Methods and compositions for treating liver disease

Abstract

Methods of treatment are provided that include administering a therapeutically effective amount of activated stem cells to a diseased tissue or organ in a subject in need thereof. The methods described herein are methods of purifying and formulating MSCs that include "activating" or "preconditioning" the stem cells.

Description

[Technical field]

[0001] The present disclosure relates to methods and compositions for treating liver disease using stem cells.More specifically described herein are methods of treatment using mesenchymal stem cells (MSCs) in mammalian treatment, and methods of purifying and formulating MSCs, including stem cell "activation" or "preconditioning". [Background technology]

[0002] Stem cells are specialized cells that can self-replicate through cell division and differentiate into cells of multiple lineages. These cells are classified as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from humans and animals. Human MSCs (hMSCs or huMSCs) are non-hematopoietic, multipotent stem cells that have the ability to differentiate into mesodermal lineages such as bone cells, adipocytes, and chondrocytes, as well as ectodermal (neuronal cells) and endodermal (hepatic cells) lineages. MSCs express cell surface markers including cluster of differentiation (CD)29, CD44, CD73, CD90, CD105, and lack expression of CD14, CD34, CD45, and HLA (human leukocyte antigen)-DR. hMSCs have been isolated from various tissues including adipose tissue, amniotic fluid, endometrium, dental tissue, umbilical cord, and Wharton's jelly. hMSCs have been cultured long-term in specific media without any severe abnormalities.

[0003] MSCs exhibit immunomodulatory functions and can secrete cytokines and immune receptors that regulate the microenvironment in host tissues. Their multipotency, immunomodulatory, and secretion of anti-inflammatory molecules make MSCs an effective tool for the treatment of chronic diseases. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure is based, at least in part, on the non-limiting theory that MSCs may treat various conditions, such as liver disease, that affect mammals by using MSCs to produce factors beneficial in the treatment of liver disease. Such factors may include cytokines, such as IL-6. IL-6 is a pleiotropic cytokine that exerts various effects on inflammation, liver regeneration, and defense against infection through the regulation of adaptive immunity. IL-6 is often considered a harmful cytokine because of its abundance in inflammatory environments. However, accumulating evidence supports the view that IL-6 exerts beneficial effects in many liver pathologies due to its role in liver regeneration and promoting anti-inflammatory responses in certain conditions. IL-6 promotes proliferation, angiogenesis, and metabolism, and downregulates apoptosis and oxidative stress. Both of these functions are important for mediating hepatoprotection. IL-6 is also a key regulator of adaptive immunity where it induces T cell differentiation and controls autoimmunity. It can enhance antiviral adaptive immune responses and alleviate T cell exhaustion during chronic infection.

[0005] Disclosed embodiments include compositions for treating a patient, e.g., a human or non-human mammal, suffering from a liver disease or a symptom thereof, comprising MSCs, e.g., derived from progenitor cells isolated from adipose tissue, umbilical cord, placental tissue, bone marrow, dental tissue, testicular tissue, uterine tissue, umbilical cord tissue, or skin tissue, which are allogeneic or autologous to the target patient, and saline, which can prevent, reduce, or eliminate a symptom of the liver disease in the target patient.

[0006] Disclosed embodiments include therapeutic uses of "activated" MSCs. For example, embodiments include purifying MSCs with different capabilities, such as by using magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS), to maximize their therapeutic benefits for specific applications. Further embodiments include activating MSCs with specific stimuli, including, for example, cytokines, reactive proteins, chemicals, small molecules, and combinations thereof. These stimuli can enhance or suppress MSC functions, such as MSC-induced immunosuppression that can be induced by proinflammatory cytokines.

[0007] The disclosed embodiments include frozen and thawed MSCs. The MSCs can include inactivated or activated MSCs. In embodiments that include the use of activated and then frozen MSCs, the MSCs can be reactivated. In embodiments that include the use of activated and then frozen MSCs, the activated MSCs do not require further activation.

