Priming media and methods for stem cell culture and therapy

Abstract

To provide a priming medium for producing primed, activated, or induced stem cells used in cell-based therapy, providing significant advantages over known culture media and methods, to selectively suppress inflammation and promote immunity.SOLUTION: The priming medium includes a serum-free medium, a functional activator of a Type I interferon (IFN) pathway and a Type II IFN pathway, and at least two pro-inflammatory cytokines. The functional activator and the at least two pro-inflammatory cytokines may be present in an amount sufficient to promote induction of stem cells having an anti-inflammatory phenotype. The cells having the anti-inflammatory phenotype are marked by increased expression and / or secretion of one or more anti-inflammatory or immune modulatory mediators as compared to the unprimed population of stem cells. Another aspect of the present disclosure may include stem cells produced by the present disclosure, therapeutic compositions including the stem cells, and use of the stem cells.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 870,832, filed Jul. 5, 2019, and U.S. Provisional Patent Application No. 62 / 908,762, filed Oct. 1, 2019. The entire contents of each of the above U.S. provisional patent applications are incorporated herein by reference for all purposes of the present invention.

[0002] The present disclosure generally relates to priming media and methods for stem cell culture and stem cell therapy, and more specifically, to inducing, activating, or priming discrete and uniform cell phenotypes in order to selectively suppress inflammation and promote immunity, thereby providing significant advantages over known media and methods for producing primed, activated, or induced stem cells for use in cell-based therapies.

Background Art

[0003] For the clinical application of stem cells (e.g., mesenchymal stem cells or MSCs), there is a need for reproducible cell culture and cell expansion methods that can provide a sufficient amount of cells with appropriate quality and consistent therapeutic advantages. Different media and methods have already achieved varying degrees of success. However, further improvements to stem cell media and stem cell culture methods are needed to ensure an improved yield of primed, activated, or induced cells for use in cell therapies with safe and consistently reproducible therapeutic effects.

Summary of the Invention

[0004] According to various aspects of the present disclosure, it is an object to provide a more uniform and predictable population of ex vivo expanded and induced, primed or activated stem cells (e.g., mesenchymal stem cells or MSCs), and such a stem cell population can be used in cell-based therapies. In this field, there has long been a need for improved methods for providing large quantities of uniform and effective stem cells required for cell-based therapies. As an advantage according to various aspects of the present disclosure, stem cells produced by the priming media and priming methods disclosed herein can be used to induce, activate or prime a stem cell culture into a uniform and discrete anti-inflammatory phenotype that functions in a predictable manner after being introduced into a subject.

[0005] According to one aspect of the present disclosure, it is an object to provide improved therapeutic methods, cell culture methods and media for inducing, activating or priming a uniform population of MSCs derived from various adult tissues. The effects or therapeutic advantages of induced, activated or primed MSCs over non-induced or non-primed general MSCs have been demonstrated in vitro and in preclinical models of diseases.

[0006] Surprisingly, it has been found that stem cells (e.g., MSCs) are specifically primed to regulate immune cell activity and improve immune cell dysfunction when exposed to a priming medium having a specific combination of components, particularly after induction of type I and type II interferon pathways by autoimmunity. Primed stem cells (e.g., MSCs) exhibit safety and efficacy in in vivo models of induced interferon activation (e.g., pristane-induced systemic lupus erythematosus) mouse models and their complications. Preferably, stem cells (e.g., MSCs) exposed to the priming medium of the present invention can acquire a state or phenotype that promotes an effective anti-inflammatory response, controls innate and adaptive immune cell activity, and / or regulates interferon, and produces factors that promote angiogenesis and tissue regeneration. Without being bound by theory, the primed stem cells (e.g., MSCs) according to the present invention may be CD4+ T cells and CD8 +By expanding and activating T cells, NK cells, and regulatory T cells (Tregs), the above-described effective immunomodulatory effects are exhibited, and by controlling the production and activity of type I (α, β) and type II interferons (γ) and their downstream effectors, the response to molecules, and other inflammatory signaling pathways, it is recognized that dendritic cells (DCs) and B cells are inhibited.

[0007] Based at least in part on the above findings, one aspect of the present disclosure may include a priming medium for producing an isolated stem cell population having an anti-inflammatory phenotype from a non-primed stem cell population (hereinafter also referred to as the "non-primed stem cell population"). The priming medium may include a serum-free medium, a functional activator of the type I interferon (IFN) pathway and the type II IFN pathway, and at least two inflammatory cytokines. The functional activator and at least two inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be characterized by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the non-primed stem cell population.

[0008] Another aspect of the present disclosure may include a composition comprising a priming medium and stem cells. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. The functional activator and at least two inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be characterized by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the non-primed stem cell population.

[0009] Another aspect of the present disclosure may include an in vitro method for producing an isolated stem cell population having an anti-inflammatory phenotype. The method may include contacting a non-primed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. The cells having an anti-inflammatory phenotype may be characterized by an increased expression of one or more anti-inflammatory or immunomodulatory mediators as compared to non-primed stem cells.

[0010] Another aspect of the present disclosure may include an isolated stem cell population having an anti-inflammatory phenotype produced by an in vitro method. The method may include contacting a non-primed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. The cells having an anti-inflammatory phenotype may be characterized by an increased expression of one or more anti-inflammatory or immunomodulatory mediators as compared to non-primed stem cells.

[0011] The stem cells produced by the in vitro method of the present disclosure are significantly different from naturally occurring stem cells. The reason is that the level or degree of expression of anti-inflammatory genes (for example, indoleamine 2,3-dioxygenase, or IDO) in the stem cells according to the present disclosure is significantly higher than the level or degree of those existing in nature (for example, 50,000 to 150,000 times in the case of the stem cells of the present disclosure compared to the stem cells found in nature). The same is true for the level or degree of expression of surface markers in the primed stem cells (for example, MSC) produced by the in vitro method of the present disclosure. Here, for example, CD146 is expressed at a level significantly higher than the level existing in nature, and as a result, the stem cells can have a pericyte-like phenotype. Furthermore, the stem cells produced by the method of the present disclosure are significantly different from the naturally existing stem cells exposed because the exposure to the priming medium of the present disclosure results in structural and functional distinctions in the (present disclosure's) primed stem cells. The priming medium includes a unique combination of components added to a serum-free medium (and optionally a synthetic medium), and such a state is different from the in vivo inflammatory state where unspecified molecules (for example, proteins, carbohydrates, metabolites, etc.) are present in large quantities.

[0012] Another aspect of the present disclosure may include a method for treating a systemic inflammatory disease or disorder in a subject. This method may include administering to the subject a therapeutically effective amount of stem cells having an anti-inflammatory phenotype. The stem cells are produced by an in vitro method prior to administration. This method may include contacting a non-primed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The priming medium may include a serum-free medium, functional activators of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. The cells having an anti-inflammatory phenotype may be characterized in that the expression of one or more anti-inflammatory or immunomodulatory mediators is increased compared to non-primed stem cells.

[0013] Another aspect of the present disclosure may include a priming medium for producing an isolated stem cell population having an anti-inflammatory phenotype from an unprimed stem cell population. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The functional activator and at least four inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be characterized by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators as compared to an unprimed stem cell population.

[0014] Another aspect of the present disclosure may include a composition comprising a priming medium and stem cells. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The functional activator and at least four inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be characterized by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators as compared to an unprimed stem cell population.

[0015] Another aspect of the present disclosure may include an in vitro method for producing an isolated stem cell population having an anti-inflammatory phenotype. The method may include contacting an unprimed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. Cells having an anti-inflammatory phenotype may be characterized by an increase in the expression of one or more anti-inflammatory or immunomodulatory mediators as compared to unprimed stem cells.

[0016] Another aspect of the present disclosure may include an isolated stem cell population having an anti-inflammatory phenotype produced by an in vitro method. This method may include contacting a non-primed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The cells having an anti-inflammatory phenotype may be characterized by an increased expression of one or more anti-inflammatory or immunomodulatory mediators as compared to non-primed stem cells.

[0017] Another aspect of the present disclosure may include a method for treating a systemic inflammatory disease or disorder in a subject. This method may include administering to the subject a therapeutically effective amount of stem cells having an anti-inflammatory phenotype. The stem cells are produced by an in vitro method prior to administration. This method may include contacting a non-primed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The cells having an anti-inflammatory phenotype may be characterized by an increased expression of one or more anti-inflammatory or immunomodulatory mediators as compared to non-primed stem cells.

[0018] Another aspect of the present disclosure may include a method for adoptive immunotherapy targeting a subject. The method may include exposing non-primed (not primed) CAR-T cells and / or CAR-NK cells for a certain period of time under conditions sufficient to prime, expand, and increase the cytotoxic activity of the CAR-T cells and / or CAR-NK cells. The primed CAR-T cells and / or CAR-NK cells may be produced by the in vitro methods of the present disclosure. The in vitro method may include contacting a non-primed CAR-T cell and / or CAR-NK cell population with a priming medium for a predetermined period of time under conditions sufficient to prime, expand, and increase the cytotoxic activity of the CAR-T cells and / or CAR-NK cells as compared to non-primed CAR-T cells and / or CAR-NK cells. In some cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. In other cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. A therapeutically effective amount of the primed CAR-T cells and / or CAR-NK cells may be administered to the subject targeted for adoptive immunotherapy.

[0019] Another aspect of the present disclosure may include a method for drug discovery. This method may include evaluating the effect of a reagent on the ability of stem cells with an anti-inflammatory phenotype produced by an in vitro method to affect inflammation by exposing the stem cell population with the anti-inflammatory phenotype to the reagent in vitro. The in vitro method may include contacting an unprimed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells with an anti-inflammatory phenotype. In some cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. In other cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. Stem cells with an anti-inflammatory phenotype may be characterized by an increased expression of one or more anti-inflammatory or immunomodulatory mediators compared to unprimed stem cells.

[0020] Another aspect of the present disclosure may include the use of stem cells with an anti-inflammatory phenotype produced by an in vitro method for the development and study of an inflammatory disease model. The in vitro method may include contacting an unprimed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction of stem cells with an anti-inflammatory phenotype. In some cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. In other cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. Cells with an anti-inflammatory phenotype may be characterized by an increased expression of one or more anti-inflammatory or immunomodulatory mediators compared to unprimed stem cells.

Brief Description of the Drawings

[0021] The above-described features and other features of the present disclosure will be understood by those skilled in the art by the following description with reference to the accompanying drawings.

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Mode for Carrying Out the Invention

[0048] Unless otherwise specified, the methods and means described in this specification may generally be carried out according to conventional methods well known in the art and are described in various general and more specific reference documents cited and discussed throughout this specification. For example, reference may be made to "Molecular Cloning: A Laboratory Manual", Sambrook et al., 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); "Current Protocols in Molecular Biology", Ausubel et al., Greene Publishing Associates (1992); and "Antibodies: A Laboratory Manual", Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990).

[0049] Definition For clarity of understanding and ease of reference, a list of terms used throughout this specification and in other parts of this application is gathered here. Some of these terms are well known in the art and are defined herein for clarity, while some of these terms are used specifically herein and thus must be defined in order to accurately understand the present invention.

[0050] In this specification, the singular form of the English language means one or more than one, at least one. When the plural form of the English language is used in this specification, it generally also includes the singular meaning.

[0051] As used herein, the term "cell bank" may mean stem cells (e.g., mesenchymal stem cells, MSCs or multipotent stromal cells) that have been cultured in a priming medium according to the present disclosure and then stored for future use. Such cells may be dispensed and stored as aliquots. They may be used as they are without storage, or may be expanded in culture after storage. This can provide "ready-made" cells useful for administration during research, clinical trials or clinical therapy, thus bringing convenience. These cell lines may already be stored in a pharmaceutically acceptable excipient, in which case they may be administered directly, or may be mixed with a suitable excipient when released from storage. The cells may be frozen or otherwise preserved to maintain viability. The bank may be established using cells from a subject to be treated. Alternatively, the bank may contain cells intended for use in allografts.

[0052] As used herein, the terms "cell therapy" or "therapy by cells" may mean transplanting human or animal-derived stem cells according to the present disclosure (i.e., cells cultured in the priming medium according to the present disclosure or cells contacted or exposed thereto) in order to prevent, treat or improve one or more symptoms associated with a disease or disorder (e.g., systemic inflammation or autoinflammation), and examples of the prevention, treatment or improvement of the one or more symptoms include, but are not limited to, replacement or repair of damaged tissues or organs, regulation of immune responses, reduction of inflammatory symptoms, etc.

[0053] As used herein, the term "repair", when used directly with respect to damaged tissues, may mean improving such damage by direct mechanisms (e.g., regeneration of damaged tissues) and indirect mechanisms (e.g., enabling the formation of tissues as a result of alleviating inflammation by stem cells according to the present disclosure (i.e., cells cultured in the priming medium according to the present disclosure or cells contacted or exposed thereto)).

[0054] As used herein, the term "cell growth medium" may mean a growth or expansion culture aqueous medium containing factors and nutrients suitable for supporting the growth of cells such as stem cells according to the present disclosure (i.e., cells cultured in the priming medium according to the present disclosure or cells contacted or exposed thereto) as described herein. For initial seeding and culturing (e.g., until confluent), a standard or off-the-shelf medium such as DMEM containing 10% FBS may be used. After reaching confluence, a maintenance medium such as low-glucose serum-free DMEM may be used for cell maintenance. The cell media applied are well-known in the field of cell culture and may all be commercially available products.

[0055] Unless otherwise specifically limited, the term "comprising" means that the object is necessarily included and there is no intention to impose any restrictions or exclusions on other possible contents. For example, a "composition comprising X and Y" includes any composition containing X and Y, regardless of the presence of any other components in the composition. Similarly, a "method comprising step X" includes any method in which step X is carried out, whether step X is the only step of the method or one of a plurality of steps, and regardless of the number of other steps present and whether step X is simpler or more complex than the other steps. The term "comprising" and similar terms using the root "comprise" are used herein as synonyms of "comprising" and have the same meaning as "comprising".

[0056] As used herein, the term "conditioned medium" or "CM" may mean a medium in which stem cells according to the present disclosure (i.e., cells cultured in the priming medium according to the present disclosure or cells contacted or exposed thereto) are cultured for a certain period of time. In one embodiment, the conditioned medium can be prepared by culturing stem cells produced by the priming method disclosed herein and then collecting the supernatant or conditioned medium after the culture period.

[0057] As used herein, the term "synthetic medium" may mean a cell culture growth medium to which specific designated factors are added. This term may also refer to a cell culture growth medium in which all components can be represented by their chemical formulas and are included at known concentrations, having a meaning opposite to that of a non-synthetic medium.

