Immortalized cardiomyocytes for heart repair

Abstract

The present invention provides compositions and methods for treating cardiac medical conditions such as heart failure caused by damaged myocardial tissue, as well as compositions and methods for addressing inflammatory and fibrotic processes observed in a wide range of medical conditions. [Solution] Embodiments of the present disclosure relate to compositions and methods of use relating to specific immortalized cells, including cardiomyocytes, obtained from pediatric or neonatal individuals. In specific embodiments, immortalized cells, or cell-derived conditioning media, or a partial or whole secretome thereof, are provided to individuals requiring it in an effective amount, either alone or in combination with cardiomyocytes.

Description

[Technical Field]

[0001] The fields of this disclosure relate, at a minimum, to 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 increase in the number of children with heart failure (Go et al., 2014; Go (et al., 2014). Two important biological processes known to contribute to a wide range of medical conditions, including many of the underlying causes of heart disease, are inflammation and fibrosis. For centuries, the heart was considered a permanently differentiated organ that could not regenerate, in contrast to other tissues such as the liver and skin (outlined in Buja 2019). This paradigm has recently been overturned when it was revealed that cardiomyocytes in adult human hearts are replaced at a small but detectable rate of about 1-2% per year (outlined in Vujic et al 2019). This has motivated scientists and pharmaceutical companies alike to identify and target the fundamental mechanisms of cardiac regeneration for the treatment of various heart diseases. There are two possible mechanisms for cardiac regeneration: 1) cardiomyocyte replication and 2) the presence of endogenous cardiomyocyte stem cells that can proliferate and differentiate into cardiomyocytes. Currently, cardiomyocyte proliferation occurs in certain situations, such as in amphibians, certain fish, and neonatal mammals, but it is clear that it does not contribute to cardiac regeneration in adult mammals. In contrast, recent clinical trials have shown that transplantation of resident cardiomyocyte stem cells can repair / regenerate / remodel human cardiomyocytes, resulting in improved cardiac function, as indicated by improved ejection fraction, reduced scar size, reduced end-diastolic and end-systolic volume, and improved quality of life and NYHA class (Garbern et al., 2013).

[0003] Populations of cardiomyocyte stem cells expressing the cell surface marker c-kit (also known as CD117) were described nearly 15 years ago, and homogeneous, healthy populations of c-kit+ cells have been shown to confer anti-inflammatory and anti-fibrotic properties. Magnet selection of c-kit-expressing cardiac cells to enrich these cells, followed by cardiac delivery of these cells in animal models of heart 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 revealed two significant problems. First, these c-kit+ cardiomyocyte stem cells are a mixture of many different progenitor cells, most of which (about 90%) are hematopoietic and endothelial cells, and not cardiogenic stem cells (Vicinanza et al. 2017). Second, most cardiac c-kit+ cells obtained from adult human hearts are senescent, thus contributing little to cardiac repair and potentially contributing to cardiac damage through various inflammatory factors secreted by such senescent cells (Lewis-McDouggal et al.). (al. 2019).

[0004] Therefore, 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 inflammatory and fibrotic processes observed in a wide range of medical conditions. The present invention satisfies these needs and provides other relevant advantages. [Overview of the project] [Means for solving the problem]

[0005] Embodiments of this disclosure relate to the use of certain human cardiomyocyte stem cells (hCSCs), particularly neonatal cardiomyocyte stem cells (nCSCs), immortalized nCSCs (Im-nCSCs), for use in the treatment of medical conditions. For example, it relates to cloned isolates), and methods and compositions related to conditioned media produced by Im-nCSC. In certain embodiments, the medical condition is a cardiac medical condition. Any inflammatory, fibrotic or cardiac medical condition can be treated using such compositions and methods, but in certain embodiments, the condition is a cardiac condition that benefits from repair, regeneration or remodeling of the heart muscle (myocardium). In certain embodiments, the methods and compositions promote or enhance the repair, regeneration or remodeling capabilities of nCSC. In certain embodiments, the medical condition is an inflammatory condition or disease. In more particular embodiments, the inflammatory condition or disease is selected from, but not limited to, ischemic stroke, acute and chronic kidney disease, arthritis 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 include methods and compositions related to immortalized cells derived from mammalian myocardium, particularly human, such as neonatal human. In certain embodiments, the cells include immortalized cloned isolates of immortalized neonatal CD117+ cardiac stem cells, particularly neonatal CD117+ cardiac stem cells.

[0007] In further embodiments, 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 having 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 cardiomyogenic stem cells are provided to an individual for treating one or more cardiac medical conditions. In other embodiments, conditioned media derived from immortalized cardiomyogenic stem cells are provided to an individual for treating cardiac medical conditions. In still other embodiments, cell-derived secretomes are provided to an individual for treating cardiac medical conditions. In still other embodiments, trophic factors derived from one or more immortalized cell clonal isolates are provided to an individual for treating cardiac medical conditions. In certain other embodiments, any combination of the above may be used in treating cardiac or other medical conditions.

[0010] In some embodiments, the present disclosure provides a composition comprising conditioned media (CM) derived from one or more immortalized neonatal cardiomyogenic stem cells (Im-nCSC), such as CD117+ immortalized neonatal cardiomyogenic stem cells. In certain specific embodiments, the immortalized neonatal cardiomyogenic stem cells are immortalized clonal isolates. In other embodiments, the immortalized neonatal cardiomyogenic 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 negative for tryptase. In still other embodiments, the immortalized neonatal cardiomyogenic stem cells have the following characteristics: CD90 + , CD105 + , CD117 + , CD44 + , CD73 + , CD47 + , CD31 - , CD34 <( - , CD45 -and have all of them being tryptase negative. In yet other embodiments, the immortalized neonatal cardiomyoblasts 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 cardiomyoblasts have all of the following characteristics: GATA4-, CD44+, tryptase negative, CD80-, CD86-. In still further embodiments, the immortalized neonatal cardiomyoblasts have one or more, two or more, or all of the following characteristics: CD117+, CD45- and Lin - In yet further embodiments, the immortalized neonatal cardiomyoblasts have one or more, two or more, or all of the following characteristics: CD117 + and CD45 - and have them.

[0011] In some embodiments of the present invention, prior to immortalization, the neonatal cardiomyoblasts 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 process typically performed in the literature. In some embodiments, the individual from whom 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, the immortalization is achieved by exogenous expression of the human telomerase (hTERT) gene using a delivery vector, such as a lentiviral expression vector for example.

[0012] In other embodiments of the present invention, a composition comprising a plurality of immortalized human neonatal cardiomyoblast clonal isolates is provided.

[0013] In further embodiments, the Disclosure provides a method for treating an individual for, for example, a medical condition of the heart or other condition as described herein, comprising the step of providing the individual with a composition comprising a therapeutically effective amount of a composition comprising a condition medium derived from immortalized neonatal cardiomyocytes. In some embodiments, the composition is selected from any one of the compositions provided herein.

[0014] In some embodiments, the Disclosure provides a method for treating an individual for a medical condition of the heart, comprising the step of providing the individual with a composition comprising a therapeutically effective amount of a condition medium derived from immortalized neonatal cardiomyocytes, in combination with a composition comprising neonatal cardiomyocytes, such as immortalized neonatal cardiomyocytes. In some embodiments, the composition comprising the condition medium is selected from any one of the compositions provided herein.

[0015] In further embodiments, the disclosure provides a method for isolating cell clones from immortalized neonatal cardiomyocyte stem cells, the method comprising isolating one or more cells from neonatal heart tissue or having one or more cells isolated from neonatal heart tissue, and culturing one or more cells in a suitable culture medium to promote proliferation. In further specific embodiments, the isolation step does not involve contacting the cells with a moiety that binds to a specific cell surface protein such as CD117.

[0016] In additional embodiments, the immortalized isolated cell clonal isolates according to the present disclosure, when isolated, have one, two, three, four, or five of the following characteristics: GATA4-, CD44+, CD47+, CD31-, CD34-, CD45-, tryptase-, CD80-, CD86-, and immortalized cells that maintain 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 pediatric or non-pediatric individuals, prior to immortalization. In certain embodiments, the individual has a medical condition of the heart. In certain embodiments, the individual has normal cardiomyopathy. In some embodiments, the cells may be derived from the cardiomyopathy of a pediatric individual with end-stage heart failure. The cardiomyopathy may be derived from a neonatal individual with congenital heart disease.

