Primed medium and priming method for stem cell culture and stem cell therapy

Abstract

One embodiment of the present disclosure may include a priming medium for producing an isolated population of stem cells having an anti-inflammatory phenotype from an unprimed population of stem cells. The priming medium comprises a serum-free medium, functional activators of the type I interferon (IFN) pathway and the type II IFN pathway, and at least two pro-inflammatory cytokines. The functional activators and the at least two pro-inflammatory cytokines may be present in amounts sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The cells having an anti-inflammatory phenotype are marked by increased expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the unprimed stem cell population. Another embodiment of the present disclosure may include stem cells produced according to the present disclosure, therapeutic compositions comprising the stem cells, and uses of the stem cells.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 870,832, filed on 5 July 2019, and U.S. Provisional Patent Application No. 62 / 908,762, filed on 1 October 2019. The entire contents of each of the aforementioned U.S. Provisional Patent Applications are incorporated herein by reference for use in all purposes of the present invention.

[0002] This disclosure relates, in general, to primed media and priming methods for stem cell culture and stem cell therapy, and more specifically, to novel stem cell culture methods and stem cell therapies, as well as stem cell culture media compositions, which aim to produce primed, activated, or induced stem cells for use in cell therapy by inducing, activating, or priming discrete and homogeneous cellular phenotypes to selectively suppress inflammation and promote immunity, thereby providing significant advantages over known media and methods. [Background technology]

[0003] The clinical application of stem cells (e.g., mesenchymal stem cells or MSCs) requires reproducible cell culture and proliferation methods that can provide sufficient quantities of cells with appropriate quality and consistent therapeutic benefits. 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 higher yields of primed, activated, or induced cells for use in cell therapies that are safe, consistently reproducible, and have therapeutic effects. [Overview of the Initiative]

[0004] Various aspects of this disclosure aim to provide a more uniform and predictable population of ex vivo-grown and induced, primed, or activated stem cells (e.g., mesenchymal stem cells or MSCs), such a stem cell population that can be used in cell-based therapies. There has long been a need in the art for improved methods to provide large quantities of uniform and effective stem cells required for cell-based therapies. An advantage of various aspects of this disclosure is that stem cells produced by the primed media and primed methods disclosed herein can be used to induce, activate, or prime stem cell cultures into a uniform, discrete anti-inflammatory phenotype that functions in a predictable manner after introduction into a subject.

[0005] According to one aspect of this disclosure, the aim is to provide improved therapeutic methods, cell culture methods, and media for inducing, activating, or priming a homogeneous population of MSCs derived from various adult tissues. The efficacy or therapeutic benefits of induced, activated, or primed MSCs compared to common, uninducible or unprimed MSCs have been demonstrated in vitro and in preclinical disease models.

[0006] Surprisingly, we have found that when stem cells (e.g., MSCs) are exposed to a primed medium having a specific combination of components, they are primed to specifically regulate immune cell activity and improve immune cell dysfunction, particularly after autoimmune induction of type I and type II interferon pathways. Primed stem cells (e.g., MSCs) demonstrate 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 primed medium of the present invention can acquire a state or phenotype that promotes effective anti-inflammatory responses, controls innate and adaptive immune cell activity, and / or modulates interferon, as well as producing factors that promote angiogenesis and tissue regeneration. While not limited to theory, primed stem cells (e.g., MSCs) according to the present invention are CD4+ T cells and CD8 +It is recognized that expanding culture and activating T cells, NK cells, and regulatory T cells (Treg) exhibits the effective immunomodulatory effects described above, and inhibits dendritic cells (DCs) and B cells by regulating the production and activity of type I (α, β) and type II (γ) interferons and their downstream effectors, molecular responses, and other inflammatory signaling pathways.

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

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

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

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

[0011] Stem cells produced by the in vitro method of this disclosure are significantly different from naturally occurring stem cells. This is because the level or degree of expression of anti-inflammatory genes (e.g., indoleamine 2,3-dioxygenase, or IDO) in the stem cells of this disclosure is significantly higher than that found in nature (for example, 50,000 to 150,000 times higher in the stem cells of this disclosure compared to naturally occurring stem cells). The same applies to the level or degree of expression of surface markers in primed stem cells (e.g., MSCs) produced by the in vitro method of this disclosure, where, for example, CD146 is expressed at a significantly higher level than that found in nature, resulting in the stem cells exhibiting a pericyte-like phenotype. Furthermore, the significant difference between stem cells produced by the method of this disclosure and naturally occurring stem cells is due to the structural and functional distinctions that arise in the primed stem cells (of this disclosure) upon exposure to the primed medium of this disclosure. The aforementioned primed media contain a unique combination of components added to serum-free media (and, in some cases, synthetic media), and this state differs from the in vivo inflammatory state in which large amounts of unspecified molecules (e.g., proteins, carbohydrates, metabolites, etc.) are present.

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

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

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

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

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

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

[0018] Another aspect of the present disclosure may include a method for adoptive immunotherapy involving a subject. The method may include priming and expanding the culture of CAR-T cells and / or CAR-NK cells, and exposing unprimed (not primed) CAR-T cells and / or CAR-NK cells for a certain period of time under conditions sufficient to increase the cytotoxic activity of the CAR-T cells and / or CAR-NK cells. Primed CAR-T cells and / or CAR-NK cells may be produced by the in vitro method of the present disclosure. The in vitro method may include priming and expanding the culture of CAR-T cells and / or CAR-NK cells, and contacting the unprimed CAR-T cell and / or CAR-NK cell population with a primed medium for a predetermined period of time under conditions sufficient to increase the cytotoxic activity of the CAR-T cells and / or CAR-NK cells compared to unprimed CAR-T cells and / or CAR-NK cells. In some cases, the primed medium may contain serum-free medium, functional activators of the type I and type II IFN pathways, and at least two inflammatory cytokines. In other cases, the primed medium may contain serum-free medium, functional activators of the type I and type II IFN pathways, at least four inflammatory cytokines, and essential vitamins. Subjects undergoing adoptive immunotherapy may be administered a therapeutically effective dose of primed CAR-T cells and / or CAR-NK cells.

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

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

[0021] The above-mentioned features and other features of this disclosure will be explained below with reference to the attached drawings, so that those skilled in the art can understand them.

[0022] [Figure 1A] A schematic diagram showing the cytokine pathway network (red = upregulation, green = downregulation) after exposing mesenchymal stem cells to Poly(I:C). [Figure 1B] A schematic diagram showing the cell migration pathway network (Figure 1B) after exposing mesenchymal stem cells to Poly(I:C).

[0023] [Figure 2] Flow cytometry data showing the increase in CD146+ MSCs in mixed bone marrow isolated MSC cultures (MSCs from two different healthy bone marrow donors are shown) by Poly(I:C) treatment.

[0024] [Figure 3] A schematic diagram showing the effectiveness of the priming medium of the present disclosure. MSCs were cultured using the priming medium of the present disclosure by robotic cell culture technology. As an efficacy measurement means, the difference in the fold change of the expression of the indoleamine 2,3-dioxygenase (IDO) gene in primed MSCs was verified using RT-PCR. Isolated RNA was collected from MSCs at different time points (6 hours, 12 hours, 18 hours, 24 hours) of the priming period. Maximum IDO gene expression was measured for three unrelated prime-type MSC sources (passage 3 cells from healthy donors numbered 2183, 1427; and a commercially available MSC product of unknown passage number from Rooster Bioscience). Condition 1 indicates the priming medium condition, and condition 2 is the control condition. <000,0108> [Figure 4] A series of figures showing that the difference in the fold change of the expression of the IDO gene and the activity level (N-formyl-kynurenine enzyme level (mcg / ml) in the cell culture medium) increased significantly after 18 hours of exposure to the priming medium of the present disclosure.

[0026] <.,000115>This figure shows the difference in gene expression levels of perivascular MSC markers (PDGFBP and CD146) measured using RT-PCR (green = after 18 hours of exposure to the primed medium of this disclosure).

[0027] [Figure 6] The results of sorting cells in peritoneal lavage fluid in a Pristane mouse model one week after treatment with primed MSCs (after 18 hours of treatment with the primed medium of this disclosure) (n=7) and unprimed MSCs (n=6). The results show that in the treated subset (primed; Pristane-HXB-319), CD3+CD69+CD4+ T cells (blue bars) and CD3+CD69+CD8+ T cells (red bars) were significantly (*) increased compared to unprimed subsets (Pristane-MSCs) and sham surgery (Pristane-sham surgery) mice injected with Pristane.

[0028] [Figure 7A] This figure shows the results of cell sorting experiments on human peripheral blood monocytes (PBMCs). Figure 7A shows the results of unprimed MSCs (A), MSCs exposed to the 6-component primed medium according to this disclosure (B), MSCs exposed to the 3-component primed medium according to this disclosure (C), and MSCs exposed to 2× 3-component primed medium (D). [Figure 7B] This figure shows the results of a cell sorting experiment for natural killer (NK) cells. Figure 7B shows the percentage of NK cells in peritoneal lavage fluid 9 months after priming MSCs with the primed medium according to this disclosure, and demonstrates that MSC treatment has a dose-effect relationship with the primed medium and a sustainable effect on peritoneal NK cells.

[0029] [Figure 8A]The results of exposing MSCs to primed medium on different T cell subsets in peritoneal lavage fluid from an in vivopristane-induced SLE mouse model are shown. Figure 8A shows a significant increase in the percentage of CD4+FoxP3+ regulatory T cells (shown as a 10-fold increase in the figure) after MSC priming (OHA in Figure 8A). [Figure 8B] This shows the results of exposing MSCs to primed medium on different T cell subsets in in vivo peritoneal lavage fluid of a pristane-induced SLE mouse model. Figure 8B shows sustained immune system regulation (suppression of the percentage of Th17 cells in peritoneal lavage fluid) to Th17 cells (CD4+RORγ+T cells) 9 months after a single injection of primed MSCs (1 × 10⁶ cells).

[0030] [Figure 9] This figure shows that plasmacytoid dendritic cell antigen-1+ cells (indicated as pDCs) were significantly reduced one week after administration of primed MSCs (Pristane-HXB-319 in Figure 9) into the peritoneal lavage fluid of an in vivo pristane-induced SLE mouse model, compared to administration of unprimed MSCs (Pristane-MSC in Figure 9). Furthermore, a significant dose-effect was observed when cells were used at a dose of 2 × 10⁶ per mouse (Pristane-HXB-3192X).

[0031] [Figure 10] This figure shows the positive effect of administering primed MSCs on the percentage of PD-L1+ (CD-274) cells (an anti-inflammatory and immune checkpoint cell marker) in peritoneal lavage fluid (shown as a 10-fold increase in the figure), providing evidence of anti-inflammatory activity in a pristane-inducible mouse model.

[0032] [Figure 11]This series of figures shows the suppression of systemic levels of serum IFN-α (left) and IFN-γ (right) in an acute pristane mouse model by a single injection of MSCs (pristane-HXB-319) (1 × 10⁶ cells) treated with the primed medium of this disclosure.

[0033] [Figure 12] A series of figures showing the suppression of systemic levels of serum IFN-α (top figure) and IFN-γ (bottom figure) by a single injection of MSCs (pristane-HXB-319;-2X or -2.5M) (1×10⁶, 2×10⁶, or 2.5×10⁶ cells) treated with the primed medium according to this disclosure, compared with pristane-MSC injection (1×10⁶ or 2×10⁶ cells) in a chronic pristane mouse model (9 months after pristane induction).

[0034] [Figure 13] This is a series of figures showing serum inflammatory cytokine levels one week after injection of MSCs exposed to the primed medium relating to this disclosure.