[0058] As used herein, the term "effective amount" may mean an amount capable of providing a desired local or systemic effect. Examples of the "desired local or systemic effect" include effectively improving an undesirable condition (e.g., inflammation associated with a systemic inflammatory or autoinflammatory disease such as graft-versus-host disease, systemic lupus erythematosus), and achieving specific desired effects described in the present disclosure. For example, an effective amount may be an amount sufficient to achieve a beneficial or desired clinical outcome. The effective amount may be provided all at once in a single administration or may be provided in several administrations. The amount considered effective may be accurately determined based on individual factors of each subject, such as the body size, age, injury and / or disease or injury to be treated of each subject, and the amount of time elapsed since the injury or disease occurred. One of ordinary skill in the art may determine the effective amount to be administered to a given subject based on these conventional considerations in the art. The term "effective dose" may have the same meaning as "effective amount".

[0059] As used herein, the term "effective route" may mean a route for delivering an agent (e.g., a stem cell) to a desired compartment, system, or location in the body. For example, an effective route may be a route by which an agent can be administered to provide a sufficient amount of the agent at the desired site of action to achieve a beneficial or desired clinical outcome.

[0060] As used herein, the term "exogenous addition" may refer to adding a compound, growth factor, differentiation factor, etc. to a culture or medium in the context of a culture or conditioned medium to supplement any compound or growth factor that may already be present in the culture or medium.

[0061] As used herein, the term "isolated" may refer to a target cell becoming unassociated with one or more cells or one or more cell components that are associated with it in vivo. The term "expanded population" may mean that the number of desired cells is relatively increased as compared to one or more other cell types in vivo or in primary culture. However, as used herein, the term "isolated" does not mean that only the cells described herein are present. On the other hand, the term "isolated" may imply that the cells described herein are removed from their native tissue environment and are present at a higher concentration as compared to the normal tissue environment. Thus, an "isolated" cell population may further contain cell types other than the cells described herein and may further contain other tissue components. This can also be expressed, for example, in terms of cell doubling. Cells can be concentrated by doubling 10, 20, 30, 40 or more times in vitro or ex vivo as compared to their original amount in vivo or in their original tissue environment (e.g., bone marrow, peripheral blood, placenta, umbilical cord, umbilical cord blood, adipose tissue, etc.).

[0062] As used herein, the term "mesenchymal stem cell" or "MSC" may mean a cell that can be isolated from many sources such as adult bone marrow, peripheral blood, adipose, placenta and umbilical cord blood, which are derived from the embryonic mesoderm. MSC can differentiate into many mesodermal tissues including muscle, bone, cartilage, adipose, tendon. There is a large amount of literature on these cells. See, for example, U.S. Patent Nos. 5,486,389; 5,827,735; 5,811,094; 5,736,396; 5,837,539; 5,837,670; and 5,827,740. Also see Pittenger M et al., "Science, 284:143-147 (1999)". In some cases, MSC may be derived from apparently healthy subjects (e.g., human subjects), i.e., subjects without signs and / or symptoms of disease. The term may also be used interchangeably with "multipotent stromal cells".

[0063] As used herein, the term "multiponent" or "multiponent cell" refers to a cell type capable of producing other specific cell types of limited variety. Since multiponent cells are involved in the fate of one or more embryonic cells, unlike pluriponent cells, they cannot produce any of the three embryonic cell lineages and cannot produce extraembryonic cells either.

[0064] The term "partial", when used in combination with a cell culture and / or a cell population, may mean any non-zero amount of a cell culture or cell population, covering a range from one cell to the entire cell culture or cell population. In some cases, the term "partial" means at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 94%, or at least 95% of a cell culture or cell population.

[0065] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions (e.g., cells), and / or dosage forms that, within the scope of reasonable medical judgment, do not cause excessive toxicity, irritation, allergic response, or other problems or complications, and exhibit a reasonable benefit / risk ratio, and are suitable for use in contact with human and animal tissues.

[0066] As used herein, in the context of cells, the term "pluripotent" or "pluripotency" may refer to cells that can give rise to multiple tissues derived from any of the three embryonic cell lineages and extraembryonic cells. This term is not limited to the inner cell mass state and, in any case, is not used to describe lineage-specific progenitor cells formed during the development period or present from the perinatal to the adult stage. The state of pluripotent cells is a natural state but can also be proliferated in vitro under certain conditions known to those skilled in the art. Under such culture conditions, the permanent maintenance or pluripotent state of stem cells occurs. The properties for the definition of pluripotent cells are not limited to their functional characteristics but are also represented by gene expression patterns, so the identification of pluripotent cells can be achieved by the staining patterns and quantitative measurements of the markers they exhibit. The presence of the protein transcription factors Oct3 / 4, Sox2, and NANOG represents a set of core transcription factors involved in the maintenance of the pluripotent state, and the downregulation of these factors is a prerequisite for differentiation. All of the above three transcription factors show interactions, and there are many overlapping genes among those transcriptionally regulated by them. Furthermore, the transcription factors responsible for pluripotency maintenance are usually identified using some surface markers present thereon, and the most common surface markers are stage-specific embryonic antigens 3 and 4 (SSEA3 and SSEA4), TRA-1-61, and TRA-1-81. Pluripotent cells such as embryonic stem cells can also exhibit alkaline phosphatase activity, which is used for the identification of normal pluripotent cells.

[0067] As used herein, the term "progenitor cell" may in any case refer to a normal cell state that represents the direct lineage ancestor of a differentiated cell. By definition, a progenitor cell does not have to irreversibly assume the above-defined fate, but may be pluripotent with respect to multiple defined states. Thus, it shows the ability to differentiate into the fates of two or more progeny. As development proceeds in any living organism, the gradual loss of ability and thus pluripotency becomes an indicator of formation that is temporarily defined as the differentiation of tissues and organs. An example of a progenitor cell is an induced pluripotent stem cell (iPSC), which is a type of pluripotent stem cell artificially induced from non-pluripotent cells (usually adult somatic cells) by the insertion of a certain gene.

[0068] As used herein, the term "self-renewal" may refer to the ability of a stem cell to produce replicated daughter stem cells, and the replicated daughter stem cells have the same differentiation potential as the stem cells that produced them.

[0069] As used herein, the terms "expansion culture" and "proliferation" may mean the process of achieving an increase in the number of cells. This term may also refer to the balance between cell division and cell loss due to cell death and cell differentiation.

[0070] As used herein, the term "stem cell" may refer to a cell that can self-renew (i.e., produce progeny with the same differentiation potential) and can also produce progeny cells with more restricted differentiation potential. In the context of the present disclosure, stem cells also include, for example, more differentiated cells that have been dedifferentiated by nuclear transfer, fusion with more primitive stem cells, introduction of specific transcription factors, or culture under specific conditions. See, for example, Wilmut et al., Nature, 385:810-813 (1997); Ying et al., Nature, 416:545-548 (2002); Guan et al., Nature, 440:1199-1203 (2006); Takahashi et al., Cell, 126:663-676 (2006); Okita et al., Nature, 448:313-317 (2007); Takahashi et al., Cell, 131:861-872 (2007).

[0071] Dedifferentiation can also be induced by administering specific compounds or exposing to an in vitro or in vivo physical environment capable of inducing dedifferentiation, and this process substantially reflects the acquisition of pluripotency. Stem cells can also be obtained from other sources such as abnormal tissues like teratocarcinomas and embryoid bodies (which are not directly derived from the inner cell mass but are considered embryonal stem cells in that they are ultimately derived from embryonal tissue). The pluripotent stem cell state can also be produced by introducing genes related to stem cell functions into non-stem cells (e.g., induced pluripotent stem cells).

[0072] As used herein, the term "subject" may refer to a vertebrate or a mammal. Examples of subjects include domestic animals, test animals, and pets, such as mammals like sheep, cows, pigs, dogs, cats, and mice, as well as reptiles, birds, and fish. The terms "patient" and "subject" may be used interchangeably herein. In one example, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow.

[0073] As used herein, with respect to cells in a cell culture or cell population, the term "substantially free" means that the subject cell type is present in an amount of less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total number of cells present in the cell culture or cell population.

[0074] As used herein, the term "therapeutically effective amount" may mean the amount of a reagent (e.g., one or more cells cultured in or contacted or exposed to the priming medium according to the present disclosure) that can be confirmed to produce some therapeutic response in a subject. A therapeutically effective treatment within the meaning of the terms used herein may be a treatment that improves the quality of life of a subject even if it does not itself improve the outcome of the disease. Such a therapeutically effective amount can be readily determined by one of ordinary skill in the art. Thus, "treatment" may mean delivering such an amount. Thereby, treatment can prevent or ameliorate any pathological symptoms of a disease or disorder such as a systemic inflammatory or autoinflammatory disease.

[0075] As used herein, the term "treatment" includes preventing, ameliorating, suppressing, or curing a defect, dysfunction, disease, or other harmful process, and examples of the other harmful processes include those that can interfere with the treatment and / or those that result from the treatment. In some cases, the symptoms of a disease or disorder are reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0076] As used herein, "prevent" or "preventively" may mean completely or partially (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%) preventing or suppressing at least one symptom of a disease or disorder (e.g., a systemic inflammatory or autoinflammatory disease or disorder), or the frequency with which such a symptom appears, completely or partially (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%) preventing or suppressing the symptoms of the disease or disorder, or the frequency with which such a symptom appears.

[0077] As used herein, when referring to one stem cell (or a plurality of stem cells) in contact with the priming medium of the present disclosure, the term "induce" may refer to a change in the phenotype (e.g., surface marker type and / or presence and / or absence) and / or genotype (e.g., mRNA expression level) of one stem cell (or a plurality of stem cells) resulting from contact with the priming medium.

[0078] As used herein, the term "uniform induction" may mean that at least some of the stem cells in a stem cell population are induced to obtain one or more changes in the phenotype and / or genotype of the stem cells by contact with the priming medium of the present disclosure. In some cases, substantially uniform induction may occur. Substantially uniform induction may mean that most of the stem cells (>50%) are induced to obtain one or more changes in the phenotype and / or genotype of the stem cells. Alternatively, substantially identical induction may mean that most of the stem cells, e.g., about 50-55%, about 55-60%, about 60-65%, about 65-70%, about 70-75%, about 75-80%, about 80-85%, about 85-90%, about 90-95%, about 96%, about 97%, about 98%, or about 99% of the stem cells, are induced to obtain one or more changes in the phenotype and / or genotype of the stem cells.

[0079] As used herein, the term "verification" may mean confirmation. In the context of the present disclosure, it can be confirmed that a stem cell is a specific cell type (e.g., MSC) having a desired function and / or phenotype. Thereby, when a person skilled in the art uses the stem cell (for uses such as treatment, banking, drug screening, etc.), it is reasonably expectable to expect the efficacy of the stem cell. Therefore, verification may mean confirming that a stem cell found and established to have a desired activity and / or phenotype indeed retains that activity and / or phenotype. Therefore, verification may be a proof event in a two-event process regarding an initial determination and a subsequent determination. The second event may be referred to as "verification" herein.

[0080] As used herein, the term "ready-made medium" may mean a medium composition containing a serum-containing basal medium and other compounds (e.g., ascorbic acid, dexamethasone, etc.). Serum is essentially an undefined component in the medium. Therefore, due to the heterogeneity of different lots of serum, the exact composition of commercially available "ready-made" serum-containing media is unknown. An example of a "ready-made medium" is Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / F-12 1:1; Invitrogen) supplemented with 1% L-glutamine (Glutamax; Invitrogen), 10% fetal bovine serum, 100 nM dexamethasone, 50 μM ascorbic acid-2P, and 10 mM β-glycerophosphate.

[0081] As used herein, the term "systemic autoimmune disease or disorder" or "auto-inflammatory disease or disorder" may mean a condition of a subject characterized by damage to cells, tissues, and / or organs caused by an immune reaction against the subject's own cells, tissues, and / or organs. Exemplary and non-limiting examples of autoimmune diseases treatable by the immunomodulatory cells produced by the present disclosure include alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behçet's disease, bullous pemphigoid, cardiomyopathy, aphthous dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Chagas' disease, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, Ménière's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, sarcoidosis, scleroderma, progressive systemic sclerosis, Sjögren's syndrome, Goodpasture's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis (e.g., dermatitis herpetiformis type vasculitis), vitiligo, Wegener's granulomatosis, anti-glomerular basement membrane disease, antiphospholipid syndrome, nervous system autoimmune diseases, familial Mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmia, polyendocrinopathy, psoriasis, and the like.

[0082] In one example, systemic autoimmune or autoinflammatory diseases treatable by the present disclosure include SLE, mixed connective tissue disease, Sjögren's syndrome, overlap syndromes (rheumatoid arthritis with SLE and / or Sjögren's syndrome), graft-versus-host disease, adult or juvenile idiopathic psoriatic arthritis, dermatomyositis, polymyositis, other myositis, systemic vasculitis, CNS vasculitis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic juvenile idiopathic arthritis, juvenile arthritis, adult Still's disease, and systemic scleroderma.

[0083] Other examples of systemic autoimmune or autoinflammatory diseases include monogenic interferonopathies, where gene variants cause activation of the type I interferon pathway, leading to complex rheumatic diseases. These diseases are thought to be based on an autoimmune-autoinflammatory spectrum that depends on the driving factors of dysregulated type I interferon production. These diseases include Aicardi-Goutières syndrome (AGS), chronic atypical neutrophilic dermatosis with lipodystrophy and fever (CANDLE), and neonatal-onset STING-associated vasculitis (SAVI). Other diseases associated with vasculitis and high interferon levels include retinal vasculopathy with cerebral leukodystrophy (RVCL) and Singleton-Merten syndrome, where the clinical symptoms are thought to be related to chronic inflammation and at least partly result from constitutive activation of RIG-I, causing increased type I interferon activity and ISG expression.

[0084] As used herein, the term "immunomodulation" may mean modification, amplification, inhibition, or reduction of one or more biological activities of the immune system, such as downregulation of the immune response, enhancement of the immune response, and changes in inflammatory states through changes in the cytokine profile, cytotoxic activity, and antibody production, as well as effects on immune and immune-related cells in each of the above situations, but is not limited thereto.

[0085] As used herein, the term "cell population" may mean any number of stem cells (e.g., MSC) greater than 1, but preferably at least 1×10 3 cells, at least 1×10 4 cells, at least 1×10 5 cells, at least 1×10 6 cells, at least 1×10 7 cells, at least 1×10 8 cells, or at least 1×10 9 cells. In some cases, in the initial stem cell population, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the cells (percentage of cell number) (e.g., stem cells such as MSC) are undifferentiated stem cells (e.g., undifferentiated MSC).

[0086] As used herein, the terms "significant expression" or its synonyms "positive" and "+", when used with respect to cell surface markers, mean that in a cell population, more than 20% of the cells, preferably more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or 100% of the cells express the cell surface marker.

[0087] The expression of cell surface markers can be confirmed, for example, by flow cytometry of specific cell surface markers using conventional methods and apparatuses (e.g., commercially available antibodies and the BECKMAN COULTER EPICS XL FACS system used with standard protocols known in the art), and when using the conventional methods and apparatuses, in flow cytometry, a signal of a specific cell surface marker higher than the background signal is shown. The background signal is defined as the signal intensity by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker in conventional FACS analysis. For markers regarded as positive, when using conventional methods and apparatuses, the observed specific signal is 20% stronger than the background signal intensity, preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, 500%, 1000%, 5000%, 10000% or more stronger. Further, relevant cells can be identified using commercially available or known monoclonal antibodies against the cell surface markers (e.g., cell receptors and transmembrane proteins).

