital heart disease and postoperative ICU care have led to an increasing number of children with heart failure (Go et al., 2014; Go et al., 2014). Two key biological processes known to contribute to a wide range of medical conditions, including many of the underlying causes of heart disease, include inflammation and fibrosis. For centuries, the heart, in contrast to other tissues such as the liver and skin, was considered a fully differentiated organ unable to regenerate (reviewed in Buja 2019). This paradigm was recently overturned when it was revealed that cardiomyocytes in the adult human heart are replaced at a small but detectable rate of approximately 1–2% per year (reviewed in Vujic et al., 2019). This has motivated scholars and pharmaceutical companies alike to identify and target fundamental mechanisms of cardiac regeneration for the treatment of various cardiac diseases. There are two potential mechanisms for cardiac regeneration: 1) cardiomyocyte replication and 2) the presence of endogenous cardiac stem cells that can proliferate and differentiate into cardiomyocytes. It is now clear that cardiomyocyte proliferation occurs in certain contexts, such as amphibians, certain fish, and neonatal mammals, but does not contribute to cardiac regeneration in adult mammals. In contrast, clinical trials have recently shown that transplantation of resident cardiac stem cells can repair / regenerate / remodel human myocardium, resulting in improved cardiac function as indicated by improved ejection fraction, reduced scar size, reduced end-diastolic and end-systolic volumes, and improved quality of life and NYHA class (Garbern et al., 2013).

[0003] A population of cardiac stem cells expressing the cell surface marker c-kit (also known as CD117) was described nearly 15 years ago, and a homogenous, healthy population of c-kit+ cells has been shown to confer anti-inflammatory and anti-fibrotic properties. Enrichment of these cells by magnetic selection of c-kit-expressing cardiac cells, followed by cardiac delivery of the cells in animal models of cardiac disease such as myocardial infarction, demonstrated consistent improvements in cardiac function. Similar promising results were observed in early clinical trials of these cells. However, continued scientific investigation of these cells has clarified two issues. First, these c-kit+ cardiac stem cells are a mixture of many different progenitor cells, most of which (approximately 90%) are hematopoietic and endothelial cells, not cardiogenic stem cells (Vicinanza et al. 2017). Second, most cardiac c-kit+ cells obtained from adult human hearts are senescent, which may contribute little to cardiac repair and may also contribute to cardiac injury via various inflammatory factors secreted by such senescent cells (Lewis-McDouggal et al. 2019).

[0004] Thus, there is a need in the art for compositions and methods for treating cardiac medical conditions, such as heart failure, caused by damaged myocardial tissue, as well as for compositions and methods for addressing the inflammatory and fibrotic processes observed in a wide range of medical conditions. The present invention fulfills these needs and provides other related advantages. Summary of the Invention [Means for solving the problem]

[0005] Embodiments of the present disclosure relate to methods and compositions related to certain human cardiac stem cells (hCSCs), particularly neonatal cardiac stem cells (nCSCs), immortalized nCSCs (Im-nCSCs) (e.g., clonal isolates), and conditioned medium produced by Im-nCSCs, for use in treating medical conditions. In certain embodiments, the medical condition is a cardiac medical condition. While any inflammatory, fibrotic, or cardiac medical condition can be treated using such compositions and methods, in certain embodiments, the condition is a cardiac condition that would benefit from repair, regeneration, or remodeling of cardiac muscle (myocardium). In certain embodiments, the methods and compositions promote or enhance the repair, regeneration, or remodeling capabilities of nCSCs. In certain embodiments, the medical condition is an inflammatory condition or disease. In more specific embodiments, the inflammatory condition or disease is selected from, but is not limited to, ischemic stroke, acute and chronic kidney disease, arthritic conditions, dermatological conditions, and COVID-19. In yet other embodiments, the anti-fibrotic features of the present invention can improve wound healing and conditions characterized by chronic fibrosis.

[0006] Embodiments of the present disclosure encompass methods and compositions relating to immortalized cells derived from mammalian, such as human, particularly neonatal, human cardiac muscle. In certain particular embodiments, the cells comprise immortalized neonatal CD117+ cardiac muscle stem cells, particularly immortalized clonal isolates of neonatal CD117+ cardiac muscle stem cells.

[0007] In a further embodiment, the immortalized stem cells of the present invention have the following cell surface marker characteristics: CD90 + , CD105 + , CD117 + , CD44 + , CD73 + , CD47 + , CD31 - , CD34 - , CD45 - and have one or more, two or more, three or more, four or more, or five or more of tryptase negative (e.g., Figure 6 ).

[0008] In other embodiments, the immortalized stem cells of the present invention can optionally be reversibly immortalized with a kill switch.

[0009] In some embodiments, immortalized cardiac stem cells are provided to an individual to treat one or more cardiac medical conditions. In other embodiments, conditioned medium from immortalized cardiac stem cells is provided to an individual for treatment of a cardiac medical condition. In still other embodiments, a secretome derived from the cells is provided to an individual for treatment of a cardiac medical condition. In still other embodiments, trophic factors derived from one or more immortalized cell clonal isolates are provided to an individual for treatment of a cardiac medical condition. In certain other embodiments, any combination of the above may be used in the treatment of cardiac or other medical conditions.

[0010] In some embodiments, the present disclosure provides compositions comprising conditioned medium (CM) from one or more immortalized neonatal cardiomyocyte stem cells (Im-nCSCs), such as CD117+ immortalized neonatal cardiomyocyte stem cells. In certain specific embodiments, the immortalized neonatal cardiomyocyte stem cells are immortalized clonal isolates. In other embodiments, the immortalized neonatal cardiomyocyte stem cells exhibit the following characteristics: CD90 + , CD105 + , CD117 + , CD44 + , CD73 + , CD47 + , CD31 - , CD34 - , CD45 - and tryptase negative. In yet other embodiments, the immortalized neonatal cardiac stem cells have one or more, two or more, three or more, four or more, or five or more of the following characteristics: CD90 + , CD105 + , CD117 + , CD44 + , CD73 + , CD47 + , CD31 - , CD34 - , CD45 -and tryptase negative. In yet other embodiments, the immortalized neonatal cardiac stem cells have one or more, two or more, three or more, or four or more of the following characteristics: GATA4-, CD44+, tryptase negative, CD80-, CD86-. In further embodiments, the immortalized neonatal cardiac stem cells have all of the following characteristics: GATA4-, CD44+, tryptase negative, CD80-, CD86-. In yet further embodiments, the immortalized neonatal cardiac stem cells have the following characteristics: CD117+, CD45-, and Lin - In still further embodiments, the immortalized neonatal cardiac stem cells have one or more, two or more, or all of the following characteristics: CD117 + and CD45 - It has.

[0011] In some embodiments of the present invention, prior to immortalization, neonatal cardiac stem cells are isolated from the heart of a neonatal individual (e.g., from a biopsy from the individual). In certain related embodiments, the stem cells are isolated by single-cell cloning. In yet other embodiments, the stem cells are isolated by single-cell cloning without any cell selection steps typically performed in the literature. In some embodiments, the individual from which the cells are derived is less than 30 days old when the cells are obtained from the individual's heart (e.g., when the biopsy is taken). In some embodiments, the isolation does not include contacting the cells with an antibody for cell selection. In some embodiments, the isolation does not include a cell enrichment step using antibody-based selection. In some embodiments, the cells are isolated by limiting dilution culture. In some embodiments, immortalization is achieved by exogenous expression of the human telomerase (hTERT) gene using a delivery vector, such as a lentiviral expression vector.

[0012] In another embodiment of the present invention, a composition is provided comprising a plurality of immortalized human neonatal cardiac stem cell clonal isolates.

[0013] In yet other embodiments, the present disclosure provides methods of treating an individual for, e.g., a cardiac medical condition described herein or other condition, comprising providing to the individual a therapeutically effective amount of a composition comprising conditioned medium derived from immortalized neonatal cardiac stem cells. In some embodiments, the composition is selected from any one of the compositions provided herein.

[0014] In some embodiments, the present disclosure provides methods of treating an individual for a cardiac medical condition, comprising providing to the individual a therapeutically effective amount of a composition comprising conditioned medium from immortalized neonatal cardiac stem cells in combination with a composition comprising neonatal cardiac stem cells, such as immortalized neonatal cardiac stem cells, hi some embodiments, the composition comprising conditioned medium is selected from any one of the compositions provided herein.

[0015] In yet other embodiments, the present disclosure provides methods for isolating cell clones from immortalized neonatal cardiac stem cells, the methods comprising isolating one or more cells from neonatal cardiac tissue or having one or more cells isolated from neonatal cardiac tissue, and culturing the one or more cells in an appropriate culture medium to promote proliferation. In more specific embodiments, the isolating step does not include contacting the cells with a moiety that binds to a specific cell surface protein, such as CD117.

[0016] In additional embodiments, the isolated clonal cell isolates immortalized according to the present disclosure have, when isolated, one or more, two or more, three or more, four or more, or five or more of the following characteristics: GATA4-, CD44+, CD47+ CD31-, CD34-, CD45-, tryptase-, CD80-, CD86- and the immortalized cells maintained such expression patterns upon immortalization.

[0017] In further embodiments, the immortalized cells of the present disclosure are originally obtained from the heart of a neonatal individual, including a pediatric or non-pediatric individual, prior to immortalization. In certain embodiments, the individual has a cardiac medical condition. In certain embodiments, the individual has normal myocardium. In some embodiments, the cells may be derived from the myocardium of a pediatric individual with end-stage heart failure. The myocardium may be derived from a neonatal individual with congenital heart disease.

[0018] In other specific embodiments, the immortalized cells of the present disclosure are CD63 + , CD73 + , CD47 + , CD45 - , CD31 - Immortalized cells secrete exosomes that are pro-angiogenic and pro-angiogenic cytokines, such as VEGF-A, HGF, SCF, SDF-1α, IGF, PDGF-B, and ANG-1 (e.g., Table 1).

[0019] In yet other embodiments, methods are provided for treating an individual for a medical condition, such as a cardiac medical condition or any other condition described herein, comprising providing to the individual a therapeutically effective amount of a composition of the present disclosure comprising immortalized neonatal cardiac muscle stem cells, conditioned medium by immortalized neonatal cardiac muscle stem cells, exosomes secreted by immortalized neonatal cardiac muscle stem cells, and / or any combination thereof. In more particular embodiments, the composition independently comprises proteins and / or exosomes secreted from immortalized neonatal cardiac muscle stem cells.

[0020] In other specific embodiments, cardiac medical conditions are treated using compositions of the invention, such as by intramyocardial injection, intravenous injection, delivering the composition with encapsulated cells, e.g., in a device that retains the cells but allows secretion of paracrine factors produced by the cells into the circulation, etc. In yet other specific embodiments, the cardiac medical condition is heart failure, cardiomyopathy, or congenital heart disease.

