Mesenchymal stem cell-mediated treatments for the prevention and targeted treatment of injured tissues and promotion of tissue repair, healing, and regeneration

Abstract

Disclosed herein are compositions, formulations, and / or methods of using mesenchymal stem cells (MSCs) and / or MSC-derived products (such as, for instance, MSC-derived exosomes (MSC-Exos) and / or one or more MSC-sourced biological compounds) for the prevention and targeted treatment of injured tissues, and / or the promotion of tissue repair, healing, and / or regeneration. The MSCs and / or MSC-derived products can be administered to a subject in a pharmaceutical composition and / or formulation, which may include one or more additional agents and / or one or more pharmaceutical excipients. The MSCs and / or MSC-derived products can promote tissue repair, healing, and / or regeneration by, for instance, (1) mediating the pericyte-to-MSC transition, (2) suppressing one or more inflammatory immune cells and / or inflammatory immune responses in injured tissues, and (3) supporting the activation, proliferation, migration, and / or differentiation of tissue-resident stem cells.

Description

[0001] MESENCHYMAL STEM CELL-MEDIATED TREATMENTS FOR THE PREVENTION AND TARGETED TREATMENT OF INJURED TISSUES AND PROMOTION OF TISSUE REPAIR, HEALING, AND REGENERATION

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to U.S. Provisional Application No. 63 / 587,294, filed October 2, 2023, which is hereby incorporated by reference in its entirety.

[0004] FIELD OF THE DISCLOSURE

[0005] The disclosure relates generally to compositions, formulations, and methods for immunotherapy and / or regenerative medicine. In particular, embodiments of the disclosure relate to mesenchymal stem cell-mediated (e.g., one or more types of mesenchymal stem cells and / or one or more types of mesenchymal stem cell-derived products) and / or pericyte-mediated treatments that can be used as a targeted therapy for the prevention and targeted treatment of injured tissues, as well as the promotion of tissue repair, healing, and / or regeneration. Such therapy can be used for any condition, disease, and / or disorder that causes tissue injury, including, for instance, cancers, tumors, and other disorders. In certain embodiments, one or more compositions and / or formulations described herein may include, for example, macromolecular proteins, growth factors, mesenchymal stem cells (MSCs), MSC-derived products, and various immunoregulatory biomolecules. One or more such compositions and / or formulations may be used in combination with, or formulated with, one or more additional active agents.

[0006] BACKGROUND

[0007] During the last three decades, immunosuppressive drugs have been frequently used in clinical practice due to the increase of autoimmune and inflammatory diseases. However, longterm use of immunosuppressive agents may result in the development of severe infections due to the inhibition of anti-microbial immune response. As a result, one area of interest, especially in the treatment of injured tissues, is the development of novel immunomodulatory compounds that can selectively inhibit detrimental immune responses without causing life-threatening i immunosuppression.

[0008] Mesenchymal stem cells (“MSC” or “MSCs”) are self-renewable, multipotent stem cells that regulate innate and / or adaptive immune responses in various human tissues. For instance, MSCs play a role in responding to tissue injury and reducing inflammation. Moreover, due to their immunosuppressive properties, MSCs have therapeutic potential in alleviating various diseases (e.g., autoimmune diseases, specific cancers).

[0009] MSCs may originate from different sources (e.g., bone marrow, amniotic fluid, placental tissue, etc.) and contain a variety of biological compounds (e.g., carbohydrates, proteins and peptides, lipids, lactate, pyruvate, electrolytes, enzymes, hormones, and various growth factors).

[0010] MSCs are also constituents of the cellular environment existing around various types of tissues. Thus, in the specific context of injured tissues and / or wound sites, MSCs may have the potential to modulate the phenotype and / or function of one or more types of immune cells that participate in immune responses to tissue injuries.

[0011] Given the foregoing, there exists a significant need for systems and methods that treat injured tissues, and / or one or more diseases that result in injured tissues, using MSCs and / or MSC- derived products (e.g., MSC-sourced exosomes, MSC-sourced nucleic acids, MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration). In particular, there is a need for methods that provide for the clinical use of MSCs and / or MSC- derived products in immunotherapy and / or regenerative medicine treatments.

[0012] SUMMARY

[0013] It is to be understood that both the following summary and the detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Neither the summary nor the description that follows is intended to define or limit the scope of the invention to the particular features mentioned in the summary or in the description.

[0014] In certain embodiments, the disclosed embodiments may include one or more of the features described herein.

[0015] Embodiments of the present disclosure are directed towards compositions, formulations, and methods for using one or more types of mesenchymal stem cells (MSCs) and / or MSC-derived products (e.g., exosomes derived from MSCs, nucleic acids (including micro-ribonucleic acids (miRNAs)) derived from MSCs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) for preventing and / or treating injured tissues, including, but not limited to, in the context of one or more diseases that cause injured tissues (e.g., cancer). The MSCs and / or MSC-derived products contain significant numbers of biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration, including, for instance, growth factors, anti-inflammatory cytokines, and the like, and are amenable for long-term storage without the loss of biological potency. In at least one embodiment, various types of MSCs and / or MSC-derived products are shown to improve tissue injuries and / or wound sites. In at least another embodiment, one or more types of MSCs and / or MSC-derived products are used in combination with, or formulated with, one or more additional active agents. As a non-limiting example, exosomes derived from MSCs can be used to deliver one or more MSC-derived biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration.

[0016] A non-limiting example of one of the aforementioned MSC-derived products described herein is d-MAPPS™ regenerative biologies platform technology (“d-MAPPS”), which contains large numbers of MSC-sourced growth factors, anti-inflammatory cytokines and chemokines. Notably, the acronym d-MAPPS in d-MAPPS™ regenerative biologies platform technology stands for “derived Multiple Allogeneic Proteins Paracrine Signaling”. In vitro, d-MAPPS™ regenerative biologies platform technology efficiently inhibits proliferation of activated human peripheral blood mononuclear cells (“pbMNCs”). d-MAPPS™ regenerative biologies platform technology further suppresses production of inflammatory cytokines and promotes secretion of immunosuppressive factors in pbMNCs. In addition, d-MAPPS™ regenerative biologies platform technology favors development of tolerogenic and regulatory phenotype in activated monocytes and lymphocytes, indicating its potential for therapeutic use in treating injured tissues and / or various diseases that result in tissue injury.

[0017] In at least a further embodiment, one or more types of MSCs and / or MSC-derived products suppress the production of inflammatory cytokines and promote the secretion of immunosuppressive immune responses and / or immune cell phenotypes. In at least another embodiment, the one or more types of MSCs and / or MSC-derived products favor the development of tolerogenic and / or regulatory phenotypes in activated monocytes and lymphocytes, indicating its potential for therapeutic use to treat one or more injured tissues and / or one or more disorders or conditions that result in injured tissues (e.g., various cancers).

[0018] In at least another embodiment, the aforementioned one or more types of MSCs and / or MSC-derived products contain biological compounds (e.g., various cytokines) that can either enhance or suppress one or more immune responses (e.g., T-cell driven responses) in a tissue microenvironment (e.g., in a microenvironment around one or more types of injured tissues, in a microenvironment around one or more tumors, etc.). In at least another embodiment, a method for prevention and treatment of tissue injury is disclosed, which includes, for instance, altering the response of endogenous immune cells in the subject provided. The method may therefore comprise administering to a subject an effective amount of one or more types of MSCs and / or MSC-derived products (e.g., exosomes derived from MSCs, nucleic acids derived from MSCs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration), which may be composed within one or more MSC compositions and / or formulations, thereby altering the response of one or more endogenous immune cells (e.g., dendritic cells, macrophages, natural killer cells, T cells, and the like) in the subject. In at least another embodiment, administration of an effective amount of such one or more types of MSCs and / or MSC-derived products increases the likelihood of survival of the subject and / or decreases the incidence of tissue injury, wound sites, and / or one or more disorders (e.g., cancers and / or tumors) in the subject.

[0019] In at least another embodiment, the one or more types of MSCs and / or MSC-derived products may be administered in combination with one or more agents, such as, for instance, one or more antimicrobial agents, one or more analgesic agents, one or more chemotherapeutic agents and / or drugs, one or more local anesthetic agents, one or more anti-inflammatory agents, one or more anti-oxidant agents, one or more immunosuppressant agents, one or more anti-allergenic agents, one or more enzyme cofactors, one or more essential nutrients, one or more growth factors, and combinations thereof.

[0020] In at least another embodiment, the one or more types of MSCs and / or MSC-derived products are used as a delivery vehicle for one or more other agents, including, for instance, bispecific T-cell engaging antibodies, glypican 3, one or more treatment compounds (e.g., prodrugs, anti-cancer drugs, including, for instance, one or more chemotherapeutic drugs, and the like), one or more cytokines (e.g., IL-2, IL-12, IL-21 , and TRAIL), one or more interferons (e.g., IFN-a, IFN-P, and IFN-y), and combinations thereof.

[0021] In at least another embodiment, a pharmaceutical composition comprises one or more types of MSCs and / or MSC-derived products. As a non-limiting example, a pharmaceutical composition comprising d-MAPPS™ regenerative biologies platform technology (also referred to herein as “d- MAPPS pharmaceutical composition”) is disclosed herein. Such d-MAPPS pharmaceutical compositions may be formulated in various formulations, including, for example, as one or more liquid solutions (also referred to herein as “d-MAPPS solution” or “d-MAPPS solutions”). At least one such d-MAPPS solution is suitable for administration to the eyes of a subject as eye drops. Other formulations (e.g., gels, solids) are possible. The d-MAPPS pharmaceutical compositions (e.g., one or more d-MAPPS solutions) may comprise, for instance, one or more types of MSCs, one or more types of exosomes derived from one or more types of MSCs (“MSC-Exos”) (e.g., exosomes generated ex vivo from mesenchymal stem cells, wherein the mesenchymal stem cells may be, for instance, placental tissue-derived mesenchymal stem cells), one or more MSC-sourced growth factors and / or immunoregulatory proteins, and / or sterile de-cellularized human amniotic fluid (D-HAF).

[0022] In at least one embodiment, a d-MAPPS pharmaceutical composition (e.g., a d-MAPPS solutions) may include a sterile de-cellularized human amniotic fluid (D-HAF), preferably diluted with a pharmaceutically accepted carrier, and typically administered using a standard eye dropper apparatus. D-HAF contains over 300 human growth factors. D-HAF is devoid of amniotic stem cells and elements of micronized membrane or chorion particles. The dilution ratio of the D-HAF is dependent on the severity of the disorder or injury. For example, surface inflammation may be best treated with a low concentration, whereas more severe inflammation will typically utilize a higher concentration of D-HAF. Daily applications of a d-MAPPS pharmaceutical composition (e.g., one or more d-MAPPS solutions containing D-HAF) can deliver a sustainable level of beneficial growth factors.

[0023] D-HAF can be prepared from human amniotic fluid (“AF”) from which the amniotic stem cells and particulate matter have been removed. In at least one embodiment, the process includes separating the cells from the AF using centrifugation and utilizing a series of filtration devices to remove all remaining cells and bioburden. Each lot is tested for bioburden and is certified sterile to contain <1 harmful organisms. The purified fluid is sterilized without the use of harsh terminal irradiation, e-beam, or Ethylene Oxide (EO).

[0024] In at least a further embodiment, any one or more of the pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients. Such a composition may comprise one or more agents selected from the group consisting of adjuvants, antioxidants, anti-inflammatory agents, growth factors, neuroprotective agents, antimicrobial agents, local anesthetics, and combinations thereof. In at least another embodiment, the composition may comprise exosomes generated ex vivo from MSCs. Such exosomes may be used as a delivery vehicle for one or more MSC-sourced biological molecules (e.g., nucleic acids (such as, for instance, miRNAs, messenger RNAs (mRNAs)), enzymes, cytokines, chemokines, growth factors, immunomodulatory factors, small-molecule drugs, proteins, compounds that enhance and / or promote tissue repair, healing, and / or regeneration, and combinations thereof).

[0025] Methods for the prevention and targeted treatment of injured tissues, and / or the promotion of tissue repair, healing, and / or regeneration using one or more pharmaceutical compositions (e.g., one or more compositions including MSCs, MSC-derived products, d-MAPPS, etc.) are disclosed. Such compositions can be used in combination with one or more therapeutic, prophylactic or diagnostic agents. In some embodiments, one or more such pharmaceutical compositions is administered prior to, in conjunction with, subsequent to, or alternation with treatment with one or more therapeutic, prophylactic or diagnostic agents. In some embodiments, the one or more therapeutic, prophylactic or diagnostic agents are selected from the group consisting of an antiglaucoma agent, an anti-angiogenesis agent, an anti-infective agent, an anti-inflammatory agent, an analgesic agent, a local anesthetic, a growth factor, an immunosuppressant agent, an antiallergic agent, an anti-oxidant, a cytokine, and combinations thereof. In some embodiments, the one or more diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, radionuclides, x-ray imaging agents, contrast media, and combinations thereof.

[0026] These and further and other objects and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification, as well as the drawings.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

[0029] Figure 1 shows various biological compounds that play a role in influencing the pericyte- to-MSC transition, according to at least one embodiment of the disclosure.

[0030] Figure 2 shows various pericyte changes that occur during the pericyte-to-MSC transition, according to at least one embodiment of the disclosure.

[0031] Figure 3 shows various MSC-mediated mechanisms that result in the suppression of inflammation in one or more injured tissues, according to at least one embodiment of the disclosure.

[0032] Figure 4 shows various MSC-sourced and / or MSC-derived compounds that can either suppress the generation of an inflammatory phenotype in one or more types of immune cells and / or induce the generation of an immunosuppressive phenotype in one or more types of immune cells, according to at least one embodiment of the disclosure.

[0033] Figure 5 shows various MSC-sourced and / or MSC-derived compounds that can contribute to the interaction and / or cross-talk between MSCs and tissue-resident stem cells in injured tissues, according to at least one embodiment of the disclosure.

[0034] DETAILED DESCRIPTION

[0035] The present invention is more fully described below with reference to the accompanying figures. The following description is exemplary in that several embodiments are described (e.g., by use of the terms “preferably,” “for example,” or “in one embodiment”); however, such should not be viewed as limiting or as setting forth the only embodiments of the present invention, as the invention encompasses other embodiments not specifically recited in this description, including alternatives, modifications, and equivalents within the spirit and scope of the invention. Further, the use of the terms “invention,” “present invention,” “embodiment,” and similar terms throughout the description are used broadly and not intended to mean that the invention requires, or is limited to, any particular aspect being described or that such description is the only manner in which the invention may be made or used. Additionally, the invention may be described in the context of specific applications; however, the invention may be used in a variety of applications not specifically described.

[0036] The embodiment(s) described, and references in the specification to “one embodiment”, “an embodiment”, “an example embodiment”, etc., indicate that the embodiment(s) described may include a particular feature, structure, or characteristic. Such phrases are not necessarily referring to the same embodiment. When a particular feature, structure, or characteristic is described in connection with an embodiment, persons skilled in the art may affect such feature, structure, or characteristic in connection with other embodiments whether or not explicitly described.

[0037] In the several figures, like reference numerals may be used for like elements having like functions even in different drawings. The embodiments described, and their detailed construction and elements, are merely provided to assist in a comprehensive understanding of the invention. Thus, it is apparent that the present invention can be carried out in a variety of ways and does not require any of the specific features described herein. Also, well-known functions or constructions are not described in detail since they would obscure the invention with unnecessary detail. Any signal arrows in the drawings / figures should be considered only as exemplary, and not limiting, unless otherwise specifically noted. Further, the description is not to be taken in a limiting sense, but is made merely for the purpose of illustrating the general principles of the invention, since the scope of the invention is best defined by the appended claims.

[0038] It will be understood that, although the terms “first,” “second,” etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Purely as a non-limiting example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of example embodiments. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. As used herein, “at least one of A, B, and C” indicates A or B or C or any combination thereof. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be noted that, in some alternative implementations, the functions and / or acts noted may occur out of the order as represented in at least one of the several figures. Purely as a non-limiting example, two figures shown in succession may in fact be executed substantially concurrently or may sometimes be executed in the reverse order, depending upon the functionality and / or acts described or depicted.

[0039] As used herein, ranges are used herein in shorthand, so as to avoid having to list and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range.

[0040] Unless indicated to the contrary, numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0041] The words “comprise,” “comprises,” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including,” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. The terms “comprising” or “including” are intended to include embodiments encompassed by the terms “consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include embodiments encompassed by the term “consisting of.” Although having distinct meanings, the terms “comprising,” “having,” “containing,” and “consisting of’ may be replaced with one another throughout the description of the invention.

[0042] Conditional language, such as, among others, “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or steps. Thus, such conditional language is not generally intended to imply that features, elements and / or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements and / or steps are included or are to be performed in any particular embodiment.

[0043] Terms such as, among others, “about,” “approximately,” “approaching,” or “substantially,” mean within an acceptable error for a particular value or numeric indication as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. The aforementioned terms, when used with reference to a particular non-zero value or numeric indication, are intended to mean plus or minus 10% of that referenced numeric indication. As an example, the term “about 4” would include a range of 3.6 to 4.4. All numbers expressing dimensions, velocity, and so forth used in the specification are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0044] “Typically” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0045] Wherever the phrase “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.

[0046] Definitions

[0047] The following is a non-exhaustive and non-limiting list of terms used herein and their respective definitions.

[0048] The terms “agent” or “active agent,” which are used interchangeably herein, refer to a physiologically or pharmacologically active substance that acts locally and / or systemically in a subject’s body. An “agent” or “active agent” is a compound or substance that is administered to an individual for the treatment (e.g., therapeutic agent, cancer therapeutic agent, and the like), prevention (e.g., prophylactic agent), or diagnosis (e.g., diagnostic agent) of a disease or disorder. Such agents may also include therapeutics that prevent or alleviate symptoms, such as, for instance, symptoms associated with one or more cancers or treatments for such cancer(s).

[0049] The term “administering” or “administration” refers to providing or giving a subject one or more agents and / or formulations, such as one or more types of MSCs, either alone or in conjunction with any other compound and / or agent (including, e.g., cancer prophylactic or anticancer therapeutic agents), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as, e.g., subcutaneous, subdermal, intramuscular, intradermal, intraperitoneal, intracerebroventricular, intraosseous, intratumoral, intraprostatic, and intravenous), transdermal, intranasal, oral, vaginal, rectal, and inhalation.

[0050] The term “amniotic factor” generally refers to one or more compounds naturally present in the amniotic fluid. These include, for example, carbohydrates, proteins and peptides (e.g., enzymes, hormones), lipids, metabolic substrates and products (e.g., lactate, pyruvate), and electrolytes.

[0051] The term “antigen” refers to a compound, composition, and / or substance that can stimulate the production of antibodies or an immune response in a subject, including compositions (such as one that includes a tumor-specific protein) that are injected or absorbed into a subject. An “antigen” may react with the products of specific humoral and / or cellular immunity, including, for example, those induced by heterologous antigens.

[0052] The term “cancer” refers to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. A malignant cancer is one in which a group of tumor cells display one or more of uncontrolled growth e.g., division beyond normal limits), invasion (e.g., intrusion on and destruction of adjacent tissues), and / or metastasis (e.g., spread to other locations in the subject’s body via lymph or blood). As used herein, the terms “metastasis” or “metastasize” refer to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are similar to those in the original tumor (i.e., the tumor at the primary site of tumor growth). A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A “tumor” refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancers form tumors, but some, e.g., leukemia, and some blood cancers, do not necessarily form tumors. For those cancers that form tumors, the terms “cancer,” “cancer cell,” “tumor,” and “tumor cell” are used interchangeably. The amount of a tumor in a given subject is the “tumor burden,” which can be measured as the number, volume, and / or weight of the tumor.

[0053] Exemplary tumors, such as cancers, that can be treated using the disclosed one or more agents, formulations, and / or methods (e.g., including one or more types of MSCs, either alone or in conjunction with one or more other agents) include solid tumors, such as breast carcinomas (e.g., lobular and duct carcinomas, such as a triple negative breast cancer), sarcomas, carcinomas of the lung (e.g., non-small cell carcinoma, large cell carcinoma, blood cancers, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (e.g., serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (e.g., transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma, endometrial carcinomas (e.g., adenocarcinomas and mixed Mullerian tumors (carcinosarcomas)), carcinomas of the endocervix, ectocervix, and vagina (e.g., adenocarcinoma and squamous carcinoma of each of same), tumors of the skin (e.g., squamous cell carcinoma, basal cell carcinoma, malignant melanoma, skin appendage tumors, Kaposi sarcoma, cutaneous lymphoma, skin adnexal tumors, and various types of sarcomas and Merkel cell carcinoma), esophageal carcinoma, carcinomas of the nasopharynx and oropharynx (e.g., squamous carcinoma and adenocarcinomas of same), salivary gland carcinomas, brain and central nervous system tumors (e.g., tumors of glial, neuronal, and meningeal origin), tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and cartilage, head and neck squamous cell carcinoma, and lymphatic tumors (e.g., B-cell and T-cell malignant lymphoma). In one example, the tumor is an adenocarcinoma. In another example, the cancer is pancreatic adenocarcinoma. In yet another example, the cancer is colorectal adenocarcinoma. The disclosed methods and / or formulations can also be used to treat liquid tumors, such as a lymphatic, white blood cell, or other type of leukemia. In a specific example, the tumor treated is a tumor of the blood, such as a leukemia (for example acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), a lymphoma (such as Hodgkin’s lymphoma or non-Hodgkin’s lymphoma), or a myeloma.

[0054] The term “combination therapy” refers to the administration of different compounds, agents, and / or individual therapies in a sequential and / or simultaneous manner. Individual elements of a “combination therapy” may be administered at different times and / or by different routes, but act in combination to provide a beneficial effect on the subject.

[0055] The term “compound” refers to a substance formed from one or more chemical elements, arranged together in any proportion or structural arrangement. The one or more chemical elements may be either naturally occurring and / or non-naturally occurring. As used herein, the term “biological compound” refers to a compound of biological origin and / or having one or more effects on a subject’s local and / or systemic biological functions. Accordingly, “compounds” or “biological compounds” include, as non-limiting examples, various proteins (e.g., growth factors, hormones, enzymes), nucleic acids, and pharmaceutical products (e.g., drugs, prodrugs). The term “drug” generally refers to a medicine or other substance that has a physiological effect when introduced into a subject. The term “prodrug” generally refers to a biologically and / or chemically inactive compound that can be metabolized by a subject to produce a drug.

[0056] The terms “decrease,” “lower,” “lessen,” “reduce,” and “abate,” which are used interchangeably herein, refer generally to the ability of a compound, formulation, or therapy (including those disclosed herein) to produce, elicit, and / or cause a lesser physiological response (e.g., downstream effects) compared to the response caused by a respective control compound, formulation, or therapy. A “decrease” or “reduced” amount is typically a “statistically significant” amount, and may include a decrease that is, for instance, 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.).

[0057] The term “dendritic cell” refers to a type of specialized antigen-presenting cell (“APC”) involved in innate and / or adaptive immunity. Dendritic cells may also be referred to herein as “DC” or “DCs.” Dendritic cells may be present in the tumor microenvironment, and these are referred to as “tumor-associated dendritic cells” (“tDC” or “tDCs”).

[0058] The terms “effective amount” or “therapeutically effective amount,” which are used interchangeably herein, refer to the amount of an agent (e.g., including one or more types of MSCs described herein) that is sufficient to effect beneficial or desired therapeutic result, including clinical results. An “effective amount” may vary depending upon one or more of: the subject and disease condition being treated, the sex, weight and age of the subject, the severity of the disease condition, the manner of administration, the ability of one or more formulations to elicit a desired response in the subject, and the like. The beneficial therapeutic effect can include, but is not limited to, enablement of diagnostic determinations; prevention of disease or tumor formation; amelioration of a disease, symptom, disorder, and / or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, and / or pathological condition; and generally counteracting a disease, symptom, disorder, and / or pathological condition. The term “effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient or individual). When a therapeutic amount is indicated, the precise amount of one or more formulations described in the present disclosure to be administered can be determined by a physician, based on, for instance, considerations such as individual differences in age, weight, tumor size, extent of infection or metastasis, and / or condition of the subject (individual).

[0059] In at least one embodiment, an “effective amount” (e.g., of one or more agents and / or formulations described herein, including one or more types of MSCs, either alone or in conjunction with one or more other agents) may be an amount sufficient to increase the rate of survival of a subject, reduce the volume / size of a tumor, reduce the weight of a tumor, reduce the number / extent of metastases, reduce the volume / size of a metastasis, reduce the weight of a metastasis, and combinations thereof, for example by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% (as compared to no administration of the therapeutic agent and / or formulation). In at least a further embodiment, an “effective amount” (e.g., of one or more agents and / or formulations described herein, including one or more types of MSCs, either alone or in conjunction with one or more other agents) may be an amount sufficient to increase the survival time of a subject, such as a subject with cancer, for example by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, 100%, 200%, 300%, 400%, or 500% (as compared to no administration of the therapeutic agent and / or formulation).

[0060] The terms “enhance,” “induce,” “induction,” and “increase,” which are used interchangeably herein, refer generally to the ability of a compound, formulation, or therapy (including those disclosed herein) to produce, elicit, and / or cause a greater physiological response (e.g., downstream effects) compared to the response caused by a respective control compound, formulation, or therapy. A non-limiting example of a measurable physiological response includes inducing one or more responses of cancer-associated endogenous immune cells in the subject and / or an increase in cytotoxic and / or cancer cell death killing ability, among others apparent from the description herein. An “enhanced” or “increased” amount is typically a “statistically significant” amount, and may include an increase that is, for instance, 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.). The term “growth factor” refers to any compound (e.g, one or more groups of proteins or hormones) that stimulate cellular growth. Generally, growth factors play an important role in promoting cellular differentiation and cell division, and they occur in a wide range of organisms, including humans.

[0061] The term “immune cell” refers to any cell of the immune system that has one or more effector functions (e.g, cytotoxic cell killing activity, secretion of cytokines, induction of antibody-dependent cell-mediated cytotoxicity (ADCC), and / or induction of complementdependent cytotoxicity (CDC)).

[0062] The terms “immunologic,” “immunological,” or “immune” response, which are used interchangeably herein, refer to the development of a beneficial humoral (z.e., antibody-mediated) and / or a cellular (e.g., mediated by immune cells, such as antigen-specific T cells, or their secretion products) response directed against an antigen and / or immunogen in a specific subject. Such a response can be an active response induced by administration of an antigen and / or immunogen, or a passive response induced by administration of antibodies or primed T-cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II major histocompatibility complex (MHC) molecules to activate antigen-specific CD4+ healer T cells and / or cos+ cytotoxic T cells. The response may also involve, for instance, activation of monocytes, macrophages, natural killer (NK) cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils, and / or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (e.g, CD4+ T cells) or cytotoxic T lymphocyte (CTL) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an antigen and / or immunogen can be distinguished by, for example, separately isolating antibodies and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

[0063] The term “ionizing radiation” refers to radiation, traveling as a particle or electromagnetic wave, that carries sufficient energy to detach electrons from atoms or molecules, thereby ionizing an atom or a molecule. Generally, ionizing radiation is made up of energetic subatomic particles, ions, or atoms moving at high speeds and electromagnetic waves on the high-energy end of the electromagnetic spectrum. Radiation has been demonstrated to induce adaptive immune responses to mediate tumor regression. In addition, the induction of type I interferons (“IFNs”) by radiation is essential for the function of CD8+ T cells. Radiation induces cell stress and causes excess deoxyribonucleic acid (DNA) breaks, indicating that the nucleic acid-sensing pathway likely accounts for the induction of type I IFNs upon radiation. Type I IFN responses in DCs dictate the efficacy of antitumor radiation. In contrast, chemotherapeutic agents and anti-human epidermal growth factor receptor 2 (HER2) antibody treatments have been demonstrated to depend on a distinct immune mechanism to trigger adaptive immune responses. In general, therapeutic radiation-mediated antitumor immunity depends on a proper cytosolic DNA sensing pathway. In at least one embodiment, one or more agents, formulations, and / or methods (e.g., including one or more types of MSCs, either alone or in conjunction with one or more other agents) described herein is administered in combination with radiation therapy.

[0064] The term “macrophage” refers to a type of white blood cell of the immune system that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and the like. These phagocytes include various subtypes (e.g., histiocytes, Kupffer cells, alveolar macrophages, microglia, and others), but all are part of the mononuclear phagocyte system. Besides phagocytosis, macrophages play a critical role in both innate and adaptive immunity by recruiting other endogenous immune cells (e.g., lymphocytes). For example, they are important as antigen presenters to T cells. In humans, dysfunctional macrophages can cause severe diseases (e.g., chronic granulomatous disease) that result in frequent infections. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of various compounds (e.g., cytokines). Macrophages that encourage inflammation may be termed “Ml macrophages” because they have the so-called “Ml phenotype,” whereas those that decrease inflammation and encourage tissue repair may be termed “M2 macrophages” because they have the so-called “M2 phenotype.”