[0035] [Figure 14] This figure shows the IL-10 levels one week after injection of MSCs exposed to the primed medium relating to this disclosure.

[0036] [Figure 15] This figure shows serum levels of IL-17A 9 months after treatment of MSCs that were injected with pristane and exposed to the primed medium of this disclosure (shown as OHA in Figure 15).

[0037] [Figure 16] This figure shows the changes in double-stranded DNA titer. Nine months after injection, treatment with MSCs exposed to the primed medium according to this disclosure significantly controlled double-stranded DNA at higher doses (the dose effect is significant at doses of 2× or 2.5 million cells or more per mouse; OHA in the figure indicates MSCs primed with HXB-319).

[0038] [Figure 17] This figure shows that in the case of MSCs exposed to the primed medium according to this disclosure, serum B-cell activating factor (BAFF) levels were reduced.

[0039] [Figure 18] This figure shows the pathological evaluation score for chronic glomerulonephritis in pristane-injected mice treated with the primed medium according to this disclosure.

[0040] [Figure 19] This figure shows the pathological evaluation scores of overall and local scar formation in pristane-injected mice treated with the primed medium according to this disclosure.

[0041] [Figure 20A] and [Figure 20B] This is a series of figures showing the IDO expression levels and activity after treating MSCs with modified subsets of the two- and three-component primed media according to this disclosure.

[0042] [Figure 21A] This is a series of figures showing the difference in gene expression multipliers between primed and unprimed MSCs with altered subsets of the two- and three-component primed media according to this disclosure. [Figure 21B] This is a series of figures showing the difference in IL-6 / IL-8 level multipliers between primed and unprimed MSCs with variations in the two- and three-component subsets of the primed medium relating to this disclosure.

[0043] [Figure 22] This figure shows the expression of IL-10 and CD274 in MSCs treated with modified subsets of the two and three components of the primed medium according to this disclosure.

[0044] [Figure 23]This figure shows the expression of TLR-7 and TLR-4 in MSCs treated with modified subsets of the two and three components of the primed medium according to this disclosure.

[0045] [Figure 24] This series of figures shows that the RT-PCR results for IDO and CD274 expression in MSCs treated with HXB-319 and a subset of its three molecules are dose-dependent.

[0046] [Figure 25] This figure shows the differences between HXB-319 and a subset of its three components in terms of fractalkine inhibition, AKT, and CTLA-4.

[0047] [Figure 26] This figure shows the difference in IL-6 and IL-8 expression levels in MSCs primed with HXB-319 and a subset of its three components. [Modes for carrying out the invention]

[0048] Unless otherwise specified, the methods and means described herein may also be carried out in accordance with conventional methods commonly known in the art, as described in the various general and more specific references cited and discussed throughout this specification. For example, see "Molecular Cloning: A Laboratory Manual," Sambrook et al., 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (1990).

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

[0050] In this specification, the singular form of English means one or more, or at least one. The plural form of English, as used herein, 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 pluripotent stromal cells) that have been cultured in the primed medium relating to this disclosure and stored for future use. Such cells may be stored in aliquots. They may be used immediately without storage or expanded cultured after storage. This can provide convenience by enabling the provision of “ready-made” cells useful for administration in research, clinical trials, or clinical therapy. These cell lines may already be stored in pharmaceutically acceptable excipients, in which case they may be administered directly or mixed with appropriate excipients when released from storage. Cells may be preserved by freezing or other means to maintain viability. The bank may be established using cells derived from the subject of treatment. Alternatively, the bank may contain cells intended for use by allogenes.

[0052] As used herein, the terms “cell therapy” or “cell-based therapy” may also mean the transplantation of human or animal stem cells relating to the Disclosure (i.e., cells cultured in or in contact with or exposed to the Primed Medium relating to the Disclosure) to prevent, treat or improve one or more symptoms associated with a disease or disorder (e.g., systemic inflammation or autoinflammation), and such prevention, treatment, or improvement of one or more symptoms may include, but are not limited to, replacement or repair of damaged tissue or organ, modulation of immune responses, or reduction of inflammatory symptoms.

[0053] In this specification, the term “repair” may, when used directly in relation to damaged tissue, mean improvement of such damage by direct mechanisms (e.g., regeneration of damaged tissue) and indirect mechanisms (e.g., enabling tissue formation as a result of inflammation being alleviated by the stem cells relating to this disclosure (i.e., cells cultured in or in contact with the primed medium relating to this disclosure)).

[0054] As used herein, the term “cell growth medium” may mean an aqueous growth or expansion culture medium containing factors and nutrients suitable for supporting the growth of cells such as stem cells relating to this disclosure (i.e., cells cultured in or in contact with the primed medium relating to this disclosure). For initial inoculation and culture (e.g., until confluence), standard or commercially available media such as DMEM containing 10% FBS may be used. After confluence, supporting media such as low glucose serum-free DMEM may be used for cell maintenance. The cell media used are all well-known in the field of cell culture and may all be commercially available.

[0055] The term “contains” means, unless otherwise specified, that the subject matter is necessarily included, but is not intended to imply any restriction or exclusion of other potentially containing substances. For example, “a composition containing X and Y” encompasses any composition containing X and Y, regardless of whether any other components are present in the composition. Similarly, “a method comprising step X” encompasses any method by which X is carried out, regardless of whether step X is the sole step of the method or one of several steps, and regardless of how many other steps there are or how simple or complex step X is compared to the other steps. The similar phrases using the root “contains” are used herein as synonyms for “contains” and have the same meaning as “contains.”

[0056] As used herein, the terms “condition medium” or “CM” may mean a medium in which stem cells relating to the present disclosure (i.e., cells cultured in or in contact with or exposed to the primed medium relating to the present disclosure) are cultured for a certain period of time. In one example, the condition medium can be prepared by culturing stem cells produced by the priming method disclosed herein, and then collecting the supernatant or condition 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 have been added. The term may also mean a cell culture growth medium in which all components can be expressed by their chemical formulas and are present in known concentrations, and is the opposite of “non-synthetic medium.”

[0058] As used herein, the term “effective dose” may mean an amount capable of providing the desired local or systemic effect. Such “desired local or systemic effect” may include, for example, effectively improving an undesirable condition (e.g., inflammation associated with systemic inflammatory or autoinflammatory diseases such as graft-versus-host disease or systemic lupus erythematosus) and achieving a specific desired effect as described herein. For example, the effective dose may be an amount sufficient to achieve a beneficial or desired clinical outcome. The effective dose may be provided in a single dose or in several doses. The amount considered effective may be precisely determined based on individual factors of each subject, such as the subject’s body type, age, injury and / or disease or injury being treated, and the amount of time since the injury or disease occurred. A person skilled in the art may determine the effective dose to be administered to a given subject based on these conventional considerations of the art. The term “effective dose” may have the same meaning as “effective dose.”

[0059] As used herein, the term “effective route” may mean a route for delivering a drug (e.g., stem cells) to a desired compartment, system, or location within the body. For example, an effective route may be a drug-administering route for delivering a sufficient amount of the drug at a desired site of action to achieve a beneficial or desired clinical outcome.

[0060] As used herein, the term “external addition” may mean, in the context of a culture or conditioning medium, adding a compound, growth factor, differentiation factor, etc., to a culture or 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 mean that the cells of interest are unrelated to one or more cells or cellular components associated with them in vivo. “Expanded population” may mean that the number of desired cells is relatively increased compared to one or more other cell types in vivo or in a primary culture. However, as used herein, the term “isolated” does not mean that only the cells described herein are present. Rather, the term “isolated” may suggest that the cells described herein have been removed from their native tissue environment and are present at a higher concentration compared to a normal tissue environment. Thus, an “isolated” cell population may further include cell types other than those described herein, and may also include other tissue components. This can also be expressed, for example, by cell doubling. Cells can be enriched by doubling 10, 20, 30, 40 or more times in vitro or ex vivo, 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 cells” or “MSCs” may mean cells derived from embryonic mesoderm that can be isolated from many sources, including adult bone marrow, peripheral blood, adipose tissue, placenta, and umbilical cord blood. MSCs can differentiate into many mesodermal tissues, including muscle, bone, cartilage, adipose tissue, and 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. See also Pittenger M et al., “Science, 284:143-147 (1999).” In some cases, MSCs may be derived from obviously 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 terms “multiponent” or “multipotent cell” refer to a cell type capable of producing a limited number of other specific cell types. Unlike pluriponent cells, multipotent cells are involved in the fate of one or more embryonic cells and therefore cannot produce any of the three embryonic cell lineages, nor can they produce extraembryonic cells.

[0064] When used in conjunction with cell cultures and / or cell populations, the term “partial” may mean any non-zero amount of any cell culture or cell population, and can range from a single cell to an entire cell culture or cell population. In some cases, the “partial” contract refers to 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% of the cell culture or cell population. This may mean 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%.

[0065] As used herein, the term “pharmaceutically acceptable” may mean a compound, material, composition (e.g., cells), and / or dosage form that, within reasonable medical judgment, does not cause excessive toxicity, irritation, allergic reactions, or other problems or complications, and exhibits a reasonable benefit / risk ratio, and is suitable for use in contact with human and animal tissues.

[0066] As used herein, in the context of cells, the terms “pluripotent” or “multipotency” may refer to cells capable of producing multiple tissues derived from any of the three embryonic cell lineages and extraembryonic cells. The term is limited to the internal cell mass state and is not used in any case to describe lineage-specific progenitor cells formed during development or present from the perinatal to adult stages. While the pluripotent state is native, it can also be grown in vitro under specific conditions known to those skilled in the art. Under such culture conditions, the persistent maintenance of stem cells or the pluripotent state arises. Since the properties for defining pluripotent cells are not limited to their functional characteristics but are also represented by gene expression patterns, the identification of pluripotent cells can be achieved by staining patterns of markers they exhibit or by quantitative measurements. The presence of the protein transcription factors Oct3 / 4, Sox2, and NANOG represents a set of core transcription factors involved in maintaining the pluripotent state, and the downregulation of these factors is a prerequisite for differentiation. The three transcription factors mentioned above all interact with each other, and there is considerable overlap in the genes they regulate. Furthermore, transcription factors responsible for maintaining pluripotency are usually identified using several surface markers present on them, the most common of which 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 to identify typical pluripotent cells.

[0067] As used herein, the term “progenitor cell” may in any case refer to a normal cellular state representing the direct ancestor of a differentiated cell. By definition, a progenitor cell is not necessarily required to irreversibly take on the deterministic fate described above, but may be pluripotent for multiple deterministic states. Thus, it exhibits the ability to differentiate into two or more progeny destinies. In any living organism, the gradual loss of ability, and therefore loss of pluripotency, as development progresses is an indicator of the differentiation and transiently defined formation of tissues and organs. An example of a progenitor cell is an induced pluripotent stem cell (iPSC), 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 also refer to the ability of a stem cell to produce replicating daughter stem cells, which have the same differentiation potential as the stem cell that produced them.

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

[0070] As used herein, the term “stem cell” may also refer to a cell that is capable of self-renewal (i.e., producing offspring with the same differentiation potential) and can also produce offspring cells with more limited differentiation potential. In the context of this disclosure, stem cells also include more differentiated cells that have been dedifferentiated, for example, 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 by exposure 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 teratoma or embryoid bodies (which, although not directly derived from the inner cell mass, can certainly be considered embryonic stem cells as they originate from embryonic tissue). The pluripotent stem cell state can also be produced by introducing genes related to stem cell function into non-stem cells (e.g., induced pluripotent stem cells).

