WO2025199431A1 - Innate immune pathway activated mesenchymal stromal cells for treatment of musculoskeletal disorders or conditions - Google Patents

Abstract

Embodiments of the present disclosure generally relate to compositions and methods for treating musculoskeletal disorders or conditions, such as osteoarthritis (OA). Specifically, the compositions and methods include activating mesenchymal stromal cell (MSC) with a stimulator of interferon gene (STING) agonist or a toll like receptor (TLR) agonist. A method of treating a musculoskeletal disorder or condition in a subject is disclosed. The method includes administering to the subject a composition including a MSC. The MSC include an activated signaling pathway downstream of (i) STING and/or (ii) TLR3. In another embodiment, a composition for treating musculoskeletal disorders or conditions in a patient is disclosed. The composition includes a MSC with pattern recognition receptors (PRR), wherein the PRR comprises an activated STING pathway, an activated a TLR3 pathway, or a combination thereof.

Description

INNATE IMMUNE PATHWAY ACTIVATED MESENCHYMAL STROMAL CELLS FOR TREATMENT OF MUSCULOSKELETAL DISORDERS OR CONDITIONS

BACKGROUND

Field

[0001] Embodiments of the present disclosure generally relate to compositions and methods for treating musculoskeletal disorders or conditions, such as osteoarthritis (OA). Specifically, the compositions and methods include activating mesenchymal stromal cells (MSCs) with an agonist for a pattern recognition receptor (PRR).

Description of the Related Art

[0002] Osteoarthritis (OA) is a progressive, degenerative, condition that affects over 550 million people worldwide - a 113% increase since 1990. The prevalence has doubled in the US over the last ten years with an economic impact of $136 billion annually. OA refers to a type of arthritis, or swelling, tenderness, and/or inflammation of one or more joints, that occurs when the flexible tissue at the ends of bones wears down. The process of wearing down occurs gradually and worsens over time. This process of wearing down causes joint pain in the hands, neck, lower back, knees, or hips. Despite this high prevalence, there remains a lack of effective treatment options that improve quality of life without risk of adverse effects, with current therapies including nonsteroidal anti-inflammatories, intra-articular injections, or arthroscopic debridement and cartilage resurfacing techniques. A recent option for OA treatment are cellular therapies. However, there are mixed results regarding the efficacy of the cellular therapies. One cellular therapy involves mesenchymal stromal cells (MSCs). Metanalysis of human randomized MSC trials showed that treatments using MSC based therapies reduced joint inflammation, improved pain scores, and preserved cartilage integrity.

[0003] However, challenges remain for the MSC based therapies. For example, there is a variability in efficacy of the MSC based therapies. Heterogeneity within stromal cell populations has been proposed to be partially responsible for the observed variability in therapeutic responses, particularly in the context of variably inflamed recipient environments such as that seen in OA. [0004] A proposed solution for the variability in efficacy is activation of MSCs. Activating specific pattern recognition receptors (PRRs) with specific agonists associated with PRRs as a means to generate a homogenous population of immunomodulatory MSCs. Accordingly, there is a need for compositions and methods to activate MSCs.

SUMMARY

[0005] In a first embodiment, a method of treating a musculoskeletal disorder or condition in a subject is disclosed. The method includes administering to the subject a composition comprising a mesenchymal stromal cell (MSC). The MSC comprise an activated signaling pathway downstream of (i) stimulator of interferon gene (STING) and/or (ii) toll-like receptor 3 (TLR3).

[0006] In another embodiment, a method of treating of treating a musculoskeletal disorder or condition in a subject is disclosed. The method includes administering to the subject a composition including a mesenchymal stromal cell (MSC). The MSC has been cultured in the presence of a stimulator of interferon gene (STING) agonist and/or a toll-like receptor 3 (TLR3) agonist.

[0007] In another embodiment, a composition for treating musculoskeletal disorders or conditions in a patient is disclosed. The composition includes a mesenchymal stromal cell (MSC) with pattern recognition receptors (PRR), wherein the PRR comprises an activated stimulator of interferon genes (STING) pathway, an activated a toll-like receptor 3 (TLR3) pathway, or a combination thereof.

[0008] In another embodiment, a composition for treating a musculoskeletal disease in a patient is disclosed. The composition including a mesenchymal stromal cell (MSC). The MSC including an activated stimulator of interferon gene (STING) pathway. The STING pathway is activated by an agonist. The agonist may include a cyclic dinucleotide (CDN), a amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, a BMS-986301, a Cyclic GMP-AMP synthase (cGAS), or combinations thereof.

[0009] In another embodiment, a method of forming a composition for treating musculoskeletal disorders or conditions is disclosed. The method includes activating a pattern recognition receptors (PRR) of a mesenchymal stromal cell (MSC). The PRR comprises a stimulator of interferon gene (STING) pathway, a toll-like receptor 3 (TLR3) pathway, or a combination thereof. The method further includes forming the composition for treating musculoskeletal disorders or conditions where the composition includes an activated MSC.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] So that the manner in which the above recited features of the present disclosure can be understood in detail, a more particular description of the disclosure, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only exemplary embodiments and are therefore not to be considered limiting of its scope, may admit to other equally effective embodiments.

[0011] Figure 1A shows a schematic overview of the study design, according to certain embodiments.

[0012] Figure IB is a flow diagram of a method for activating MSCs, according to certain embodiments.

[0013] Figures 2A, 2B, 2C, 2D, 2E, and 2F show exemplary data of activated mesenchymal stromal cell treatment viability with ANY-maze™ cage monitoring parameters, according to certain embodiments.

[0014] Figures 3A, 3B, 3C, and 3D show exemplary data of TLR3 activated mesenchymal stromal cell treatment viability with ANY-maze™ cage monitoring parameters, according to certain embodiments.

[0015] Figures 4A, 4B, and 4C show exemplary data of RNA sequencing analysis comparing transcriptomes of joint tissues from mice treated with STING activated MSC or MSC, according to certain embodiments

[0016] Figures 5 A, 5B, and 5C show exemplary data of RNA sequencing analysis comparing transcriptomes of joint tissues from mice treated with TLR3 activated MSC or MSC, according to certain embodiments.

[0017] Figures 6A, 6B, 6C, and 6D show exemplary data of DEG analysis for STING and TLR3 activated equine MSCs, according to certain embodiments.

[0018] Figure 7 shows exemplary data of RNA sequencing analysis comparing joint tissues from mice treated with STING activated MSC vs. TLR3 activated MSC, according to certain embodiments. [0019] Figure 8 shows exemplary data of overlapping gene signatures from joint tissues from mice treated with STING activated MSC, TLR3 activated MSC, or MSC, according to certain embodiments.

[0020] Figures 9A and 9B show exemplary data of the pathways that are either upregulated or downregulated in joint tissues from mice treated with STING activated MSC, TLR3 activated MSC, or MSC, according to certain embodiments.

[0021] Figure 10 shows exemplary data of sequencing analysis comparing gene expression of equine MSCs treated with STING activated MSC vs. TLR3 activated MSC, according to certain embodiments.

[0022] Figure 11 shows exemplary data of overlapping gene signatures from equine bone marrow derived MSC, according to certain embodiments.

[0023] Figures 12 A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, 12 J, 12K, and 12L show histologic representative images of mouse knees after treatment, according to certain embodiments.

[0024] Figures 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H, and 131 show exemplary data of OARSI histopathology scores that was collected following DMM surgery and treatment, according to certain embodiments.

[0025] Figure 14 shows an example schematic of the stimulation of equine bone- marrow MSCs stimulated with agonists, assayed, extracted, and mapped to equCab3.0, according to certain embodiments.

[0026] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements and features of one embodiment may be beneficially incorporated in other embodiments without further recitation.

DETAILED DESCRIPTION

[0027] Embodiments of the present disclosure generally relate to compositions and methods for treating musculoskeletal disorders and conditions, such as osteoarthritis (OA) and other chronic musculoskeletal disorders such as rheumatoid arthritis, psoriatic arthritis, gout, spondylarthritis, osteoporosis, osteopenia, tendinitis, tendonitis, bursitis, fibromyalgia, joint cartillage injury, myopathy, ligament injuries, fragility fractures, traumatic bone fractures, sarcopenia, or inflammatory diseases (including, but not limited to connective tissue diseases, connective tissue vasculitis, systemic lupus erythematosus, or prior amputation). Specifically, the compositions and methods include activating a pattern recognition receptor (PRR) of mesenchymal stromal cells (MSCs). Expression of PRRs represent a key link between the immune system and sensory nervous system in response to inflammation or injury, such as that described in OA. PRRs are specialized proteins that detect exogenous pathogens and endogenous ligands. PRRs play a key role in immune and sensory nervous system regulation of pain and inflammation in injury. For example, when an activated PRR begins a signaling pathway downstream to the PRR within the cell (e.g., the MSC). The PRR includes, but is not limited to, Toll-like receptors (TLR) (e.g., nucleotide oligomerization domain-like receptors, C-type lectin receptors, RIG-I-like receptors, and retinoic acid-inducible gene I receptors) and/or cytosolic DNA sensors (e.g., STimulator of Interferon Genes or STING). The STING agonist to induce the STING signaling pathway may include cGAS-STING or a Cyclic GMP-AMP synthase (cGAS). A primary TLR receptor to induce the TLR pathway is TLR3, the TLR3 agonist to induce the TLR3 signaling pathway may include polyinosinic-polycytidylic acid (pIC).