[0065] The terms “MSC-sourced” or “MSC-derived,” which are used interchangeably herein, refer to an agent or compound obtained, sourced, and / or derived from one or more types of mesenchymal stem cells (“MSC” or “MSCs”). Such agents or compounds include, but are not limited to, biological compounds such as, for instance, microscopic ribonucleic acids (also referred to herein as “micro-ribonucleic acids”) (“miRNA” or “miRNAs”). Thus, as a non-limiting example, miRNAs sourced from MSCs are referred to herein as “MSC-miRNAs,” “MSC-sourced miRNAs,” or “MSC-derived miRNAs,” all of which are used interchangeably. The term “parenteral administration” refers to a type of administration by any method other than through the digestive tract or non-invasive topical or regional routes. As a non-limiting example, parenteral administration may include administration to a subject via intravenous, intradermal, intraperitoneal, intrapleural, intratracheal, intraarticular, intrathecal, intramuscular, subcutaneous, subjunctival, injection, and / or infusion.

[0066] The term “peptide” refers to a polymer of amino acid residues. The amino acid residues may be naturally occurring and / or non-naturally occurring. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein. The terms apply to, for instance, amino acid polymers of one or more amino acid residues, an artificial chemical mimetic of a corresponding naturally occurring amino acid, naturally occurring amino acid polymers, and non-naturally occurring amino acid polymers.

[0067] The terms “subject,” “individual,” or “patient,” which are used interchangeably herein, refer to a vertebrate, such as a mammal (e.g., a human). Mammals include, but are not limited to, murines (e.g., mice), simians, humans, farm animals, sport animals, and pets. In at least one embodiment, the subject is a non-human mammal, such as a monkey or other non -human primate, mouse, rat, rabbit, guinea pig, pig, goat, sheep, dog, cat, horse, or cow. In at least one example, the subject has a tumor, such as a cancer, that can be treated using one or more agents, formulations, and / or methods (e.g., including one or more types of MSCs, either alone or in conjunction with one or more other agents) disclosed herein. In at least an additional example, the subject is a laboratory animal / organism, such as, for example, a mouse, rabbit, guinea pig, or rat. In at least a further example, a subject includes, for instance, farm animals, domestic animals and / or pets (e.g., cats, dogs). In at least a still further example, a subject is a human patient that has a cancer, has been diagnosed with a cancer, and / or is at risk of having a cancer. A “patient” can specifically refer to a subject that has been diagnosed with a particular disease, condition, and / or indication that can be treated with refers to a subject that has been diagnosed with a particular indication that can be treated with one or more agents, formulations, and / or methods (e.g., including one or more types of MSCs, either alone or in conjunction with one or more other agents) disclosed herein.

[0068] The term “topical administration” refers to a type of non-invasive administration to the skin, orifices, and / or mucosa of a subject. Topical administrations can be administered locally; that is, they are capable of providing a local effect in the region of application without systemic exposure. Topical formulations can, however, provide one or more systemic effects via, e.g., adsorption into the blood stream of the individual. Routes of topical administration include, but are not limited to, cutaneous and transdermal administration, buccal administration, intranasal administration, intravaginal administration, intravesical administration, ophthalmic administration, pulmonary administration, and rectal administration.

[0069] The terms “treating,” “treatment,” and “therapy” refer, either individually or in any combination, to any success or indicia of success in the attenuation or amelioration of an injury, disease, symptom, disorder, pathology, and / or condition, and / or pathological condition, including any objective or subjective parameter such as, for instance, abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, and / or prolonging the length of survival. Treatment does not necessarily indicate complete eradication or cure of the injury, disease, symptom, disorder, pathology, and / or condition, and / or pathological condition, or any associated symptom(s) thereof. The treatment may be assessed by one or more objective or subjective parameters, including, for example, the results of a physical examination, blood and other clinical tests (e.g., imaging), and the like. In at least one example, treatment with the disclosed one or more agents, formulations, and / or methods (e.g., including one or more types of MSCs, either alone or in conjunction with one or more other agents) results in a decrease in the number, volume, and / or weight of a tumor and / or metastases.

[0070] The term “molecular weight” generally refers to the relative average chain length of a bulk polymer or protein, unless otherwise specified. In practice, molecular weights can be estimated or characterized using various methods including, for example, gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight-average molecular weight (MW), as opposed to the number-average molecular weight (MN). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0071] Further, unless otherwise noted, technical terms are generally used according to conventional usage. Aspects of the disclosed methods employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and / or cell biology, many of which are described below solely for the purpose of illustration. Such techniques are explained fully in technical literature sources. General definitions of common terms in the aforementioned fields, including, for instance, molecular biology, may be found in references such as, e.g., Krebs et al., Lewin ’s Genes X, Jones & Bartlett Learning (2009) (ISBN 0763766321); Redei, Encyclopedic Dictionary of Genetics, Genomics, Proteomics and Informatics (3rd ed.), Springer (2008) (ISBN: 1402067532); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons (updated July 2008) (ISBN: 047150338X); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology (2nd ed.), Wiley-Interscience (1989) (ISBN 0471514705); Glover, et al., DNA Cloning: A Practical Approach, Vol. I-II, Oxford University Press (1985) (ISBN 0199634777); Anand et al., Techniques for the Analysis of Complex Genomes, Academic Press (1992) (ISBN 0120576201); Hames et al., Transcription and Translation: A Practical Approach, Oxford University Press (1984) (ISBN 0904147525); Perbal et al ., A Practical Guide to Molecular Cloning (2nd ed.), Wiley-Interscience (1988) (ISBN 0471850713); Kendrew et al., Encyclopedia of Molecular Biology, Wiley -Blackwall (1994) (ISBN 0632021829); Meyers et al., Molecular Biology and Biotechnology: A Comprehensive Desk Reference, Wiley-VCH (1996) (ISBN 047118571X); Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988) (ISBN 0879693746); Coligan et al., Current Protocols in Immunology, Current Protocols (2002) (ISBN 0471522767); Annual Review of Immunology , articles and / or monographs in scientific journals (e.g., Advances in Immunology),' and other similar references.

[0072] Tissue injury

[0073] The terms “tissue injury” or “injured tissues,” which are used interchangeably herein, refer to one or more of the following effects in one or more cells of an organism: cellular damage, disruption of normal tissue architecture, impaired organ function, and activation of systemic and severe, life-threatening inflammatory responses. Tissue injury may occur as a result of, for instance, physical trauma, infection, inflammation, ischemia, chemical exposure, autoimmune reactions, and / or diseases or disorders (e.g., one or more types of cancer).

[0074] As explained in further detail below, cross-talk between pericytes, mesenchymal stem cells (“MSC” or “MSCs”), tissue-resident stem cells, and immune cells has an important role in the regeneration of injured tissues and the promotion of healing and repair of such injured tissues. Upon tissue injury, and under the influence of various growth factors and chemotactic factors, tissue-resident stem cells can be mobilized from their niches towards the site(s) of injury. Stem cells can secrete various bioactive factors that promote tissue repair, healing, and / or regeneration, as well as modulate the phenotype and / or function of inflammatory immune cells that may be attracted to the site of injury. Such attraction can be mediated by, for instance, damage-associated molecular patterns (DAMPs) and alarmins, which can be released from damaged parenchymal cells and endothelial cells (“EC” or “ECs”).

[0075] DAMPs and alarmins can induce the generation of an inflammatory phenotype in tissue- infiltrated immune cells (e.g., macrophages, neutrophils, dendritic cells (DCs), T lymphocytes, natural killer (NK) cells, natural killer (NKT) cells, and the like), thereby enabling increased production of vasoactive factors and inflammatory cytokines. This can induce changes to the phenotype and / or function of pericytes, which are perivascular cells that wrap around the ECs that line the inner walls of capillaries. Under the influence of inflammatory mediators, pericytes can increase the synthesis of contractile proteins, detach from blood vessels, and differentiate in MSCs.

[0076] Thus, the therapeutic potential of MSCs, MSC-derived products, and / or MSC-containing compositions and / or formulations described herein can be seen with respect to MSC-dependent tissue repair, tissue regeneration, and wound healing. Large numbers of chemokine receptors are expressed on the membrane of MSCs, enabling their rapid recruitment to the site of any injury or wound site (e.g., skin wounds). Alarmins and DAMPS can then bind to alarmin / DAMPs-specific receptors on tissue-resident macrophages and induce the production of inflammatory cytokines and / or chemokines. These compounds then recruit MSCs from their respective niches or areas towards and / or into the injury or wound site. Upon migrating to the injury or wound site, MSCs can integrate into damaged tissues (e.g., skin tissues) and modulate the viability of injured parenchymal cells, induce differentiation of resident progenitor cells, and alter the phenotype and / or function of tissue-infiltrated immune cells.

[0077] Accordingly, MSCs, MSC-derived products, and / or MSC-containing compositions and / or formulations described herein can be used in different treatments for the prevention and targeted treatment of injured tissues, and / or the promotion of tissue repair, healing, and / or regeneration.

[0078] Role of MSCs in enhancing repair, healing, and / or regeneration of injured tissues

[0079] MSCs are self-renewable, multipotent stem cells that are “plastic,” a term which, at least as used herein, means that MSCs are capable of exhibiting adaptability in response to one or more changes and / or alterations in their environment. As a non-limiting example, MSCs can adapt their phenotype and / or function in response to certain characteristics (e.g., the cytokine profile) of neighboring cells, including, for instance, tumor and / or cancer cells.

[0080] MSCs can be derived from multiple sources within the human body, including, for instance, bone marrow (also referred to as “BM-MSC” or “BM-MSCs”), adipose tissue (also referred to as “AT-MSC” or “AT-MSCs”), muscles, skin, the placenta, the femoral shaft, the iliac crest, umbilical cord blood, the umbilical cord itself, Wharton’s jelly, the endometrium, menstrual blood, dental pulp, periodontal ligaments, the gingiva, apical papilla, and dental follicles.

[0081] MSCs can play a role in various immune responses. For example, after injury, alarmins, endogenous molecules released from damaged cells, activate tissue-resident MSCs, which express PD-L1 and produce various immunoregulatory factors that modulate the cytokine milieu of the local environment. This can alter the phenotype and function of different immune cells. Additionally, MSCs can produce various immunosuppressive, pro-angiogenic, and / or trophic factors that provide one or more of the following effects: (1) inhibiting one or more detrimental immune responses, (2) attenuating ongoing inflammation, (3) inducing the generation of new blood vessels, (4) stimulating the proliferation and differentiation of tissue-specific, resident stem cells, and (5) enabling enhanced repair, healing, and / or regeneration of injured tissues.

[0082] MSCs can also affect the antigen-presenting properties of immune cells, including, for example, DCs, B cells, and macrophages. Additionally, MSCs can modulate the phagocytic ability of neutrophils and monocytes, change the polarization of macrophages, modify the cytotoxic properties of NK cells, and regulate the proliferation, activation and / or effector functions of CD4+ and CD8+ T cells.

[0083] Additionally, MSCs, via juxtracrine and / or paracrine signaling, can induce the generation and / or expansion of immunosuppressive Tregs and MDSCs, which results in alleviation of ongoing inflammation.

[0084] Juxtracrine signaling (also referred to as “contact-dependent signaling”) is a type of intercell signaling that requires close contact. Such signaling can occur when a ligand on one surface binds to a receptor on another adjacent surface. There are at least three different types of juxtracrine signaling, including, for instance (1) interaction between a membrane compound (c. ., lipid) of a first cell and a membrane protein of a second, nearby cell, (2) junctions between two nearby cells that permit passage of specific molecules, and (3) interaction between a membrane protein of a first cell and a biological compound in the extracellular matrix. Further, juxtracrine signaling can occur for specific growth factors and cytokines, including growth factors that play a role in the immune response.

[0085] Paracrine signaling is another type of intercell signaling in which a given cell produces one or more signals, thereby inducing a change in one or more nearby cells. Paracrine signaling can proceed via specific paracrine factors, which diffuse over the distance between the given cell and the one or more nearby cells. Thus, a cell engaging in paracrine signaling can produce, and excrete, the aforementioned paracrine factors into the extracellular matrix. Many paracrine factors bind to specific receptors (e.g., receptors in the TGF- family).

[0086] Regulation of MSCs can proceed via several pathways and biological factors, including, for example, various cytokines, transcription factors, and nucleic acids. For instance, transcription factors such as Runt-related transcription factor 2 (Runx2), SRY-related high-mobility group-box gene 9 (Sox9), peroxisome proliferation-activated receptor y (PPARy), various members of the helix-loop-helix family transcription factors (e.g., myoblast determination protein 1 (MyoD)), and various members of the GATA zinc finger transcription factor family e.g., GATA4, GATA6) can play a role in MSC differentiation.

[0087] As mentioned further herein, the interaction between pericytes, MSCs, tissue-resident stem cells, and immune cells is important for the generation of healing processes in injured tissues. Such cross-talk includes, for instance, various signaling pathways involved in (1) the activation, contraction, and / or migration of pericytes, (2) pericyte-to-MSC transitions, and (3) immunoregulatory, pro-angiogenic, and / or regenerative properties of MSCs that can be responsible for the attenuation of immune-cell driven inflammation and the enhanced regeneration of injured tissues. As a result, MSCs and / or products derived therefrom can be used for the prevention and targeted treatment of injured tissues, and / or the promotion of tissue repair, healing, and / or regeneration.

[0088] MSCs and MSC-derived products in tissue repair, healing, and / or regeneration

[0089] The therapeutic potential of MSCs and MSC-derived products can be seen with respect to MSC-dependent tissue repair, healing, and / or regeneration, as well as wound healing. Large numbers of chemokine receptors are expressed on the membrane of MSCs, enabling their rapid recruitment to the site of any injury or wound site (e.g., skin wounds). Alarmins and DAMPs, which are released from injured parenchymal cells and / or ECs, bind to alarmin / DAMPs-specific receptors on tissue-resident macrophages and induce the production of inflammatory cytokines and / or chemokines. These compounds then recruit MSCs from their respective niches or areas towards and / or into the injury or wound site. Upon migrating to the injury or wound site, MSCs can integrate into damaged tissues (e.g., skin tissues) and modulate the viability of injured parenchymal cells, induce differentiation of resident progenitor cells, and alter the phenotype and / or function of tissue-infiltrated immune cells.

[0090] Tissue repair, healing, and / or regeneration

[0091] Neutrophils

[0092] Neutrophils, in a time-dependent manner, contribute to tissue repair and / or regeneration via multiple mechanisms. Within minutes after tissue damage, neutrophils migrate to the site of injury and, as professional phagocytes, clear necrotic tissue and cellular debris by phagocytosis. After the elimination of microbial pathogens and cellular debris, neutrophils participate in the restoration of tissue homeostasis. By removing cellular remnants, neutrophils prevent DAMPs- driven recruitment of inflammatory cells in healing tissues. Additionally, neutrophils release neutrophil extracellular traps (NETs) that capture monocyte and lymphocyte-attracting chemokines and express chemokine receptors that can function as scavengers to reduce the availability of pro-inflammatory chemokines for the recruitment of additional circulating leucocytes. Moreover, neutrophils produce matrix metalloproteinase (MMP)-9 which is capable of degrading DAMPs, particularly HMGB1 and HSP90, further dampening the recruitment of leucocytes into the site of injury.

[0093] A sub-population of CXCR4+VEGFR+CD49d+ neutrophils, which is abundantly present in the injured tissues, release large amounts of pro-angiogenic factors, including, for instance, VEGF, TGF-P, and IL-6, which stimulate neo-angiogenesis and promote tissue repair. These pro- angiogenic neutrophils generate new blood vessels and enable the better delivery of oxygen, growth factors, and / or trophic factors in ischemic regions, facilitating tissue regrowth and regeneration.

[0094] During the healing phase of tissue repair, the majority of neutrophils acquire the immunosuppressive N2 phenotype. Anti-inflammatory N2 neutrophils produce immunosuppressive cytokines (e.g., IL- 10 and TGF-P) and release microvesicles containing annexin Al, which induces macrophage phenotype switching toward an immunosuppressive and “pro-repair” M2 phenotype. Additionally, after the removal of cellular remnants, neutrophils undergo apoptosis, expose phosphatidyl-serine on their surfaces, and become phagocyted by resident macrophages. The phagocytosis of apoptotic neutrophils further induces macrophage phenotype switching towards the anti-inflammatory M2 phenotype. M2 macrophages, in turn, release various pro-resolving mediators, contributing to the enhanced repair of injured tissue.

[0095] MSCs and / or MSC-derived products (e.g., one or more of the MSCs and / or MSC- containing compositions and / or formulations described herein) modulate the phenotype and / or function of neutrophils in all phases of tissue repair and regeneration. During an initial phase of tissue healing, resident MSCs, in an IL-8 and macrophage migration inhibitory factor (MIF)- dependent manner, enhance the phagocytic ability of neutrophils, contributing to the efficient removal of necrotic tissue and cellular debris. Additionally, MSCs, via up-regulation of the extracellular superoxide dismutase (SOD3), prevent neutrophil death and enhance neutrophildependent elimination of microbial pathogens and cellular remnants. On the contrary, during the resolution phase of tissue repair, MSCs reduce the presence of pro-inflammatory N1 neutrophils and induce their conversion in anti-inflammatory N2 cells. Intercellular adhesion molecule 1 (IC AM- Independent engulfment of neutrophils is mainly responsible for the MSC-based elimination of N1 neutrophils. Additionally, MSCs produce TSG-6, which reduces the production of reactive oxygen species (ROS) and induces the enhanced expression of IL- 10 in neutrophils, favoring their polarization in an immunosuppressive N2 phenotype. The inhibition of the extracellular signal regulated kinase (ERK) pathway may be responsible for the MSC-dependent generation of an N2 phenotype in neutrophils. For instance, by using dextran sodium sulphate (DSS)-induced colitis, murine models of ulcerative colitis in which N1 neutrophils play an important role in disease progression and N2 neutrophils in disease regression, MSCs can induce the polarization of N1 neutrophils in N2 immunosuppressive cells. MSCs perform such induction by modulating modulating ERK phosphorylation, resulting in enhanced mucosal healing of DSS- injured colons. Indeed, significantly reduced numbers of ICAM-1, FAS, and CCL3 -expressing N1 neutrophils, as well as increased numbers of CCL2 and CXCR4-expressing N2 neutrophils may be present in colon tissue samples of DSS-treated mice that received MSCs. Additionally, MSCs and / or MSC-derived products (e.g., one or more of the MSCs and / or MSC-containing compositions and / or formulations described herein) can induce an increased production of VEGF in neutrophils, contributing to the better neovascularization of healing tissues. Similarly, pro- angiogenic neutrophils promote the expression of, for instance, PDGF, angiopoietin-1, IL-6, and HGF in MSCs, which can enhance their pro-angiogenic properties. Accordingly, crosstalk between pro-angiogenic neutrophils and MSCs can result in the enhanced proliferation of endothelial cells and vascular regeneration in healing tissues.

[0096] Macrophages

[0097] Macrophages are also critically involved in normal tissue homeostasis and exhibit an important regulatory activity at all stages of repair and regeneration of damaged tissues. For instance, immediately after injury, tissue-resident macrophages act as scavenger cells which phagocyte cellular debris, pathogens, apoptotic neutrophils, and / or dying cells. DAMPs and pathogen associated molecular patterns (PAMPs) activate toll-like receptors (TLRs) in macrophages, which acquire a pro-inflammatory Ml phenotype and orchestrate the initial cellular response following injury. Ml macrophages secrete, among other compounds, CCL2, MMP-12, nitric oxide (NO), inflammatory cytokines (e.g., TNF-a, IL-1 , IL-12) and various other inflammatory chemokines, enabling the increased recruitment of circulating leucocytes to the site of injury. In the initial phase of tissue healing, macrophages act as phagocytes and clear apoptotic cells and cellular debris. After the early inflammatory phase subsides, the predominant macrophage population assumes a wound healing M2 phenotype characterized by the low expression of Ly6C and CCR2 and the high expression of CX3CR1, CD206, and CD163. M2 macrophages, through the production of numerous growth factors (e.g., PDGF, TGF-01, insulin growth factor (IGF)-l, and VEGF-a), promote cellular proliferation, neo-angiogenesis and, in the case of severe injury, activation and differentiation of tissue resident stem cells and progenitor cells. M2 macrophages also produce soluble mediators (e.g., IL-13, TGF-01) that induce the differentiation of fibroblasts into myofibroblasts which, through the increased synthesis of extracellular matrix (ECM) components, enable wound contraction and closure. In order to prevent the excessive deposition of collagen and ECM proteins, at the final phase of tissue healing, the majority of the M2 macrophages obtain an anti-inflammatory phenotype, characterized by an increased capacity for the production of the immunosuppressive cytokine IL-10. Additionally, anti-inflammatory M2 macrophages secrete ECM-degrading MMPs (e.g., MMP-2, MMP-9, MMP-13) and prevent fibrosis. These anti-inflammatory macrophages express program death ligand (PD-L)l and PD-L2, which play major roles in suppressing other pro-inflammatory and pro-fibrotic immune cells, enabling alleviation of on-going inflammation and fibrosis.

[0098] Since Ml and M2 macrophages play critical roles at different stages of tissue repair and regeneration, MSCs and MSC-derived products (e.g., one or more of the MSCs and / or MSC- containing compositions and / or formulations described herein) can support the phagocytic properties of Ml macrophages in the initial phase of tissue healing, while, at the final stage of tissue repair, promote the generation and expansion of anti-inflammatory M2 macrophages.

[0099] At the initial stage of tissue injury, microbial invasion activates tissue resident MSCs. After sensing pathogens in inflamed tissues, MSCs can produce monocyte-attracting chemokines (e.g., CCL2, CCL3, CXCL2, CCL12), which promote the egression of monocytes from the bone marrow and enable their recruitment into the site of injury and inflammation. After the phagocytosis of microbial pathogens and apoptotic cells, macrophages obtain a pro-inflammatory Ml phenotype and produce MSC-attracting chemokines and cytokines (e.g., CCL5, CCL2, CXCL12, IL-8). These inflammatory mediators activate c-JunNH2-terminal kinase (JNK)-dependent signaling pathway in MSCs and induce their conversion in inflammatory, IFN-y- and TNF-a-producing MSC1 cells, which, together with N1 neutrophils and Ml macrophages, participate in the elimination of microbial pathogens and cellular debris.

[0100] During the healing phase of tissue repair, MSCs, in a TSG-6, PGE2, and IDO-dependent manner, induce the conversion of TNF-a and IL-ip producing inflammatory Ml macrophages into immunosuppressive, IL- 10 producing M2 cells that attenuate on -going inflammation and promote tissue regeneration. MSC-derived TSG-6 can interact with CD44 on macrophages to decrease TLR2 / NFK-B signaling and consequently alleviate the secretion of inflammatory mediators (e.g., NO, TNF-a, and IL-ip). Ml macrophage-sourced IL-ip is considered as an important regulator of persistent inflammation and fibrosis. Importantly, IL-ip, released from activated macrophages, can induce the generation of the immunosuppressive phenotype in tissue resident MSCs. IL- 1 P- primed MSCs can increase the production of anti-inflammatory cytokines (e.g., IL-10, IL-IRa). MSC-derived IL-IRa, a naturally occurring inhibitor of IL-ip, has an important role in the MSC- based suppression of Ml macrophages-driven inflammation. When IL-IRa binds to the IL-1 receptor (IL-1R), the interaction between inflammatory IL-1 and IL-1R is prevented. The apoptosis of parenchymal cells, synthesis and release of matrix-degrading enzymes and chemokines, as well as other inflammatory events, which are initiated by IL-LIL-IR interaction, can be inhibited by MSC-sourced IL-IRa.

[0101] In addition to TSG-6 and IL-IRa, MSC-sourced IL-6 and PGE2 may also have the ability to transform inflammatory, TNF-a and IL-1 -producing Ml macrophages into IL-10-secreting, anti-inflammatory M2 cells. MSC-derived IL-6 and PGE2 can bind to IL-6R and EP2 and EP4 receptors on macrophages, which can promote the production of immunosuppressive IL-10. This, in turn, in autocrine and paracrine manners, favors the generation of M2 macrophages that participate in tissue repair and regeneration.

[0102] The interplay between M2 macrophages and MSCs plays an essential role in the vascular regeneration of injured tissues. Since MSCs represent a valuable source of pro-angiogenic VEGF and angiopoentin-1, the transplantation of autologous MSCs and / or MSC-derived products (e.g., one or more of the MSCs and / or MSC-containing compositions and / or formulations described herein) may repair corneal wounds by promoting local tissue neo-vascularization. Macrophage depletion can abrogate MSC-based beneficial effects, while the administration of peritoneal macrophages can restore MSC-driven neovascularization in macrophage-depleted animals. This suggests that cooperation between MSCs and / or MSC-derived products on one hand, and macrophages on the other hand, is important for successful vascular regeneration. MSCs and / or MSC-derived products can promote the growth of endothelial cells and induce vascular sprouting in a VEGF-dependent manner, while M2 macrophage-derived IL-8 can induce the increased expression of VEGFR on endothelial cells, enhancing the pro-angiogenic effects elicited by MSC- sourced VEGF. The generation of smooth muscle cells (SMCs) and pericytes and their recruitment in regenerative vessels are necessary for the development of the mature and functional vasculature. The crosstalk between M2 macrophages and MSCs and / or MSC-derived products can regulate the differentiation of MSCs in SMCs and pericytes. M2 macrophage-sourced TGF- and prostaglandin F2a are considered as essential paracrine signaling factors for the successful differentiation of MSCs in SMCs, while M2 macrophage-derived PDGF-P is necessary for the optimal differentiation of MSCs in functional pericytes. Crosstalk between MSCs and macrophages can be important for the successful engraftment of transplanted MSCs. The survival of exogenously injected MSCs can be dependent on the phenotype and / or function of tissue resident macrophages. At least in murine models of myocardial infarction and spinal cord injury, anti-inflammatory M2 macrophages can provide a more favorable environment for the engraftment of MSCs than pro-inflammatory Ml macrophages. The conversion of pro-inflammatory Ml macrophages to an anti-inflammatory M2 phenotype appears to be critical for the long-term survival of MSCs in healing tissues, suggesting that a mutually beneficial feedback loop exists between M2 macrophages and MSCs, and that the interplay between these cells may be important for efficient tissue regeneration.

[0103] NK cells, innate lymphoid cells, and DCs

[0104] NK cells are innate immune cells which, due to their potent cytotoxic properties, efficiently eliminate infected and stressed cells at the initial phase of tissue injury and inflammation. However, at the healing phase of tissue repair, under the influence of M2 macrophage-derived immunosuppressive IL-10 and TGF-P, the majority of NK cells acquire an anti-inflammatory, regulatory NKreg phenotype, and participate in tissue repair and regeneration through the secretion of immunosuppressive IL- 10.

[0105] MSCs can enhance NK cell cytotoxicity at the induction phase of tissue healing, while, at later time points, induce regulatory phenotype or senescence in inflammatory NK cells. At the early stages of tissue injury, upon activation of TLR-3, TLR-7, and / or TLR-9 by viral antigens, MSCs can obtain a pro-inflammatory (MSC1) phenotype and secrete anti-viral cytokines (e.g., IFN-a, IFN-P) that up-regulate the cytotoxic potential of NK cells. On the contrary, during the resolution phase of tissue injury and inflammation, MSCs, in a PGE2- and IDO-dependent manner, can induce the polarization of inflammatory NK cells into IL-10-producing, anti-inflammatory NKregs. Additionally, MSCs can produce TGF-P and IL-6, which can act synergistically to induce senescence of inflammatory NK cells. Importantly, MSC-generated senescent NK cells can exert feedback on MSCs. Senescent NK cells can induce a highly significant increase in VEGF gene expression in MSCs which, in turn, in a VEGF-dependent manner, promotes endothelial cell proliferation and improves vascular regeneration in healing tissues.

[0106] Innate lymphoid cells (ILCs), a recently discovered heterogeneous group of hematopoietic cells of the innate immune system, are abundant at the mucosal barriers, where they serve as the first responders to tissue injury. GAT A-3 -expressing type 2 ILCs (ILC2) and RORytT-expressing type 3 ILCs (ILC3) respond rapidly to alarmins (e.g., IL-25, IL-33) released from injured epithelial cells and participate in tissue repair at the barrier surfaces of the skin, airways, and intestine. ILC2 produces Amphiregulin (AREG), a protein that promotes repair of injured lung epithelial cells. By producing PDGF and IL-7, MSCs can induce differentiation of common lymphoid progenitors (CLPs) into AREG-expressing ILC2 cells. MSC-primed ILC2 cells, in an AREG-dependent manner, can maintain the integrity of the epithelial barrier in the lungs and enhance repair and regeneration of injured lung epithelial cells.

[0107] RORyt+ ILC3 cells produce IL-22, which protects the integrity of the epithelial cell barrier in the lungs and the gut. MSCs, in a juxtracrine manner (e.g., through the activation of aryl hydrocarbon receptor (AhR)) and in a paracrine manner (e.g., through the secretion of IL-7) induce the proliferation and activation of AhR and IL-7R-expressing ILC3. MSC-primed ILC3 may have an increased capacity for IL -22 secretion. IL-22, derived from MSC-activated ILC3, can increase the synthesis of anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL) and proteins that regulate cell cycle (e.g., c-Myc, cyclin DI, CDK4) in epithelial cells. Accordingly, MSC-primed ILC3, in an IL-22- dependent manner, can contribute to wound healing and tissue homeostasis in the lungs and intestines by enhancing the viability and proliferation of epithelial cells.