[0018] In other specific embodiments of the Disclosure, the immortalized cells of the Disclosure are CD63 + CD73 + CD47 + CD45 - CD31 - Immortalized cells secrete exosomes (e.g., Figure 8). Immortalized cells secrete pro-angiogenic and angiogenic cytokines such as VEGF-A, HGF, SCF, SDF-1α, IGF, PDGF-B, and ANG-1 (e.g., Table 1). .

[0019] In yet another embodiment, a method is provided for treating an individual for a medical condition, such as a medical condition of the heart described herein or any other condition, comprising the step of providing the individual with a therapeutically effective amount of the composition of this disclosure, for example, immortalized neonatal cardiomyocytes, a conditioning medium with immortalized neonatal cardiomyocytes, exosomes secreted by immortalized neonatal cardiomyocytes and / or any combination thereof. In a more specific embodiment, the composition independently comprises proteins and / or exosomes secreted from immortalized neonatal cardiomyocytes.

[0020] In other specific embodiments, a medical condition of the heart is treated using the composition of the present invention, for example, by delivering the composition by intramyocardial injection, intravenous injection, or in a device that holds encapsulated cells, for example, a device that retains cells but allows the secretion of paracrine factors produced by the cells into circulation. In yet another specific embodiment, the medical condition of the heart is heart failure, cardiomyopathy, or congenital heart disease.

[0021] In additional embodiments of the present invention, compositions comprising a total condition medium (TCM) and / or components derived from immortalized cells of the present disclosure, and methods for using the same in therapeutic doses, are provided for the treatment of the medical conditions described herein. Further embodiments also provide compositions comprising exosomes and / or components derived from immortalized cells of the present disclosure, and methods for using the same in therapeutic doses, for the treatment of the medical conditions described herein. [Brief explanation of the drawing]

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

[0023] [Figure 2] Figure 2 shows a typical phase-contrast image of a single-cell culture in a 96-well plate initiated using cells diluted by the method described in Figure 1.

[0024] [Figure 3] Figure 3 shows the phenotypic characterization of seven clonal isolates of nCSC obtained by flow cytometry analysis using the methods shown in Figures 1 and 2.

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

[0026] [Figure 5] Figure 5 shows typical phase-contrast microscope images of nCSCs and Im-nCSCs.

[0027] [Figure 6] Figure 6 shows the results of flow cytometry analysis of Im-nCSCs grown from 16 passages of single-cell culture.

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

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

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

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

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

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

[0034] [Figure 13] Figure 13 shows typical normal karyotypes observed in Im-nCSCs. [Modes for carrying out the invention]

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described. For the 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 more (i.e., at least one) of the grammatical objects of the article. For example, "element" means one element or more elements.

[0037] The terms “and / or” are used in this disclosure to mean either “and” or “or” unless otherwise indicated.

[0038] The term "e.g." is used herein to mean "for example" and is understood to include a given process or component or group of processes or components, but not to exclude any other process or component or group of processes or components.

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

[0040] As used herein, the term “administer” refers to any mode of delivery, transfer, introduction, or transport of a substance, such as a compound, a pharmaceutical compound, or another agent, such as an antigen, to a subject. Modes of administration include oral, topical, intravenous, intraperitoneal, intramuscular, intranasal, or subcutaneous administration. Administration “in combination with” one or more substances, such as therapeutic agents, includes simultaneous (concurrent) and sequential administration in any order.

[0041] Throughout this specification, unless the context otherwise requires, the phrases “comprise,” “comprises,” and “comprise” are understood to mean that a given process or component or group of processes or components is included, but not that any other process or component or group of processes or components is excluded. “Consists of” means that anything following the phrase “consists of” is included and limited to such things. Therefore, the phrase “consists of” indicates that the enumerated components are required or essential, and that no other components are present. “Essentially consists of” means that any components enumerated after the phrase, and any other components that do not interfere with or contribute to the activity or action identified in this disclosure for the enumerated components. Therefore, the phrase “essentially consists of” indicates that the enumerated components are required or essential, and that the other components are optional. This indicates whether a component may or may not be present, depending on whether it substantially affects the activity or function of the enumerated components.

[0042] When used in relation to a compound, "effective dose" refers to the amount of the compound required to elicit a desired response. In some embodiments, the desired response is, for example, a biological response in the subject. In some embodiments, the compound may be administered to the subject in an effective dose that acts on the biological response in the subject. In some embodiments, the effective dose is a "therapeutic effective dose."

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

[0044] The term “modulate” typically includes “increase,” “enhance,” or “stimulate,” as well as “decrease” or “reduce,” 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 may include increases of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) (including all integers and decimals in between, as well as amounts greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) that are produced by the non-composition or control composition, sample, or test subject. The amount “reduced” or “reduced” is typically a “statistically significant” amount and may include a reduction 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 the non-composition (absence of the drug or compound) or the control composition, including all integers between these.

[0045] As used herein, “subject” or “patient” includes any animal that exhibits or is at risk of exhibiting symptoms that can be treated with the compositions disclosed herein (e.g., conditional media derived from neonatal CSCs, such as immortalized hCSCs disclosed herein). Appropriate subjects (patients) include human patients. Appropriate subjects also include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock (such as pigs, horses, or cattle), and domesticated 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 almost entirely or completely, for example, 95% or more of a 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 even include minimal change or improvement in one or more measurable markers of the disease or condition being treated. “Treatment” or “treating” does not necessarily indicate the complete eradication or cure of the disease or condition or its associated symptoms. The subject receiving this treatment is any subject who needs it. Illustrative 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 this disclosure to refer to the same protein, which proceeds synonymously by both names. The c-kit / CD117 protein, as well as the gene encoding it, is extensively characterized and well known in the art (e.g., https: / / www.uniprot.org / unip (rot / P10721 and UNIPROT accession number P10721).

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Any methods, compositions, reagents, or cells similar or equivalent to those described herein may be used in the practice or testing of the present invention, but 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 whole as if each individual publication or reference were specifically and individually indicated to be incorporated herein by whole as if it were described in full. Any patent application for which this application claims priority is also incorporated herein by whole as in the same manner as described above for publications and references.

[0050] As used herein, the term “cardiac stem cells” may be defined as cells derived from cardiac tissue that are clonal, pluripotent, and self-renewing. In certain embodiments, these cardiomyocytes 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 This disclosure provides, in particular, compositions and methods relating to human neonatal cardiomyocyte stem cells (nCSCs), methods for isolating and immortalizing them, conditional media taken from cultures of such cardiomyocyte stem cells, and methods for using such cells, media, and / or cells resuspended in conditional media therapeutically, for example, to repair, regenerate and / or remodel cardiac tissue, and to treat medical conditions (e.g., medical conditions of the heart) caused by inflammatory and / or fibrotic processes.

[0052] I. Cells and their conditions, culture medium, and secretome of the present disclosure. Embodiments of this disclosure relate to mammalian cells or cell-derived conditional media that provide therapeutic functions to individuals in need thereof. In certain embodiments, the cells are immortalized human neonatal cardiomyocyte stem cells (Im-nCSCs) useful for achieving therapeutic functions or effects in the mammalian heart. In some embodiments, the cells are immortalized human neonatal cardiomyocyte stem cells (Im-nCSCs) that secrete one or more agents useful for anti-inflammatory, anti-fibrotic, pro-angiogenic, and / or therapeutic functions or effects in the mammalian heart. In certain embodiments, the cells are immortalized human neonatal cardiac CSCs that, upon in vivo administration to the heart, possess their own repair, regenerative, or remodeling activity, and / or activity for promoting the repair, regenerative, or remodeling activity of endogenous cells and / or tissues of human cardiomyocytes upon in vivo administration to the heart. In certain embodiments, the cells are immortalized neonatal human CSCs, which secrete one or more proteins that are anti-inflammatory, anti-fibrotic, pro-angiogenic, and / or have repair, regenerative, or remodeling activity upon in vivo delivery to the heart, and / or have activity to promote the repair, regenerative, or remodeling capacity of endogenous cells and / or tissues in human cardiomyocytes upon in vivo delivery to the heart. In certain embodiments, the cells secrete one or more proteins that can induce a bystander effect such that delivery of a cell-derived conditioning medium to the cardiomyocyte in vivo enhances the repair, regeneration, or remodeling capacity of endogenous cells and / or tissues.