[0072] As used herein, the term “subject” may refer to a vertebrate or mammal. Examples of subjects include livestock, test animals, and pets, which may be mammals such as sheep, cattle, pigs, dogs, cats, and mice, or reptiles, birds, and fish. The terms “patient” and “subject” may be used interchangeably herein. In one example, a subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle.

[0073] As used herein, the term “substantially absent” with respect to cells in a cell culture or cell population may mean that the cell species in question is present in amounts less than approximately 10%, less than approximately 9%, less than approximately 8%, less than approximately 7%, less than approximately 6%, less than approximately 5%, less than approximately 4%, less than approximately 3%, less than approximately 2%, or less than approximately 1% of the total number of cells present in the cell culture or cell population.

[0074] As used herein, the term “therapeutic dose” may mean the amount of reagent (e.g., one or more types of cells cultured in, or in contact with or exposed to, the primed medium relating to this disclosure) that can be confirmed to produce some therapeutic response in a subject. Within the scope of the terms as used herein, a therapeutically effective treatment may be a treatment that improves the quality of life of a subject, even if they do not improve the outcome of the disease themselves. Such a therapeutic dose can be readily determined by those skilled in the art. Thus, “treatment” may mean delivering such a quantity. In this way, the treatment can prevent or improve any pathological symptoms of a disease or disorder, such as systemic inflammatory or autoinflammatory diseases.

[0075] As used herein, the term “treatment” includes preventing, improving, suppressing or curing a defect, dysfunction, disease, or other harmful process, such other harmful processes including those that may interfere with and / or result from the treatment. In some cases, the symptoms of the 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, “prevention” 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 of such symptom occurrence 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 the symptoms of a disease or disorder, or the frequency of such symptom occurrence.

[0077] As used herein, when referring to one or more stem cells in contact with the primed medium of this disclosure, the term “induction” may also refer to a change in the phenotype (e.g., surface labeling and / or presence and / or absence) and / or genotype (e.g., mRNA expression level) of one or more stem cells in contact with the primed medium.

[0078] As used herein, the term “homogeneous induction” may mean that at least a portion of the stem cells in a stem cell population are induced to obtain one or more changes in stem cell phenotype and / or genotype upon contact with the primed medium of this disclosure. In some cases, substantially homogeneous induction may occur. Substantially homogeneous induction may mean that the majority of stem cells (>50%) are induced to obtain one or more changes in stem cell phenotype and / or genotype. Alternatively, substantially homologous induction may mean that the majority of stem cells are induced to obtain one or more changes in stem cell phenotype and / or genotype, for example, 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.

[0079] As used herein, the term “verification” may also mean “confirmation.” In the context of this disclosure, stem cells can be confirmed to be a specific cell type (e.g., MSC) having a desired function and / or phenotype. This allows a person skilled in the art to reasonably expect efficacy from such stem cells when using them (for applications such as therapy, bank establishment, or drug screening). Thus, verification may mean confirming that stem cells discovered and established to have a desired activity and / or phenotype indeed retain that activity and / or phenotype. Accordingly, verification may be a proof event in a two-event process relating to an initial determination and a subsequent determination. The second event may refer to “verification” as used herein.

[0080] As used herein, the term “prepared medium” may mean a culture medium composition comprising serum-containing basal medium and other compounds (e.g., ascorbic acid, dexamethasone, etc.). Serum is inherently an ambiguous component in the medium. Therefore, due to the heterogeneity of serum from different lots, the exact composition of commercially available “prepared” serum-containing media is unknown. An example of a “prepared medium” is Dulbecco-modified Eagle medium / Ham 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 terms “systemic autoimmune disease or disorder” or “autoinflammatory disease or disorder” may mean a condition of a subject characterized by damage to cells, tissues and / or organs caused by an immune response against the subject’s own cells, tissues and / or organs. Exemplary and non-limiting examples of autoimmune diseases treatable by immunomodulatory cells produced by this 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, Befçett's disease, bullous pemphigoid, cardiomyopathy, stomatitis, chronic fatigue immunodeficiency syndrome (CF1DS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigoid, CREST syndrome, cold agglutinin disease, discoid lupus erythematosus, primary mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, and Hashimoto's thyroiditis. Thyroiditis, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA Neuropathy, Juvenile Arthritis, Lichen Planus, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Type 1 or Immune-mediated Diabetes Membrane Disease, 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, Nodule, Scleroderma, Progressive Systemic Scleroderma, Sjögren's Syndrome, Good Examples include Pasture syndrome, rigidity syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis (e.g., herpetiform dermatitis), vitiligo, Wegener's granulomatosis, anti-glomerular basement disease, antiphospholipid syndrome, autoimmune diseases of the nervous system, familial Mediterranean fever, Lambert-Eaton myasthenic syndrome, sympathetic ophthalmitis, polyendocrine disorders, and psoriasis.

[0082] For example, systemic autoimmune or autoinflammatory diseases treatable by this disclosure may include SLE, mixed connective tissue disease, Sjögren's syndrome, overlapping syndromes (rheumatoid arthritis and 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 Steer's disease, and systemic scleroderma.

[0083] Other examples of systemic autoimmune or autoinflammatory diseases include monogenic interferonopathy, in which a gene variant triggers activation of the type I interferon pathway, leading to complex rheumatic diseases. These diseases are thought to be based on an autoimmune-autoinflammatory spectrum dependent on the driving factors of impaired type I interferon production. These diseases include Aicardi-Goutieres syndrome (AGS), chronic atypical neutrophilic dermatosis with lipodystrophy and fever (CANDLE), and infant-onset STING-associated vasculitis (SAVI). Other diseases associated with vasculitis and high interferon levels include retinal vascular disorder with cerebral white matter atrophy (RVCL) and Singleton-Merten syndrome, in which clinical signs are thought to be related to chronic inflammation, at least in part, attributable to constitutive activation of RIG-I, leading to 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, including, but not limited to, downregulation of the immune response, enhancement of the immune response, and alteration of the inflammatory state through changes in cytokine characteristics, cytotoxic activity and antibody production, as well as the effects on the immune system and immune-related cells resulting from each of the above situations.

[0085] As used herein, the term “cell population” may mean any number of stem cells (e.g., MSCs) greater than one, but preferably at least 1 × 10⁶ 3 A single cell, at least 1 × 10⁶ 4 A single cell, at least 1 × 10⁶ 5 A single cell, at least 1 × 10⁶ 6 A single cell, at least 1 × 10⁶ 7 A single cell, at least 1 × 10⁶ 8 A single cell, or at least 1 × 10⁶ 9 These are individual cells. In some cases, in an early 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 individual cells) (e.g., stem cells such as MSCs) are undifferentiated stem cells (e.g., undifferentiated MSCs).

[0086] As used herein, the terms “significant expression” or its synonyms “positive” and “+” mean, when used in relation to cell surface markers, 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 equipment (e.g., the BECKMAN COULTER EPICS XL FACS system used with commercially available antibodies and standard protocols known in the art). When using such conventional methods and equipment, the flow cytometry results in a signal for the specific cell surface marker that is higher than the background signal. The background signal is defined as the signal intensity from a nonspecific antibody of the same isotype as the specific antibody used to detect each surface marker in conventional FACS analysis. For markers considered positive, when using conventional methods and equipment, 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. Furthermore, the associated 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 “primed medium” may refer to a cell medium containing novel or specific components, the combination of which 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) modulate the type I interferon pathway. Type I IFN production is induced after the detection of pattern recognition receptors (PRRs). Type I interferons modulate the innate immune response in a balanced manner, inhibiting pro-inflammatory pathways and cytokine production while promoting antigen presentation and natural killer cell function. Type I interferons also promote the development of high-affinity antigen-specific T and B cell responses and immunological memory by activating the adaptive immune system, initiating a series of events that can lead to autoimmune diseases (e.g., Sjögren's syndrome or SLE).

[0090] As used herein, the term “type II interferon pathway” may also refer to an inflammatory pathway primarily regulated by interferon-gamma (IFN-γ). IFN-γ signals primarily via the JAK-STAT pathway, which influences gene regulation. JAK-STAT signaling involves the recruitment and activation of a set of receptors for members of the Janus family of kinases (JAKs: JAKs1-3 and TYK2) and STATs (STATs1-6, including STAT5a and STAT5b), and regulates the transcription of target genes through specific responsive factors. IFN-γ is a cytokine that plays a crucial role in adaptive and innate immunity.

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

[0092] As used herein, the term “anti-inflammatory phenotype” may mean a collection of multiple cellular processes involved in gene and protein expression, which can result in specific morphological and functional characteristics of cells, which, as a result of culturing with the primed medium of the present disclosure, label or characterize cells that show increased expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators or markers compared to cells not in contact with the primed medium of the present disclosure.

[0093] As used herein, the term “inflammatory cytokine” may mean a type of signaling molecule secreted by 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 or more interferons are actively involved in the pathogenesis or pathogenesis of the disease and actively act on the progression of the autoimmune disease. In other words, if interferon activity is an indicator of the disease, then the disease is an interferon-regulated autoimmune disease.

[0095] As used herein, the term “abnormal activation of the type I interferon pathway” may mean congenital and adaptive immune responses that differ from the generally expected responses 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 congenital and adaptive immune responses.

[0096] As used herein, the term “abnormal activation of the type II interferon pathway” may mean congenital and adaptive immune responses that differ from the generally expected responses during the activation of the type I interferon pathway. For example, in the absence of pathogen induction, excessive or harmful congenital and adaptive immune responses may occur, leading to inflammatory changes or damage to terminal organs.

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

[0098] As used herein, the term "CAR-NK cells" may mean NK cells collected from a subject, modified in vitro to add CAR proteins to them, and then re-administered to the subject. Methods for priming CAR-NK cells before 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, immunohistocompatibility 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., increased protein levels and / or activity, or increased proliferation and / or activity of immune cells) or negative (e.g., decreased protein levels and / or activity, or decreased proliferation and / or activity of immune cells). Non-limiting examples of immunomodulatory mediators may include IDO, IL-1β, IL-17A, TNF-α, and TNF-γ.

[0100] As used herein, the term “cytotoxic activity” may also mean the ability of a cell (e.g., a CAR-T cell or a CAR-NK cell) to kill different types of cells, which in the case of lymphocytes typically accomplish this killing by synthesizing porins. This ability can be measured by co-culturing lymphocytes with target cells. Other methods for determining the cytotoxic activity of cells are known in the art.

[0101] As used herein, the terms “downregulate” or “downregulation,” “decrease,” or “reduction” 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 downregulated, reduced, or decreased to 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 “downregulation” may mean complete inhibition.

[0102] As used herein, the terms “upregulate” or “upregulate,” “improve” or “increase” 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 upregulated or increased to 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] Primed culture medium

[0104] In one aspect of this disclosure, a priming medium may be provided for producing an isolated stem cell population having an anti-inflammatory phenotype from an unprimed stem cell population compared to the unprimed stem cell population. The priming medium may include a serum-free medium, functional activators of type I and type II IFN pathways, and at least two inflammatory cytokines. The functional activators and at least two inflammatory cytokines may be present in amounts sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be labeled with increased expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the unprimed stem cell population.

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

[0106] The primed medium may be a serum-free medium. Examples of serum-free media that can be contained in the primed medium of this 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 that the serum-free medium avoids the possibility of heterologous contamination of stem cells. A medium without serum substitutes is a medium that is not supplemented with any commercially available serum substitute preparations. The serum-free medium may be provided in the primed medium such that its content (e.g., w / v) in the primed 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 primed media of this disclosure may contain other components necessary for cell survival and contamination prevention, such as glucose and antimicrobial agents to reduce contamination by bacteria, mycoplasma, and fungi. Accordingly, the primed media of this disclosure may contain one or more antimicrobial agents or antibiotics to prevent contamination. Commonly used antibiotics or antifungal compounds are penicillin / streptomycin mixtures, but may also include, but are not limited to, amphotericin® (registered trademark: amphotericin B), ampicinicillin, gentamicin, boremycin, hydromycin, kanamycin, and mitomycin.