[0108] At the healing phase of tissue repair, MSC-derived immunomodulatory factors (e.g., IL-6, PGE2, IL-10, galectin-3) can induce the generation of tolerogenic phenotype in DCs. Tolerogenic DCs are characterized by the reduced expression of co-stimulatory molecules (e.g., CD80, CD86, CD40), down-regulated production of inflammatory cytokines (e.g., IL-ip, IL-12, TNF-a), and increased expression of PDL-1 and PDL-2. Importantly, tolerogenic DCs produce antiinflammatory cytokines IL- 10 and IL-35 and, in an IDO-dependent manner, induce the differentiation of naive CD4+T cells in immunosuppressive Tregs. This induction contributes to tissue repair and regeneration.

[0109] Tregs

[0110] Cells of adaptive immunity, particularly Tregs, also participate in tissue healing. CD3+CD4+CD127lowCD25hlghFoxp3+ Tregs, both thymus-derived (tTregs) and peripherally derived (pTregs), can mediate tissue repair by dampening inflammation. This can occur via modulation of the phenotype and / or function of N1 neutrophils, Ml macrophages, cytotoxic NK cells, and / or pro-inflammatory DCs. Tregs, in TGF-P- and TL-10-dependent manners, can induce the apoptosis ofNl neutrophils. Additionally, Treg-sourced immunosuppressive IL-10, IL-35, and TGF-P can modulate neutrophil and macrophage phenotype and / or function by promoting their polarization in IL-10, TGF-P, IDO-producing N2 and M2 anti-inflammatory cells. In a contactdependent manner, through the expression of CTLA-4 and LAG-3, Tregs can induce the generation of a tolerogenic phenotype in DCs which, in turn, in an IDO-dependent manner, can promote the expansion of Tregs and create a “positive healing loop” in injured tissues. In addition to their immunosuppressive properties, Tregs can mediate tissue repair by synthesizing “prorepair” molecules, such as AREG and keratinocyte growth factor (KGF), that directly promote tissue regeneration. KGF secreted by activated Tregs can promote alveolar epithelial repair, while Treg-derived AREG, an epidermal growth factor receptor (EGFR) ligand, can induce mitogenic and cell differentiation signals, enabling repair of injured muscles, lungs, and / or colons by promoting differentiation of tissue resident stem cells and progenitor cells. Additionally, Tregs may promote tissue regeneration by inducing the proliferation of endothelial and parenchymal cells. Treg-sourced AREG, CCL24, and growth arrest specific 6 (GAS6) can also regulate neonatal heart regeneration by promoting the proliferation of neonatal cardiomyocytes. Treg-derived AREG and IL- 10 can induce the proliferation of endothelial cells and may be responsible for Treg- mediated revascularization and the regeneration of ischemic tissues in diabetic patients.

[0111] Efficient tissue repair and / or regeneration may be dependent on the crosstalk between MSCs and Tregs. MSCs, in an IDO-dependent manner, can induce the degradation of tryptophan (TRP) and the generation of immunosuppressive kynurenine (KYN). KYN promotes the expression of Treg lineage-defining transcription factor (forkhead box P3-FoxP3) in CD4+T cells, enabling the generation of immunosuppressive CD4+FoxP3+Tregs. The IDO-mediated degradation of TRP yields a series of KYN catabolites that can act as ligands for AhR. The binding of KYN catabolites to AhR can induce conformational changes in AhR that promote its nuclear translocation. In the nucleus, AhR can itself induce the enhanced transcription of target genes, including FoxP3. Accordingly, MSC-sourced IDO, through the activation of the KYN / AhR axis, can result in the increased generation of FoxP3+Tregs, contributing to the creation of an immunosuppressive microenvironment, which can enable efficient tissue healing.

[0112] Additionally, during the resolution of tissue injury, MSC-derived IDO can prevent the trans-differentiation of IL-10 and IL-35-producing immunosuppressive Tregs in IL-17-producing inflammatory Thl7 cells. During initial TCR-mediated activation of resting Tregs, signals via the protein kinase B (PKB / Akt) and mammalian target of rapamycin (mTOR) pathways destabilize the immunoregulatory phenotype of Tregs and cause their reprogramming into a pro-inflammatory helper-like phenotype (“ex-Tregs”). This phenotype is characterized by the enhanced production of inflammatory cytokine IL- 17. Low levels of TRP in the local microenvironment activate stressresponse pathways, including the activation of control nonderepressible 2 (GCN2) kinase, which suppresses Akt / mT0R2 signaling. MSC-sourced IDO can induce low TRP levels, enabling the activation of GCN2 kinase, which inhibits Akt / mT0RC2 signaling in Tregs, preventing their conversion in inflammatory IL-17-producing Thl7 cells. Additionally, MSC-derived IDO and TGF-P may act synergistically to induce conversion of inflammatory Th 17 cells in immunosuppressive Tregs. Since Th 17 cells can activate pro-fibrogenic hepatic stellate cells, the IDO-dependent suppression of liver Thl7 cells may contribute to the attenuation of fibrosis in MSC-treated livers.

[0113] In addition to IDO, MSC-sourced IL-6, PGE2, NO, TGF-P, and IL-10 may be responsible for the MSC-dependent expansion of Tregs in healing tissues. MSC-sourced NO and PGE2 can significantly increase the expression of CD62L and CCR7 in Tregs, which may enable their increased migration into injured tissues. Additionally, MSCs, in an IL-6, TGF-P, and IL-10- dependent manner, can induce the generation of M2 macrophages. These macrophages, in turn, in an IL-10 and CCL18-dependent manner, recruit Tregs to inflamed tissues, contributing to the creation of an immunosuppressive and “pro-healing” microenvironment in injured tissues.

[0114] Tregs can also enhance the survival and / or engraftment of MSCs in ischemic tissues, indicating that the crosstalk between MSCs and Tregs may be a bidirectional process that enables efficient tissue repair. Tregs may improve pro-angionic properties of MSCs by increasing their capacity for VEGF production. Indeed, certain animal models (e.g., ischemic hearts of Yorkshire pigs) show a significantly increased number of newly generated endothelial cells after administration of both Tregs and MSCs, compared to experimental animals that were transplanted with MSCs only. These newly generated cells may be associated with improved myocardial function, suggesting that Tregs improve the pro-angiogenic properties and therapeutic potential of MSCs. Wound healing

[0115] Wound healing response processes are especially relevant for tumors and associated cancers, since tumors are wounds that never fully heal. MSCs can be recruited to tumor sites by wound-associated chemokines and inflammatory cytokines produced by tumor-associated macrophages and neutrophils. Within the tumor microenvironment (“TME”), MSCs are constantly exposed to growth factors and / or cytokines released by tumor-infiltrating immune cells, endothelial cells, and / or tumor cells. Although MSCs have some pro-tumorigenic potential, there is no indication that MSCs are natively or constitutively immunosuppressive cells. Rather, MSCs act as a double-edged sword with respect to anti-tumor immunity. As plastic cells, MSCs may adopt the phenotype and / or function of various immune system cells, depending on the influence of one or more biological factors to which they are exposed. Thus, MSCs may obtain either pro- inflammatory (e.g., MSC1) or anti-inflammatory (e.g, MSC2) phenotypes, depending on the local tissue concentration of various inflammatory cytokines, such as, for instance, TNF-a and IFN-y.

[0116] Specifically, when MSCs engraft in a specific tissue that has low levels of TNF-a and IFN- y, they obtain a pro-inflammatory MSC1 phenotype and secrete a large number of inflammatory factors (e.g., reactive oxygen species (ROS), IL-10, interferon alpha and beta (IFN-a, IFN-0), TNF-a, and IFN-y). These factors can enhance the phagocytic properties of neutrophils and macrophages, as well as enhancing the cytotoxicity of CTLs and NK cells. By contrast, in at least one embodiment, when MSCs are exposed to high levels of inflammatory cytokines (e.g., TNF-a, IFN-y), they acquire an immunosuppressive MSC2 phenotype characterized by, for instance, the increased production of anti-inflammatory factors (e.g., TGF-0, IL-10, PGE2, NO, IDO, IL-IRa). These anti-inflammatory factors can suppress the effector function of inflammatory immune cells and attenuate on-going inflammation. Additionally, TNF-a and fFN-y-primed MSC2 express and secrete PD-L1 and PD-L2, which suppress the proliferation of TNF-a and IFN-y-producing T cells and promote the generation and / or expansion of immunosuppressive Tregs.

[0117] Thus, within the TME, MSCs can regulate the viability, growth, and / or invasiveness of malignant cells. MSCs can also modulate the phenotype and / or function of tumor-infdtrated immune cells. Additionally, MSC-dependent biological effects in the TME can be due, at least in part, to MSC-sourced exosomes (“MSC-Exos”), which are extracellular vesicles that, due to their lipid envelope and nano-sized dimensions, easily bypass all biological barriers and deliver their cargo directly to one or more target cells. MSC-Exos can contain a variety of biological compounds, including one or more proteins, factors, cytokines, microRNAs, messenger RNAs, DNAs, and the like.

[0118] Molecular mechanisms responsible for changing the phenotype and / or function of pericytes in injured tissues

[0119] MSCs can play a role in various molecular mechanisms that are responsible for changes in pericytes’ phenotype and / or function in injured tissues.

[0120] For example, DAMPs and alarmins, including, for instance, high-mobility group box 1 (HMGB1), heat shock proteins (HSPs), SI 00 proteins, uric acid, adenosine triphosphate (ATP), and fragments of DNA and / or RNA, which can be released from injured epithelial, parenchymal, and ECs, act as danger signals. These signals can alert tissue-resident stem cells and / or immune cells to cellular stress, tissue damage, and / or tissue injury. DAMPs and alarmins can bind directly to pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs)), which are expressed on the membrane of perivascular cells (e.g., pericytes) and / or immune cells. Such binding can trigger one or more intracellular signaling cascades that lead to the initiation of repair, healing, and / or regeneration processes in injured tissues.

[0121] Specifically, DAMPs-dependent activation of TLRs and NLRs can initiate the phosphorylation and consequent activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). This, in turn, can result in the phosphorylation of, for instance, c-Jun, ATF-2, and CREB transcription factors, which can activate transcriptional factors (e.g., nuclear factor-kappa B (NF-KB), c-Jun, ATF-2, CREB) that translocate into the nucleus, bind to DNA, and initiate the transcription and / or translation of various inflammatory cytokines. Specific non-limiting examples of these cytokines include, for instance, tumor necrosis factor alpha (TNF-a), interleukin (IL)-ip, IL-6, IL-18, and one or more chemokines (e.g., C-C motif chemokine ligand (CCL)-2, CCL3, CCL5, C-X-C motif chemokine ligand (CXC)-8, CXC10). Inflammatory cytokines and chemokines can induce conformational changes in G-protein-coupled receptors (GPCRs), voltage-gated calcium channels, thereby promoting intracellular calcium release from the sarcoplasmic reticulum.

[0122] Additionally, TNF-a, IL-p, and / or IL-18 can induce the activation of several signaling pathways that can enhance the synthesis of contractile proteins in pericytes. For instance, the RhoA-ROCK (Rho-associated protein kinase) cascade activates myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1), which can increase actomyosin contractility and pericyte constriction. Protein kinase C (PKC) can also be activated in pericytes in response to inflammatory cytokines and DAMPs PRRs-mediated signaling. PKC can phosphorylate MLC and MYPT1, and increase pericyte contractility. Further, ATP-dependent activation of purinergic receptors can enhance calcium influx in pericytes. An increase in calcium levels can then trigger the activation of myosin light chain kinase (MLCK), which phosphorylates MLC and can enable interactions between actin and myosin filaments, also resulting in enhanced pericyte contraction. Pericyte contraction leads to the narrowing of the blood vessel lumen, resulting in vasoconstriction, thereby reducing blood flow to the injured area, limiting bleeding, and / or preventing excessive fluid leakage into the surrounding tissue. By constricting blood vessels, pericytes can therefore restrict the entry of immune cells and / or inflammatory mediators into the injured tissue, thereby modulating the inflammatory cascade and preventing excessive inflammation. Additionally, constricted blood vessels can provide a favorable environment for platelet aggregation and clot formation, promoting hemostasis and preventing further bleeding.

[0123] In addition to their effects on pericytes’ contraction, immune cell-derived cytokines and / or chemokines can play a key role in promoting (1) pericyte motility, (2) detachment from blood vessels, and / or (3) the pericyte-to-MSC transition. Transforming growth factor beta (TGF- ) and TNF-a can downregulate the expression of adhesion molecules (e.g., integrins) on pericytes, promoting their detachment from the blood vessel wall. TNF-a and / or IL-ip can induce increased synthesis and release of matrix metalloproteinases (MMPs) from injured ECs. MMPs can degrade the extracellular matrix components that anchor pericytes to ECs, facilitating detachment and / or migration of the pericytes away from the blood vessels. Various chemokines (e.g., CXCL12, CXCL8, CCL2, and platelet-derived growth factor (PDGF)) can act as chemoattractants that bind to their respective receptors on pericytes (e.g., CXCR4, CCR1, CCR2, PDGFRP) and activate MAPK- and ERK-driven intracellular signaling pathways that promote cytoskeletal rearrangement and / or cell motility.

[0124] Further, pericytes primed by immune cell-derived cytokines and / or chemokines can differentiate into MSCs in injured tissues. This differentiation process, known as the pericyte-to- MSC transition, or pericyte plasticity, can be important for the repair, healing, and / or regeneration of injured tissues (e.g., the scarless healing of injured tissues).

[0125] The pericyte-to-MSC transition and initial steps in tissue repair, healing, and / or regeneration

[0126] The pericyte-to-MSC transition is influenced by several cytokines and growth factors (e.g., TGF-P, IL-ip, IL-10, IL-6, Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), fibroblast growth factor (FGF)-2)) which play important roles in regulating the differentiation, proliferation, migration, and / or immunomodulatory properties of pericytes during their transition to MSCs.

[0127] In particular, PDGF and FGF2 can be involved in various cellular processes, including cell proliferation and / or differentiation. IL-6 and VEGF are multifunctional cytokines that may be responsible for neo-angiogenesis in injured tissues. PDGF, FGF2, IL-6, and VEGF can promote the proliferation, migration, and / or differentiation of pericytes, facilitating their transition into MSCs. FGF2 and IL-6 may induce the up-regulation of one or more MSC markers (e.g., CD73 (ecto-5'-nucleotidase), CD90 (Thy-1), CD105 (endoglin), and CD44 (hyaluronan receptor)) in pericytes. Up-regulation of these markers may be associated with pericytes acquiring specific MSC characteristics, such as, for example, multipotency and / or tissue repair capabilities. IL- 10 is an anti-inflammatory cytokine that has immunosuppressive effects. Specifically, IL- 10 can influence the pericyte-to-MSC transition by promoting the acquisition of immunomodulatory properties in pericytes. IL-10 can further stimulate the up-regulation of immunomodulatory molecules and enhance the immunosuppressive functions of MSCs derived from pericytes. In addition to IL-10, pro-inflammatory cytokines (e.g., IL-ip and IL-6) may also be able to enhance the immunomodulatory properties of pericytes during their transition to MSCs, enabling MSC- dependent attenuation of ongoing inflammation in injured tissues.

[0128] Additionally, TGF-P, a multifunctional cytokine involved in cell proliferation, differentiation, migration, and / or extracellular matrix (ECM) production, may be important for the pericyte-to-MSC transition in injured tissues. Macrophages and DCs can massively produce TGF- P in response to injury-related signals. Inflammatory cytokines (e.g., TNF-a, IL-P), oxidative stress, and / or hypoxia can activate the NF-kB and / or hypoxia-inducible factor (HIF) pathways, which stimulate TGF-P secretion. Epigenetic modifications, such as, for example, DNA methylation and histone modifications, which occur in injured tissues, can significantly increase TGF-P expression in tissue-resident macrophages and DCs.

[0129] TGF-P can be secreted in an inactive, latent form, bound to latency-associated peptides (LAPs). MMPs, plasmin, and thrombospondin- 1 can cleave LAPs, releasing the active form of TGF- . Injured tissues undergo ECM remodeling, which can induce the release of TGF-P from its latent form, contributing to increased TGF-P availability in the injured tissue. Once secreted, TGF- P may act in an autocrine and paracrine manner to further stimulate its own production and secretion. TGF-P may also bind to its receptors and activate, for instance, Smad and non-Smad signaling pathways, which are both involved in the pericyte-to-MSC transition. In the Smad- dependent pathway, interaction between TGF-P and its type II receptor (TGFBR2) can activate activin receptor-like kinase 5 (ALK5), which phosphorylates and activates the type I receptor (TGFBRl)ZALK receptor complex. The phosphorylated TGFBR1 / ALK5 complex can then phosphorylate receptor-regulated Smads (R-Smads), specifically Smad2 and Smad3. Phosphorylated R-Smads can form complexes with the common mediator Smad (“Co-Smad”), Smad4. The Smad complex may then translocate into the nucleus, where, along with other transcriptional regulators, the complex binds to specific DNA sequences (e.g., Smad-binding elements) in the promoter regions of target genes, leading to the transcriptional regulation of those genes. In addition to the Smad-dependent pathway, TGF-p / TGF-PRs interactions can elicit activation of non-Smad signaling, including the activation of phosphoinositide 3 -kinase (PI3K), protein kinase B (Akt), TAK1, MAPK kinases (e.g., ERK, INK, p38 MAPK), and Rho-like GTPases (e.g., RhoA, Rael, Cdc42). These proteins can be involved in cytoskeletal rearrangement, cell migration, and / or ECM remodeling. One or more of the TGF-P-dependent signaling pathways may also work together to mediate the diverse cellular responses in pericytes during their transition to MSCs. While non-Smad pathways can regulate pericytes’ interaction with neighboring cells (e.g., detachment from blood vessels, migration, interaction with ECs and / or ECM), the Smad- dependent pathway may primarily affect genes that regulate the sternness, pluripotency, and / or secretory profde of the newly formed MSCs.

[0130] Turning now to Figure 1, various biological compounds that play a role in influencing the pericyte-to-MSC transition 102 are shown. First, as shown in block 104, PDGF, FGF2, IL-6, and / or VEGF can promote the proliferation, migration, and / or differentiation of pericytes, shown at block 106. This can facilitate these pericytes’ transition to MSCs. Second, as shown in block 108, FGF2 and / or IL-6 may produce one or more of the following effects, as shown in block 110: (1) up-regulation of one or more MSC markers (e.g., CD73 (ecto- 5 '-nucleotidase), CD90 (Thy-1), CD 105 (endoglin), and CD44 (hyaluronan receptor)) in pericytes, and (2) acquisition of one or more MSC-specific characteristics (e.g., multipotency, tissue repair capabilities). Additionally, as shown in block 112, one or more anti-inflammatory cytokines (e.g., IL- 10) can produce one or more of the following effects, as shown in block 114: (1) promoting the acquisition of immunomodulatory properties in pericytes, (2) stimulating the up-regulation of immunomodulatory molecules, and (3) enhancing the immunosuppressive functions of MSCs derived from pericytes. Further, as shown in block 116, one or more pro-inflammatory cytokines (e.g., IL-ip, IL-6) can enhance the immunomodulatory properties of pericytes during their transition to MSCs, as shown in block 118. These immunomodulatory properties may then enable MSC-dependent attenuation of ongoing inflammation in injured tissues. Finally, as shown in block 120, cytokines such as TGF-P can activate Smad and non-Smad signaling pathways, as shown in block 122. For instance, in the Smad-dependent pathway, phosphorylated receptor-regulated Smads (R-Smads) (e.g., Smad2 and Smad3) can form complexes with Smad4. Such complexes may then translocate into the nucleus, where, along with other transcriptional regulators, the complex binds to specific DNA sequences (e.g., Smad-binding elements) in the promoter regions of target genes, leading to the transcriptional regulation of those genes. As a further example, in the non-Smad signaling pathway, various proteins (e.g., phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), TAK1, MAPK kinases (e.g., ERK, JNK, p38 MAPK), and Rho-like GTPases (e.g., RhoA, Rael, Cdc42)) can be involved in cytoskeletal rearrangement, cell migration, and / or ECM remodeling. Thus, non-Smad pathways can regulate pericytes’ interaction with neighboring cells (e.g., detachment from blood vessels, migration, interaction with ECs and / or ECM), while Smad- dependent pathways can affect genes that regulate the sternness, pluripotency, and / or secretory profile of newly formed MSCs.

[0131] During the pericyte-to-MSC transition, pericytes generally undergo significant changes in gene expression as they acquire the characteristics of MSCs. Common pericyte-related markers, such as, for example, neuron-glial antigen 2 (NG2), platelet-derived growth factor receptor-beta (PDGFR-P), and regulator of G protein signaling 5 (RGS5), can play important roles in pericyte functions (e.g., attachment to blood vessel walls, proliferation, survival, angiogenesis, ECM remodeling, cell signaling). The markers can be reduced in expression as pericytes shift towards MSCs. These changes in gene expression can be responsible for the altered pericytes’ morphology, phenotype, and / or function. As pericytes transition into MSCs, their spindle-like, elongated shape may become more rounded or fibroblast-like. This change in cell shape can reflect the shift from a contractile and supportive role around blood vessels into a more migratory and tissue-repairing phenotype. During the transition, pericytes may undergo an increase in cell size, becoming larger and more similar in size to MSCs. The change in cell size is associated with the acquisition of a more proliferative and multipotent phenotype. Pericytes undergoing the transition can exhibit changes in cytoplasmic characteristics, including, for instance, increased cytoplasmic volume and / or alterations in organelle distribution. For example, their cytoplasms can contain more mitochondria, endoplasmic reticulum, and / or Golgi apparatus, reflecting the increased metabolic and secretory activities associated with the MSC phenotype. During the transition, pericytes can exhibit increased migratory behavior due to changes in cytoskeletal organization, including the reorganization of actin filaments, reduced expression of integrins, and / or decreased production of ECM components (e.g., collagen and fibronectin). These changes therefore reflect the shift in pericyte function from ECM support to a more dynamic, regenerative role.

[0132] Significantly enhanced expression of various transcriptional factors (e.g., Sox9, Oct4, Nanog, and Klf4) are observed in pericytes during their transition to MSCs. These transcriptional factors can be associated with the sternness and / or pluripotency of MSCs, and / or the increased expression of genes that encode growth factors capable of inducing neo-angiogenesis and / or differentiation of tissue-resident stem cells (e.g., vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), fibroblast growth factor 2 (FGF2), TGF-P). Up-regulation of these factors can crucially contribute to the generation of potent regenerative properties in newly formed MSCs. Additionally, during their transition to MSCs, pericytes can exhibit changes in the expression of genes involved in ECM remodeling. These changes can include the up-regulation of genes that are responsible for ECM degradation (e.g., MMP2, MMP9) and ECM synthesis (e.g., collagen (COL)lAl, C0L3A1, fibronectin 1 (FN1)). Additionally, the pericyte-to-MSC transition generally involves up-regulation of genes involved in TGF-P (e.g., TGFBR2, SMAD2-7: ID1-3, CDKN1A), Wnt (Wnt3A, Wnt5A, WntlOB, AXIN2, TCF7, FZD4), and Notch signaling (e.g., NOTCH!, NOTCH3, JAG1 -2, DLL1 , DLL4, HES1, HES5, HEY1-2), which regulate the selfrenewal, osteogenic, chondrogenic, and / or adipogenic differentiation of MSCs. Accordingly, pericytes undergoing the transition to MSCs are multipotent cells capable of spontaneously differentiating into mesodermal cell lineages.

[0133] Finally, during the pericyte-to-MSC transition, pericytes undergo alterations in the gene expression of cytokines, chemokines, and / or immunomodulatory molecules. Up-regulated expression of genes that are responsible for the synthesis of immunoregulatory molecules (e.g., IL-10, TGF- , PGE2, indoleamine 2, 3-dioxigenase (IDO), programmed death-ligand 1 (PD-L1)) can be observed in pericytes during their transition to MSCs. Additionally, down-regulated expression of genes that encode inflammatory cytokines (e.g., TNF-a, IL-ip, IL-6) can also be observed during this transition. Further, these pericytes can exhibit changes in the expression of chemokines that are involved in immune cell recruitment and / or migration. For example, upregulation of CCL2, CCL5, and / or CXCL12 can attract monocytes, lymphocytes, and / or DCs to the site of tissue injury and / or injured tissues. In this way, newly formed MSCs are able to directly interact with tissue-infiltrated immune cells and, in a juxtacrine and paracrine manner, modulate their inflammatory phenotypes and / or functions.

[0134] Turning now to Figure 2, various pericyte changes 202 that occur during the pericyte-to- MSC transition are shown. For instance, the expression of one or more pericyte-related markers is reduced, as shown in block 204. These markers include, for instance, neuron-glial antigen 2 (NG2), platelet-derived growth factor receptor-beta (PDGFR-P), and regulator of G protein signaling 5 (RGS5). These changes in gene expression can be responsible for the altered pericytes’ morphology, phenotype, and / or function, as shown in block 206. Additionally, pericytes undergo changes in shape and / or size, as shown in block 208. Such changes are associated with one or more of the following, as shown in block 210: (1) a shift into a more migratory and tissue-repairing phenotype, and (2) the acquisition of a more proliferative and / or multipotent phenotype. Further, pericytes can undergo changes in cytoplasmic characteristics, as shown in block 212. Such changes include, for instance, (1) increased cytoplasmic volume, and (2) alterations in organelle distribution (e.g., more mitochondria, endoplasmic reticulum, and / or Golgi apparatus). Still further, pericytes may exhibit increased changes in cytoskeletal reorganization and / or migratory behavior, as shown in block 214. Non-limiting examples of such changes include, for example, the reorganization of actin filaments, the reduced expression of integrins, and / or the decreased production of ECM components (e.g., collagen and fibronectin).

[0135] Still further, an increased expression of various transcription factors can be seen in pericytes during the pericyte-to-MSC transition, as shown in block 216. Non-limiting examples of such transcription factors include, for instance, Sox9, Oct4, Nanog, and Klf4. Increased expression of these transcription factors can result in one or more of the following effects, as shown in block 218: (1) increased induction of neo-angiogenesis, (2) increased differentiation of tissue-resident stem cells, and (3) increased generation of regenerative properties (e.g., in the newly formed MSCs). Finally, various genetic changes can be seen in pericytes during the pericyte-to-MSC transition, as shown in block 220. These changes include, for instance, (1) increased expression of genes responsible for ECM degradation (e.g., MMP2, MMP9), (2) increased expression of genes responsible for ECM synthesis (e.g., collagen (COL)lAl, COL3A1, fibronectin 1 (FN1)), (3) increased expression of genes involved in TGF-P signaling (e.g., TGFBR2, SMAD2-7: ID1-3, CDKN1A), (4) increased expression of genes involved in Wnt signaling (e.g., Wnt3A, Wnt5A, WntlOB, AXIN2, TCF7, FZD4), (5) increased expression of genes involved in Notch signaling (e.g, NOTCH1, NOTCH3, JAG1-2, DLL1, DLL4, HES1, HES5, HEY1-2), and / or (6) increased alterations in the gene expression of cytokines, chemokines, and / or immunomodulatory molecules. One or more of these changes can result in one or more of the following effects, as shown in block 222: (1) increased attraction of immune cells (e.g., monocytes, lymphocytes, DCs) to injured tissues, (2) increased interaction with tissue-infiltrated immune cells, and (3) increased modulation of inflammatory phenotypes and / or functions of these tissue-infiltrated immune cells.

[0136] MSC-dependent suppression of inflammatory immune cells in injured tissues

[0137] As mentioned herein, MSCs are immunoregulatory stem cells with potent migratory characteristics. MSCs express various chemokine receptors (e.g., CXCR4, c-Met, PDGFRs, VEGFRs, CCR2, CXCR1, CXCR2, C5aR) that enable their rapid recruitment from the periphery to site(s) of tissue injury and / or inflammation. Stromal-Derived Factor-1 (SDF-1 / CXCL12), which can play a crucial role in attracting MSCs, is a chemokine produced by injured cells. SDF-1 binds to its receptor, CXCR4, expressed on MSCs, thereby promoting their migration towards the site(s) of tissue injury. In a similar manner to SDF-1, HGF also acts as a chemoattractant for MSCs. HGF binds to its receptor, c-Met, on MSCs, thereby activating signaling pathways that induce cytoskeletal rearrangement and / or promote migration and homing of MSCs to injured tissue. PDGF and VEGF are growth factors released by platelets and pericytes in injured blood vessels. PDGF and VEGF can bind to their receptors (e.g., PDGFR-ot, PDGFR- , VEGFR1, VEGFR2) on MSCs, thereby triggering signaling pathways that promote migration and / or recruitment of MSCs. DAMPs-dependent activation of tissue-resident macrophages can result in massive secretions of, for instance, CCL2 and IL-8, which, in a CCR2, CXCR1, and CXCR2-dependent manner, can attract circulating MSCs to the site(s) of tissue injury. In addition to these inflammatory cytokines, C5a, a protein fragment released from cleavage of complement component C5, can attract MSCs to the site(s) of tissue injury. C5a can bind to its receptor, C5aR, on MSCs, thereby stimulating their migration towards the injured tissues.

[0138] MSCs are generally not constitutively immunosuppressive cells. Their phenotype and / or function depends on the microenvironment to which they are exposed. In injured tissues, various inflammatory cytokines (e.g., TNF-a, IL-ip, IFN-y, IL-6), immunoregulatory cytokines (e.g., TGF-P, PGE2), hypoxia, and components ofECM (e.g, fibronectin and collagen) can collectively contribute to the generation of an immunosuppressive phenotype in both pericyte-derived MSCs and chemokine-recruited MSCs. These bioactive molecules can induce an increased synthesis of various immunomodulatory molecules (e.g, IL-10, TGF-P, IDO, PGE2), thereby enabling MSCs to suppress detrimental immune responses and / or promote tissue repair, healing, and / or regeneration.