[0008] Further embodiments include the use of MSCs in combination therapy, e.g., the use of MSCs in combination with a drug or pharma- ceutical active agent, or a pharmaceutical composition. For example, disclosed embodiments include the administration of MSCs in combination with exosomes, e.g., purified exosomes. [Brief description of the drawings]

[0009] [Figure 1] MSC treatment improved mortality in humanized FRG mice with alcoholic hepatitis (overall survival). The alcohol-overloading cohort shows higher survival in MSC-treated mice compared to the PBS control group. Log-rank (Mantel-Cox) test P<0.0001. Gehan-Breslow-Wilcoxon test P<0.0001. [Diagram 2]ALT and AST in cohort 1 are shown. ALT and AST were measured by biochemical assay. After repeated injection of non-activated MSCs in mice with alcoholic hepatitis, both ALT and AST concentrations were decreased. Student's T-test analysis shows that ALT and AST after treatment showed significantly lower values ​​in the MSC repeated injection group, while PBS injection did not significantly change ALT and AST levels. [Diagram 3] Figure 1 shows that activated MSC treatment improved the mortality rate of humanized FRG mice with alcoholic hepatitis (overall survival). Survival data of FRG mice with and without MSC injection are shown. P value of the log-rank (Mantel-Cox) test was 0.0130, P value of the log-rank trend test was 0.0032, and P value of the Gehan-Breslow-Wilcoxon test was 0.0270. [Figure 4] Histological findings at the time of death or sacrifice of humanized FRG mice with alcoholic hepatitis are shown. (A) PBS control mouse showing mild (1+) steatosis (HE x100x). (B) PBS control mouse showing mild (1+ steatosis) (HE x100x). (C) Mice treated with non-activated IP showing mild (1+ steatosis) and 30% necrosis (HE x100x). (D) Mice treated with non-activated MSC IP showing no steatosis (HE x100x). (E) Mice treated with activated MSC IP showing no significant steatosis (<5%) (HE x100x). (F) Mice treated with activated MSC IV showing mild (1+ steatosis) and 20% necrosis (HE x100x). [Diagram 5] Figure 1 shows the concentrations of AST and ALT in cohort 2. ASL and ALT were measured at the start of treatment and at the time of death, including euthanasia. Student's T-test analysis shows that ALT and AST after treatment were significantly lower in the MSC repeated injection group, whereas PBS injection did not significantly change the ALT and AST levels in mouse serum. [Figure 6] 1 shows cytokine expression by MSCs after MSC activation. [Figure 7] Baseline chemistry panel data (pig) are shown. [Figure 8] Baseline chemistry panel data (pig) are shown. [Figure 9] Chemistry panel data for porcine endpoints are shown in Figures 7 and 8. [Figure 10] Chemistry panel data for porcine endpoints are shown in Figures 7 and 8. [Figure 11] Baseline chemistry panel data (pig) are shown. [Figure 12] Baseline chemistry panel data (pig) are shown. [Figure 13] Figures 11 and 12 show chemistry panel data for pig endpoints. [Figure 14] Figures 11 and 12 show chemistry panel data for pig endpoints. [Figure 15] Baseline chemistry panel data (pig) are shown. [Figure 16] Baseline chemistry panel data (pig) are shown. [Figure 17] Figures 15 and 16 show chemistry panel data for pig endpoints. [Figure 18] Figures 15 and 16 show chemistry panel data for pig endpoints. [Figure 19] Baseline complete blood count data indicating the health status of the test animals are shown. [Figure 20] The number of cells harvested per flask is shown as described in Example 8. [Figure 21] 1 shows the doubling time of cell populations after 48 hours of culture as described in Example 6. [Figure 22] 1 shows the average viability of activated cells from Example 8. [Diagram 23]It shows that the "original" P.5 cells (Example 8) released higher concentrations per cell. When pre-activated cells were frozen / thawed and cultured for 48 hours, the cells were still able to produce high levels of key cytokines (IL6, MCP1, TGFB, etc. cytokines). The graph also shows that pre-activated cells were frozen / thawed and cultured for 48 hours and re-activated a second time. The cells were still able to produce key cytokines similar to cells that had already been activated once, or cells that had already been activated, frozen / thawed, and cultured for 48 hours. [Figure 24] Figure 1 shows that mesenchymal stem cell (MCS) treatment improved mortality in humanized Fah- / -, Rag2- / -, Il2rgc- / - (FRG) mice with alcoholic hepatitis. (A) Schematic showing relevant time points for the ASH1 cohort. (B) Survival of mice treated with non-activated MSCs was significantly better than mice treated with phosphate-buffered saline (PBS) (Wilcoxon p<0.0001). HSC, hematopoietic stem cells; Hep, hepatocytes; IP, intraperitoneal; IV, intravenous; qRT-PCR, quantitative real-time polymerase chain reaction. [Diagram 25] Figure 1 shows that MSC treatment improved mortality in humanized FRG mice with alcoholic hepatitis. (A) Schematic showing relevant time points for the ASH2 cohort. (B) Survival of mice treated with activated MSCs by either route was significantly better than mice treated with non-activated MSCs or PBS alone (Wilcoxon p<0.0001). The activated MSC IP+IV, activated MSC IV, activated MSC IP, and activated MSC IP+IV groups overlapped and all had similar 100% survival lines. HFCD, high fat diet. *=p<0.05. [Figure 26]Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations are shown for cohort 2. AST and ALT were measured at the start of treatment and at time of death, including sacrifice. (A) Post hoc analysis with Bonferroni correction