[0088] As used herein, the term "priming medium" may refer to a cell culture medium containing new or specific components, and the combination of each component synergistically induces an anti-inflammatory phenotype in stem cells (e.g., MSCs) in contact with the medium under effective culture conditions.

[0089] As used herein, the term "type I interferon pathway" may mean an inflammatory pathway that functions in the regulation of innate and adaptive immunity. Type I interferons (IFNs) (primarily IFN-α and IFN-β) regulate the type I interferon pathway. Type I IFN production is induced after detection by pattern recognition receptors (PRRs). Type I interferons balance the innate immune response by promoting antigen presentation and natural killer cell function while inhibiting pro-inflammatory pathways and cytokine production. Type I interferons also promote the development of high-affinity antigen-specific T and B cell responses and immune memory by activating the adaptive immune system, initiating a series of events that can cause autoimmune diseases (e.g., Sjögren's syndrome or SLE).

[0090] As used herein, the term "type II interferon pathway" may refer to an inflammatory pathway primarily regulated by interferon gamma (IFN-γ). IFN-γ signals mainly through the JAK-STAT pathway, which affects gene regulation. JAK-STAT signaling involves the recruitment and activation of a series of receptors of the Janus family of kinases (JAKs: JAKs1-3 and TYK2) and STATs (STATs1-6 including STAT5a and STAT5b), and controls the transcription of target genes through specific response factors. IFN-γ is a cytokine that plays an important role in adaptive and innate immunity.

[0091] As used herein, the term "functional activator of type I and type II interferon pathways" may mean any one or a combination of molecules that stimulate both type I and type II interferon pathways. In one example, the expression level and / or activity of IDO can be used as an indicator of the activation of type I and type II interferon pathways (e.g., increased IDO expression and / or activity compared to a control value may indicate activation of type I and type II interferon pathways). Methods for measuring the expression and activity of IDO are known in the art.

[0092] As used herein, the term "anti-inflammatory phenotype" may mean an aggregate of multiple cellular processes involved in gene and protein expression, which can result in specific morphological and functional characteristics of cells, and the specific morphological and functional characteristics of the cells are characterized by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators or markers as compared to cells not in contact with the priming medium of the present disclosure as a result of culturing using the priming medium of the present disclosure.

[0093] As used herein, the term "inflammatory cytokine" may mean a type of signaling molecule secreted from immune cells (e.g., helper or cytotoxic T cells (Th), monocytes, dendritic cells, and macrophages) that stimulates inflammation.

[0094] As used herein, the term "interferon-regulated autoimmune disease" may mean an autoimmune disease in which one type of interferon or a combination of multiple types of interferons is actively involved in the etiology or pathogenesis of the disease and actively acts on the progression of the autoimmune disease. In other words, if interferon activity is an indicator of the disease, the disease is an interferon-regulated autoimmune disease.

[0095] As used herein, the term "abnormal activation of the type I interferon pathway" may mean responses of the innate and adaptive immune systems that are different from the responses generally expected during the activation process of the type I interferon pathway. For example, abnormal activation of the type I interferon pathway, such as a cytokine storm, may result in excessive innate and adaptive immune responses.

[0096] As used herein, the term "abnormal activation of the type II interferon pathway" may mean an innate and adaptive immune system response that is different from the response generally expected during the activation of the type I interferon pathway. For example, in the absence of pathogen induction, excessive or harmful innate and adaptive immune responses may occur, which may cause inflammatory changes and damage to end organs.

[0097] As used herein, the term "CAR-T cell" may mean a T cell collected from a subject, modified in vitro to add a chimeric antigen receptor (CAR) protein thereto, and then re-administered to the subject. Methods for priming CAR-T cells prior to re-administration to a subject are disclosed herein.

[0098] As used herein, the term "CAR-NK cell" may mean an NK cell collected from a subject, modified in vitro to add a CAR protein thereto, and then re-administered to the subject. Methods for priming CAR-NK cells prior to re-administration to a subject are disclosed herein.

[0099] As used herein, the term "immunomodulatory mediator" may mean a molecule (e.g., a molecule secreted by a cell) that affects one or more components of the immune system. Examples of immune system components include proteins (e.g., cytokines, interleukins, antibodies, complement, cell receptors, ligands, major histocompatibility complexes, etc.), and immune cells (e.g., lymphocytes such as NK cells, T cells, and B cells, neutrophils, macrophages / monocytes). In some cases, the effect may be positive (e.g., an increase in protein level and / or activity, or an increase in the proliferation and / or activity of immune cells) or negative (e.g., a decrease in protein level and / or activity, or a decrease in the proliferation and / or activity of immune cells). Non-limiting examples of immunomodulatory mediators can include IDO, IL-1β, IL-17A, TNF-α, and TNF-γ.

[0100] As used herein, the term "cytotoxic activity" may mean the ability of a cell (e.g., a CAR-T cell or a CAR-NK cell) to kill different types of cells, and usually, in the case of lymphocytes, the killing is carried out by synthesizing perforin. This ability can be measured by co-culturing lymphocytes and target cells. Other measurement methods for confirming the cytotoxic activity of cells are known in the art.

[0101] As used herein, the terms "down-regulate" or "down-regulation", or "decrease" or "reduce" of the activity and / or amount of a molecule (e.g., an immunomodulatory mediator) may mean that the activity and / or amount of the molecule is down-regulated, decreased, or reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control value. In some cases, the term "down-regulation" may mean complete inhibition.

[0102] As used herein, the terms "up-regulate" or "up-regulation", or "enhance" or "increase" of the activity and / or amount of a molecule (e.g., an immunomodulatory mediator) may mean that the activity and / or amount of the molecule is up-regulated or increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a control value.

[0103] Prime medium

[0104] In one aspect of the present disclosure, a priming medium for producing an isolated stem cell population having an anti-inflammatory phenotype from an unprimed stem cell population may be included. The priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. The functional activator and the at least two inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be marked by an improvement in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators as compared to the unprimed stem cell population.

[0105] In some cases, the priming medium of the present disclosure may be a synthetic medium.

[0106] The priming medium may be a serum-free medium. Examples of serum-free media that may be contained in the priming medium of the present disclosure may include DMEM-F12, DMEM, and α-MEM. A serum-free medium is a medium that does not contain any type of animal serum. It is preferable to avoid the possibility of cross-contamination of stem cells with a serum-free medium. A medium without a serum substitute is a medium that is not supplemented with any commercially available serum substitute preparation. The serum-free medium may be provided in the priming medium such that its content (e.g., w / v) in the priming medium is at least about 80-99%, for example, about 80-82%, about 82-84%, about 84-86%, about 86-88%, about 88-90%, about 90-92%, about 92-94%, about 94-96%, about 96-98%, or about 98-99%.

[0107] The priming medium of the present disclosure may contain other components necessary for cell survival and contamination prevention. Such components include, for example, glucose and antibacterial agents for reducing contamination by bacteria, mycoplasma, and fungi. Therefore, the priming medium of the present disclosure may contain one or more antibacterial agents or antibiotics to prevent contamination. Commonly used antibiotics or antifungal compounds are a mixture of penicillin / streptomycin, but may include, but are not limited to, amphotericin (registered trademark: amphotericin B), ampicillinicillin, gentamicin, boremicin, hydromycin, kanamycin, mitomycin, etc.

[0108] In some cases, the priming medium of the present disclosure may contain one or more functional activators of the type I IFN pathway and the type II IFN pathway. In one example, the priming medium of the present disclosure contains only one functional activator (e.g., poly(I:C)) of the type I IFN pathway and the type II IFN pathway. In some cases, the functional activators of the type I IFN pathway and the type II IFN pathway may include Toll-like receptor (TLR) ligands such as TLR3. The TLR ligand may include any molecule, compound, or reagent that can activate TLR signaling and induce an inflammatory response. The TLR ligand can activate TLR signaling by binding or interacting with any known TLR (e.g., TLR1-TLR10). The functional activators of the type I IFN pathway and the type II IFN pathway may include synthetic molecules (e.g., synthetic analogs of TLR ligands) that stimulate or activate the type I IFN pathway and the type II IFN pathway. In some cases, the TLR ligand may include Poly(I:C), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). In one example, the TLR3 receptor may be induced by poly(I:C). In another example, the TLR4 receptor may be induced by LPS.

[0109] In one example, the priming medium of the present disclosure may include poly(I:C) as a functional activator of the type I IFN pathway and the type II IFN pathway. As described in Example 1 below, surprisingly, exposing human MSC cultures to poly(I:C) results in an increase in IL-1β production (which induces CCR-8 activation), activation of five classical pathways, downregulation of three, and a marked proliferation of CD146+ / pericyte-like MSCs was discovered.

[0110] One or more functional activators of the type I IFN pathway and the type II IFN pathway are included in the priming medium of the present disclosure at a concentration sufficient to activate the type I and type II IFN pathways. For example, the functional activator of the type I IFN pathway and the type II IFN pathway may be included in the priming medium at a concentration of at least about 0.1 μg / mL to about 500 μg / mL, about 0.2 μg / mL to about 450 μg / mL, about 0.3 μg / mL to about 400 μg / mL, about 0.4 μg / mL to about 350 μg / mL, about 0.5 μg / mL to about 300 μg / mL, about 0.6 μg / mL to about 250 μg / mL, about 0.7 μg / mL to about 200 μg / mL, about 0.8 μg / mL to about 150 μg / mL, about 0.9 μg / mL to about 100 μg / mL, about 0.5 μg / mL to about 50 μg / mL, about 0.5 μg / mL to about 25 μg / mL, about 0.5 μg / mL to about 10 μg / mL, or about 0.5 μg / mL to about 5 μg / mL. In one example, poly(I:C) may be provided in the priming medium of the present disclosure at a concentration of 1 μg / mL.

[0111] In some cases, the priming medium of the present disclosure may contain at least 2 inflammatory cytokines, at least 3 inflammatory cytokines, at least 4 inflammatory cytokines, at least 5 inflammatory cytokines, at least 6 inflammatory cytokines, at least 7 inflammatory cytokines, at least 8 inflammatory cytokines, or more than 8 inflammatory cytokines. Non-limiting examples of inflammatory cytokines include IFN-α, -β, -γ, TNF-α, -β, IL-1β, IL-1Ra, GM-CSF, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-13, IL-15, IL-22, -23, MIP-1α / β, MCP-1, IP-10, TGF-β1, SCM-1, and IL-17A.

[0112] At least 2 inflammatory cytokines may be included in the priming medium of the present disclosure at a concentration sufficient to promote the induction of stem cells (e.g., MSCs) with an anti-inflammatory phenotype compared to stem cells not in contact with the priming medium of the present disclosure. For example, at least 2 inflammatory cytokines may be at a concentration of at least about 1 ng / ml to about 200 ng / ml, such as about 1 - 10 ng / ml, about 10 - 20 ng / ml, about 20 - 30 ng / ml, about 30 - 40 ng / ml, about 40 - 50 ng / ml, about 50 - 60 ng / ml, about 60 - 70 ng / ml, about 70 - 80 ng / ml, about 80 - 90 ng / ml, about 90 - 100 ng / ml, about 100 - 125 ng / ml, about 125 - 150 ng / ml, about 150 - 175 ng / ml, or about 175 - 200 ng / ml in the priming medium. In one example, the priming medium of the present disclosure may contain IFN-γ (100 ng / ml) and TNF-α (20 ng / ml). In another example, the priming medium of the present disclosure may contain IFN-γ (100 ng / ml), TNF-α (20 ng / ml), IL-1β (10 ng / ml), and IL-17A (50 ng / ml).

[0113] In some cases, the priming medium of the present disclosure may contain only a serum-free medium, a functional activator of the type I and type II IFN pathways, at least two inflammatory cytokines (e.g., at least four inflammatory cytokines), and / or essential vitamins. "Only" means that, as described herein (e.g., in Examples 1 and 2), the priming medium does not contain any other immunomodulatory mediators (e.g., growth factors and cytokines) that can substantially affect the efficacy (e.g., basic and novel properties) of the priming medium. However, even when using the term "only", it is still acceptable to include some components that do not substantially affect the basic and novel properties of the priming medium of the present application. Examples of such components include substances necessary for the survival of cells in culture (e.g., amino acids, trace metals, inorganic salts, carbon energy, buffers, etc.). In such cases, the priming medium of the present application can be said to consist essentially of a serum-free medium, a functional activator of the type I and type II IFN pathways, at least two inflammatory cytokines (e.g., at least four inflammatory cytokines) and / or essential vitamins.

[0114] In one example of the present disclosure, the priming medium may contain a serum-free medium, a functional activator of the type I and type II IFN pathways (e.g., poly(I:C)), and two inflammatory cytokines (e.g., IFN-γ and TNF-α). Such a priming medium is also referred to herein as a "four-component priming medium". In some cases, the four-component priming medium may contain only a serum-free medium, a functional activator of the type I and type II IFN pathways (e.g., poly(I:C)), and two inflammatory cytokines (e.g., IFN-γ and TNF-α).

[0115] In another aspect, the priming medium of the present disclosure may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The functional activator and at least four inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be marked by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators as compared to a non-primed stem cell population.

[0116] In some cases, the priming medium of the present disclosure may include at least one essential vitamin and other vitamins known in the art, and examples of the essential vitamins include ascorbic acid, thiamine, folic acid, and riboflavin. The at least one essential vitamin may be present in the priming medium at a concentration of about 50 - 300 μM, about 50 - 75 μM, about 75 - 100 μM, about 100 - 125 μM, about 125 - 150 μM, about 150 - 175 μM, about 175 - 200 μM, about 200 - 225 μM, about 225 - 250 μM, about 250 - 275 μM, or about 275 - 300 μM. In one example, the priming medium of the present disclosure may include ascorbic acid (200 μM).

[0117] In one example of the present disclosure, the priming medium may include a serum-free medium, a functional activator of the type I and type II IFN pathways (e.g., Poly(I:C)), four inflammatory cytokines (e.g., IFN-γ, TNF-α, IL-1β, and IL-17A), and essential vitamins (e.g., ascorbic acid). Such a priming medium is also referred to herein as a "six-component priming medium". In some cases, the six-component priming medium may include only a serum-free medium, a functional activator of the type I and type II IFN pathways (e.g., Poly(I:C)), four inflammatory cytokines (e.g., IFN-γ, TNF-α, IL-1β, and IL-17A), and essential vitamins (e.g., ascorbic acid).

[0118] In some cases, the priming medium of the present disclosure may be prepared with deionized distilled water. The priming medium of the present disclosure may be sterilized by means such as ultraviolet rays, heating, radiation irradiation, or filtration before use to prevent contamination. The priming medium of the present disclosure may be frozen (for example, at -20°C or -80°C) for storage or transportation.