[0139] MSC-derived IL-10, TGF-P, and / or PGE2 can suppress the production of pro- inflammatory cytokines (e.g., TNF-a, IL-ip, IL-12) in macrophages and promote their transition to an alternatively activated, anti-inflammatory and tissue-regenerative phenotype. MSC-primed, alternatively activated (M2) macrophages can produce, for instance, IL-10, TGF-P, IL-4, IL-13, VEGF, FGF-2, FGF-7, and PDGF, which can contribute to the resolution of inflammation and / or the restoration of tissue homeostasis. TGF-P, PDGF, FGF-2, and / or FGF-7 can promote the production of ECM components and / or stimulate the differentiation of fibroblasts into myofibroblasts, which are involved in wound healing and tissue contraction. VEGF may promote the formation of new blood vessels, facilitating the delivery of oxygen and nutrients to the injured tissue. IL-13 can promote tissue repair by stimulating collagen synthesis, promoting extracellular matrix remodeling, and enhancing angiogenesis, while IL-10, IL-4, and / or TGF-P can act as anti- inflammatory cytokines to suppress the generation of an inflammatory phenotype in neutrophils, DCs, and / or T lymphocytes.

[0140] MSC-sourced IL-10 and / or TGF-P can be primarily responsible for the MSC-dependent induction of an immunosuppressive, tolerogenic phenotype in DCs in injured tissues. Tolerogenic DCs can secrete immunosuppressive cytokines (e.g., IL-10, PGE2, TGF-P, IL-4, IDO), which can promote the generation and / or expansion of immunosuppressive T regulatory cells and alternatively activated macrophages.

[0141] MSC-derived IDO can deplete tryptophan in injured tissues. The reduced availability of tryptophan can affect the metabolic balance and alter the proliferation of inflammatory T lymphocytes, NK, and / or NKT cells. Additionally, IDO-dependent reduction ofL-tryptophan may inhibit the mechanistic target of the rapamycin (mTOR) signaling pathway, which reduces activation of the NLRP3 inflammasome in inflammatory macrophages and / or neutrophils. Additionally, metabolites generated from tryptophan metabolism, such as, for example, kynurenine and quinolinic acid, can suppress the production of reactive oxygen species in inflammatory immune cells. Further, kynurenine and its downstream metabolite 3- hydroxyanthranilic acid, may activate the aryl hydrocarbon receptor (AhR), which, in turn, can attenuate the production of IL-1J3 and IL-18 in DCs, macrophages, and / or neutrophils. Finally, MSC-sourced IDO can enhance the generation and / or expansion of immunosuppressive Tregs, since kynurenine may promote the expression of Treg lineage-defining transcription factor FoxP3 in naive CD4+ T cells. Upon activation of TCRs, intracellular signaling pathways driven by, for example, protein kinase B and mTOR, can destabilize the immunoregulatory phenotype of resting Tregs and cause their reprogramming into a pro-inflammatory helper-like phenotype (“ex-Tregs”). This phenotype can be characterized by increased production of Thl and Thl7-related inflammatory cytokines (e.g., IFN-y and IL-17). By inducing low levels of tryptophan in the local microenvironment, MSC-derived IDO and kynurenine can activate stress-response-induced general control nonderepressible 2 (GCN2) kinase, which can suppress protein kinase B / mTOR signaling in Tregs and prevent the trans-differentiation of Tregs in inflammatory Thl and Th 17 cells. Additionally, since GCN2 kinase down-regulates expression of TCR zeta chain in CD8+ cytotoxic T cells (CTLs) by increasing the activity of GCN2 kinase in CTLs, MSC-sourced IDO and kynurenine can attenuate the cytotoxic properties of CTLs and suppress CTL-dependent tissue damage.

[0142] Turning now to Figure 3, various MSC-mediated mechanisms 302 that result in the suppression of inflammation in one or more injured tissues are shown. Specifically, MSC-derived IL-10, TGF-P, and / or PGE2, as shown in block 304, can suppress and / or decrease the production of pro-inflammatory cytokines (e.g., TNF-a, IL-ip, IL-12), as shown in block 306. As mentioned above herein, such suppression can result in promoting the transition of macrophages to an antiinflammatory and / or tissue-regenerative phenotype. Additionally, MSC-primed M2 macrophages, shown in block 308, can contribute to one or more of the following effects, as shown in block 310:

[0143] (1) an increased resolution of inflammation, and (2) an increased restoration of tissue homeostasis. TGF-P, PDGF, FGF-2, and / or FGF-7, shown in block 312, can contribute to one or more of the following effects, as shown in block 314: (1) promoting the production of ECM components, and

[0144] (2) stimulating the differentiation of fibroblasts into myofibroblasts. Further, VEGF, shown in block 316, may promote the formation of new blood vessels, as shown in block 318. This can result in facilitating the delivery of oxygen and nutrients to one or more injured tissues. Finally, IL- 13, shown in block 320, can contribute to one or more of the following effects, as shown in block 322: (1) stimulating collagen synthesis, (2) promoting extracellular matrix remodeling, and

[0145] (3) enhancing angiogenesis. One or more such effects can result in the promotion of the repair, healing, and / or regeneration of injured tissues.

[0146] Other MSC-sourced and / or MSC-derived compounds can either suppress the generation of an inflammatory phenotype in one or more types of immune cells (e.g., neutrophils, DCs, T lymphocytes) and / or induce the generation of an immunosuppressive phenotype in one or more types of immune cells (e.g, DCs in injured tissues). Figure 4 shows various non-limiting examples of these MSC-sourced compounds 402. Specifically, IL- 10, IL-4, and / or TGF-P, shown in block 404, can act as anti-inflammatory cytokines to suppress the generation of an inflammatory phenotype in various immune cells, as shown in block 406. Non-limiting examples of such immune cells include, for instance, neutrophils, DCs, and / or T lymphocytes. Additionally, MSC- sourced IL- 10 and / or TGF-P, shown in block 408, can contribute to one or more of the following effects, as shown in block 410: (1) inducing an immunosuppressive, tolerogenic phenotype in one or more immune cells (e.g., DCs in injured tissues), and (2) promoting the generation and / or expansion of immunosuppressive T regulatory cells and M2 macrophages. Further, MSC-derived IDO, shown in block 412, can deplete tryptophan in injured tissues, as shown in block 414. The reduced availability of tryptophan can alter the proliferation of inflammatory cells (e.g., inflammatory T lymphocytes, NK cells, NKT cells), as shown in block 416. The reduction of tryptophan, as shown in block 414, can also result in one or more of the following effects, as shown in block 418: (1) inhibition of the mechanistic target of the rapamycin (mTOR) signaling pathway, and (2) reduction of activation of the NLRP3 inflammasome in inflammatory macrophages and / or neutrophils. MSC-sourced IDO, shown in block 412, and / or the reduction in tryptophan, shown in block 414, can affect the metabolites generated from tryptophan metabolism, shown in block 420. These metabolites (e.g., kynurenine, quinolinic acid, kynurenine’s downstream metabolite 3- hydroxyanthranilic acid) can contribute to one or more of the following effects, as shown in block 422: (1) suppression of the production of reactive oxygen species in inflammatory immune cells, (2) activation of the aryl hydrocarbon receptor (AhR), (3) attenuation of the production of IL- 10 and / or IL- 18 in DCs, macrophages, and / or neutrophils, (4) activation of stress-response-induced general control nonderepressible 2 (GCN2) kinase, which can suppress protein kinase B / mTOR signaling in Tregs and prevent the trans-differentiation of Tregs in inflammatory Thl and Th 17 cells, (5) attenuation of the cytotoxic properties of CTLs (e.g., since GCN2 kinase down-regulates expression of TCR zeta chain in CD8+ CTLs by increasing the activity of GCN2 kinase in CTLs), and (6) suppression of CTL-dependent tissue damage.

[0147] Cross-talk between MSCs and tissue-resident stem cells in injured tissues

[0148] The interaction between MSCs and tissue-resident stem cells in injured tissues is a complex and dynamic process that plays a crucial role in tissue repair, healing, and / or regeneration. This process generally involves a combination of paracrine signaling, growth factor / cytokine crosstalk, direct cell-to-cell contact, and ECM remodeling. These interactions can create a microenvironment that supports the activation, proliferation, migration, and / or differentiation of tissue-resident stem cells, facilitating more efficient tissue repair, healing, and / or regeneration.

[0149] MSCs can secrete a variety of growth factors (e.g., TGF-0, FGF, HGF, PDGF, VEGF, and insulin-like growth factor (IGF)) that can act synergistically to promote the proliferation, migration, and / or differentiation of tissue-resident stem cells, thereby enhancing their regenerative capacity. Notably, the biological effects of MSC-derived growth factors on survival, proliferation, and / or differentiation of stem cells can vary depending on the specific tissue microenvironment and the extent of any tissue injury.

[0150] MSC-derived TGF-P can be primarily responsible for the MSC-dependent suppression of activated T lymphocytes and / or NKT cells in injured tissues since it causes G1 cell cycle arrest of proliferated lymphocytes. Within the central nervous system, MSC-sourced TGF- can activate neural stem cells (NSC), regulate neurogenesis, and promote neuronal survival. MSC-sourced TGF-P can also be important for cardiac regeneration since it activates the Smad-dependent pathway in cardiac stem cells (CSCs) and / or cardiac progenitor cells, thereby promoting their activation and / or proliferation. Additionally, MSC-sourced TGF-P can induce the hypertrophic growth of cardiomyocytes, leading to the enlargement of individual cardiac cells. MSCs, in a TGF- P-dependent manner, may interact with endothelial progenitor stem cells (EPC) to promote their proliferation and / or differentiation, which can crucially contribute to the generation of new blood vessels in injured tissues. Within the bone marrow, MSC-derived TGF-P can play a role in maintaining the hematopoietic stem cell (HSC) niche and / or regulating immune cell differentiation.

[0151] Additionally, MSC-sourced FGF can bind to FGF receptors (FGFRs) on tissue-resident stem cells, thereby affecting their phenotype and / or function. Upon FGF binding, FGFRs can undergo conformational changes that lead to receptor dimerization. This dimerization brings the intracellular kinase domains of the receptors into close proximity, allowing them to phosphorylate each other on specific tyrosine residues. This autophosphorylation event can further activate the kinase activity of the FGFRs. The activated FGFRs can recruit adaptor protein, FGFR substrate 2 (FRS2), enabling activation of the Grb2-Sos complex, which leads to the activation of the Ras- Raf-MEK cascade. This cascade activates ERK kinase, which can translocate to the nucleus and phosphorylate various transcription factors, thereby modulating gene expression and / or promoting cellular proliferation and differentiation of tissue-resident stem cells. The FGF:FGFRs complex can also activate PI3K kinase, leading to the production of phosphatidylinositol (3,4,5)- trisphosphate (PIP3). PIP3 may then recruit and activate Akt, a serine / threonine kinase that regulates various cellular processes in tissue-resident stem cells, including, for example, cell survival, metabolism, and / or migration. FGFR activation can stimulate the phosphorylation of specific tyrosine residues on the receptor, which serves as docking sites for members of the STAT family of transcription factors. Phosphorylated STAT proteins can translocate to the nucleus, where they regulate gene expression and mediate cellular responses, such as, for example, proliferation and / or differentiation of stem and / or progenitor cells. Accordingly, FGF released by MSCs can induce the proliferation and / or differentiation of tissue-resident stem cells into specific cell lineages necessary for tissue repair, healing, and / or regeneration. Further, MSC-sourced FGF can act as chemoattractants, guiding the migration of tissue-resident stem cells towards the area of tissue damage and / or tissue injury. MSC-sourced FGF can also stimulate the synthesis and / or deposition of ECM components e.g., collagen and fibronectin), thereby creating a favorable microenvironment for engraftment of stem cells by providing structural support and / or guidance cues for their migration, proliferation, and / or differentiation.

[0152] HGF, also known as scatter factor, is a paracrine factor secreted by MSCs that can modulate survival, proliferation, migration, and / or differentiation of tissue-resident stem cells during tissue repair, healing, and / or regeneration. MSC-derived HGF can have anti-apoptotic effects, promoting the survival of stem cells and / or protecting them from cell death induced by injury-related factors. Upon binding to the c-Met receptor on stem cells, MSC-sourced HGF can trigger the activation of the PI3K / Akt pathway. Akt can phosphorylate and inactivate pro-apoptotic proteins (e.g., Bad, Bax), thereby preventing them from triggering the apoptotic cascade. Additionally, HGF can up- regulate the expression of anti-apoptotic Bcl-2 family members, promoting cell survival by, for instance, preventing the release of cytochrome c from mitochondria. MSC-derived HGF also up- regulates the expression of VEGF and IGF-1, which can have anti-apoptotic effects. These induced factors further contribute to the protection of stem cells from apoptosis. Further, HGF can enhance the expression of integrins, which may mediate cell-ECM interactions. By promoting cell adhesion and providing survival signals, MSC-sourced HGF can protect stem cells from apoptotic cell death. HGF-mediated anti-apoptotic effects can enable tissue-resident stem cells to resist injury-related factors and successfully carry out their healing and / or regenerative functions. In addition to its anti-apoptotic effects, MSC-derived HGF can promote the activation and / or proliferation of tissueresident stem cells by activating the MAPK / ERK pathway. Finally, MSC-sourced HGF may act as a chemoattractant by stimulating cytoskeletal rearrangements in stem cells, thereby enabling their migration to the site of injury.

[0153] MSC-derived VEGF and PDGF can stimulate the proliferation and / or migration of endothelial progenitor cells (EPCs) and / or ECs, leading to the formation of a functional vascular network in the injured tissue(s). Both VEGF and PDGF signaling pathways in EPCs converge on common downstream targets, such as, for instance, Akt and ERK, which regulate cell survival, proliferation, and / or migration. VEGF binds to its specific VEGF2 receptor while PDGF binds to PDGFRa and / or PDGFRp, leading to receptor dimerization and activation. Activated VEGFR2 and PDGFRs can recruit and activate PI3K, which generates PIP3. PIP3, in turn, activates Akt, a serine / threonine kinase that may promote cell survival, proliferation, and / or migration in EPCs. VEGF and PDGF can also activate the Ras / Raf / MEK / ERK-signaling cascade in EPCs and ECs, resulting in the phosphorylation and / or activation of the NF-kB transcription factor that regulates ECs’ proliferation, survival, and / or angiogenesis. VEGF and PDGF-driven neo-angiogenesis can be crucial for supplying oxygen and / or nutrients to EPCs, promoting their survival and / or regenerative functions.

[0154] In injured tissues, MSC-derived IGF may promote the differentiation of tissue-resident stem cells by, for instance, (1) activating intracellular signaling pathways, (2) modulating gene expression, (3) interacting with other growth factors and / or cytokines, (4) regulating cell cycle and / or proliferation, and (5) modulating epigenetic modifications. In a similar manner to VEGF and PDGF, MSC-derived IGF can activates the PI3K / Akt signaling pathway, thereby enhancing the proliferation of stem cells. MSC-derived IGF can promote the transition of stem cells from a quiescent state (GO phase) to an active proliferative state (G1 phase). Additionally, MSC-sourced IGF can activate Smad proteins, which can bind to target gene promoters and / or initiate gene transcription. The activated genes can encode proteins that drive stem cells towards specific lineages, promoting their differentiation. Additionally, MSC-derived IGF can influence the activity of enzymes involved in epigenetic modifications, leading to changes in chromatin structure and / or accessibility. This modulation of epigenetic marks can promote or suppress the expression of specific genes associated with stem cell differentiation. For this purpose, MSC-derived IGF can interact with other growth factors (e.g, bone morphogenetic proteins (BMPs), FGFs, and TGF-P) that may be present in the microenvironment of injured tissues. These growth factors can collectively contribute to the commitment and / or differentiation of resident stem cells into specific lineages, enabling enhanced tissue repair, healing, and / or regeneration.

[0155] Turning now to Figure 5, various MSC-sourced and / or MSC-derived compounds 502 are shown that can contribute to the interaction and / or cross-talk between MSCs and tissue-resident stem cells in injured tissues. Specifically, MSC-derived TGF-0, shown in block 504, can produce one or more of the following effects, as shown in block 506: (1) suppressing one or more types of immune cells (e.g., activated T lymphocytes, NKT cells) in injured tissues by, for instance, causing G1 cell cycle arrest of proliferated lymphocytes, (2) activating neural stem cells (NSC), (3) regulating neurogenesis, (4) promoting neuronal survival, (5) promoting activation and / or proliferation of cardiac cells (e.g., cardiac stem cells, cardiac progenitor cells) by, for instance, activating the Smad-dependent pathway, (6) inducing hypertrophic growth of cardiomyocytes, (7) promoting proliferation and / or differentiation of endothelial progenitor stem cells (EPC), (8) maintaining the hematopoietic stem cell (HSC) niche e.g., in the bone marrow), and (9) regulating immune cell differentiation.

[0156] Additionally, MSC-sourced FGF, shown in block 508, can bind to FGF receptors (FGFRs) on tissue-resident stem cells, thereby affecting their phenotype and / or function. FGF binding can result in one or more of the following effects, as shown in block 510: (1) activating the kinase activity of FGFRs, (2) phosphorylation of transcription factors in the nucleus (e.g., via the activation of the Ras-Raf-MEK cascade, which leads to the activation of ERK kinase), (3) promoting cellular proliferation and / or differentiation of tissue-resident stem cells, (4) regulating cellular processes (e.g., cell survival, metabolism, migration) in tissue-resident stem cells by, for instance, PI3K kinase and Akt, (5) regulating proliferation and / or differentiation of stem and / or progenitor cells (e.g., via phosphorylated STAT proteins), (6) guiding migration of tissue-resident stem cells towards injured tissues, and (7) stimulating the synthesis and / or deposition of ECM components (e.g., collagen, fibronectin), thereby creating a favorable microenvironment for engraftment of stem cells by providing structural support and / or guidance cues for their migration, proliferation, and / or differentiation.

[0157] Further, MSC-derived HGF (also known as scatter factor), shown in block 512, can contribute to one or more of the following effects, as shown in block 514: (1) modulating the survival, proliferation, migration, and / or differentiation of tissue-resident stem cells, (2) promoting the survival of stem cells (e.g., via anti-apoptotic effects and / or protecting them from cell death), (3) mediating cell-ECM interactions (e.g., via the expression of integrins), (4) enabling tissueresident stem cells to resist injury-related factors, and (5) enabling migration of stem cells to injured tissues (e.g., by stimulating cytoskeletal rearrangements). Still further, MSC-derived VEGF and / or PDGF, shown in block 516, can contribute to one or more of the following effects, as shown in block 518: (1) stimulating the proliferation and / or migration of EPCs and / or ECs (e.g., via Akt, ERK, VEGF2, PDGFRa, PDGFRP, PI3K, PIP3, the Ras / Raf / MEK / ERK-signaling cascade, NF-kB), which can lead to the formation of a functional vascular network in injured tissues, and (2) promoting angiogenesis of EPCs and / or ECs, resulting in an increased supply of oxygen and / or nutrients.

[0158] Finally, MSC-derived IGF, shown in block 520, can promote the differentiation of tissueresident stem cells, as shown in block 522. This can occur by one or more of the following, as shown in block 524: (1) activating intracellular signaling pathways, (2) modulating gene expression, (3) interacting with other growth factors and / or cytokines, (4) regulating cell cycle and / or proliferation, and (5) modulating epigenetic modifications. MSC-derived IGF, shown in block 520, can also contribute to one or more of the following effects, as shown in block 526: (1) enhancing the proliferation of stem cells (e.g., via the PI3K / Akt signaling pathway), (2) promoting the transition of stem cells from a quiescent state (e.g., GO phase) to a more active, proliferative state (e.g., G1 phase), (3) promoting stem cell differentiation (e.g., via activation of Smad proteins, which can bind to target gene promoters and / or initiate gene transcription), (4) changing chromatin structure and / or accessibility (e.g., via influencing the activity of enzymes involved in epigenetic modifications), and (5) interacting with other growth factors (e.g., bone morphogenetic proteins (BMPs), FGFs, and TGF-P) to trigger differentiation of resident stem cells into specific lineages, which can lead to enhanced tissue repair, healing, and / or regeneration.

[0159] As described herein, the crosstalk between pericytes, MSCs, and immune cells is important and, in some cases, essential for the successful repair, healing, and / or generation of injured tissues. This interaction is generally driven by paracrine signaling. Pericytes, MSCs, and immune cells can secrete bioactive factors that influence each other’s behaviors and / or functions. For instance, immune cells may produce inflammatory cytokines and / or chemokines that influence pericytes’ migration, proliferation, and / or transition to MSCs. Pericyte-derived MSCs can then release immunoregulatory factors, which may induce the generation of an immunosuppressive phenotype in inflammatory immune cells, alleviating one or more detrimental immune responses in injured tissues. MSCs may also produce various growth factors, which can influence the survival, proliferation, and / or differentiation of tissue-resident stem cells into specific cell lineages, thereby enhancing and / or enabling the successful repair, healing, and / or regeneration of injured tissues.

[0160] Animal studies, in particular, can provide further evidence of the molecular mechanisms that regulate crosstalk between pericytes, MSCs, and / or immune cells in tissue repair, healing, and / or regeneration. For instance, juxtacrine interactions may occur between one or more of these cells. Additionally, there may be effects of temporal dynamics and / or microenvironmental influences on the aforementioned crosstalk.

[0161] Given the above, one or more types of MSCs and / or one or more products extracted and / or derived therefrom (e.g., MSC-Exos, one or more MSC-sourced microribonucleic acids (miRNAs), anti-tumor proteins, cytokines, etc.) can be used as part of new therapeutic strategies to enhance tissue repair, healing, and / or regeneration, resulting in new and improved outcomes in regenerative medicine, especially with respect to injured tissues. Such therapeutic strategies may include, for instance, administration of one or more of the MSCs and / or MSC-containing compositions and / or formulations described herein (e.g., any d-MAPPS composition and / or formulation).

[0162] MSC Compositions

[0163] In at least one embodiment of the present disclosure, one or more compositions are disclosed that comprise one or more types of MSCs and / or one or more products extracted and / or derived therefrom (e.g., MSC-Exos, one or more MSC-sourced microribonucleic acids (miRNAs), anti-tumor proteins, cytokines, etc.) (also referred to herein as “MSC Composition” or “MSC Compositions”). Such MSC Compositions can include, in at least one embodiment, any d-MAPPS composition and / or any composition having D-HAF.

[0164] The MSCs themselves may be derived from various sources within the human body and / or subject, including, for example, bone marrow (also referred to as “BM-MSC” or “BM-MSCs”), adipose tissue (also referred to as “AT -MSC” or “AT-MSCs”), muscles, skin, the placenta, the femoral shaft, the iliac crest, umbilical cord blood, the umbilical cord itself, Wharton’s jelly, the endometrium, menstrual blood, dental pulp, periodontal ligaments, the gingiva, apical papilla, and dental follicles.

[0165] In at least an additional embodiment, other active agents may be co-administered with one or more MSC Compositions, including, for example, secondary anti-cancer agents, antiinflammatories, exogenous immune cells, small molecules, therapeutic proteins, and the like. Nonlimiting examples include chemotherapeutic compounds and / or drugs, nucleic acids, and the like, which will be discussed further below. Tn at least one embodiment, the MSC Compositions retain most, if not all, of the biological compounds (including anti-tumor compounds) after short-term or long-term storage under temperature-controlled conditions. The MSC Compositions may be stored under any such conditions known in the art, e.g., as a liquid, as a lyophilized powder, etc. The total protein content of the MSC Compositions when compared to MSCs extracted from a subject is, for example, at least 60%, 70%, 80%, and preferably more than 85%.

[0166] In at least one embodiment, one or more of the MSC Compositions disclosed herein exhibits any of the aforementioned promotion of tissue repair, healing, and / or regeneration effects when administered into a subject, including, for instance, (1) enhancing and / or enabling the pericyte-to-MSC transition, (2) inducing the generation of an immunosuppressive phenotype in inflammatory immune cells, (3) alleviating one or more detrimental immune response in injured tissues, (4) enabling and / or enhancing the production of various growth factors that influence the survival, proliferation, and / or differentiation of tissue-resident stem cells into specific cell lineages, and (5) any other tissue repair, healing, and / or regeneration effect described herein.

[0167] In at least another embodiment, one or more of the MSC Compositions can also suppress tumor growth and / or progression when administered into a subject, including a subject that has one or more types of injured tissues. Such suppression of tumor growth and / or progression can occur by, for instance, (i) up-regulating expression of chemoresistance-related genes in one or more types of tumor cells, (ii) reducing viability and / or invasiveness of one or more types of malignant cells and / or tumor cells, (iii) suppressing neo-angiogenesis in the TME, and / or (iv) inducing the generation, proliferation, and / or tumorotoxicity of one or more types of immune cells e.g., CTLs, NKT cells, etc.).

[0168] In at least an additional embodiment, one or more of the MSC Compositions are not heat- treated, chemical-treated, or fractionated to produce any of the formulations described herein. In at least one embodiment, one or more formulations that include one or more of the MSC Compositions (also referred to “MSC Formulations”) retain more than 50%, more than 60%, more than 70%, more than 80%, or preferably more than 90%, of the biological compounds (including, for instance, miRNAs, anti -turn or compounds, compounds that promote tissue repair, healing, and / or regeneration, and the like) present in MSCs freshly extracted from a subject. In at least a further embodiment, one or more MSC Formulations are not diluted with any additional solution. In at least another embodiment, one or more MSC Formulations are not concentrated.

[0169] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations can be stored for long periods of time, allowing for a variety of modes of application, including distribution and storage as aerosols, solutions, powders, etc. In at least an additional embodiment, one or more MSC Compositions and / or one or more MSC Formulations are refrigerated at about 1° C. to about 10° C. for long-term storage. In at least a further embodiment, the one or more MSC Compositions and / or one or more MSC Formulations are refrigerated at 4° C. for up to 12 months or more. Preferably, long-term storage does not reduce the quantity and / or quality of the total soluble proteins and / or biological compounds present. For at least one embodiment, the total soluble proteins and / or biological compounds retained after long-term storage in refrigerated conditions is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to MSCs extracted from a given subject.

[0170] In at least one embodiment, one or more MSC Formulations can be supplied as a clear one- part solution in a suitable container for storage at 4° C., or for storage at -20° C., or at -80° C. As non-limiting examples, liquid formulations in prefdled aliquots can be suitable for storage at 1-5° C., or for storage at -20° C., or at -80° C. The liquid formulation can be suitable for topical application in, e.g., a nebulizer or an inhaler. In at least an additional embodiment, the fluid can be supplied as a kit that can be stored at 4° C., at -20° C., or at -80° C. until needed.

[0171] In at least one embodiment, one or more MSC Formulations use a final filtration through a 0.2 pm filter. In at least an additional embodiment, such filtration is necessary to optimize sterile conditions without the requirement for irradiation (e.g., e-beam treatment). In at least a further embodiment, the one or more MSC Formulations have a 106sterility assurance level without irradiation. In at least another embodiment, lyophilisate versions of the one or more MSC Formulations may also be irradiated by e-beam irradiation or gamma ray irradiation to fully sterilize the lyophilisate.

[0172] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise various growth factors. Non-limiting examples of such growth factors include TGF-0, VEGF, and others as described further below.

[0173] In at least one embodiment, one or more types of MSCs and / or one or more MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) comprised in one or more MSC Compositions and / or one or more MSC Formulations can be genetically modified to express various biological factors, including one or more miRNAs, interleukins (e.g., IL- 12), and the like. Such modified MSCs and / or MSC-derived products (e.g., MSC-Exos) may exhibit stronger and / or more sustained expressions and / or secretions of, for example, miRNAs, IL-12, IFN-y, and the like. Accordingly, exogenous administration and / or injection of these genetically modified MSCs and / or MSC-derived products may result in stronger anti-tumor T cell responses.

[0174] As a further non-limiting example, in murine metastatic models of lung cancer, intravenously injected BM-MSCs significantly augmented lung cancer metastasis by downregulating the anti-tumor immune response. Thus, in at least one embodiment, one or more types of MSCs and / or one or more MSC-derived products (e.g, MSC-Exos, one or more MSC- sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) comprised in one or more MSC Compositions and / or one or more MSC Formulations suppress production of TNF-a in DCs and macrophages, as well as inducing polarization of TNF-a-producing CD4+ Thl cells and IL-17-producing Thl7 cells in IL-10-producing Tregs. Accordingly, serum levels of various anti-tumorigenic cytokines (e.g., TNF-a and IL-17) were decreased, and the serum concentration of immunosuppressive IL-10 was increased in MSC-treated animals with tumors. In at least an additional embodiment, one or more types of MSCs and / or one or more MSC-derived products (e.g, MSC-Exos, one or more MSC- sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) comprised in one or more MSC Compositions and / or one or more MSC Formulations suppress cytotoxicity of CTLs and NK cells in metastatic lungs by (1) downregulating the expression of, for instance, FASL and NKG2D, and (2) reducing exocytosis of, for instance, perforins and granzymes. Additionally, pharmacological inhibition of IDO and iNOS activity completely abrogated MSC-driven suppression of anti-tumor immunity in tumor-bearing mice. This suggests that MSC-sourced IDO and NO were mainly responsible for the pro-tumorigenic effects of MSCs, at least in the context of the aforementioned murine metastatic model.