[0021] In additional embodiments of the present invention, there are provided compositions comprising whole conditioned medium (TCM) and / or components thereof from the immortalized cells of the present disclosure, and methods of using the same in therapeutic amounts, for the treatment of the medical conditions described herein. In yet further embodiments, there are provided compositions comprising exosomes and / or components thereof from the immortalized cells of the present disclosure, and methods of using the same in therapeutic amounts, for the treatment of the medical conditions described herein. This application includes the following inventions. [Item 1] One or more immortalized neonatal CD117 + A composition comprising a conditioned medium derived from cardiac stem cells. [Item 2] The cells have the following characteristics: CD90 + , CD105 + , CD31 - , CD34 - , CD45 - and / or tryptase negative, and a pharmaceutically acceptable carrier. [Item 3] The cells have the following characteristics: CD44 + , CD47 + and / or CD73 + 3. The composition according to item 1 or 2, having one or more of: [Item 4] The cells have the following characteristics: GATA4, CD80 - , CD86 - and / or Lin - The composition according to any one of items 1 to 3, comprising one or more of the following: [Item 5] Prior to immortalization, the neonatal CD117 + 2. The composition of item 1, wherein the cardiac cells are isolated from the heart of a neonatal individual that is less than 30 days old. [Item 6] The composition described in Item 5, wherein the isolation does not include contacting the cells with an antibody. [Item 7] The composition described in Item 5, wherein the isolation does not include a cell enrichment step using antibody-based selection. [Item 8] The composition described in Item 1, wherein the cells are isolated by limiting dilution culture. [Item 9] The composition described in Item 1, wherein the immortalization is achieved by exogenous expression of hTERT. [Item 10] Immortalized human neonatal CD117 + A composition comprising cardiac stem cells. [Item 11] The cells have the following characteristics: CD90 + , CD105 + , CD31 - , CD34 - , CD45 - and / or tryptase negative, and a pharmaceutically acceptable carrier. [Item 12] The cells have the following characteristics: CD44 + , CD47 + and / or CD73 + 12. The composition according to any one of items 10 to 11, comprising one or more of the following: [Item 13] The cells have the following characteristics: GATA4, CD80 - , CD86 - and / or Lin - 13. The composition according to any one of items 10 to 12, comprising one or more of: [Item 14] Prior to immortalization, the neonatal CD117 + 11. The composition of item 10, wherein the cardiac cells are isolated from the heart of a neonatal individual that was less than 30 days old. [Item 15] The composition described in Item 14, wherein the isolation does not include contacting the cells with an antibody. [Item 16] The composition described in Item 14, wherein the isolation does not include a cell enrichment step using antibody-based selection. [Item 17] The composition of any one of Items 10, wherein the cells are isolated by limiting dilution culture. [Item 18] The composition described in Item 10, wherein the immortalization is achieved by exogenous expression of hTERT. 19. A method of treating an individual for a cardiac medical condition, comprising administering to the individual a therapeutically effective amount of immortalized neonatal CD117. + A method comprising the step of providing a composition comprising conditioned medium derived from cardiac stem cells. [Item 20] The method according to Item 19, wherein the composition is selected from any one of the compositions provided in any one of Items 1 to 9. 21. A method of treating an individual for a cardiac medical condition, comprising administering to the individual a therapeutically effective amount of immortalized neonatal CD117. + A composition comprising conditioned medium from cardiac stem cells was prepared by injecting a plurality of neonatal CD117 + A method comprising providing the method in combination with a composition comprising cardiac stem cells. [Item 22] The method according to Item 21, wherein the composition comprising the conditioned medium is selected from any one of the compositions provided in any one of Items 1 to 9. [Item 23] Multiple neonatal CD117 + 23. The method according to item 21 or 22, wherein the composition comprising cardiac stem cells is selected from any one of the compositions provided in any one of items 10 to 18. [Item 24] A method for repairing or remodeling myocardial tissue in a subject in need thereof, comprising contacting the myocardial tissue of the subject with a composition provided in any one of Items 1 to 9, a composition provided in any one of Items 10 to 18, or a combination thereof. [Item 25] CD117 from neonatal cardiac tissue + A method for isolating stem cells, comprising isolating or having isolated one or more cells from neonatal cardiac tissue, and culturing the one or more cells in an appropriate culture medium to promote proliferation, wherein the isolation does not include contacting the cells with a moiety that binds to CD117. [Item 26] The composition described in Item 25, wherein the isolation does not include contacting the cells with an antibody. [Item 27] ​​The method according to Item 25, wherein the isolation does not include contacting the cells with a CD117 antibody. [Item 28] The method according to Item 25, wherein the isolation is achieved by direct cloning without the need for negative selection against endothelial cells and / or hematopoietic cells. [Brief explanation of the drawings]

[0022] [Figure 1] Figure 1 shows an exemplary methodology for cell dilution of nCSCs to initiate single-cell culture.

[0023] [Figure 2]FIG. 2 shows a representative phase contrast image of a single cell culture in a 96-well plate initiated with cells diluted by the method described in FIG.

[0024] [Figure 3] FIG. 3 shows the phenotypic characterization by flow cytometry analysis of seven clonal isolates of nCSCs obtained using the methods shown in FIGS.

[0025] [Figure 4] FIG. 4 shows an exemplary lentiviral vector carrying the hTERT gene used to immortalize nCSCs isolated via the methods disclosed herein.

[0026] [Figure 5] Figure 5 shows representative phase-contrast microscopic images of nCSCs and Im-nCSCs.

[0027] [Figure 6] FIG. 6 shows the results of flow cytometry analysis of Im-nCSCs expanded from single-cell cultures for 16 passages.

[0028] [Figure 7] Figure 7 shows the improvement of cardiac function after myocardial infarction in rat hearts. Left ventricular ejection fraction (EF) and fractional shortening were analyzed by echocardiography.

[0029] [Figure 8] FIG. 8 shows the results of characterization of Im-nCSC-derived total conditioned medium (Im-nCSC TCM).

[0030] [Figure 9] FIG. 9 shows that Im-nCSC TCM protects neonatal rat cardiomyocytes from hydrogen peroxide-induced apoptosis.

[0031] [Figure 10]FIG. 10 shows that Im-nCSC TCM promotes angiogenesis.

[0032] [Figure 11] FIG. 11 shows that Im-nCSC TCM promotes cell migration and in vitro wound healing.

[0033] [Figure 12] FIG. 12 shows the in vivo functional activity of nCSC and Im-nCSC TCM in a rat myocardial infarction model by intravenous injection, as measured by echocardiogram.

[0034] [Figure 13] FIG. 13 shows a representative normal karyotype observed in Im-nCSCs. DETAILED DESCRIPTION OF THE INVENTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0036] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0037] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0038] The term "eg" is used herein to mean "for example," and is understood to mean the inclusion of a stated step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components.

[0039] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0040] As used herein, the term "administering" refers to any manner of communicating, delivering, introducing, or transporting a substance, such as a compound, e.g., a pharmaceutical compound, or other agent, such as an antigen, to a subject. Modes of administration include oral administration, topical contact, intravenous, intraperitoneal, intramuscular, intranasal, or subcutaneous administration. Administration "in combination with" additional substances, such as one or more therapeutic agents, includes simultaneous (concurrent) and consecutive administration in any order.

[0041] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of a specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to whatever follows the word "consisting of." Thus, the word "consisting of" indicates that the listed components are required or essential, and that no other components are present. "Consisting essentially of" means including any components listed after the word, and any components limited to other components that do not interfere with or contribute to the activity or action specified in this disclosure for the listed components. Thus, the word "consisting essentially of" indicates that the listed components are required or essential, but that other components are optional and may or may not be present depending on whether they substantially affect the activity or action of the listed components.

[0042] An "effective amount," when used in reference to a compound, is the amount of compound necessary to elicit a desired response. In some embodiments, the desired response is, for example, a biological response in a subject. In some embodiments, a compound may be administered to a subject in an amount effective to affect a biological response in the subject. In some embodiments, the effective amount is a "therapeutically effective amount."

[0043] The terms "therapeutically effective amount" and "therapeutic dose" are used interchangeably herein to refer to an amount of a composition (e.g., conditioned medium derived from neonatal CSCs, such as immortalized CSCs disclosed herein) that is effective after administration to a subject to treat a disease or disorder in the subject as described herein.

[0044] The term "modulating" includes "increasing," "enhancing," or "stimulating," as well as "decreasing" or "reducing," typically by a statistically significant or physiologically significant amount compared to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30-fold or more (e.g., 500, 1000-fold) (including all integers and decimal points therebetween, and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the amount produced by a non-composition or control composition, sample, or test subject. A "decreased" or "reduced" amount is typically a "statistically significant" amount and may include a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the amount produced by no composition (absence of agent or compound) or a control composition, including all integers in between.

[0045] As used herein, a "subject" or "patient" includes any animal exhibiting or at risk of exhibiting a condition that can be treated with the compositions disclosed herein (e.g., conditioned medium derived from neonatal CSCs, such as immortalized hCSCs, as disclosed herein). Suitable subjects (patients) include human patients. Suitable subjects also include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock (such as pigs, horses, cows), and domestic animals or pets (such as cats or dogs). Non-human primates (such as monkeys, chimpanzees, baboons, or lizards) are also included.

[0046] "Substantially" or "essentially" means nearly entirely or completely, eg, 95% or more of some given amount.

[0047] As used herein, "treatment" or "treating" includes any desired effect on the symptoms or pathology of a disease or condition, and may include even a minimal change or improvement in one or more measurable markers of the disease or condition being treated. "Treatment" or "treating" does not necessarily indicate a complete eradication or cure of the disease or condition, or its associated symptoms. The subject receiving this treatment is any subject in need thereof. Exemplary markers of clinical improvement will be apparent to those skilled in the art.

[0048] The terms "CD117" and "c-kit" may be used interchangeably in accordance with the present disclosure to refer to the same protein, which is referred to synonymously by both names. The c-kit / CD117 protein, as well as the gene encoding it, have been extensively characterized and are well known in the art (e.g., https: / / www.uniprot.org / uniprot / P10721 and UNIPROT accession number P10721).

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method, composition, reagent, cell similar or equivalent to those described herein can be used in carrying out or testing the present invention, preferred methods and materials are described herein.All publications and references cited herein, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated herein by reference as if fully set forth.Any patent application to which this application claims priority is also incorporated herein by reference in its entirety, in the manner described above for publications and references.

[0050] As used herein, the term "cardiomyocyte stem cells" may be defined as cells derived from cardiac tissue, which are clonogenic, multipotent, and self-renewing. In certain embodiments, these cardiac stem cells express one or more of the following: CD117, CD90, CD105, CD73, CD44, and CD47, and are negative for one or more of the following: CD31, CD34, CD45, and tryptase.

[0051] overview In particular, the present disclosure provides compositions and methods relating to and methods for isolating and immortalizing human neonatal cardiac stem cells (nCSCs), as well as conditioned medium harvested from cultures of such cardiac stem cells, and methods of using such cells, medium, and / or cells resuspended in the conditioned medium therapeutically, e.g., to repair, regenerate, and / or remodel cardiac tissue and to treat medical conditions caused by inflammatory and / or fibrotic processes (e.g., cardiac medical conditions).

[0052] I. Cells of the Present Disclosure and Their Conditioned Media and Secretomes Embodiments of the present disclosure relate to mammalian cells or conditioned medium derived from the cells that provide a therapeutic function to an individual in need thereof. In certain embodiments, the cells are immortalized human neonatal cardiac stem cells (Im-nCSCs) useful for achieving a therapeutic function or effect in a mammalian heart. In some embodiments, the cells are immortalized human neonatal cardiac stem cells, Im-nCSCs, that secrete one or more agents that are anti-inflammatory, anti-fibrotic, pro-angiogenic, and / or useful for a therapeutic function or effect in a mammalian heart. In certain embodiments, the cells are immortalized human neonatal cardiac CSCs that, upon administration to the heart in vivo, possess their own repair, regeneration, or remodeling activity and / or have activity to promote the repair, regeneration, or remodeling activity of endogenous cells and / or tissues of human myocardium upon administration to the heart in vivo. In certain embodiments, the cells are immortalized neonatal human CSCs that are anti-inflammatory, anti-fibrotic, pro-angiogenic, and / or have repair, regeneration, or remodeling activity upon administration to the heart in vivo, and / or secrete one or more proteins that have activity upon administration to the heart in vivo to promote the repair, regeneration, or remodeling capacity of endogenous cells and / or tissues in human myocardium. In certain embodiments, the cells secrete one or more proteins that can elicit a bystander effect, such that delivery of conditioned medium from the cells to myocardium in vivo enhances the repair, regeneration, or remodeling of endogenous cells and / or tissues.

[0053] In certain embodiments, immortalized human neonatal cardiac CSCs are generated by obtaining clonal isolates of human neonatal cardiac CSCs and immortalizing them by any suitable immortalization method. Many such methods are known that are suitable for use in the present invention, including, but not limited to, immortalization by introduction of simian virus 40 large T antigen (Kobayashi et al., (2000) Science 287:1258-62; Nakamura et al., (1997) Transplantation 63 (11):1541-47), transfection of antisense constructs against p53 and retinoblastoma proteins (Werner et al., (2000) Biotechnol Bioeng 68 (1):59-70), transgenic introduction of truncated Met protein (Amicone et al., (1997) EMBO J. 16 (3):495-503), and expression of hepatitis C virus core protein (Ray et al., (2000) Virology 271:197-204).