[0028] Musculoskeletal disorders and conditions are a diverse group of conditions affecting the bones, joints, muscles, and connective tissues of a subject. This group of disorders and conditions also affect the locomotor functions of the subject typically characterized by either acute or chronic pain with limited mobility functions. Musculoskeletal disorders and conditions are relevant throughout all stages of life from childhood to old age, and range from sudden, short-lived disorders and conditions to long-term disorders and conditions. Musculoskeletal disorders and conditions may include chronic disorders and conditions such as osteoarthritis, or acute disorders and conditions such as injury. The term “osteoarthritis” refers to a type of arthritis, or swelling, tenderness, and/or inflammation of one or more joints, that occurs when the flexible tissue at the ends of bones wears down. The process of wearing down occurs gradually and worsens over time. This process of wearing down causes joint pain in the hands, neck, lower back, knees, or hips. In some embodiments, the musculoskeletal disorder or condition is a chronic musculoskeletal disorder or condition. In some embodiments, the musculoskeletal disorder or condition is an acute musculoskeletal disorder or condition. In some embodiments, the acute or chronic musculoskeletal disorder or condition is osteoarthritis. In some embodiments, the acute or chronic musculoskeletal disorder or condition includes, but is not limited to rheumatoid arthritis, psoriatic arthritis, gout, spondylarthritis, osteoporosis, osteopenia, tendinitis, tendonitis, bursitis, fibromyalgia, joint cartillage injury, myopathy, ligament injuries, fragility fractures, traumatic bone fractures, sarcopenia, or inflammatory diseases (including, but not limited to connective tissue diseases, connective tissue vasculitis, systemic lupus erythematosus, or prior amputation).

[0029] Mesenchymal stromal cells (MSC) are also known in the art as mesenchymal stem cells, mesenchymal progenitor cells, medicinal signaling cells, and non-hematopoietic stem/stomal cells and these terms may be used interchangeably. MSC are adherent cells during in vitro expansion, possess variable differentiation potential, and produce bioactive agents and have immunomodulatory properties. MSC are multipotent cells with the capacity to proliferate and differentiate into mature cells from the mesenchymal lineage, such as osteoblasts, adipocytes, and chondrocytes. As used herein, MSC comprise both MSC derived from tissue or tissue fluid (tissue- derived MSC) and MSC derived from stem cells (stem cell-derived MSC). Tissue- derived MSC are obtained by dissociating a solid mesenchymal tissue (such as, but not limited to, bone marrow, adipose tissue, placenta, umbilical cord tissue, umbilical cord blood, peripheral blood, muscle, uterine tissue, corneal stroma, skin, and/or dental pulp) to create a cell suspension or by isolating cells from a tissue fluid (such as, but not limited to, blood, synovial fluid, lymphatic fluid, thoracic fluid, pericardial fluid, peritoneal fluid, and/or cerebrospinal fluid), culturing the cells obtained from the dissociated solid tissue or tissue fluid, and isolating the adherent cell fraction (i.e. the cells adherent to a culture substrate). Tissue-derived MSCs are thus isolated as a cell fraction adherent to a culture substrate. In some embodiments, a culture substrate is a culture vessel, a microbead, a microtube, or a culture matrix, and these culture substrates may be uncoated or coated with a suitable culture coating including but not limited to collagen or any other suitable polymer. The process of obtaining and/or isolating MSC from tissue or tissue fluid may be performed according to methods known in the art. Stem cell-derived MSC can be generated and differentiated into MSC by methods known in the art. In some embodiments, the MSC are derived from induced pluripotent stem cells (iPSC) (iPSC-derived MSC) or from embryonic stem cells (ESC) (ESC-MSC), or from other cell types capable of being directed to an MSC phenotype or MSC-like phenotype.

[0030] An agonist is a chemical composition or compound that activates a receptor protein (e.g., a PRR) to produce a biological response (e.g., a signaling pathway). MSCs are activated with a toll-like receptor 3 (TLR3) pathway and/or the cGAS- STING pathway. Activating the MSCs with agonists of the endosomal TLR3 pathway or the cytoplasmic STING pathway improved the efficacy of MSCs when treating OA. For example, the TLR3 pathway is activated when an agonist interacts with a TLR3 receptor. An intra-articular therapy in a rodent destabilization of the medial meniscus (DMM) model of OA is used herein as an example. The DMM model revealed significant improvements in post-operative voluntary movement parameters, improved histological outcomes with STING activated MSC, and improved histological outcomes with TLR3 activated MSC. Activation of TLR3 pathways and/or STING pathways in MSCs prior to injection into a subject activates multiple interferon (IFN) pathways. The stimulation of IFN pathways in MSCs is an effective method of activating MSCs for orthopedic applications, such as treating OA.

[0031] Following tissue damage, PRRs on immune cells are activated to initiate an inflammatory response and sensory neurons concurrently sense these signals PRR expression themselves. In other words, PRRs on immune cells are activated to initiate a downstream inflammatory response. Activation of both STING and TLR3 results in production of type I interferons (IFN-Is) (IFN-a, IFN-P, and IFN-K) in immune cells and sensory neurons following tissue injury or infection. The activation of both STING and TLR3 is an inflammatory and/or antinociceptive depending on the disease process. Stimulation of MSCs with TLR pathway and the STING pathway enhanced their immunomodulatory properties associated with OA. Specifically, the STING pathway enhances both functional outcomes and structural joint integrity while promoting transcriptional pathways (genes) favoring tissue repair.

Methods and Materials

[0032] Embodiments of the present disclosure generally relate to methods for treating osteoarthritis (OA) by activating MSCs with a STING agonist or a TLR3 agonist. The activated MSCs (e.g., the STING activated MSCs or the TLR3 activated MSCs) can be used as at least a portion of a treatment for OA. As shown in Figure IB, a method 100 of preparing the activated MSCs is disclosed herein.

[0033] In one or more embodiments, a method of forming an activated MSC described herein includes activating an MSC with an agonist for a PRR. MSCs are multipotent cells with the capacity to proliferate and differentiate into mature cells from the mesenchymal lineage (e.g., osteoblasts, adipocytes, and chondrocytes). Activation (e.g., preactivation) of MSCs involves exposing or surrounding the MSCs with an activating composition, compound, molecule, or substance that will enhance the antiinflammatory, immunomodulatory, and angiogenetic properties of the MSCs.

[0034] In one or more embodiments, the STING pathway can be activated by an agonist. In one or more embodiments, the STING pathway agonist is stimulator of interferon (IFN) genes, cyclic dinucleotides (CDN), including both natural and synthetic CDNs, amidobenzimidazole (ABZI), 5,6-dimethylxanthenone-4-acetiv acid (DMXAA), 2’3’-cGAMP, c-di-GMP, c-di-AMP3’3’-cGAMP, ADU-S100, SB 11285, GSK 3745417, MK-1454, BMS-986301, Cyclic GMP-AMP synthase (cGAS), or combinations thereof.

[0035] In one or more embodiments, the TLR pathway is a TLR3 pathway. The TLR3 pathway can be activated by an agonist such as a polyinosinic-polycytidylic acid (pIC), polyadenylic-polyuridic acid (pAU), polyinosinic-polycytidylic acid stablilized with polylysine and carboxymethylcellulose (Poly-ICLC), Rintatolimod, Resiquimod, UV-inactivated viral particles, ARNAX, RGC100, or combinations thereof. In one or more embodiments, the TLR agonist is a TLR4 agonist, a lipopolysaccharide agonist, a TLR9 agonist, a TLR1 agonist, a TLR2 agonist, or a CpG oligodeoxynucleotide (ODN).

[0036] For the examples disclosed herein the MSCs were harvested from age- matched male C57BL/6Nci mice served as adipose MSC (adMSC) donors. Murine adMSC was generated from abdominal and inguinal adipose tissue aseptically collected immediately following euthanasia via CO2 inhalation and cervical dislocation. In one or more embodiments involving a mammalian subject, the MSCs are isolated from a bone marrow sample, adipose tissue, umbilical cord blood or tissue, peripheral blood, embryonic tissue, or iPSC-derived cells. In one or more embodiments, the MSCs can be isolated from tissues including, but not limited to dental pulp, uterine tissue, skin biopsies, and other organ biopsies. In one or more embodiments, the MSCs are generated from the patient (autologous MSCs), from unrelated, healthy donors (allogeneic MSCs), or from a different species (xenogeneic MSCs). In one or more embodiments, the MSCs are derived from bone marrow, adipose tissue, placenta, umbilical cord tissue, umbilical cord blood, peripheral blood, muscle, uterine tissue, corneal stroma, skin, and/or dental pulp.

[0037] Reference is made herein to the non-limiting, exemplary embodiment illustrated in Figure IB and operations of expanding 102, activating 104, and administering 106 the MSCs provided therein. References to the operations of Figure IB, and those demonstrated in the examples herein, are provided as non-limiting, exemplary embodiments of the invention, and the skilled practitioner in view of the common general knowledge in the art will understand that variations of these operations may be substituted in using the invention.

[0038] At operation 102, the MSCs are expanded. For example, the adipose tissue is isolated, pooled, and cultured. The cells generated from the adipose tissue are plasticadherent and display typical adipose tissue MSC (adMSC) morphology and expansion properties. AdMSCs are expanded in culture in complete growth media (Dulbecco modified eagle medium (DMEM), 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 p.g/mL), and 1 mol/L HEPES) until injection. The MSCs are expanded for 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks. In one or more embodiment, the MSCs are expanded until the desired amount of cells are within the culture. For example, the number of cells required for treatment is dependent on the treatment type, as the treatment type determines the concentration of cells required for effectiveness. Once an appropriate amount of MSCs is reached, the MSCs are trypsinized. After the MSCs are trypsinized, the MSCs are collected. For example, the MSCs are trypsinized at week 3 and week 5.