[0175] In at least one embodiment of the disclosure, a method for treating a disease (e.g., injured tissue(s), cancer) in a subject comprises one or more of: determining that the subject is in need of treatment with one or more MSC Compositions and / or one or more MSC Formulations, the one or more MSC Compositions and / or one or more MSC Formulations, administering the one or more MSC Compositions and / or one or more MSC Formulations via one or more administration pathways, such as, for instance, intravenous injection. Such administration may result in (1) any of the promotion of tissue healing, repair, and / or regeneration effects described herein, and / or (2) any of the immune cell-related effects herein, including, but not limited to, suppressing the production of TNF-a in DCs and / or macrophages, inducing polarization of TNF-a-producing CD4+ Thl cells and IL-17-producing Thl7 cells in IL-10-producing Tregs, decreasing serum concentration and / or levels of TNF-a and / or IL-17, increasing serum concentration and / or levels of IL-10, and / or suppressing one or more immune cell responses (e.g, cytotoxicity of CTLs and NK cells).

[0176] In at least a further embodiment, the aforementioned suppression of cytotoxicity of CTLs and NK cells may be achieved by, for instance, (1) downregulating the expression of one or more biological compounds selected from the group consisting of: FASL, NKG2D, and combinations thereof, and / or (2) reducing exocytosis of one or more biological compounds selected from the group consisting of: one or more perforins, one or more granzymes, and combinations thereof.

[0177] In at least an additional embodiment, the method further comprises inhibiting IDO and / or iNOS activity, leading to a reduction in measurable levels of IDO and / or NO.

[0178] In at least one embodiment, one or more types of MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) comprised in one or more MSC Compositions and / or one or more MSC Formulations are administered during an initial phase of injury or disease (e.g., during or after one or more tissues have been injured). The administering may be achieved using one or more processes, including, but not limited to, intravenous, intraosseous, intraperitoneal, submandibular, and / or one or more other injection processes. The administered one or more MSC Compositions and / or one or more MSC Formulations thereby results in (1) any of the promotion of tissue healing, repair, and / or regeneration effects described herein, and / or (2) any of the immune cell-related effects herein, including, but not limited to, influencing one or more immune cell responses (e.g., enhancing or decreasing immune cell cytotoxicity, increasing or decreasing the production of one or more chemokines and / or cytokines, and the like). The aforementioned one or more cytokines may be selected from the group consisting of TNF-a, IFN-y, IL- 17, and combinations thereof.

[0179] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered to a subject in combination with one or more additional therapeutic, diagnostic, and / or prophylactic agents to alleviate pain (e.g., pain associated with one or more injured tissues and / or one or more diseases, such as cancer), facilitate healing, and / or to reduce or inhibit scarring. In at least an additional embodiment, one or more MSC Compositions comprise one or more additional compounds to prevent or treat tissue injury, and / or to relieve symptoms such as inflammation. Such one or more additional compounds may be, in at least one aspect, administered to the subject via MSC-Exos. Non-limiting examples of such one or more additional compounds include antimicrobial agents, analgesics, local anesthetics, anti-inflammatory agents, antioxidants, immunosuppressants, anti-allergenic agents, enzyme cofactors, essential nutrients, and growth factors.

[0180] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered to a subject for prevention or treatment of injured tissues and / or to promote tissue repair, healing, and / or regeneration. In one example, an effective amount of one or more MSC Formulations are administered adjacent to a site (e.g., a wound site) in need thereof. In at least another embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered with one or more therapeutics (e.g., chemotherapeutic agent, humanized molecular antibody, etc.) to a subject for prevention or treatment of injured tissues and / or to promote tissue repair, healing, and / or regeneration. Accordingly, in at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations may be considered a targeted adjuvant therapy, serving to complement traditional therapeutic approaches for injured tissues, while minimizing adverse side effects. Additional secondary therapeutic agents include, but are not limited to, antibiotics, cytokines, and growth factors (e.g., fibroblast growth factor, hepatocyte growth factor, cell-cycle checkpoint inhibitors, platelet-derived growth factor, vascular endothelial cell growth factor, and insulin-like growth factor). In at least another embodiment, secondary therapeutic agents include, for instance, hyaluronic acid or glycosaminoglycans. In at least one embodiment, additional active agents may be administered with one or more MSC Compositions and / or one or more MSC Formulations, the active agents including, for instance, small molecules, biomolecule, peptides, sugar, glycoproteins, polysaccharides, lipids, nucleic acids, and / or combinations thereof. Suitable small molecule active agents include, but are not limited to, organic and organometallic compounds. In at least one instance, the aforementioned small molecule active agent has a molecular weight of less than about 2000 g / mol, more preferably less than about 1500 g / mol, and most preferably less than about 1200 g / mol. The small molecule active agent can be a hydrophilic, hydrophobic, or amphiphilic compound. In at least one example, one or more additional agents may be dispersed, dissolved, and / or suspended in one or more MSC Compositions and / or one or more MSC Formulations.

[0181] Volume of administration of one or more MSC Compositions and / or one or more MSC Formulations is tissue-specific and dependent on the stage of the disease or disorder. Dosages can be readily determined by those of skill in the art. See, e.g., Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th ed.), Williams and Wilkins (1995). Additionally, one or more MSC Compositions and / or one or more MSC Formulations may be administered in conjunction with other types of cells, e.g., other exogenous stem cells, pluripotent cells, somatic cells, and / or combinations thereof. In at least one embodiment, one or more therapeutic, prophylactic, and / or diagnostic agents is administered prior to, in conjunction with, and / or subsequent to treatment with one or more MSC Compositions and / or one or more MSC Formulations.

[0182] In at least one embodiment, the aforementioned therapeutic, prophylactic and / or diagnostic agents may be administered in a neutral form, or in the form of a pharmaceutically acceptable salt. In at least one example, it may be desirable to prepare a formulation containing a salt of an agent due to one or more of the salt's advantageous physical properties, such as, for example, enhanced stability, a desirable solubility, and / or a desirable dissolution profde.

[0183] In at least one embodiment, pharmaceutically acceptable salts are prepared by reaction of the free acid or base forms of an active agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media such as, for example, ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an active agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts, as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Adejare et al., Remington: The Science and Practice of Pharmacy (23rd ed.), Academic Press (2020).

[0184] In at least one embodiment, the aforementioned secondary agent administered with one or more MSC Compositions and / or one or more MSC Formulations comprises a diagnostic agent such as, for example, paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, X-ray imaging agents, and / or contrast media.

[0185] In at least one embodiment, one or more MSC Formulations comprises one or more local anesthetics. Non-limiting examples of such local anesthetics include tetracaine, lidocaine, amethocaine, proparacaine, lignocaine, and bupivacaine. In at least one example, one or more additional agents, such as, e.g., a hyaluronidase enzyme, are also added to the one or more MSC Formulations to accelerate and / or improve dispersal of the local anesthetic.

[0186] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are used in combination with one or more antimicrobial agent. An antimicrobial agent, at least in the context of the present disclosure, is a substance that inhibits the growth of microbes including, for instance, bacteria, fungi, viruses, and / or parasites. Accordingly, antimicrobial agents include, for example, antiviral agents, antibacterial agents, antiparasitic agents, and anti-fungal agents. Non-limiting examples of antiviral agents include, e.g., ganciclovir and acyclovir. Non-limiting examples of antibiotic agents include, for example, aminoglycosides (e.g., streptomycin, amikacin, gentamicin, and tobramycin), ansamycins (e.g., geldanamycin and herbimycin), carbacephems, carbapenems, cephalosporins, glycopeptides (e.g., vancomycin, teicoplanin, and telavancin), lincosamides, lipopeptides (e.g., daptomycin, macrolides such as azithromycin, clarithromycin, dirithromycin, and erythromycin), monobactams, nitrofurans, penicillins, polypeptides (e.g., bacitracin, colistin, and polymyxin B), quinolones, sulfonamides, and tetracyclines.

[0187] Other exemplary antimicrobial agents include, for instance, iodine, silver compounds, moxifloxacin, ciprofloxacin, levofloxacin, cefazolin, tigecycline, gentamycin, ceftazidime, ofloxacin, gatifloxacin, amphotericin, voriconazole, natamycin.

[0188] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered in combination with one or more local anesthetics. A local anesthetic, at least in the context of the present disclosure, is a substance that causes reversible local anesthesia and has the effect of loss of sensation of pain. Non-limiting examples of local anesthetics include ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta- eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p- ami nobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and combinations thereof. In at least another aspect of this embodiment, the one or more MSC Compositions and / or one or more MSC Formulations include an anesthetic agent in an amount of, e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8% about 0.9%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10% by weight of the total composition and / or formulation.

[0189] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered in combination with one or more anti-inflammatory agents. Antiinflammatory agents reduce inflammation and include, for instance, steroidal and non-steroidal drugs. Suitable steroidal active agents include, for example, glucocorticoids, progestins, mineralocorticoids, and corticosteroids. Other non-limiting examples of anti-inflammatory agents include triamcinolone acetonide, fluocinolone acetonide, prednisolone, dexamethasone, loteprednol, fluoromethoIone, ibuprofen, aspirin, and naproxen. Non-limiting examples of immune-modulating drugs include cyclosporine, tacrolimus, and rapamycin. Non-limiting examples of non-steroidal anti-inflammatory drugs (NSAIDs) include ketorolac, nepafenac, and diclofenac. In at least one embodiment, anti-inflammatory agents are anti-inflammatory cytokines. Non-limiting examples of such cytokines include IL-10, IL-17, TNF-a, TGF-P, IL-35, and others described below. In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered in combination with one or more growth factors. As mentioned above herein, growth factors are proteins and / or glycoproteins capable of stimulating cellular growth, proliferation, and / or cellular differentiation. Non-limiting examples of growth factors include transforming growth factor beta (TGF-P), transforming growth factor alpha (TGF-a), granulocyte-colony stimulating factor (GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), myostatin (GDF8), growth differentiation factor-9 (GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF).

[0190] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered in combination with one or more enzyme cofactors, and / or one or more essential nutrients. Non-limiting examples of such cofactors include vitamin C, biotin, vitamin E, and vitamin K. Non-limiting examples of such essential nutrients include amino acids, fatty acids, etc.

[0191] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise at least one eukaryotic cell type other than one or more types of MSCs. Non-limiting examples of such eukaryotic cell types include non-mesenchymal stem cells, immune cells (e.g, T lymphocytes, B lymphocytes, natural killer cells, macrophages, dendritic cells), and combinations thereof. In at least an additional embodiment, the cells used are cells that dampen one or more inflammation responses (e.g, regulatory T cells). In at least a further embodiment, exosomes are generated ex vivo from one or more types of MSCs.

[0192] One or more types of MSCs and / or MSC-derived products (e.g, MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) can be exogenously administered and / or injected either alone or in conjunction with other cells and / or compounds, as discussed further below. Any of the below-mentioned other cells and / or compounds may be administered in combination with, for instance, one or more of types of MSCs and / or MSC-derived products described herein. In at least one embodiment of the disclosure, the one or more MSC Compositions and / or one or more MSC Formulations comprise MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are purposefully infected with one or more oncolytic viruses, such as ICOVIR-5. Generally, oncolytic viruses (also referred to as “OV” or “OVs”) are viruses, either genetically engineered or naturally occurring, that can help treat one or more diseases or disorders without harming the subject’s own healthy cells. OVs are usually administered using a delivery vehicle (e.g., MSCs and / or MSC-Exos) since direct administration of OVs generally results in the immune system clearing the OVs. Various OVs exist, including, for instance, oncolytic adenovirus (also referred to as “OAV” or “OAVs”), oncolytic herpes simplex virus (HSV) (also referred to as “OHSV” or “OHSVs”), and oncolytic measles virus (also referred to as “OMV” or “OMVs”).

[0193] In at least one embodiment, MSCs and / or MSC-Exos are used as a delivery vehicle for OAVs (also referred to as “MSC-OAV” or “MSC-OAVs”). In at least an additional embodiment, such MSCs and / or MSC-Exos can inhibit IFN-y production by activated T cells. Exogenously administered and / or injected MSC-OAVs may also provide additional immune system-related effects (e.g., in cancer patients, inhibiting tumor growth and / or development). In at least a further embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations that comprise MSC-OAVs to treat a subject with one or more diseases and / or disorders, non-limiting examples of which include any disease and / or disorder that produces injured tissue(s), such as, for instance, one or more types of cancer (e.g., hepatocellular carcinoma (HCC), lung cancer, breast cancer, pancreatic cancer, neuroblastoma, colorectal cancer, and prostate cancer).

[0194] In at least one embodiment, the specific OAV is ICOVIR-5, a virus that may exhibit increased replication in tumor cells when compared with other OAVs. Generally, ICOVIR-5 acts by controlling expression of the Ela-A24 gene under an E2F Transcription Factor 1 (E2F1) promoter that is insulated with DM-1, the myotonic dystrophy locus insulator. ICOVIR-5 further contains the so-called Kozak consensus sequence (also referred to as the “Kozak consensus” or “Kozak sequence”) immediately before the first codon of the Ela gene. The Kozak sequence is a nucleic acid sequence that functions as a protein translation initiation site that optimizes translation of mRNA by ribosomes. This may result in increased oncolytic and anti-tumor activity in patients with one or more injured tissues (e.g., due to one or more types of cancer).

[0195] In at least one embodiment, MSCs and / or MSC-derived products (e.g., MSC-Exos) can also be used to deliver OHSVs. Such delivery can result in lysis of cancer cells (e.g., glioblastoma cells), thereby resulting in increased repair, healing, and / or regeneration of the subject’s own healthy tissues and / or cells. Further, MSC-OHVs may, at least in animal models, stimulate apoptosis of cancer cells, leading to reduced tumor growth and reduced and / or absent metastases. In at least an additional embodiment, a specific OHSV used is HF10, a mutant form of HSV-1. In at least a further embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations comprising MSC-OHSVs to treat a subject with one or more diseases and / or disorders, non-limiting examples of which include any disease and / or disorder that produces injured tissue(s), such as, for instance, one or more types of cancer (e.g., pancreatic cancer, melanomas, and ovarian cancer). The method may further comprise administration in combination with other agents (e.g., the tyrosine kinase inhibitor erlotinib). Without wishing to be bound by theory, such administration may result in high levels of cytotoxicity towards specific tumor cells and / or cell lines (e.g., human pancreatic cell lines) when compared to controls. Moreover, the combination of MSCs with HF 10 and erlotinib may further result in more persistent viral presence and / or replication in tumor sites, leading to more prolonged uptake of the virus by tumor cells. Additional non-limiting examples of tumors and / or cancers that may be inhibited by exogenous administration and / or injection of MSC- OHSVs include melanomas and ovarian cancer.

[0196] In at least one embodiment, MSCs and / or MSC-derived products (e.g., MSC-Exos) are used to encapsulate and / or deliver OMVs (also referred to as “MSC-OMV” or “MSC-OMVs”). In at least one example, exogenously administered and / or injected MSC-OMVs can home to specific tumors (e.g., peritoneal tumors) and cause viral infection in those tumors, resulting in increased repair, healing, and / or regeneration of the subject’s own healthy tissues and / or cells. At least in animal models, such infections may occur regardless of whether the animals were previously immunized against the measles virus. Moreover, administration of MSC-OMVs may provide antitumor benefits that are not provided by either (1) administration of MSCs and / or MSC-Exos alone, or (2) administration of OMVs alone. Such anti-tumor benefits may be due to, for instance, increased induction of apoptosis. Additional non-limiting examples of tumors and / or cancers that may be inhibited by exogenous administration and / or injection of MSC-OMVs include ovarian cancer, HCC, and acute lymphocytic leukemia (ALL). Accordingly, in at least an additional embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations comprising MSC-OMVs to treat a subject with one or more diseases and / or disorders, non-limiting examples of which include any disease and / or disorder that produces injured tissue(s), such as, for instance, one or more types of cancer (e.g., ovarian cancer, HCC, and ALL).

[0197] In at least one embodiment, MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) can be used as vehicles for delivering bi-specific T-cell engaging antibodies. Without wishing to be bound by theory, such MSCs and / or MSC-derived products can be used as such vehicles due to their low immunogenicity and tumorhoming properties, including in patients that have injured tissues due to, for instance, one or more types of cancer. The aforementioned antibodies are protein engagers that simultaneously bind to tumor antigens and the appropriate ligand on one or more T lymphocytes, thereby enabling specific T cell-mediated elimination of tumor cells. Accordingly, in at least an additional embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations comprising MSCs and / or MSC-derived products that encapsulate one or more bi-specific T-cell engaging antibodies.

[0198] In at least one embodiment, MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) are used to encapsulate and / or deliver glypican 3 (GPC3), a protein that regulates the proliferation of hepatocellular carcinoma cells. Hedgehog (“Hh”) pathway signaling can regulate one or more aspects of hepatocellular carcinoma tumorigenesis, and GPC3 can regulate Hh signaling. In at least one example, the delivered GPC3 can inhibit expression of one or more genes in the Hh pathway. Such inhibitory effects can be themselves reduced by heparin, a glycosaminoglycan that is a competitor for GPC3 binding.

[0199] In at least one embodiment, MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) can be genetically modified with one or more viral vectors encoding a GPC3 / cluster of differentiation 3 (CD3) bi-specific T cell engager. In at least an additional embodiment, MSCs that express the GPC3 -specific single chain variable fragment (“scFv”) and the CD3-specific scFv (“MSCGPC3-CD3” or “MSCsGPC3- CD3”) can direct GPC3-specific CD4+T helper cells and CD8+ CTLs towards GPC3 -expressing hepatocellular carcinoma cells. In at least a further embodiment, co-cultures of GPC3+ tumor cells, MSCsGPC3-CD3s, and T lymphocytes can lead to an increased production of IFN-y in GPC3- specific CD4+ T cells, as well as an enhanced activation and expansion of GPC3-specific CTLs. These effects resulted in CTL-dependent killing of GPC3 -expressing malignant cells. Similar results can occur in vivo. In at least another embodiment, in MSCsGPC3-CD3 -treated tumorbearing mice, there may be an increased activation of GPC3-specific T cells and a concomitant significant reduction in hepatocellular carcinoma growth. Thus, MSCsGPC3-CD3 have the potential for treating, either alone or in combination with other compounds and / or treatments, one or more diseases and / or disorders, non-limiting examples of which include any disease and / or disorder that produces injured tissue(s), such as, for instance, one or more types of cancer (e.g., hepatocellular carcinoma).

[0200] Accordingly, in at least an additional embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations comprising MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that encapsulate GPC3 to treat a subject with one or more diseases and / or disorders, non-limiting examples of which include any disease and / or disorder that produces injured tissue(s), such as, for instance, one or more types of cancer (e.g., hepatocellular carcinomas).

[0201] In at least one embodiment, administration of one or more MSC Compositions and / or one or more MSC Formulations is combined with one or more low doses of ultraviolet (UV) radiation and / or X-ray irradiation, thereby generating the anti-tumorigenic MSC1 phenotype in MSCs. In at least an additional embodiment, such radiation is used for MSC priming, and irradiated MSCs can be used as an immunotherapy in combination with other radiation-based therapies. Irradiated BM- MSC1 cells can secrete large amounts of TNF-a and / or IFN-y which result in several effects, including, for instance, (1) inhibiting the proliferation of tumor cells by deregulating Wnt and TGF-p / Smad signaling, and (2) inducing apoptosis of tumor cells by, for instance, blocking their cell cycle in the G1 phase. Further, irradiation of MSCs can (1) induce cleavage of caspase-3, a protein that, along with other caspase proteins, plays a role in apoptosis, (2) attenuate the phosphorylation of phosphatidylinositol 3-kinase (PI3K) / protein kinase B (PKB, also referred to as AKT), and (3) attenuate the phosphorylation of extracellular signal-regulated kinase. Accordingly, in at least an additional embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations comprising MSCs in combination with one or more low doses of UV radiation and / or X-ray irradiation. In at least another embodiment, one or more types of MSCs are primed with such irradiation before administration and / or injection into a subject.

[0202] In at least one embodiment, MSC-sourced TNF-a can induce necrosis of tumor cells in patients with one or more injured tissues (e.g., due to one or more types of cancer) and enhance the expression of specific selectins (e.g., E-family selections, P-family selectins) on tumor endothelial cells, enabling an influx of immune cells. In at least an additional embodiment, MSC- sourced IFN-y can also induce the generation of the anti-tumorigenic Ml phenotype in TAMs and can enhance the cytotoxicity of tumor-infiltrated CTLs and / or NK cells. Upon activation by MSC- derived IFN-y, CD8+ CTLs and / or NK cells can upregulate the expression of, for instance, FASL and TRAIL, and increase the release of perforins and / or granzymes that induce apoptosis of tumor cells, up-regulate expression of FASL and TRAIL and increase release of perforin and granzymes that induce apoptosis of tumor cells. IFN-y-primed Ml macrophages can either (1) phagocyte apoptotic tumor cells, or (2) secrete ROS, NO, and TNF-a, which have direct cytotoxic effects on malignant cells. Accordingly, in at least another embodiment, a method comprises exogenous administration and / or injection of one or more MSC Compositions and / or one or more MSC Formulations comprising (1) MSC-sourced TNF-a, and / or (2) MSC-sourced IFN-y. In at least another embodiment, the method comprises exogenous administration of (1) MSC-sourced TNF- a, and / or (2) MSC-sourced IFN-y.

[0203] Since MSCs may have a high affinity for tumor tissue, in at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are used as targeted delivery vehicles and / or agents of one or more treatment compounds (e.g., one or more compounds that enhance and / or promote tissue repair, healing, and / or regeneration, one or more MSC-sourced miRNAs, prodrugs, anti-cancer drugs, including, for instance, one or more chemotherapeutic drugs, and the like). Non-limiting examples of such compounds include any of the MSC-sourced miRNAs described herein, gemcitabine (GCB), paclitaxel (PTX), doxorubicin (DOX), 5 -fluorocytosine (5-FC), ganciclovir (GCV), and one or more immune-activating cytokines. In at least an additional embodiment, a method comprises exogenous administration and / or injection of one or more types of MSCs and / or MSC- derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) loaded with the anti-cancer drug PTX, which can result in reduced numbers of lung metastases, at least in melanoma-bearing animals. MSCs and / or MSC-Exos loaded with PTX may also exhibit anti-tumor properties against other types of cancer as well (e.g., ovarian cancer), including in patients with one or more injured tissues (e.g., due to one or more types of cancer). Moreover, MSCs and / or MSC-Exos may be able to uptake and secrete chemotherapeutic agents (e.g., PTX, DOX, GCB). In leukemia models, MSCs and / or MSC-Exos secreting PTX can reduce the ability of leukemia cells to adhere to the microvascular endothelium (MEC) by negatively regulating, for instance, MEC expression of intercellular adhesion molecule- 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).

[0204] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC- Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are used as targeted delivery vehicles and / or agents for DOX. MSCs and / or MSC-Exos primed with DOX may likewise induce anti-tumor effects against various tumor cells (e.g., breast cancer cells, anaplastic thyroid cancer cells). In at least an additional embodiment, DOX is loaded into one or more engineered particles (e.g., nanoparticles) coated with MSC or MSC-like membranes. Without wishing to be bound by theory, such coated particles can distribute DOX more effectively, and with fewer side effects, than general systemic administration. In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC- Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are used as targeted delivery vehicles and / or agents for one or more prodrugs. In at least an additional embodiment, one or more types of MSCs and / or MSC-derived products are engineered to express particular enzymes (e.g., HSV-thymidine kinase (also referred to as “HSV-TK”), cytosine deaminase) that can convert various prodrugs (e.g., 5-FC, GCV) into their active cytotoxic forms. For instance, MSCs modified to express HSV-TK can phosphorylate GCV into its cytotoxic metabolites, thereby resulting in anti-tumor effects, including in patients with one or more injured tissues (e.g., due to one or more types of cancers). In at least a further embodiment, one or more types of MSCs are transduced with one or more vectors (e.g., lentivectors) expressing HSV-TK. In at least another embodiment, a method comprises exogenous administration and / or injection of such modified MSCs and / or MSC-derived products, either alone or in combination with subsequent administration of GCV. Without wishing to be bound by theory, such combination treatment can result in anti-tumor effects, at least in murine cancer models. Such anti-tumor effects may result from, for instance, activating NK cell and / or CTL anti-tumor functions. In at least another embodiment, one or more types of MSCs and / or MSC-derived products that are modified to express HSV-TK can be synergistically combined with other agents (e.g., valproic acid (VP A)). Such combination therapy can cause induction of apoptosis in glioma cells; this effect may occur via, for instance, caspase activation. In at least another embodiment, one or more types of MSCs and / or MSC-derived products are genetically engineered to express both HSV-TK and TRAIL, which can reduce tumor nodule frequencies, at least in murine lung cancer models, when compared to treatment with controls. Such effects can be sustained and / or increased via routine, serial injections. In at least another embodiment, at least with respect to pancreatic cancer models, one or more types of MSCs and / or MSC-derived products engineered to express HSV-TK can home into primary pancreatic tumor stroma and induce C-C motif chemokine ligand 5 (CCL5) promoter activation. Since CCL5 expresses a chemokine that functions as a chemoattractant for various immune system cells (e.g., memory helper T cells), administration MSCs engineered to express HSV-TK can result in anti-tumor effects in patients with one or more injured tissues (e.g., due to one or more cancers, including, for example, pancreatic cancer).

[0205] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC- Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are engineered to express cytosine deaminase, which can convert 5-FC into the cytotoxic antineoplastic 5 -fluorouracil (5- FU). In at least another embodiment, a method comprises exogenous administration and / or injection of BM-MSCs expressing cytosine deaminase. This can result, at least in murine models, in lower tumor masses and weights when the mice are subsequently treated with 5-FC, as compared to treatment with 5-FC alone. In at least a further embodiment, such MSCs are administered in combination with one or more other agents (e.g., temozolomide (TMZ), an alkylating agent used to treat glioblastoma multiforme). Indeed, MSCs expressing cytosine deaminase may synergistically interact with TMZ to hinder glioma cell proliferation by, for instance, inducing cell cycle arrest and / or DNA breakage. In at least another embodiment, other combination therapies are used, including, for example, administering MSCs and / or MSC-derived products expressing cytosine deaminase with lysomustine, a nitrosourea derivative of lysine, followed by administration of 5-FC. Such a treatment protocol, at least in murine models, can result in a reduction of late-stage Lewis lung carcinoma (LLC) tumor volume and / or tumor growth, including in patients with one or more injured tissues (e.g., due to one or more types of cancer).

[0206] It should be appreciated that using one or more types of MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) for targeted drug and / or prodrug delivery, according to embodiments described herein, can have several advantages to other drug administration protocols and / or routes. In patients with one or more injured tissues (e.g., due to one or more types of cancer), MSCs and / or MSC-Exos can be administered at the site of both primary and metastatic tumors, and their targeted nature minimizes side effects that are common with other cancer treatments (e.g., systemic application of chemotherapeutic drugs). In other words, drug loaded MSCs and / or MSC-Exos can release chemotherapeutic drugs directly at the tumor site without affecting neighboring tissues. This may result in an increased half-life for the chemotherapeutic compounds, as well as more significant anti -tumor effects.

[0207] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC- Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are used as targeted delivery vehicles and / or agents for one or more cytokines, including, for instance, IL-2, IL-12, IL- 21, and TRAIL. In at least another embodiment, the one or more types of MSCs and / or MSC- derived products produce IL-2. This can assist CD8+ cells in anti-tumorigenic responses, at least in murine models of melanoma and glioma. In at least an additional embodiment, exogenous administration and / or injection of MSCs and / or MSC-derived products producing IL-12 can produce anti-tumor effects in murine models of various cancers (e.g., melanoma, cervical cancer, renal cell carcinoma (RCC), breast cancer, and glioma. IL-12-producing MSCs and / or MSC- derived products may have several effects, including, for example, activating NK cells and increasing IFN-y secretion. MSCs and / or MSC-derived products producing IL-21 may also promote IFN-y secretion and NK cell cytotoxicity, including in patients with one or more types of injured tissues (e.g., due to one or more types of cancer).

[0208] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC- Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) genetically engineered to express TRAIL. Such MSCs and / or MSC-derived products, as discussed above herein, are a potentially interesting immunotherapy treatment option for patients with one or more injured tissues (e.g., due to one or more types of cancer). This is because TRAIL uniquely targets cancer cells without harming nearby, non-cancerous cells. As mentioned above herein, the presence of TRAIL-specific receptors also referred to as death receptors, is much higher in cancer cells than non-cancer cells. Accordingly, in at least an additional embodiment, TRAIL can be used in various immunotherapies, including, but not limited to, the therapies discussed herein, in either a full- length and membrane-bound form or a modified form generally referred to as “soluble TRAIL” or “sTRAIL.” MSCs and / or MSC-derived products expressing TRAIL can migrate to tumor sites, including lung tumors, where they can induce apoptosis. Such apoptotic effects may also occur in other cancers, including, for example, pancreatic cancers, mesothelioma, renal cancer, breast cancers, neuroblastomas, and non-small cell lung cancers. In at least a further embodiment, MSCs and / or MSC-derived products expressing TRAIL may be able to target certain cancer stem cells (e.g., cluster of differentiation 133 (CD133)-positive cancer stem cells), at least in the context of non-small cell lung cancer, resulting in reduction of their proliferation and / or promotion of apoptosis. Such effects may be due to, for instance, modification of the expression of various factors (e.g., nuclear factor-KBl (NF-KB1), BAG cochaperone 3 (BAG3), myeloid cell leukemia- 1 (MCL1), DNA damage-inducible alpha (GADD45A), and harakiri (HRK)). In at least another embodiment, MSCs and / or MSC-derived products expressing TRAIL can be administered either alone or in combination with one or more other agents, including small-molecule drugs. Administration of both (1) MSCs and / or MSC-derived products expressing TRAIL and (2) smallmolecule drugs can result in increased tumor sensitivity to TRAIL.