[0053] In certain embodiments, immortalized human neonatal cardiac CSCs are produced by obtaining clonal isolates of human neonatal cardiac CSCs and immortalizing them by any suitable immortalization method. For example, non-limitingly, immortalization by introduction of simian virus 40 large T antigen (Ko Many such methods are known that are suitable for use in the present invention, including bayashi et al., (2000) Science 287:1258-62, Nakamura et al., (1997) Transplantation 63 (11):1541-47, transfection of antisense constructs against p53 and retinoblastoma protein (Werner et al., (2000) Biotechnol Bioeng 68 (1):59-70), transgenic introduction of cleaved 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 particular embodiment, the human neonatal cardiac CSCs of this disclosure are immortalized by stably transgenicating them with a vector expressing human telomerase (hTERT) (e.g., a lentiviral vector). The mechanism by which human fibroblast cells limit proliferation in vitro has been shown to be progressive telomere shortening at each cell division (Hayflicket al., (1961) Exp. Cell Res. 25:585-621). Telomeres constitute the terminal regions of chromosomes, and shortened telomeres cause limitation of proliferation. However, stem cells can circumvent telomere-dependent proliferation limitation by adding telomere repeat sequences to the chromosome ends using telomerase reverse transcriptase (Greider et al., (1985) Cell 43:405-413). The ability to achieve telomerase rearrangement of human neonatal cardiac CSCs and generate stable neonatal human cardiac clonal stem cell lines with the phenotypic characteristics of in vitro passaged neonatal cardiomyocytes for use in cardiac-targeted 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 may function as cells capable of producing unlimited amounts of conditioned medium containing one or more secreted factors, e.g., secreted proteins, which have repair, regenerative, or remodeling activity when administered in vivo to the heart, and / or have activity to promote the repair, regenerative, or remodeling ability of endogenous cells and / or tissues in human cardiomyocytes when administered in vivo to the heart. In other embodiments, such cells may function as cell factories capable of producing unlimited amounts of conditioned medium containing one or more secreted factors, e.g., secreted proteins, which have anti-inflammatory, anti-fibrotic, and pro-angiogenic properties that can provide therapeutic benefits 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 genetically modified 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 the cell. 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 a suitable promoter (e.g., the CMV promoter), an exemplary example of which is shown in Figure 4.

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

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

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

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

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

[0061] Neonatal cardiomyocytes may be obtained from any suitable source prior to immortalization. In certain embodiments, the source of cardiomyocytes to be immortalized is from a neonatal individual or an individual in utero. In certain embodiments, the cells are not adult cardiomyocytes. The cells may be obtained from an individual that requires therapeutic use with the offspring of the same cells, or the cells may be obtained from a different individual. The cells may be derived from a donated heart of a neonatal individual or from the heart of a living neonatal individual. The cells may be commercially provided to an individual that needs them, or provided to a medical facility or practitioner that oversees the medical care of an individual that needs them. In certain embodiments, the cells are obtained from human subjects aged 1 to 30 days. For example, a 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. A human subject may also be less than 1 day old.

[0062] Embodiments of this disclosure encompass immortalized neonatal cardiac cells (CSCs) derived from mammalian cardiomyocytes, including human cells. In certain embodiments, human neonatal cardiac CSCs may have a specific genotype and / or phenotype. In certain embodiments, immortalized human neonatal cardiac CSCs may be provided to individuals requiring them after determining a specific genotype or phenotype of cells suitable for the intended function of the cells, for therapeutic use. However, in some preferred embodiments, the conditioned medium produced from culturing immortalized human neonatal cardiac CSCs is provided to individuals requiring them after determining a specific genotype or phenotype of cells suitable for producing a conditioned medium having the intended function. In certain embodiments, immortalized human neonatal cardiac CSCs are CD117+ cells, and in certain embodiments, immortalized human neonatal cardiac CSCs are CD117+, CD90+, CD105+, CD73+, CD44+, CD47+ These are CD31- and CD45-. In other embodiments, immortalized human neonatal cardiac CSCs also have one, two, three, four or five or more of the following features: GATA4-, CD44+, CD31-, tryptase-, CD80-, CD45-, CD86-, HLA class I+, and HLA class II-. In other embodiments, immortalized human neonatal cardiac CSCs also have one, two, three, four or five or more of the following features: 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 (CSCs) spontaneously secrete one or more proteins or factors beneficial to the repair, regeneration, or remodeling of localized cells or tissues. In some embodiments, the Disclosure provides a conditional medium for culturing immortalized human neonatal cardiac CSCs under conditions in which they produce by secreting 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 kind, 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, cells secrete factors derived from one or more of VEGF-A, HGF, SCF, SDF-1α, ANG-1, bFGF, PDGFB, and IGF-1, and in some embodiments, the Disclosure provides a conditioned medium containing one or more of such factors. In certain embodiments, cells are manipulated to increase the secretion of SDF-1α, VEGF-A, PGDF-A, and / or FGF-2, or a combination thereof. Such manipulation is by any method, but in certain embodiments, it involves cell manipulation by recombinant techniques to increase the expression of these factors and / or one or more other factors that have therapeutic value but are not expressed by these cells. Thus, in some embodiments, a conditioned medium derived from such genetically modified cells is provided by the Disclosure. Another manipulation intended for use in the Invention is the exposure of immortalized human neonatal cardiac cytoscotyledons (CSCs) to other agents that increase the secretion of heat shock factors and / or cytokines.

[0064] In some embodiments, immortalized human neonatal cardiac CSCs spontaneously express one or more proteins or factors that are beneficial, directly or indirectly, for the repair, regeneration, or remodeling of localized cells or tissues. For example, immortalized human neonatal cardiac CSCs may express VEGF-A and / or SDF-1a. In addition, similar to human neonatal cardiac CSCs, immortalized human neonatal cardiac CSCs may have activated HSF-1, HSP60, and / or HSP70 expression, either spontaneously or by cell manipulation to increase HSF-1, HSP60, and / or HSP70 expression (Sharma et al.). (al, 2017). Accordingly, in some embodiments, the Disclosure provides a condition medium produced by such cells, the condition medium comprising VEGF-A and / or SDF-1α. In some embodiments, the Disclosure provides a condition medium produced by such cells comprising the expression of HSF-1, HSP60, and / or HSP70, either naturally or artificially. In some embodiments, the condition medium (CM) for immortalized neonatal cardiomyocytes comprises a specific combination of paracrine factors (e.g., cytokines and growth factors) within a pre-specified range detailed in Table 1.

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

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

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

[0068] Immortalized human neonatal cardiac cytoplasmic stem cells (CSCs) may be used in 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 the immune system of another patient. In such cases, the cells can be used as stock cells for clinical application.

[0069] In certain embodiments, CSCs derived from young individuals exhibited stronger repair and / or regenerative capabilities compared to similarly derived cells from adult hearts (Sharma et al., 2017). These increased capabilities are, in part, due to a more potent secretome than those produced by young cells in certain embodiments. This means that young cells may be a preferred alloproduct, at least in certain embodiments. Furthermore, in some embodiments, these superior repair and / or regenerative properties are maintained after immortalization of human neonatal cardiac CSCs according to this disclosure. Therefore, 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 the repair and / or regeneration of damaged cardiac tissue. Accordingly, this disclosure provides a method for inducing the repair and / or regeneration of damage to cardiac tissue, comprising the step of administering to a patient in need a composition comprising a condition medium derived from one or more immortalized human neonatal cardiac CSCs. In some embodiments, a population of human neonatal cardiac CSCs is resuspended in a composition containing a conditioning medium derived from one or more immortalized human neonatal cardiac CSCs, and the composition containing the conditioning medium and the resuspended human neonatal cardiac CSCs is administered to a patient. II. Isolation Method for nCSCs

[0070] This disclosure also provides a novel method for isolating CD117+ CSCs from neonatal tissue, which does not require exposing collected cells to a substrate containing a surface having a CD117 (i.e., c-kit) binding moiety. This method is in contrast to 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) containing a CD117 binding moiety (e.g., an antibody) in order 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 adult human hearts, neonatal human hearts contain a homogeneous population of cardiogenic CD117+ stem cells with little to no contamination of hematopoietic progenitor cells and endothelial progenitor cells. Furthermore, the inventors have found that CD117+ cells from neonatal human hearts, even if initially seeded as single cells, However, we were able to produce clones that could grow in culture, and we found that their growth rate was related to the level of CD117 positivity in the cells. Therefore, cells with a high degree of CD117+ expression generally grow faster than cells with lower CD117 expression, although all cells whose growth we observed expressed CD117 to at least some degree. Furthermore, CD117+ cells from neonatal cardiac tissue did not show evidence of aging and did not expand to large cell numbers with significant cardiac repair and regenerative activity when tested in an animal model of myocardial infarction (see, for example, Figure 7).