[0039] At operation 104, the MSCs are activated with the TLR3 agonist (e.g., polyinosinic-polycytidylic acid (pIC)) or a STING agonist (e.g., a stimulator of IFN genes, e.g., a 2’3’-cGAMP molecule). For example the TLR3 pathway or the STING pathway is activated with the respective agonist. In one or more embodiments, the concentration of agonist to MSCs is about lOpg/mL agonist to about IxlO⁶ cells/mL in growth media. However, it should be understood the concentration of agonist to MSC may be adjusted based on the biological readout of interest. In one or more embodiments, the MSCs are activated prior to trypsinization. Activating the MSCs includes in vitro activation via incubation. All activations are performed at about 37°C. The MSCs are activated for about 0.25 hours to about 48 hours in the presence of the TLR3 agonist or STING agonist. In one or more embodiments, the MSCs are activated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or more hours in the presence of the TLR3 agonist or STING stimulator. For example, the MSCs are activated for about 2 hours. For example the MSCs are activated for about 0.25 hours to about 0.5 hours. The activated MSCs are washed about 3 times with sterile phosphate buffered saline (PBS) or cell culture medium to remove the agonists. The activated MSCs are prepared and stored at a concentration of about 1.6 x 10⁶ cells per mL to about 5 x 10⁶ cells per mL. The activated cells are stored in PBS, dimethyl sulfoxide (DMSO), another commercially available freezing medium, or combinations thereof. In one or more embodiments, the activated MSCs are preserved using a cryogenic, hypothermic, lyophilization or dehydration methods. For example, when the cells are stored in DMSO as the freezing medium, the cells are typically frozen in about 8% to about 10% weight to volume ratio. In one or more embodiments, a commercial freezing medium and method are used to preserve the activated MSCs. The preserved MSCs can be frozen in N2 indefinitely. The preserved MSCs are administered to the subject after thawing or rehydration. In one or more embodiments, the activated MSCs are administered after preparation (e.g., without a storage step).

[0040] Culture” or “cell culture” is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under controlled conditions. These conditions may vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Adherent cells require a surface or a substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. Many suitable methods and materials for cell culture are known and available in the art. In some embodiments, the MSCs are cultured in the presence of a STING agonist and/or a TLR3 agonist, such as by providing the agonist in the culture medium. Culture of MSCs in the presence of agonists may activate STING and/or TLR3 receptors, resulting in activation of the signaling pathway downstream from these receptors.

[0041] At operation 106, the activated MSCs are administered to a subject. In one or more embodiments, the activated MSCs formed during operation 104 and a pharmaceutically acceptable carrier are administered to a subject. The pharmaceutically acceptable carrier is a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. The pharmaceutically acceptable carrier is an excipient, a diluent, a buffer, a stabilizer, a lipid, an emulsion, and/or a nanoparticle. In one or more embodiments, the activated MSCs are administered by an injection into the affected area, an intravenous injection, or topically. In one or more embodiments, the injection is administered directly to an affected tissue or administered near the affected tissue. In one or more embodiments, an additional treatment is administered to the subject before, during, or after the administration of the activated MSCs. For example, the additional treatment comprises an anti-inflammatory compound, an anesthetic, or a sedative.

[0042] In one or more embodiments, the composition for treating musculoskeletal disorders or conditions, such as osteoarthritis (OA), in a patient includes a mesenchymal stromal cell (MSC) with pattern recognition receptors (PRR), wherein the PRR includes at least one activated TLR3 receptor, at least one activated stimulator of interferon genes (STING), or a combination thereof. In one or more embodiments, the activated MSCs are part of a composition for treating a musculoskeletal disease in a patient, the composition includes a mesenchymal stromal cell (MSC) comprising an activated stimulator of interferon gene (STING) pathway where the STING pathway was activated by an agonist. The agonist may be a cyclic dinucleotides (CDN), including both natural and synthetic CDNs, an amidobenzimidazole (ABZI), a 5,6- dimethylxanthenone-4-acetiv acid (DMXAA), a 2’3’-cGAMP, c-di-GMP, a c-di- AMP3’3’-cGAMP, an ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, BMS- 986301, a Cyclic GMP-AMP synthase (cGAS), or combinations thereof.

[0043] The activated MSCs are administered in such amounts, time, and route deemed necessary in order to achieve the desired result. If the MSCs are preserved, the MSCs are thawed before administering. The exact amount of the activated MSCs will vary from subject to subject depending on the species, age, and general condition of the subject, the severity of the musculoskeletal condition, the particular activated MSCs, the activated MSCs mode of administration, the activated MSCs mode of activity, or other similar characteristics. The activated MSCs are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It should be understood, however, that the total daily usage of the activated MSCs will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the musculoskeletal condition being treated, the severity of said condition, the activity of the activated MSCs employed, the specific cell employed, the age, body weight, general health, sex and diet of the patient, the time of administration, the route of administration, the rate of excretion of the specific MSC employed, the duration of the treatment, the drugs used in combination or coincidental with the specific cell employed, and similar factors well known in the medical arts. The exact amount of activated MSCs required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular cell(s), mode of administration, and the like. The amount of activated MSCs to be administered to, for example, a juvenile, a child, or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0044] The term “subject” may be used interchangeably with “patient” and refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can be any recreational or agricultural animal including but not limited to a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician or a veterinarian.

[0045] In one or more embodiments, the MSC is administered in one or more doses to the subject. In one or more embodiments, the MSC is administered repeatedly, depending on the severity of the musculoskeletal disorder or condition (e.g., OA) and the patient’s condition, at time intervals of days, weeks, months, or years, as established by a medical practitioner or specialist. In some embodiments, the MSC is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,

73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

96, 97, 98, 99, 100, or more times. In one or more embodiments, the MSC is administered daily. In some embodiments, the MSC is administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some embodiments, the activated MSCs is administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In one or more embodiments, the activated MSCs are administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In one or more embodiments, the MSC is administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more. For example, the subject is administered as many doses as required to treat the musculoskeletal disorder or condition.

[0046] The terms “treat,” “treating,” and grammatical variations thereof as used herein include partially or completely delaying, alleviating, mitigating, or reducing the intensity of one or more attendant symptoms of a musculoskeletal disorder or condition and/or alleviating, mitigating, or impeding one or more causes of a musculoskeletal disorder or condition. Treatments according to the disclosure may be applied preventatively, prophylactically, palliatively, or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of inflammation, swelling, pain, or loss of function), during early onset (e.g., upon initial signs and symptoms of inflammation, swelling, pain, or loss of function), or after an established development of the musculoskeletal disorder or condition.

[0047] In one example, the activated MSCs are administered to an affected tissue or near an affected tissue at about 10² to about 10⁷ activated per cm³ of tissue. In one example, administration of the activated MSCs may be done by injection into an affected joint or tissue or near an affected joint or tissue (e.g., the knee joint of a human or a horse) in a therapeutically effective amount ranging from about 1 x 10⁶ to about 1 x 10⁸ cells per injection in a physiologically acceptable medium. The physiologically acceptable medium includes, but is not limited to saline, PBS, or the freezing medium (e.g., the freezing medium disclosed above). In one example, where the subject is a mouse, the affected joint was aseptically prepared in a routine fashion and injected with 2.5 x 10⁵ activated MSCs suspended in 10 pL phosphate buffered saline (PBS). Injections are performed with a #27 needle and Hamilton syringe. For treatment administered to injured tendons, the MSCs are administered in a therapeutically effective amount ranging from about 1 x 10⁶ to about 5 x 10⁶ cells per injured tendon in a physiologically acceptable medium. In another example, if the affected tissue or joint is small, fewer cells are administered per injection. In another example, the activated MSCs are injected intravenously, intra-peritoneally, or intra-thoracically. In another example, an IV injection include about 2 x 10⁶ to about 10 x 10⁶ cells per kilogram of body weight of the subject. It is contemplated that MSCs can be administered systemically, by injection, or topically. In another example, activated MSCs are administered by intra-articular injections, direct intra-tendinous injections, or peri -tendinous injections into the affected tissues or near the affected tissues.

Rodent Destabilization of the Medial Meniscus Study and Data

[0048] The proceeding examples are studies involving an intra-articular therapy in a rodent destabilization of the medial meniscus (DMM) model of OA. The DMM model revealed significant improvements in post-operative voluntary movement parameters and histological outcomes with STING activated MSC and TLR3 activated MSC. Some examples include data collected from equine bone marrow derived MSCs. The materials, methods, and data are disclosed below. Example Materials and Method for OA Study

[0049] As shown in Figure 1A, the overview of the study disclosed herein as an example is summarized. The example used 12-week-old male C57BL/6Nci mice (Charles River Laboratories, Wilmington, MA, USA). The mice (n=10 per group for a total of forty mice) received unilateral destabilization of the medial meniscus surgery in the right knee and were randomly assigned to receive either control treatment (intraarticular needle insertion alone), non-activated MSCs, TLR3 activated MSCs, or STING activated MSCs for two treatments at weeks three and five postoperatively . The TLR3 activated MSCs are activated by the TLR3 agonist, polyinosinic-polycytidylic acid (pIC). The STING activated MSCs are activated by the stimulator of IFN genes (STING). The activated MSCs (e.g., the TLR3 activated MSCs or the STING activated MSCs) were formed using method 100, as disclosed above. Activity monitoring was performed weekly on all mice in each group. At endterm, five mice per group were assessed for histologic scoring and joint tissues from the remaining five mice were processed for transcriptomic analyses of joint tissues.

[0050] To induce osteoarthritis in in the right femorotibial joint (knee) of the mice at time 0 (week 0), established methods of destabilization of the medial meniscus (DMM) in the right hindlimb were used. The mice were induced under general anesthesia using isoflurane (1-5%, to effect) and the right femorotibial joint (knee) was clipped and aseptically prepared in routine fashion. To induce OA, a medial parapatellar arthrotomy using a #11 scalpel blade was performed and the infrapatellar fat pad (IFP) temporarily repositioned laterally to allow access to the anterior medial meniscotibial ligament. This ligament was severed using the #11 scalpel blade. The IFP was repositioned, and the surgical incision closed in simple interrupted fashion using 6-0 monofilament absorbable suture. The mice were administered buprenorphine SR (slow release) 0.6-0.8 mg/kg subcutaneously under anesthesia at time of surgery.

[0051] Intra-articular injections of MSCs were performed at weeks three and five following DMM operation. For the injections, mice were induced under general anesthesia using 3% isoflurane with oxygen, followed by 1-1.5% isoflurane to maintain plane of anesthesia. The right (operated) femorotibial joint was aseptically prepared in routine fashion and injected with 2.5xl0⁵ murine adipose-derived MSC, TLR3 activated MSC or STING activated MSC suspended in 10 pL phosphate buffered saline (PBS), or controls of needle insertion alone. Injections were performed with #27 needle and Hamilton syringe.