[0119] The priming medium of the present disclosure may be a 1× formulation or a concentrated formulation, for example, a 2× to 250× concentrated medium formulation. In the 1× formulation, each component in the priming medium is at a concentration suitable for cell induction. In the concentrated formulation, one or more components are present at a concentration higher than the concentration for cell induction. The priming medium of the present disclosure may be concentrated using known methods such as salting out or selective filtration. The concentrated priming medium may be diluted with water (preferably deionized and distilled water) or any suitable solution (for example, a saline solution, an aqueous buffer, or a medium) for use.

[0120] According to another aspect of the present disclosure, a composition containing a priming medium and stem cells (for example, MSCs) may be included. In some cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. In other cases, the priming medium may include a serum-free medium, a functional activator of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The functional activator and the inflammatory cytokines may be present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be marked by an increase in the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to a non-primed stem cell population.

[0121] In one example, the composition may include a four-component priming medium (for example, only the four-component priming medium) and stem cells (for example, MSCs).

[0122] In another example, the composition may comprise a 6-component priming medium (e.g., only the 6-component priming medium) and stem cells (e.g., MSCs).

[0123] Cultivation method

[0124] According to another aspect of the present disclosure, an in vitro method of producing an isolated stem cell population having an anti-inflammatory phenotype from an unprimed stem cell population may be included. The method may include contacting the unprimed stem cell population with a priming medium for a predetermined time under conditions sufficient to promote the induction (e.g., uniform or substantially uniform induction) of stem cells having an anti-inflammatory phenotype. In some cases, the priming medium may include a serum-free medium, functional activators of the type I IFN pathway and the type II IFN pathway, and at least two inflammatory cytokines. In other cases, the priming medium may include a serum-free medium, functional activators of the type I IFN pathway and the type II IFN pathway, at least four inflammatory cytokines, and essential vitamins. The produced cells may have an anti-inflammatory phenotype, and the increased expression of one or more anti-inflammatory or immunomodulatory mediators compared to unprimed stem cells may be used as a marker.

[0125] In one example of the in vitro method, the priming medium may include a 4-component priming medium (e.g., only the 4-component priming medium).

[0126] In another example of the in vitro method, the priming medium may include a 6-component priming medium (e.g., only the 6-component priming medium).

[0127] In some cases, the non-primed stem cell population may include an isolated stem cell population that has not been in contact with, exposed to, or cultured in the priming medium of the present disclosure. The stem cells used in this method may be autologous, allogeneic, or xenogeneic. Alternatively, the stem cells may be obtained from a commercial source (e.g., an established cell line). Other sources of stem cells are known to those skilled in the art. In one example, the non-primed stem cells include an isolated MSC population or multipotent stromal cells derived from the tissue (e.g., adipose tissue, bone marrow, or umbilical cord blood) of an apparently healthy subject (e.g., a human).

[0128] The non-primed stem cells may be cultured using the priming medium of the present disclosure in a general cell culture method that can be understood by those skilled in the art. For example, the non-primed stem cells are isolated from a subject or obtained from a commercial source and may be inoculated into a culture vessel at a desired density under appropriate environmental conditions (e.g., 37°C, humid atmosphere, 5% CO2). Cell culture can be performed using any suitable cell culture vessel as a support. Cell culture vessels of various shapes and sizes (e.g., flasks, single-well plates or multi-well plates, single-well dishes or multi-well dishes, bottles, cans, vials, bags, bioreactors) composed of various materials (e.g., plastic, glass) are known in the art. Those skilled in the art can easily select a suitable cell culture vessel.

[0129] The non-primed stem cells can be contacted with, exposed to, or cultured using the priming medium of the present disclosure for a desired period sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The desired period may be 1 day or less (e.g., 6 hours or 12 hours), about 2 days (e.g., 18 hours), 2 days, 3 days, 4 days, or more than 5 days.

[0130] In some cases, in order to verify the phenotype of non-primed stem cells, it is necessary to measure them before contacting the non-primed stem cells with the priming medium. For example, if the non-primed stem cells contain MSCs, one or more known measurements may be performed to verify the presence of MSCs. In one example, flow cytometry assessment of the presence of cell surface markers CD90, CD73, CD105 and the absence of CD34, HLA-DR can be used to verify the presence of MSCs.

[0131] Methods for verifying the identity of a cell population (whether non-primed stem cells or primed stem cells) are known in the art. For example, the expression of some markers can be confirmed by measuring the level of the marker present in the cells of a cell culture or cell population. In such a process, the measurement of marker expression may be qualitative or quantitative. One example of a method for quantifying the expression of a marker produced from a marker gene is quantitative PCR (Q-PCR). The method of performing Q-PCR is well known in the art. Other methods known in the art may also be used for quantifying marker gene expression. For example, the expression of the marker gene product may be detected using an antibody specific for the marker gene product to be measured. Qualitative or semi-quantitative techniques such as blotting and immunocytochemistry can be used for measuring marker expression. It is also understood that techniques for measuring the content of extracellular markers such as ELISA may be used.

[0132] In another aspect, the primed stem cells (primed stem cells) produced by the methods of the present disclosure may be concentrated, isolated, and / or purified according to techniques known to those of skill in the art. In some cases, a cell population in which the primed stem cells are concentrated, isolated, and / or purified may be produced by isolating such cells from a cell culture. The primed stem cells produced by the methods described herein may be concentrated, isolated, and / or purified using an affinity tag specific for such cells. Examples of affinity tags specific for the primed stem cells of the present disclosure include antibodies, antibody fragments, ligands, or other binding agents specific for a marker molecule (e.g., a polypeptide), wherein the marker molecule is present on the cell surface of the primed stem cells (e.g., CD146) while not present (or substantially not present) on other cell types found in the cell cultures produced by the methods described herein.

[0133] Methods for preparing antibodies and methods for using antibodies to isolate cells are known to those of skill in the art, and such methods may be practiced for use with antibodies and the primed stem cells described herein. In one process, an antibody capable of binding to a marker expressed by the primed stem cells is attached to magnetic beads and then bound to the primed stem cells in an enzymatically treated cell culture to reduce cell-cell and cell-culture substrate adhesion. The cell / antibody / bead complex is then placed in a movable magnetic field to separate the primed stem cells bound to the beads from the unbound cells. After the primed stem cells are physically separated from other cells in the culture, the binding to the antibody is disrupted and the cells are re-inoculated into an appropriate tissue culture medium. Optionally, the isolated cell composition may be further purified by alternative affinity-based methods or additional rounds of enrichment using the same or different markers specific for the primed stem cells.

[0134] Optionally, any steps and procedures for isolating the primed stem cells produced by the methods of the present disclosure may be performed manually. Alternatively, the process of isolating such cells may be facilitated and / or automated by, for example, one or more suitable devices known to those of skill in the art.

[0135] By the methods described herein, the content of primed stem cells in a cell population or cell culture can be enriched by at least about 2- to about 1000-fold as compared to a cell population or cell culture that is not primed or enriched. In some cases, the primed stem cells may be enriched by at least about 5-fold to about 500-fold as compared to a cell population or cell culture that is not primed or enriched. In other cases, the primed stem cells may be enriched by at least about 10-fold to about 200-fold as compared to a cell population or cell culture that is not primed or enriched. In other cases, the primed stem cells may be enriched by at least about 20-fold to about 100-fold as compared to a cell population or cell culture that is not primed or enriched. In other cases, the primed cells may be enriched by at least about 40-fold to about 80-fold as compared to a cell population or cell culture that is not primed or enriched. In some cases, the primed stem cells may be enriched by at least about 2-fold to about 20-fold as compared to a cell population or cell culture that is not primed or enriched.

[0136] In another aspect, there is provided a composition produced by the methods described herein, which composition includes, as a major cell type, a cell culture or cell population comprising primed stem cells. In some cases, the cell culture and / or cell population produced by the methods described herein comprises at least about 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least about 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least about 62%, at least about 61%, at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51%, or at least about 50% primed stem cells. In other cases, the cell culture or cell population produced by the methods described herein comprises at least about 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 24%, at least 23%, at least 22%, at least about 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, at least 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% primed stem cells. In some cases, the percentage of primed stem cells in the cell culture or cell population is calculated regardless of the remaining cells in the culture.

[0137] In some cases, the in vitro methods of the present disclosure may be performed in the presence or absence of a feeder cell layer (e.g., a cell layer that typically cannot divide and provides extracellular secretions to assist the growth of other cells; e.g., fibroblasts).

[0138] It should be noted that each step in the methods disclosed herein may, in some cases, be performed in any suitable order or simultaneously, and does not necessarily have to be performed in the order described. For example, in the above method, the step of supplying a non-primed stem cell population may be performed before, after, or simultaneously with the step of supplying a priming medium.

[0139] Primed cells (primed cells)

[0140] According to another aspect of the present disclosure, it includes an isolated stem cell population having an anti-inflammatory phenotype by contacting or exposing to the priming medium of the present disclosure or culturing in the priming medium of the present disclosure. Such cells (also referred to as primed stem cells (e.g., primed MSCs).) are characterized by an increase in the expression and / or secretion of one or more anti-inflammatory mediators and / or unique surface markers compared to a non-primed stem cell population. As described above, the primed stem cells of the present disclosure are significantly different from naturally occurring stem cells, at least because the primed stem cells are exposed to or contacted with a priming medium containing a unique combination of components in a serum-free medium (in some cases, a synthetic medium), which is different from the in vivo inflammatory state with a large number of uncertain molecules (e.g., proteins, carbohydrates, metabolites, etc.). Therefore, surprisingly, the primed stem cells of the present disclosure have unique functional and structural characteristics and effects (identified below) that are significantly distinguishable from naturally occurring stem cells.

[0141] In some cases, the present disclosure may include an isolated population of primed MSCs that are CD146+ (e.g., those obtained using a 4-component or 6-component priming medium). In other cases, the present disclosure may include an isolated population of primed MSCs that are CD146+ and Lep-R+ (e.g., those obtained using a 4-component or 6-component priming medium). In other cases, the present disclosure may include an isolated population of primed MSCs that are CD146+, Lep-R+, nestin+, and SDF-1+ (e.g., those obtained using a 4-component or 6-component priming medium). In other cases, the present disclosure may include an isolated population of primed MSCs that are at least one or a combination of CD146+, Lep-R+, nestin+, and SDF-1+ (e.g., those obtained using a 4-component or 6-component priming medium).

[0142] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSCs) having an anti-inflammatory phenotype marked by an increase in the expression of IDO, CD274, CD146, and platelet-derived growth factor receptor beta (PDGFRB) and a decrease in the expression of C-X3-C motif chemokine ligand 1 (CX3CL1) as compared to unprimed stem cells (e.g., unprimed MSCs). In one example, primed MSCs (e.g., those obtained using a 6-component priming medium) exhibit an IDO expression that is about 50,000 to about 150,000 times higher than the IDO expression in unprimed MSCs.

[0143] In some embodiments, an isolated population of primed stem cells (e.g., MSCs) (e.g., those obtained using a 4-component or 6-component priming medium) can upregulate or downregulate the production of immune cells and / or immunomodulatory mediators.

[0144] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSCs) (e.g., obtained using a 6-component priming medium) that upregulate the production of cytotoxic T cells by at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or at least 8-fold compared to unprimed stem cells. In one example, primed MSCs (e.g., obtained using a 6-component priming medium) upregulate the production of cytotoxic T cells by at least 6-fold compared to unprimed MSCs.

[0145] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSCs) (e.g., obtained using a 6-component priming medium) that upregulate the production of TH1 cells by at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold compared to unprimed stem cells. In one example, primed MSCs (e.g., obtained using a 6-component priming medium) upregulate the production of cytotoxic T cells by at least 12-fold compared to at least unprimed MSCs.

[0146] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSCs) (e.g., obtained using a 6-component priming medium) that upregulate the production of CD4+ T cells by at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, or at least 15-fold compared to unprimed stem cells. In one example, primed MSCs (e.g., obtained using a 6-component priming medium) upregulate the production of CD4+ T cells by at least 12-fold compared to at least unprimed MSCs.

[0147] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSC) that upregulate the production of NK cells to be higher than that of unprimed stem cells (e.g., those obtained using a 6-component priming medium).

[0148] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSC) that more strongly suppress the production of Th17 cells than unprimed stem cells (e.g., those obtained using a 6-component priming medium).

[0149] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSC) that suppress the production of double-stranded DNA autoantibodies in vivo compared to unprimed stem cells (e.g., those obtained using a 6-component priming medium).

[0150] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSC) that suppress the production of B cell-activating factor (BAFF) compared to unprimed stem cells (e.g., those obtained using a 6-component priming medium).

[0151] In some cases, the present disclosure may include an isolated population of primed stem cells (e.g., MSC) that decrease the production of IL-17 compared to unprimed stem cells (e.g., those obtained using a 6-component priming medium).

[0152] Furthermore, the isolated population of primed stem cells (e.g., MSC) may have an anti-inflammatory phenotype that labels any one or a combination of the aforementioned structural and / or functional effects.

[0153] Another aspect of the present disclosure may include conditioned media produced by culturing stem cells (e.g., MSCs) in the priming medium of the present disclosure for a predetermined time, or contacting or exposing them to the priming medium of the present disclosure for a predetermined time, and collecting the supernatant or conditioned media after the culture period. Preferably, the conditioned media of the present disclosure may include immunomodulatory mediators (e.g., IDO) with improved levels and other components such as exosomes from primed stem cells, compared to conditioned media prepared from unprimed stem cells.

[0154] Use

[0155] In another aspect, methods of using an isolated population of primed stem cells and / or methods of using conditioned media prepared from an isolated population of primed stem cells are described herein. In one example, a population of primed stem cells and / or conditioned media prepared from an isolated population of primed stem cells are useful for preparing a pharmaceutical composition, which can be used in a subject in need of treatment, such as a subject suffering from or at risk of developing a systemic inflammatory or autoinflammatory disease or disorder.

[0156] Compositions comprising a primed stem cell population as disclosed herein and / or conditioned media prepared from an isolated population of primed stem cells have various uses in clinical treatment, research, development, and commercial purposes. For therapeutic purposes, for example, to treat a subject suffering from or at risk of developing a systemic inflammatory or autoinflammatory disease or disorder (e.g., an interferon-regulated autoimmune disease), a primed stem cell population as disclosed herein and / or conditioned media prepared from an isolated population of primed stem cells may be administered. In some cases, a primed stem cell population as disclosed herein, its cell membrane exosomes, and / or conditioned media prepared from an isolated primed stem cell population may be administered to a subject, which can not only contribute to restoring the function of damaged or otherwise unhealthy tissue, but also promote the reconstruction of damaged tissue.

[0157] A primed stem cell population as disclosed herein and / or conditioned media prepared from an isolated population of primed stem cells can be used in the development and study of disease models specifically related to systemic inflammatory or autoinflammatory diseases or disorders for research and development purposes.