[0054] In one specific embodiment, the human neonatal cardiac CSCs of the present disclosure are immortalized by stably transfecting them with a vector (e.g., a lentiviral vector) expressing human telomerase (hTERT). It has been shown that the mechanism that limits the proliferation of human fibroblast cells in vitro is the progressive shortening of telomeres with each cell division (Hayflick et al., (1961) Exp. Cell Res. 25:585-621). Telomeres constitute the terminal regions of chromosomes, and shortened telomeres cause proliferation restriction. However, stem cells can circumvent telomere-dependent proliferation restriction by using telomerase reverse transcriptase to add telomere repeat sequences to the chromosome ends (Greider et al., (1985) Cell 43:405-413). The ability to achieve telomerase reconstitution of human neonatal cardiac CSCs to generate stable neonatal human cardiac-derived clonal stem cell lines with phenotypic characteristics of neonatal cardiac stem cells after in vitro passaging for use in, for example, cardiac-directed cell therapy and cardiac research ensures cellular consistency and eliminates the need for multiple donors to produce large quantities of these cells for therapeutic use. For example, in some embodiments, such cells can function as cells capable of producing unlimited quantities of conditioned medium containing one or more secreted factors, e.g., secreted proteins, that have repair, regeneration, or remodeling activity upon administration to the heart in vivo and / or have activity to promote the repair, regeneration, or remodeling capacity of endogenous cells and / or tissues in human myocardium upon administration to the heart in vivo. In other embodiments, such cells can function as cell factories capable of producing unlimited quantities of conditioned medium containing one or more secreted factors, e.g., secreted proteins with anti-inflammatory, anti-fibrotic, and pro-angiogenic properties, that can provide therapeutic benefit in wound healing and / or medical conditions other than cardiac disease.

[0055] The telomerase used for immortalization may be encoded by the human TERT (hTERT) gene, for example. Human neonatal cardiac CSCs may be transfected with hTERT using any suitable method. For example, human neonatal cardiac CSCs may be infected with a recombinant virus capable of introducing the hTERT gene into cells. In another example, human neonatal cardiac CSCs may be infected with a lentiviral viral vector, and individual CSC clones containing hTERT may be isolated and expanded. The lentiviral vector may contain hTERT under the control of an appropriate promoter (e.g., a CMV promoter), an illustrative example of which is shown in Figure 4.

[0056] In one aspect, the invention provides a population of immortalized human cells expressing human telomerase, which exhibit phenotypic characteristics of neonatal human cardiac stem cells at early passages and continue to express said phenotypic characteristics at later passages in vitro (e.g., very high population doubling levels (PDL) in the range of 70 or more). In another aspect, the invention provides immortalized human cells expressing human telomerase, which exhibit phenotypic characteristics of neonatal human cardiac stem cells at early passages in vitro and continue to express said phenotypic characteristics at later passages in vitro. In one embodiment, the immortalized cells can be induced to differentiate in vitro into the major types of cardiac cells (i.e., endothelial cells, smooth muscle cells, cardiomyocytes).

[0057] In additional embodiments of the invention, the immortalized cells may be used to produce conditioned medium, which in certain embodiments may be used in any of a variety of indications, for example, to treat cardiac and other medical conditions, induce angiogenesis, inhibit inflammation, promote cardiomyocyte rescue, and reduce cardiac fibrosis in vivo (Ongstad et al. 2019).

[0058] In some embodiments, the immortalized cells express CD117. In other embodiments, the immortalized cells express CD117 at high levels. In various embodiments, reference herein to a "high level" of CD117 expression means that >80% of the cells analyzed (e.g., by flow cytometry) express CD117. In yet other embodiments, reference herein to a "low level" of CD117 expression means that <80% of the cells analyzed (e.g., by flow cytometry) express CD117.

[0059] In certain embodiments, the morphology of the cells before and after immortalization is characterized by being substantially unchanged and / or showing no evidence of senescence (Figure 5).

[0060] In other embodiments, the immortalized cells do not express CD31. In one embodiment, the immortalized cells do not express CD45. In yet other embodiments, the immortalized cells express CD117 but do not express CD31 or CD45. In other specific embodiments, the immortalized cells express high levels of CD117 but do not express CD31 or CD45 (Figure 6). In other specific embodiments, the immortalized cells express CD117, CD90, CD105, CD44, CD47, and CD73 but do not express CD31 or CD45 (Figure 6).

[0061] Neonatal cardiac stem cells may be obtained from any suitable source prior to immortalization. In certain embodiments, the source of the cardiac stem cells to be immortalized is from a newborn individual or from an individual in utero. In certain embodiments, the cells are not adult cardiac stem cells. The cells may be obtained from an individual in need of therapeutic use using progeny of the same cell, or the cells may be obtained from another individual. The cells may be derived from the donated heart of a newborn individual or the heart of a living newborn individual. The cells may be provided commercially to an individual in need, or may be provided to a medical facility or practitioner overseeing the medical care of an individual in need. In certain embodiments, the cells are obtained from a human subject between 1 and 30 days old. For example, the human subject may be 1 day old, 2 days old, 3 days old, 4 days old, 5 days old, 6 days old, 7 days old, 8 days old, 9 days old, 10 days old, 11 days old, 12 days old, 13 days old, 14 days old, 15 days old, 16 days old, 17 days old, 18 days old, 19 days old, 20 days old, 21 days old, 22 days old, 23 days old, 24 days old, 25 days old, 26 days old, 27 days old, 28 days old, 29 days old, or 30 days old or younger. The human subject may also be less than 1 day old.

[0062] Embodiments of the present disclosure encompass immortalized neonatal CSCs derived from cells derived from mammalian, including human, cardiac muscle. In certain embodiments, the human neonatal cardiac CSCs may have a specific genotype and / or phenotype. In certain embodiments, the immortalized human neonatal cardiac CSCs may be provided to an individual in need thereof after determining the specific genotype or phenotype of the cells appropriate for the intended function of the cells during therapeutic use. However, in some preferred embodiments, the conditioned medium produced from culturing the immortalized human neonatal cardiac CSCs is provided to an individual in need thereof after determining the specific genotype or phenotype of the cells appropriate for producing conditioned medium having the intended function. In certain embodiments, the immortalized human neonatal cardiac CSCs are CD117+ cells, and in certain embodiments, the immortalized human neonatal cardiac CSCs are CD117+, CD90+, CD105+, CD73+, CD44+, CD47+, and CD31-, CD45-. In other embodiments, the immortalized human neonatal cardiac CSCs also have one, two, three, four, or five or more of the following characteristics: GATA4-, CD44+, CD31-, tryptase-, CD80-, CD45-, CD86-, HLA class I+, and HLA class II-. In other embodiments, the immortalized human neonatal cardiac CSCs also have one, two, three, four, or five or more of the following characteristics: GATA4-, CD44+, CD73+, CD47+, CD31-, tryptase-, CD80-, CD45-, CD86-, HLA class I+, and HLA class II-.

[0063] In some embodiments, immortalized human neonatal cardiac CSCs naturally secrete one or more proteins or factors beneficial to the repair, regeneration, or remodeling of localized cells or tissues. In some embodiments, the present disclosure provides conditioned medium produced by culturing immortalized human neonatal cardiac CSCs under conditions whereby they secrete one or more proteins or factors beneficial to the repair, regeneration, or remodeling of localized cells or tissues. Such proteins or factors may be of any type, but in certain embodiments, they are cytokines, pro-angiogenic factors, growth factors, transcription factors, miRNAs, etc. In some embodiments, the cells secrete one or more, or any combination of, VEGF-A, HGF, SCF, SDF-1α, ANG-1, bFGF, PDGFB, and IGF-1. In some embodiments, the cells secrete factors secreted from one or more of VEGF-A, HGF, SCF, SDF-1α, ANG-1, bFGF, PDGFB, and IGF-1, and in some embodiments, the present disclosure provides conditioned media containing one or more of such factors. In certain embodiments, the cells are engineered to increase secretion of SDF-1α, VEGF-A, PGDF-A, and / or FGF-2, or a combination thereof. Such engineering can be by any method, but in certain embodiments, includes recombinantly engineering cells to increase expression of one or more of these factors and / or other factors that have therapeutic value but are not expressed by these cells. Thus, in some embodiments, the present disclosure provides conditioned media derived from such engineered cells. Another engineering technique contemplated for use in the present invention is exposure of immortalized human neonatal cardiac CSCs to heat shock factors and / or other agents that increase cytokine secretion.

[0064] In some embodiments, immortalized human neonatal cardiac CSCs naturally express one or more proteins or factors beneficial to direct or indirect localized cell or tissue repair, regeneration, or remodeling. For example, immortalized human neonatal cardiac CSCs may express VEGF-A and / or SDF-1α. In addition, similar to human neonatal cardiac CSCs, immortalized human neonatal cardiac CSCs may have activated expression of HSF-1, HSP60, and / or HSP70, either naturally or by engineering the cells to increase expression of HSF-1, HSP60, and / or HSP70 (Sharma et al., 2017). Thus, in some embodiments, the present disclosure provides conditioned media produced by such cells, wherein the conditioned media comprises VEGF-A and / or SDF-1α. In some embodiments, the present disclosure provides conditioned media produced by such cells that comprise expression of HSF-1, HSP60, and / or HSP70, either naturally or by engineering. In some embodiments, the conditioned medium (CM) of the immortalized neonatal cardiac stem cells comprises a unique combination of paracrine factors (e.g., cytokines and growth factors) within pre-specified ranges detailed in Table 1.

[0065] Furthermore, analysis of the exosome content in the secretome of immortalized neonatal cardiac stem cells revealed that the number of exosomes with a diameter of 137.6 nM (mode value, SD = 49.1 nM) was in the range of 1.1e+9 ± 3.81e+7 particles / ml, and that these exosomes expressed surface markers CD63, CD73, and CD83. + , CD47 + and showed that they were negative for CD31 and CD45 (see Figure 8).

[0066] The immortalized human neonatal cardiac CSCs may exist as a plurality of cells, and the plurality of cells may be 100% homogeneous with respect to the desired cardiac stem cells. Alternatively, in some embodiments, the plurality of cells may have less than 100% homogeneity, such as 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25% homogeneity, or at least that percentage of homogeneity with respect to the desired immortalized human neonatal cardiac CSCs. Immortalized human neonatal cardiac CSCs can be utilized in the methods when present in plural with 100% homogeneity or with less than 100% homogeneity, such as 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25% homogeneity.

[0067] The immortalized human neonatal cardiac CSCs may be stored for a period of time prior to use (e.g., for use in culture to produce the conditioned medium disclosed herein), including under appropriate medium, temperature, and oxygen level conditions, or may be used without significant storage time. In certain embodiments, the storage cell medium includes one or more heat shock response inducers.

[0068] Immortalized human neonatal cardiac CSCs may be used for allogeneic therapy because they express low levels of MHC class II or the costimulatory proteins CD88 and CD80. These cells do not initiate an immune response when transplanted into another patient's immune system. In such cases, the cells can be used as a stock for clinical applications.

[0069] In certain embodiments, CSCs derived from young individuals have demonstrated stronger repair and / or regeneration capabilities compared to similarly derived cells from adult hearts (Sharma et al., 2017). These increased capabilities are due, in part, to a stronger secretome, in certain embodiments, than that of young cells. This means that young cells may, at least in certain embodiments, be a preferred allogeneic product. Furthermore, in some embodiments, these superior repair and / or regeneration properties are maintained after immortalization of human neonatal cardiac CSCs according to the present disclosure. Thus, for example, in some embodiments, the secretome produced by immortalized human neonatal cardiac CSCs is superior to the secretome produced by CSCs derived from adult cardiac cells for use in inducing repair and / or regeneration of damaged cardiac tissue. Accordingly, the present disclosure provides a method for inducing repair and / or regeneration of cardiac tissue damage, comprising administering to a patient in need thereof a composition comprising conditioned medium derived from one or more immortalized human neonatal cardiac CSCs. In some embodiments, the population of human neonatal cardiac CSCs is resuspended in a composition comprising conditioned medium derived from one or more immortalized human neonatal cardiac CSCs, and the composition comprising the conditioned medium and the resuspended human neonatal cardiac CSCs is administered to the patient. II. Methods for isolating nCSCs

[0070] The present disclosure also provides a novel method for isolating CD117+ CSCs from neonatal tissue that does not require exposing collected cells to a substrate comprising a surface bearing a moiety that binds to CD117 (i.e., c-kit). This method contrasts with prior art methods for isolating CD117+ hCSCs, all of which involve exposing collected cells to such a substrate (e.g., a plate, beads, or column) that comprises a CD117-binding moiety (such as an antibody) to select CD117+ cells from a heterogeneous population of cells present in the sample.