[0072] Therefore, based at least in part on these remarkable findings, this disclosure provides a method for isolating and growing human neonatal cardiac CSCs (e.g., a homogeneous population of CD117+ neonatal cardiac CSCs) directly from neonatal cardiac tissue without prior selection with CD117 magnetic beads (or any other CD117+ cell enrichment method). This method is not prior to the disclosure of phenotypic CD90 + CD105 + CD117 + CD44 + CD73 + CD47 + CD31 - CD34 - CD45 -This provides the selection of a specific cell population defined by tryptase negativity. In addition, such methods offer significant advantages over prior art methods by resulting in single-cell donor and donor independence in the acquisition of large quantities of cells, ensuring cell homogeneity, and avoiding regulatory concerns associated with the exposure of cells intended for therapeutic purposes to non-GMP quality materials (i.e., CD117 magnetic beads). Furthermore, they reduce the level of processing that must be performed between the acquisition of neonatal cardiac samples and the provision of population CD117+ cells for, for example, therapeutic purposes, for the production of conditional media and / or immortalization according to the methods disclosed herein.

[0073] In various embodiments, the disclosure also provides a method for isolating CD117+ CSCs from neonatal cardiac tissue, the method comprising the steps of isolating one or more clonal isolate cells from neonatal cardiac tissue or having one or more isolated clonal isolate cells, and culturing one or more cells in a suitable culture medium to promote growth, wherein the isolation step does not involve contacting the cells with a CD117-binding moiety (e.g., a CD117 antibody). In some embodiments, the method comprises the step of initiating a culture or a group of cultures, each culture comprising one or more cells derived from cardiac tissue. In some embodiments, each of each culture or group of cultures is initially seeded with only one cell derived from cardiac tissue. This can be achieved by any suitable method known in the art, e.g., FACS sorting or limited dilution culture of cells obtained from a neonatal cardiac tissue sample.

[0074] The method may include a step of monitoring the growth rate of one or more cultures or a group of cultures. The method may also include a step of approximating the level of CD117 positivity in one or more cultures or a group of cultures by comparing the growth rate of the culture or a group of cultures with the growth rate of a known CD117+ expression reference sample or reference value. 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, the characterization of 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 marker CD105 or CD90, stem cell marker CD117, endothelial cell marker CD31, mast cell marker tryptase, and / or hematopoietic agent cell lineage marker CD45); aging analysis; and / or characterization of function in an in vivo cardiac injury model, such as angiogenesis, resistance to oxidative stress, and / or ability to promote therapeutic efficacy. In some embodiments, such methods may involve cells isolated using methods known in the art or methods disclosed herein. The method may further include immortalization. Immortalization may be by any suitable means. In some embodiments, immortalization is by telomerase expression. Exemplarily, telomerase may be encoded by the human TERT (hTERT) gene. Human neonatal cardiac cytoscoplasmic sperm cells (CSCs) may be transgenic with hTERT, infected with a recombinant virus capable of transmitting the hTERT gene to cells, or delivered to the CSCs using any suitable method known and available in the art.

[0075] In some embodiments, the Disclosure provides a method for approximating the level of CD117 positivity in a culture of neonatal CSCs or a plurality of cultures of neonatal CSCs, the method comprising the steps of determining the growth rate of cells in culture and comparing the rate with the growth 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 (as disclosed herein) are used for therapeutic purposes, such as those disclosed in U.S. Patent Publication No. US2015 / 0328263, which is incorporated herein by reference in whole, and include, but are not limited to, the administration of CD117+ or otherwise immortalized CSCs to subjects requiring them; the administration of conditional media derived from immortalized CSCs to subjects requiring them; and / or the administration of a combination of immortalized CSCs and conditional media derived from immortalized CSCs to subjects requiring them. Such therapeutic purposes typically include the administration of any of the above compositions to subjects requiring them after immortalization of neonatal CSCs according to methods known in the art or methods disclosed herein.

[0077] Those skilled in the art will understand that, in addition to the above method for isolating CD117+ CSCs from neonatal cardiac tissue, other routine methods exist 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 having a surface having a CD117-binding portion, such as a CD117 antibody, in order to isolate CD117+ cells from a population of cells present in the sample. In some embodiments, the present disclosure does not use such methods.

[0078] Those skilled in the art will also understand that there are routine methods for obtaining cells from human cardiomyocytes and then further processing the cells. In certain embodiments, there are methods for isolating desired human neonatal cardiac CSCs by obtaining tissue from human cardiomyocytes, including neonatal cardiomyocytes (e.g., obtained from the right atrial appendage or "RAA" of the heart), such as by biopsy. The cardiomyocytes may be from individuals without known cardiac abnormalities. The cardiomyocytes may be from individuals with end-stage heart failure or congenital heart disease, in which case the cardiomyocytes may be normal or not. The extracted tissues may be exposed to certain culture media while isolating individual cells from those tissues, including by tissue cutting, such as in the presence of collagenase. In certain embodiments, the tissues and tissue fragments are allowed to settle in the culture medium to obtain a supernatant. Cells may be collected from the supernatant and suspended in culture medium for an appropriate period. After this, the desired CD117+ human neonatal cardiac CSCs are isolated therefrom, for example. The isolation of desired human neonatal cardiac CSCs may be achieved by means disclosed herein or by any means known in the art. In some embodiments, isolation is performed via a method disclosed herein that does not involve the step of exposing the collected cells to a substrate having a surface having a CD117-binding portion, 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 passaging. The cell culture medium may or may not be substantially identical to the medium used when the cells are delivered to the individual. The medium may or may not contain one or more heat shock response inducers.

[0080] III. Methods of using the cells disclosed herein, as well as their conditions, culture media, and secretome. The methods of the present disclosure include the use of certain nCSCs (and, in certain specific embodiments, CD117+ mesenchymal-like cells) or conditional media produced by culturing such cells for the therapy of at least one medical condition in an individual requiring it. In some embodiments, the cells are isolated via the methods disclosed herein, and isolation does not involve exposing the collected cells to a substrate having a CD117-binding portion, such as a CD117 antibody, or a portion that binds to any other surface protein. In some embodiments, clonal cell isolates are immortalized (e.g., via hTERT immortalization). In certain embodiments, the cells, conditional media produced by culturing the cells, or cells resuspended in those conditional media are useful for a medical condition, and the creation of anti-inflammatory or anti-fibrotic effects and / or promotion of tissue repair, regeneration, and / or replacement are therapeutically useful.

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

[0082] During isolation from a source organism of desired cells, for example via a method disclosed herein, prior to their delivery to an organism requiring it or delivery of a cell-derived condition medium to an organism, the collected cells may be further modified, for example, through genetic engineering for the recombinant expression of one or more expression constructs, and further through culture and / or enrichment. Such further modifications may also include the immortalization of cells as described herein. Such practices are commonplace in the art. The expression constructs introduced into the cells may be of any kind, but in certain embodiments, the constructs express cytokines, pro-angiogenic factors, growth factors, transcription factors, etc. In certain embodiments, the constructs express VEGF-A, HGF, SCF, SDF-1α, ANG-1, HSF-1, PGDF-A, FGF-2, or a combination thereof, and / or express other proteins that directly or indirectly increase their expression. 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 used in one or more therapeutic methods, but in some preferred embodiments, the organism is instead a therapeutically effective amount of cell-derived conditional medium; a portion of the cell-derived secretome; and one or more cell-derived secreted proteins or other factors (e.g., exosomes, extracellular vehicles, miRNs). The recipient may receive A, etc.); or a combination thereof. In some embodiments, a cell-derived condition medium 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 promotes the proliferation of endogenous cardiomyocytes in certain embodiments, upon their use. In certain embodiments, the use of any one of these components or combinations enables the repair, regeneration, or remodeling of the cardiomyocyte. 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 individual being treated.