of ALT on the last day: activated vs. PBS: <0.0001; non-activated vs. PBS: <0.0001. (B) Post hoc analysis with Bonferroni correction of AST on the last day: activated vs. PBS: <0.0001; non-activated vs. PBS: <0.0001. Post hoc analysis with Bonferroni correction of change in AST: activated vs. PBS: <0.0001; non-activated vs. PBS: <0.0001. [Figure 27] Figure 1 shows that vimentin validates the location of human MSCs in the liver. (A) Vimentin (human specific) immunohistochemistry (IHC) shows minimal expression only in the activated MSC treatment group. Scale bar = 10 μm. (B) Quantitative PCR shows a significant 2-fold increase in vimentin MSC marker compared to the PBS control group. (C) Ki-67 marker was significantly elevated in the activated MSC group compared to the PBS and non-activated MSC groups. (D) Myeloperoxidase (MPO) expression marker was significantly decreased in both activated and non-activated MSC compared to the PBS treatment group. (E) Activated MSCs expressed human serum albumin at significantly lower levels compared to the PBS control. DAPI, 4',6-diamidino-2-phenylindole. *=p<0.05, **:P<0.01, ****:p<0.0001. [Figure 28]Receptor-interacting protein kinase (RIPK3) IHC provides insight into the necroptosis pathway. (A) Confocal fluorescence images showing the expression of DAPI (blue) and RIPK3 (red) in PBS, non-activated MSCs, and activated MSCs. Scale bar = 10 μm. (B) Immunoreactivity score (IRS) shows that the expression of RIPS3 was significantly decreased in activated MSC tissues. Three representative paraffin-embedded liver tissues were stained for each group. (C) Western blotting showing the expression of RIP3 in activated MSC-treated and PBS-treated mouse groups. The expression of RIP3 was decreased in the activated MSC group compared to the PBS control group. (D) Western blotting showing the expression of B-cell lymphoma 2 (BCL-2). The activated MSC-treated group shows the highest expression of BCL-2 compared to the PBS and non-activated MSC groups. (E) The BCL-2 promoter is regulated by signal transducer and activator of transcription 3 (Stat3) and cyclic adenosine monophosphate response element-binding protein (CREB1) binding sites. (F) Western blotting showing decreased cleaved GSDMD (GSDMD) expression in activated MSC-treated mice. Cleaved GSDMD was highly expressed in non-activated and PBS-treated mice. (G) Proposed hypothetical mechanism. *: p<0.05, **: P=<0.01, ***: p<0.001. [Figure 29] Bioluminescence images showing the reduction of MSCs after short hairpin CD44 (sh-CD44) transduction. (A) Image of a mouse after IP injection of sh-scramble activated MSCs. (B) Image of a mouse after sh-CD44 transduced activated MSCs. Images show lower amounts of luciferase compared to sh-scramble. ROI, region of interest. [Diagram 30] Overall survival based on gender. Kaplan-Meier plot shows minimal difference in survival between male and female mice. [Diagram 31] Human mitochondrial staining showing FRG replacement rate. IHC of human mitochondrial DNA shows that humanized FRG mice have a replacement rate of 60-70%. [Diagram 32] 4 shows Elisa assays revealing the importance of IL-6, IL-10, and MCPs in activated MSCs. [Diagram 33] Male mice had significantly higher survival rates than female mice (p=0.03), although these were comparable between groups. [Diagram 34] Survival rates by treatment group are shown. Mice receiving 1 million cells were the only cohort with significantly better survival than the placebo group (p=0.03). [Diagram 35]A comparison of liver histology between mice that died during each dose treatment group and mice that survived 28 days after treatment is shown. (A) shows placebo-injected mouse #604, which died with moderate (2+) steatosis (HE x 100x). (B) shows placebo-injected mouse #606, which survived 28 days after treatment with moderate (2+) steatosis (HE x 100x). (C) shows 28,000 activated MSC-injected mouse #645, which died with moderate (2+) steatosis (HE x 100x). (D) shows 28,000 activated MSC-injected mouse #654, which survived 28 days after treatment with mild (1+) steatosis (HE x 100x). (E) shows mouse #658 injected with 100,000 activated MSCs, which showed moderate (2+) steatosis and died (HE×100x). (F) shows mouse #119 injected with 100,000 activated MSCs, which showed mild (1+) steatosis and survived 28 days after treatment (HE×100x). (G) shows mouse #701 injected with 250,000 activated MSCs, which showed marked (3+) steatosis and necrosis and died (HE×100x). (H) shows mouse #607 injected with 250,000 activated MSCs, which showed mild (1+) steatosis and survived 28 days after treatment (HE×100x). (I) shows mouse #691 injected with 500,000 activated MSCs, which showed significant (3+) steatosis and necrosis and died (HE x 100x). (J) shows mouse #632 injected with 500,000 activated MSCs, which showed mild (1+) steatosis and survived 28 days after treatment (HE x 100x). (K) shows mouse #727 injected with 1,000,000 activated MSCs, which showed significant (3+) steatosis and died (HE x 100x). (L) shows mouse #601 injected with 1,000,000 activated MSCs, which showed mild (1+) steatosis and survived 28 days after treatment (HE x 100x). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The liver is a vital organ located in the upper right corner of the abdomen. It weighs 2-3 pounds and performs many functions in the body, including metabolizing and detoxifying harmful substances, transforming nutrients from food, regulating blood clotting, maintaining hormone balance, storing vitamins, producing immune system components, and producing bile, which is essential for digestion.