[0071] The methods disclosed herein are based, in part, on the surprising discovery that, in contrast to the adult human heart, the neonatal human heart contains a homogenous population of cardiogenic CD117+ stem cells with little or no contamination from hematopoietic and endothelial progenitor cells. Furthermore, we discovered that CD117+ cells from neonatal human hearts, even when initially seeded as single cells, give rise to clones that can expand in culture, and that their proliferation rate is related to the level of CD117 positivity of the cells. Thus, although cells with a high degree of CD117+ expression generally proliferate faster than cells with lower CD117 expression, all cells we observed expanding expressed CD117 to at least some degree. Furthermore, CD117+ cells from neonatal cardiac tissue, when tested in an animal model of myocardial infarction, do not show evidence of senescence and do not expand to large numbers of cells with significant cardiac repair and regenerative activity (see, e.g., Figure 7).

[0072] Thus, based at least in part on these surprising discoveries, the present disclosure provides a method for isolating and expanding human neonatal cardiac CSCs (e.g., a homogenous population of CD117+ neonatal cardiac CSCs) directly from neonatal cardiac tissue without prior selection with CD117 magnetic beads (or any other CD117+ cell enrichment technique). This method allows for the isolation and expansion of phenotypically CD90+ CSCs, which has not been disclosed prior to the present disclosure. + , CD105 + , CD117 + , CD44 + , CD73 + , CD47 + , CD31 - , CD34 - , CD45 -and tryptase negativity. In addition, such methods offer significant advantages over prior art methods because they provide a single cell donor and donor independence in obtaining large quantities of cells, ensuring cell homogeneity and avoiding regulatory concerns associated with exposing cells intended for therapy to non-GMP-quality materials (i.e., CD117 magnetic beads). Furthermore, they reduce the level of processing that must occur between obtaining a neonatal heart sample and providing a population of CD117+ cells for, e.g., therapeutic purposes, for production of conditioned medium and / or immortalization according to the methods disclosed herein.

[0073] In various embodiments, the present disclosure also provides methods for isolating CD117+ CSCs from neonatal cardiac tissue, the methods comprising isolating or having isolated one or more clonal isolate cells from neonatal cardiac tissue and culturing the one or more cells in an appropriate culture medium to promote proliferation, wherein the isolating step does not include contacting the cells with a moiety that binds CD117 (e.g., a CD117 antibody). In some embodiments, the methods comprise initiating a culture or multiple cultures, each culture involving one or more cells derived from cardiac tissue. In some embodiments, each culture or each of the multiple cultures is initially seeded with only one cell derived from cardiac tissue. This can be accomplished by any suitable method known in the art, such as FACS sorting or limiting dilution culture of cells obtained from a neonatal cardiac tissue sample.

[0074] The method may include monitoring the growth rate of one or more of the cultures. The method may include approximating the level of CD117 positivity of one or more of the cultures by comparing the growth rate of the culture or cultures to the growth rate of a reference sample or reference value of known CD117+ expression. Clones that grow more rapidly than other clones may be selected for further expansion, characterization, and / or cryopreservation. Clones that express higher levels of CD117 positivity than other clones may be selected for further expansion, characterization, and / or cryopreservation. Further characterization may include, but is not limited to, secretome analysis (e.g., by ELISA, MSD, etc.), such as analysis for human VEGFA, SDF-1α, PDGFB, IGF-1, ANG-1, bFGF, SCF, and / or HGF; phenotypic characterization (e.g., by flow cytometry), such as analysis of cell surface expression of markers (e.g., mesenchymal stem cell markers CD105 or CD90, stem cell marker CD117, endothelial cell marker CD31, mast cell marker tryptase, and / or hematopoietic cell lineage marker CD45); senescence analysis; and / or characterization of function, e.g., angiogenesis, resistance to oxidative stress, and / or ability to promote therapeutic efficacy, for example, in an in vivo cardiac injury model. In some embodiments, such methods may further include immortalizing the isolated cells using methods known in the art or disclosed herein. Immortalization may be by any suitable means. In some embodiments, immortalization is by expression of telomerase. Illustratively, telomerase may be encoded by the human TERT (hTERT) gene. Human neonatal cardiac CSCs may be transfected with hTERT, infected with a recombinant virus capable of transferring the hTERT gene to cells, or delivered to CSCs using any suitable method known and available in the art.

[0075] In some embodiments, the present disclosure provides a method for approximating the level of CD117 positivity in a culture of neonatal CSCs or in a plurality of cultures of neonatal CSCs, the method comprising determining the proliferation rate of the cells in culture and comparing that rate to the proliferation rate of a reference sample or a known reference value of CD117+ expression.

[0076] In some embodiments, such methods for isolating CD117+ neonatal cardiac CSCs (disclosed herein) are utilized for therapeutic purposes, such as those disclosed in U.S. Patent Publication No. US2015 / 0328263, the entire contents of which are incorporated herein by reference, including, but not limited to, administering CD117+ or otherwise immortalized CSCs to a subject in need thereof; administering conditioned medium derived from immortalized CSCs to a subject in need thereof; and / or administering a combination of immortalized CSCs and conditioned medium derived from immortalized CSCs to a subject in need thereof. Such therapeutic purposes typically involve administering any of the above compositions to a subject in need thereof following immortalization of neonatal CSCs according to methods known in the art or disclosed herein.

[0077] Those skilled in the art will understand that in addition to the above-described method for isolating CD117+ CSCs from neonatal cardiac tissue, there are other routine methods for isolating desired nCSCs, and isolation can be performed by any suitable means. For example, a conventional method for isolating CD117+ adult CSCs involves exposing collected cells to a substrate comprising a surface having a moiety that binds to CD117, such as a CD117 antibody, to remove CD117+ cells from a population of cells present in a sample. In some embodiments, the present disclosure does not use such a method.

[0078] Those skilled in the art will also understand that there are routine methods for obtaining cells from human myocardium and then further processing the cells. In certain embodiments, methods exist for isolating desired human neonatal cardiac CSCs by obtaining tissue from human myocardium, including neonatal myocardium (e.g., obtained from the right atrial appendage or "RAA"), such as by biopsy. The myocardium may be from an individual without a known cardiac abnormality. The myocardium may be from an individual with end-stage heart failure or from an individual with congenital heart disease, in which case the myocardium may or may not be normal. The extracted tissue may be exposed to a specific medium while separating single cells from the tissue, including by cutting the tissue, such as in the presence of collagenase. In certain embodiments, the tissue and tissue fragments are allowed to settle in the medium, and a supernatant is obtained. Cells may be collected from the supernatant and suspended in culture medium for an appropriate period of time. Thereafter, the desired CD117+ human neonatal cardiac CSCs are isolated therefrom, for example. Isolation of the desired human neonatal cardiac CSCs may occur by any means disclosed herein or known in the art. In some embodiments, isolation is via a method disclosed herein that does not include exposing the collected cells to a substrate comprising a surface having a moiety that binds to CD117, such as a CD117 antibody.

[0079] In certain embodiments, once the desired human neonatal cardiac CSCs are isolated, they may be cultured under standard conditions, including appropriate subculture. The culture medium for the cells may or may not be substantially the same as the medium used when the cells are delivered to an individual. The medium may or may not contain one or more heat shock response inducers.

[0080] III. Methods of Using the Disclosed Cells and Their Conditioned Media and Secretomes The disclosed methods include the use of certain nCSCs (and, in certain embodiments, CD117+ mesenchymal-like cells), or the use of conditioned medium produced by culturing such cells, for therapy of at least one medical condition in an individual in need thereof. In some embodiments, the cells are isolated via the methods disclosed herein, and the isolation does not include exposing the collected cells to a substrate comprising a surface having a moiety that binds to CD117, such as a CD117 antibody, or a moiety that binds to any other surface protein. In some embodiments, the clonal cell isolates are immortalized (e.g., via hTERT immortalization). In certain embodiments, the cells, the conditioned medium produced by culturing the cells, or cells resuspended in those conditioned medium are useful for a medical condition, and producing an anti-inflammatory or anti-fibrotic effect and / or promoting tissue repair, regeneration, and / or replacement are therapeutically useful.

[0081] In certain embodiments, the medical condition is a cardiac medical condition. In certain embodiments, a therapeutically effective amount of the immortalized human neonatal cardiac CSCs, conditioned medium derived from immortalized human neonatal cardiac CSCs, or cells resuspended in the conditioned medium derived from immortalized human neonatal cardiac CSCs described herein is provided to an individual; in certain embodiments, the conditioned medium is delivered to the individual via a catheter, direct injection, or in some cases, locally to the area requiring treatment by local administration. In other embodiments, a therapeutically effective amount of the immortalized human neonatal cardiac CSCs, conditioned medium derived from immortalized human neonatal cardiac CSCs, or cells resuspended in the conditioned medium derived from immortalized human neonatal cardiac CSCs described herein is provided to an individual intravenously. The individual receiving therapy may be of any gender or age. An individual with a cardiac medical condition may or may not be diagnosed by a physician. In certain embodiments, the individual has a personal or family history of a cardiac medical condition. The individual may be at risk for a cardiac medical condition, such as smoking, high low-density lipoprotein (LDL) plasma levels and / or low high-density lipoprotein (HDL) plasma levels, uncontrolled high blood pressure, obesity (more than 20% of an individual's ideal body weight), uncontrolled diabetes, high C-reactive protein plasma levels, or a combination thereof. In certain embodiments, upon diagnosis of a particular genotype or phenotype for the individual, the individual is provided with one or more methods of the present disclosure.

[0082] Upon isolation from a source individual of desired cells, e.g., via the methods disclosed herein, where the isolation does not involve exposing the collected cells to a substrate comprising a surface bearing a moiety that binds CD117 (i.e., c-kit) or another cell surface protein, such as a CD117 antibody, prior to their delivery to an individual in need thereof or delivery of conditioned medium derived from the cells to the individual, the cells may be further modified, e.g., through genetic engineering for recombinant expression of one or more expression constructs, further culturing and / or enrichment. Such further modification may also include immortalization of the cells as described herein. Such practice is routine in the art. The expression construct introduced into the cells may be of any type, although in certain embodiments, the construct expresses a cytokine, a pro-angiogenic factor, a growth factor, a transcription factor, or the like. In certain embodiments, the construct expresses VEGF-A, HGF, SCF, SDF-1α, ANG-1, HSF-1, PGDF-A, FGF-2, or a combination thereof, and / or another protein that directly or indirectly increases their expression. The cells may be exposed to one or more agents to increase the secretion and / or expression of certain proteins.

[0083] In certain embodiments, a therapeutically effective amount of the cells described herein is utilized in one or more treatment methods, although in some preferred embodiments, the individual instead receives a therapeutically effective amount of conditioned medium derived from the cells; a portion of the entire secretome derived from the cells; one or more secreted proteins or other factors (e.g., exosomes, extracellular vehicles, miRNAs, etc.) derived from the cells; or a combination thereof. In some embodiments, the conditioned medium derived from the cells is administered to the individual in combination with one or more human neonatal cardiac CSCs. The use of any one of these components or combinations, in certain embodiments, promotes the proliferation of endogenous cardiomyocytes upon their use. In certain embodiments, the use of any one of these components or combinations enables myocardial repair, regeneration, or remodeling. In certain embodiments, the use of any one of these components or combinations promotes or enhances the proliferation, repair, regeneration, or remodeling capacity of endogenous CSCs in the treated individual.

[0084] In certain embodiments, a therapeutically effective amount of cells, conditioned medium produced by culturing cells (or immortalized human neonatal cardiac CSCs), or cells resuspended in such conditioned medium may be administered multiple times over a period of hours, days, weeks, or months. The therapeutically effective dose may be increased or decreased over the course of successive administrations.