[0209] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSCs and / or MSC-derived products (e.g., MSC- Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that are used as targeted delivery vehicles and / or agents for one or more interferons, including, for example, IFN-a, IFN-P, and IFN-y. IFN-P-producing MSCs and / or MSC-derived products can downregulate various factors, including, for instance, STAT3, Src, Akt, cMyc, MMP2, VEGF, and IL-6. Additionally, in at least a further embodiment, exogenous administration and / or injection of MSCs and / or MSC- derived products expressing IFN-a can inhibit tumor growth in patients with one or more injured tissues (e.g., due to one or more cancers), including lung cancer metastases, at least in murine models. Such inhibitory effects may result from, for example, activation of NK cells and CD8+ T cells. Further, MSCs and / or MSC-derived products expressing IFN-y can activate the TRAIL pathway, which is responsible for inducing apoptosis. MSCs and / or MSC-derived products expressing IFN-y may also upregulate caspase-3 activation, leading to apoptosis. MSCs and MSC- derived products expressing IFN-y can polarize macrophages to the Ml phenotype in vitro, as well as inducing cell cycle arrest of tumor cells in the G1 phase.

[0210] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations comprise one or more types of MSC-Exos. In at least a further embodiment, a method comprises administration of such MSC-Exos In at least an additional embodiment, the MSC-Exos contain one or more MSC-sourced products described herein, including, for instance, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration. Generally, exosomes are a subset of extracellular vesicles (“EV” or “EVs”), which are membrane-bound vesicles that can be released extracellularly. Such vesicles can contain various biological compounds, including, for example, proteins, lipids, nucleic acids, metabolites, growth factors, and cytokines. EVs may play a role as intercellular communication regulators in various biological processes.

[0211] Without wishing to be bound by theory, MSCs can, given their plastic nature, either encourage or suppress immune system responses via MSC-Exos. This can occur via, for instance, paracrine signaling. As with MSCs themselves, MSC-derived Exos can exert immune system responses (e.g., to promote tissue repair, healing, and / or regeneration) effects by, for instance, mimicking their parental cells. Further, MSC-Exos are EVs which, due to their nanosized dimensions and lipid envelope, can bypass biological barriers and deliver their cargo directly into the target cells. As a result, in at least a further embodiment, MSC-Exos can be genetically engineered to deliver a variety of anti-tumorigenic compounds to patients with one or more injured tissues (e.g., due to one or more types of cancer). Such compounds include MSC-sourced biological molecules (e.g., anti-tumorigenic miRNAs, messenger RNAs (mRNAs), enzymes, cytokines, chemokines, growth factors, immunomodulatory factors). In at least another embodiment, additional biological compounds (e.g., small -molecule drugs, proteins) are also carried by MSC-Exos. Delivery of anti-tumorigenic compounds and / or molecules directly into a tumor could result in the alteration of tumor cell viability, proliferation rate, and / or invasive characteristics, thereby treating the one or more tumors and encouraging the repair, healing, and / or reproduction of the patient’s native, healthy tissues and / or cells.

[0212] In at least one embodiment, the MSC-Exos are engineered to deliver one or more immunoregulatory miRNAs (e.g., any one or more of the miRNAs described herein) and / or one or more immunomodulatory proteins in one or more immune system cells (e.g., Ml macrophages, DCs, CD4+ Thl, CD4+ Thl7 cells), thereby enabling their phenotypic conversion into immunosuppressive M2 macrophages, tolerogenic DCs, and regulatory T cells, respectively. In at least an additional embodiment, MSC-Exos can, via delivery of one or more mRNAs and / or miRNAs, activate autophagy, inhibit apoptosis, necrosis, and / or oxidative stress. Such effects can be seen in a variety of cells, including, for example, injured hepatocytes, neurons, retinal cells, and lung, gut, and renal epithelial cells.

[0213] In at least one embodiment, the MSC-Exos contain miRNA-16-5p and miRNA-3940-5p. Such MSC-Exo-sourced miRNA-16-5p and miRNA-3940-5p can, in patients with one or more injured tissues (e.g., due to one or more cancers), inhibit the migratory properties and metastatic potential of tumor cells by, for instance, downregulating the expression of Integrin Subunit Alpha 2 (ITGA2) and Integrin Subunit Alpha 6 (ITGA6) on their membranes. MSC-Exos overexpressing miR-16-5p can inhibit proliferation, migration, and / or invasion of tumor cells (e.g., colorectal cancer cells), as well as repressing general tumor growth. Upregulation of miRNA-3940-5p can inhibit invasion of tumor cells as well; additionally, it can suppress tumor metastasis. Since miRNA-3940-5p can bind directly to ITGA6, overexpression of ITGA6 can promote tumor cell invasion and tumor progression via upregulating TGF-pi signaling.

[0214] In at least one embodiment, the MSC-Exos contain miRNA-4461. Such MSC-Exo- delivered miRNA-4461 can, in patients with one or more injured tissues (e.g., due to one or more cancers), suppress the proliferation and / or invasive properties of tumor cells (e.g., colorectal cancer cells) by, for example, reducing expression of COPI coat complex subunit beta 2 (COPB2), which is essential for Golgi budding and vesicular trafficking. miRNA-4461, which may be underexpressed in tumor cells relative to normal cells, can directly target COPB2.

[0215] In at least one embodiment, the MSC-Exos contain miRNA-15a. Such MSC-Exos carrying miRNA-15a can, in patients with one or more injured tissues (e.g., due to one or more cancers), inhibit immune escape of tumor cells by, for instance, regulating the expression of homeobox C4 (H0XC4), which binds to the promoter sequence of PD-L1, controlling its synthesis and membrane expression. MSC-derived miRNA-15a can also induce the apoptosis of tumor cells by, for example, inhibiting the activity of histone lysine demethylase 4B (KDM4B), which epigenetically regulates chromatin structure.

[0216] In at least one embodiment, one or more types of genetically engineered MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC- sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that express one or more bi-specific T-cell engaging antibodies (e.g., GPC3 -specific scFv, CD3-specific scFv) and / or produce one or more anti-tumorigenic miRNAs (e.g., any of the miRNAs described herein) can be used as therapeutic agents in the immunotherapy of malignant diseases in patients with one or more tissues (e.g., due to various types of cancers). Since MSCs can alter their phenotype and / or function in the tumor microenvironment, MSC-mediated treatments and / or MSC-Exos mediated treatments can be further tested to address potential safety concerns related to plasticity of MSCs and their possible pro-tumorigenic effects.

[0217] Accordingly, in at least a further embodiment of the disclosure, a method for treating a disease (e.g., injured tissue(s), cancer) comprises administering one or more MSC Compositions and / or one or more MSC Formulations comprising one or more types of MSCs and / or MSC- derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) that express one or more bi-specific T-cell engaging antibodies, thereby resulting in at least one of (1) directing GPC3-specific CD4+T helper cells and CD8+ CTLs towards GPC3 -expressing tumor cells, (2) increasing production of IFN-y in GPC3-specific CD4+ T cells, (3) enhancing activation of GPC3-specific CTLs, and (4) enhancing expansion of GPC3-specific CTLs. The administering may be achieved using one or more processes, including, but not limited to, intravenous, intraosseous, intraperitoneal, submandibular, and / or one or more other injection processes. The aforementioned bi-specific T-cell engaging antibodies may include, for instance, GPC3-specific scFv and / or CD3-specific scFv.

[0218] In at least an additional embodiment, the method comprises administering TNF-o. and / or ZFN-y sourced from MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC- sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) to a patient with one or more injured tissues (e.g., due to one or more types of cancer). Such administering can result in at least one of (1) deregulating Wnt signaling, (2) deregulating TGF-0 / Smad signaling, (3) blocking the cell cycle of one or more tumor cells in the G1 phase, (4) inducing necrosis of the one or more tumor cells, (5) enhancing expression of one or more selections (e.g., E-family selections, P-family selectins) on the one or more tumor cells, (6) inducing generation of an anti-tumorigenic Ml phenotype in TAMs, (7) activating CD8+ CTLs and / or NK cells to upregulate expression of FASL and / or TRAIL, (8) activating CD8+ CTLs and / or NK cells to increase release of perforins and / or granzymes, (9) priming Ml macrophages to phagocyte apoptotic tumor cells, and (10) priming Ml macrophages to secrete one or more of ROS, NO, and TNF-a. The aforementioned administering may be achieved using one or more processes, including, but not limited to, intravenous, intraosseous, intraperitoneal, submandibular, and / or one or more other injection processes.

[0219] In at least a further embodiment, the method comprises administering one or more MSC- Exos that comprise one or more of the miRNAs described herein). The administering may be achieved using one or more processes, including, but not limited to, intravenous, intraosseous, intraperitoneal, submandibular, and / or one or more other injection processes.

[0220] In at least one aspect, administration of MSC-Exo-derived miRNA-16-5p and / or MSC- Exo-derived miRNA-3940-5p (e.g., in the context of one or more MSC Compositions and / or one or more MSC Formulations) in a patient with one or more injured tissues e.g., due to one or more types of cancer) results in at least one of (1) downregulating expression of ITGA2 on tumor cell membranes, and (2) downregulating expression of ITGA6 on the tumor cell membranes. In at least an additional aspect, administration of MSC-Exo-derived miRNA-4461 results in reducing expression of COPB2 in one or more tumor cells. In at least a further aspect, administration of MSC-Exos-derived miRNA-15a results in at least one of (1) regulating expression of homeobox C4 (HOXC4), which binds to the promoter sequence of PD-L1, thereby controlling PD-L1 synthesis and membrane expression, (2) inhibiting activity of histone lysine demethylase 4B (KDM4B), thereby inducing apoptosis of the one or more tumor cells. In at least a further aspect, administration of MSC-Exos-derived miRNA-100 results in downregulation of VEGF production in the one or more tumor cells, thereby preventing generation of new blood vessels within, and / or in-between, the one or more tumor cells.

[0221] MSC Formulations

[0222] As described above herein, the exogenous administration and / or injection of MSCs and / or MSC-derived products (e.g., MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC- sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) has the potential to treat various diseases, including, but not limited to, one or more types of tissue injury and / or different types of cancers. First, as a practical matter, MSCs generally do not express costimulatory molecules, which are cell surface molecules that can either amplify or inhibit activating signals provided by the TCR to T cells, thereby influencing T cell differentiation. Accordingly, MSCs have relatively low immunogenicity, meaning that there is little or no need to administer immunosuppressive agents in conjunction with, or after, exogenous administration of MSCs and / or MSC-derived products.

[0223] In at least one embodiment, one or more of the MSC Formulations are packaged into sterile dosage units, which can be stored and distributed for use by attending physicians and / or other healthcare professionals. These formulations, which may be in various forms (e.g., fluid, lyophilized), can be administered through, for instance, sterile packaged syringes for injection, dropper bottles, tubes, or vials of solution. The dosages for injectables generally will be 0.1 cubic centimeter (cc), 0.25 cc, 0.5 cc, 1.0 cc, 2 cc, 5 cc, 10 cc, and 20 cc. The injectables can be administered at, for example, the site of the tumor. In at least one embodiment, one or more formulations described herein are sprayed onto, soaked into, or powder-dispersed onto the tumor site or cancer lesion. Efficacy of administration can generally be determined by, for instance, physician evaluations, patient self-evaluations, and / or quality of life evaluations.

[0224] In the aforementioned at least one embodiment, the sterile one or more MSC Formulations can be administered in concentrated form, diluted with sterile water or buffer, or formulated as a solution or suspension. The one or more MSC Formulations may be administered with additional therapeutic, prophylactic, and / or diagnostic agents, either in solution or suspension, or as particles (e.g., nanoparticles, liposomes, microparticles), or directly at tumor sites.

[0225] Non-limiting examples of excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe (GRAS) and may be administered to an individual without causing undesirable biological side effects or unwanted interaction(s).

[0226] In at least one embodiment, one or more MSC Formulations are in a solution or suspension. In at least one embodiment, the solutions may include sterile filtered liquids, diluted liquids, buffers, lipids, and / or oils. Emulsions are generally dispersions of oily droplets in an aqueous phase. In at least one example, there should be no evidence of breaking or coalescence in an emulsion. Suspensions generally contain solid particles dispersed in a liquid vehicle; in at least another example, such suspensions must be homogeneous when shaken gently and remain sufficiently dispersed to enable the correct dose to be removed from the container. A sediment may occur, but this should disperse readily when the container is shaken, and the size of the dispersed particles should be controlled. The active ingredient and any other suspended material must be reduced to a particle size small enough to prevent irritation and damage to the site of administration. Suspensions may comprise suitable additives, such as, for instance, antimicrobial agents, antioxidants, and stabilizing agents. In at least one embodiment, when the solution is dispensed in a multidose container that is to be used over a period of time longer than 24 hours, a preservative must be added to ensure microbiologic safety over the period of use.

[0227] In at least one embodiment, the aforementioned solution or suspension is physiological, for example, at pH 7.4. In at least an additional embodiment, the pH is optimized for both stability of the active pharmaceutical ingredient and physiological tolerance. If a buffer system is used, it must not cause precipitation or deterioration of the active ingredient. The normal useful pH range is 6.5 to 8.5, although lower pHs may be used. Buffers and / or pH adjusting agents or vehicles can be added to adjust and stabilize the pH at a desired level. In at least a further embodiment, one or more such buffers are included to minimize any change in pH during storage. Changes in pH can affect the solubility and stability of drugs; consequently, it is important to minimize fluctuations in pH. The buffer system should be sufficient to maintain the pH throughout the expected shelflife of the product. Low concentrations of buffer salts are used to prepare buffers of low buffer capacity.

[0228] Aqueous solution preparation can be optimized and / or supplemented for isotonicity, pH, antimicrobial agents, antioxidants, and / or viscosity-increasing agents. Solutions are generally considered isotonic when the tonicity is equal to that of a 0.9% solution of sodium chloride (NaCl). Tissues can usually tolerate solutions equivalent to 0.5-2% of sodium chloride. Solutions that are isotonic are therefore preferred. An amount equivalent to 0.9% NaCl is used in at least one embodiment. In at least a further embodiment, hypertonic solutions are prepared to facilitate solubility of one or more other agents co-administered with the one or more MSC Compositions and / or one or more MSC Formulations. A widely used buffer solution is Sorensen’s modified phosphate buffer, which is generally used to modulate pH values between the range of 6.5-8.0. This buffer comprises two stock solutions, one acidic containing NaH2PO4, and one basic containing Na2HPC>4. Other suitable buffers known in the art include, for example, acetate, borate, carbonate, citrate, and phosphate buffers.

[0229] In at least one embodiment, one or more MSC Formulations are packaged and / or distributed in liquid form. Alternatively, one or more such formulations can be packed as a solid, which can be obtained by, for example, lyophilization of a suitable liquid formulation. In at least an additional embodiment, the solid can be reconstituted with an appropriate carrier or diluent prior to administration. Solutions, suspensions, and / or emulsions for administration to a subject may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are known in the art, non-limiting examples of which include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

[0230] Solutions, suspensions, aerosols, sprays, and / or emulsions may also contain one or more preservatives to prevent contamination (e.g., bacterial contamination). Suitable preservatives are known in the art, non-limiting examples of which include polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as PURITE®), phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and combinations and / or mixtures thereof.

[0231] Solutions, suspensions, and / or emulsions may also contain one or more excipients known in the art, non-limiting examples of which include dispersing agents, wetting agents, and suspending agents.

[0232] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are provided in a kit. Specific formulations can be prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable side effects. These formulations (e.g., in lyophilized or fluid form) can be in sterile packaged syringes for injection, and / or tubes or jars of solution. The dosages for the injectables can be 0.1 cc, 0.25 cc, 0.5 cc, 1.0 cc, 2 cc, 5 cc, 10 cc, and 20 cc. Typically sterile kits also comprise at least one liquid to rehydrate any dry components. The kit may also include various elements facilitating the administration of prophylactics or treatments of cancer, tumors, and other disorders, such as, for example, syringes and one or more applicators (e.g., needles).

[0233] Methods of administration

[0234] Methods of using and / or administering one or more MSC Compositions and / or one or more MSC Formulations to a subject for therapeutic, diagnostic, and / or prophylactic applications, especially with respect to one or more injured tissues, one or more wound sites, and / or one or more other disorders (e.g., cancers, tumors, and other related disorders).

[0235] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered to a mammalian subject (e.g., terrestrial mammal, aquatic mammal, and the like). Such administration is performed using a suitable dosing regimen, as described above herein, and for a period of time effective to prevent formation of tumors and / or to promote healing, repair, and / or regeneration of tissues.

[0236] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations experience limited perfusion and therefore may be retained at the site of application and / or injection for an extended period of time. In at least an additional embodiment, after administration, the one or more MSC Compositions and / or one or more MSC Formulations remain at the site of application for at least 6 hours, at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least one week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 2 months, at least 3 months, at least 6 months, at least 9 months, or at least 1 year or more.

[0237] Methods of using one or more MSC Compositions and / or one or more MSC Formulations to prevent and / or treat one or more injured tissues and / or one or more other disorders (e.g., any type of cancer) are described herein. In at least one embodiment, the methods, compositions, and / or formulations are effective in preventing and / or treating one or more injured tissues, including, for instance, promoting tissue repair, healing, and / or regeneration. In at least a further embodiment, the methods, compositions, and / or formulations are also effective in preventing and / or treating cancers (e.g., breast cancer) and other related disorders, including in patients with one or more injured tissues (e.g., due to one or more types of cancer). In at least an additional embodiment, the one or more MSC Compositions and / or one or more MSC Formulations are administered in one or more amounts effective to restore tissues impacted by tissue injury, cancer, and / or tumor growth to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more than 300% of the damage present at the time of treatment, as measured by endogenous tissue regrowth.

[0238] In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered by injection near the site of tissue injury and / or tumor infarction. In at least another embodiment, one or more MSC Compositions and / or one or more MSC Formulations is sprayed onto, soaked into, and / or powder-dispersed onto the site of tissue injury and / or tumor growth.

[0239] The compositions, formulations, and / or methods of use thereof that are described herein are suitable for managing and / or treating any type of tissue injury or wound site, in addition to other associated diseases and disorders (e.g., any type of cancer or tumor). As a non-limiting example, administration of one or more MSC Compositions and / or one or more MSC Formulations may prevent and / or treat tissue injury in patients with cancer and / or a degenerative disease, contributing to the reduction of symptoms of both the cancer and the degenerative disease.

[0240] Additionally disclosed herein are methods of preventing and / or treating one or more types of tissue injury and / or other disorders (e.g., one or more types of cancer such as, for example, breast cancer, blood cancers, pancreatic adenocarcinomas, colorectal adenocarcinomas) via the administration of one or more MSC Compositions and / or one or more MSC Formulations. In at least one embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered to a patient in combination with one or more additional therapies (e.g., radiation therapy and / or chemotherapy). In at least an additional embodiment, the methods include administering to the subject one or more MSC Compositions and / or one or more MSC Formulations in conjunction with a pharmaceutically acceptable carrier. In at least one example, the methods include administering to the subject a pharmaceutical composition including an expression vector expressing one or more co-stimulatory molecules, one or more MSC Compositions and / or one or more MSC Formulations, and a pharmaceutically acceptable carrier.

[0241] In at least one embodiment, methods of promoting tissue repair, healing, and / or regeneration in a patient with one or more types of cancer include measuring a tumor sample or tumor volume from a subject, determining an appropriate dosage of one or more MSC Compositions and / or one or more MSC Formulations, and treating the subject. In at least an additional embodiment, treating the subject may include administering to the subject an effective amount of ionizing radiation in combination with an effective amount of one or more MSC Compositions and / or one or more MSC Formulations. In at least a further embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered in combination with one or more adjuvants, antigens, vaccines, allergens, antibiotics, gene therapy vectors, vaccines, kinase inhibitors, co-stimulatory molecules, Toll-like receptor (TLR) agonists, and / or TLR antagonists. In at least another embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered in combination with a second anti-cancer therapeutic agent (e.g., a chemotherapeutic nucleic acid, an immunostimulatory protein, an inflammatory molecule, an immunostimulatory molecule). In at least another embodiment, one or more MSC Compositions and / or one or more MSC Formulations are administered systemically and / or at specific sites of tissue injury and / or tumor locations in the subject.

[0242] In at least one embodiment, a method is disclosed for treating a subject with one or more types of injured tissues and / or other disorders (e.g., cancer) by enhancing or inducing response of one or more cells in the subject (e.g., one or more cancer-associated endogenous immune cells). In at least an additional embodiment, enhancing or inducing response of cancer-associated endogenous immune cells may include administering to the subject an effective amount of one or more MSC Compositions and / or one or more MSC Formulations as a prophylactic (e.g., an amount effective at preventing the appearance and / or growth of tumors). In at least a further embodiment, enhancing or inducing response of cancer-associated endogenous immune cells may include administering to the subject an effective amount of one or more MSC Compositions and / or one or more MSC Formulations to treat a subject with one or more types of injured tissues (e.g., due to a cancer or a tumor). In at least another embodiment, enhancing or inducing response of cancer- associated endogenous immune cells may include administering to the subject an effective amount of ionizing radiation, then administering to the subject an effective amount of one or more MSC Compositions and / or one or more MSC Formulations, thereby enhancing or inducing the response of cancer-associated endogenous immune cells in the subject. In at least another embodiment, the cancer-associated endogenous immune cells may include, for instance, dendritic cells, macrophages, T cells, natural killer cells, and the like.

[0243] In at least one embodiment, the compositions, formulations, and / or methods of use thereof that are described herein are used to prevent and / or treat multiple types of tissue injury and / or other disorders (e.g., multiple types of cancers). In at least an additional embodiment, the one or more MSC Compositions and / or one or more MSC Formulations is administered to a subject with both one or more types of tissue injury and another one or more disorders (e.g., cancer, systemic inflammation, a neurodegenerative disease, etc.). A cell, tissue, or target may be a cancer cell, a cancerous tissue, harbor cancerous tissue, or be a subject or patient diagnosed or at risk of developing a disease or condition. In at least one example, a cell may be an epithelial, an endothelial, a mesothelial, a glial, a stromal, and / or a mucosal cell. The cancer cell population can include, but is not limited to, a brain, a neuronal, a blood, an endometrial, a meninges, an esophageal, a lung, a cardiovascular, a liver, a lymphoid, a breast, a bone, a connective tissue, a fat, a retinal, a thyroid, a glandular, an adrenal, a pancreatic, a stomach, an intestinal, a kidney, a bladder, a colon, a prostate, a uterine, an ovarian, a cervical, a testicular, a splenic, a skin, a smooth muscle, a cardiac muscle, and / or a striated muscle cell. In at least a further example, cancer includes, but is not limited to, astrocytoma, acute myeloid leukemia, anaplastic large cell lymphoma, acute lymphoblastic leukemia, angiosarcoma, B-cell lymphoma, Burkitt’s lymphoma, breast carcinoma, bladder carcinoma, carcinoma of the head and neck, cervical carcinoma, chronic lymphoblastic leukemia, chronic myeloid leukemia, colorectal carcinoma, endometrial carcinoma, esophageal squamous cell carcinoma, Ewing’s sarcoma, fibrosarcoma, glioma, glioblastoma, gastrinoma, gastric carcinoma, hepatoblastoma, hepatocellular carcinoma, Kaposi's sarcoma, Hodgkin lymphoma, laryngeal squamous cell carcinoma, larynx carcinoma, leukemia, leiomyosarcoma, lipoma, liposarcoma, melanoma, mantle cell lymphoma, medulloblastoma, mesothelioma, myxofibrosarcoma, myeloid leukemia, mucosa-associated lymphoid tissue B cell lymphoma, multiple myeloma, high-risk myelodysplastic syndrome, nasopharyngeal carcinoma, neuroblastoma, neurofibroma, high-grade non-Hodgkin lymphoma, non-Hodgkin lymphoma, lung carcinoma, non-small cell lung carcinoma, ovarian carcinoma, oesophageal carcinoma, osteosarcoma, pancreatic carcinoma, pheochromocytoma, prostate carcinoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland tumor, Schwanomma, small cell lung cancer, squamous cell carcinoma of the head and neck, testicular tumor, thyroid carcinoma, urothelial carcinoma, and / or Wilms’ tumor.

[0244] Other non-limiting examples of cancers that may affect patients (e.g., patients with one or more types of injured tissues) include hematological malignancies such as, for example, leukemias, including acute leukemias (e.g., 1 lq23 -positive acute leukemia, acute lymphocytic leukemia (ALL), T-cell ALL, acute myelocytic leukemia, acute myelogenous leukemia (AML), and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), lymphoblastic leukemia, polycythemia vera, lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, T cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin disease, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. The compositions, formulations, and / or methods of use thereof that are described herein are also used to treat non-small cell lung cancer (NSCLC), pediatric malignancies, cervical and other tumors caused or promoted by human papilloma virus (HPV), melanoma, Barrett’s esophagus (pre-malignant syndrome), adrenal and skin cancers, and auto-immune, neoplastic cutaneous diseases.

[0245] Non-limiting examples of solid tumors that may affect patients (e.g., patients with one or more types of injured tissues) include sarcomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas), synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, peritoneal cancer, esophageal cancer, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma, and lobular breast carcinoma), lung cancer, ovarian cancer, prostate cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, squamous cell carcinoma (including head and neck squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms’ tumor, cervical cancer, fallopian tube cancer, testicular tumor, seminoma, bladder cancer (such as renal cell cancer), melanoma, and central nervous system (CNS) tumors (e.g., a glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, and retinoblastoma). Solid tumors also include tumor metastases (e.g., metastases to the lung, liver, brain, and / or bone).

[0246] In at least one embodiment, tumors (e.g., in patients with one or more types of injured tissues) comprise non-cancerous tumors such as, for instance, benign soft tissue tumors. Nonlimiting examples of benign soft tissue tumors include lipoma, angiolipoma, fibroma, benign fibrous histiocytoma, neurilemmoma, hemangioma, giant cell tumor of tendon sheath, myxoma, and the like. In at least an additional embodiment, one or more MSC Compositions and / or one or more MSC Formulations may be administered as a prophylactic or treatment for other non- cancerous soft tissue tumors, including fat tissue tumors (e.g., lipoblastoma, hibernoma), fibrous tissue tumors (e.g., elastofibroma, superficial fibromatosis, desmoid-type fibromatosis, and deep benign fibrous histiocytoma), muscle tissue tumors (e.g., leiomyomas, and rhabdomyoma), blood and lymph vessel tumors (e.g., hemangioma, glomus tumors, and lymphangioma), and nerve tissue tumors (e.g., neurofibroma and schwannoma).

[0247] In at least one embodiment, the methods described herein may include identifying and / or selecting a subject in need of treatment and / or a subject that would benefit from administration of one or more MSC Compositions and / or one or more MSC Formulations. In at least an additional embodiment, the subject to be treated is a mammal (e.g., a human, domestic animal, livestock, aquatic mammal, and the like).

[0248] One or more of various pharmaceutically acceptable carriers can be used with one or more MSC Compositions and / or one or more MSC Formulations described herein. As a non-limiting example, buffered saline and the like may be used with the one or more MSC Compositions and / or one or more MSC Formulations described herein. Optionally, these solutions may be sterilized prior to use. In at least one example, the one or more MSC Compositions and / or one or more MSC Formulations include pharmaceutically acceptable auxiliary substances such as, for example, pH adjusting and buffering agents, toxicity adjusting agents, and preservatives (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like). The concentration of these auxiliary substances and / or formulations can vary depending on individual differences in age, weight, tumor size, extent of metastasis, and condition of the subject (patient).

[0249] Methods related to the one or more MSC Compositions and / or one or more MSC Formulations and their use are provided. The one or more MSC Compositions and / or one or more MSC Formulations may be prepared as one or more pharmaceutical compositions (e.g., compositions or formulations in combination with a pharmaceutically acceptable buffer, carrier, diluent, and / or excipient) for use in one or more methods described herein. As a non-limiting example, methods are disclosed herein for administration of the one or more MSC Compositions and / or one or more MSC Formulations, methods for decreasing, downregulating, inducing, and / or increasing the expansion and / or function of one or more types of immune cells (e.g., CD4+ regulatory T cells), either ex vivo or in vivo. Additionally disclosed herein are methods of decreasing, downregulating, inducing, or increasing a population of one or more types of immune cells (e.g., DC cells, NK cells) in a subject in need thereof. The methods of treatment can include administering to a subject (e.g., a human patient) an effective amount of one or more MSC Compositions and / or one or more MSC Formulations to one or more tissues in the subject (e.g., one or more types of injured tissues, one or more types of cancerous or tumorigenic tissues).