[0108] In some cases, the primed media of this disclosure may contain one or more functional activators of the type I and type II IFN pathways. For example, the primed media of this disclosure may contain only one functional activator of the type I and type II IFN pathways (e.g., poly(I:C)). In some cases, the functional activators of the type I and type II IFN pathways may include Toll-like receptor (TLR) ligands such as TLR3. The TLR ligands may include any molecule, compound, or reagent that can activate TLR signaling and induce an inflammatory response. The TLR ligands can activate TLR signaling by binding to or interacting with any known TLRs (e.g., TLR1-TLR10). The functional activators of the type I and type II IFN pathways may include synthetic molecules (e.g., synthetic analogs of TLR ligands) that stimulate or activate the type I and type II IFN pathways. In some cases, TLR ligands 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] For example, the primed medium of this disclosure may contain poly(I:C) as a functional activator of the type I and type II IFN pathways. Surprisingly, as described in Example 1 below, exposure of human MSC cultures to poly(I:C) resulted in increased IL-1β production (which induces CCR-8 activation), activation of five classical pathways, downregulation of three, and significant proliferation of CD146+ / pericyte-like MSCs.

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

[0111] In some cases, the primed media of this disclosure may contain at least two inflammatory cytokines, at least three inflammatory cytokines, at least four inflammatory cytokines, at least five inflammatory cytokines, at least six inflammatory cytokines, at least seven inflammatory cytokines, at least eight inflammatory cytokines, or more than eight 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 two inflammatory cytokines may be included in the primed medium of this disclosure at concentrations 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 primed medium of this disclosure. For example, at least two inflammatory cytokines may be included in the primed medium at concentrations of at least about 1 ng / ml to about 200 ng / ml, for example, about 1 to 10 ng / ml, about 10 to 20 ng / ml, about 20 to 30 ng / ml, about 30 to 40 ng / ml, about 40 to 50 ng / ml, about 50 to 60 ng / ml, about 60 to 70 ng / ml, about 70 to 80 ng / ml, about 80 to 90 ng / ml, about 90 to 100 ng / ml, about 100 to 125 ng / ml, about 125 to 150 ng / ml, about 150 to 175 ng / ml, or about 175 to 200 ng / ml. In one example, the primed medium of the present disclosure may contain IFN-γ (100 ng / ml) and TNF-α (20 ng / ml). In another example, the primed 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 primed media of this disclosure may consist only of serum-free media, functional activators of 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 the primed media does not contain any other immunomodulatory mediators (e.g., growth factors and cytokines) that could substantially affect the efficacy (e.g., basic and novel properties) of the primed media, as described herein (e.g., in Examples 1 and 2). However, even using the term "only," it is still permissible to include some components that do not substantially affect the basic and novel properties of the primed media of this application, such components being 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 primed media of this application can be said to consist substantially of serum-free media, functional activators of 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 this disclosure, the primed medium may comprise a serum-free medium, a functional activator of 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 primed medium is also referred to herein as a "four-component primed medium." In some cases, the four-component primed medium may comprise only a serum-free medium, a functional activator of 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 embodiment, the primed medium of the present disclosure may comprise serum-free medium, functional activators of type I and type II IFN pathways, at least four inflammatory cytokines, and essential vitamins. The functional activators and at least four inflammatory cytokines may be present in amounts sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be labeled with increased expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the unprimed stem cell population.

[0116] In some cases, the primed medium of this disclosure may contain at least one essential vitamin and other vitamins known in the art, such as ascorbic acid, thiamine, folic acid, and riboflavin. At least one essential vitamin may be present in the primed medium at concentrations 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 primed medium of this disclosure may contain ascorbic acid (200 μM).

[0117] In one example of this disclosure, the primed medium may comprise serum-free medium, functional activators of 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 an essential vitamin (e.g., ascorbic acid). Such a primed medium is also referred to herein as a "six-component primed medium." In some cases, the six-component primed medium may comprise only serum-free medium, functional activators of 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 an essential vitamin (e.g., ascorbic acid).

[0118] In some cases, the primed media of this disclosure may be prepared with deionized distilled water. The primed media of this disclosure may be sterilized before use by means of ultraviolet light, heating, radiation, or filtration to prevent contamination. The primed media of this disclosure may be frozen (e.g., at -20°C or -80°C) for storage or transport.

[0119] The primed medium of this 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 primed 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 primed medium of this disclosure may be concentrated by known methods such as salting out or selective filtration. The concentrated primed medium may be diluted with water (preferably deionized and distilled water) or any suitable solution (e.g., aqueous saline solution, aqueous buffer, or culture medium) before use.

[0120] In another aspect of this disclosure, the composition may include a primed medium and stem cells (e.g., MSCs). In some cases, the primed medium may include a serum-free medium, functional activators of the type I and type II IFN pathways, and at least two inflammatory cytokines. In other cases, the primed medium may include a serum-free medium, functional activators of the type I and type II IFN pathways, at least four inflammatory cytokines, and essential vitamins. The functional activators and inflammatory cytokines may be present in amounts sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. Cells having an anti-inflammatory phenotype may be labeled with increased 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 primed medium (e.g., four-component primed medium only) and stem cells (e.g., MSCs).

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

[0123] Culture method

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

[0125] In one example of an in vitro method, the primed medium may include a four-component primed medium (for example, only a four-component primed medium).

[0126] In another example of an in vitro method, the primed medium may consist of a six-component primed medium (for example, a six-component primed medium only).

[0127] In some cases, the unprimed stem cell population may include an isolated stem cell population that has not been in contact with, been exposed to, or cultured in the primed media of the Disclosure. The stem cells used in this method may be autologous, allogeneic, or heterologous. 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 unprimed stem cells may include an isolated MSC population or pluripotent stromal cells derived from the tissue (e.g., adipose tissue, bone marrow, or umbilical cord blood) of a seemingly healthy subject (e.g., human).

[0128] Unprimed stem cells may be cultured using the primed medium of this disclosure in a general cell culture method that is understandable to those skilled in the art. For example, unprimed stem cells may be isolated from a subject or obtained from a commercial source and inoculated into a culture vessel at a desired density under suitable 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 made of various materials (e.g., plastic, glass) (e.g., flasks, single-well plates or multi-well plates, single-well dishes or multi-well dishes, bottles, cans, vials, bags, bioreactors) are known in the art. Those skilled in the art can easily select a suitable cell culture vessel.

[0129] Unprimed stem cells can be contacted with, exposed to, or cultured in the primed medium of the Disclosure for a desired period sufficient to promote the induction of stem cells having an anti-inflammatory phenotype. The desired period may be one day or less (e.g., 6 or 12 hours), about two days (e.g., 18 hours), two days, three days, four days, or more than five days.

[0130] In some cases, to verify the phenotype of unprimed stem cells, it is necessary to measure them before contacting them with primed medium. For example, if unprimed stem cells contain mesoplastic stem cells (MSCs), one or more known measurements may be performed to verify the presence of MSCs. For example, the presence of MSCs can be verified using flow cytometry evaluation for the presence of cell surface markers CD90, CD73, and CD105 and the absence of CD34 and HLA-DR.

[0131] Methods for verifying the identity of cell populations (whether unprimed or primed stem cells) are known in the art. For example, the expression of several markers can be confirmed by measuring the level of markers present in 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 markers produced from marker genes is quantitative PCR (Q-PCR). The method for performing Q-PCR is well known in the art. Other methods known in the art may also be used to quantify marker gene expression. For example, the expression of a marker gene product may be detected using an antibody specific to the marker gene product to be measured. Qualitative or semi-quantitative techniques such as blot transcription and immunocytochemistry can be used to measure marker expression. It is also understood that techniques for measuring the content of extracellular markers, such as ELISA, may be used.

[0132] In another embodiment, primed stem cells produced by the method of the present disclosure may be concentrated, isolated, and / or purified according to techniques known to those skilled in the art. In some cases, the cell population from which primed stem cells have been concentrated, isolated, and / or purified may be produced by isolating such cells from a cell culture. Primed stem cells produced by the method herein may be concentrated, isolated, and / or purified using an affinity tag specific to such cells. Examples of affinity tags specific to primed stem cells of the present disclosure include antibodies, antibody fragments, ligands, or other binders specific to a marker molecule (e.g., polypeptide), the marker molecule being present on the cell surface of primed stem cells (e.g., CD146), while being absent (or substantially absent) on other cell types found in the cell culture produced by the method herein.

[0133] Methods for preparing antibodies and for isolating cells using antibodies are known to those skilled in the art, and such methods may be carried out for use with antibodies and primed stem cells as described herein. In one process, antibodies capable of binding to markers expressed by primed stem cells may be attached to magnetic beads and then bound to primed stem cells in a cell culture that has been enzymatically treated to reduce cell-cell and culture substrate adhesion. The cell / antibody / bead complex is then placed in a movable magnetic field to separate the bead-bound primed stem cells from the unbound cells. After the primed stem cells are physically separated from other cells in the culture, the binding to the antibody is broken, and the cells are re-inoculated into a suitable tissue medium. If necessary, the isolated cell composition may be further purified by an affinity-based alternative method or by further enrichment using the same or different markers specific to primed stem cells.

[0134] If necessary, any steps and procedures for isolating primed stem cells produced by the method of this disclosure may be carried out artificially. Alternatively, the process of isolating such cells may be facilitated and / or automated, for example, by one or more suitable devices known to those skilled 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 at least about 2 to about 1000 times compared to a cell population or cell culture that is not primed or enriched. In some cases, primed stem cells may be enriched at least about 5 to about 500 times compared to a cell population or cell culture that is not primed or enriched. In other cases, primed stem cells may be enriched at least about 10 to about 200 times compared to a cell population or cell culture that is not primed or enriched. In other cases, primed stem cells may be enriched at least about 20 to about 100 times compared to a cell population or cell culture that is not primed or enriched. In other cases, primed cells may be enriched at least about 40 to about 80 times compared to a cell population or cell culture that is not primed or enriched. In some cases, primed stem cells may be enriched at least about 2 to about 20 times compared to a cell population or cell culture that is not primed or enriched.

[0136] In another embodiment, the present invention relates to a composition produced by the method herein, wherein the composition includes a cell culture or cell population comprising primed stem cells as the main cell type. In some cases, the cell culture and / or cell population produced by the method herein contains 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%, and at least The mixture contains 75%, at least 74%, at least 73%, at least 72%, at least approximately 71%, at least approximately 70%, at least approximately 69%, at least approximately 68%, at least approximately 67%, at least approximately 66%, at least approximately 65%, at least approximately 64%, at least approximately 63%, at least approximately 62%, at least approximately 61%, at least approximately 60%, at least approximately 59%, at least approximately 58%, at least approximately 57%, at least approximately 56%, at least approximately 55%, at least approximately 54%, at least approximately 53%, at least approximately 52%, at least approximately 51%, or at least approximately 50% primed stem cells. In other cases, the cell cultures or cell populations produced by the methods described herein contain 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 residual cells in the culture.

[0137] In some cases, the in vitro methods of the present disclosure may be carried out in or without the presence of a feeder cell layer (e.g., a cell layer that is normally unable to divide and provides extracellular secretions to aid the proliferation of other cells; e.g., fibroblasts).