[0084] In certain embodiments, therapeutically effective doses of cells, conditioned media produced by culturing cells (or immortalized human neonatal cardiac CSCs), or cells resuspended in those conditioned media may be administered multiple times over periods of several hours, several days, several weeks, or several months. The therapeutically effective dose may be increased or decreased during the course of continuous administration.

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

[0086] In some cases, one or more drugs are delivered to the organism substantially simultaneously with the cells, but in certain embodiments, the cells are exposed to one or more drugs prior to their delivery to the organism, and the drugs enhance the function of the cells upon in vivo delivery. In certain embodiments, the cells are exposed to one or more drugs during culture. The cells may be exposed to the drugs once or more times, and when the cells are passaged in culture, the drugs may or may not be present in the subsequent culture medium. In certain embodiments, the drugs enhance the therapeutic use of the cells by increasing the expression of one or more genes, or by 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 drug is a heat shock response inducer.

[0087] In one embodiment, a specific and therapeutically effective number of cells is provided to an individual. For example, in some embodiments, fewer than 100 million cells are provided to an individual, e.g., 1 million to 40 million or 1 million to 50 million cells. In certain embodiments, 1 million to 20 million cells are provided, but in some embodiments, the millions digit of the cell number 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, 2-1 7, 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, 4-1 8, 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, 6-1 6, 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 These include 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 a condition medium prepared by culturing immortalized human neonatal cardiac CSCs as disclosed herein. In certain embodiments, from the condition medium, the secretome itself or one or more components of the secretome, such as proteins and exosomes, are provided to an organism. The exosomes themselves may have very potent repair and / or regenerative capabilities and may be provided therapeutically to the organism rather than to cells. In certain embodiments, the condition medium is provided therapeutically to the organism rather than to cells.

[0089] IV. Medical condition of the heart Embodiments of this disclosure relate to the treatment of one or more cardiac medical conditions using neonatal cardiac CSCs isolated via conditioned media prepared from cultures of immortalized neonatal CSCs as described herein and / or methods disclosed herein. Certain aspects of such embodiments result in the reversal of one or more cardiac medical conditions or the improvement of at least one symptom of one or more cardiac medical conditions. In exemplary embodiments, the cardiac medical condition is heart failure. Heart failure may be the result of one or more causes, including coronary artery disease, heart attack, hypertension, heart valve disorder, cardiomyopathy (e.g., caused by disease, infection, alcohol abuse and the toxic effects of drugs such as cocaine or certain drugs used in chemotherapy), idiopathic cardiomyopathy, congenital heart disease and / or genetic factors.

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

[0091] If an individual has heart failure, the patient may have either preserved or reduced ejection fraction (EF). Ejection fraction is characterized by microcirculation dilution and extensive cardiomyopathy, and since previous studies using an isoproterenol-induced cardiomyopathy model have shown that CSCs improve cardiac function in rodent models (Sharma et al; 2017), both may be improved with therapeutically effective amounts of cells, conditional medium derived from immortalized human neonatal cardiac CSCs, or cells resuspended in conditional medium derived from immortalized human neonatal cardiac CSCs.

[0092] When an individual has cardiomyopathy, the cardiomyopathy may be ischemic or non-ischemic. Cardiomyopathy can be caused by long-term hypertension, heart valve problems, cardiac tissue damage from a previous heart attack, chronic palpitations, metabolic disorders, nutritional deficiencies, pregnancy, alcohol abuse, drug abuse, chemotherapy drugs, viral infections, hemochromatosis, genetic conditions, elevated cholesterol levels, or a combination thereof. Cardiomyopathy can also be caused by unspecified causes, i.e., specific Patients may have cardiomyopathy.

[0093] A therapeutically effective dose or number of cells, a condition medium derived from immortalized human neonatal cardiac CSCs, or cells resuspended in a condition medium derived from immortalized human neonatal cardiac CSCs may prevent chemotherapy-induced cardiotoxicity. The disclosed invention may also be used to extend 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 media derived from neonatal cardiac CSCs, or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned media derived from c-neonatal cardiac CSCs or immortalized human neonatal CSCs, each possessing anti-inflammatory and anti-apoptotic properties, and which can be used to treat one or more diseases in which the major components of the disease process are involved in an inflammatory process. Non-limiting examples of such diseases that contain inflammatory components and can be treated with such compositions of the present disclosure include renal impairment (both acute and chronic kidney disease), ischemic stroke, arthritis, dry eye, neurodegenerative diseases including Parkinson's disease, severe limb ischemia, COVID-19, and other inflammatory diseases. Accordingly, in some embodiments, the present disclosure provides compositions comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditioned media derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in conditioned media derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs for use in the treatment of inflammatory diseases. In some embodiments, the Disclosure provides compositions comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, neonatal cardiac CSCs or a conditioned medium derived from immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in a conditioned medium derived from neonatal cardiac CSCs, for use in the treatment of diseases selected from renal impairment (both acute and chronic kidney disease), ischemic stroke, arthritis, dry eye, neurodegenerative diseases, Parkinson's disease, and critical limb ischemia. In some embodiments, the Disclosure provides a method for treating inflammatory diseases, comprising the step of administering to a subject requiring it an effective dose of a composition comprising neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, neonatal cardiac CSCs or a conditioned medium derived from immortalized neonatal cardiac CSCs, and neonatal cardiac CSCs resuspended in a conditioned medium derived from neonatal cardiac CSCs.In some embodiments, the Disclosure provides a method for treating renal impairment (both acute and chronic kidney disease), ischemic stroke, arthritis, dry eye, neurodegenerative disease, Parkinson's disease, or critical limb ischemia, comprising the step of administering to a subject requiring it a composition comprising an effective dose of neonatal cardiac CSC, immortalized neonatal cardiac CSC, a conditioned medium derived from neonatal cardiac CSC or immortalized neonatal cardiac CSC, and neonatal cardiac CSC resuspended in a conditioned medium derived from neonatal cardiac CSC or immortalized neonatal cardiac CSC.

[0095] VI. Wound healing Fibrosis and inflammation are two important pathways in wound healing. Accordingly, some embodiments of this disclosure provide neonatal cardiac CSCs, immortalized neonatal cardiac CSCs, conditional media derived from neonatal cardiac CSCs or immortalized neonatal cardiac CSCs, and compositions comprising neonatal cardiac CSCs resuspended in conditional media derived from neonatal cardiac CSCs or immortalized neonatal CSCs, each of which can be used to treat wounds and promote wound healing. In certain embodiments, proteins, miRNAs, or any other factors can be isolated from conditional media prepared by culturing immortalized neonatal CSCs disclosed herein, and such isolated proteins, miRNAs, or factors may be administered to a subject having a wound to treat the wound. In certain embodiments, immortalized neonatal CSCs, conditional media, secretomes themselves, or one or more components, such as proteins and exosomes, which are responsible for regulating tissue fibrosis and cellular functions such as proliferation, migration, and substrate synthesis, can be used to advance wound progression and isolation.

[0096] VII. Combination Therapy In some embodiments, individuals who have received, are receiving, or are scheduled to receive the treatments of the Disclosure may also be provided with additional treatments for a target medical condition. For example, in some embodiments, individuals who have received, are receiving, or are scheduled to receive the treatments of the Disclosure for a medical condition of the heart may also be provided with additional treatments for a medical condition of the heart. The treatments of the Disclosure may precede or follow other treatments. The treatments of the Disclosure may precede or follow other treatments at intervals ranging from minutes to hours, days, weeks, or months. In embodiments where other drugs and immediate therapies are administered to the individual separately, it is generally ensured that a significant period of time does not expire between the times of each delivery so that the treatments of the Disclosure and the additional therapies can still produce a combined effect favorably on the individual. In such examples, it is intended that the individuals may be in contact with each other simultaneously, within minutes, or within approximately 1–12, 6–12, or 12–24 hours between each other, in both manner. In some situations, it may be desirable to significantly extend the duration of treatment, but the interval between each dose should be between a few days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8).