[0011] Thus, liver disease can have a significant impact on quality of life. Causes can include infection, injury, exposure to drugs or toxic compounds, autoimmune processes, drug or alcohol overdose, etc. Effects of liver disease can include inflammation, scarring, blockages, blood clotting abnormalities, and liver failure.

[0012] The present disclosure is based, at least in part, on the benefits of treating patients with MSCs, such as umbilical cord-derived, placenta-derived, adipose-derived MSCs. Treatment includes methods of improving or reducing pain or other disease or condition symptoms, such as reducing at least one symptom of liver disease, such as reducing pain, nausea, fatigue, loss of appetite, yellowing of the skin, and combinations thereof. Subjects suitable for treatment of the present disclosure include, for example, mammals, such as humans or animals. Treatment disclosed herein can include administration of other bioactive agents, such as immunosuppressants.

[0013] Disclosed herein are methods of isolating and purifying MSCs, e.g., umbilical cord MSCs, placental MSCs, adipose-derived MSCs, etc. Further embodiments include methods of expanding and storing MSCs, e.g., umbilical cord MSCs, placental MSCs, or adipose-derived MSCs, etc. Embodiments include purifying MSCs based on the distinct capabilities of the cells, such as by using magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS), to maximize their therapeutic benefits for a particular use.

[0014] Further embodiments include activating MSCs to modulate their therapeutic benefit, for example, to increase their ability to suppress or enhance immune responses. In further embodiments, MSCs can be activated prior to administration to a patient by contacting them with at least one cytokine, for example, interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-8 (IL-8), macrophage inflammatory protein-1 beta (MIP-1b), or interleukin-17 (IL-17).

[0015] (definition) ALD: Alcoholic Liver Disease ALT: Alanine aminotransferase AST: Aspartate aminotransferase HFCD: High-fat, high-cholesterol diet HSC: Human hematopoietic stem cell hUCMSC: human umbilical cord mesenchymal stem cells LPS: lipopolysaccharide MSC: Mesenchymal stem cell PBS: Phosphate-buffered saline

[0016] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of its object. For example, "an element" means one element or more than one element.

[0017] As used herein with respect to MSCs, "activation" (or "preconditioning") refers to the use of stimulatory agents, including, for example, cytokines, reactive proteins, chemicals, small molecules, and combinations thereof, to enhance MSC function by contacting the MSCs with the stimulatory agent.