[0138] Furthermore, the steps in the methods disclosed herein may be performed in any suitable order or simultaneously, and do not necessarily have to be performed in the order described. For example, in the above method, the step of supplying an unprimed stem cell population may be performed before, after, or simultaneously with the step of supplying the primed medium.

[0139] Primed cells (cells that have been primed)

[0140] Another aspect of this disclosure includes an isolated stem cell population having an anti-inflammatory phenotype obtained by contact with or exposure to the primed medium of this disclosure, or by culturing in the primed medium of this disclosure. Such cells (also referred to as primed stem cells (e.g., primed MSCs)) are labeled by increased expression and / or secretion of one or more anti-inflammatory mediators and / or unique surface markers compared to a non-primed stem cell population. Thus, the primed stem cells of this disclosure are remarkably different from naturally occurring stem cells, at least because they are obtained by contact with or exposure to a primed medium containing a unique combination of components in a serum-free medium (or possibly a synthetic medium), which is different from the in vivo inflammatory state in which many uncertain molecules (e.g., proteins, carbohydrates, metabolites, etc.) are present, or by culturing in such a primed medium. Therefore, surprisingly, the primed stem cells of this disclosure have remarkably distinctive and unique functional and structural features and effects (identified below) that are remarkably different from naturally occurring stem cells.

[0141] In some cases, the disclosure may include an isolated primed MSC population that is CD146+ (e.g., obtained using a four- or six-component primed medium). In other cases, the disclosure may include an isolated primed MSC population that is CD146+ and Lep-R+ (e.g., obtained using a four- or six-component primed medium). In other cases, the disclosure may include an isolated primed MSC population that is CD146+, Lep-R+, nestin+, and SDF-1+ (e.g., obtained using a four- or six-component primed medium). In other cases, the disclosure may include an isolated primed MSC population that is at least one of CD146+, Lep-R+, nestin+, and SDF-1+ or a combination thereof (e.g., obtained using a four- or six-component primed medium).

[0142] In some cases, the disclosure may include isolated populations of primed stem cells (e.g., MSCs) having an anti-inflammatory phenotype characterized by increased expression of 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 unprimed stem cells (e.g., unprimed MSCs) (e.g., obtained using a 6-component primed medium). In one example, primed MSCs (e.g., obtained using a 6-component primed medium) exhibit IDO expression approximately 50,000 to 150,000 times higher than that of unprimed MSCs.

[0143] In some embodiments, an isolated population of primed stem cells (e.g., MSCs) obtained using, for example, four- or six-component primed media can upregulate or downregulate the production of immune cells and / or immunomodulatory mediators.

[0144] In some cases, the disclosure may include an isolated population of primed stem cells (e.g., MSCs) (e.g., obtained using a six-component primed medium) that upregulate cytotoxic T cell production by at least four-fold, at least five-fold, at least six-fold, at least seven-fold, or at least eight-fold compared to unprimed stem cells. In one example, primed MSCs (e.g., obtained using a six-component primed medium) upregulate cytotoxic T cell production by at least six-fold compared to unprimed MSCs.

[0145] In some cases, the disclosure may include an isolated population of primed stem cells (e.g., MSCs) (e.g., obtained using a 6-component primed medium) that upregulate TH1 cell production 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 primed medium) upregulate cytotoxic T cell production by at least 12-fold compared to at least unprimed MSCs.

[0146] In some cases, the disclosure may include an isolated population of primed stem cells (e.g., MSCs) (e.g., obtained using a 6-component primed medium) that upregulate CD4+ T cell production 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 primed medium) upregulate CD4+ T cell production by at least 12-fold compared to unprimed MSCs.

[0147] In some cases, the disclosure may include an isolated population of primed stem cells (e.g., MSCs) that upregulate NK cell production to a higher level than that of unprimed stem cells (e.g., obtained using a six-component primed medium).

[0148] In some cases, the disclosure may include an isolated population of primed stem cells (e.g., MSCs) that suppress Th17 cell production to a lower level than that of unprimed stem cells, e.g., obtained using a six-component primed medium.

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

[0150] In some cases, the disclosure may include an isolated population of primed stem cells (e.g., MSCs) that suppress the production of B cell activator (BAFF) compared to unprimed stem cells (e.g., obtained using a six-component primed medium).

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

[0152] Furthermore, isolated populations of primed stem cells (e.g., MSCs) may have an anti-inflammatory phenotype labeled with one or more of the aforementioned structural and / or functional effects.

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

[0154] use

[0155] In another aspect, this specification describes methods for using an isolated population of primed stem cells and / or a conditional medium prepared from an isolated population of primed stem cells. For example, a primed stem cell population and / or a conditional medium prepared from an isolated population of primed stem cells are useful for preparing a pharmaceutical composition which can be used in subjects in need of treatment, such as subjects suffering from or at risk of developing a systemic inflammatory or autoinflammatory disease or disorder.

[0156] Compositions comprising primed stem cell populations and / or conditioned media prepared from isolated primed stem cell populations, as disclosed herein, have a variety of applications in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, for example, primed stem cell populations and / or conditioned media prepared from isolated primed stem cell populations, as disclosed herein, may be administered to subjects suffering from or at risk of developing systemic inflammatory or autoinflammatory diseases or disorders (e.g., interferon-regulated autoimmune diseases). In some cases, subjects may be administered primed stem cell populations, their cell membrane exosomes, and / or conditioned media prepared from isolated primed stem cell populations, as disclosed herein, which may not only contribute to restoring the function of damaged or otherwise unhealthy tissues but also promote the reconstruction of damaged tissues.

[0157] Conditional media prepared from primed stem cell populations and / or isolated primed stem cell populations as disclosed herein can be used for research and development purposes to develop and study disease models specifically associated with systemic inflammatory or autoinflammatory diseases or disorders.

[0158] One aspect of this disclosure may include a method for treating a subject who has, is suspected of having, or is 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 interferon-regulated autoimmune diseases regulated by abnormal activation of type I and type II IFN-mediated pathways. Non-limiting examples of interferon-regulated autoimmune diseases treatable by this 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 one of the aforementioned interferon-regulated autoimmune diseases are publicly known in the art.

[0159] This method may include administering a therapeutically effective dose to a subject a primed stem cell, i.e., a stem cell having an anti-inflammatory phenotype, which is labeled as having increased expression of one or more anti-inflammatory or immunomodulatory mediators compared to unprimed stem cells. Preferably, administration of a therapeutically effective dose of primed stem cells may result in 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 Th17 cell production; (7) suppression of double-stranded DNA production; (8) suppression of BAFF production; and (9) decreased production of IL-17.

[0160] The inventors of this application have surprisingly discovered that a single administration of primed stem cells (e.g., primed MSCs) results in the above-described cellular effects (e.g., cellular effects (1) to (9)) persisting for a long period of time. Therefore, in some cases, a therapeutically effective dose of primed stem cells (e.g., primed MSCs) may be administered to a subject as a single dose, and the duration for which one or more combinations of cellular effects (1) to (9) persists thereafter may include, but are not limited to, 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.

[0161] For 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. Pathogenic T cells and B cells recognize autoantigens, resulting in an overreaction of the immune response and production of autoantibodies, ultimately leading to a multisystem chronic inflammatory disease. The clinical manifestations of SLE exhibit a high degree of heterogeneity, ranging from mild systemic inflammation affecting the skin or joints to severe organ damage (brain, kidneys, etc.). Unfortunately, there is still no unified and effective treatment for the cellular and humoral autoimmunity of SLE. Multicenter clinical trials have shown that therapies targeting components of the cellular or humoral immune system have not yet been able to induce sustained mitigation of disease activity.

[0162] Preferably, primed stem cells may be administered in a therapeutically effective dose to individuals who have, are suspected of having, or are at risk of having SLE. Clinical methods for determining whether a subject is suspected of having, or is at risk of having, a systemic inflammatory or autoinflammatory disease or disorder are known in the art. In one example, the primed stem cells may include primed MSCs that have been in contact with or cultured in a six-component primed medium. The primed medium may be administered alone or as part of a pharmaceutical composition (described below). Preferably, the administered primed MSCs may exhibit one or more combinations of the cellular effects (1) to (9) (described above), and further have a positive modulatory effect on immune cells known to be responsible for the abnormal modulation of type I and type II interferon pathways in SLE. Specifically, the primed MSCs of this disclosure can expand and activate CD4+ T cells and CD8+ T cells, NK cells and regulatory T cells (Treg) while suppressing dendritic cells (DCs) and B cells by controlling the production and activity of type I (-α, -β) and type II interferons (-γ).

[0163] In another example, because primed stem cells (e.g., MSCs) can favorably prevent or mitigate nephrosclerosis and inflammation, a therapeutically effective dose 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 donor's age and health status, the organ's time in vitro (and storage conditions), and the recipient's characteristics. A large number of organs will be discarded because they do not meet the surgeon's transplantation criteria (usually the ischemic time after brain death of Toner, or the metastasis time from Toner's removal from the body to transplantation into the recipient). This is clearly a devastating loss each time, especially for organs with very short out-of-body timelines. Currently, physicians perfusing organs with specific culture medium cocktails during transport (extracorporeally for both donors and recipients) to reduce endothelial inflammation / activation and improve patient prognosis. Active research is also being conducted on cocktails that include the use of stem cells or stem cell byproducts. Research is also being conducted on recipient therapies at the time of transplantation (or immediately before transplantation) to contribute to reducing delayed graft function (kidney) and ischemic inflammation / damage, and / or to reduce the demand for toxic immunosuppressants by improving tolerance (Treg), etc. Preferably, primed stem cells (e.g., MSCs) have great potential in this field. This is because we have found that primed stem cells (e.g., MSCs) can 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 embodiment, the disclosure may include a method used for adoptive immunotherapy in a subject. Adoptive immunotherapy is a cell therapy comprising removing immune cells from a subject, treating them ex vivo (i.e., activating, purifying, and / or expanding the cells), and subsequently injecting the resulting cells 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 cultured tumor influx 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), and 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), antigen-specific CD8+ CTL T cells produced ex vivo and cultured in large quantities using anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) in the presence of IL-2 (Oelke et al. (2000) Clinical Cancer Research Examples include a method for producing and using (6:1997); and a method comprising inoculating a subject with autologous tumor cells mixed with BCG vaccine (BCG) and irradiated by injection, collecting T cells from the influx region lymph nodes 7 days later, activating the influx region lymph node T cells with an anti-CD3 mAb, and then culturing them in IL-2 (Chang et al. (1997) Journal of Clinical Oncology 15:796).

[0165] In some cases, the method may include contacting an unprimed population of CAR-T cells and / or CAR-NK cells with the primed medium of the Disclosure for a predetermined time under conditions sufficient to expand culture the CAR-T cells and / or CAR-NK cells and increase their cytotoxic activity. Primed CAR-T cells and / or CAR-NK cells may be produced by the in vitro method of the Disclosure (e.g., a method using a four-component or six-component primed medium). The primed CAR-T cells and / or CAR-NK cells may then be administered in therapeutically effective doses to subjects in need of adoptive immunotherapy (e.g., subjects who have, are suspected of having, or are at risk of having, a systemic inflammatory or autoinflammatory disease or disorder (e.g., SLE)).

[0166] In some cases, suitable routes of administration of compositions comprising a condition medium prepared from primed stem cells and / or isolated primed cell populations may include intra-arterial or intravenous administration, parenteral administration, intrathecal administration, intraventricular administration, intraparenchymal administration, intracisional administration, intrastriatal administration, intrasubstantia nigra administration, intramuscular administration, intraspinal administration, subcutaneous administration, transdermal administration, intrapulmonary administration, intranasal administration, rectal administration, and topical administration (including oral and sublingual administration).