[0097] In certain embodiments, the Therapy of the Disclosure and the additional Therapy are provided simultaneously. In certain embodiments, the Therapy of the Disclosure and the additional Therapy are provided at different times. The distinct entities may be in the same composition, or they may be contained in separate compositions. If the Therapy of the Disclosure and the second Therapy are provided at different times, they may be separated by any suitable range of time, such as minutes, hours, days, weeks, or months. In embodiments where they are provided separately, the order of delivery of the two (or more) Therapies may be any suitable order, including the delivery of cells, secretomes, and / or conditioning media before, simultaneously with, or after the other Therapy.

[0098] Examples of other treatments used in conjunction with the therapies described herein include: 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, anticoagulants, aspirin, surgery, VAD implantation, VAT, coronary artery bypass, percutaneous coronary intervention (PCI), or any combination thereof.

[0099] VIII. Kits Disclosed The kit may include either immortalized neonatal cardiac cytoplasmic stem cells (CSCs) as described herein, or conditioned media derived from such CSCs. The kit may also include other agents for the treatment of the medical condition of the heart.

[0100] The components of the kit may be packaged either in aqueous culture medium or in lyophilized form. The kit's container means generally include at least one vial, test tube, flask, bottle, syringe or other container means in which the components are placed and preferably appropriately dispensed. If there are more than one components in the kit, the kit also generally includes a second, third, or other additional container in which the additional components may be placed separately. However, various combinations of components may be contained in vials. The kits of this disclosure also typically include means for containing one or more compositions in a tightly sealed state for commercial use. Such containers may include syringes or blow-molded plastic containers in which the desired vials are held. In certain embodiments, cells may or may not be delivered in a frozen state and provided in plastic vials.

[0101] The composition may be formulated into a syringe-able composition. If the container means is itself a syringe, pipette, and / or other such similar device, then the formulation may 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 be provided as dry powder. When the reagents and / or components are provided as dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is assumed that the solvent may also be provided in a separate container means.

[0102] The kits of this disclosure also typically include means for housing vials in a tightly sealed state for commercial use, such as a syringe and / or a blow-molded plastic container that holds the desired vial.

[0103] In certain embodiments, the kit includes reagents and / or tools for determining whether an individual has a cardiac medical condition. In some embodiments, the kit includes one or more additional therapies for cardiac-related medical conditions, such as ACE inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs); beta-blockers, calcium channel blockers, cholesterol-lowering agents, digoxin, diuretics, inotropic therapies, potassium, magnesium, vasodilators, anticoagulants, aspirin, TGF-beta inhibitors, and one or more combinations thereof.

[0104] VII. Pharmaceutical Compositions Embodiments of the pharmaceutical compositions of this disclosure include an effective amount of neonatal cardiac CSCs or a conditioned medium derived from such cells dispersed in a pharmaceutically acceptable carrier. The effective amount of neonatal CSCs may contain any appropriate number of cells. In some embodiments, the effective amount is less than 100 million cells, for example, 1 million to 40 million or 1 million to 50 million cells. In some embodiments, the effective dose contains 1 million to 20 million cells, but in some embodiments, the millions digit of the cell count 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, 12-15,12-14, 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, These ranges from 18 to 20, 18 to 19, or 19 to 20, for example. In one particular embodiment, the pharmaceutical composition of the Disclosure comprises 10 million neonatal cardiac CSCs dispersed in a pharmaceutically acceptable carrier. In one particular embodiment, the pharmaceutical composition of the Disclosure comprises 10 million neonatal CD117+ CSCs and a conditioned medium derived from cultured neonatal CSCs. In some embodiments, the cultured neonatal cardiac CSCs from which the conditioned medium is recovered are immortalized neonatal human neonatal cardiac CSCs.

[0105] In one particular embodiment, the pharmaceutical composition of the present disclosure comprises an effective amount of neonatal CSCs (e.g., 10 million cells) and a concentrated preparation of secreted factors present in a conditioned medium derived from the cultured neonatal CSCs. Such a concentrated preparation of secreted factors may be prepared, for example, by filtering the cell-derived conditioned medium to remove some or all of the medium while retaining all or some of the secreted factors present in the medium. Such a concentrated factor may be stored in pellet form. Such a concentrated factor may be resuspended in a pharmaceutically acceptable carrier, excipient, diluent, surfactant, and / or vehicle for storage or administration to a subject. The concentrated factor 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 concentrated 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, the 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 term "pharmaceutically or pharmacologically acceptable" means molecular entities and compositions that, when administered to animals, such as humans as appropriate, do not produce adverse reactions, allergic reactions, or other adverse reactions. The preparation of pharmaceutical compositions containing cells is incorporated herein by reference in Remington: The Science and Practice of Pharmacy, 21st As illustrated by Ed. Lippincott Williams and Wilkins, 2005, this is known to those skilled in the art in light of this disclosure. Furthermore, for administration to animals (e.g., humans), it will be understood that preparations should meet the sterility, pyrogenicity, general safety, and purity requirements of the FDA Secretariat's biological standards.

[0107] As used herein, “pharmaceutically acceptable carrier” may include any solvent, dispersion medium, coating, surfactant, antioxidant, preservative (e.g., antimicrobial, antifungal), isotonic agent, absorption retarder, salt, preservative, drug, drug stabilizer, gel, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, and similar substances and combinations thereof, which are known to those skilled in the art. Any conventional carrier is intended for use in a pharmaceutical composition unless it is incompatible with the active ingredient.

[0108] In some embodiments, the Disclosure provides a pharmaceutical composition comprising one or more human neonatal cardiac CSCs isolated according to a method disclosed herein, the composition further comprising 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 a method disclosed herein, prepared in PlasmaLyte (Baxter, Deerfield, IL). In some embodiments, the human neonatal cardiac CSCs are immortalized. In some embodiments, immortalization is via hTERT expression.

[0109] In some embodiments, this disclosure relates to human novel coronavirus isolated according to the methods disclosed herein. A pharmaceutical composition is provided comprising a condition medium derived from a culture of live cardiac CSCs, the composition further comprising one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles. The condition medium may be formulated for direct administration to a subject. In certain embodiments, the condition medium is enriched with bioactive factors by filtration, and the enriched factors are reformulated in a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, diluents, surfactants, and / or vehicles. For example, in some embodiments, the composition comprises enriched bioactive factors derived from a condition medium derived from a culture of multiple neonatal cardiac CSCs isolated according to a method disclosed herein, prepared in PlasmaLyte (Baxter, Deerfield, IL). In some embodiments, human neonatal cardiac CSCs are immortalized. In some embodiments, immortalization is via hTERT expression. [Examples]

[0110] The following embodiments are included to illustrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors to function well in the implementation of the present invention and can therefore be considered to constitute a preferred form for its implementation. However, those skilled in the art will understand that many modifications may be made in particular embodiments of the present disclosure to obtain similar or similar results without departing from the spirit and scope of the present 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 embodiment describes a novel method for isolating and immortalizing CD117+nCSCs, which can be used to produce a conditional culture medium that can be administered to patients who require it to treat damaged myocardium.

[0113] The inventors have found that, in contrast to adult human hearts, neonatal human hearts contain a homogeneous population of cardiogenic CD117+ stem cells with little to no contamination of hematopoietic progenitor cells and endothelial progenitor cells. Furthermore, the inventors have found that these neonatal human heart-derived stem cells do not show evidence of aging and, when tested in an animal model of myocardial infarction, produce clones that can proliferate and expand to large cell numbers with significant cardiac repair and regenerative activity. Therefore, based in part on these findings, the inventors have determined that these neonatal cardiac stem cells can be obtained directly from neonatal heart tissue without prior selection with CD117 magnetic beads, as has been done in the literature. This is a significant advantage in that it avoids regulatory concerns regarding the exposure of cells intended for therapy to non-GMP quality materials (i.e., CD117 magnetic beads).