[0085] In certain embodiments, when cells are provided to an individual, they may be provided in conjunction with (in the same or a different composition) or simultaneously with another therapeutic moiety. That is, in certain embodiments, the cells are administered to an individual substantially simultaneously with one or more agents that enhance the function of the cells upon administration in vivo. Such agents may be of any type, but in certain embodiments, the agents are one or more heat shock response inducers.

[0086] While in some cases, one or more agents are provided to an individual substantially simultaneously with the cells, in certain embodiments, the cells are exposed to one or more agents prior to their delivery to the individual, where the agents enhance the function of the cells upon in vivo delivery. In certain embodiments, the cells are exposed to one or more agents during culture. The cells may be exposed to the agents once or multiple times, and the agents may or may not be present in the subsequent culture medium when the cells are passaged in culture. In certain embodiments, the agents enhance the therapeutic use of the cells by increasing the expression of one or more genes and increasing the secretion of one or more proteins or other factors (such as miRNAs), or a combination thereof. In certain embodiments, the genes or proteins are cytokines, pro-angiogenic factors, growth factors, transcription factors, miRNAs, and exosomes (small transport vehicles containing concentrated proteins and miRNAs), etc. In certain embodiments, the agent is a heat shock response inducer.

[0087] In one embodiment, a specific therapeutically effective number of cells is provided to an individual, e.g., in some embodiments, less than 100 million cells, e.g., 1 to 40 million or 1 to 50 million cells, are provided to an individual. In certain embodiments, between 1 million and 20 million cells are provided, although in some embodiments, the number to the millionth place of the cells is 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6- 19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12 , 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19,14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20, etc.

[0088] In certain embodiments, proteins, miRNAs, or any other factors can be isolated from the conditioned medium produced by culturing the immortalized human neonatal cardiac CSCs disclosed herein. In certain embodiments, the secretome itself or one or more components of the secretome, such as proteins and exosomes, from the conditioned medium are provided to an individual. Exosomes themselves have very strong repair and / or regenerative capabilities and may be provided therapeutically to an individual rather than to cells. In certain embodiments, the conditioned medium is provided therapeutically to an individual rather than to cells.

[0089] IV. Cardiac Medical Conditions Embodiments of the present disclosure relate to the treatment of one or more cardiac medical conditions using conditioned medium produced from the culture of immortalized neonatal CSCs described herein and / or neonatal cardiac CSCs isolated via the methods disclosed herein. Certain aspects of such embodiments result in the reversal of one or more cardiac medical conditions or the amelioration of at least one symptom of one or more cardiac medical conditions. In exemplary embodiments, the cardiac medical condition is heart failure. Heart failure can be the result of one or more causes, including coronary artery disease, heart attack, high blood pressure, heart valve disorders, cardiomyopathy (e.g., caused by disease, infection, alcohol abuse, and the toxic effects of drugs such as cocaine or some drugs used in chemotherapy), idiopathic cardiomyopathy, congenital heart disease, and / or genetic factors.

[0090] Specific, but exemplary, indications for embodiments of the present disclosure include at least heart failure, including congestive heart failure; prevention of ventricular remodeling; and / or application for cardiomyopathy. Other indications may also include coronary artery disease, ischemic heart disease, valvular heart disease, stroke secondary to structural heart intervention, and the like. In certain embodiments, the methods and compositions of the present disclosure provide myocardial repair and / or regeneration sufficient to treat, including reversing, established cardiac medical conditions such as cardiomyopathy or congestive heart failure.

[0091] When an individual has heart failure, the patient may have preserved or reduced ejection fraction (EF). EF is characterized by microcirculatory rarefaction and extensive myocardial fibrosis, and previous research using an isoproterenol-induced cardiomyopathy model has shown that CSCs improve cardiac function in rodent models (Sharma et al., 2017). Therefore, both can be improved with a therapeutically effective amount of cells, conditioned medium derived from immortalized human neonatal cardiac CSCs, or cells resuspended in conditioned medium derived from immortalized human neonatal cardiac CSCs.

[0092] If an individual has cardiomyopathy, the cardiomyopathy may be ischemic or non-ischemic. Cardiomyopathy may be caused by long-term high blood pressure, heart valve problems, heart tissue damage from a previous heart attack, chronic palpitations, metabolic disorders, nutritional disorders, pregnancy, alcohol abuse, drug abuse, chemotherapy drugs, viral infections, hemochromatosis, genetic conditions, elevated cholesterol levels, or a combination thereof. Cardiomyopathy may also have an unidentified cause, i.e., idiopathic cardiomyopathy.

[0093] A therapeutically effective amount or number of cells, conditioned medium derived from immortalized human neonatal cardiac CSCs, or cells resuspended in conditioned medium derived from immortalized human neonatal cardiac CSCs, can prevent chemotherapy-induced cardiotoxicity. The disclosed invention may also be used for the extension of organ preservation during transport in organ transplant surgery.

[0094] V. Inflammatory diseases Embodiments of the present disclosure provide compositions comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditioned medium derived from neonatal cardiac CSCs, or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned medium derived from c-neonatal cardiac CSCs or immortalized human neonatal CSCs, each of which has anti-inflammatory and anti-apoptotic properties and can be used to treat one or more diseases in which a key component of the disease process involves an inflammatory process. Non-limiting examples of diseases that contain an inflammatory component and can be treated with such compositions of the present disclosure include kidney damage (both acute kidney injury and chronic kidney disease), ischemic stroke, arthritis, dry eye, neurodegenerative diseases including Parkinson's disease, critical limb ischemia, COVID-19, and other inflammatory diseases. Thus, in some embodiments, the present disclosure provides compositions comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal CSCs for use in treating inflammatory diseases. In some embodiments, the present disclosure provides a composition comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, for use in treating a disease selected from renal disorders (both acute kidney injury and chronic kidney disease), ischemic stroke, arthritis, dry eye, neurodegenerative diseases, Parkinson's disease, and critical limb ischemia. In some embodiments, the present disclosure provides a method of treating an inflammatory disease, comprising administering to a subject in need thereof an effective dose of a composition comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, neonatal cardiac CSCs, or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs.In some embodiments, the present disclosure provides methods for treating kidney damage (both acute kidney injury and chronic kidney disease), ischemic stroke, arthritis, dry eye, neurodegenerative disease, Parkinson's disease, or critical limb ischemia, comprising administering to a subject in need thereof an effective dose of neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, and a composition comprising neonatal cardiac CSCs resuspended in conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs.

[0095] VI. Wound healing Fibrosis and inflammation are two important pathways in wound healing. Accordingly, some embodiments of the present disclosure provide compositions comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned medium derived from neonatal cardiac CSCs or immortalized neonatal CSCs for use in treating inflammatory diseases, each of which can be used to treat wounds and promote wound healing. In certain embodiments, proteins, miRNAs, or any other factors in the conditioned medium produced by culturing the immortalized neonatal CSCs disclosed herein can be isolated, and such isolated proteins, miRNAs, or factors can be administered to a subject with a wound to treat the wound. In certain embodiments, immortalized neonatal CSCs, conditioned medium, the secretome itself, or one or more components of the secretome, such as proteins and exosomes, responsible for regulating tissue fibrosis and cellular functions such as proliferation, migration, and matrix synthesis, can be used to promote wound progression and separation.

[0096] VII. Combination Therapy In some embodiments, an individual who has received, or is receiving, or will receive a treatment of the present disclosure is also provided with another therapy for the target medical condition. For example, in some embodiments, an individual who has received, or is receiving, a therapy of the present disclosure for treating a cardiac medical condition is also provided with another therapy for the cardiac medical condition. The therapy of the present disclosure may precede or follow the other therapy. The therapy of the present disclosure may precede or follow the other therapy by intervals ranging from minutes to hours, days, weeks, or months. In embodiments in which the other agent and the immediate therapy are administered separately to an individual, it is generally ensured that a significant period of time does not expire between the time of each delivery so that the therapy of the present disclosure and the additional therapy can still exert a beneficial combined effect on the individual. In such instances, it is contemplated that both modalities may contact the individual, for example, simultaneously, or within minutes of each other, or within about 1-12, 6-12, or 12-24 hours of each other. In some circumstances, it may be desirable to extend the duration of treatment significantly, with several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between each administration.

[0097] In certain embodiments, the therapy of the present disclosure and the additional therapy are provided simultaneously. In certain embodiments, the therapy of the present disclosure and the additional therapy are provided at different times. The separate entities may be in the same composition, or they may be included in separate compositions. When the therapy of the present disclosure and the second therapy are provided at different times, they may be separated by any suitable time period, such as minutes, hours, days, weeks, or months. In embodiments in which they are provided separately, the order of delivery of the two (or more) therapies may be any suitable order, including delivery of cells, secretome, and / or conditioned medium before, simultaneously with, or after another therapy.

[0098] Examples of other treatments utilized in conjunction with the therapies of the present disclosure include one or more of the following: ACE inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs); beta blockers, calcium channel blockers, cholesterol-lowering drugs, digoxin, diuretics, inotropic therapy, potassium or magnesium, vasodilators, anticoagulant medications, aspirin, surgery, VAD implantation, VAT, coronary artery bypass, percutaneous coronary intervention (PCI), or combinations thereof.

[0099] VIII. Kits of the Present Disclosure Any of the immortalized neonatal cardiac CSCs described herein or conditioned medium derived from such CSCs may be included in the kit, which may additionally include other agents for the treatment of cardiac medical conditions.

[0100] The components of the kit may be packaged either in aqueous medium or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, and preferably, appropriately dispensed. Where more than one component is present in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be separately disposed. However, various combinations of components may be included in vials. The kits of the present disclosure will also typically include means for containing one or more compositions in close confinement for commercial sale. Such containers may include syringes or blow-molded plastic containers into which the desired vials are retained. In certain embodiments, the cells are delivered in a frozen state and may or may not be provided in plastic vials.

[0101] The compositions may be formulated into syringeable compositions. Where the container means may itself be a syringe, pipette, and / or other such similar device, the formulation may then be applied to an affected area of ​​the body, injected into an animal, and / or applied to and / or mixed with other components of the kit. However, the components of the kit may also be provided as dry powders. When reagents and / or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in a separate container means.

[0102] The kits of the present disclosure will also typically include a means for containing the vials in close confinement for commercial sale, such as, for example, a syringe and / or blow-molded plastic container into which the desired vials are retained.

[0103] In certain embodiments, the kit includes reagents and / or tools for determining that an individual has a cardiac medical condition. In some embodiments, the kit includes one or more additional therapies for the cardiac-related medical condition, such as one or more of an ACE inhibitor, an aldosterone inhibitor, an angiotensin II receptor blocker (ARB); a beta-blocker, a calcium channel blocker, a cholesterol-lowering drug, digoxin, a diuretic, an inotropic therapy, potassium, magnesium, a vasodilator, an anticoagulant medication, aspirin, a TGF-beta inhibitor, and combinations thereof.

[0104] VII. Pharmaceutical Compositions

[0013] Embodiments of the pharmaceutical compositions of the present disclosure comprise an effective amount of neonatal cardiac CSCs or conditioned medium from such cells dispersed in a pharmaceutically acceptable carrier. The effective amount of neonatal CSCs may comprise any suitable number of cells. In some embodiments, the effective amount is less than 100 million cells, e.g., 1 to 40 million or 1 to 50 million cells. In some embodiments, an effective amount comprises between 1 million and 20 million cells, and in some embodiments, the number to the millionth place of the cells is 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, , 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12 , 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-1 0, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15,and the like. For example, in one specific embodiment, the pharmaceutical composition of the present disclosure comprises 10 million neonatal cardiac CSCs dispersed in a pharmaceutically acceptable carrier. In one specific embodiment, the pharmaceutical composition of the present disclosure comprises 10 million neonatal CD117+ CSCs and conditioned medium from the cultured neonatal CSCs. In some embodiments, the cultured neonatal cardiac CSCs from which the conditioned medium is collected are immortalized neonatal human neonatal cardiac CSCs.