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

[0168] In some cases, a therapeutic composition may contain a primed stem cell population alone, and / or a condition medium prepared from a primed cell population alone, and / or an isolated primed stem cell population and / or a condition medium prepared from an isolated primed cell population formulated as a pharmaceutical composition. The pharmaceutical composition may also contain, on top of an effective amount of primed stem cells and / or a condition medium prepared from an isolated population of primed stem cells, a pharmaceutically acceptable carrier (additive), additive, diluent or excipient (e.g., sterile saline solution, Hanks equilibrium salt solution (HBSS), or Isolyte S, pH 7.4). For example, the isolated population of primed stem cells and / or a condition 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 sufficient sterile conditions for administration to humans. For 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 TherapyYou may also refer to EDBall, J. Lister & P. ​​Law, Churchill Livingstone, 2000. The selection of cell excipients and any accompanying components of the composition is adjusted according to the route and apparatus for administration.

[0169] Pharmacoherent carriers useful for preparing primed stem cells for administration to subjects are well known in the field and may include, for example, aqueous solutions such as water or buffered saline; or other solvents or media such as diols, glycerol, oils (e.g., olive oil), or injectable organic esters. The pharmaceutically acceptable carrier may also include, for example, physiologically acceptable compounds that act to stabilize or increase the absorption of the conjugate. Such physiologically acceptable compounds 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, the isolated population of primed stem cells and / or the conditioning medium prepared from the isolated population of primed stem cells may be packaged in a suitable container containing written instructions for any desired purpose, such as reconstituting or thawing (if frozen) the primed stem cells and / or the conditioning medium before administration to a subject, as necessary.

[0171] In some cases, subjects may be administered a therapeutically effective dose of primed stem cells and / or a condition 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. Determining the therapeutically effective dose can be achieved entirely within the capabilities of those skilled in the art. Typically, the therapeutically effective dose varies depending on the subject's medical history, age, condition, sex, and the severity and type of the subject's medical condition, as well as the administration of other pharmacoactive agents.

[0172] A composition comprising an isolated population of primed stem cells and / or a conditioning medium prepared from an isolated population of primed stem cells may be administered to a subject in one or more divided doses. When administered in divided doses, the composition for administration to a subject 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 single dose. When the composition is administered in the form of different pharmaceutical compositions, the route of administration may differ. In some cases, a composition comprising primed stem cells alone and / or a composition comprising primed stem cells as a pharmaceutical composition is administered to the subject. In other cases, a composition comprising a conditioning medium prepared from an isolated population of primed stem cells alone and / or a composition comprising a conditioning medium prepared from an isolated population of primed stem cells as a pharmaceutical composition is administered to the subject. In another embodiment, a composition comprising an isolated population of primed stem cells mixed with a conditioning medium prepared from an isolated population of primed stem cells is administered to the subject. In yet another embodiment, such multiple compositions may be administered substantially simultaneously or separately and sequentially.

[0173] In other cases, the isolated population of primed stem cells and / or the conditioned medium prepared from the isolated population of primed stem cells may be stored for subsequent transplantation / infusion. 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, with some reserved for subsequent use and some applied immediately to the subject. Medium- to long-term storage of all or some primed stem cells in a cell bank is also within the scope of this application, for example, disclosed in U.S. Patent Publication No. 2003 / 0054331, the contents of which are incorporated herein by reference. At the end of processing, the conditioned medium prepared from the concentrated primed stem cells and / or the isolated population of primed stem cells may be loaded into a delivery device (e.g., a syringe) and placed in the recipient 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 an agent 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 inhibit 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 at a dosage sufficient to achieve the desired therapeutic effect. In some cases, the dosage may be calculated using the actual body weight measured immediately prior to the start of the treatment process. For the dosage calculated in this way, the body surface area (m 2 ) is calculated by the Dubois method using the following formula 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 effect of a composition containing an isolated population of primed stem cells and / or a condition medium prepared from an isolated population of primed stem cells may be measured by standard pharmaceutical procedures for cell cultures or experimental animals, for example, to confirm the LD50 and ED50. Compositions containing an isolated population of primed stem cells and / or a condition medium prepared from an isolated population of primed stem cells that exhibit a large therapeutic index are preferred. The amount of a composition containing an isolated population of primed stem cells and / or a condition medium prepared from an isolated population of primed stem cells can be tested using several well-established animal models. In some cases, a suitable dosage range for humans can be established using data obtained from cell culture assays and animal model studies. The dosage of such a composition is preferably within a blood concentration range that includes the ED50 and has very little or no toxicity. The dosage may vary within the above range depending on the dosage form used and the route of administration utilized.

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

[0177] Regarding the duration and frequency of treatment, an experienced clinician will typically monitor the subject's condition to determine when the treatment has produced a therapeutic benefit, and then decide whether to increase or decrease the dose, increase or decrease the frequency of administration, interrupt or resume treatment, or make other changes to the treatment plan. The administration plan may vary in frequency from once a week to once a day, depending on many clinical factors, such as the subject's sensitivity to primed stem cells and / or primed stem cell-derived conditioning media. The desired dose may be administered as a single dose or divided into sub-dose periods, such as 2 to 4 times, administered at appropriate intervals throughout the day or on other appropriate schedules, over a period of time. Such sub-dose periods may 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 administration plans include administering the drug twice, three times, or four or more times a day over periods of one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or longer.

[0178] In another embodiment, 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, genetic recombination may be performed on the primed stem cell population for purposes such as improving viability or controlling proliferation. The primed stem cell population disclosed herein may be genetically modified to express a target gene by transfection or transduction with an appropriate vector, homologous recombination, or other appropriate technique. In one example, one or more primed stem cells may be transfected with a gene encoding a telomerase catalytic component (TERT), such a gene being acted upon by a heterologous promoter that can produce telomerase expression that is normally increased compared to the telomerase expression produced under the action of an endogenous promoter (see also International Patent Application WO98 / 14592, which is incorporated herein by reference). In another example, a selection marker is introduced to provide a higher purity primed stem cell population. In another example, a population of primed stem cells may be genetically modified using the supernatant containing the vector for a period of 8–16 hours, and then replaced with growth medium for 1–2 days. Genetically modified primed stem cells may be selected and cultured using a drug selector such as puromycin, G418, or blastosidine.

[0179] Numerous vectors useful for introducing exogenous genes into primed stem cells are available as disclosed herein. The vectors may be episomal vectors, such as plasmids or viral vectors such as cytomegalovirus and adenovirus. Alternatively, the vectors may be incorporated into the primed stem cell genome by homologous recombination or random integration, such as retrovirus vectors like MMLV, HIV-1, and ALV. In some cases, a combination of retrovirus and a suitable packaging cell system is also available, where the capsid protein functions to infect the primed stem cells disclosed herein. Typically, primed stem cells are incubated in culture medium for at least approximately 24 hours. In some cases, and in some application areas, primed stem cells may be grown in culture medium for a short period (e.g., 24–73 hours), or at least two weeks, and even longer than five weeks, before analysis. Typical retroviral vectors are "incomplete," meaning they cannot produce the viral proteins necessary for proliferation and infection. Vector replication requires growth within the packaging cell system.

[0180] The host cell specificity of retroviruses is determined by the envelope protein Env(P120). Envelope proteins are provided by the packaging cell system. There are at least three types of envelope proteins: homotropic, bitropic, and heterotropic. Retroviruses packaged with homotropic envelope proteins, such as MMLV, can infect most cell types in mice and rats. An example of a homotropic packaging cell system is BOSC23 (Pear et al. (1993) PNAS 90:8392-8396). Retroviruses containing bitropic envelope proteins (e.g., 4070A) can infect most mammalian cell types, including humans, dogs, and mice. Examples of bispecific packaging cell systems include pAL2 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:28 95-2902); and GRIP (Danos et al. (1988) PNAS 85:6460-6464). Retroviruses packaged with heterospecific envelope proteins (e.g., AKREnv) can infect most mammalian cell types except mouse cells. In some cases, the vector may contain genes that must be subsequently removed using recombinant enzyme systems such as Cre / lox, or it may contain cells in which the expression of such genes has been disrupted by including genes capable of producing selective toxicity, such as herpesvirus TK or BC1-XS.

[0181] Appropriate inducible promoters are activated in the desired primed stem cell (target) species (transfected cells or their offspring). Transcriptional activation is expected to increase transcription in target cells by at least approximately 100-fold, and more generally, at least approximately 1000-fold, compared to basal levels. Various promoters that are induced in different cell types are known.

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

[0183] By testing the effects of potential therapeutic agents on the proliferation and function of primed stem cells, it is also possible to screen for potential drug side effects on primed stem cells.

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

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

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

[0187] <Poly(I:C) exposure of human MSC cultures that induce anti-inflammatory responses>

[0188] The inventors isolated a mixed MSC population from healthy donors using standard methods, and then 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) during the second passage. Next, the MSC population was placed in serum after changing the medium, and collected within 24 hours for measurement of secreted proteins. A Ray Biotech protein array was used for protein arraying. Secreted proteins from primed and unprimed MSCs in tissue medium were analyzed using pathway analysis for biomarker detection (Ingenuity.com). The inventors identified five classical pathways activated and three classical pathways downregulated after cell exposure 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 times), IL-13 (3.7 times), CCl4 (2.8 times), IL-1β (2.5 times), and TNF-α (2.15 times) increased, while IL-10 (-4.3 times), CXCL1 (-5.4 times), and IL-3 (-1.6 times) decreased. The cytokine signaling pathways that regulate IL-17A, B, and F, as well as the interferon-gamma pathway (the NFκB complex, interferon-gamma, IL-17 dimer, and IL-17 receptor together regulate a range of chemokines and cytokines in this pathway, such as CX3CL, CCL1, CCL4, CXCL6, CCL2, CCL11, CCL18, and IL1 / IL6 / TNF-α), were downregulated, while cytokine signaling pathways that assist in the differentiation of T helper cells were upregulated (IFN-γ (1.6x), IL-4 (23x), IL-5 (3.1x), IL-6 (1.6x), IL-13 (3.7x), TGF-β (2.3x), IL-10 (-4.3x)).

[0190] (2) After poly(I:C) treatment, cell motility, hematopoiesis, and immune cell transport pathways were downregulated. In this pathway analysis, the inventors found that important proteins that function in the pathogenesis 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 on the activation of both T cells and B cells, was significantly suppressed.

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

[0192] (4) After poly(I:C) treatment, the activation of TLR2 / TLR4 by MAPK Erk1 / 2 activity, and the network related to the activation of FGF, FGF6, -4, -7, IL-1, TNFRSF11B and CSF (Figure 1B) were 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] 0] Therefore, surprisingly, it was discovered that MSC priming with poly(I:C) can control the abnormally elevated INF-γ-related pathways in vitro and in vivo, and can control the interferon-regulated autoimmune disease activities 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, we discovered that hMSC can change the composition of its surface markers in vitro and acquire a pericyte phenotype 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 those with CD146+ and Lep-R+ cell surface receptors) in unfractionated MSC samples and the stimuli used to induce immunomodulatory effects in vitro. Stimulation of MSCs with Poly(I:C) significantly increased the percentage of CD146+-MSCs compared to unstimulated MSCs. Conversely, stimulation of MSCs with LPS showed a decrease in CD146+-MSCs (Figure 2).