[0158] One aspect of the present disclosure may include a method of treating a subject suffering from, suspected of suffering from, or at risk of developing a systemic inflammatory or autoinflammatory disease or disorder. In some cases, the systemic inflammatory or autoinflammatory disease or disorder may be an interferon-regulated autoimmune disease, such as an interferon-regulated autoimmune disease regulated by abnormal activation of the type I IFN-mediated pathway and the type II IFN-mediated pathway. Non-limiting examples of interferon-regulated autoimmune diseases treatable by the present disclosure include SLE, Sjögren's syndrome, organ transplant rejection (e.g., graft-versus-host disease), adult or juvenile idiopathic psoriatic arthritis, dermatomyositis, polymyositis, CNS vasculitis, scleroderma, inflammatory bowel disease, rheumatoid arthritis, and systemic scleroderma. Methods for determining whether a subject has, is suspected of having, or is at risk of developing any of the aforementioned interferon-regulated autoimmune diseases are known in the art.

[0159] This method may include administering to the subject a therapeutically effective amount of primed stem cells, i.e., stem cells having an anti-inflammatory phenotype marked by increased expression of one or more anti-inflammatory or immunomodulatory mediators as compared to non-primed stem cells. Preferably, administration of a therapeutically effective amount of primed stem cells may have any one or a combination of the following cellular effects: (1) increased expression of IDO, CD274, CD146, and PDGFRB and decreased expression of CX3CL1; (2) increased production of cytotoxic T cells; (3) increased production of TH1 cells; (4) increased production of CD4+ T cells; (5) increased production of NK cells; (6) suppression of production of Th17 cells; (7) suppression of production of double-stranded DNA; (8) suppression of production of BAFF; and (9) decreased production of IL-17.

[0160] The inventors of the present application surprisingly discovered that when primed stem cells (e.g., primed MSCs) are administered once, the above-described cell effects (e.g., cell effects (1) to (9)) persist over a long period of time. Thus, in some cases, a therapeutically effective amount of primed stem cells (e.g., primed MSCs) may be administered to a subject as a single dose, and thereafter, as a sustainable period of any one or a combination of cell effects (1) to (9), it may be about 1 to 12 months, about 1 to 2 months, about 1 to 3 months, about 1 to 4 months, about 1 to 5 months, about 1 to 6 months, about 1 to 7 months, about 1 to 8 months, about 1 to 9 months (e.g., about 9 months), about 1 to 10 months, about 1 to 11 months, or about 1 year, but is not limited thereto.

[0161] In one example, the methods described herein can be used to treat human subjects who have, are suspected of having, or are at risk of developing SLE. SLE is a type of autoimmune disease characterized by abnormalities in cellular and humoral autoimmunity. As a result of pathogenic T cells and B cells recognizing self-antigens, it causes an overactive immune response and the production of autoantibodies, ultimately leading to a multi-system chronic inflammatory disease. The clinical manifestations of SLE show a high degree of heterogeneity, ranging from mild systemic inflammation affecting the skin or joints to severe organ damage (such as the brain and kidneys). Unfortunately, there is still no unified and effective treatment for the cellular and humoral autoimmunity of SLE. In multi-center clinical trials, therapies targeting components of the cellular or humoral immune system have not yet been able to induce a sustained remission of disease activity.

[0162] Preferably, primed stem cells may be administered in a therapeutically effective amount to a human being suffering from, suspected of suffering from, or at risk of suffering from SLE. Clinical methods for determining whether a subject is suspected of suffering from, or at risk of suffering from, a systemic inflammatory or autoinflammatory disease or disorder are known in the art. In one example, the primed stem cells may comprise primed MSCs contacted with or cultured in a six-component priming medium. The priming medium may be administered alone or as a pharmaceutical composition (described below). Preferably, the administered primed MSCs can exhibit any one or a combination of cell effects (1) to (9) (described above), and further have a positive regulatory effect on immune cells known to cause abnormal regulation of type I and type II interferon pathways in SLE. Specifically, the primed MSCs of the present disclosure can expand and activate CD4+ T cells, CD8+ T cells, NK cells, and regulatory T cells (Tregs) while suppressing dendritic cells (DCs) and B cells by controlling the production and activity of type I (-α, -β) and type II interferon (-γ).

[0163] In another example, primed stem cells (e.g., MSCs) can advantageously prevent or reduce renal sclerosis and inflammation, and thus a therapeutically effective amount of primed stem cells (e.g., primed MSCs) may be administered to a subject before, during, or after an organ (e.g., kidney, liver, lung) transplantation procedure. In the context of organ transplantation, primed stem cells (e.g., MSCs) can be used in various ways. The quality of the obtained organ varies greatly depending on the age and health status of the donor, the in vitro time of the organ (and storage conditions), and the characteristics of the recipient. A large number of organs are discarded because they do not meet the surgeon's transplantation criteria (usually, the ischemia time after brain death of the donor, or the transfer time from when the donor is removed from the body until transplantation into the recipient). This is clearly a destructive loss every time, especially important for organs with a very short ex vivo detachment timeline. Currently, physicians perfuse the organ with a specific media cocktail mixture during transport (performed ex vivo for both the donor and the recipient) to reduce endothelial inflammation / activation and improve the patient's prognosis. Also, research on cocktail mixtures containing the use of stem cells or stem cell by-products is being actively conducted. Research is also being done on therapies for the recipient at the time of transplantation (or immediately before transplantation) to contribute to reducing delayed graft function (kidney) and ischemia-induced inflammation / damage, and / or to reduce the need for toxic immunosuppressants by improving tolerance (Treg), etc. Preferably, primed stem cells (e.g., MSCs) have great potential in the art. This is because the primed stem cells (e.g., MSCs) of the present disclosure have been found to be able to reduce the IFN-γ response and promote regulatory T cell / tolerance, and this finding can be utilized in various ways to contribute to organ utilization, organ health, and successful organ transplantation.

[0164] In another aspect, the present disclosure may include a method for adoptive immunotherapy in a subject. Adoptive immunotherapy is a cell therapy that includes removing immune cells from a subject, processing them ex vivo (i.e., activating, purifying, and / or expanding the cells), and then injecting the resulting cells back into the same subject. Examples of adoptive immunotherapy include LAK cells (Rosenberg, U.S. Patent No. 4,690,915), TIL cells (Rosenberg, U.S. Patent No. 5,126,132), cytotoxic T cells (Cai et al., U.S. Patent No. 6,255,073; Celis et al., U.S. Patent No. 5,846,827), expanded tumor-infiltrating regional lymph node cells (Terman, U.S. Patent No. 6,251,385), various lymphocyte preparations (Bell et al., U.S. Patent No. 6,194,207; Ochoa et al., U.S. Patent No. 5,443,983; Riddell et al., U.S. Patent No. 6,040,180; Babbitt et al., U.S. Patent No. 5,766,920; Bolton, U.S. Patent No. 6,204,058), CD8+ TIL cells (Figlin et al., (1997) Journal of Urology 158: 740), CD4+ T cells activated with anti-CD3 monoclonal antibody in the presence of IL-2 (Nishimura (1992) J. Immunol. 148: 285), T cells co-activated with anti-CD3 and anti-CD28 in the presence of IL-2 (Garlie et al. (1999) Journal of Immunotherapy 22:336), methods for producing and using antigen-specific CD8+ CTL T cells expanded in vitro with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) in the presence of IL-2 produced ex vivo (Oelke et al. (2000) Clinical Cancer Research 6:1997); and methods including inoculating a subject by injecting autologous tumor cells mixed with BCG vaccine (BCG) and irradiated, then collecting regional lymph node T cells 7 days later, activating the regional lymph node T cells with anti-CD3 mAb, and then expanding them in IL-2 (Chang et al. (1997) Journal of Clinical Oncology 15:796).

[0165] In some cases, the method may include contacting a non-primed population of CAR-T cells and / or CAR-NK cells with the priming medium of the present disclosure for a predetermined time under conditions sufficient to expand the CAR-T cells and / or CAR-NK cells and increase the cytotoxic activity of the CAR-T cells and / or CAR-NK cells. The primed CAR-T cells and / or CAR-NK cells may be produced by the in vitro methods of the present disclosure (e.g., methods using a 4-component or 6-component priming medium). Thereafter, a subject in need of adoptive immunotherapy (e.g., a subject suffering from, suspected of suffering from, or at risk of suffering from a systemic inflammatory or autoinflammatory disease or disorder (e.g., SLE)) may be administered the primed CAR-T cells and / or CAR-NK cells in a therapeutically effective amount.

[0166] In some cases, suitable routes of administration of a composition comprising conditioned medium prepared from primed stem cells and / or isolated primed cell populations may include intraarterial administration or intravenous administration, parenteral administration, intrathecal administration, intraventricular administration, intrasubstantial administration, intracranial administration, intracisternal administration, intrastriatal administration, intranigral administration, intramuscular administration, intraspinal administration, subcutaneous administration, transdermal administration, intralung administration, intranasal administration, rectal administration, and topical (including oral and sublingual) administration.

[0167] Compositions comprising an isolated primed stem cell population and / or a conditioned medium prepared from an isolated primed cell population may be administered to a subject using an implantable device. Implantable devices and related techniques are known in the art and are useful as delivery systems necessary for continuous or sustained release delivery of the compositions described herein. Also, an implantable device delivery system may be used to determine a predetermined location of delivery of a compound or composition (e.g., a local site, an organ, a bone, etc.) (Negrin et al., Biomaterials, 22(6):563 (2001)). According to some aspects of the present disclosure, sustained release techniques for alternative delivery methods may also be used. For example, sustained release formulations based on polymer technology, sustained release technology, and encapsulation technology (e.g., those using polymers, liposomes) can also be used to deliver the compositions described herein.

[0168] In some cases, the therapeutic composition may comprise a primed stem cell population alone and / or a conditioned medium prepared from a primed cell population alone and / or an isolated primed stem cell population formulated as a pharmaceutical composition and / or a conditioned medium prepared from an isolated primed cell population. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier (additive), additive, diluent or excipient (e.g., sterile saline, Hank's balanced salt solution (HBSS) or Isolyte S, pH 7.4) over an effective amount of primed stem cells and / or a conditioned medium prepared from an isolated population of primed stem cells. For example, an isolated population of primed stem cells and / or a conditioned medium prepared from an isolated population of primed stem cells disclosed herein may be provided in the form of a pharmaceutical composition containing an isotonic excipient prepared under aseptic conditions sufficient for human administration. For the general principles of pharmaceutical formulations, Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy , edited by G. Morstyn & W. Sheridan, Cambridge University Press, 1996; and, Hematopoietic Stem Cell Therapy,Reference may also be made to E.D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The choice of the cellular excipient and any accompanying components of the composition will be adjusted according to the route and device for administration.

[0169] Pharmaceutically acceptable carriers useful for preparing primed stem cells for administration to a subject are well known in the art and may include, for example, aqueous solutions such as water or buffered saline; or other solvents or media such as diols, glycerin, oils (e.g., olive oil), or injectable organic esters. The pharmaceutically acceptable carrier may include, for example, physiologically acceptable compounds that act to stabilize or increase the absorption of the conjugate. Such physiologically acceptable compounds may include, for example, carbohydrates (e.g., glucose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers or excipients.

[0170] In some cases, an isolated population of primed stem cells and / or conditioned medium prepared from an isolated population of primed stem cells may be packaged in a suitable container containing written instructions for any desired purpose, such as reconstructing or thawing (if frozen) the primed stem cells and / or conditioned medium prior to administration to a subject.

[0171] In some cases, a therapeutically effective amount of primed stem cells and / or conditioned medium prepared from primed stem cells sufficient to produce a statistically significant and measurable change in at least one symptom associated with a systemic inflammatory or autoinflammatory disease or disorder may be administered to a subject. Determination of a therapeutically effective amount is within the complete ability of one of ordinary skill in the art. Generally, the therapeutically effective amount will vary depending on the subject's medical history, age, condition, gender, and the severity and type of the subject's medical condition, as well as the administration of other pharmaceutically active agents.

[0172] A composition comprising an isolated population of primed stem cells and / or a conditioned medium prepared from an isolated population of primed stem cells may be administered to a subject once or in several divided doses. When the composition is administered in several divided doses, it may be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, or 24 hours from another administration. When the composition is administered in different pharmaceutical composition forms, the administration routes may be different. In some cases, a composition containing primed stem cells alone and / or a composition containing primed stem cells as a pharmaceutical composition is administered to the subject. In other cases, a composition containing a conditioned medium prepared from an isolated population of primed stem cells alone and / or a composition containing a conditioned medium prepared from an isolated population of primed stem cells as a pharmaceutical composition is administered to the subject. In another aspect, a composition containing an isolated population of primed stem cells mixed with a conditioned medium prepared from the isolated population of primed stem cells is administered to the subject. In another aspect, such multiple compositions may be administered substantially simultaneously or separately in succession.

[0173] In other cases, an isolated population of primed stem cells and / or a conditioned medium prepared from an isolated population of primed stem cells may be stored for subsequent transplantation / injection. The isolated population of primed stem cells and / or the conditioned medium prepared from the isolated population of primed stem cells may be divided into two or more aliquots or units such that a portion is reserved for subsequent use and a portion is immediately applied to the subject. Also, medium-term to long-term storage of all or part of the primed stem cells in a cell bank is within the scope of the present application and is disclosed, for example, in U.S. Patent Publication No. 2003 / 0054331, the content of which is incorporated herein by reference. At the end of the treatment, the concentrated primed stem cells and / or the conditioned medium prepared from the isolated population of primed stem cells may be loaded into a delivery device (e.g., a syringe) and placed into the recipient's body by any method known to those skilled in the art.

[0174] In some cases, a composition for administration to a subject, comprising an isolated primed stem cell population and / or a conditioned medium prepared from an isolated primed stem cell population, may further comprise a pharmaceutically active agent (e.g., an immunomodulatory agent), and such pharmaceutically active agents include agents for treating systemic inflammatory or autoinflammatory diseases or disorders known in the art. As used herein, the term "immunomodulatory agent" may include agents that can activate or enhance normal immune function, or that can suppress or interfere with normal immune function. Examples of immunomodulatory agents as immunosuppressive agents include, for example, reagents that suppress the T cell / B cell costimulatory pathway, specifically, reagents that interfere with the binding of T cells and B cells via the CTLA4 and B7 pathways disclosed in U.S. Patent No. 7,094,874, which is incorporated herein by reference. The immunomodulatory agent may be administered in a formulation suitable for the route of administration and administered to the subject in a dose sufficient to achieve the desired therapeutic effect. In some cases, the dose may be calculated using the actual body weight measured immediately before the start of the treatment process. For the dose calculated in this way, the body surface area (m 2 ) is calculated by the following formula according to the Dubois method before the start of the treatment process: m 2 =(weight in kg 0.425 ×height in cm 0.725 )×0.007184

[0175] The toxicity and therapeutic effects by administration of a composition comprising an isolated population of primed stem cells and / or a conditioned medium prepared from the isolated population of primed stem cells may be measured by standard pharmaceutical procedures for cell cultures or experimental animals, for example, to confirm LD50 and ED50. Compositions comprising an isolated population of primed stem cells and / or a conditioned medium prepared from the isolated population of primed stem cells that exhibit a large therapeutic index are preferred. The amount of the composition comprising the isolated primed stem cell population and / or the conditioned medium prepared from the isolated primed stem cell population can be tested using several well-established animal models. In some cases, the data obtained from cell culture assays and animal model studies can be used to establish a dosage range suitable for humans. The dosage of such a composition preferably includes the ED50 and is within a blood concentration range with very low or no toxicity. The dosage may vary within the above range depending on the dosage form used and the administration route utilized.