[0052] The mice were monitored prior to injury and for eight weeks following surgery using individual cage monitoring to determine general animal behavior and mobility. Cage monitoring was performed for 10 minutes weekly during the experimental time-course. The mice were placed in their primary enclosure and/or resident cage with their environmental enrichment hut for the duration of the assessment. Prior to taking the baseline measurement, mice were acclimated to the system over one week. After acclimation, two baseline measurements were collected immediately before the start of the study. The training and data collection occurred during the same time of day (8am to 12pm) and involved the same handlers throughout the course of the study to minimize circadian rhythm cycle variations. The video analysis software used (ANY-maze™, Wood Dale, IL, USA) automatically collected mobility parameters including total distance traveled, time mobile, mean speed, maximum speed, time in hut, and entries to the top of the hut. Parameters of interest were assessed both cross-sectionally amongst groups’ normalized to preoperative baseline and longitudinally over time.

[0053] Mouse femorotibial (knee) tissues (n=5 biological replicates per treatment group of 10 mice) were removed immediately following euthanasia, minced, and stored in RLT lysis buffer (Qiagen) at -80°C. RNA was extracted by first using TRIzol Reagent (Invitrogen, Waltham, MA) following manufactures instructions, with a 1 :3 RLT lysis buffer to TRIzol ratio. RNA precipitate pellet was then cleaned and concentrated using RNeasy MinElute Cleanup Kit (Qiagen Germantown, MD), according to manufacturer’s instructions and sent to Novogene Corporation Inc. (Sacramento, CA) for bulk RNA sequencing. RNA quality was determined by bioanalyzer (Agilent Technologies, Santa Clara, CA). RIN (RNA integrity number) was determined to be >7.5 for all samples. For bulk RNA sequencing, mRNA was enriched using oligo (dT) beads, followed by cDNA library generation using TruSeq RNA Library Prep Kit (Illumina, San Diego, CA). Sequencing was performed on Illumina Novaseq 6000 machine using 150 bp paired end reads. [0054] At eight weeks after surgery, femorotibial (knee) joints (n=5 biological replicates per treatment group of 10 mice) were fixed in 10% neutral buffered formalin for 24 hours, then decalcified in ethylenediamine tetra-acetic acid (EDTA) and paraffin embedded. Coronal sections (5 pm) were taken from the center of the medial tibial plateau and stained with toluidine blue. Histological grading of joint tissues was performed using an established criteria for OA by two blinded, independent individuals trained in using this scoring method via consensus. Joint tissues were semi- quantitatively graded for osteoarthritic damage including cartilage fibrillation, cartilage loss including clefts/erosions and calcification, synovitis, and proteoglycan content for the whole joint and medial and lateral joint compartments.

[0055] The experimental sample size was calculated by a-priori power analysis using GPower Version 3.1.1, using pilot gait data (stride length) obtained using this injury model in mice as the primary outcome measure for calculating group sizes. Cross-sectional activity monitoring data was normalized to baseline values and compared using repeated-measures analysis of variance (ANOVA) with Tukey’s correction. Longitudinal data were compared using repeated-measures ANOVA and Dunnett’ s correction, comparing each timepoint to baseline values. Histological scoring at end-term was evaluated using a one-way non-parametric ANOVA (Kruskal-Wallis test) with Dunn’s multiple comparisons test. Statistical analysis was performed using GraphPad Prism v9.3.1 (GraphPad Software Inc., La Jolla, CA, USA). The significance for enclosure monitoring and histological outcomes was assessed at p<0.1 as described.

[0056] Sequence data were analyzed on Partek Flow software, version 10.0 (Partek Inc. Chesterfield, MO). Raw data were filtered by removing reads containing adapters and reads containing N > 10% and for Phred scores > 30. Filtered reads were then aligned with STAR 2.7.3a using GRCm39 Genome Reference. Aligned reads were annotated and counted using HT-seq with Ensembl 108. Differentially expressed genes were identified using DEseq2. Biological interpretations included gene ontology and gene set enrichment analysis (GSEA), which were performed using GSEA.

Example 1: Impact of the TLR3 Activated MSC Treatment on Mouse Behavior

[0057] FIGS. 3A, 3B, 3C, and 3D show the impact of immune activated MSC on mobility and knee function in a mouse model of trauma-induced osteoarthritis. Studies were done in mice where surgical injury to the knee triggers the development of osteoarthritis. After the surgery, mice were treated twice by injection of either nonactivated MSC, or immune-activated MSC (e.g., TLR3 activated MSC) and the impact of cell injection on 4 parameters associated with joint function and mobility was assessed over 8-weeks of video monitoring of the 10 animals. These studies demonstrated that the TLR3 activated MSC were significantly more effective than nonactivated MSC in improving mobility and joint function in the treated animals.

Example 2: Comparing the Impact of the STING Activated MSC Treatment, the TLR3 Activated MSC Treatment, and Controls on Mouse Behavior

[0058] Figures 2A, 2B, 2C, 2D, 2E, and 2F show exemplary data of activated mesenchymal stromal cell treatment viability with ANY-maze™ cage monitoring parameters. Loss of mobility and articular associated pain are primary reasons for individuals with OA to seek treatment. Accordingly, the functional activity of the mouse subjects was monitored with cage-monitoring or “open field testing.” The voluntary behavior and mobility differences relative to a baseline activity level were measured as an indication of pain following surgery. Various parameters were tested. For example, some of the parameters reported include adjusted time mobile, maximum speed, time spent in hut.

[0059] As shown in Figure 2A and 2B, the mice treated with STING pathway activated MSC exhibited greater adjusted time mobile compared to MSC treated mice (p=0.04) or control (p=0.07) at week 8. As shown in Figure 2C and 2D, the mice treated with STING activated MSC spent less time in their security huts relative to baseline compared to MSC treated mice (p=0.09) at week 8. As shown in Figure 2E and 2F, mice treated with STING activated MSC completed fewer hut entries relative to baseline compared to control at week 4 (p=0.04) or TLR3 activated MSC treated mice at week 6 (p=0.1). There were no significant differences between TLR3 activated MSC treated mice compared to needle or MSC alone treated mice.

[0060] As shown in Figures 2A-2F, after about eight weeks post-DMM surgery, the mice injected with two doses of STING activated MSCs demonstrated increased activity. The increased activity is shown by a greater adjusted time mobile and less time in their security huts. This indicates differences in clinical or pain responses between the different treatment groups. Additionally, STING activated MSCs treated animals exhibited the greatest number of entries to the top of their hut compared to other treatment groups, which reached significance compared to TLR activated MSC treated mice at week 6, as shown in Figure 2E and Figure 2F. In this hindlimb injury model, this indicates an increased willingness to climb, use, and propel off the hut, which indicates an earlier return to full hind limb function for mice treated with the STING activated MSCs. The significant improvement in voluntary movement parameters (adjusted time mobile, maximum speed, time spent in hut) in mice treated with the STING activated MSCs indicates the STING pathway enhances both functional outcomes and structural joint integrity.

Example 3: RNA Sequencing Analysis of Joint Tissues Treated with STING Activated MSCs and MSCs.

[0061] Figures 4A, 4B, 4C, Table 1 and Table 2 show exemplary data of RNA sequencing analysis comparing transcriptomes of joint tissues from mice treated with STING activated MSC or MSC. As shown in Figure 4A, the knee joint tissues from STING activated MSC and MSC treated subjects demonstrated significant upregulation of 24 genes and downregulation of 103 genes (significance defined as fold change > 2 or < -2 or P value <0.05). As shown in Table 1, differential gene expressions (list of genes, description, unadjusted p-value and fold-change of top 20 genes) for upregulated genes are listed. As shown in Table 2, differential gene expressions (list of genes, description, unadjusted p-value and fold-change of top 20 genes) for downregulated genes are listed. Table 1 and Table 2 show the difference in upregulated and downregulated genes from the STING activated MSC and MSC treated joints. Figure 4B shows the pathway analysis of the top 15 upregulated gene pathways. Figure 4C shows the pathway analysis for the top 15 downregulated gene pathways. Several gene signatures emerged as particularly relevant to how the two immune pathways (e.g., the TLR3 pathway and the STING pathway) function to improve joint function.

[0062] Expressed genes and transcriptomic pathway analyses between treatment groups (e.g., treatment of joints with STING activated MSC) resulted in upregulation of gene pathways associated with tissue remodeling, cell motility and invasion, cell proliferation and angiogenesis and downregulation of genes associated with inflammatory cytokine production and epidermal barrier formation. For example, in joints of STING activated MSC treated mice, one of the most upregulated genes was encoding tryptophan 5-hydroxylase. Tryptophan 5-hydroxylase is an enzyme essential to synthesis of the neurotransmitter serotonin. Tryptophan metabolite disturbances are associated with erosive hand osteoarthritis in humans. Supplementation of tryptophan metabolites, including 5-hydroxytryptophan, suppresses inflammation and arthritis through suppression of pro-inflammatory mediator production in a rodent inflammatory collagen-induced arthritis model.

Table 1 Table 2 Example 4: RNA Sequencing Analysis of Joint Tissues Treated with TLR3

Activated MSCs and MSCs

[0063] Figures 5A, 5B, 5C, Table 3 and Table 4 show exemplary data of RNA sequencing analysis comparing transcriptomes of joint tissues from mice treated with TLR3 activated MSC or MSC. Figure 4A shows the comparison of knee joint tissues from TLR3 activated MSC to MSC treated animals demonstrated significant upregulation of 16 genes and downregulation of 26 genes (significance defined as fold change > 2 or < -2 or P value <0.05). Table 3 shows the differential gene expression (list of genes, description, unadjusted p-value and fold-change of top 20) for upregulated genes in differential analysis results from TLR3 activated MSC vs. MSC treated joints. Table 4 shows the differential gene expression (list of genes, description, unadjusted p-value and fold-change of top 20) for downregulated genes in differential analysis results from TLR3 activated MSC vs. MSC treated joints. Figure 5B shows a pathway analyses of the top 15 upregulated pathways. Figure 5C shows a pathway analysis of the top 15 downregulated pathways.