[0203] Development of the HXB-319

[0204] <Cell culture>

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

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

[0207] The hMSCs used in the development of HXB-319 were obtained from a hematopoietic stem cell organization established as an independent regulatory body based on its own protocol (09-90-195). At Case Western Reserve University (Cleveland, OH), bone marrow was collected from healthy donors in accordance with the currently approved IRB protocol #09-90-195. Samples were anonymized as described in the implementation details of HIPAA Rule 45 CFR §164.514(b)(2)(i). The inventors conducted experiments in mice using five donor MSCs 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 perturbation 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 a 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 Experimental Design (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, endothelial growth factor (EGF), TGF-β, and vitamin D3. Subsequently, the inventors prepared simultaneous tests using 96 run plans and test applications. Complete cell culture, including robotic manipulation in addition to artificial pre-expansion culture and inoculation, was incorporated into a modular X-Vivo system (Biospherix, NY, USA) that provides process analysis technology (PAT) for cell culture conditions (providing %N2, %O2, %CO2, and temperature control). For each experimental condition, a total of 53 custom gene expression data were obtained using Open Array technology (Life Technologies, Quant Studio FLEX12). Mathematical modeling of the culture space was performed using QPCR data included in MODDE software, which utilized multivariate tools for variance minimization and model fitting. This process identified key effect and response parameters and enabled the optimization of queries for amplification of target genes, such as the expression of anti-inflammatory cytokines or pericyte-like genes in MSCs. By performing this process, the inventors determined the combination of six components that maximized the expression of IDO, CD274, CD146, and PDGFRB, while minimizing the expression of CX3CL1 and PDL1.

[0214] Using Trailhead Biosystems (Cleveland, OH), we performed a series of complex multidimensional analyses and analyzed 53 candidate gene targets based on genes involved in the pathway analysis experiments we performed and found to play a crucial role in the interferon-activated signaling pathway. The 53 candidate gene targets listed above 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 MSCs, the expression levels of three housekeeping genes (GAPDH, YWHAZ, TBP) were standardized using the following cytokines and their concentrations: 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 diphosphate (200 μM), 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, we designed a complex multidimensional experiment consisting of 96 conditions that simultaneously examined 12 different effectors in different combinations and concentrations. MSCs derived from bone marrow obtained from healthy donors were inoculated into 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. This generated over 5000 data points. Each response was fitted to a statistical regression model to analyze how different perturbations affect gene expression. Next, we extracted optimization conditions to maximize the gene expression of IDO1, PDGFRB, LEPR, IL-10, MCAM (CD146), and CD274 (PDL-1), while minimizing CX3CL1 (fractalkine activity). The resulting MSC population expressed MSC-specific cytokines at high levels, ensuring that they had not differentiated into different cell types. The cytokines characteristic of the above-mentioned MSCs were CD44, CD59, CD73, CD90, CD105, PDGFRB, TNFAIP6, and CD146. The supercharged MSC population 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] The inventors tested the priming method on cells of different origins using the same method as described above. Cells were cultured using the same robotic cell culture technology, under the same culture conditions, and with the same priming technology (i.e., HXB-319). As an efficacy test, the inventors studied the differences in IDO gene expression multipliers. RNA was collected and isolated from MSCs at multiple time points during the priming period (6 hours, 12 hours, 18 hours, and 24 hours), and maximum IDO gene expression was observed in three unrelated primed MSC sources (healthy donor cells number 2183 and 1427, third passaged cells; and a Rooster Bioscience cell product with an unknown passage number). Condition 1 represents the HXB-319 condition, and Condition 2 is the control condition (Figure 3). IDO gene expression in primed MSCs showed a consistent multiplier increase in all MSC sources from the three different healthy donor cell lines (Figure 3).

[0218] Based on the above experimental results, we were surprised to discover that using HXB-319 with the following formulation, it is possible to induce or prime MSCs, thereby inducing type I and type II interferon pathways (e.g., autoimmunely) and obtaining an anti-inflammatory phenotype, 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 (9582-5 mg);

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

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

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

[0224] (6) ASC-AC-21, 200 μM (Sigma-Alrich (L4524-5MG)).

[0225] In vitro and in vivo validation experiments

[0226] <In vitro bone marrow donor hMSC IDO gene expression and kynurenin enzyme activity in tissue culture medium>

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

[0228] RT-PCR results showed a 50,000-fold increase in IDO expression compared to untreated MSCs, and IDO enzyme activity, measured by kynurenine levels (mcg / ml), was significantly improved (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 proven and approved mouse model in which 0.5 cc of tetramethylpentadecane (pristane) was injected intraperitoneally, and type I interferon activation was induced over a period of 4 weeks (Freitas, EC et al., Clin Rheumatol, 2017.36(11):p.2403-2414; Gardet, A. et al., PLoS One, 2016.11(10):p.e0164423; and Richards, HB et al., Kidney Int, 2001.60(6):p.2173-80).

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

[0232] The inventors investigated how mice injected with pristane responded to HXB-319 during the acute phase of interferon activation and the subsequent chronic phase of autoimmune disease activity mimicking SLE. Type I interferon activation was induced over four weeks after intraperitoneal injection of 0.5 cc of pristane. Subsequently, it was expected that interferon response genes (IRGs) would be activated, generating an immune response profile consisting of further interferon activation. The activation pathways included type I IFN-α and -β, type II IFN-γ, elevated inflammatory cytokines, and IL-6 and IL-12. In short, the above led to a reduction in 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) were used in acute experiments. Unlike the untreated mice, the inventors treated these mice with MSCs or HXB-319MSCs approximately four weeks after pristane injection. One week after treatment, the inventors evaluated the cell repertoire in the peritoneal lavage fluid from the acute experiments.

[0234] Simply put, Day 0: Pristane was injected to induce type I IFN activation; Day 28: MSCs or primed MSCs (1 x 10⁶) 6 ) was injected within 4 weeks; Day 35: One week after MSC injection, peritoneal lavage fluid and peripheral blood were collected from all mice.

[0235] Another group of animals (67 female BALBc / j and 20 C57BL / 6) was injected at the same time point but euthanized after 270 days (approximately 9 months) 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 kidneys, liver, lungs, heart, lymph nodes, and spleen. Splenocytes were also isolated from the spleen and cell sorting experiments were performed.

[0236] Our study of an acute pristane-injected mouse model surprisingly showed that, unlike unprimed MSCs used in other groups in the current human clinical trials, MSCs primed with HXB-319 could safely, significantly, and potently improve interferon-activated inflammation in a lupus mouse model.

[0237] <Cell sorting of peritoneal lavage fluid>

[0238] The inventors separated the peritoneal lavage fluid, centrifuged and dissolved the red blood cells, and immobilized them with 4% PFA. Next, the inventors placed 3 × 10⁶ cells into two separate tubes. 7 The individual cells (dendritic cells vs. lymphocyte panel) were divided equally and used in the flow (see Table 2). [Table 2]

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

[0240] In vitro and in vivo evidence demonstrates that MSC priming with HXB-319 can proliferate, activate, and regulate innate and adaptive immune system cytotoxic cells 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 IL-2-stimulated cells in in vivo cell sorting experiments (Figure 6). In vitro experiments also showed that CD-25+ was expressed in T cell subsets with regulatory and inhibitory capabilities. CTLA-4+ T cells also proliferated in vivo and in vitro, indicating that they are an anti-inflammatory costimulatory molecule.

[0242] The expression of cell surface receptors revealed that these cells were functionally activated. In PBMCs co-cultured with HXB-319, CD-69+ expression was significantly elevated, suggesting activation of immune cells. CD69 was expressed in multiple subsets of tissue-resident immune cells, such as resident memory T (TRM) cells and γδ T cells, and is therefore 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 its activation was significantly enhanced. CD56+ T cells did not proliferate as strongly as CD56- T cells, but showed enhanced intrinsic cytotoxicity. CD56+ T cells released interferon-γ (IFN-γ) and interleukin-13 (IL-13) after TCR stimulation, but did not release IL-10. A higher percentage of CD56+ T cells expressed IFN-γ, IL-4, and IL-13 within hours of activation. Because CD56+ T cells acquired these cytolytic and cytokine secretion activities, they are potential candidates for immunotherapy against immune-mediated diseases.

[0243] NK cells In the case of HXB-319 treatment, the number of CD25+ NK cells increased significantly in mice, which was confirmed in vitro by NK cell surface markers such as NKp46, NKp30, and NKp44. Figures 7A-B show the results of cell sorting experiments performed on PBMCs 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 as follows: B is HXB-319, C is HXB-319-3, and D is 2×HXB-319-3). CD-56 is a prototype marker for NK cells, and NK cells proliferated significantly even after HXB-319 treatment. Figure 7B shows the percentage of NK cells in peritoneal lavage fluid 9 months after HXB-319 and MSC treatment, demonstrating the dose-effect and sustained effects of HXB-319.

[0244] Tregs Regulatory T cells (Tregs) are a specialized subpopulation of T cells that exert a suppressive effect on the immune response, thereby maintaining homeostasis and autoimmune tolerance. Tregs can inhibit T cell proliferation and cytokine production, and have been shown to play a crucial role in preventing autoimmunity. Surprisingly, CD4+ Tregs were significantly increased in in vivo experiments after HXB-319 (OHA in Figure 8A).

[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 controlled Th17 cell activation and significantly suppressed Th17 cell proliferation, and the above results suggest that HXB-319 (1 × 10⁻¹⁴) 6 This was demonstrated by sustained regulation of the immune system against Th17 cells up to 9 months after a single injection of individual cells (Figure 8B).

[0247] Th17 expression in spleen cells 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, with p<0.06 (mean value 3.33 for untreated SLE controls, 1.32 for HXB-319, higher coefficient of variation). On the other hand, in the chronic experiment, Group 2: p<0.018, f=3.84, HXB-319, CD8+RORGT+ cells in 1 million injected arms were not significantly higher than pristane controls.

[0248] Plasmacytoid dendritic cells (pDCs)Interferon activation of MSCs primed with HXB-319 was tested in an in vivo model and demonstrated that it suppresses the proliferation / activation of spleen pDCs. The inventors observed similar results for myeloid dendritic cells (mDCs) (Table 3: Selection results of MSCs and spleen cells 9 months after HXB-319 treatment in chronic model group 2 and their significance). [Table 3]

[0249] Plasmacytoid dendritic cells are major producers of type I IFNs, directly influencing T cell responses through antigen presentation, which is 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, the Pristan model is 2.5 x 10 6 When treated with one primed MSC (HXB-319), the frequency of PDCA-1+ splenic cells and peritoneal cells was significantly reduced compared to treatment with 1 million normal (unprimed) MSCs and 1 million HXB-319 cells (Table 3, Figure 9). PDCA-1+ is a cellular 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 plays a crucial role in immunomodulation by binding to PD-1 expressed on effector T cells and inducing apoptosis or unresponsiveness, thereby preventing autoimmune diseases. This molecule is a checkpoint molecule that requires a balance of normal immune defense. Its levels should be low during inflammation and normalized or increased during treatment of autoimmune diseases. PD-L1 is known to be CD274, and HXB-319, considered an anti-inflammatory marker, actively affects the counting of PD-L1-expressing cells and spleen cells in peritoneal lavage fluid (Figure 10), demonstrating evidence of its anti-inflammatory effect. However, this level should not be too high, and in acute experiments with Group 1, there was no statistically significant difference in PD-L1 levels between wild-type untreated mice and pristane-inducing HXB-319-treated mice.

[0253] In Group 1 (acute study, 1 week after cell treatment 4 weeks after pristane induction), CD4+PDL1+ cells showed differences using ANOVA, with untreated MSCs being more numerous than healthy controls, and the single treatment with OHA × 1,000,000 (HXB-319) resulted in a higher quantity.