[0176] The therapeutically effective dose or amount of a composition comprising an isolated population of primed stem cells and / or a conditioned medium prepared from the isolated population of primed stem cells may also be initially estimated from cell culture assays. Alternatively, the effect of any particular dosage may be monitored by appropriate bioassays.

[0177] With regard to the duration and frequency of treatment, a skilled clinician typically monitors the subject's condition to determine when the treatment is producing a therapeutic benefit and then decides whether to increase or decrease the dose, increase or decrease the dosing frequency, discontinue or resume treatment, or make other changes to the treatment plan. The dosing schedule may vary from once a week to once a day depending on a number of clinical factors such as the subject's responsiveness to primed stem cells and / or conditioned medium derived from primed stem cells. The desired dose may be administered as a single bolus or divided into sub-doses, for example 2 - 4 sub-doses, and administered over a period of time at appropriate intervals throughout the day or other appropriate schedule. Such sub-doses can be administered as a unit dosage form. In some cases, administration is carried out over a long period, for example once or multiple times a day over a period of several weeks or months. Examples of dosing schedules include administration two, three, or four or more times a day within a period of one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months or more.

[0178] In another aspect, the primed stem cell population disclosed herein may be genetically modified to introduce genes useful for suppressing inflammation (e.g., genes encoding functional activators or inhibitors of type I and type II interferon pathways, or genes encoding anti-inflammatory mediators). In some cases, the primed stem cell population may be genetically engineered for improved survival, growth control, etc. The primed stem cell population as disclosed herein may be genetically modified by transfection, transduction with a suitable vector, homologous recombination, or other suitable techniques to be able to express the target gene. In one example, one or more primed stem cells may be transfected with a gene encoding the telomerase catalytic component (TERT), and such a gene is typically operably linked to a heterologous promoter that can result in increased telomerase expression compared to telomerase expression that occurs under the action of the endogenous promoter (see International Patent Application WO98 / 14592, which is incorporated herein by reference). In another example, a selectable marker is introduced to provide a higher purity primed stem cell population. In another example, after genetically modifying the primed stem cell population using the supernatant containing the vector over a period of 8 - 16 h, it may be switched to growth medium for 1 - 2 days. Genetically modified primed stem cells may be selected and then cultured using a drug selection agent such as puromycin, G418, or blasticidin.

[0179] A number of vectors useful for introducing foreign genes into primed stem cells disclosed herein are available. The vector may be an episomal vector, for example, a plasmid or a virus-derived vector such as a cytomegalovirus or adenovirus vector. On the other hand, the vector may be integrated into the primed stem cell genome by homologous recombination or random integration, for example, a retrovirus-derived vector such as MMLV, HIV-1, or ALV. In some cases, a combination of a retrovirus and an appropriate packaging cell line can also be used, where the capsid protein functions to infect the primed stem cells disclosed herein. Usually, the primed stem cells are incubated in the medium for at least about 24 hours. In some cases, in some applications, the primed stem cells may be grown in the medium for a short period (e.g., 24 to 73 hours) or at least 2 weeks, and ultimately, for more than 5 weeks before analysis. General retroviral vectors are "incomplete," i.e., they cannot produce the viral proteins necessary for productive infection. Replication of the vector requires growth within a packaging cell line.

[0180] The host cell specificity of retroviruses is determined by the envelope protein Env (P120). The envelope protein is provided from a packaging cell line. There are at least three types of envelope proteins: xenotropic, amphotropic, and heterotropic. Retroviruses packaged with xenotropic envelope proteins, such as MMLV, can infect most cell types of mice and rats. Examples of xenotropic packaging cell lines include BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses containing amphotropic envelope proteins (e.g., 4070A) can infect most mammalian cell types, including human, canine, and murine cells. Examples of amphotropic packaging cell lines include pAL2 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902); GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with heterotropic envelope proteins (e.g., AKR Env) can infect most mammalian cell types except murine cells. In some cases, the vector may contain genes that must be subsequently removed, for example, using a recombinase system such as Cre / lox, or may contain genes capable of producing selective toxicity, such as herpesvirus TK, Bcl-XS, and include cells in which their expression has been disrupted.

[0181] Suitable inducible promoters are activated in the desired primed stem cell (target) species (transfected cells or their progeny). It is expected that transcription activation results in at least about 100-fold, more typically at least about 1000-fold, increase in transcription in target cells compared to basal levels. A variety of promoters induced in different cell types are known.

[0182] In another aspect, the primed stem cells are useful for screening potential therapeutic agents. The potential therapeutic agent may be administered to the primed stem cells in the culture at different doses and the response of the primed stem cells at different time periods may be monitored. The physical and / or functional characteristics of the primed stem cells can be elucidated, for example, by microscopic observation of cell growth. The induction of the expression of new or improved levels of proteins (such as enzymes, receptors, and other cell surface molecules) or amino acids may be analyzed using any technique known in the art capable of identifying such molecular level changes. Such techniques include immunohistochemical or biochemical analysis techniques using antibodies against such molecules. Such biochemical analysis techniques include protein assays, enzyme assays, receptor binding assays, enzyme-linked immunosorbent assays (ELISA), electrophoretic assays, assays using high performance liquid chromatography (HPLC), Western blot, and radioimmunoassays (RIA). Nucleic acid analysis methods such as Northern blot can be used to measure the levels of these molecules, or the mRNA encoding the enzymes synthesizing these molecules.

[0183] By testing the effect of potential therapeutic agents on the cell proliferation and function of the primed stem cells, the side effects of potential drugs on the primed stem cells can also be screened.

[0184] The following examples are provided for illustrative purposes only and are not intended to limit the appended claims.

[0185] Example 1 In Example 1, experiments were conducted to develop an MSC priming medium designated "HXB-319" to control the inflammatory activities induced by the activation of type I and type II interferon pathways in various autoimmune diseases (such as SLE, Sjogren's syndrome, systemic scleroderma, graft-versus-host disease, inflammatory bowel disease, multiple sclerosis, psoriatic arthritis). The term "HXB-319" used in Example 1 may also refer to MSCs treated or primed with the MSC priming medium discovered in Example 1.

[0186] In vitro experiment of healthy bone marrow-derived MSCs using danger signal stimulation

[0187] <Poly(I:C) Exposure of Human MSC Cultures That Elicit an Anti-Inflammatory Response>

[0188] The inventors isolated a mixed MSC population from healthy donors using standard methods and then, at the second passage, treated the MSCs overnight (18 hours) with danger signals such as Poly(I:C) (30 ng / ml), lipopolysaccharide (LPS) (10 μg / ml), TNF-α (50 ng / ml), and IL-1β (25 ng / ml). Next, the medium was changed and the MSC population was left in serum, and they were harvested within 24 hours to measure secreted proteins. A Ray Biotech protein array was used for the protein array. The secreted proteins of primed and unprimed MSCs in tissue culture were analyzed using pathway analysis (Ingenuity.com) for biomarker detection. The inventors identified five classical pathways that were activated and three classical pathways that were downregulated after the cells were exposed to Poly(I:C). The five activated pathways are as follows.

[0189] (1) Upregulation of cytokine production known to be produced by macrophages and T helper cells: CSF-2 (16.5-fold), IL-13 (3.7-fold), CCl4 (2.8-fold), IL-1β (2.5-fold), and TNF-α (2.15-fold) increased, while IL-10 (-4.3-fold), CXCL1 (-5.4-fold), and IL-3 (-1.6-fold) decreased. The cytokine signaling pathways that regulate IL-17A, B, F and the interferon γ pathway (NFκB complex, interferon γ, IL-17 dimer and IL-17 receptor together regulate a series of chemokines and cytokines in this pathway, such as CX3CL, CCL1, CCL4, CXCL6, CCL2, CCL11, CCL18 and IL1 / IL6 / TNF-α) were downregulated, while the cytokine signaling pathways that assist in the differentiation of T helper cells were upregulated (IFN-γ (1.6-fold), IL-4 (23-fold), IL-5 (3.1-fold), IL-6 (1.6-fold), IL-13 (3.7-fold), TGF-β (2.3-fold), IL-10 (-4.3)).

[0190] (2) After poly(I:C) treatment, cell motility, hematopoiesis, and immune cell transport pathways were downregulated. The inventors found in this pathway analysis that important proteins that function in the pathogenesis mechanism of SLE, such as interferon and its regulators, such as IL-12 and interferon γ, were downregulated. Furthermore, the inflammatory cytokine IL-6, which acts together in the activation of T cells and B cells, was significantly suppressed.

[0191] (3) Cell growth and proliferation, development and function of connective tissue, and the structure and developmental network of lymphoid tissue (Figure 1A) showed significant suppression of CSF, IGF, HGF, FGF, and LTA.

[0192] (4) After poly(I:C) treatment, the network related to the activation of TLR2 / TLR4 by the activity of MAPK Erk1 / 2 and the activation of FGF, FGF6, -4, -7, IL-1, TNFRSF11B and CSF (Figure 1B) was suppressed.

[0193] (5) MSC primed with poly(I:C) increased the production of IL-1β and induced the activation of CCR-8 ligand. CCR-8 is a biomarker for confirming that Foxp3(+) T-regulatory cells involved in immunosuppressive delivery are concentrated in CD4(+) memory cells.

[0194] Also, the downregulated pathways are as follows.

[0195] (1) Signaling pathways functioning in psoriasis,

[0196] (2) Signaling pathways functioning in atherosclerosis, and

[0197] (3) Arthritis signaling pathways (IL-18 was downregulated 11-fold, CXCL5 was downregulated 1.7-fold, and CXCL1 was downregulated 5-fold.).

[0198] Therefore, surprisingly, it was discovered that MSC priming with poly(I:C) can control abnormally elevated INF-γ-related pathways in vitro and in vivo and control the interferon-regulated autoimmune disease activity such as SLE.

[0199] <MSC cell sorting experiment after Poly(I:C) exposure>

[0200] Cell sorting experiments showed that exposure of human MSC cultures (thought to be a mixed population after bone marrow isolation) to Poly(I:C) significantly increased the CD146+ / pericyte-like MSC phenotype. Based on our observations, surprisingly, it was discovered that hMSCs can change the composition of their surface markers and acquire a pericyte phenotype in vitro when exposed to specific danger signals (e.g., inflammatory cytokines found in damaged tissues).

[0201] Pericyte-like MSCs in MSC mixed cultures have effective and important immunomodulatory activities.

[0202] There is evidence demonstrating a direct relationship between the percentage of pericytes (defined as CD146+ and Lep-R+ cell surface receptors) contained in the unfractionated sample of MSCs and the stimuli used to induce immunomodulatory effects in vitro. When MSCs were stimulated with Poly(I:C), the percentage of CD146+-MSCs increased significantly compared to unstimulated MSCs. Conversely, when MSCs were stimulated with LPS, a decrease in CD146+-MSCs was shown (Figure 2).

[0203] Development of HXB-319

[0204] <Cell culture>

[0205] The inventors verified the immunostimulatory effect of HXB-319 by comparing it with non-primed primary bone marrow-derived MSCs commonly used in conventional clinical trials.

[0206] <Preparation of human mesenchymal stem cell cultures>

[0207] The hMSCs used for the development of HXB-319 were obtained from a hematopoietic stem cell facility established as an independent entity based on its own protocol (09-90-195). At Case Western Reserve University (Cleveland, OH), bone marrow was collected from healthy donors according to the currently approved IRB protocol #09-90-195. As described in the implementing regulations of HIPAA rule 45 CFR §164.514(b)(2)(i), the samples are anonymized. The inventors conducted experiments in mice using MSCs from five donors in vitro.

[0208] Human bone marrow-derived multipotent stromal cells (mesenchymal stem cells - MSC) obtained from healthy donors (2183 cell line) were cultured in the logarithmic growth phase using StemPro MSC SFM serum-free medium (Thermo Fisher Scientific) in BioSpherix (aseptic condition environment, 37 °C, humid atmosphere, 5% CO2). In the experiment, the cells were starved and maintained in StemPro basal medium for 48 hours, and then treated with perturbing substrates.

[0209] <RNA Extraction and PCR Analysis>

[0210] Total cellular mRNA was extracted using the MagMax RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The quality and quantity of the collected RNA were evaluated by measuring the absorption at 260 / 280 nm using a Nano Drop. Subsequently, the RNA was transcribed into first-strand cDNA using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific) for gene expression analysis. A custom-designed open array gene chip was constructed using 53 candidate genes and 3 housekeeping genes. Real-time polymerase chain reaction (qPCR) was performed using SYBR® Green I master mix on a Quant Studio (Thermo Fisher Scientific). The relative expression (RQ) of the transcripts was calculated using the 2 ΔΔ -CT method.

[0211] <Process Planning Based on Drug Substrates and Design of Experiments (DOE)>

[0212] The D-optimal experimental design was generated by software (Umetrics MODDE) and executed on a Freedom EVO150 liquid handling robot (TECAN, CH) in a 96-well format.

[0213] The inventors selected 12 factors: Poly(I:C), TNF-α, IL-1β, IL-17, IFN-α, IFN-β, IFN-α, PDGF-BB, ascorbic acid 2-phosphate, epidermal growth factor (EGF), TGF-β, and vitamin D3. Subsequently, the inventors prepared a simultaneous test using a 96-run design and test application. A complete cell culture including robotic operation in addition to artificial pre-expansion culture and inoculation is included in the modular X-Vivo system (Biospherix, NY, USA) that provides process analytical technology (PAT) for cell culture conditions (%N2, %O2, %CO2, and temperature control). For each experimental condition, a total of 53 custom gene expression data were obtained using the Open Array technology (Life Technologies, Quant Studio FLEX12). Mathematical modeling of the culture space was performed using the QPCR data included in the MODDE software, and the MODDE software used multivariate tools for dispersion minimization and model fitting. This process enabled the identification of main effects and response parameters and also allowed for query optimization of the amplification of target genes such as the expression of anti-inflammatory cytokines or perivascular cell-like genes of MSCs. By implementing this process, the inventors determined a six-component combination condition that maximizes the expression of IDO, CD274, CD146, and PDGFRB and minimizes the expression of CX3CL1 and PDL1.