[0018] The terms "including," "comprising," and "having" are used in an inclusive and open sense, meaning that additional elements may be included. As used herein, the terms "such as," "for example," and "for example" are non-limiting and for illustrative purposes only, and are used interchangeably with "including," "including but not limited to," and "including."

[0019] "Effective," "effective amount," and "therapeutically effective amount" refer to an amount of MSCs or a pharmaceutical composition thereof that produces a beneficial result following administration.

[0020] "In vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments include, but are not limited to, test tubes and cell cultures. "In vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0021] "Liver disease" is any condition that can cause inflammation or damage to the liver and affect liver function.

[0022] As used herein, "or" should be understood to mean "and / or" unless the context clearly dictates otherwise.

[0023] "Parenteral administration" and "administered parenterally" are terms well known in the art and include forms of administration such as injection excluding enteral administration and topical administration, including, but not limited to, retroorbital, intraocular, intravenous, intramuscular, intraperitoneal, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0024] A "patient," "subject," or "host" to be treated by the subject method can mean either a human or a non-human animal, such as a mammal, fish, bird, reptile, or amphibian.

[0025] "Pharmaceutically acceptable" or "therapeutically acceptable" refers to a material that does not interfere with the effectiveness or biological activity of the active ingredient and that is not toxic to the patient.

[0026] "Pharmaceutically acceptable carriers" are well known in the art and include, for example, pharma- ceutically acceptable materials, compositions, or vehicles (such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials) involved in carrying or transporting any of the subject compositions from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the subject composition and not deleterious to the patient. In certain embodiments, pharma-ceutically acceptable carriers are non-pyrogenic. Exemplary materials which may serve as pharma- ceutically acceptable carriers include sugars (such as lactose, glucose, and sucrose), starches (such as corn starch and potato starch), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (such as cocoa butter and suppository wax), oils (such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (such as propylene glycol), polyols (such as glycerin, sorbitol, mannitol, and polyethylene glycol), esters (such as ethyl oleate and ethyl laurate), agar, buffers (such as magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffers, and other non-toxic compatible substances employed in pharmaceutical formulations.

[0027] A "pharmaceutical composition" refers to a formulation containing a therapeutically active agent as described herein in a form suitable for administration to a subject. In an embodiment, the pharmaceutical composition is in bulk or unit dose form. The amount of active ingredient (e.g., MSCs) in a unit dose of the composition is an effective amount and may vary according to the specific treatment involved. Those skilled in the art will understand that routine modifications to the dosage are sometimes required depending on the age and condition of the patient. The dosage will also depend on the route of administration. In a preferred embodiment, the active ingredient is mixed under sterile conditions with a pharma- ceutically acceptable carrier and with any preservatives, buffers, or propellants, as required.

[0028] "Treatment" refers to any therapeutic intervention in an animal, such as a mammal, e.g., a human or a pet, including (i) prevention, i.e., preventing clinical symptoms from developing, e.g., infection or inflammation from occurring and / or developing into a harmful condition, (ii) inhibition, i.e., arresting the development of clinical symptoms, e.g., halting an ongoing infection so that the infection is completely eliminated or to the extent that it is no longer harmful, and / or (iii) alleviation, i.e., causing regression of clinical symptoms, e.g., causing reduction in fever and / or inflammation caused by or associated with a microbial infection. Treatment may include multiple administrations of a composition described herein.

[0029] "Reduce," "suppress," and "inhibit" have their commonly understood meanings of making less or decreasing.

[0030] Isolation of MSCs Disclosed embodiments may include methods of harvesting and isolating MSCs.

[0031] In disclosed embodiments, MSCs are harvested and isolated from a variety of tissues, including, but not limited to, placenta, skeletal muscle, adipose tissue, umbilical cord, synovial membrane, circulatory system (e.g., blood), dental pulp, amniotic fluid, fetal blood, lung, liver, gonadal tissue, and bone marrow.

[0032] In embodiments, such methods may include aseptically collecting tissue from a qualified mammalian donor. For example, in embodiments using umbilical cord or placental MSCs, tissue may be collected from a normal term fetus or during the third trimester of pregnancy. In embodiments, placenta is collected from a specific pathogen-free donor or from a healthy donor with known health and travel history, free of adventitious pathogenic agents. Multiple parts of the placenta may be used for the derivation of MSCs, including, for example, endothelial chorion, chorioallantoic membrane, amniotic membrane, umbilical cord, and Wharton's jelly.