[0105] In one specific embodiment, the pharmaceutical composition of the present disclosure comprises an effective amount of neonatal CSCs (e.g., 10 million cells) and an enriched preparation of secreted factors present in the conditioned medium from the cultured neonatal CSCs. Such an enriched preparation of secreted factors may be prepared, for example, by filtering the conditioned medium from the cells to remove some or all of the medium while retaining all or a portion of the secreted factors present in the medium. Such enriched factors may be stored in pellet form. Such enriched factors may be resuspended in a pharmaceutically acceptable carrier, excipient, diluent, surfactant, and / or vehicle for storage or administration to a subject. The enriched factors may be resuspended in a pharmaceutically acceptable carrier, excipient, diluent, surfactant, and / or vehicle alone or in combination with a neonatal cardiac CSC population (e.g., an effective dose of CSCs). In some embodiments, the cultured neonatal CSCs from which the conditioned medium is recovered to prepare the enriched preparation of secreted factors are immortalized neonatal CSCs. In some embodiments, the CSCs administered to a subject and / or contained in the pharmaceutical compositions disclosed herein are not immortalized.

[0106] The conditioned medium may be formulated for administration to a subject. In some embodiments, bioactive factors present in the conditioned medium are concentrated (e.g., by filtration of the conditioned medium), and the bioactive factors are reformulated in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, e.g., a human, as appropriate. The preparation of pharmaceutical compositions comprising cells is known to those skilled in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Furthermore, it will be understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0107] As used herein, "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and similar substances and combinations thereof, known to those skilled in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.

[0108] In some embodiments, the present disclosure provides a pharmaceutical composition comprising one or more human neonatal cardiac CSCs isolated according to the methods disclosed herein, wherein the composition further comprises one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles. For example, in some embodiments, the composition comprises a plurality of human neonatal cardiac CSCs isolated according to the methods disclosed herein formulated in PlasmaLyte (Baxter, Deerfield, IL). In some embodiments, the human neonatal cardiac CSCs are immortalized. In some embodiments, the immortalization is via hTERT expression.

[0109] In some embodiments, the present disclosure provides a pharmaceutical composition comprising conditioned medium from a culture of human neonatal cardiac CSCs isolated according to the methods disclosed herein, wherein the composition further comprises one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles. The conditioned medium may be formulated for direct administration to a subject. In certain embodiments, the conditioned medium is concentrated for bioactive factors by filtration, and the concentrated factors are reformulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles. For example, in some embodiments, the composition comprises concentrated bioactive factors from conditioned medium from a culture of a plurality of neonatal cardiac CSCs isolated according to the methods disclosed herein, formulated in PlasmaLyte (Baxter, Deerfield, IL). In some embodiments, the human neonatal cardiac CSCs are immortalized. In some embodiments, the immortalization is via hTERT expression. [Example]

[0110] The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art should understand that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and can therefore be considered to constitute preferred modes for its practice. However, those of skill in the art will appreciate that, in light of this disclosure, many changes can be made in the specific embodiments of this disclosure and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0111] Example 1: Isolation of neonatal CD117-positive cardiac progenitor cells Cardiac medical conditions, including heart failure, and inflammatory diseases require effective therapies for patients of all ages, including infants, children, and adults. In particular, compositions and methods for regenerating functional myocardium are needed in adults and children with cardiac medical conditions, such as heart failure, caused by damaged myocardial tissue.

[0112] This example describes a novel method for isolating and immortalizing CD117+ nCSCs that can be used to produce conditioned medium that can be administered to patients in need to treat damaged myocardium.

[0113] We have found that, in contrast to adult human hearts, neonatal human hearts contain a homogenous population of cardiogenic CD117+ stem cells with little or no contamination from hematopoietic and endothelial progenitor cells. Furthermore, we have found that these neonatal human cardiac-derived stem cells give rise to clones that show no evidence of senescence and, when tested in an animal model of myocardial infarction, can proliferate and expand to large cell numbers with significant cardiac repair and regeneration activity. Based in part on these findings, we have determined that these neonatal cardiac stem cells can be obtained directly from neonatal cardiac tissue without prior selection with CD117 magnetic beads, as previously described in the literature. This represents a significant advantage in that it avoids regulatory concerns regarding exposing cells intended for therapy to non-GMP-quality materials (i.e., CD117 magnetic beads).

[0114] Here, we describe the methodology and certain data regarding the isolation and functional characterization of human neonatal cardiac stem cell clones obtained without CD117 antibody enrichment.

[0115] This study was approved by the Institutional Review Board and Animal Care and Use Committee (IACUC) of the University of Maryland School of Medicine. After obtaining parental or patient consent, right atrial appendage (RAA) specimens (20 ± 40 mg) were obtained from neonates (1–30 days of age) during routine cardiac surgery. 1. The RAA tissue was transferred to a 100 mm Petri dish filled with saline and washed. This process was repeated twice. Fibrotic tissue and fat were removed from the cardiac specimen using forceps sterilized with Steri 250 (Inotech). The specimen was then transferred to Ham's F12 medium and separated by 1-2 mm. 2 The tissue was cut into slices. 2. The tissue fragments were transferred to a 50 ml tube and allowed to settle. The supernatant was removed, and the sediment was resuspended in 5-10 ml of collagen type II CSL2 (Worthington #4177). Collagenase was dissolved in Ham's F12 medium at a concentration of 1-2 mg / ml, depending on the tissue size and type. The samples were then incubated on a shaker at 200 rpm, 37°C, for 30-45 minutes. After collagenase treatment, the tube was removed from the shaker, undigested debris was allowed to settle, and the supernatant containing the released cells was centrifuged at 1000 rpm for 10 minutes at 15°C. The cells were resuspended in growth medium (Ham's F12 nutrient mixture supplemented with 10% FBS, 0.2 mM L-glutionone, 10 ng / ml bFGF, and 0.005 U / ml EPO) and seeded into a T25 flask containing growth medium. The flask was placed in an incubator at 37°C and 5% CO2. After 72 hours, non-adherent cells were removed by aspiration, and adherent cells were washed with PBS and fresh growth medium was added. When the cells reached 90-95% confluence, the growth medium was removed and the cells were detached using 3 ml of TrypLE™. After cell detachment, growth medium was added, the cell suspension was transferred to a 50 ml test tube, and centrifuged at 1000 rpm for 10 minutes at 15°C. The supernatant was discarded, and the cell pellet was collected and a cell count was obtained. 4. As shown in Figure 1, 10,000 cells were used for serial dilution to obtain single cell isolates of clones. Approximately 8 cells / mL were used to obtain single cell isolates. 50 μl of the 8 cell / mL suspension was dispensed into individual wells in a 96-well plate. Wells with more than one cell were excluded from the experiment by visual inspection under a microscope. 5. Cell numbers were manually counted and the population doubling level (PDL) was calculated using the formula: PDL = 3.32 (log (total cells at harvest / total cells at seeding)). Clones were subcultured until they reached senescence, and no change in PDL was observed from one subculture to the next.

[0116] Of the 80 wells in total, 24 wells containing a single cell were detected. Of the 24 seeded single cells, 18 formed actively growing clones (cloning efficiency 75%). Figure 2 shows a representative image of a portion of the single-cell culture.

[0117] Growing clones were fixed with 4% paraformaldehyde and labeled with fluorochrome-conjugated primary antibodies specific for mesenchymal stem cell markers CD105 and CD90, stem cell marker CD117, endothelial cell marker CD31, mast cell marker tryptase, hematopoietic cell lineage marker CD45, and CD44 and CD47, and evaluated by flow cytometry on a Becton-Dickinson Fortessa, collecting 10,000 events / sample.

[0118] FACS analysis showed that all 18 growing clones were CD117 positive, but only 7 were highly (>80%) CD117 positive (Fig. 3 ), and the remaining 11 clones were weakly positive (<74%).

[0119] Furthermore, these high CD117-positive clones proliferated approximately two-fold faster than low CD117-positive clones (data not shown) and were negative for CD31 and CD45.

[0120] Taken together, these results show that, in contrast to animal- and adult human cardiac-derived CD117+ cells, human neonatal cardiac CD117+ cells surprisingly avoid contaminating endothelial and hematopoietic progenitor cells, which is not only biologically unique and unexpected, but also in line with current practice in the literature.

[0121] Example 2: Immortalization of neonatal CD117-positive cardiac progenitor cells Neonatal CD117+ CSCs secrete proteins that induce repair, regeneration, and / or remodeling of damaged myocardium and improve cardiac function. Immortalization of these CD117+ neonatal CSCs allows for the production of an unlimited supply of conditioned medium containing such secreted proteins. This example provides such immortalization.

[0122] Neonatal CD117+ CSC clones are isolated according to the methods disclosed above in Example 1, and the clones are expanded in culture. Typically, clones expressing high levels of CD117 were immortalized, although in some cases, clones expressing low levels of CD117 were also immortalized.

[0123] Immortalization was achieved by transfection of human neonatal CD117+ cardiac CSC clones with a lentiviral vector expressing hTERT, and the hTERT-expressing clones were isolated, expanded, and stored for long-term use. Figure 4 shows non-limiting examples of constructs for use in immortalizing neonatal CD117+ human neonatal cardiac CSCs according to the present invention.

[0124] Additionally, reversibly immortalized neonatal CD117+ CSC clones can be constructed by flanking the immortalization gene shown in Figure 4 with sequences that can be used for gene excision, for example, by the method described in Hu, X., et al., Oncotarget, 2017, Vol. 8, (No. 67), pp: 111847-111865, which is incorporated herein by reference in its entirety. In one example, the immortalization gene is flanked by FRT sites, one 5' to the CMV promoter and one 3' to the puromycin resistance cassette. FLP recombinase may then be used to promote excision of the immortalization cassette, thereby reversing immortalization. In addition to their utility for producing large quantities of conditioned medium, such cells can be used for direct administration to patients, optionally after reversing immortalization prior to delivery of the cells to the patient, combined with conditioned medium made from the cells.

[0125] Killing switch control is optionally incorporated into the vector, for example, by incorporating the thymidine kinase gene of herpes simplex virus (HSV-TK). HSV-TK phosphorylates the prodrug ganciclovir (GCV), a guanosine nucleoside analog. The phosphorylated GCV is incorporated into host DNA, terminating DNA chain elongation and thereby causing cell death. Therefore, treatment of patients administered with such reversibly immortalized neonatal cardiac CSCs with GCV results in the death of administered cells expressing HSV-TK, ensuring the safety of the drug. Other killing switches are known in the art and may be used in the constructs disclosed herein.

[0126] Example 3 Characterization of neonatal CSCs and their culture medium Telomeres and telomere length To calculate the telomere length of human neonatal cardiac CSC clones, flow cytometry analysis was performed using fluorescent in situ hybridization and a fluorescein-conjugated PNA probe (Telomere PNA Kit / FITC) for flow cytometry from Dako (Cat. No. K5327). Cell line 1301, which is tetraploid and has long telomeres (>30 kbp), was used as a control. Relative telomere length (RTL) was calculated using the following formula:

number

[0127] b. Senescence-associated β-galactosidase staining Cell senescence is assessed using a β-galactosidase staining kit (Cat. No. 9860, Cell Signaling Technology, Boston, MA) according to the manufacturer's instructions. Briefly, human neonatal cardiac CSCs (5.0 × 10) were cultured before and after immortalization. 4) are seeded into 24-well plates. After 24 hours, the growth medium is removed from the cells, washed with PBS, and fixed with 1x fixation solution at room temperature for 15 minutes. The cells are incubated overnight with 1 ml of β-galactosidase staining solution and imaged the following day. The results show that these neonatal human cardiac-derived stem cells show no evidence of senescence (Figure 5).

[0128] C. paracrine factor secretion Immortalized neonatal CSCs were grown in completely xeno-free medium until they reached 85-90% confluence. Cells were washed twice with warm, serum- and growth factor-free Ham's F12 medium, then added with Ham's F12 and incubated at 37°C for 48 hours to obtain total conditioned medium (TCM). Cell debris and particulate matter were removed from TCM by centrifugation at 1,000 x g for 30 minutes, followed by 20,000 x g for 30 minutes to remove microparticles (MVs). The TCM was then concentrated using a 3 KDa filter (Millipore Inc., Billerica, MA). Total protein content was quantified using the TCA-NLS method followed by the bicinchoninic assay (BCA) method (Thermofisher, Waltham, MA). The appearance of im-nCSC-derived TCM was clear and transparent, and it contained no apoptotic bodies (Figure 8). IM-nCPC TCM is dsDNA-free and has a concentration of 0.2-0.4ng / ml (maximum allowable 200ng / ml). TCM is 90-99.0% CD63 + and are negative for CD45 and CD31 (Figure).