[0214] Trailhead Biosystems (Cleveland, OH) performed a series of complex multi-dimensional analyses, participated in the executed pathway analysis experiments, and analyzed 53 candidate gene targets based on genes found to have an important role in the interferon activation signaling pathway. The above 53 candidate gene targets were ANG, BCL2, C3AR1, C5AR1, CCL17, CCL2, CCL5, CD274, CD36, CD44, CD55, CD59, CSF2, CSPG4, CX3CL1, CXCL1, CXCL12, CXCL3, CXCR4, ENG, ENTPD1, HSPA5, IDO1, IFNG, IL10, IL12A, IL17A, IL2, IL4, IL6, IL8, IRF3, LAMA4, LEPR, LIF, MCAM, NES, NGFR, NT5E, PDGFRB, PPBP, PTGS1, PTGS2, RGS5, SEMA3A, TGFB1, THY1, TLR3, TLR4, TLR7, TLR9, TNFAIP6 and VEGFA. In bone marrow-derived mesenchymal stem cells from healthy donor MSC lines, the expression levels of these gene complexes were normalized for the following cytokines and their concentrations against three housekeeping genes (GAPDH, YWHAZ, TBP): TNF-α (20 ng / ml), IFN-α (20 ng / ml), IFN-β (10 ng / ml), PDGF-BB (10 ng / ml), IFN-γ (100 ng / ml), IL-1β (10 ng / ml), IL-17A (50 ng / ml), ascorbic acid 2-phosphate (200 uM), poly I:C (1 μg / ml), TGF-β (10 ng / ml), EGF (20 ng / ml), and vitamin D3 (10 ng / ml).

[0215] Using the DoE method, a complex multi-dimensional experiment consisting of 96 conditions was designed to simultaneously examine 12 effectors with different combinations and concentrations. Bone marrow-derived MSCs obtained from healthy donors were seeded in 96-well plates (12,000 cells / well), and qPCR analysis was performed 6, 12, 18, and 24 hours after drug substrate treatment. The results were output to the Modde software platform for further analysis. As a result, more than 5,000 data points were generated. Each reaction was fitted to a statistical regression model to analyze how different perturbations affect gene expression. Next, the inventors extracted the optimization conditions for maximizing the gene expression of IDO1, PDGFRB, LEPR, IL-10, MCAM (CD146), CD274 (PDL-1) and minimizing CX3CL1 (fractalkine activity). The obtained MSC population expressed characteristic cytokines of MSCs at high levels, ensuring that they did not differentiate into different cell types. The cytokines characteristic of the above MSCs were CD44, CD59, CD73, CD90, CD105, PDGFRB, TNFAIP6, and CD146. The MSC population after priming expressed TLR-3 and TLR-4, IL6, IL8, IL12A, CCL2, CCL5 (RANTES), GMCSF, CXCL1, CXCL12, CXCL3, and ENG at higher levels (Table 1).

Table 1

[0216] <Verification of priming technology using commercially available bone marrow-derived MSCs (e.g., Rooster Bioscience®; and different bone marrow donors)>

[0217] Using the same method as above, the inventors tested the priming method for cells from different origins. Using the same robotic cell culture technique, cells were cultured using the same culture conditions and priming technique (i.e., HXB-319). As an efficacy test, the inventors studied the difference in the fold change of IDO gene expression. The inventors collected and isolated RNA from MSCs at multiple time points (6 hours, 12 hours, 18 hours, and 24 hours) during the priming period and observed the maximum IDO gene expression in three unrelated primed MSC sources (healthy donors numbered 2183 and 1427, passage 3 cells; and Rooster Bioscience cell products of unknown passage number). Condition 1 represents the HXB-319 condition, and Condition 2 is the control condition (Figure 3). A consistent fold increase in the expression of the IDO gene in primed MSCs was confirmed in all MSC sources from three different healthy donor cell lines (Figure 3).

[0218] Based on the above experimental results, surprisingly, it was found that by using HXB-319 having the following formulation, it is possible to induce or prime MSCs to obtain an anti-inflammatory phenotype after induction of type I and type II interferon pathways (e.g., due to autoimmunity), thereby specifically regulating immune cell activity and improving immune cell dysfunction:

[0219] (1) Interferon-γ, 100 ng / ml (eBioscience (BMS303));

[0220] (2) Poly(I:C), 1 μg / ml (Sigma-Alrich (9582-5mg);

[0221] (3) Tumor necrosis factor-α, 20 ng / ml (Peprotech (300-01A-50μg));

[0222] (4) Interleukin 1-β, 10 ng / ml (Peprotech (200-01B-10μg));

[0223] (5) Interleukin-17-α, 50 ng / ml (Peprotech (200-17)); and

[0224] (6) ASCOR® (Ascorbic Acid Injection; USP), 200 μM, (500 mg / mL, 50 mL vial, McGuff Medical Products)

[0225] In vitro and in vivo verification experiments

[0226] <In Vitro Bone Marrow Donor hMSC IDO Gene Expression and Kynurenine Enzyme Activity in Tissue Culture Medium>

[0227] MSCs after passage 3 were inoculated in triplicate into tissue culture flasks. After reaching confluence, the inventors removed DMEM containing 10% FBS, washed the cells three times with PBS, and then treated them with HXB-319 for 18 hours. Next, HXB-319 was washed away, serum-free DMEM was added to the culture plates, and the plates were incubated in a standard incubator. After 24 hours, the MSCs were harvested, RNA was isolated, and then the tissue culture medium was collected and the secreted products were measured. The results shown here were replicated using at least three different MSC donors.

[0228] RT-PCR results showed that compared with untreated MSCs, IDO expression increased 50,000-fold, and IDO measurement showed a significant improvement in IDO enzyme activity measured by the amount of kynurenine (mcg / ml) (Figure 4). Furthermore, perivascular MSC markers such as PDGFBP and CD146 were significantly upregulated (Figure 5).

[0229] <Efficacy of HXB-319 in a Pristane-Induced Interferon-Activated Mouse Model>

[0230] To prove the concept, the inventors used a validated and approved mouse model and injected 0.5 cc of tetramethylpentadecane (pristan) intraperitoneally into the mouse model, after which type I interferon activation was induced over a period of four weeks (Freitas, E.C. et al., Clin Rheumatol, 2017. 36(11): p. 2403-2414; Gardet, A. et al., PLoS One, 2016. 11(10): p.e0164423; and Richards, H.B. et al., Kidney Int, 2001. 60(6): p. 2173-80).

[0231] The inventors obtained 12- to 14-week-old BALBC / cj female mice from Jackson Labs and obtained age- and sex-matched 57BL / 6 mice as a control group. The inventors conducted two experiments using two sets of mice.

[0232] The inventors tested how mice injected with pristane would respond to HXB-319 during the acute phase of interferon activation and the chronic phase of autoimmune disease activity that mimicked subsequent SLE. Type I interferon activation was induced for four weeks after injecting 0.5 cc of pristane intraperitoneally. Thereafter, interferon response genes (IRGs) were activated, and an immune response profile consisting of further interferon activation was expected to be generally generated. The activation pathways were type I IFN-α and -β, type II IFN-γ, elevated inflammatory cytokines, IL-6 and IL-12. In short, the above caused depletion of innate immune system responders, namely cytotoxic T cells (CD8+), T helper cells (CD4+) and NK cells.

[0233] The first group of animals (16 BALBC / j mice and 11 C57BL / 6 mice) was used in the acute experiment. The inventors treated these mice with MSC or HXB-319MSC approximately four weeks after pristane injection, unlike the untreated mice. One week after treatment, the inventors evaluated the cell repertoire in the peritoneal lavage fluid in the acute experiment.

[0234] Briefly, on day 0: pristane was injected to induce type I IFN activation; on day 28: MSC or primed MSC (1×10 6 ) was injected within 4 weeks; on day 35: one week after MSC injection, peritoneal lavage fluid and peripheral blood were collected from all mice.

[0235] Another group of animals, group 2 (67 female BALBc / j and 20 C57BL / 6), was injected at the same time points but sacrificed 270 days (about 9 months) later to obtain the chronic effects of the pristane model and the results of HXB-319 treatment. The inventors collected urine, blood, peritoneal lavage fluid, and tissues from the kidney, liver, lung, heart, lymph nodes, and spleen. Also, splenocytes were isolated from the spleen and cell sorting experiments were performed.

[0236] In our study of the acute pristane-injected mouse model, surprisingly, unlike non-primed MSC used in other groups during current human clinical trials, MSC primed with HXB-319 was shown to be able to safely, significantly, and potently improve inflammation regulated by interferon activation in the lupus mouse model.

[0237] <Cell sorting of peritoneal lavage fluid>

[0238] The inventors separated the peritoneal lavage fluid, centrifuged and lysed the red blood cells, and fixed them with 4% pfa. Next, the inventors aliquoted 3×10 7 cells (dendritic cell vs lymphocyte panel) into two independent tubes for use in flow (see Table 2).

Table 2

[0239] In the pristane-induced mouse model, due to interferon-related inflammatory activity, the cytotoxic T cell subset (CD4−CD8+) in the peritoneal lavage fluid was lower than that in normal WT. Surprisingly, the primed MSCs of the present invention not only corrected the deficiency of cytotoxic T cells (CD3+CD4−CD8+), but also upregulated their numbers up to 7-fold (F = 103.8, p = 0.003), showing a significant effect by multivariate analysis compared to non-primed MSCs (F = 3.9, p = 0.11). Furthermore, the number of Th1 cells, CD4+ T cells (CD3+CD4+), was significantly upregulated up to 4-fold at most (p < 0.05). These data were collected 1 week after treatment with HXB-319 (primed MSCs) of the present invention. Furthermore, surprisingly, in in vitro studies, MSCs primed with HXB-319 were shown to express indoleamine 2,3-dioxygenase (IDO) more than 50,000-fold compared to MSCs currently in clinical trials.

[0240] Priming of MSCs with HXB-319 shows in vitro and in vivo evidence that can prove to proliferate, activate, and regulate cytotoxic cells of the innate and adaptive immune systems in specific target signaling pathways.

[0241] T cells : CD3+CD4+ T cells, CD3+CD8+ T cells, PD-L1+, CD25+CD4+ & CD25+CD8+ regulatory T cells; all CD25+ cells proliferated significantly more than the IL-2 stimulation of the same cells by in vivo cell sorting experiments (Figure 6). Also, in in vitro experiments, CD-25+ was shown to be expressed in T cell subsets with regulatory and inhibitory capabilities. CTLA-4+ T cells were also proliferated in vivo and in vitro and are anti-inflammatory costimulatory molecules.

[0242] From the expression of cell surface receptors, it was found that these cells were functionally activated. In PBMC co-cultured with HXB-319, the expression of CD-69+ was significantly increased, suggesting the activation of immune cells. Since CD69 was expressed in multiple subsets of tissue-resident immune cells such as resident memory T (TRM) cells and γδT cells, it was recognized as a marker of tissue retention. CD69 regulates the differentiation of regulatory T (Treg) cells and the secretion of IFN-γ, IL-17, and IL-22 (Eur. J. Immunol. 2017. 47:946-953). CD56+ was expressed in T cells even in the HXB-319 co-culture environment without IL-2, and the activation was significantly improved. CD56+ T cells do not proliferate as strongly as CD56- T cells, but show enhanced natural cytotoxicity. CD56+ T cells released interferon-γ (IFN-γ) and interleukin-13 (IL-13) after TCR stimulation, but did not release IL-10. The increased proportion of CD56+ T cells expressed IFN-γ, IL-4, and IL-13 within hours of activation. CD56+ T cells have acquired these cell lysis activities and cytokine secretion activities, making them potential candidates for immunotherapy against immune-mediated diseases.

[0243] NK cells : In the case of HXB-319 treatment, the number of CD25+ NK cells was significantly increased in mice, and this was demonstrated in vitro by cell surface markers of NK cells such as NKp46, NKp30, and NKp44. Figures 7A-B show the results of cell sorting experiments performed on PBMC after exposure to HXB-319 and its three-component derivative (also referred to as HXB-319-3 in Example 2 below). The symbols in Figure 7A are shown as follows. B is HXB-319, C is HXB-319-3, and D is 2×HXB-319-3. CD-56 is a prototype marker of NK cells, and NK cells were significantly proliferated after HXB-319 treatment. Figure 7B shows the percentage of peritoneal lavage NK cells 9 months after HXB-319 and MSC treatment, showing the dose effect of HXB-319 and the sustainable effect of HXB-319.

[0244] Tregs : Regulatory T cells (Tregs) are a special subset of T cells that play a role in suppressing the immune response, thereby maintaining homeostasis and self - immune tolerance. Tregs can inhibit T cell proliferation and cytokine production, indicating that they play an important role in preventing autoimmunity. Surprisingly, in in - vivo experiments after treatment with HXB - 319 (OHA in Figure 8A), CD4+ Tregs significantly increased.

[0245] Th17 cells (RORgT+) : Th17 cells are a specific subset of T - helper lymphocytes identified by high secretion of IL - 17 and other inflammatory cytokines.

[0246] Surprisingly, MSCs primed with HXB - 319 control the activation of Th17 cells and significantly suppress the proliferation of Th17 cells. The above results were demonstrated by the sustainable regulation of the immune system against Th17 cells up to 9 months after a single injection of HXB - 319 (1×10 6 cells) (Figure 8B).

[0247] Th17 expression in splenocytes : Group 1: p < 0.011, F = 5.0, HXB - 319, 1 million CD8+RORGT+ cells (Th17 cells) were significantly lower than healthy controls and numerically lower than pristane - SLE mice, p < 0.06 (mean value of untreated SLE controls was 3.33, 1.32 for HXB - 319, with a high coefficient of variation). On the other hand, in the chronic experiment, Group 2: p < 0.018, f = 3.84, HXB - 319, the CD8+RORGT+ cells in the 1 million injection arm were not significantly higher than the pristane control.

[0248] Plasmacytoid dendritic cells pDC:The interferon activation of MSCs primed with HXB-319 was tested in an in vivo model, and it was shown to suppress the proliferation / activation of splenic pDCs. The inventors observed similar results for myeloid dendritic cells mDCs (Table 3: Results of spleen cell sorting and their significance 9 months after MSC and HXB-319 treatment in the chronic model group 2).

Table 3

[0249] Plasmacytoid dendritic cells are the main producers of type I IFN, directly affect T cell responses through antigen presentation, and are represented by the presence of PDCA-1+.

[0250] PDCA-1 is a type II transmembrane glycoprotein with a molecular weight of 29-33 kD. It is mainly expressed in type I IFN-producing cells (IPCs) of naive mice, but is upregulated in most cell types after stimulation with type I IFN and IFNs-γ. It is highly expressed in terminally differentiated normal plasmacytoid dendritic cells.

[0251] Surprisingly, when the pristane model was treated with 2.5×10 6 primed MSCs (HXB-319), the frequencies of PDCA-1+ splenocytes and peritoneal cavity cells were significantly reduced compared to treatment with 1 million normal MSCs (unprimed) and 1 million HXB-319 (Table 3, Figure 9). PDCA-1+ is also a cell marker for plasmacytoid dendritic cells (pDCs), but is also found in B cells. It is mainly expressed in type I IFN-producing cells (IPCs), but is upregulated in most cell types after stimulation with type I IFN and IFNs-γ.