[0250] In at least one embodiment, administration of one or more MSC Compositions and / or one or more MSC Formulations to a subject results in a decrease or increase in the proliferation and / or number of endogenous immune cells (e.g., anti-inflammatory cells). Generally, any such decrease or increase is observed within days, weeks, or months after the initial treatment, with an observed increase up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more than 500%.

[0251] Examples

[0252] Example 1: Effect of exogenous administration of MSCs and / or MSC-derived products in a murine melanoma model

[0253] This example describes the exogenous administration of MSCs (which can include MSC- derived products such as, for instance, any of the MSC-derived compounds described herein including MSC-Exos, one or more MSC-sourced miRNAs, one or more MSC-sourced biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) in a murine model of melanoma to determine whether timing of such administration affected MSC-mediated anti-tumor responses and / or anti-tumor immunity.

[0254] Cells

[0255] MSCs were isolated from bone marrow of C57BL / 6 mice, were purchased from Gibco. The murine melanoma cell line B16F10, which is syngeneic to the C57BL / 6 background, was obtained from the American Type Culture Collection (ATCC, USA). Both types of cells were grown in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 lU / mL penicillin G, and 100 pg / mL streptomycin (Sigma- Aldrich, Munich, Germany). The cells were grown at 37°C in a 5% CO2 incubator. MSCs in passage 4 and B16F10 cells in passage 4 were used in the experiments below. Animals

[0256] C57BL / 6 mice, eight to ten weeks old, were used. Mice were maintained in animal breeding facilities of the Faculty of Medical Sciences, University of Kragujevac, Serbia. All procedures were performed in accordance with the guidelines for the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, and all animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, 1985 revision). All experiments were approved by the Animal Ethical Review Board of the Faculty of Medical Sciences, University of Kragujevac, Serbia. Mice were housed in a temperature-controlled environment with a 12-hour light-dark cycle. All mice were fed with standard laboratory chow and were provided water ad libitum. At least eight mice per group were used in each experiment.

[0257] Experimental design

[0258] B16F10 cells (specifically 5xl05cells suspended in 200 pL of phosphate-buffered saline (PBS)) were subcutaneously injected in the left flank of C57BL / 6 mice. The mice were then immediately divided into four experimental groups. The first experimental group of mice intravenously received MSCs (specifically 5xl05cells suspended in 200 pL of PBS; B16F10+MSCld-treated mice) one day after injection of B16F10 cells. The second experimental group of B 16F10-treated animals intravenously received MSCs (specifically 5xl05cells suspended in 200 pL of PBS; B16F10+MSC14d-treated mice) 14 days after administration of B16F10 cells. Mice from the third and fourth experimental groups intravenously received 200 pL of PBS at comparable time points (z.e., either one day (B16F10+PBSld-treated mice) or 14 days after Bl 6F 10 administration (B16F10+PBS14d-treated animals)). All animals were sacrificed 28 days after the injection of Bl 6F 10 cells.

[0259] Measurement of tumors

[0260] Once the tumors were palpable, they were measured daily. Tumor volume was calculated using the following formula: V = 4 / 3?r * a / 2 * b / 2 * c / 2, where a = length, b = width, and c = thickness.

[0261] Measurement of cytokines

[0262] To measure cytokines in the plasma samples of tumor-bearing mice, blood samples were collected from the facial vein at days 1, 14, and 28 after injection with the B16F10 cells. Mouse blood was kept in tubes with anticoagulant and then centrifuged for 10 minutes at 2000 g at 4°C. Supernatants were then stored at -20°C until needed. The concentrations of (1) tumor necrosis factor alpha (TNF-a), (2) interferon gamma (IFN-y), (3) transforming growth factor beta (TGF-0), and (4) interleukin- 10 (IL- 10) in mouse plasma samples were measured by using enzyme-linked immunosorbent assay (ELISA) sets (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

[0263] Isolation of leucocytes

[0264] Forceps and scissors were used to resect subcutaneous tumors en bloc, including any overlying and surrounding skin. After the removal of surrounding skin, tumors were measured and weighed. The tumors were then minced using scissors until all large sections were processed into 1-2 mm pieces, which were digested in 5 mL of DMEM containing 1 mg / mL collagenase I, 1 mM EDTA, and 2% FBS (all from Sigma- Aldrich, Munich, Germany). After incubation for 2 hours at 37°C, the digested tumor tissue was incubated with 4 mL of trypsin and DNase I (Roche Diagnostics), followed by passing through a 40 pm nylon filter. Single-cell suspensions were then processed for flow cytometry analysis.

[0265] Flow cytometry analysis

[0266] The tumor-infiltrating leucocytes were investigated for different cell surface and intracellular markers using flow cytometry. Briefly, cells were incubated with anti-mouse F4 / 80, CD4, CD8, CDl lc, NK1.1, CD80, LA, granzyme B, and Fas ligand (FASL) monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions. Immune cells derived from the tumors were concomitantly stained for the intracellular content of TNF-a, IFN-y, IL- 12, IL-4, and IL- 17 by using the fixation / permeabilization kit and anti-mouse monoclonal antibodies conjugated with FITC, PE, PerCP, and APC (BD Biosciences). For intracellular cytokine staining, cells were stimulated with 50 ng / mL PMA and 500 ng / mL ionomycin for 5 hours, and GolgiStop (BD Biosciences) was added. Cells were then fixed in Cytofix / Cy toperm, permeated with 0.1% saponin, and stained with fluorescent antibodies. Flow cytometric analysis was then conducted on a BD Biosciences’ FACSCalibur machine and analyzed by using the Flowing Software analysis program. Statistical analysis

[0267] Statistical data was analyzed using statistical package SPSS, version 21. The normality of the distribution was tested with the Kolmogorov-Smirnov test. Results were then analyzed using the Student’s T-test. All data were then expressed as the mean ± standard error of the mean (SEM). The difference was considered significant when p < 0.05.

[0268] Results

[0269] Results generally showed that MSCs intravenously injected 24 hours after melanoma induction efficiently suppressed tumor growth and progression. However, MSCs intravenously injected 14 days after melanoma induction promoted tumor growth.

[0270] Specifically, tumors become palpable in B16F10+MSCld-treated mice eight days later compared with other experimental groups. Starting from day 18, average tumor volumes were significantly lower in B16F10+MSCld-treated mice than in B16F10+PBSld-treated animals (p < 0.05). Further, at day 28, the average volume and weight of tumors removed from B16F10+MSCld-treated mice were significantly lower than melanomas taken from B16F10+PBSld-treated animals.

[0271] By contrast, starting from day 18 (that is, four days after MSC injection), the average tumor volumes in B16F10+MSC14d-treated mice were significantly greater than in B16F10+PBS14d- treated mice. Thus, at day 28, the average volume and weight of tumors removed from B16F10+PBS14d-treated mice were significantly lower than those of melanomas of B16F10+MSC14d-treated mice. Further, While the lowest survival rate was observed in B16F10+MSC14d-treated mice, all of the melanoma-bearing animals that received MSCs 24 h after tumor induction survived till the end of the experiment.

[0272] Since, as described above herein, MSCs can adopt pro-inflammatory (i.e., MSC1) or immunosuppressive (i.e., MSC2) phenotypes in response to the inflammatory and immunosuppressive cytokines to which they are exposed, the concentration of inflammatory (TNF-a, IFN-y) and immunosuppressive cytokines (IL- 10, TGF-P) in plasma samples of melanoma-bearing mice at the time of MSC administration were analyzed and compared. The ratios of pro-inflammatory to anti-inflammatory cytokines (TNF-a : IL-10, TNF-a : TGF-P, IFN- y : IL-10, IFN-y : TGF-P, IL-12 : IL-10, and IL-12 : TGF-P) were significantly lower in plasma samples ofB16F10+PBSld-treated mice compared to B16F10+PBS14d-treated animals (p < 0.001). This suggests that MSCs, administered one day after the injection of tumor cells, were exposed to a higher concentration of immunosuppressive cytokines, while MSCs transplanted 14 days after tumor induction were exposed to a higher concentration of inflammatory cytokines. Thus, MSCs administered during the initial phase of melanoma growth adopted a pro-inflammatory (MSC1) phenotype, while MSCs administered during the progressive stage of melanoma growth adopted an immunosuppressive (MSC2) phenotype.

[0273] Further supporting this conclusion is the fact that MSCs administered 24 hours after tumor induction significantly enhanced NK and T-cell driven antitumor immunity. Specifically, the cellular makeup of tumors obtained from B16F10+PBSld-treated mice and B16F10+MSCld- treated mice revealed that MSCs, when injected 24 hours after melanoma induction, significantly increased the total number of tumor-infiltrating cytotoxic NK1.1+NK cells (p < 0.05). Additionally, there were significantly higher numbers of IFN-y-producing (p < 0.05) and FASL- and granzyme B-expressing (p < 0.05) NK cells in the tumors of B16F10+MSCld-treated mice. This result indicates that MSCs, when administered 24 hours after melanoma induction, enhanced the cytotoxic and antitumorigenic potential of NK cells in tumor-bearing animals.

[0274] Additionally, the tumors of B16F10+MSCld-treated mice contained significantly higher numbers of both CD4+ helper T cells (p < 0.05) and CD8+ CTLs (p < 0.05) than in melanomas of B16F10+PBSld-treated mice. The phenotype and function of these CD4+ helper T cells and CD8+ CTLs revealed that MSCs, when injected 24 hours after melanoma induction, significantly increased the presence of (1) antitumorigenic and IFN-y- and TNF-a-producing CD4+ Thl cells (p < 0.05 for IFN-y and p < 0.001 for TNF-a), (2) IL-17-producing CD4+ Thl7 cells (p < 0.001), and (3) IFN-y- and TNF-a-producing CD8+ CTLs in melanoma-bearing animals.

[0275] Similarly, significantly higher plasma levels of the inflammatory and antitumorigenic cytokines TNF-a (p < 0.05) and IFN-y (p < 0.05), and significantly lower plasma levels of immunosuppressive cytokines TGF- (p < 0.05) and IL-10 (p < 0.05) were observed in B16F10+MSCld-treated mice. These results further indicate that, when MSCs are transplanted during the initial phase of melanoma growth, MSCs enhance the anti-tumor immune response in melanoma-bearing animals.

[0276] By contrast, MSCs, when transplanted 14 days after melanoma induction, attenuated tumoricidal capacity of NK cells, as evidenced by the lower number of tumor-infiltrating granzyme B-expressing NK1.1 + cells in B16F10+MSC14d-treated mice (p < 0.05). Further, MSCs injected 14 days after melanoma induction suppressed the tumoricidal capacity of CD8+ CTLs, CD4+ Thl, and CD4+ Th 17 lymphocytes. Intracellular staining revealed that MSCs suppressed production of tumoricidal cytokines (e.g., IFN-y and IL-17) in CD4+ Thl and Thl7 cells (p < 0.05 for TNF-a and IL-17) and in CTLs (p < 0.05 for IFN-y and IL-17) of B16F10+MSC14d-treated mice. This may have prevented generation of optimal TNF-a, IFN-y, and IL-17-driven anti-tumor immune responses. Additionally, a significantly lower number of granzyme B-expressing CD8+ CTLs were observed in the tumors of B16F10+MSC14d-treated mice (p < 0.05), indicating that MSCs injected 14 days after tumor induction significantly reduced the presence of cytotoxic and pro- apoptotic CD8+ CTLs in the tumors of melanoma-bearing animals.

[0277] Finally, significantly lower levels of antitumorigenic cytokines TNF-a and IFN-y (p < 0.05) and significantly higher levels of TGF-P and IL-10 (p < 0.001) were found in plasma samples of B16F10+MSC14d-treated mice. This result indicated that MSCs and / or MSC-derived products (including, for instance, any of the MSC-derived compounds described herein such as, e.g., MSC- Exos and / or one or more MSC-sourced miRNAs), when injected during the progressive stage of melanoma development, attenuated anti-tumor immunity by increasing production of immunosuppressive cytokines in tumor-bearing animals.

[0278] Example 2: Effects of MSCs and MSC-derived products

[0279] This example describes generation and therapeutic effects of MSCs and MSC-derived products (e.g., products with biomaterials, growth factors, immunomodulatory cytokines, and other compounds that enhance and / or promote tissue repair, healing, and / or regeneration). One such product is Exosomes Derived Multiple Allogeneic Proteins Paracrine Signaling (Exosomes d-MAPPS).

[0280] Sample acquisition

[0281] Exosomes d-MAPPS is an engineered biologic product obtained from placental tissue, previously collected from healthy human donors. Blood samples were provided by the donor prior to, or at the time of, collection and were tested by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) and were found negative using United States (U.S.) Food and Drug Administration (FDA) licensed tests for detection of, at minimum, hepatitis B virus, hepatitis C virus, human immunodeficiency virus types 1 / 2, and Treponema Pallidum. Placental tissue samples were obtained with patient consent as well as institutional ethical approval and kept at 4°C until processed. Samples were engineered as a sterile product, manufactured under current Good Manufacturing Practices (cGMP) regulations and reviewed by the FDA.

[0282] Presence of cytokines, chemokines, and growth factors

[0283] The concentrations of cytokines, chemokines, growth factors and their receptors in Exosomes d-MAPPS samples were determined. Briefly, about fifty milliliters of sample was concentrated to 1.0 ml protein with trichloroacetic acid. The acetone-washed protein pellet was then resolubilized in urea, and proteins were processed with dithiothreitol and iodoacetamide and digested with trypsin. Tryptic peptides were quantified and 10 pg was loaded through pressure cell onto a biphasic column for online two-dimensional high-performance liquid chromatography (HPLC) separation (strong-cation exchange and reversed-phase) and concurrent analysis by nanospray using a hybrid mass spectrometer. Three salt cuts of 50, 100, and 500 mM ammonium acetate were performed per sample run, with each followed by a 120-min organic gradient to separate the peptides.

[0284] Resultant peptide fragmentation spectra were compared with proteome database concatenated with common contaminants and reversed sequences to control false discovery rates. Peptide spectrum matches (PSMs) were filtered and assigned matched-ion intensities (MITs) based on observed peptide fragment peaks. PSM MITs were summed on a per-peptide basis, and only those uniquely and specifically matching a particular protein were moved onto subsequent analysis. Briefly, peptide intensity distributions were log-transformed, normalized across biological replicates by LOESS, and standardized by median absolute deviation and mean centering across samples as suggested. Peptides were then filtered to maintain at least two hits in one replicate set, and missing values were imputed using a random distribution of low-level values. Peptide abundance trends for each protein were scaled to a specific, well-sampled reference peptide. Sample-to-sample variation was visualized by PC A, Pearson’s correlation and hierarchically clustered using the Ward agglomeration method to generate a heat map of protein abundance trends normalized by z-score.

[0285] Results

[0286] The concentrations of major MSC-derived immunomodulatory molecules were analyzed, specifically, levels of IDO, IL-lra, IL-10, IL-4, IL-13, IL-18 binding protein (IL-18 Bpa), TGFpi and Latency associated peptide of TGFpi (LAP (TGFpi), were measured. IL-IRa was found in high concentrations (1000 pg / pl); MSC-derived IL-IRa is a naturally occurring cytokine which acts as an inhibitor of inflammatory cytokine IL-l. When IL-IRa binds to the IL-1 receptor (IL- 1R), binding of IL-1 is blocked and pro-inflammatory signal from IL-1 receptor is stopped. In line with these findings, a high concentration of IL-IRa indicates strong anti-inflammatory and immunomodulatory potential.

[0287] Additionally, the main inflammatory cytokines of innate immunity (e.g. TNF-a, IL-ip, IL- 12, IL-18) were not detected. Similarly, Thl (IFN-y), Th2 (IL-4, IL-5, IL-10, IL-13) and Thl7 (IL- 17 and IL-22) cytokines were present in non-detectable concentrations, indicating that neither one of the T cell-dependent inflammatory pathways could be elicited by the sample.

[0288] Promoting migration of CXCR6, CCR7, and CXCR4 expressing cells

[0289] As described above herein, MSCs have a capacity to home towards the site of injury or inflammation where they, in a juxtacrine and / or paracrine manner, suppress detrimental immune responses and ongoing inflammation. MSCs express chemokine-specific receptors (CXCR4, CX3CR1, CXCR6, CCR1, and CCR7) and are attracted by chemokines (CXCL12, CXCL14, CX3CL1, CXCL16, CCL3, CCL19, and CCL21) released from damaged tissues and inflammatory immune cells. MSCs themselves are also able to produce chemokines which, in autocrine manner, enable migration of MSCs towards the site of injury or inflammation. In line with these observations, high concentrations ofMSCs-derived chemokine CXCL16 were found in the sample (1500 pg / pl). Since CXCR6, the ligand for CXCL16, is highly expressed on MSCs and immune cells (e.g., memory / effector T cells, NK cells, NKT cells, and plasma cells), high concentrations of this chemokine strongly indicates that MSC-Exos and exosome-derived products such as Exosomes d-MAPPS can be used as a chemoattractant, enabling migration of CXCR6 expressing cells into inflamed or injured tissues.

[0290] Similarly, 6Ckine (CCL21) (ligand for CCR7 receptor) was measured in the sample (500 pg / pl. Bearing in mind that CCL21 :CCR7 axis is important for migration of MSCs in wounds, homing of naive T cells in peripheral lymph nodes and for migration of antigen processing, activated DCs into peripheral lymph nodes and T cell-rich fields within injured lungs, synovia, and eyes, high levels of CCL21 could be used for recruitment of CCR7 expressing MSCs and immune cells for treatment of skinjoint, eye, and / or lung inflammatory diseases. In line with these findings, high concentrations (2000pg / ml) of platelet factor 4 (PF4), which is involved in tissue regeneration and wound repair, was found in the sample as well.

[0291] CXCL14 was also detected in the sample (500 pg / pl). CXCL14 specifically binds to CXCR4 and, in a similar manner as CXCL12, is involved in CXCR4-dependant migration of MSCs into injured or inflamed tissues.

[0292] In addition to elevated levels of CXCL16, CCL21, PF4, and CXCL14, GRO-well known MSC-derived chemokine with strong immunosuppressive properties was detected (500 pg / pl). Human MSCs secrete GRO-y which, accompanied with GRO-a, promote conversion of monocyte derived DCs (MDDCs) towards a myeloid suppressive phenotype, enabling generation of tolerogenic myeloid derived suppressor cells (MDSCs). In line with these findings, presence of GRO in the sample strongly indicates potential for generation of MDSCs and MDSCs-based cell therapy of, e.g., autoimmune and chronic inflammatory diseases.

[0293] Inducing neo-vascul arization in a VEGF-dependent manner

[0294] Since the generation of new blood vessels and re-vascularization are mainly responsible for MSC-dependent regeneration of ischemic tissues, the presence of angiogenesis-related growth factor receptors in the sample was determined. Results indicate the capacity of MSC-Exos and exosome-derived products (e.g., MSC-sourced miRNAs) to induce neo-angiogenesis-based tissue regeneration. High concentrations of VEGFR1 (20000 pg / pl) were found in the sample. VEGFR1 plays a critical role in the migration of MSCs and MSC-based neo-angiogenesis. VEGFR1 also binds VEGF and is expressed by multiple bone marrow-derived cell types, including endothelial progenitor cells and MSCs. BM-derived endothelial progenitor cells and MSCs are mobilized into peripheral blood and_recruited to the sites of ischemia in a VEGFR1 -dependent manner, where they participate in tissue repair and revascularization. Based on these results, MSC-Exos and exosome-derived products (e.g., Exosomes d-MAPPS, which may contain one or more MSC- sourced miRNAs) can modulate generation and maturation of BM-derived cells. In line with these observations, high concentrations of granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) were also noticed in the sample (20000 pg / pl). Since signaling from GM-CSFR can promote a variety of cellular functions, including protection from apoptosis, progression through the cell cycle, early commitment to myelopoiesis, differentiation / maturation of committed progenitors, and multiple activation and motility functions in mature immune cells, MSC-Exos and exosome-derived products e.g., Exosomes d-MAPPS, which may contain one or more MSC- sourced miRNAs) can be used for controlled differentiation of BM-derived, GM-CSFR expressing cells.

[0295] Example 3: Treatment of COPD patients with MSCs and MSC-derived products

[0296] This example describes therapeutic effects of MSCs and MSC-derived products (e.g., Exosomes d-MAPPS, which may contain one or more biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration), in patients with one or more injured tissues, specifically, COPD patients.

[0297] Sample acquisition

[0298] Sterile Exosomes d-MAPPS, which may include one or more biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration, is an engineered biological product obtained from placenta MSCs (PL-MSCs) previously collected from healthy human donors. PL-MSC samples were obtained with patient consent as well as institutional ethical approval and kept at 4°C until processed. All donors prior to, or at the time of, collection were tested by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) and were found negative using United States (U.S.) Food and Drug Administration (FDA) licensed tests for the detection of, at minimum, hepatitis B virus, hepatitis C virus, human immunodeficiency virus types 1 / 2, and Treponema pallidum.

[0299] Exosomes d-MAPPS was engineered as a sterile product and manufactured under current Good Manufacturing Practices (cGMP) regulated and reviewed by the FDA. Briefly, PL-MSCs were grown in complete MSC Dulbecco’s Modified Eagle's Medium (DMEM). Low passage (<5) PL-MSCs were grown to 60%-80% confluence in multiflasks before isolation. Fresh PL-MSC media were layered and collected after 48 to 72 hours (conditioned medium). Exosomes (Exos) were isolated by the ultracentrifugation protocol (100,000 g at 4°C for 70 min). The isolation of Exos was performed by positive selection using the pMACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany) and the Exosome Isolation Kit Pan, human (Miltenyi Biotec, Bergisch Gladbach, Germany) which contained a cocktail of MicroBeads conjugated to the tetraspanin proteins CD9, CD63, and CD81. Briefly, Exos were magnetically labeled and loaded onto a i column, which was placed in the magnetic field of a pMACS™ Separator. The magnetically labeled Exos were retained within the column, while the unlabeled vesicles and cell components run through the column. After removing the column from the magnetic field, the intact Exos were collected by elution. Exos were stored at -70°C until use.

[0300] Animals

[0301] For animal studies, eight- to ten-week-old male BALB / c mice were used. Mice were maintained in animal facilities of the Faculty of Medical Sciences, University of Kragujevac, Serbia. All animals received humane care, and all experiments were approved by and conducted in accordance with the Guidelines of the Animal Ethics Committee of the Faculty of Medical Sciences of the University of Kragujevac. Mice were housed in a temperature-controlled environment with a 12-hour light-dark cycle and were administered with standard laboratory chow and water ad libitum.

[0302] Experimental design

[0303] Animals were randomly divided into control and experimental groups (n = 8 mice per group). Mice from the experimental group underwent whole-body exposure to cigarette smoke (CS) of 5 cigarettes in a CS chamber with 30-minute smoke-free intervals, every day for four weeks. The smoke exposure experimental box, adapted for a group of 8 mice, consisted of a box body and a cover. CS was drawn through an exposure chamber by negative pressure using an extraction pump. Between draws of CS, room air was continuously drawn through the chamber. The smoke-to-air ratio was 1 : 12 to protect mice from acute smoke toxicity and death.

[0304] After four weeks of CS treatment, mice were randomly divided into two groups and received either vehicle or Exosomes d-MAPPS (0.1 mL / intraperitoneally / 5 days per week for three weeks). Mice from the control group were exposed to air only and received either vehicle or Exosomes d-MAPPS.

[0305] Histopathological analysis

[0306] All mice were sacrificed 8 weeks after initial CS exposure, and the lungs were isolated for histopathological analysis. The isolated lungs were fixed in 10% formalin, embedded in paraffin, and consecutive 4 pm tissue sections were mounted on slides. Sections were stained with hematoxylin and eosin (H&E) and examined under a low-power (lOOx) light microscope-equipped digital camera (Zeiss Axioskop 40, Jena, Germany). Blood gas analysis

[0307] In order to explore whether Exosomes d-MAPPS treatment managed to improve extracellular acid-base status and gas exchange in CS-exposed mice, blood gas parameters (partial pressure of oxygen in arterial blood (PaCh), partial pressure of carbon dioxide (PaCCh) in arterial blood, oxygen saturation (SaCL), and pH) were analyzed. For this purpose, arterial blood samples were obtained from control and experimental animals and analyzed within a few minutes using a test cartridge blood analysis system (Premier GEM 3500, Instrumentation Laboratory, Bedford, Massachusetts, USA).

[0308] Isolation of lung-infiltrated immune cells

[0309] Lungs obtained from control and CS-exposed mice were washed with sterile phosphate- buffered saline (PBS) and placed in Petri dishes with DMEM supplemented with 10% FBS. The dissected lung tissues were incubated in a medium that contained collagenase type IV (0.5 mg / mL) and type IV bovine pancreatic DNAse (Roche Diagnostics; 1 mg / mL) at 37°C for 45 minutes. The cells were filtered through a 100 pm nylon cell strainer into a clean 50 mL conical tube. Then, cells were pelleted by centrifuging for 10 min at 300 g at 10°C. Red blood cells were depleted with a lysis buffer (0.144 M NH4CI, 0.0169 M TRIS base, pH 7.4) at 37°C in a 5% CO2 atmosphere for 5 minutes.

[0310] Flow cytometry analysis and intracellular staining

[0311] Lung-infiltrated immune cells were screened for various cell surface and intracellular markers by flow cytometry. Since a combination of mechanical and enzymatic dissociations of lung tissue may result in cell damage and death, the MACS® Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for magnetic cell separation of viable cells. Briefly, a single-cell suspension of lung-infiltrated cells was resuspended in 100 pL of the Dead Cell Removal MicroBeads (per 107 of cells), mixed, and incubated for 15 minutes at room temperature. Cells were applied on MS columns within lx MACS Binding Buffer. Effluent that passed through the column contained live cells. To reduce nonspecific binding of antibodies, viable lung-infiltrated cells were incubated with an anti-Fc block (anti-mouse CD16 / CD32). For that purpose, the cell suspension was incubated with 1 pg of the BD Fc Block / 106 cells in 100 pL of staining buffer (Dulbecco's PBS (DPBS) without Mg2+or Ca2+, 1% heat-inactivated FCS, and 0.09% (w / v) sodium azide) for 15 minutes at 4°C. The cells were then washed and stained with fluorochrome-conjugated antibodies. Briefly, IxlO6cells were incubated with anti -mouse CD45, F4 / 80, I-A, CD80, CD206, CDl lc, NKp46, Gr-1, CD3, CD4, CD8, CXCR3, monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC) (all from BD Biosciences, San Jose, CA, USA) in a staining buffer for 30 minutes in the dark at 4°C. Cells were washed twice in a staining buffer and pelleted by centrifugation. For intracellular cytokine staining, cells were stimulated with 50 ng / mL phorbol 12-myristate 13-acetate (PMA) and 500 ng / mL ionomycin for 5 hours and GolgiStop (BD Biosciences, San Jose, CA, USA) was added. Cells were then incubated in a BD fixation / permeabilization solution (BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit) for 20 minutes at 4°C. Afterwards, cells were washed two times in lx BD Perm / Wash™ buffer (BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit) and pelleted. Fixed / permeabilized cells were concomitantly resuspended in 50 pF of BD Perm / Wash™ buffer containing a predetermined optimal concentration of fluorochrome-conjugated antibodies specific for FoxP3, TNF-a, IL-12, IL- 10, IL- 10, IFN-y, and IL- 17 by using appropriate anti-mouse monoclonal antibodies conjugated with FITC, PE, PerCP, and APC (BD Biosciences, San Jose, CA, USA). Cells were incubated with fluorochrome-conjugated antibodies at 4°C for 30 minutes in the dark. Afterwards, cells were washed 2 times with lx BD Perm / Wash™ buffer and resuspended in a staining buffer prior to flow cytometric analysis. In experiments in which the phenotype and function of T cells were analyzed, CD3+ T lymphocytes were isolated from the population of viable lung-infiltrated cells by magnetic separation. Forthat purpose, the MACS Separator, the MACS Columns, and the CD3s MicroBead Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, Germany) were used. Afterwards, CD3+ T cells were stained with fluorochrome-conjugated anti-mouse antibodies specific for CD4, CD8, CXCR3, FoxP3, TNF-a, IL-10, IFN-y, and IL-17, following the procedure that was described above. Flow cytometric analysis was conducted on a BD Biosciences’ FACSCalibur and analyzed by using the Flowing Software analysis program.

[0312] Determination of cytokines

[0313] Commercial ELISA sets (R&D Systems, Minneapolis, MN, USA) were used to determine the concentration of TNF-a, IL- 12, IL- 10, IL- 10, and IFN-y in serum samples of control and experimental animals.

[0314] COPP patients Thirty COPD patients were recruited with the aim to receive an Exosomes d-MAPPS inhalation solution. Patients enrolled in this study were men (n = 20) or postmenopausal women (n = 10) aged between 50 and 75 years, having a postbronchodilator forced expiratory volume in 1 s (FEV1) > 30% and < 80% predicted, a postbronchodilator FEVl / forced vital capacity (FVC) < 70%, a smoking history of > 10 packs per year, and lung hyperinflation defined as a functional residual capacity (FRC) greater than 120%. Subjects with past or current history of abnormal vital signs, abnormal laboratory findings, clinically relevant ECG abnormalities, or cardiovascular conditions prior to screening were excluded from the study. All subjects provided written informed consent prior to study participation.