[0114] Here, we describe a methodology and certain data relating to 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 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 to 30 days old) 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. Using forceps sterilized with Steri 250 (Inotech), fibrous tissue and fat were removed from the cardiac specimens. The samples were then subjected to Ham's F12. Transfer to culture medium, 1-2 mm 2 It was cut into slices. 2. The tissue fragments were transferred to 50 ml tubes and allowed to precipitate. The supernatant was removed, and the sediment was resuspended in 5-10 ml of collagen type II CSL2 (Worthington No. 4177). Collagenase was dissolved at a concentration of 1-2 mg / ml in Ham's F12 medium, depending on the tissue size and type. The samples were then incubated on a shaker at 200 rpm at 37°C for 30-45 minutes. 3. After collagenase treatment, the tube was removed from the shaker, undigested fragments were allowed to settle, and the supernatant containing the released cells was centrifuged at 1000 rpm for 10 minutes at 15°C. The supernatant was resuspended in growth medium (ham's F12 nutrient mixture supplemented with 10% FBS, 0.2 mM L-gluchion, 10 ng / ml bFGF, and 0.005 U / ml EPO), seeded onto T25 containing the growth medium, and the flask was placed in an incubator at 37°C under 5% CO2. After 72 hours, non-adherent cells were removed by aspiration, 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, centrifuged at 1000 rpm at 15°C for 10 minutes, the supernatant was discarded, and the cell palette was collected to obtain the cell count. 4. As shown in Figure 1, 10,000 cells were used for serial dilution to obtain single-cell isolates of clones. Single-cell isolates were obtained using approximately 8 cells / mL. 50 μl of the 8-cell / mL suspension was dispensed into individual wells in a 96-well plate. Wells containing more than one cell were removed from the experiment by visual inspection under a microscope. 5. Cell counts were manually counted, and the population doubling level (PDL) was calculated using the formula: PDL = 3.32 (log(total cells at collection / total cells at seeding)). Subculturing was performed until the clones reached senescence, and no change in PDL was observed between subculturing cycles.

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

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

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

[0119] Furthermore, these high-CD117-positive clones proliferated approximately twice as fast as low-CD117-positive clones (data not shown) and were negative for CD31 and CD45.

[0120] In summary, these results show that, in contrast to CD117+ cells derived from animal and adult human hearts, human neonatal cardiac CD117+ cells surprisingly avoided contamination with endothelial cells and hematopoietic progenitor cells, which is not only biologically unique and unexpected but also consistent with current practices in the literature.

[0121] Example 2: Immortalization of neonatal CD117-positive cardiac progenitor cells Neonatal CD117+ CSCs are involved in the repair, regeneration, and / or remodeling of damaged myocardium. It induces the secretion of proteins that improve cardiac function. Immortalization of these CD117+ neonatal CSCs allows for the production of such secreted proteins, which is an unlimited supply of the conditioned medium containing them. This embodiment provides such immortalization.

[0122] Neonatal CD117+ CSC clones were isolated according to the method disclosed above in Example 1, and the clones were expanded in culture. Typically, clones expressing high levels of CD117 were immortalized, but 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. The hTERT-expressing clones were isolated, propagated, and stored for long-term use. Figure 4 shows a non-limiting example of a construct for use in the immortalization of neonatal CD117+ human neonatal cardiac CSCs according to the present invention.

[0124] In addition, reversible immortalized neonatal CD117+ CSC clones can be constructed, for example, by flanking the immortalization gene shown in Figure 4 with a sequence that can be used for gene excision, 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 at the FRT site, i.e., one at 5' to the CMV promoter and the other at 3' to the puromycin resistance cassette. The excision of the immortalization cassette may then be promoted using FLP recombinase, thereby reversing immortalization. In addition to its usefulness for producing large quantities of conditioned medium, such cells can be optionally used for direct administration to patients after reversing immortalization, in combination with conditioned medium prepared from the cells, prior to delivery of the cells to the patient.

[0125] A killing switch control is selectively incorporated into the vector, for example, by incorporating the thymidine kinase gene of herpes simplex virus (HSV-TK). HSV-TK phosphorylates ganciclovir (GCV), a prodrug analogue of guanosine nucleoside. Phosphorylated GCV is incorporated into host DNA, terminating DNA strand elongation and thereby inducing cell death. Therefore, treatment of patients administered with GCV in such reversibly immortalized neonatal cardiac CSCs results in the death of administered cells expressing HSV-TK, ensuring drug safety. 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 media a. Telomeres and telomere length To calculate telomere length in human neonatal cardiac CSC clones, flow cytometry analysis is performed using fluorescence insight hybridization and the Dako (catalog number K5327) fluorescein-conjugated PNA probe for flow cytometry (Telomere PNA Kit / FITC). Cell line 1301, which is tetraploid and has long telomeres (>30 kbp), is used as a control. Relative telomere length (RTL) is calculated using the following formula.

number

[0127] b. Staining with aging-related β-galactosidase Cellular senescence can be detected using the β-galactosidase staining kit (catalog number 9) according to the manufacturer's instructions. The evaluation is performed using Cell Signaling Technology (860, Boston, Massachusetts). In short, the CSC (5.0 × 10⁻¹) of human neonatal cardiac CSC before and after immortalization is evaluated. 4The cells were seeded in 24 wells. After 24 hours, the growth medium was removed from the cells, the cells were washed with PBS, and fixed with 1× fixative solution for 15 minutes at room temperature. The cells were incubated overnight with 1 ml of β-galactosidase staining solution and imaged the following day. The results showed that these neonatal human cardiac-derived stem cells did not show evidence of aging. (Figure 5)

[0128] c. Paracrine factor secretion Immortalized neonatal CSCs were grown in a completely foreign-free medium until they reached 85–90% confluence. The cells were washed twice with warm, serum- and growth factor-free Ham's F12 medium, then Ham's F12 was added, and the medium was incubated at 37°C for 48 hours to obtain total condition medium (TCM). Cell debris and particulate matter were pre-removed from the TCM by removing microparticulate matter (MV) by centrifugation at 1000 g for 30 minutes, followed by 20,000 g for 30 minutes, and then by concentration using a 3 kDa filter (Millipore Inc., Billerica, Massachusetts). Total protein content was quantified using the TCA-NLS method, followed by the bicinconine assay (BCA) method (Thermofisher, Waltham, MA). The appearance of the TCM derived from Im-nCSCs was clear and transparent, and free of apoptotic bodies (Figure 8). IM-nCPC TCM is dsDNA-free and has a concentration of 0.2-0.4 ng / ml (maximum permissible 200 ng / ml). TCM is 90-99.0% CD63. + The results were negative for CD45 and CD31 (Figure).

[0129] To normalize the protein content, the inventors used the following formula: The formula used was (concentration factor) × (total volume of culture medium) / total protein content of the conditional culture medium.

[0130] The conditional medium is quantified using the TCA-NLS method followed by the BCA method and normalized to a total protein of 1 mg. Eight paracrine factors, VEGFA, SDF-1α, PDGFB, IGF-1, ANG-1, bFGF, SCF, and HGF, are analyzed using a mesoscale discovery device according to the manufacturer's protocol. The results are processed using Im-nCSC. This shows that 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 angiogenic effect of TCM on angiogenesis, HMEC cells were subjected to an in vitro standard angiogenesis assay using Im-nCPC-derived TCM (Im-nCSC TCM), IMDM basal medium as a negative control, and HMEC complete medium as a positive control. In short, a tubular assay is performed to evaluate the angiogenic capacity of CMs. The formation of tubular structures is evaluated in a 24-well plate coated with Matrigel (BD Biosciences, San Jose, California) as described above. In short, human microvascular endothelial cells (HMEC-1, ATCC® CRL-3243®) are counted and seeded at a density of 20,000 cells / mm2 on reducing growth factor containing Matrigel (product number 354230, BD Biosciences, San Jose, California). (i) Complete endothelial cell medium (Lonza) is added as a positive control, (ii) Im-nCSC-derived conditional medium, or (iii) basal medium is added as a negative control. Cells are imaged after 6-12 hours, and complete images of each well are reconstructed. The total tubular length is then measured using ImageJ64, NIH (http: / / rsb.info.nih.gov / ij).

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

[0133] a2. Wound healing activity To evaluate the wound healing capacity of Im-nCSC TCM, an in vitro wound healing assay was performed to assess the relative migratory ability of cells treated with total condition media. HMEC (HMEC-1 ATCC® CRL-3243®) was seeded in 12-well plates to generate a confluent monolayer. After 12 hours of serum depletion with basal medium, a wound was simulated by making a linear scratch along the cell monolayer using a 1 mL pipette tip. Cell debris was removed by washing the cells once with basal medium. Cells were treated with total condition media derived from Im-nCSC, 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 post-treatment. HMECs were stained with calcein AM cell-permeable dye (ThermoFisher Scientific, Inc.) and imaged before and after treatment using conditional medium derived from Im-nCSC TCMs. Total wound area was measured before and after treatment using ImagePro software, and the percentage change in wound closure was calculated. The results showed that Im-nCSC-derived TCMs significantly increased the wound healing process compared to negative controls (Figure 11).