Table 3

Table 4

Example 5: Comparing Joint Tissues Treated with TLR3 activated MSCs, STING activated MSCs, and MSCs [0064] Intra-articular injections affect immune transcriptomes of the joints in the subjects. The subjects were treated by an intra-articular injection of activated or nonactivated MSC. 24h later RNA was extracted from harvested joint tissues and analyzed via bulk RNA sequencing. The joint tissues are from mouse subjects or equine subjects.

[0065] Figures 6A, 6B, 6C, and 6D show differential gene expression (DEG) analysis for TLR3 activated MSCs, STING activated MSCs, and MSCs. MSCs are non-activated MSCs. The data shown in 6A, 6B, 6C, and 6D are results from activated equine MSCs. Figure 6A shows STING activated MSCs vs. MSCs in a volcano plot of DEG (log2FC = 1.5) top 10 labeled. Figure 6B shows a TLR3 activated MSCs vs. MSCs: volcano plot of DEG (log2FC = 1.5) top 10 labeled. Figure 6C shows a Venn diagram of DEG in STING activated MSCs & TLR activated MSCs compared to (nonactivated) MSCs. Figure 6D shows a STING activated MSCs vs. TLR activated MSCs: heat map of top 10 DEG and their corresponding biological processes. The positive fold changes indicated upregulation while the negative fold change indicates downregulation. Some of the upregulated genes are associated with antiviral immune responses. Upregulation of the genes indicates the activated MSC may secrete chemokines and cytokines in the tested joint tissue, which recruit immune cells (e.g., monocytes) that serve to stimulate joint healing and reduce inflammation.

[0066] Figure 7, Table 5 and Table 6 show exemplary data of RNA sequencing analysis comparing transcriptomes of joint tissues from mice treated with STING activated MSC or TLR3 activated MSC. As shown in Figure 7, gene expression of mouse joints treated with STING activated MSC and mouse joints treated with TLR activated MSC are compared. A volcano plot of DESeq analysis shows the results. The x-axis shows fold change on a Log2 scale, positive values for upregulated in STING activated MSC and negative values for downregulated STING activated MSC. The y- axis shows a -LoglO p-value. Significant genes (p-value <=0.05 or fold change >=2 or <=-2) are shown in red representing upregulated and blue representing downregulated.

[0067] Table 5 and Table 6 show a differential gene expression list from mouse joints with experimentally induced OA that was treated with an MSC treatment. Table 5 and Table 6 show the gene ID, the gene description, the p value, and the fold change in Log2. The mice compared were injected with STING activated MSCs or TLR activated MSCs (e.g. TLR3 activated MSCs). RNA was extracted from whole joints of the mice and sequenced. The top 20 upregulated genes (e.g., the upregulation was statistically significant) in joints treated with STING activated MSCs are shown in Table 5. Table 6 shows the differential gene expression from mouse joints treated with STING activated MSCs compared to TLR3 activated MSCs. The top 20 downregulated genes (e.g., the downregulation was statistically significant) in joints treated with STING activated MSCs are shown in Table 6. Many of the downregulated genes (e.g., Protein S100-A14 or Complement Clq tumor necrosis factor-related protein 3) are related to inflammation. The downregulation of genes related to inflammation indicates a reduction in inflammation in the joint tissue of the subjects treated with the activated MSCs.

Table 5

Table 6 [0068] Figure 8 shows exemplary data of overlapping gene signatures from differential expression analysis in a Venn diagram. A total of 34 significant differentially expressed genes (DEGs) are unique when comparing joints of mice treated with TLR3 activated MSCs to untreated joints of mice, as shown in Figure 8. A total of 119 DEGs are unique when comparing joints of mice treated with STING activated MSCs to untreated joints of mice, as shown in Figure 8. There are 9 genes that are significant in both comparison groups. The difference between the gene signatures of the TLR3 activated MSCs and the STING activated MSCs indicate the two pathways are not identical in terms of their downstream immune effects. Accordingly, the activation of both the TLR3 pathway and the STING pathway are not redundant and provide a significant level of activation of different gene signatures.

[0069] Table 7, Table 8, Figure 9A and Figure 9B show pathway analysis using Gene Set Enrichment Analysis (GSEA). Table 7 and Table 8 show the pathway names, the number of genes mapped to the listed pathway, the enrichment score, and the P- value for significance. A higher enrichment score indicates the most upregulated pathway in the joints of mice treated with the STING activated MSC. The data in Table 7 and Table 8 was derived from RNA extracted from the joints of mice. Specifically, the top 20 upregulated pathways (e.g., the upregulation was statistically significant) comparing the STING activated MSC treated mouse joints and the TLR3 activated MSC treated mouse joints are shown in Table 7. Many of the upregulated pathways are related to cell division, indicating that at least some of the dividing cells are related to repairing a joint injury (e.g., a join injury related to OA). Specifically, the top 20 downregulated pathways (e.g., the downregulation was statistically significant) comparing the STING activated MSC treated mouse joints and the TLR3 activated MSC treated mouse joints are shown in Table 8. Many of the downregulated pathways are related to the formation of collagen, which is associated with fibrosis of an injured joint overtime and inflammation. The downregulation of the pathways indicates a reduction in inflammation and/or fibrosis in the tested joint cells.

Table 7

Table 8

[0070] Figure 10 shows the gene expression of in vitro treated STING activated equine MSC and in vitro treated TLR activated equine MSC are compared. A volcano plot of differential gene expression is shown in Figure 10. The x-axis shows fold change on a Log2 scale, positive values for upregulated in STING activated equine MSC and negative values for downregulated STING activated equine MSC. The y- axis shows a -LoglO p-value. Significant genes (p-value <=0.05 or fold change >=2 or <=-2 are shown in diamonds representing upregulated genes and circles representing downregulated genes. [0071] Table 9 and Table 10 show a differential gene expression list from in vitro tested equine bone MSCs. Table 9 and Table 10 show the gene ID, the gene description, the p value, and the fold change in Log2. The equine MSCs were grown from healthy horse bone marrow and then activated in vitro with a STING agonist or a TLR3 agonist. The differential gene expression list compares the STING activated equine MSCs to the TLR activated equine MSCs using DEseq. The top 20 upregulated genes (e.g., the upregulation was statistically significant) of the STING activated equine MSCs are shown in Table 9. The 19 statistically significant downregulated genes in the STING activated equine MSCs are shown in Table 10.

Table 9

Table 10

[0072] Figure 11 shows exemplary data of overlapping gene signatures from differential expression analysis in a Venn diagram. A total of 10 significant DEGs are unique when comparing TLR3 activated equine MSCs to untreated equine MSCs. A total of 78 DEGs are unique when comparing STING activated equine MSCs to untreated equine MSCs. There are 76 genes that are significant in both comparison groups. The difference between the gene signatures of the TLR3 activated MSCs and the STING activated MSCs indicate the two pathways are not identical in terms of their downstream immune effects. Accordingly, the activation of both the TLR3 pathway and the STING pathway are not redundant and provide a significant level of activation of different gene signatures.

[0073] Table 11 and Table 12 show pathway analysis using Gene Set Enrichment Analysis (GSEA). Table 11 and Table 12 show the pathway names, the number of genes mapped to the listed pathway, the enrichment score, and the P-value for significance. A higher enrichment score indicates the most upregulated pathway in the STING activated equine MSCs. The data in Table 11 and Table 12 was derived from RNA extracted in vitro activated equine MSCs. The statistically significant upregulated pathways comparing the STING activated equine MSCs and the TLR3 activated equine MSCs are shown in Table 11. The top 20 downregulated pathways (e.g., the downregulation was statistically significant) comparing the STING activated equine MSCs and the TLR3 activated equine MSCs are shown in Table 12.

Table 11

Table 12

[0074] Expressed genes and transcriptomic pathway analyses between treatment groups (e.g., treatment of joints with STING activated MSCs, the TLR3 activated MSCs, or non-activated MSCs) resulted in upregulation of gene pathways associated with tissue remodeling, cell motility and invasion, cell proliferation and angiogenesis and downregulation of genes associated with inflammatory cytokine production and epidermal barrier formation.

[0075] One common denominator between the two pathways discussed above in Figures 3A-3D, 4A-4C, 5A-5C, 6A-6D, 7, 8, 9A-9B, 10, 11 and Tables 1-12 is a strong stimulation of interferon pathways, including all 3 major pathways (type I, type II, and type III IFN pathways). IFN-related genes have been previously implicated in the resolution of inflammatory pain. Upregulated IFN pathways provide other functions such as slowing joint degeneration and stimulation of counter-regulatory immune pathways (e.g., IDO pathway, PD-L1 and other checkpoint molecule expression, expansion of Tregs, recruitment of immune suppressive monocytes). The upregulated IFN pathways contribute to reducing joint inflammation and preserving cartilage integrity. Further, the upregulated IFN pathways contribute to the suppression of deleterious vascular responses by suppressing abnormal angiogenesis. In one or more embodiments, IFN-g is associated with stimulating cartilage regeneration. Other IFN- regulated pathways are involved in improved joint function following injection of activated MSC. For example, alterations in tryptophan metabolism have been previously associated with occurrence and development of OA.

[0076] STING and TLR3 pathway activated-MSC exert a therapeutic effect in early post-traumatic OA subjects. Differential gene expression on the cellular level relates to the improvement seen functionally and structurally in the joint material collected from the subjects. Accordingly, multiple different IFN pathways are involved in reducing joint inflammation, improving mobility, and improving cartilage integrity.