[0129] To normalize the protein content, we used the following formula: The following formula was used: (concentration factor) x (total volume of medium) / total protein content of conditioned medium.

[0130] Conditioned medium was quantified using the TCA-NLS method followed by the BCA method and normalized to 1 mg of total protein. Eight paracrine factors, VEGFA, SDF-1α, PDGFB, IGF-1, ANG-1, bFGF, SCF, and HGF, were analyzed using the Mesoscale Discovery Device according to the manufacturer's protocol. Results show that Im-nCSC TCM secretes all eight paracrine factors at the levels shown in Table 1. [Table 1]

[0131] Example 4 - Functional activity of immortalized neonatal CSCs and their culture medium a. In vitro activity a1.Angiogenic activity To test the proangiogenic effect of TCM on blood vessel formation, HMEC cells were subjected to a standard in vitro angiogenesis assay using Im-nCPC-derived TCM (Im-nCSC TCM) and IMDM basal medium as a negative control and HMEC complete medium as a positive control. Briefly, a tube formation assay was performed to evaluate the angiogenic potential of CM. The formation of tube-like structures was assessed in Matrigel-coated 24-well plates (BD Biosciences, San Jose, CA) as previously described. Briefly, human microvascular endothelial cells (HMEC-1, ATCC® CRL-3243™) were counted and seeded at a density of 20,000 cells / mm on reduced growth factor containing Matrigel (Product No. 354230, BD Biosciences, San Jose, CA) and added (i) endothelial complete cell medium (Lonza) as a positive control, (ii) Im-nCSC-derived conditioned medium, or (iii) basal medium as a negative control. After 6–12 h, cells were imaged and a complete image of each well was reconstructed. Total tube length was then measured using ImageJ64, NIH (http: / / rsb.info.nih.gov / ij).

[0132] The results showed that the ability of HMECs to form complex and mature endothelial tube networks was absent in IMDM, but in the presence of Im-nCSC TCM, HMECs formed mature tubes as shown in Figure 10 .

[0133] a2. Wound healing activity To evaluate the wound healing ability of Im-nCSC TCM, an in vitro wound healing assay was performed to assess the relative migration ability of cells treated with total conditioned medium. HMECs (HMEC-1 ATCC® CRL-3243™) were seeded in 12-well plates to generate confluent monolayers. After 12 hours of serum starvation with basal medium, a linear scratch was made along the cell monolayer using a 1 mL pipette tip to simulate a wound. Cell debris was removed by washing the cells once with basal medium. Cells were treated with total conditioned medium derived from Im-nCSCs, PBS as a negative control, and VEGF-A (3.0 μg / μl) as a positive control. Images of each wound were taken at specific reference points along the scratch at 0 and 22 hours after treatment. HMECs were stained with calcein AM cell-permeable dye (ThermoFisher Scientific, Inc.) and imaged before and after treatment with conditioned medium obtained from Im-nCSC TCM. ImagePro software was used to measure the total wound area before and after treatment, and the percent change in wound closure was calculated. Results showed that Im-nCSC-derived TCM significantly increased the wound healing process compared to the negative control (Figure 11).

[0134] a3.Protection against H2O2-induced cell apoptosis Assessment of apoptosis in response to oxidative stress is performed using the Annexin V Apoptosis Detection Kit (Cat. No. 556547, BD Pharmingin). Briefly, neonatal rat cardiomyocytes (NRCM) were purchased from Lonza Walkersville, Inc. (RCM-561) and cultured according to the manufacturer's instructions. Briefly, all components of rat cardiomyocyte growth medium (RCGM) were thawed overnight in a cold room and mixed. Ten wells of a 24-well plate were coated with a nitrocellulose / methanol mixture (0.1 cm2 of nitrocellulose dissolved in 1.0 ml of methanol), and each vial of rat cardiomyocytes was suspended in 10 ml of complete RCGM. 1 ml of cell suspension was added to each well (3 × 10 cells) after overnight incubation. 5 Im-nCSC TCM were transferred to a 500-well plate (100 cells / well) and treated with 100 μM hydrogen peroxide in serum-free basal medium in the presence or absence of Im-nCSC-derived conditioned medium (50 μM) for 6 hours (n = 4 technical replicates), followed by flow analysis for annexin V / PI using the BD Pharmingen FITC Annexin V Apoptosis Detection Kit I (catalog no. 556547). The data showed that Im-nCSC TCM significantly reduced the expression of the early apoptotic marker annexin V (Figure 9). These results indicate that Im-nCSC-derived TCM reduced oxidative stress-induced apoptosis.

[0135] b. In vivo activity b1. Cell transplantation in a rat myocardial infarction (MI) model Myocardial infarction was induced in immunodeficient male rats (body weight, 250-300 g) by permanent ligation of the left anterior descending (LAD) coronary artery. The heart was exposed via a left thoracotomy, and the proximal LAD was ligated. One million nCSC and Im-nCSC cells suspended in 100 μL of vehicle (IMDM) were then injected into the myocardium at four adjacent sites of the infarction. A baseline echocardiogram was obtained 1 day before the myocardial infarction surgery. Echocardiography was also performed 7 and 28 days after myocardial infarction. Two-dimensional and M-mode echocardiography was performed using a VisualSonics Vevo 2100 ultrasound unit (VisualSonics, Toronto, Canada, www.visualsonics.com) to evaluate fractional area change (FAC). Images were acquired from the parasternal long axis and parasternal short axis at the mid-papillary level. Myocardial viability was assessed as follows: To calculate infarct size, Masson's trichrome-stained sections at various levels along the longitudinal axis were analyzed for collagen deposition. The midline technique for determining infarct size was used as described above. Stained sections were analyzed using ImagePro software. Briefly, infarct size was calculated using Masson's trichrome-stained sections at various levels along the longitudinal axis. To calculate viable and non-viable tissue, the number of red pixels (viable tissue) and blue pixels (non-viable tissue) was counted, and the ratio of non-viable tissue to total number of pixels was shown. The data showed that both nCSCs and nCSC-derived immortalized clones were functional, as shown in the figure, by a significant increase in ejection fraction and a shortening of the ejection fraction after transplantation of nCSCs or nCSC-derived immortalized clones. These results suggest that immortalization did not affect the function of the nCSC-derived clones (Figure 7).

[0136] b2. Functional activity of Im-nCPC-derived TCM in a rat MI model To determine the function of Im-nCSC-derived total conditioned medium (TCM), rats were subjected to anterior septal MI by LAD ligation with a suture. Echocardiography was performed with the rats anesthetized and in the supine position, with the ultrasound probe placed directly on the chest wall. Prior to MI, the left ventricular ejection fraction (LVEF) was approximately 80%. Approximately 10 minutes after MI, treatments (nCSCs, Im-nCSC-derived TCM, and IMDM) were administered intravenously via the tail vein. 24 hours later, cardiac function was assessed by baseline echocardiography. Five days later, the treatment groups were administered another dose of treatment. Animals injected with nCSCs and Im-nCSC-derived TCM showed no significant deterioration in LV function from 2 days post-MI to 4 weeks post-MI. At 4 weeks, LVEF was significantly higher in the nCSC-treated group than in the placebo group (Figure 12). Other parameters, including fractional shortening (FS) and reduced end-systolic volume (ESV), also significantly improved compared to the placebo group. Other LV function parameters, including cardiac output / mass and posterior wall thickness, also improved and trended toward normal remodeling. The benefits of nMSCs were significantly sustained through the 4-week endpoint (Figure 12).