[0252] PD-L1+ cells (Figure 10): PD-L1 binds to PD-1 expressed on effector T cells and plays an important role in immune regulation by preventing autoimmune diseases by inducing apoptosis or anergy. This molecule is a checkpoint molecule that requires a balance of normal immune defense. Its level is low during inflammation and is desirably normalized or increased during the treatment of autoimmune diseases. PD-L1 is known as CD274 and is considered an anti-inflammatory marker. HXB-319 has a positive effect on counting the PD-L1 expression cell rate and spleen cell rate in peritoneal lavage fluid (Figure 10), indicating evidence of an anti-inflammatory effect. However, this level is not too high, and in the acute experiment of the first group, there was no statistical difference in the PD-L1 level between wild-type untreated mice and pristane-induced HXB-319-treated mice in the arms.

[0253] The CD4+PDL1+ cells in Group 1 (acute test, 1 week after cell treatment 4 weeks after pristane induction) showed a difference using ANOVA. Untreated MSCs showed a higher number than healthy controls, and a single treatment of OHA×1 million (HXB-319) had a higher quantity.

[0254] The CD4+PDL1+ cells in Group 2 (chronic experiment, 9 months after cell treatment 4 weeks after pristane induction) showed a difference using ANOVA. OHA-treated MSCs×1 (HXB-319)×1 million were significantly higher than healthy controls and numerically higher than SLE control mice (pristane sham surgery) (p<0.030, F=3.33).

[0255] <Serum interferon levels>

[0256] In the acute pristane mouse model, a single treatment with MSCs primed with HXB-319 (2×10 6 cells) of pristane-induced mice surprisingly inhibited the systemic levels of IFN-α (p<0.05) and IFN-γ (p<0.001) in the serum (Figure 11).

[0257] In the chronic pristane mouse model, a single treatment with MSCs primed with HXB-319 (1× or 2×10 6 cells) of pristane-induced mice surprisingly showed a systemic level of sustained inhibition of IFN-α (p<0.05) and IFN-γ (p<0.001) (Figure 12).

[0258] Furthermore, surprisingly, it was found that treatment with MSCs primed with HXB-319 successfully inhibited inflammation in which type I and type II IFN pathways were activated in acute or chronic pristane-induced inflammatory diseases, showing evidence that type I and type II interferon activation pathways were regulated. Furthermore, the amount of inflammatory cytokines was significantly improved after treatment with HXB-319.

[0259] <Examination of reducing serum cytokine levels in the pristane chronic infection model>

[0260] Cytokines such as TNF-α, IL-1β, and IL-6 increased after treatment with normal non-primed MSCs, but MSCs primed with HXB-319 did not show a significant increase in inflammatory cytokines, so there was no problem with the increase in cytokine storm (Figure 13). In Figures 13-14, these cytokines were examined in the sera of pristane-induced mice 1 week after treatment with non-primed MSCs and MSCs primed with HXB-319.

[0261] <Serum IL-17 level>

[0262] Also, the serum levels of IL-17A in mouse sera were examined 270 days after pristane injection. Surprisingly, when treated with non-primed MSCs and MSCs primed with HXB-319 (OHA in Figure 15), treatment with HXB-319 showed a dose-dependent significant decrease in cytokine levels.

[0263] <Change in titer of double-stranded DNA>

[0264] Double-stranded DNA is a marker for nephritis and represents the clinical improvement of SLE nephritis. MSC treatment primed with HXB-319 shows significant control of this marker at higher doses. The effect was significant after using X2 or at doses exceeding 2.5 million per mouse (Figure 16).

[0265] <Serum BAFF>

[0266] Also, the serum levels of BAFF were measured in the chronic test group (Figure 17). As a result, it was shown that the BAFF level was decreased using MSC primed with HXB-319, which is a molecule that activates and stimulates B cell activation and is considered one of the major therapeutic targets in SLE. Belimumab (a drug recently approved by the FDA) also affected the B cell-activating factor BLyS.

[0267] <Pathological evaluation and scoring of chronic injection experiments of BALB / cj pristane>

[0268] The inventors fixed all organs removed from the mice in formalin containing 10% PBS. H, E, and PAS staining were performed for all kidney biopsies, and the inventors hired an independent pathology expert in kidney pathology (Figures 18 - 19). The previously published kidney pathology score has been used for the numerical evaluation of inflammatory cells and sclerosis (Kidney Int, September 2009; 76(5):534 - 45, Epub July 1, 2009). Surprisingly, MSC primed with HXB-319 improved glomerulosclerosis and overall scar formation in the pristane-induced BalbCJ human SLE mouse model, and it was also found that MSC primed with HXB-319 improved the dermatitis seen in the pristane-induced BALBcj human SLE mouse model.

[0269] In short, through the experiments outlined in Example 1, the surprising finding was as follows:

[0270] (1)In pristane-induced BALBcj interferon-activated acute models and chronic pristane-induced human SLE mouse models, improving serological and cytological signs of interferon activation by MSCs primed with HXB-319;

[0271] (2)MSCs primed with HXB-319-3 are more effective than IDO gene expression and are also effective in NK cell amplification. In in vitro tissue culture, after priming MSCs with HXB-319, there was no equal increase in IL-6 and IL-8. Therefore, MSCs primed with HXB-319-3 can be used when cytokine storms are observed in macrophage activation syndrome (MAS) where immune activation cannot be controlled, a serious complication observed in many autoimmune diseases, including SLE active disease;

[0272] (3)MSCs primed with HXB-319 improve glomerulosclerosis and overall scarring in pristane-induced BALBcj human SLE mouse models, and thus prevent advanced SLE nephritis;

[0273] (4)MSCs primed with HXB-319 improve dermatitis observed in pristane-induced BALBcj human SLE mouse models, and thus prevent skin findings in SLE;

[0274] (5)MSCs primed with HXB-319 improve the amplification and activity of NK, CD4+ and CD8+ T cells when interferon activation is present; and

[0275] (6) MSCs primed with HXB-319, or CCM isolated from MSCs primed with HXB-319 (including MSC secretions and exosomes), when exposed to CAR-T and CAR-NK cells, induce their amplification and cytotoxic activity, thereby contributing to improved efficacy and patient outcomes. Cell therapy products such as CAR-T, CAR-NK, and further engineered variants, genetic mutants, secreted products, and exosomes can be primed with HXB-319 to improve their efficacy and lifespan. Chimeric antigen receptor-modified T cells (CART) or CAR-natural killer cells are immunotherapies that have been shown to induce remission in some patients suffering from hematological malignancies refractory to chemotherapy. HXB-319 can improve the effects of CAR-T / CAR-NK therapy and patient treatment outcomes by enhancing the anti-inflammatory effects of CAR-T and CAR-NK cells.

[0276] Example 2 To control the inflammatory activities induced by the activation of type I and type II interferon pathways in various autoimmune diseases (e.g., SLE, Sjogren's syndrome, systemic scleroderma, graft-versus-host disease, inflammatory bowel disease, multiple sclerosis, psoriatic arthritis), experiments were conducted in Example 2 to develop an MSC priming medium designated "HXB-319-3". The term "HXB-319-3" used in Example 2 can also mean MSCs treated or primed with the MSC priming medium discovered in Example 2.

[0277] The inventors, in the same experiment as described in Example 1, tested different subsets of priming media (compared to HXB-319) to maximize the gene expression of IDO1, IL-10, PDGFR, MCAM (CD146), and CD274 (PDL-1) and minimize the expression of CX3CL1 (chemokine). As in Example 1, the inventors first evaluated the expression and activity of IDO. ASCOR® (ascorbic acid injection; USP), 200 μM, (500 mg / mL, 50 mL vial, McGuff Medical Products) was added to the serum-free DMEM of all study groups. The different combinations of tested media components are shown in FIGS. 20A-B.

[0278] The inventors used the following genes (CTLA4, IL-4, IL-6, IL-8, AKT, IL-17, TLR4, TLR-7, CX3CR3, and PDGF-R) and MSC isolated from at least 5 different bone marrow donors to evaluate the in vivo efficacy of HXB-319-3 using IDO enzyme assay and RT-PCR. As described in Example 1, MSC were grown and amplified and treated with a serum-free medium containing four components of IFN-γ (100 ng / ml), poly(I:C) (1 μg / ml), and TNF-α (20 ng / ml) (collectively referred to as "HXB-319-3") for 18 hours. At the end of 18 hours, the cells were washed three times with PBS and then RNA was isolated using an RNA isolation Qiagen kit. Subsequently, RT-PCR was repeated twice, and at the same time, two sets of treated cells were maintained in serum-free medium for 24 hours, and the secretions of MSC were collected for ELISA. The results of the MSC responses of two donors in kynurenine activity (IDO) are shown in FIGS. 20A-B. Surprisingly, it was revealed that HXB-319-3 has the most potent anti-inflammatory effect and an effect similar to the expression of anti-inflammatory genes in vitro.

[0279] The inventors also performed RT-PCR on the study groups of untreated MSCs: 15 (HXB-319); 11 (TNF-α + IFN-γ); 12 (TNF-α + INF-γ + IL-1β); 14 (TNF-α + IFN-γ + Poly I:C) to verify whether the gene target expression is similar to or greater than the gene target expression expressed in the HXB-319 treatment group. The inventors specifically focused on the combination of IDO1, IL-10, PDGRF, and CD274 (PDL-1) with minimized CX3CL1 (Figures 21A-23).

[0280] After the inventors found that the three culture components of TNF-α + IFN-γ + Poly I:C (HXB-319-3) showed anti-inflammatory results and gene expression similar to HXB-319, further experiments were conducted. When MSCs treated with HXB-319-3 were used, the inflammatory cytokines IL-6 and IL-8 were not increased. Next, the inventors demonstrated the in vitro dose effect of HXB-319-3. The inventors performed IDO measurements and RT-PCR (Figures 24-26).

[0281] The present disclosure is further described by the claims and documents provided herein.

[0282] The present disclosure has been specifically shown and described with reference to preferred embodiments thereof, but it will be understood by those skilled in the art that various forms and detailed modifications can be made without departing from the scope of the present disclosure covered by the appended claims. All patents, publications, and references cited in the foregoing specification are hereby incorporated by reference in their entirety into this specification.

Claims

1. A priming medium for producing an isolated stem cell population having an anti-inflammatory phenotype from a non-primed stem cell population, comprising: a serum-free medium, poly(I:C), two inflammatory cytokines, wherein poly(I:C) and the inflammatory cytokines are present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype, the two inflammatory cytokines are IFN-γ and TNF-α, the isolated stem cell population having the anti-inflammatory phenotype is labeled by the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators increased as compared to the non-primed stem cell population, a priming medium.

2. A priming medium for producing an isolated stem cell population having an anti-inflammatory phenotype from a non-primed stem cell population, comprising: a serum-free medium, poly(I:C), four inflammatory cytokines, essential vitamins, wherein poly(I:C) and the inflammatory cytokines are present in an amount sufficient to promote the induction of stem cells having an anti-inflammatory phenotype, the four inflammatory cytokines are IFN-γ, TNF-α, IL-1β and IL-17A, the isolated stem cell population having the anti-inflammatory phenotype is labeled by the expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators increased as compared to the non-primed stem cell population, a priming medium.

3. The priming medium according to claim 1 or 2, further comprising stem cells.

4. The priming medium according to claim 1 or 2, which is a synthetic medium.

5. An isolated stem cell population having an anti-inflammatory phenotype, in which the expression of IDO is increased 50,000 to 150,000 times as compared to a non-primed stem cell population.

6. The isolated stem cell population according to claim 5, which is administered to a subject in a therapeutically effective amount for treating a systemic inflammatory or autoinflammatory disease or disorder of the subject.

7. The isolated stem cell population according to claim 6, wherein the systemic inflammatory or autoinflammatory disease or disorder is an interferon-regulated autoimmune disease.

8. The interferon-regulated autoimmune disease is regulated by abnormal activation of the type I IFN-mediated pathway and the type II IFN-mediated pathway, and is selected from the group consisting of systemic lupus erythematosus, monogenic interferonopathy, mixed connective tissue disease, Sjogren's syndrome, overlap syndrome (rheumatoid arthritis and SLE and / or Sjogren's syndrome), graft-versus-host disease, adult or juvenile idiopathic psoriatic arthritis, dermatomyositis, polymyositis, other myositis, systemic vasculitis, CNS vasculitis, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis, systemic juvenile idiopathic arthritis, juvenile arthritis, adult Still's disease, and systemic scleroderma, the isolated stem cell population according to claim 7.

9. Administering a therapeutically effective amount of the isolated stem cell population as a single dose, The isolated stem cell population according to any one of claims 6 to 8, wherein at least one cell effect selected from the following (1) to (9) persists in the subject for at least 6 months. (1) Increased expression of CD274, CD146 and PDGFRB, and decreased expression of CX3CL1; (2) Increased production of cytotoxic T cells; (3) Increased production of TH1 cells; (4) CD4 ＋ Increased production of T cells; (5) Increased production of NK cells; (6) Suppression of the production of Th17 cells; (7) Suppression of the production of double-stranded DNA; (8) Suppression of the production of BAFF; and (9) Decreased production of IL-17.

10. The isolated stem cell population according to any one of claims 6 to 9, wherein the level of serum cytokines has not increased after administration.

11. The isolated stem cell population according to claim 10, wherein the serum levels of TNF-α, IL-1β and IL-6 have not increased after administration.

12. Labeled by increased expression of indoleamine 2,3-dioxygenase (IDO), CD274, CD146, and platelet-derived growth factor receptor β (PDGFRB) and decreased expression of C-X3-C motif chemokine ligand 1 (CX3CL1) compared to the non-primed stem cell population, the isolated stem cell population according to any one of claims 5 to 11.

13. The isolated stem cell population according to any one of claims 5 to 11, which upregulates the production of cytotoxic T cells by at least 6-fold compared to the non-primed stem cell population.

14. The isolated stem cell population according to any one of claims 5 to 11, which upregulates the production of TH1 cells by at least 12-fold as compared to the non-primed stem cell population.

15. Compared to the non-primed stem cell population, CD4 ＋ The isolated stem cell population according to any one of claims 5 to 11, which upregulates the production of T cells by at least 12-fold.

16. The isolated stem cell population according to any one of claims 5 to 11, which upregulates the production of natural killer (NK) cells higher than the non-primed stem cell population.

17. The isolated stem cell population according to any one of claims 5 to 11, which strongly suppresses the production of Th17 cells as compared to the non-primed stem cell population.

18. The isolated stem cell population according to any one of claims 5 to 11, which suppresses the production of double-stranded DNA autoantibodies as compared to the non-primed stem cell population.

19. The isolated stem cell population according to any one of claims 5 to 11, which suppresses the production of B cell-activating factor (BAFF) as compared to the non-primed stem cell population.

20. The isolated stem cell population according to any one of claims 5 to 11, which reduces the production of IL-17 as compared to the non-primed stem cell population.

21. An in vitro method for producing the isolated stem cell population according to any one of claims 5 to 8, comprising contacting a non-primed stem cell population with the priming medium according to claim 1 for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. An in vitro method.

22. An in vitro method for producing the isolated stem cell population according to any one of claims 5 to 20, comprising contacting a non-primed stem cell population with the priming medium according to claim 2 for a predetermined time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. An in vitro method.

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