[0077] Figures 12 A, 12B, 12C, 12D, 12E, 12F, 12G, 12H, 121, 12 J, 12K, and 12L show histologic representative images of mouse knees after treatment. Low magnification (4x) images of whole knee joints are shown, control (Figure 12A), MSC (Figure 12D), TLR3 activated MSC (Figure 12G), and STING activated MSC (Figure 12J) treated joints. Higher magnification (lOx) images presented for evaluation of the medial compartment are shown, control (Figure 12B), MSC (Figure 12E), TLR3 activated MSC (Figure 12H), and STING activated MSC (Figure 12K) treated joints. Additional higher magnification image for evaluation of the lateral compartment are shown, control (Figure 12C), MSC (Figure 12F), TLR3 activated MSC (Figure 121) and STING activated MSC (Figure 12L) treated joints. In small animal (i.e., rodent) species, evaluation of the entire joint in a single plane (e.g., Figures 12A-12I) allows simultaneous examination of the entire joint, including pathological changes in cartilage degradation, synovial inflammation and fibrosis, meniscal tearing and bone remodeling, which includes marginal osteophytes or enthesiophytes and subchondral sclerosis. Multiple scoring systems have been reported for histopathological evaluation of the mouse knee OA.

[0078] The single slide images of the mouse knees evaluated with the semi- quantitative mouse-OA histopathology scoring system are shown in Figures 12A-12L. The impact of intra-articular treatment on joint pathology was evaluated according to the semi-quantitative mouse-OA histopathology scoring system.

[0079] Medial and lateral scores were assigned for synovitis and for the femur and tibia individually, which were combined to obtain the overall medial and lateral scores that were added to achieve the total joint score. In untreated control animals receiving DMM, there was synovitis, fibrillation, clefts in the medial and/or lateral compartments, and, in some instances, osteophytes on the medial aspect of the joint. The MSC treated groups showed improved cartilage integrity and synovial scores compared to the control groups.

[0080] Using the semi-quantitative mouse-OA histopathology scoring system, the subjects demonstrated a significant improvement in total joint and medial compartment scores with STING activated MSC treatment compared to control (needle insertion alone) and non-activated MSC. The STING activated MSC treatment shows an improvement in the lateral compartment when compared to the control. Further, when medial and lateral compartments were examined separately, the STING activated MSC demonstrated an improvement in medial and lateral tibial and femoral cartilage as well as synovium scores when compared to the control. The TLR3 activated MSC therapy showed an improvement in overall joint and medial compartment scores compared to needle insertion alone. The results exhibited in the animal models indicate the treatments discussed provide the basis for therapeutic development for humans with OA. The data indicates that the use of treatments including an immune activated cellular therapy, particularly STING activated MSC, to mitigate structural disease progression is effective.

[0081] Figures 13A-13I show exemplary data of Osteoarthritis Research Society International (OARSI) histopathology scores performed following DMM surgery and treatment. Histopathology is used in animal models to assess structural disease in OA pathology characterized by progressive articular cartilage degradation and osteophyte development. As shown in Figures 13A-13I, significant differences are noted with p values <0.05. For example, as shown in Figure 13 A and 13B, reduced total and medial compartment OARSI histology scores in both TLR3 activated MSC and STING activated MSC groups indicate that both treatments can be used to managing OA. Specifically, as shown in Figures 13A and 13B, joint scores (p=0.0003, p=0.05) and medial compartment scores (p=0.0003, p=0.05) were improved in STING activated MSC treated joints compared to MSC treated or control joints. Improved lateral compartment score, as shown in Figure 13C, for STING activated MSCs indicates that the STING activated MSC treatment has improved efficacy compared to the control joints. Specifically, as shown in Figure 12C, lateral compartment scores were improved in STING activated MSC treated vs. control joints (p=0.001). Overall joint and medial compartment scores (p=0.05, p=0.05) were improved in TLR3 activated MSC treated joints compared to control joints. Figures 13D, 13F, 13G, 13H, and 131 show additional scored and individual OARSI parameters for tibia, femur, synovium in the medial, and lateral compartments. [0082] The destabilization of the medial meniscus (DMM) model presented herein represents a reproducible animal model that allows longitudinal evaluation of disease. A semi-quantitative mouse-OA histopathology scoring system to grade cartilage as well as synovium, meniscus, cruciate ligaments, subchondral bone and marginal osteophytes/enthesiophytes is used. The semi-quantitative mouse-OA histopathology scoring system allows for the evaluation of a single slide and allows for reproducible grading of histopathological changes in joint tissues in this model and reduced scoring time without compromising sensitivity and specificity of results.

[0083] Figure 14 shows an example schematic of the stimulation of equine bone- marrow MSCs stimulated with agonists, assayed, extracted, and mapped to equCab3.0.

[0084] Overall, embodiments of the present disclosure generally relate to compositions and methods for treating musculoskeletal disorders or conditions, such as osteoarthritis (OA). Specifically, the compositions and methods include activating mesenchymal stromal cells (MSCs) with a stimulator of interferon gene (STING) agonist or a toll like receptor (TLR) agonist. MSC treatment, STING activated MSC treatment, and TLR3 activated MSC treatment show an improved histologic outcome. However, the STING activated MSC treatment and TLR3 activated MSC resulted in further improvement in key functional parameters and induced transcriptomic changes promoting tissue repair. Without being bound by theory, activating the MSCs with stimuli that induce strong IFN responses is an effective strategy to improve the overall effectiveness of cellular therapy for OA. For example, the STING activated MSCs reduce inflammation and attenuate pain associated with OA progression.

EMBODIMENTS LISTING

[0085] The present disclosure provides, among others, the following embodiments, each of which can be considered as optionally including any alternate embodiments:

[0086] Clause 1. A method of treating a musculoskeletal disorder or condition in a subject, comprising administering to the subject a composition comprising a mesenchymal stromal cell (MSC), wherein the MSC comprise an activated signaling pathway downstream of (i) stimulator of interferon gene (STING) and/or (ii) toll-like receptor 3 (TLR3). [0087] Clause 2. The method of Clause 1, wherein the MSC has been activated by culture with a STING agonist and/or a TLR3 agonist.

[0088] Clause 3. The method of Clause 2, wherein the STING agonist comprises one or more of a cyclic dinucleotide (CDN), amidobenzimidazole (ABZI), DMXAA, 2’3’-cGAMP, c-di-GMP, c-di-AMP3’3’-cGAMP, ADU-S100, SB 11285, GSK 3745417, MK-1454, BMS-986301, and Cyclic GMP-AMP synthase (cGAS).

[0089] Clause 4. The method of Clause 2, wherein the TLR3 agonist comprises a nucleic acid.

[0090] Clause 5. The method of Clause 2, wherein the TLR3 agonist comprises one or more of polyinosinic-polycytidylic acid (pIC), polyadenylic-polyuridic acid (pAU), Rintatolimod, Resiquimod, and UV-inactivated viral particles.

[0091] Clause 6. The method of any one of Clauses 2-5, wherein the MSC has been activated by culture with a STING agonist, a TLR3 agonist, or both for at least about 0.25 hours, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or more than 2 hours.

[0092] Clause 7. The method of any one of Clauses 1-6, wherein the MSC is a stem cell-derived MSC.

[0093] Clause 8. The method of any one of Clauses 1-7, wherein the MSC is derived from an embryonic stem cell or an induced pluripotent stem cell (iPSC).

[0094] Clause 9. The method of any one of Clauses 1-6, wherein the MSC is a tissue-derived MSC.

[0095] Clause 10. The method of Clause 9, wherein the tissue-derived MSC is derived from bone marrow, adipose tissue, placenta, umbilical cord tissue, umbilical cord blood, peripheral blood, muscle, uterine tissue, corneal stroma, skin, and/or dental pulp.

[0096] Clause 11. The method of any one of Clauses 1-10, wherein the composition is administered by intra-articular injection, intra-muscular injection, intra-tendinous inj ection, intra-ligamentous injection, intra-peritoneal injection, intra-venous injection, intra-arterial injection, sub-cutaneous injection, or intra-thoracic injection.

[0097] Clause 12. The method of any one of Clauses 1-11, wherein the musculoskeletal disorder or condition is osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, spondylarthritis, osteoporosis, osteopenia, tendinitis, tendonitis, bursitis, fibromyalgia, joint cartilage injury, myopathy, ligament injuries, fragility fractures, or traumatic bone fractures.

[0098] Clause 13. The method of any one of Clauses 1-12, wherein the MSC is an autologous MSC.

[0099] Clause 14. The method of any one of Clauses 1-12, wherein the MSC is an allogeneic MSC.

[0100] Clause 15. The method of any one of Clauses 1-12, wherein the MSC is a xenogeneic MSC.

[0101] Clause 16. The method of any one of Clauses 1-15, wherein the MSC is a mammalian MSC.

[0102] Clause 17. The method of Clause 16, wherein the mammalian MSC is from a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

[0103] Clause 18. The method of any one of Clauses 1-17, wherein the subject is a mammalian subject.

[0104] Clause 19. The method of Clause 18, wherein the mammalian subject is a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

[0105] Clause 20. A method of treating of treating a musculoskeletal disorder or condition in a subject, comprising administering to the subject a composition comprising a mesenchymal stromal cell (MSC) that has been cultured in the presence of a stimulator of interferon gene (STING) agonist and/or a toll-like receptor 3 (TLR3) agonist. [0106] Clause 21. The method of Clause 20, wherein the STING agonist comprises one or more of a cyclic dinucleotide (CDN), amidobenzimidazole (ABZI), DMXAA, 2’3’-cGAMP, c-di-GMP, c-di-AMP3’3’-cGAMP, ADU-S100, SB 11285, GSK 3745417, MK-1454, BMS-986301, and Cyclic GMP-AMP synthase (cGAS).

[0107] Clause 22. The method of Clauses 20 or 21, wherein the TLR3 agonist comprises a nucleic acid.

[0108] Clause 23. The method of any one of Clauses 20-22, wherein the TLR3 agonist comprises one or more of polyinosinic-polycytidylic acid (pIC), polyadenylic- polyuridic acid (pAU), Rintatolimod, Resiquimod, and UV-inactivated viral particles.