[0033] In embodiments involving the isolation of umbilical or placental MSCs, the following steps may be performed in a certified clean room, e.g., under cGMP conditions. In embodiments, the tissue is washed extensively in a rinse buffer and then cut into small pieces (1-5 grams). In embodiments, the decidual giant cells are removed by one or a combination of steps, e.g., by mechanical peeling of the decidual surface with a sterile spoon. The tissue may then be incubated with a protease, e.g., a serine protease, e.g., trypsin, for 30-90 minutes at 37°C and 5% CO2. Filtration may then be performed, e.g., using a nylon mesh, e.g., using 20, 25, and 30 micron nylon meshes. Gradient separation of the cells may then be performed, e.g., using BSA, Percoll, or Ficoll. In embodiments, a differential adhesion method is used, allowing rapidly adhering cells to be separated from non-adhering cells that are floating in the medium. The placental tissue can then be minced for, for example, 90 seconds or 150 cutting cycles.

[0034] In disclosed embodiments, the tissue, e.g., placental tissue, is then subjected to digestion, e.g., enzymatic digestion with an enzyme, such as collagenase, at 37°C, for 60-180 minutes, with stirring, e.g., at a rate of 100-140 cycles / min. Collagenase concentrations can range from 1 mg / ml to 5 mg / ml. In embodiments, the cells are then passed through a series of cell strainers (e.g., 100 microns, 40 microns, etc.), and then through a nylon mesh, e.g., 20, 25, or 30 microns. In embodiments, the cells are passed through a gradient. Red blood cells (RBCs) are removed with RBC lysis buffer (4°C, 3 minutes). RBC lysis is neutralized by adding PBS, e.g., 15-20 times PBS. The cells are then centrifuged, e.g., at 400 g for 10 minutes. The cells are then eluted, e.g., at 200-300 x 10 3 / cm 2 The cells are cultured in culture flasks at a density of 0.1% to 1.0% for a culture period of, for example, 5-7 days. Optionally, a differential adhesion method is applied to remove residual giant cells from the mixture, and the cells are allowed to adhere for a period of, for example, 1-10 hours, after which the floating cells are separated from the adherent cells. After the culture period, the placental MSCs can be harvested from the flasks as P0 (passage zero, primary cells).

[0035] In disclosed embodiments, MSCs are derived from tissue, e.g., umbilical cord tissue, at 1×10 6 MSC / gram umbilical cord, 2 × 10 6 MSC / gram umbilical cord, 3 × 10 6 MSC / gram umbilical cord, 4 × 10 6 MSC / gram umbilical cord, 5 × 10 6 MSC / gram umbilical cord, 6 × 10 6 MSC / gram umbilical cord, 7 × 10 6 MSC / gram umbilical cord, 8 × 10 6 MSC / gram umbilical cord, 9 × 10 6 MSC / gram umbilical cord, 1 × 10 7 MSC / gram umbilical cord, 2 × 10 7 MSC / gram umbilical cord, 3 × 10 7 MSC / gram umbilical cord, or 4 × 10 6They can be isolated in quantities such as MSC / gram umbilical cord.

[0036] In embodiments, MSCs may exhibit a viability rate after isolation of more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%, etc. In embodiments, MSCs may exhibit a viability rate after isolation of 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, etc. In embodiments, MSCs may exhibit a viability rate after isolation of between 50% and 60%, between 60% and 70%, between 70% and 80%, between 80% and 90%, or between 90% and 100%.

[0037] MSCs can be identified using minimal criteria established by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy. These criteria include, first, that MSCs must be plastic-adherent when maintained under standard culture conditions, second, that MSCs must express CD105, CD73, and CD90, and not CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules, and third, that MSCs must have the potential to differentiate in vitro into osteoblasts, adipocytes, and chondroblasts.

[0038] In embodiments, MSCs can be separated based on their ability to produce therapeutic molecules, e.g., cytokines. For example, magnetic activated cell sorting (MACS) can be used to purify MSCs based on the ability of cells to produce a particular cytokine. Disclosed embodiments can also include the use of flow cytometry to purify MSCs based on the ability of cells to produce a particular cytokine. Disclosed embodiments can also include the use of chromatography, e.g., affinity chromatography, to purify MSCs based on the ability of cells to produce a particular cytokine.