[0315] Clinical study design

[0316] Patients received Exosomes d-MAPPS inhalation solution (0.5 mL / once per week for three weeks) containing a high concentration of immunosuppressive factors (soluble TNF receptors I and II (sTNFRI and sTNFRII), IL-1 receptor antagonist (IL-IRa), and soluble receptor for advanced glycation end products (sRAGE)). Pulmonary function tests and clinical findings were recorded before, and 1 month after, such treatment. Spirometry was performed according to recommendations from the American Thoracic Society guidelines. Forced expiratory volume in 1 second (FEV1) and peak expiratory flow (PEF) rate were recorded. Chest computed tomography (CT), standard clinical COPD questionnaire (CCQ) scoring, and 6-minute walking distance (6MWD) test as a submaximal test of aerobic capacity / endurance were used to determine the effects of the treatment.

[0317] Statistical analysis

[0318] The results obtained in the animal study were analyzed using the Student t-test. All data in animal studies were expressed as the mean ± standard error of the mean (SEM). The Wilcoxon signed-rank test was applied to demonstrate differences in pulmonary function of COPD patients before and after Exosomes d-MAPPS treatment. Values of P < 0.05 were considered statistically significant.

[0319] Results

[0320] Results from both the animal models and the human patients generally showed alleviation of chronic airway inflammation after treatment, as described in further detail below.

[0321] Chronic airway inflammation in mice Remarkably improved respiratory function, as evidenced by significantly elevated PaCh (P < 0.0001), O2 saturation (P < 0.0001), and pH (P < 0.0001) and decreased PaCCh (P < 0.0001), was observed in CS-treated mice that received Exosomes d-MAPPS. Accordingly, depression-like behavior and loss of locomotor activity were not seen in CS+ Exosomes d-MAPPS-treated animals.

[0322] The alveolar wall was intact, and leucocyte accumulation was not seen, in the lung parenchyma of control animals. By contrast, partial alveolar wall destruction, widened alveolar septa and expanded alveolar space, capillary dilation, and congestion with massive infiltration of neutrophils, lymphocytes, and monocytes were observed in the lungs of CS-exposed mice. Importantly, preserved alveolar and blood vessel structures and a significantly lower number of lung-infiltrated leucocytes were noticed in the lungs of CS+ Exosomes d-MAPPS-treated animals, indicating that treatment managed to attenuate inflammation-related pathological changes in the lungs of CS-exposed mice, thereby resulting in improved repair, healing, and / or regeneration of normal tissues and / or cells.

[0323] In line with these findings, a significantly lower concentration of inflammatory cytokines that play an important pathogenic role in the development and progression of CS-induced airway inflammation (e.g, TNF-a, IL-10, IL-12, and IFN-y) was observed in serum samples of Exosomes d-MAPPS-treated CS-exposed mice compared to CS+ vehicle-treated animals (P < 0.05 for TNF- a, IL-12, and IFN-y; P < 0.01 for IL-10). Additionally, treatment resulted in the elevation of antiinflammatory and immunosuppressive IL-10 (P < 0.01), which is involved in lung repair and regeneration.

[0324] Inflammatory Macrophages, Neutrophils, and NK and NKT Cells in Inflamed Lungs

[0325] Treatment managed to significantly reduce the total number of lung-infiltrated macrophages in CS-exposed mice (P < 0.001). Additionally, Exosomes d-MAPPS remarkably attenuated antigen-presenting capacities of alveolar macrophages, as evidenced by a significantly reduced number of CD80- and LA-expressing F4 / 80+ cells in the lungs of CS+ Exosomes d- MAPPS-treated animals (P < 0.001). Intracellular staining revealed that treatment significantly attenuated the production of inflammatory TNF-a (P < 0.001) and IL-12 (P < 0.01) in lung- infiltrated macrophages. Furthermore, a significantly higher number of alternatively activated, IL- 10-producing and CD206-expressing M2 macrophages were noticed in the lungs of Exosomes d- MAPPS-treated CS-exposed mice (P < 0.01), indicating that Exosomes d-MAPPS treatment suppressed inflammation and promoted the generation of an immunosuppressive phenotype in lung-infiltrated macrophages.

[0326] Additionally, treatment attenuated the capacity of NK and NKT cells and neutrophils to produce inflammatory cytokines in CS-injured lungs. A significantly lower number of IL-17A- producing NK and NKT cells (P < 0.001 for NK and P < 0.05 for NKT cells), ZFN-y-secreting NK and NKT cells (P < 0.001), and TNF-a and IL-ip-producing neutrophils (P < 0.001) were observed in the lungs of Exosomes d-MAPPS -treated CS-exposed mice.

[0327] Attenuated Activation of CD4+ and CD8+ T Lymphocytes

[0328] Exosomes d-MAPPS affected the migratory and antigen-presenting properties of DCs. A significantly lower number of F4 / 80-CD1 lc+I-A+ DCs were observed in the CS-injured lungs of treated animals (P < 0.001). The total number of lung-infiltrated F4 / 80-CD1 lc+I-A+ DCs that expressed costimulatory molecule CD80 (P < 0.01) was significantly lower in CS-treated mice that received Exosomes d-MAPPS. Furthermore, a decreased number of proinflammatory, IL-12- producing F4 / 80-CD1 lc+I-A+ DCs (P < 0.001) and an increased presence of immunosuppressive and tolerogenic, IL-10-producing F4 / 80-CD1 lc+I-A+ DCs (P < 0.001) were observed in the lungs of CS+ Exosomes d-MAPPS-treated animals, indicating that the treatment attenuated the antigen- presenting and proinflammatory properties of airway DCs.

[0329] Exosomes d-MAPPS-induced modulation of DC function resulted in alleviated activation of inflammatory, IFN-y- and IL-17-producing CD4+ and CD8+ T lymphocytes. A significantly lower number of CXCR3 -expressing and ZFN-y-producing CD4+ Thl cells (P < 0.01) and IL-17- producing CD4+ Thl7 cells (P < 0.01) were observed in the lungs of treated CS-exposed mice. Similarly, Exosomes d-MAPPS treatment attenuated the influx of CXCR-expressing, IFN-y- producing (P < 0.001), and IL-17-producing CD8+ CTLs (P < 0.01) and reduced the total number of alveolotoxic, TNF-a-producing CD8+ CTLs (P < 0.001) in CS-injured lungs. Importantly, treatment significantly increased the total number of lung-infiltrated anti-inflammatory, IL-10- producing CD4+ FoxP3+ regulatory T cells (Tregs) (P < 0.05), enabling the generation of an immunosuppressive microenvironment in the inflamed lungs.

[0330] Improved Pulmonary Status of COPP Patients

[0331] Exosomes d-MAPPS (e.g., including one or more biological compounds that enhance and / or promote tissue repair, healing, and / or regeneration) contained a high concentration of soluble immunosuppressive mediators (e.g., sTNFRI, sTNFRII, IL-IRa, and sRAGE). Clinical parameters and CT findings indicated the beneficial effects of Exosomes d-MAPPS in the alleviation of chronic lung inflammation and / or concomitant improvement in tissue repair, healing, and / or regeneration. All of the 30 treated patients showed a marked improvement in pulmonary status, as evidenced by an increase in percentage change relative to the initial value of FEV1 (%AFEV1), significantly higher PEF, decreased CCQ total score, and increased 6-minute walking distance (6MWD). Additionally, quality of life was significantly improved after treatment and all treated patients managed to perform daily activities without hindrance. Clinical findings were confirmed by CT. Inflammation-induced destruction of alveoli and air trapping caused hyperinflation of the lungs with flattening of the diaphragm in COPD patients. Exosomes d- MAPPS significantly alleviated emphysematous changes in the lungs of COPD patients. Lungs were less hyperexpanded, diaphragms were less flattened, and centrilobular and paraseptal emphysema were significantly reduced one month after Exosomes d-MAPPS administration, indicating the beneficial effects of treatment in the attenuation of emphysema in COPD patients. Importantly, Exosomes d-MAPPS was well tolerated. None of the 30 treated COPD patients reported any side effects related to Exosomes d-MAPPS administration.

[0332] Example 4: Preparation of d-MAPPS solutions

[0333] Materials and methods

[0334] A non-limiting example of a product containing MSCs and / or MSC-derived products that can be used to treat injured tissues is a d-MAPPS solution, which is an engineered biological product obtained from MSCs (e.g., placental tissue-derived MSCs (“PL-MSC” or “PL-MSCs”)) and / or the amniotic fluid from healthy human donors. Human placental tissue and amniotic fluid was collected from healthy human donor Caesarean sections, as described above. Amniotic fluid and placental tissue were stored in refrigerated condition at 2.5° C. to 6.5° C. prior to the clarification and filtration process. Regarding amniotic fluid preparation, amniotic fluid was centrifuged at 5,000 to 10,000 rpm for 20 minutes to 1 hour in 50 mL to 250 mL receptacles. The supernatant was collected. When collecting the supernatant, it was important to avoid detaching or aspirating insoluble components. If the supernatant contained residual insoluble components, they were pre-filtered with 5 to 10 p cellulose ester capsule pre-filters without TRITON® surfactant to avoid contamination in the filtration process. The liquid phase was collected and filtered with poly ether sulfone 1.0 p capsule filters and the liquid was collected. The liquid was then filtered with poly ether sulfone 0.25 p capsule filter. The filtrate was transferred to vials and sealed with stoppers aseptically. Four samples from the final filtrate were taken to test whether the sterile filtered human amniotic fluid retained exogenous immune cells of interest. Generally, the concentration of the exogenous immune cells in the sterile filtered amniotic fluid was from about 20 pg / mL to about 2400 pg / mL. The concentrations of all the exogenous immune cells in the four samples were in the range of 20-150 pg / mL.

[0335] The d-MAPPS solution may also include MSC-Exos and / or MSC-Exos-derived growth factors and / or cytokines. PL-MSC were grown in complete DMEM. Low passage (<5) PL-MSCs were grown to 60%-80% confluence in multiflasks before isolation. Fresh PL-MSC media were layered and collected after 48 to 72 h (conditioned medium). Exosomes (“Exos”) were isolated by the ultracentrifugation protocol (100,000g at 4°C for 70 min). The isolation of exosomes was performed by positive selection using the / MACS™ Separator (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No. 130-042-602) and the Exosome Isolation Kit Pan, human (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat. No. 130-110-912), which contained a cocktail of MicroBeads conjugated to the tetraspanin proteins CD9, CD63, and CD81.

[0336] The d-MAPPS solution was further prepared by collecting MSCs and / or amniotic fluid. De-cellularization was then performed to remove only cells and particulate matter by a series of centrifugation and fdtration steps (e.g., centrifuged at 5,000 to 15,000 rpm for 15 minutes to 1 hour; filtration of supernatant with 5 to 10 p cellulose ester capsule filters). Next, the de- cellularized liquid was incubated and / or stored from 1°C to 20°C, from 2°C to 8°C, at 4°C, or at room temperature, for one or more days, weeks, months, or up to a year.

[0337] Before d-MAPPS isolation, blood samples of healthy donors were tested by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) and found negative using United States (U.S.) Food and Drug Administration (FDA) licensed tests for detection of: Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus Types 1 / 2, and Treponema Pallidum. All of the d-MAPPS samples contain one or more types of MSCs, one or more MSC- Exos (e.g., AF-MSC Exosomes), and one or more MSC-derived and / or MSC-Exos-derived growth factors and / or cytokines (e.g., AF-MSC-derived growth factors and / or cytokines), manufactured under current Good Manufacturing Practices (cGMP), regulated and reviewed by the FDA.

[0338] As stated above herein, the growth factors derived from MSCs / MSC-Exos may include, for instance, IL-IRa, sTNFRI, sTNFRII, GRO-y, fatty acid-binding protein 1 (FABP1), and / or platelet factor 4 (PF4).

[0339] As stated above herein, the cytokines derived from MSCs / MSC-Exos may include, for instance, one or more members of the IL- 12 cytokine family (e.g., IL- 12, IL-23, IL-27, IL-35), one or more CSC chemokines (e.g, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17), and / or other immunostimulatory molecules known in the art.

[0340] Results

[0341] The d-MAPPS solution was provided to patients with eye injuries. Such patients had various symptoms, including pain, dryness, grittiness, scratchiness, soreness, irritation, burning, watering, and / or eye fatigue. The d-MAPPS solution contains growth factors that (1) suppress IL- ip and TNF-a-driven inflammation, (2) prevent the generation of inflammatory Thl and Th 17 cells, (3) support tear stability, and (4) reduce ocular surface epithelial damage. The d-MAPPS solution may further attenuate, prevent, and / or remedy one or more symptoms of eye injuries and / or eye disorders, such as dry eye discomfort, tear hyperosmolarity, tear hyperosmolarity- induced changes in patients suffering from dry eye discomfort.

[0342] Patients can be treated with the d-MAPPS solution for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, or at least about 3 months. The d-MAPPS solution can be administered as eye drops 3-4 times per day (e.g, at about 10 pg / drop), every day. Changes in eye condition can be observed by measuring, for instance, ocular surface disease index, conjunctiva redness scores, ocular surface staining, best corrected visual acuity, tear secretion, tear meniscus height, tear film breakup time (TBUT), and / or tear film thickness.

[0343] TBUT is preferably less than about 10 seconds or less than about 5 seconds after one or more courses of treatment with the d-MAPPS solution. Improvements in TBUT may be seen in at least some patients after, for instance, 21 days after topical administration of the d-MAPPS solution. Such improvements may indicate improvement of, and / or restoration of, meibomian gland function. Tear film thickness, which can be used to measure the liquid layer, the lipid layer, and / or a combination of the liquid layer and the lipid layer, can increase as TBUT increases. Tear film thickness can be measured by, for instance, ( 1) a slit lamp and a video camera, and / or (2) an optical interferometer (e.g., a wavelength-dependent optical interferometer), as described above herein. Specifically, multiple measurements can be taken of an area of a subject’s eye using an optical interferometer. The area has a predetermined length and width (e.g., about 20 pm, about 30 pm, about 50 pm, about 100 pm) and may be located in a suitable area of the eye (e.g., the apex of the cornea). Multiple measurements (e.g., at least about 10, at least about 20, at least about 30, at least about 50) can be taken over a window of time (e.g., 20 seconds to 1 minute). Such tear film thickness measurements may be taken both before and after d-MAPPS solution-based treatment. Before treatment, tear film thickness may be, for example, less than about 2 microns, or less than about 1 micron. After treatment, tear film thickness may be, for example, about 5 microns, about 6 microns, about 8 microns, about 10 microns, about 12 microns, or more than about 12 microns.

[0344] Overall, significantly improved visual acuity, relieved ocular pain, and complete healing of corneal epithelial defects may be noticed in at least some patients. Further, the d-MAPPS solution can improve the viability of injured corneal epithelial cells and alleviate the symptoms elicited by corneal injury. In addition, the d-MAPPS solution may (e.g., after four weeks of treatment) result in remarkably improved visual acuity and in significantly decreased ocular pain.

[0345] Example 5: Treatment of patients with eye disorders using d-MAPPS solutions

[0346] Materials and methods

[0347] The d-MAPPS solutions used in this Example are bio-engineered biologic products obtained from amniotic fluid derived MSCs (AF-MSCs), previously collected from healthy human donors. Blood samples were given by the donor prior to, or at the time of, collection. These samples were tested by laboratories certified under the Clinical Laboratory Improvement Amendments (CLIA) and found negative using United States (U.S.) Food and Drug Administration (FDA) licensed tests for detection of at minimum: Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus Types 1 / 2, Treponema Pallidum. AF samples were obtained with patient consent and stored at 4°C until processed.

[0348] The d-MAPPS solutions are then bio-engineered as an AF-MSC-derived sterile product containing, among other components, AF-MSC-Exos and AF-MSC-derived cytokines and growth factors. The d-MAPPS solutions may contain additional components e.g., osmoprotectants) as described above herein. The d-MAPPS solutions may also have specific properties (e.g., pH) as described above herein. The d-MAPPS solutions may also include one or more solutions as described specifically in Example 1. The d-MAPPS solutions are manufactured under current Good Manufacturing Practices (cGMP), regulated and reviewed by the FDA. The d-MAPPS solutions are then sterilized to provide for a safe, sterile product that can be introduced as eye drops into the eyes of patients.

[0349] Specifically, a total of 131 patients with eye disorders and / or injuries to one or more tissues of the eye were recruited (27 male and 104 female), with a median age of 62 years (ranging from 19 to 85 years of age). Patients received d-MAPPS solutions and were followed up for 12 months. The principles of Good Clinical Practice and the Declaration of Helsinki were adhered to at all times during the study. All patients were under continuous medical supervision by either their ophthalmologist or optometrist.

[0350] Results

[0351] Patients’ subjective symptoms were graded numerically using VAS (Visual Analogue pain Score). The scale ranges from 0 (defined as the absence of pain) to 10 (defined as maximum pain). Patients were asked to describe their discomfort or pain using VAS.

[0352] The Standard Patient Evaluation of Eye Dryness Questionnaire (SPEED) questionnaire was also used to evaluate patients’ dry eye-related symptoms. The SPEED questionnaire asks about symptoms including dryness or grittiness or scratchiness, soreness or irritation, burning or watering, and eye fatigue. The frequency that patients experience these symptoms is scored from 1 through 3. A score of 1 equates to a frequency of “sometimes,” a score of 2 equates to a frequency of “often,” and a score of 3 equates to a frequency of “constant.” The severity of the symptoms is scored from 0 through 4. A score of 0 represents “no problems or symptoms,” a score of 1 represents a “tolerable” severity, a score of 2 represents an “uncomfortable” severity, a score of 3 represents a “bothersome” severity, and a score of 4 represents an “intolerable” severity.

[0353] All patients treated with the d-MAPPS solutions experienced significantly reduced VAS and SPEED scores after 3 months (p < 0.001). Specifically, VAS scores declined from a baseline of between 8-9 to between 3-4 after 3 months. SPEED scores declined from a baseline score of between 15-20 to between 5-10 after 3 months. These results indicate that the d-MAPPS solutions managed to improve patient symptoms including pain, dryness, grittiness, scratchiness, soreness, irritation, burning, watering and eye fatigue.

[0354] Importantly, the d-MAPPS solutions continued to have beneficial effects after 3 months and, indeed, during the entire 12-month observational period. Specifically, these beneficial effects appear to be significantly increased during the last 6 months of this 12-month period, with the highest reduction in VAS and SPEED scores observed after 12 months of d-MAPPS solutionbased therapy. Indeed, patients’ VAS and SPEED scores were lower after 12 months (p < 0.001) than after 3 months or 6 months. These results indicate that d-MAPPS solution-based treatment provides long-lasting beneficial effects in alleviating ocular symptoms in patients with eye disorders and / or injuries in one or more eye tissues.

[0355] Example 6: Preparation of d-MAPPS compositions containing human amniotic fluid

[0356] Materials and methods

[0357] Human amniotic fluid is collected from selected Caesarean sections, which make aspiration of the amniotic fluid in clean condition possible. Then the amniotic fluid is stored in refrigerated condition at 2°C to 6°C before the clarification and filtration process. The amniotic fluid is centrifuged at 5,000 to 10,000 rpm for 30 minutes to 1 hour in 50 mL to 250 mL swing out buckets. The supernatant is collected. When collecting the supernatant it is important to avoid detaching or aspirating insoluble components possibly coming from the pellet or from the fatty overlayer. If the supernatant still contains residual insoluble components, it may be pre-filtered with 5 to 10 p cellulose ester capsule pre-filters without TRITON® surfactant to avoid contamination in the filtration process. The liquid phase is collected and filtered with poly ether sulfone 1.0 p capsule filters and the liquid is collected. The liquid is then filtered with poly ether sulfone 0.2 p capsule filter. The filtrate is transferred to vials and sealed with stoppers aseptically. Four samples from the final filtrate are taken to test whether the sterile filtered human amniotic fluid retains growth factors, such as human growth hormone, transforming growth factor beta 1, vascular endothelial growth factor, epidermal growth factor, and transforming growth factor beta 3.

[0358] The amniotic fluid from the final filtration is aseptically transferred to syringes or vials, then kept in a deep freezer at about -80° C to about -20° C for long term storage. The sterile amniotic fluid is dried in the vial via lyophilization in a built-in a sterile environment. The lyophilisate derived from the amniotic fluid is reconstituted with sterile water before its injection or topical administration. The lyophilisate can be stored at from +4°C to about +25° C (room temperature). All of this operation may be carried out in sterile condition and does not need additional sterilization methods such as a final irradiation.

[0359] If needed, the lyophilisate derived from amniotic fluid through lyophilization may be irradiated by e-beam irradiation or gamma ray irradiation to add another guarantee for the final sterility of the powder. Irradiation of a lyophilisate is much less denaturing for proteins and peptides than irradiating aqueous solutions, because the absence of water considerably reduces the production of reactive superoxide anions and their diffusion during irradiation. Such superoxide anions are the main cause of splitting peptide bonds and chemically modifying amino acids of protein and peptides. After lyophilization, the amniotic fluid is reconstituted by adding the initial volume of water. After gentle homogenization, the powder is quickly dissolved in about one minute.

[0360] Results

[0361] The results show retention of growth factors. The concentration of the growth factors in the sterile filtered amniotic fluid is from about 30 pg / mL to about 2500 pg / mL. Except the vascular endothelial growth factor in sample 2, the concentrations of all the factors in the four samples are in the range of 30-150 pg / mL. Although part of growth differentiation factor 11 is lost in centrifugation and filtration, the final sterile filtered amniotic fluid still retains about 17% to 29% of growth differentiation factor from the raw human amniotic fluid.

[0362] The reconstituted amniotic liquid is transparent and may be used for treating one or more injured tissues, one or more types of wounds, and / or one or more other disorders that include injured tissues (e.g., cosmetic, orthopedic, or ophthalmic applications, anti-cancer applications, anti-tumor applications, etc.).

[0363] Example 7: Comparative study on the treatment of corneal inflammation

[0364] Materials and methods

[0365] The d-MAPPS composition of Example 6 was dissolved in 1 mL of sterile water to reconstitute 1 mL of the initial sterile filtered amniotic fluid. Two drops were applied on each eye of ten patients suffering from eye disorders and / or injuries to one or more eye tissues. This treatment was repeated twice per day for ten days. Two other control groups of 10 patients similarly received either their own serum or a serum prepared from cord blood as described by Kyung-Chul Yoon (Umbilical cord and its blood: A perspective on its current and potential use in Ophthalmology, in “Regenerative Medicine Using Pregnancy-Specific Biological Substances,” Springer ed. 2011).

[0366] Results

[0367] 8 to 10 patients out of 10 in each group declared that they had experienced a significant benefit. For all patients, this clinical improvement was correlated with a partial or complete decrease of their initial corneal inflammation. Nine patient had “severe” dry eye, one was “moderate.” The latter is a 70 year old woman, still working, at a computer all day, who has had dry eye for many years, as well as a long history of allergies, asthma, uses an inhaler as well as an antihistamine, and has had the red eyes associated with this problem. The treatment eliminated the redness and significantly reduced light sensitivity and need for artificial tears. The treatment also restored her ability to read books.

[0368] These and other objectives and features of the invention are apparent in the disclosure, which includes the above and ongoing written specification.

[0369] The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated.

[0370] The invention is not limited to the particular embodiments illustrated in the drawings and described above in detail. Those skilled in the art will recognize that other arrangements could be devised. The invention encompasses every possible combination of the various features of each embodiment disclosed. One or more of the elements described herein with respect to various embodiments can be implemented in a more separated or integrated manner than explicitly described, or even removed or rendered as inoperable in certain cases, as is useful in accordance with a particular application. While the invention has been described with reference to specific illustrative embodiments, modifications and variations of the invention may be constructed without departing from the spirit and scope of the invention as set forth in the following claims.

Claims

What is claimed is:

1. A method for treating one or more injured tissues, the method comprising: administering to the subject an effective amount of a pharmaceutical composition and / or formulation comprising one or more types of mesenchymal stem cells (MSCs), wherein the one or more types of MSCs alter one or more responses of one or more immune cells in the subject to promote tissue healing, repair, and / or regeneration in the one or more injured tissues, and wherein the one or more types of MSCs interact with tissue-resident stem cells to induce one or more changes in phenotype and / or function of the tissue-resident stem cells.

2. The method of claim 1, wherein the one or more immune cells are selected from the group consisting of: dendritic cells, macrophages, neutrophils, T cells, natural killer (NK) cells, NK T cells, and combinations thereof.

3. The method of claim 1, wherein the one or more types of MSCs produce, in the one or more injured tissues, one or more biological compounds that result in at least one of: one or more inhibited immune responses, attenuated inflammation, and increased proliferation and differentiation of tissue-resident stem cells.

4. The method of claim 1, wherein the one or more types of MSCs induce generation of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), to alleviate inflammation in the one or more injured tissues.

5. The method of claim 1, wherein the one or more types of MSCs induce pericyte- to-MSC transitions.

6. The method of claim 5, wherein the pericyte-to-MSC transitions are caused by one or more biological compounds selected from the group consisting of: transforming growth factor beta (TGF-P), interleukin (IL)-ip, IL-10, IL-6, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-2, and combinations thereof.

7. The method of claim 5, wherein the MSCs are used as a delivery vehicle for MSC-OAVs to treat a subject with hepatocellular carcinoma (HCC), lung cancer, breast cancer,pancreatic cancer, neuroblastoma, colorectal cancer, and / or prostate cancer.

8. The method of claim 1, wherein the one or more types of MSCs reduce expression of one or more markers in pericytes, and wherein the one or more markers are selected from the group consisting of: neuron-glial antigen 2 (NG2), platelet-derived growth factor receptor-beta (PDGFR-0), regulator of G protein signaling 5 (RGS5), and combinations thereof.

9. A method for treating one or more injured tissues, the method comprising: administering to the subject an effective amount of a pharmaceutical composition and / or formulation comprising one or more types of mesenchymal stem cells (MSCs), wherein the one or more types of MSCs trigger one or more changes in pericytes in the subject.

10. The method of claim 9, wherein the one or more changes comprise at least one of: an increase in cell size of the pericytes, a change in shape of the pericytes to become more rounded and fibroblast-like, and an increase in organelle numbers.

11. The method of claim 9, wherein the one or more changes comprise cytoskeletal reorganization caused by at least one of: reorganization of actin fdaments, reduction of integrin expression, and decreased production of one or more extracellular matrix (ECM) components.

12. The method of claim 9, wherein the one or more types of MSCs induce increased expression of one or more transcription factors in the pericytes, wherein the one or more transcription factors are selected from the group consisting of: Sox9, Oct4, Nanog, and Klf4, and combinations thereof.

13. The method of claim 9, wherein the one or more types of MSCs induce increased expression of one or more genes responsible for extracellular matrix (ECM) degradation and / or ECM synthesis.

14. The method of claim 9, further comprising: administering to the subject one or more additional agents in combination with the pharmaceutical composition and / or formulation, wherein the one or more additional agents areselected from the group consisting of: an adjuvant, an antigen, an excipient, a vaccine, an allergen, an antibiotic, a gene therapy vector, a kinase inhibitor, a co-stimulatory molecule, a Toll-like receptor (TLR) agonist, a TLR antagonist, a therapeutic agent, a prophylactic agent, a diagnostic agent, an antimicrobial agent, an analgesic, a local anesthetic, an anti-inflammatory agent, an anti-oxidant agent, an immunosuppressant agent, an anti-allergenic agent, an enzyme cofactor, an essential nutrient, a growth factor, and combinations thereof.

15. A method for treating one or more injured tissues, the method comprising: administering to the subject an effective amount of a pharmaceutical composition and / or formulation comprising: one or more types of mesenchymal stem cells (MSCs), one or more biological compounds derived from the one or more types of MSCs, and one or more pharmaceutically acceptable excipients.

16. The method of claim 15, wherein the one or more biological compounds are selected from the group consisting of: transforming growth factor beta (TGF-P), interleukin (IL)- 10, IL- 13, prostaglandin E2 (PGE2), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and combinations thereof, and wherein the one or more biological compounds result in at least one of: decreased production of one or more pro-inflammatory cytokines, increased production of extracellular matrix (ECM) components, increased differentiation of fibroblasts into myofibroblasts, and increased angiogenesis.

17. The method of claim 15, wherein the one or more biological compounds (i) suppress generation of an inflammatory phenotype in one or more types of immune cells, and (ii) induce generation of an immunosuppressive phenotype in the one or more types of immune cells, wherein the one or more types of immune cells comprises dendritic cells.

18. The method of claim 15, wherein the one or more biological compounds are selected from the group consisting of: hepatocyte growth factor (HGF), insulin-like growth factor (IGF), and combinations thereof, and wherein the one or more biological compounds result in at least one of:increased survival and differentiation of stem cells, increased cytoskeletal rearrangement in stem cells to enable migration of the stem cells to the one or more injured tissues, increased proliferation of tissue-resident stem cells, and increased expression of integrins.

19. The method of claim 15, wherein the one or more biological compounds comprises indoleamine 2,3 -dioxygenase (IDO), and wherein the IDO depletes tryptophan in the one or more injured tissues.

20. The method of claim 19, wherein the IDO affects one or more metabolites of tryptophan metabolism, and wherein the one or more metabolites comprises kynurenine.I l l

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