[0109] Clause 24. The method of any one of Clauses 20-23, wherein the MSC has been cultured in the presence of a stimulator of interferon gene (STING) agonist and/or a toll-like receptor 3 (TLR3) agonist for at least about 0.25 hours, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or more than 2 hours.

[0110] Clause 25. The method of any one of Clauses 20-24, wherein the MSC is a stem cell-derived MSC.

[OHl] Clause 26. The method of Clause 25, wherein the MSC is derived from an embryonic stem cell or an induced pluripotent stem cell (iPSC).

[0112] Clause 27. The method of any one of Clauses 20-26, wherein the MSC is a tissue-derived MSC.

[0113] Clause 28. The method of Clause 27, wherein the tissue-derived MSC is derived from bone marrow, adipose tissue, placenta, umbilical cord tissue, umbilical cord blood, peripheral blood, muscle, uterine tissue, corneal stroma, skin, and/or dental pulp.

[0114] Clause 29. The method of any one of Clauses 20-28, wherein the composition is administered by intra-articular injection, intra-muscular injection, intra- tendinous injection, intra-peritoneal injection, intra-venous injection, intra-arterial injection, sub-cutaneous injection, or intra-thoracic injection. [0115] Clause 30. The method of any one of Clauses 20-29, wherein the musculoskeletal disorder or condition is osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, spondylarthritis, osteoporosis, osteopenia, tendinitis, tendonitis, bursitis, fibromyalgia, joint cartilage injury, myopathy, ligament injuries, fragility fractures, or traumatic bone fractures.

[0116] Clause 31. The method of any one of Clauses 20-30, wherein the MSC is an autologous MSC.

[0117] Clause 32. The method of any one of Clauses 20-30, wherein the MSC is an allogeneic MSC.

[0118] Clause 33. The method of any one of Clauses 20-30, wherein the MSC is a xenogeneic MSC.

[0119] Clause 34. The method of any one of Clauses 20-33, wherein the MSC is a mammalian MSC.

[0120] Clause 35. The method of Clause 34, wherein the mammalian MSC is from a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

[0121] Clause 36. The method of any one of Clauses 20-35, wherein the subject is a mammalian subject.

[0122] Clause 37. The method of Clause 36, wherein the mammalian subject is a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

[0123] Clause 38. A composition for treating musculoskeletal disorders or conditions in a patient, the composition comprising: a mesenchymal stromal cell (MSC) with pattern recognition receptors (PRR), wherein the PRR comprises an activated stimulator of interferon genes (STING) pathway, an activated a toll-like receptor 3 (TLR3) pathway, or a combination thereof. [0124] Clause 39. The composition of Clause 38, wherein the MSC is a bone marrow-derived MSC, a stem cell-derived, an adipose tissue-derived MSC, or an umbilical cord tissue-derived MSC.

[0125] Clause 40. The composition of Clause 38 or Clause 39, wherein the activated STING pathway is activated by an agonist, the agonist comprising a 2’,3’- cGAMP.

[0126] Clause 41. The composition of Clauses 38-40, wherein the activated STING pathway is activated by an agonist, the agonist comprising a cyclic dinucleotide (CDN), an amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, BMS-986301, a Cyclic GMP- AMP synthase (cGAS), or combinations thereof.

[0127] Clause 42. The composition of Clauses 38-41, wherein the activated TLR3 pathway is activated by an agonist, the agonist comprising a polyinosinic-polycytidylic acid (pIC).

[0128] Clause 43. The composition of Clause 38-42, wherein the PRR is operable to enhance an anti-inflammatory property, an immunomodulatory property, an angiogenetic property, or combinations thereof of the MSC.

[0129] Clause 44. The composition of Clause 38-43, further comprising a concentration of about 1 x 10⁶ to about 1 x 10⁸ cells per mL of a pharmaceutically acceptable carrier.

[0130] Clause 45. The composition of Clause 44, wherein the pharmaceutically acceptable carrier is a phosphate buffered saline solution, a saline solution, or a freezing medium.

[0131] Clause 46. A composition for treating musculoskeletal disorders or conditions in a patient, the composition comprising: a mesenchymal stromal cell (MSC) comprising an activated stimulator of interferon gene (STING) pathway, the STING pathway activated by an agonist, the agonist comprising: a cyclic dinucleotide (CDN), a amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, a BMS-986301, a Cyclic GMP-AMP synthase (cGAS), or combinations thereof.

[0132] Clause 47. The composition of Clause 46, wherein the MSC is a bone marrow-derived MSC, an adipose tissue-derived MSC, or an umbilical cord tissue- derived MSC.

[0133] Clause 48. The composition of Clause 46 or Clause 47, wherein the activated STING pathway is operable to enhance an anti-inflammatory property, an immunomodulatory property, an angiogenetic property, or combinations thereof of the MSC.

[0134] Clause 49. The composition of Clause 46-48, further comprising a concentration of about 1 x 10⁶ to about 1 x 10⁸ cells per mL of a pharmaceutically acceptable carrier.

[0135] Clause 50. The composition of Clause 49, the pharmaceutically acceptable carrier is a phosphate buffered saline solution, a saline solution, or a freezing medium.

[0136] Clause 51. A method of forming a composition for treating musculoskeletal disorders or conditions, comprising: activating a pattern recognition receptors (PRR) of a mesenchymal stromal cell (MSC), wherein the PRR comprises a stimulator of interferon gene (STING) pathway, a toll-like receptor 3 (TLR3) pathway, or a combination thereof; and forming the composition for treating musculoskeletal disorders or conditions, wherein the composition comprises an activated MSC.

[0137] Clause 52. The method of Clause 51, further comprising expanding the MSC in a complete growth media.

[0138] Clause 53. The method of Clause 51 and Clause 52, wherein activating the MSC comprises in vitro activation by incubation. [0139] Clause 54. The method of Clause 51, wherein the MSC is activated at a concentration of about lOpg/mL of an agonist to about IxlO⁶ MSCs/mL.

[0140] Clause 55. The method of Clause 54, wherein the agonist comprises a polyinosinic-polycytidylic acid (pIC), a cyclic dinucleotide (CDN), an amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, BMS-986301, a Cyclic GMP- AMP synthase (cGAS), or combinations thereof.

[0141] Clause 56. The method of Clause 51-55, wherein the MSC is activated with an agonist for about 0.25 hours to about 48 hours.

[0142] Clause 57. The method of Clause 51-56, further comprising: washing the activated MSC with phosphate buffered saline; and storing the activated MSC at a concentration of 1.6 x 10⁶ cells per mL to about

5 x 10⁶ cells per mL in a freezing media.

[0143] The terms “subject”, “individual” or “patient” are used interchangeably herein and refer to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans.

[0144] In the foregoing, reference is made to embodiments of the disclosure. However, it should be understood that the disclosure is not limited to specific described embodiments. Instead, any combination of the following features and elements, whether related to different embodiments or not, is contemplated to implement and practice the disclosure. Furthermore, although embodiments of the disclosure may achieve advantages over other possible solutions and/or over the prior art, whether or not a particular advantage is achieved by a given embodiment is not limiting of the disclosure. Thus, the foregoing aspects, features, embodiments and advantages are merely illustrative and are not considered elements or limitations of the appended claims except where explicitly recited in a claim(s). Likewise, reference to “the disclosure” shall not be construed as a generalization of any inventive subject matter disclosed herein and shall not be considered to be an element or limitation of the appended claims except where explicitly recited in a claim(s). [0145] As is apparent from the foregoing general description and the specific aspects, while forms of the aspects have been illustrated and described, various modifications can be made without departing from the spirit and scope of the present disclosure. Accordingly, it is not intended that the present disclosure be limited thereby. Likewise, the term “comprising” is considered synonymous with the term “including.” Likewise whenever a composition, an element or a group of elements is preceded with the transitional phrase “comprising,” it is understood that we also contemplate the same composition or group of elements with transitional phrases “consisting essentially of,” “consisting of,” “selected from the group of consisting of,” or “Is” preceding the recitation of the composition, element, or elements and vice versa, such as the terms “comprising,” “consisting essentially of,” “consisting of’ also include the product of the combinations of elements listed after the term.

[0146] For purposes of this present disclosure, and unless otherwise specified, the term “coupled” is used herein to refer to elements that are either directly connected or connected through one or more intervening elements. For example, an opening can be directly connected to the fluidic channel, or it can be connected to the fluidic channel via intervening elements.

[0147] For purposes of this present disclosure, and unless otherwise specified, all numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and consider experimental error and variations that would be expected by a person having ordinary skill in the art. For the sake of brevity, only certain ranges are explicitly disclosed herein. However, ranges from any lower limit may be combined with any upper limit to recite a range not explicitly recited, as well as, ranges from any lower limit may be combined with any other lower limit to recite a range not explicitly recited, in the same way, ranges from any upper limit may be combined with any other upper limit to recite a range not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the subranges 1 to 4, 1.5 to 4.5, 1 to 2, among other subranges. As another example, the recitation of the numerical ranges 1 to 5, such as 2 to 4, includes the subranges 1 to 4 and 2 to 5, among other subranges. Additionally, within a range includes every point or individual value between its end points even though not explicitly recited. For example, the recitation of the numerical range 1 to 5 includes the numbers 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, among other numbers. Thus, every point or individual value may serve as its own lower or upper limit combined with any other point or individual value or any other lower or upper limit, to recite a range not explicitly recited.

[0148] As used herein, the indefinite article “a” or “an” shall mean “at least one” unless specified to the contrary or the context clearly indicates otherwise. For example, aspects comprising “a monomer” include aspects comprising one, two, or more monomers, unless specified to the contrary or the context clearly indicates only one monomer is included.

[0149] While the foregoing is directed to embodiments of the present disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

Claims

CLAIMS What is claimed is:

1. A method of treating a musculoskeletal disorder or condition in a subject, comprising administering to the subject a composition comprising a mesenchymal stromal cell (MSC), wherein the MSC comprise an activated signaling pathway downstream of (i) stimulator of interferon gene (STING) and/or (ii) toll-like receptor 3 (TLR3).