[0039] In an embodiment, 100% of isolated MSC cells express IL-6. In an embodiment, as cells are passaged, the expression of IL-6 increases. In an embodiment, the expression of cytokines is upregulated. For example, in an embodiment, the expression of IL-6, IL-17A, IFNγ, TNFα, TGFβ, MCP1, HGF, IL-8, TIMP-1, TIMP-2, VEGF, IDO, IL-10, and combinations thereof may be upregulated.

[0040] In embodiments, isolated MSCs are characterized for example for expression of surface markers by flow cytometry, trilineage mesoderm differentiation potential (adipocytes, osteocytes, and chondrocytes), indoleamine-2,3-dioxygenase (IDO) activity, sterility, endotoxin, and mycoplasma testing.

[0041] (MSC expansion culture) In embodiments, cell expansion culture for cells derived from any of the above mentioned tissues is carried out in a clean room facility that meets GMP clean room classification built for cell therapy manufacturing purposes. In embodiments, cell expansion culture is carried out in a bioreactor, for example, a 40L Bioreactor.

[0042] For example, in a sterile Class II biological safety cabinet located in a Class 10,000 clean manufacturing suite, cells are thawed under controlled conditions and washed into a 15 mL conical tube containing 10 mL of complete DMEM low glucose medium (cDMEM) (GibcoBRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone) specified to have endotoxin levels of 100 EU / mL or less (routinely levels of 10 EU / mL or less) and hemoglobin levels of 30 mg / dl or less (routinely levels of 25 mg / dl or less). In an embodiment, the serum lot used is segregated and one lot is used for all experiments. In an embodiment, the medium may be supplemented with, for example, 10% human Plasmalyte or human serum albumin or a combination thereof.

[0043] In an embodiment, the cells are then plated into a T-225 flask containing 25 mL of RB complete medium composed of Rooster Nourish-MSC-XF-basal medium and Rooster Replenish-MSC-XF supplement, and cultured for 48 hours at 37° C., 5% CO2 in a fully humidified atmosphere. Non-adherent cells are washed off by gently rinsing the flask with cDMEM. In an embodiment, the number of cells plated into the flask is, for example, 2.5×10 5 From cells 3 × 10 6 Between cells, 1.5 × 10 6 2 x 10 cells 6 In embodiments, the adherent cells are then dissociated by washing the cells with PBS and the addition of, for example, 0.05% trypsin containing EDTA (Gibco, Grand Island, NY, USA) for 2 minutes at 37° C., 5% CO2 in a fully humidified atmosphere. In embodiments, the cells may be dissociated using a recombinant composition, for example, TrypLE CTS.

[0044] The cells are centrifuged, washed and placed in a T-225 containing 45 mL of cDMEM.

[0045] In embodiments, the disclosed cell expansion method may produce between 6 and 20 million cells per starting T-225 flask. The cells from the first flask may then be split, for example, into multiple flasks. The cells may then be grown, for example, for 4 days, after which there are approximately 6 million cells per flask (24 million cells total). In embodiments, the method is repeated but not expanded beyond 10 passages, and then stored in aliquots of 6 million cells in sealed vials for distribution.

[0046] In further embodiments, the cells are grown in the medium and harvested along with the medium after about 2-10 days. The cells are prepared in this "conditioned" medium at a concentration of less than about 100,000 cells / mL for infusion. In embodiments, physiological electrolyte additives may be added. In embodiments, the cell solution may be administered intravenously.

[0047] In a further method, the cells are grown in the medium for about 5-10 days. The medium is then infused intravenously without the cells or administered locally to the site of injury. Further methods involve the isolation and / or concentration of stem cell-producing factors and / or further refinement of these chemicals and / or compounds.

[0048] In embodiments, cell proliferation can be expressed in terms of viability per passage. For example, in disclosed embodiments, the number of isolated MSCs can increase by 40% per passage, 50% per passage, 60% per passage, 70% per passage, 80% per passage, 90% per passage, 100% per passage, 120% per passage, 150% per passage, 200% per passage, 250% per passage, etc.

[0049] In embodiments, the cells may be frozen after expansion and then thawed for subsequent use.