[0137] equivalent While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and other variations will be apparent to those skilled in the art. All such alternatives, modifications, and variations are intended to fall within the spirit and scope of the present invention. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from various patents, applications, and publications to provide still further embodiments. These and other changes can be made to the embodiments in light of the description set forth above. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the scope of the claims is not limited by the present disclosure. References Roger V L.Epidemiology of heart failure.Circ Res.2013;113:646-659 Go A S,Mozaffarian D,Roger V L,Benjamin E J,Berry J D,Blaha M J,Dai S,Ford E S,Fox C S,Franco S,Fullerton H J,Gillespie C,Hailpem S M,Heit J A,Howard V J,Huffman M D,Judd S E,Kissela B M,Kittner S J,Lackland D T,Lichtman J H,Lisabeth L D,Mackey R H,Magid D J,Marcus G M,Marelli A,Matchar D B,McGuire D K,Mohler E R,3rd,Moy C S,Mussolino M E,Neumar R W,Nichol G,Pandey D K,Paynter N P,Reeves M J,Sorlie P D,Stein J,Towfighi A,Turan T N,Virani S,Wong N D,Woo D,Turner M B.Heart disease and stroke statistics-2014 update:A report from the american heart association.Circulation.2014;129:e28-e292 Rossano J W,Kim J,Decker J A,Price J F,Zafar F,Graves D E,Morales D L,Heinle J S,Bozkurt B,Towbin J A,Denfield S W,Dreyer W J,Jefferies J L.Prevalence,morbidity,and mortality of heart failure-related hospitalizations in children in the united states:A population-based study.Journal of cardiac failure.2012;18:459-470 Porrello ER,Mahmoud AI,Simpson E,Hill JA,Richardson JA,Olson EN,Sadek H A.Transient regenerative potential of the neonatal mouse heart.Science.2011;331:1078-1080 Messina E, De Angelis L, Frati G, Morrone S, Chimenti S, Fiordaliso F, Salio M, Battaglia M, Latronico MV, Coletta M, Vivarelli E, Frati L, Cossu G, Giacomello A. Isolation and expansion of adult cardiac stem cells from human and murine heart. Circulation research. 2004;95:911-921 Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003;114:763-776 Smith R,Barile L,Cho HC,Leppo MK,Hare JM,Messina E,Giacomello A,Abraham MR,Marban E.Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens.Circulation.2007;115:896-908 Matsuura K,Nagai T,Nishigaki N,Oyama T,Nishi J,Wada H,Sano M,Toko H,Akazawa H,Sato T,Nakaya H,Kasanuki H,Komuro I.Adult cardiac sca-1-positive cells differentiate into beating cardiomyocytes.The Journal of biological chemistry.2004;279:11384-11391 He J Q,Vu D M,Hunt G,Chugh A,Bhatnagar A,Bolli R.Human cardiac stem cells isolated from atrial appendages stably express c-kit.PloS one.2011;6:e27719 Stolzing A,Sethe S,Scutt A M.Stressed stem cells:Temperature response in aged mesenchymal stem cells.Stem cells and development.2006;15:478-487 Mishra R,Vijayan K,Colletti E J,Harrington D A,Matthiesen T S,Simpson D,Goh S K,Walker B L,Almeida-Porada G,Wang D,Backer C L,Dudley S C,Jr.,Wold L E,Kaushal S.Characterization and functionality of cardiac progenitor cells in congenital heart patients.Circulation.2011;123:364-373 Sharma ,Mishra R,Bigham E.G,Wehman B,Khan M,Xu H,Saha P,Goo Ah.G,Datla R.S,Chen L,Tulapurkar E.M,Taylor S.B,Yang P, Karathanasis S,Goodlett R D,Kaushal S.A Deep Proteome Analysis Identifies the Complete Secretome as the Functional Unit of Human Cardiac Progenitor Cells.Circulation Res.2017;120:816-834 Bu L,Jiang X,Martin-Puig S,Caron L,Zhu S,Shao Y,Roberts D J,Huang P L,Domian I J,Chien K R.Human isl1 heart progenitors generate diverse multipotent cardiovascular cell lineages.Nature.2009;460:113-117 Bolli R,Chugh A R,D’Amario D,Loughran J H,Stoddard M F,Ikram S,Beache G M,Wagner S G,Leri A,Hosoda T,Sanada F,Elmore J B,Goichberg P,Cappetta D,Solankhi N K,Fahsah I,Rokosh D G,Slaughter M S,Kajstura J,Anversa P.Cardiac stem cells in patients with ischaemic cardiomyopathy (scipio):Initial results of a randomised phase 1 trial.Lancet.2011;378:1847-1857 Makkar R,Smith R,Cheng K,Malliaras K,Thomson L E,Berman D,Czer L S,Marban L,Mendizabal A,Johnston P V,Russell S D,Schuleri K H,Lardo A C,Gerstenblith G,Marban E.Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (caduceus):A prospective,randomised phase 1 trial.Lancet.2012;379:895-904 Simpson D L,Mishra R,Sharma S,Goh S K,Deshmukh S,Kaushal S.A strong regenerative ability of cardiac stem cells derived from neonatal hearts.Circulation.2012;126:S46-53 Kajstura J,Leri A,Finato N,Di Loreto C,Beltrami C A,Anversa P.Myocyte proliferation in end-stage cardiac failure in humans.Proceedings of the National Academy of Sciences of the United States of America.1998;95:8801-8805 Anversa P,Kajstura J.Ventricular myocytes are not terminally differentiated in the adult mammalian heart.Circulation research.1998;83:1-14 Rajabi M,Kassiotis C,Razeghi P,Taegtmeyer H.Return to the fetal gene program protects the stressed heart:A strong hypothesis.Heart failure reviews.2007;12:331-343 Malliaras K,Makkar R,Smith R,Cheng K,Wu E,Bonow R O,Marban L,Mendizabal A,Cingolani E,Johnston P V,Gerstenblith G,Schuleri K H,Lardo A C,Marban E.Intracoronary cardiosphere-derived cells after myocardial infarction:Evidence of therapeutic regeneration in the final 1-year results of the caduceus trial (cardiosphere-derived autologous stem cells to reverse ventricular dysfunction).Journal of the American College of Cardiology.2014;63:110-122 Zaruba M,Soonpaa M,Reuter S,Field L J.Cardiomyogenic potential of c-kit(+)-expressing cells derived from neonatal and adult mouse hearts.Circulation.2010;121:1992-2000 Simpson D,Liu H,Fan T H,Nerem R,Dudley S C,Jr.A tissue engineering approach to progenitor cell delivery results in significant cell engraftment and improved myocardial remodeling.Stem Cells.2007;25:2350-2357 Chimenti I,Smith R,Li T S,Gerstenblith G,Messina E,Giacomello A,Marban E.Relative roles of direct regeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted mice.Circ Res.2010;106:971-980 Zhang X,Wang X,Zhu H,Kranias E G,Tang Y,Peng T,Chang J,Fan G C.Hsp20 functions as a novel cardiokine in promoting angiogenesis via activation of vegfr2.PloS one.2012;7:e32765 Miyata S,Minobe W,Bristow M R,Leinwand L A.Myosin heavy chain isoform expression in the failing and nonfailing human heart.Circ Res.2000;86:386-390 Razeghi P,Young M E,Alcorn J L,Moravec C S,Frazier O H,Taegtmeyer H.Metabolic gene expression in fetal and failing human heart.Circulation.2001;104:2923-2931 Afanasiev S A,Falaleeva L P,Rebrova T U,Suslova T E,Popov S V,Karpov R S.Effect of stress-proteins on survival of bone marrow mesenchymal stem cells after intramyocardial transplantation against the background of postinfarction heart remodeling.Bulletin of experimental biology and medicine.2008;146:111-115 Genth-Zotz S,Bolger A P,Kalra P R,von Haehling S,Doehner W,Coats A J,Volk H D,Anker S D.Heat shock protein 70 in patients with chronic heart failure:Relation to disease severity and survival.International journal of cardiology.2004;96:397-401 Wang Y,Chen L,Hagiwara N,Knowlton A.Regulation of heat shock protein 60 and 72 expression in the failing heart.Journal of molecular and cellular cardiology.2010;48:360-366 Reynolds B A,Weiss S.Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.Science.1992;255:1707-1710 Rietze R L,Reynolds B A.Neural stem cell isolation and characterization.Methods in enzymology.2006;419:3-23 Mirotsou M,Jayawardena T M,Schmeckpeper J,Gnecchi M,Dzau V J.Paracrine mechanisms of stem cell reparative and regenerative actions in the heart.Journal of molecular and cellular cardiology.2011;50:280-289 Angelini A,Castellani C,Ravara B,Franzin C,Pozzobon M,Tavano R,Libera L D,Papini E,Vettor R,De Coppi P,Thiene G,Vescovo G.Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure:Role of paracrine and neurohormonal milieu in the remodeling process.The Journal of heart and lung transplantation:the official publication of the International Society for Heart Transplantation.2011;30:1281-1293 McGinley L M,McMahon J,Stocca A,Duffy A,Flynn A,O’Toole D,O’Brien T.Mesenchymal stem cell survival in the infarcted heart is enhanced by lentivirus vector-mediated heat shock protein 27 expression.Human gene therapy.2013;24:840-851 Feng Y,Huang W,Meng W,Jegga A G,Wang Y,Cai W,Kim H W,Pasha Z,Wen Z,Rao F,Modi R M,Yu X,Ashraf M.Heat shock improves sca-1+ stem cell survival and directs ischemic cardiomyocytes toward a prosurvival phenotype via exosomal transfer:A critical role for hsfl / mir-34a / hsp70 pathway.Stem cells.2014;32:462-472 Cesselli D,Beltrami A P,D’Aurizio F,Marcon P,Bergamin N,Toffoletto B,Pandolfi M,Puppato E,Marino L,Signore S,Livi U,Verardo R,Piazza S,Marchionni L,Fiorini C,Schneider C,Hosoda T,Rota M,Kajstura J,Anversa P,Beltrami C A,Leri A.Effects of age and heart failure on human cardiac stem cell function.The American journal of pathology.2011;179:349-366 Laflamme M A,Chen K Y,Naumova A V,Muskheli V,Fugate J A,Dupras S K,Reinecke H,Xu C,Hassanipour M,Police S,O’Sullivan C,Collins L,Chen Y,Minami E,Gill E A,Ueno S,Yuan C,Gold J,Murry C E.Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts.Nature biotechnology.2007;25:1015-1024 Kannaiyan R,Shanmugam M K,Sethi G.Molecular targets of celastrol derived from thunder of god vine:Potential role in the treatment of inflammatory disorders and cancer.Cancer letters.2011;303:9-20 Salminen A,Lehtonen M,Paimela T,Kaarniranta K.Celastrol:Molecular targets of thunder god vine.Biochemical and biophysical research communications.2010;394:439-442 Go A S,Mozaffarian D,Roger V L,Benjamin E J,Berry J D,Blaha M J,et al.Executive summary:heart disease and stroke statistics-2014 update:a report from the american heart association.Circulation.2014;129(3):399-410.Epub Jan.22,2014.doi:10.1161 / 01.cir.0000442015.53336.12.PubMed PMID:24446411. Go A S,Mozaffarian D,Roger V L,Benjamin E J,Berry J D,Blaha M J,et al.Heart disease and stroke statistics-2014 update:a report from the american heart association.Circulation.2014;129(3):e28-e292.Epub Dec.20,2013.doi:10.1161 / 01.cir.0000441139.02102.80.PubMed PMID:24352519. Garbern J C,Lee R T.Cardiac stem cell therapy and the promise of heart regeneration.Cell stem cell.2013;12(6):689-98.Epub Jun.12,2013.doi:10.1016 / j.stem.2013.05.008.PubMed PMID:23746978;PubMed Central PMCID:PMC3756309. Bolli R,Chugh A R,D’Amario D,Loughran J H,Stoddard M F,Ikram S,et al.Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO):initial results of a randomised phase 1 trial.Lancet.2011;378(9806):1847-57.Epub Nov.18,2011.doi:10.1016 / S0140-6736(11)61590-0.PubMed PMID:22088800;PubMed Central PMCID:PMC3614010. Bolli R,Tang X L,Sanganalmath S K,Rimoldi O,Mosna F,Abdel-Latif A,et al.Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy.Circulation.2013;128(2):122-31.Epub Jun.13,2013.doi:10.1161 / CIRCULATIONAHA.112.001075.PubMed PMID:23757309;PubMed Central PMCID:PMC3807652. Tang X L,Rokosh G,Sanganalmath S K,Yuan F,Sato H,Mu J,et al.Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction.Circulation.2010;121(2):293-305.Epub Jan.6,2010.doi:10.1161 / CIRCULATIONAHA.109.871905.PubMed PMID:20048209;PubMed Central PMCID:PMC2814341. Beltrami A P,Barlucchi L,Torella D,Baker M,Limana F,Chimenti S,et al.Adult cardiac stem cells are multipotent and support myocardial regeneration.Cell.2003;114(6):763-76.Epub Sep.9,2003.PubMed PMID:14505575. Chugh A R,Beache G M,Loughran R I,Mewton N,Elmore J B,Kajstura J,et al.Administration of cardiac stem cells in patients with ischemic cardiomyopathy:the SCIPIO trial:surgical aspects and interim analysis of myocardial function and viability by magnetic resonance.Circulation.2012;126(11 Suppl 1):S54-64.Epub Sep.9,2012.doi:10.1161 / CIRCULATIONAHA.112.092627.PubMed PMID:22965994;PubMed Central PMCID:PMC3448934. Pagani F D,DerSimonian H,Zawadzka A,Wetzel K,Edge A S,Jacoby D B,et al.Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans.Histological analysis of cell survival and differentiation.Journal of the American College of Cardiology.2003;41(5):879-88.Epub Dec.3,2003.PubMed PMID:12628737. Bergmann O,Bhardwaj R D,Bernard S,Zdunek S,Barnabe-Heider F,Walsh S,et al.Evidence for cardiomyocyte renewal in humans.Science.2009;324(5923):98-102.Epub Apr.4,2009.doi:10.1126 / science.1164680.PubMed PMID:19342590;PubMed Central PMCID:PMC2991140. Kajstura J,Rota M,Cappetta D,Ogorek B,Arranto C,Bai Y,et al.Cardiomyogenesis in the aging and failing human heart.Circulation.2012;126(15):1869-81.Epub Sep.8,2012.doi:10.1161 / CIRCULATIONAHA.112.118380.PubMed PMID:22955965;PubMed Central PMCID:PMC3477474 Ongstad L.E,Mishra R,Belkhodja M,Sharma S,Gaddipati R,Phipps S,Kaushal S,Karathanasis S.Immortalization of Human Neonatal C-kit+ Cardiac Progenitor Cells Preserves Their Phenotype and Function at High Passage.Circulation 2019 Buja,M.Cardiac repair and the putative role of stem cells.Journal of Molecular and Cellular Cardiology 128 (2019)96-104. Vujic,A.,et al.,Molecular mechanisms of heart regeneration.Semin Cell Dev Biol.2019 Oct 3.pii:S1084-9521(19)30170-3. Vincinanza,C,et al.,Adult cardiac stem cells are multipotent and robustly myogenic:c-kit expression is necessary but not sufficient for their identification.Cell Death Differ.2017 Dec;24(12):2101-2116. Lewis-McDougall FC,et al.,Aged‐senescent cells contribute to impaired heart Regeneration.Aging Cell.2019 Jun;18(3):e12931.

Claims

1. 1. A pharmaceutical composition for use in wound healing comprising conditioned medium from immortalized human neonatal CD117+ cardiac stem cells and one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles, Immortalized human neonatal CD117 + cardiac stem cells are positive for CD44, CD47, CD73, CD90, and CD105 protein expression; Immortalized human neonatal CD117 + cardiac stem cells are negative for expression of CD31, CD34, CD45, and tryptase proteins; The pharmaceutical composition, wherein the conditioned medium comprises IGF-1, ANG-1, HGF, SDF-1α, VEGF-A, bFGF, PDGF-B, and SCF.

2. 10. The pharmaceutical composition of claim 1, further comprising an effective amount of immortalized human neonatal CD117+ cardiac stem cells.

3. The pharmaceutical composition described in claim 1 or 2, wherein the immortalized human neonatal CD117 + cardiac stem cells are further negative for protein expression of GATA4, CD80, CD86, Lin or a combination thereof.

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