2. The method of claim 1, wherein the MSC has been activated by culture with a STING agonist and/or a TLR3 agonist.

3. The method of claim 2, wherein the STING agonist comprises one or more of a cyclic dinucleotide (CDN), amidobenzimidazole (ABZI), DMXAA, 2’3’-cGAMP, c-di-GMP, c-di-AMP3’3’-cGAMP, ADU-S100, SB 11285, GSK 3745417, MK-1454, BMS-986301, and Cyclic GMP-AMP synthase (cGAS).

4. The method of claim 2, wherein the TLR3 agonist comprises a nucleic acid.

5. The method of claim 2, wherein the TLR3 agonist comprises one or more of polyinosinic-polycytidylic acid (pIC), polyadenylic-polyuridic acid (pAU), Rintatolimod, Resiquimod, and UV-inactivated viral particles.

6. The method of any one of claims 2-5, wherein the MSC has been activated by culture with a STING agonist, a TLR3 agonist, or both for at least about 0.25 hours, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or more than 2 hours.

7. The method of any one of claims 1-6, wherein the MSC is a stem cell-derived MSC.

8. The method of any one of claims 1-7, wherein the MSC is derived from an embryonic stem cell or an induced pluripotent stem cell (iPSC).

9. The method of any one of claims 1-6, wherein the MSC is a tissue-derived MSC.

10. The method of claim 9, wherein the tissue-derived MSC is derived from bone marrow, adipose tissue, placenta, umbilical cord tissue, umbilical cord blood, peripheral blood, muscle, uterine tissue, corneal stroma, skin, and/or dental pulp.

11. The method of any one of claims 1-10, wherein the composition is administered by intra-articular injection, intra-muscular injection, intra-tendinous injection, intra-ligamentous injection, intra-peritoneal injection, intra-venous injection, intra-arterial injection, sub-cutaneous injection, or intra-thoracic injection.

12. The method of any one of claims 1-11, wherein the musculoskeletal disorder or condition is osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, spondylarthritis, osteoporosis, osteopenia, tendinitis, tendonitis, bursitis, fibromyalgia, joint cartilage injury, myopathy, ligament injuries, fragility fractures, or traumatic bone fractures.

13. The method of any one of claims 1-12, wherein the MSC is an autologous MSC.

14. The method of any one of claims 1-12, wherein the MSC is an allogeneic MSC.

15. The method of any one of claims 1-12, wherein the MSC is a xenogeneic MSC.

16. The method of any one of claims 1-15, wherein the MSC is a mammalian MSC.

17. The method of claim 16, wherein the mammalian MSC is from a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

18. The method of any one of claims 1-17, wherein the subject is a mammalian subject.

19. The method of claim 18, wherein the mammalian subject is a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

20. A method of treating of treating a musculoskeletal disorder or condition in a subject, comprising administering to the subject a composition comprising a mesenchymal stromal cell (MSC) that has been cultured in the presence of a stimulator of interferon gene (STING) agonist and/or a toll-like receptor 3 (TLR3) agonist.

21. The method of claim 20, wherein the STING agonist comprises one or more of a cyclic dinucleotide (CDN), amidobenzimidazole (ABZI), DMXAA, 2’3’-cGAMP, c-di-GMP, c-di-AMP3’3’-cGAMP, ADU-S100, SB 11285, GSK 3745417, MK-1454, BMS-986301, and Cyclic GMP-AMP synthase (cGAS).

22. The method of claim 20 or 21, wherein the TLR3 agonist comprises a nucleic acid.

23. The method of any one of claims 20-22, wherein the TLR3 agonist comprises one or more of polyinosinic-polycytidylic acid (pIC), polyadenylic-polyuridic acid (pAU), Rintatolimod, Resiquimod, and UV-inactivated viral particles.

24. The method of any one of claims 20-23, wherein the MSC has been cultured in the presence of a stimulator of interferon gene (STING) agonist and/or a toll-like receptor 3 (TLR3) agonist for at least about 0.25 hours, at least about 0.5 hours, at least about 1 hour, at least about 2 hours, or more than 2 hours.

25. The method of any one of claims 20-24, wherein the MSC is a stem cell- derived MSC.

26. The method of claim 25, wherein the MSC is derived from an embryonic stem cell or an induced pluripotent stem cell (iPSC).

27. The method of any one of claims 20-26, wherein the MSC is a tissue-derived MSC.

28. The method of claim 27, wherein the tissue-derived MSC is derived from bone marrow, adipose tissue, placenta, umbilical cord tissue, umbilical cord blood, peripheral blood, muscle, uterine tissue, corneal stroma, skin, and/or dental pulp.

29. The method of any one of claims 20-28, wherein the composition is administered by intra-articular injection, intra-muscular injection, intra-tendinous injection, intra-peritoneal injection, intra-venous injection, intra-arterial injection, sub-cutaneous injection, or intra-thoracic injection.

30. The method of any one of claims 20-29, wherein the musculoskeletal disorder or condition is osteoarthritis, rheumatoid arthritis, psoriatic arthritis, gout, spondylarthritis, osteoporosis, osteopenia, tendinitis, tendonitis, bursitis, fibromyalgia, joint cartilage injury, myopathy, ligament injuries, fragility fractures, or traumatic bone fractures.

31. The method of any one of claims 20-30, wherein the MSC is an autologous MSC.

32. The method of any one of claims 20-30, wherein the MSC is an allogeneic MSC.

33. The method of any one of claims 20-30, wherein the MSC is a xenogeneic MSC.

34. The method of any one of claims 20-33, wherein the MSC is a mammalian MSC.

35. The method of claim 34, wherein the mammalian MSC is from a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

36. The method of any one of claims 20-35, wherein the subject is a mammalian subject.

37. The method of claim 36, wherein the mammalian subject is a human, a horse, a dog, a cat, a cattle, a sheep, a goat, a pig, a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole.

38. A composition for treating musculoskeletal disorders or conditions in a patient, the composition comprising: a mesenchymal stromal cell (MSC) with pattern recognition receptors (PRR), wherein the PRR comprises an activated stimulator of interferon genes (STING) pathway, an activated a toll-like receptor 3 (TLR3) pathway, or a combination thereof.

39. The composition of claim 38, wherein the MSC is a bone marrow-derived MSC, a stem cell-derived, an adipose tissue-derived MSC, or an umbilical cord tissue-derived MSC.

40. The composition of claim 38, wherein the activated STING pathway is activated by an agonist, the agonist comprising a 2’,3’-cGAMP.

41. The composition of claim 38, wherein the activated STING pathway is activated by an agonist, the agonist comprising a cyclic dinucleotide (CDN), an amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, BMS-986301, a Cyclic GMP- AMP synthase (cGAS), or combinations thereof.

42. The composition of claim 38, wherein the activated TLR3 pathway is activated by an agonist, the agonist comprising a polyinosinic-polycytidylic acid (pIC).

43. The composition of claim 38, wherein the PRR is operable to enhance an antiinflammatory property, an immunomodulatory property, an angiogenetic property, or combinations thereof of the MSC.

44. The composition of claim 38, further comprising a concentration of about 1 x 10⁶ to about 1 x 10⁸ cells per mL of a pharmaceutically acceptable carrier.

45. The composition of claim 44, wherein the pharmaceutically acceptable carrier is a phosphate buffered saline solution, a saline solution, or a freezing medium.

46. A composition for treating musculoskeletal disorders or conditions in a patient, the composition comprising: a mesenchymal stromal cell (MSC) comprising an activated stimulator of interferon gene (STING) pathway, the STING pathway activated by an agonist, the agonist comprising: a cyclic dinucleotide (CDN), a amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, a BMS-986301, a Cyclic GMP-AMP synthase (cGAS), or combinations thereof.

47. The composition of claim 46, wherein the MSC is a bone marrow-derived MSC, an adipose tissue-derived MSC, or an umbilical cord tissue-derived MSC.

48. The composition of claim 46, wherein the activated STING pathway is operable to enhance an anti-inflammatory property, an immunomodulatory property, an angiogenetic property, or combinations thereof of the MSC.

49. The composition of claim 46, further comprising a concentration of about 1 x 10⁶ to about 1 x 10⁸ cells per mL of a pharmaceutically acceptable carrier.

50. The composition of claim 49, the pharmaceutically acceptable carrier is a phosphate buffered saline solution, a saline solution, or a freezing medium.

51. A method of forming a composition for treating musculoskeletal disorders or conditions, comprising: activating a pattern recognition receptors (PRR) of a mesenchymal stromal cell (MSC), wherein the PRR comprises a stimulator of interferon gene (STING) pathway, a toll-like receptor 3 (TLR3) pathway, or a combination thereof; and forming the composition for treating musculoskeletal disorders or conditions, wherein the composition comprises an activated MSC.

52. The method of claim 51, further comprising expanding the MSC in a complete growth media.

53. The method of claim 51, wherein activating the MSC comprises in vitro activation by incubation.

54. The method of claim 51, wherein the MSC is activated at a concentration of about lOpg/mL of an agonist to about IxlO⁶ MSCs/mL.

55. The method of claim 54, wherein the agonist comprises a polyinosinic- polycytidylic acid (pIC), a cyclic dinucleotide (CDN), an amidobenzimidazole (ABZI), a DMXAA, a c-di-GMP, a c-di-AMP3’3’-cGAMP, a ADU-S100, a SB 11285, a GSK 3745417, a MK-1454, BMS-986301, a Cyclic GMP-AMP synthase (cGAS), or combinations thereof.

56. The method of claim 51 , wherein the MSC is activated with an agonist for about 0.25 hours to about 48 hours.

57. The method of claim 51, further comprising: washing the activated MSC with phosphate buffered saline; and storing the activated MSC at a concentration of 1.6 x 10⁶ cells per mL to about 5 x 10⁶ cells per mL in a freezing media.