[0134] a3. Protection against H2O2-induced cell apoptosis Apoptosis in response to oxidative stress was evaluated using the Annexin V Apoptosis Detection Kit (catalog number 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 cooling chamber 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 the cell suspension was incubated overnight and then refrigerated into each well (3 × 10⁶). 5 Transfer the cells to a well, treat the NRCM with 100 μM hydrogen peroxide for 6 hours in serum-free basal medium, either in or without Im-nCSC-derived condition medium (50 μM) (n=4 technical replications), and then BD. Flow analysis of annexin V / PI was performed using the Pharmingen FITC Annexin V Apoptosis Detection Kit I (catalog number 556547). The data showed that Im-nCSC TCM significantly reduced the expression of the early apoptosis 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 is induced in immunodeficient male rats (body weight 250-300g) by permanent ligation of the left anterior descending (LAD) coronary artery. The heart is exposed via left thoracotomy, and the proximal LAD is ligated. Subsequently, 1 million nCSCs and Im-nCSC cells suspended in 100 μL of Vehicle (IMDM), and 100 μL of Vehicle (IMDM) as a control, are injected into the myocardium at four adjacent sites to the infarction. Baseline echocardiography is acquired one day before myocardial infarction surgery. Echocardiography is also performed 7 and 28 days after myocardial infarction. Two-dimensional and M-mode echocardiography is performed using a VisualSonics Vevo 2100 ultrasound unit (VisualSonics, Toronto, Canada, www.visualsonics.com) to evaluate segmented area changes (FAC). Images are acquired from the parasternal long axis and parasternal short axis to the middle papillary level. Myocardial viability is evaluated as follows: Masson trichrome-stained sections at various levels along the long axis are analyzed for collagen deposition to calculate infarct size. The midline technique is used to determine infarct size as described above. Stained sections are analyzed using ImagePro software. Briefly, infarct size is calculated using Masson trichrome-stained sections at various levels along the long axis. To calculate living and non-living tissue, the number of red pixels (living tissue) and blue pixels (non-living tissue) is calculated, and the ratio of non-living tissue pixels to the total number is shown. The data showed that both nCSCs and immortalized clones derived from nCSCs were functional, as shown in the figure, with a significant increase in ejection fraction and a reduction in ejection fraction after transplantation of nCSCs or immortalized clones derived from nCSCs. These results suggest that immortalization did not affect the function of nCSC-derived clones (Figure 7).

[0136] b2. Functional activity of Im-nCPC-derived TCM in a rat MI model To determine the function of total conditioning medium (TCM) derived from Im-nCSCs, rats were used as targets for anterior septal myocardial infarction (MI) induced by LAD ligation at suture. Anesthetized rats were placed supine, and echocardiography was performed with the ultrasound probe directly on the chest wall. Prior to MI, left ventricular ejection fraction (LVEF) was approximately 80%. Approximately 10 minutes after MI, the treatment (nCSC, Im-nCSC-derived TCM, and IMDM) was administered intravenously via the tail vein. After 24 hours, cardiac function was assessed by baseline echocardiography. Five days later, the treatment group received a different dose of the treatment. Animals injected with nCSC and Im-nCSC-derived TCM did not show significant deterioration in LV function from two days after MI to four weeks after MI. At four weeks, LVEF was significantly higher in the nCSC-treated group than in the placebo group (Figure 12). Other parameters, including shortening rate (FS) and reduced end-systolic volume (ESV), also significantly improved compared to the placebo group, and other LV functional parameters, including cardiac output / body weight and posterior wall thickness, also improved and showed a tendency toward a normal remodeling heart. The benefits of nMSC were significantly sustained throughout the 4-week endpoint. (Figure 12)

[0137] Equal portions While the present invention has been described in conjunction with the specific embodiments described 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. The aspects of the embodiments may be modified to provide further embodiments by adopting concepts from various patents, applications, and publications as necessary. These and other modifications may be made to the embodiments in light of the description detailed above. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed herein and therewith, but all equivalents, together with the entire scope of equivalents to which such claims are entitled. It should be construed as including possible embodiments. Therefore, the scope of the claims is not limited by this 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, Elisabeth 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. 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Claims

1. A composition comprising one or more immortalized neonatal CD117 + cardiomyocyte-derived conditioning culture media.

2. The composition according to claim 1, wherein the cells have one or more of the following characteristics: CD90+, CD105+, CD31-, CD34-, CD45- and / or tryptase-negative, and a pharmaceutically acceptable carrier.

3. The composition according to claim 1 or 2, wherein the cells have one or more of the following characteristics: CD44+, CD47+, and / or CD73+.

4. The composition according to any one of claims 1 to 3, wherein the cells have one or more of the following characteristics: GATA4, CD80-, CD86-, and / or Lin-.

5. The composition according to claim 1, wherein, prior to immortalization, the neonatal CD117+ cardiac cells were isolated from the heart of a neonatal individual less than 30 days old.

6. The composition according to claim 5, wherein the isolation does not involve contacting the cells with the antibody.

7. The composition according to claim 5, wherein the isolation does not involve a cell enrichment step using antibody-based selection.

8. The composition according to claim 1, wherein the cells are isolated by limited dilution culture.

9. The composition according to claim 1, wherein the immortalization is achieved by the exogenous expression of hTERT.

10. A composition containing immortalized human neonatal CD117 + cardiomyocyte stem cells.

11. The composition according to claim 10, wherein the cells have one or more of the following characteristics: CD90+, CD105+, CD31-, CD34-, CD45-, and / or tryptase-negative, and a pharmaceutically acceptable carrier.

12. The composition according to any one of claims 10 to 11, wherein the cells have one or more of the following characteristics: CD44+, CD47+, and / or CD73+.

13. The composition according to any one of claims 10 to 12, wherein the cells have one or more of the following characteristics: GATA4, CD80-, CD86-, and / or Lin-.

14. The composition according to claim 10, wherein, prior to immortalization, the neonatal CD117+ cardiac cells were isolated from the heart of a neonatal individual less than 30 days old.

15. The composition according to claim 14, wherein the isolation does not involve contacting the cells with the antibody.

16. The composition according to claim 14, wherein the isolation does not involve a cell enrichment step using antibody-based selection.

17. The composition according to any one of claims 10, wherein the cells are isolated by limited dilution culture.

18. The composition according to claim 10, wherein the immortalization is achieved by the exogenous expression of hTERT.

19. A method for treating an individual due to a medical condition of the heart, comprising the step of providing the individual with a therapeutically effective amount of a composition comprising an immortalized neonatal CD117 + cardiomyocyte stem cell-derived conditioning medium.

20. The method according to claim 19, wherein the composition is selected from any one of the compositions provided in any one of claims 1 to 9.

21. A method for treating an individual due to a medical condition of the heart, comprising the step of providing the individual with a therapeutically effective amount of a composition containing a condition medium derived from immortalized neonatal CD117+ cardiomyocyte stem cells, in combination with a plurality of compositions containing neonatal CD117+ cardiomyocyte stem cells.

22. The method according to claim 21, wherein the composition containing the aforementioned conditional culture medium is selected from any one of the compositions provided in any one of claims 1 to 9.

23. The method according to claim 21 or 22, wherein the composition comprising a plurality of neonatal CD117+ cardiomyocyte stem cells is selected from any one of the compositions provided in any one of claims 10 to 18.

24. A method for repairing or remodeling myocardial tissue in a subject requiring such repair, comprising contacting the myocardial tissue of the subject with a composition provided in any one of claims 1 to 9, a composition provided in any one of claims 10 to 18, or a combination thereof.

25. A method for isolating CD117+ stem cells from neonatal cardiac tissue, comprising isolating one or more cells from neonatal cardiac tissue, or having one or more isolated cells, and culturing the one or more cells in a suitable culture medium for promoting proliferation, wherein the isolation does not involve contacting the cells with a portion that binds to CD117.

26. The composition according to claim 25, wherein the isolation does not involve contacting the cells with the antibody.

27. The method according to claim 25, wherein the isolation does not involve contacting the cells with the CD117 antibody.

28. The method according to claim 25, wherein the isolation is achieved by direct cloning without requiring negative selection for endothelial cells and / or hematopoietic cells.

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