[0254] In the second group, CD4+PDL1+ cells (chronic experiment, 9 months after cell treatment 4 weeks after pristane induction), an ANOVA showed a significant difference, with OHA-treated MSCs × 1 (HXB-319) × 1 million being 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, the pristane-induced mouse HXB-319 (2 × 10) 6 A single treatment with MSCs primed with individual cells surprisingly inhibited systemic levels of IFN-α (p<0.05) and IFN-γ (p<0.001) in 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, indicating evidence that type I and type II interferon activation pathways were controlled. 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 levels of BAFF were reduced using MSCs 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, MSCs primed with HXB-319 improved glomerulosclerosis and overall scar formation in the originally induced BalbCJ human SLE mouse model, and it was also found that MSCs primed with HXB-319 improved the dermatitis seen in the originally induced BALBcj human SLE mouse model.

[0269] In summary, the experiments outlined in Example 1 surprisingly found the following:

[0270] (1) To improve the serological and cytological signs of interferon activation in primitively induced BALBCj interferon-activated acute models and chronic pristane-induced human SLE mouse models by primed MSCs with HXB-319;

[0271] (2) MSCs primed with HXB-319-3 were more effective than IDO gene expression and were also effective in NK cell amplification. In in vitro tissue culture, there was no equivalent increase in IL-6 and IL-8 after priming MSCs with HXB-319. Therefore, MSCs primed with HXB-319-3 can be used when cytokine storms are observed in macrophage activation syndrome (MAS), including active SLE, which is a serious complication observed in many autoimmune diseases;

[0272] (3) MSCs primed with HXB-319 improve glomerular sclerosis and overall scar formation in primitively induced BALBcj human SLE mouse models, and thus prevent advanced SLE nephritis;

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

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

[0275] (6) MSCs primed with HXB-319, or CCMs (including MSC secretions and exosomes) isolated from HXB-319 primed MSCs, 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 variants, secretions, and exosomes can be primed with HXB-319 to improve their potency and lifespan. Chimeric antigen receptor-modified T cells (CARTs) or CAR-natural killer cells are immunotherapies that have been shown to induce relief in some patients with chemotherapy-refractory hematological malignancies. HXB-319 can improve the efficacy of CAR-T / CAR-NK therapy and improve patient treatment outcomes by enhancing the anti-inflammatory effects of CAR-T and CAR-NK cells.

[0276] Example 2 In Example 2, experiments were conducted to develop an MSC-primed medium called "HXB-319-3" to control inflammatory activity induced by the activation of type I and type II interferon pathways in various autoimmune diseases (e.g., SLE, xerosis, systemic sclerosis, graft-versus-host disease, inflammatory bowel disease, multiple sclerosis, psoriasis-like arthritis). The term "HXB-319-3" used in Example 2 can also refer to MSCs treated with or primed in the MSC-primed medium discovered in Example 2.

[0277] The inventors tested different subsets of primed 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), similar to the experiment described in Example 1. As in Example 1, the inventors first evaluated the expression and activity of IDO. ASC-AC-21, 200 μM (Sigma-Alrich (L4524-5MG)) was added to serum-free DMEM in all study groups. Different combinations of culture medium components tested are shown in Figures 20A-B.

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

[0279] The inventors also performed RT-PCR on untreated MSCs in the study group: 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 particularly focused on the combination of IDO1, IL-10, PDGRF, and CD274 (PDL-1) with minimized CX3CL1 (Figs. 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 measurement and RT-PCR (Figs. 24-26).

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

[0282] The present disclosure has been specifically shown and described with reference to preferred embodiments thereof. However, it will be understood by those skilled in the art that various forms and detailed modifications are possible 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.

Claims

1. 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; 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 the at least two pro-inflammatory cytokines are present in amounts sufficient to promote induction of stem cells having an anti-inflammatory phenotype; A primed medium, wherein cells with the anti-inflammatory phenotype are marked by increased expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the non-primed stem cell population.

2. 2. The primed medium of claim 1, wherein the functional activator is a Toll-like receptor (TLR) ligand selected from the group consisting of poly(I:C), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β).

3. 2. The primed medium according to claim 1, wherein the at least two inflammatory cytokines are selected from the group consisting of IFN-γ, TNF-α, IL-1β, and IL-17A.

4. 2. The primed medium of claim 1, wherein the stem cells are mesenchymal stem cells (MSCs) or multipotent stromal cells.

5. The priming medium induces the MSCs to express CD146 ＋ and Lep-R ＋ The priming medium of claim 4, wherein the priming medium transforms the cells into pericyte-like MSCs that acquire surface expression of

6. 5. The primed medium of claim 4, wherein the MSCs are derived from allogeneic adipose tissue, bone marrow, or umbilical cord blood.

7. The primed medium of claim 4, wherein the MSCs are of human origin.

8. The primed medium of claim 1 further comprising stem cells.

9. The primed medium of claim 1, which is a synthetic medium.

10. 1. An in vitro method for producing an isolated stem cell population having an anti-inflammatory phenotype, comprising: The in vitro method comprises contacting a non-primed stem cell population with the priming medium of claim 1 for a period of time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype; The in vitro method, wherein cells having the anti-inflammatory phenotype are marked by increased expression of one or more anti-inflammatory or immunomodulatory mediators compared to non-primed stem cells.

11. 11. An isolated stem cell population having an anti-inflammatory phenotype produced by the method of claim 10.

12. 1. A method for treating a systemic inflammatory or autoinflammatory disease or disorder in a subject, comprising: administering to said subject a therapeutically effective amount of stem cells having an anti-inflammatory phenotype; The method of claim 10, wherein the stem cells are produced by the method of claim 10 prior to administration.

13. 13. The method of claim 12, wherein the systemic inflammatory or autoinflammatory disease or disorder is an interferon-regulated autoimmune disease.

14. 14. The method of claim 13, wherein the interferon-regulated autoimmune disease is regulated by aberrant activation of type I IFN-mediated pathways and type II IFN-mediated pathways and is selected from the group consisting of systemic lupus erythematosus, monogenic interferonopathies, mixed connective tissue disease, Sjogren's syndrome, overlap syndrome (rheumatoid arthritis with 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 sclerosis.

15. 1. A priming medium for producing an isolated stem cell population having an anti-inflammatory phenotype from a non-primed stem cell population, comprising: The primed medium comprises: a serum-free medium; a functional activator of the type I interferon (IFN) pathway and the type II IFN pathway; At least four inflammatory cytokines; Contains essential vitamins, the functional activator and the at least four pro-inflammatory cytokines are present in amounts sufficient to promote induction of stem cells having an anti-inflammatory phenotype; A primed medium, wherein the cells having an anti-inflammatory phenotype are marked by increased expression and / or secretion of one or more anti-inflammatory or immunomodulatory mediators compared to the non-primed stem cell population.

16. 16. The primed medium of claim 15, wherein the functional activator is a Toll-like receptor (TLR) ligand selected from the group consisting of poly(I:C), lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β).

17. The primed medium according to claim 15, wherein the at least four inflammatory cytokines are IFN-γ, TNF-α, IL-1β, and IL-17A.

18. 16. The primed medium of claim 15, wherein the stem cells are mesenchymal stem cells (MSCs).

19. The priming medium induces the MSCs to transform into pericyte-like MSCs and induces CD146 ＋ and Lep-R ＋ The primed medium of claim 18, wherein the primed medium achieves surface expression of

20. 19. The primed medium of claim 18, wherein the MSCs are derived from allogeneic adipose tissue, bone marrow, or umbilical cord blood.

21. 19. The primed medium of claim 18, wherein the MSCs are of human origin.

22. 16. The primed medium of claim 15, further comprising stem cells.

23. The primed medium of claim 15, which is a synthetic medium.

24. 16. The primed medium of claim 15, wherein the cells with an anti-inflammatory phenotype are marked by increased expression of indoleamine 2,3-deoxygenase (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 non-primed stem cells.

25. 25. The primed medium of claim 24, wherein IDO expression is enhanced by about 50,000 to about 150,000 fold compared to IDO expression in non-primed stem cells.

26. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype upregulate the production of cytotoxic T cells by at least six-fold compared to non-primed stem cells.

27. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype upregulate the production of TH1 cells by at least 12-fold compared to non-primed stem cells.

28. Cells with the anti-inflammatory phenotype have a CD4 ＋ 16. The priming medium of claim 15, which upregulates the production of T cells by at least 12-fold.

29. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype upregulate the production of natural killer (NK) cells more than non-primed stem cells.

30. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype suppress the production of Th17 cells to a greater extent than non-primed stem cells.

31. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype suppress the production of double-stranded DNA autoantibodies compared to non-primed stem cells.

32. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype suppress production of B-cell activating factor (BAFF) compared to non-primed stem cells.

33. 16. The primed medium of claim 15, wherein the cells with the anti-inflammatory phenotype have reduced production of IL-17 compared to non-primed stem cells.

34. 1. An in vitro method for producing an isolated stem cell population having an anti-inflammatory phenotype, comprising: The in vitro method comprises contacting a non-primed stem cell population with the priming medium of claim 15 for a period of time under conditions sufficient to promote the induction of stem cells having an anti-inflammatory phenotype; The in vitro method, wherein cells having the anti-inflammatory phenotype are marked by increased expression of one or more anti-inflammatory or immunomodulatory mediators compared to non-primed stem cells.

35. 35. An isolated stem cell population having an anti-inflammatory phenotype produced by the method of claim 34.

36. 1. A method for treating a systemic inflammatory or autoinflammatory disease or disorder in a subject, comprising: The method comprises administering to the subject a therapeutically effective amount of stem cells having an anti-inflammatory phenotype; 35. The method of claim 34, wherein the stem cells are produced by the method of claim 34 prior to administration.

37. 37. The method of claim 36, wherein the systemic inflammatory or autoinflammatory disease or disorder is an interferon-regulated autoimmune disease.

38. 38. The method of claim 37, wherein the interferon-regulated autoimmune disease is regulated by aberrant activation of type I IFN-mediated pathways and type II IFN-mediated pathways and is selected from the group consisting of systemic lupus erythematosus, monogenic interferonopathies, mixed connective tissue disease, Sjogren's syndrome, overlap syndromes (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 sclerosis.

39. administering a therapeutically effective amount of stem cells having an anti-inflammatory phenotype as a single dose; 37. The method of claim 36, wherein at least one cellular effect selected from the following (1) to (9) persists in the subject for at least six months: (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) CD4 ＋ Increased production of T cells; (5) increased production of NK cells; (6) suppression of Th17 cell production; (7) inhibition of double-stranded DNA production; (8) suppression of BAFF production; and (9) Decreased production of IL-17.

40. 37. The method of claim 36, wherein serum cytokine levels are not elevated after administration of the cells having an anti-inflammatory phenotype.

41. 41. The method of claim 40, wherein serum levels of TNF-α, IL-1β, and IL-6 are not elevated after administration of the cells having an anti-inflammatory phenotype.

42. 1. A method for use in adoptive immunotherapy in a subject, comprising: exposing an unprimed population of CAR-T cells and / or CAR-NK cells to priming medium for a period of time under conditions sufficient to prime and expand the CAR-T cells and / or CAR-NK cells and to increase the cytotoxic activity of the CAR-T cells and / or CAR-NK cells relative to the unprimed population of CAR-T cells and / or CAR-NK cells; administering a therapeutically effective amount of the primed CAR-T cells and / or CAR-NK cells to a subject in need of adoptive immunotherapy.

43. 43. The method of claim 42, wherein the primed medium comprises a serum-free medium, a functional activator of the type I interferon (IFN) pathway and the type II IFN pathway, and at least two pro-inflammatory cytokines.

44. 43. The method of claim 42, wherein the primed medium comprises 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.

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