[0050] (Activation of MSCs) In embodiments, stem cells, e.g., isolated MSCs, can be activated to produce MSCs with desired properties. For example, MSCs can be polarized toward pro-inflammatory or anti-inflammatory properties depending on stimulated Toll-like receptors (TLRs). In embodiments, MSCs are exposed to stimulatory factors, such as inflammatory cytokines. Pro-inflammatory or pro-inflammatory cytokines stimulate the production of T helper cells (T h Inflammatory cytokines are a type of signaling molecule secreted by immune cells such as inflammatory cells (IL-1), IL-12, IL-17, and IL-18, tumor necrosis factor alpha (TNF-α), interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Disclosed embodiments include activation of MSCs with at least one of IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, and GM-CSF. Disclosed embodiments include activation of MSCs with at least two of IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, and GM-CSF. Disclosed embodiments include activation of MSCs with at least three of IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, and GM-CSF. Disclosed embodiments include activation of MSCs with at least four of IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, and GM-CSF.

[0051] The disclosed embodiments are described in further detail in the following examples. In embodiments, the activation amount of each stimulating factor can be, for example, between 1 ng / mL and 5 ng / mL, or between 2 ng / mL and 4 ng / mL, etc. In embodiments, the amount of each stimulating factor can be, for example, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 12 ng / mL, 14 ng / mL, 16 ng / mL, 18 ng / mL, 20 ng / mL, 22 ng / mL, 24 ng / mL, 26 ng / mL, 28 ng / mL, 30 ng / mL, 32 ng / mL, 34 ng / mL, 36 ng / mL, 38 ng / mL, 40 ng / mL, 42 ng / mL, 44 ng / mL, 46 ng / mL, or more, etc. In embodiments, the stimulating factors are applied in equal amounts. For example, in certain embodiments, the stimulatory factors may include equal amounts of IL-17, TNF-α, and IFNγ. In certain embodiments, the stimulatory factors may include different (non-equal) amounts of, for example, IL-17, TNF-α, and IFNγ.

[0052] In embodiments, activation of MSCs includes contacting MSCs with a stimulatory factor, e.g., a cytokine, e.g., IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, or GM-CSF. In embodiments, activation is performed for a period of time, e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or longer, e.g., at 37° C. In embodiments, activation periods can be between 1 and 20 hours, between 2 and 18 hours, between 3 and 16 hours, between 4 and 14 hours, between 6 and 12 hours, between 8 and 10 hours, etc. In embodiments, the activation period may be, for example, between 10 and 12 hours. In embodiments, the activation period may be different for different stimulating agents.

[0053] In embodiments, activation of MSCs comprises contacting MSCs with a stimulatory factor, e.g., a cytokine, e.g., IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, or GM-CSF, in embodiments carried out at 37° C. for a period including, e.g., at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, etc.

[0054] In embodiments, activation of MSCs comprises contacting MSCs with a stimulatory factor, e.g., a cytokine, e.g., IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, or GM-CSF, in embodiments, at 37° C. for a period including, e.g., at most 1 hour, at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, at most 8 hours, at most 9 hours, at most 10 hours, at most 11 hours, at most 12 hours, at most 13 hours, at most 14 hours, at most 15 hours, at most 16 hours, at most 17 hours, at most 18 hours, at most 19 hours, at most 20 hours, at most 21 hours, at most 22 hours, at most 23 hours, at most 24 hours, etc.

[0055] In embodiments, activation of the MSCs comprises contacting the MSCs with a stimulatory factor, e.g., a cytokine, e.g., IL-1, IL-8, MIP-1b, IL-12, IL-17, IL-18, TNF-α, IFNγ, or GM-CSF, in embodiments, at room temperature for a period including, e.g., between 1 and 24 hours, between 2 and 22 hours, between 4 and 18 hours, between 6 and 16 hours, between 8 and 14 hours, between 10 and 12 hours, etc.

[0056] In embodiments, the MSCs may be frozen after activation and then thawed for subsequent use, in embodiments, the MSCs may be frozen prior to activation and then thawed and activated.

[0057] (MSC Collection) In embodiments, cell harvesting from a T225 flask can be performed as follows: The medium, e.g., Rooster Broth, is removed and the flask is filled with 10 mL of D-PBS. - / - (Gibco), the PBS is removed, and then 10 mL of CTS-TrypLE (Gibco) is added to the flask and incubated at 37° C. for 5-6 minutes. Then, a medium, for example, 10 mL of Rooster medium, is added to quench trypsin activity. In an embodiment, the cell suspension is removed and the culture vessel is refilled with 25 mL of D-PBS. - / - In embodiments, the cell suspension mixture is then centrifuged, for example, at 280×g at 4° C. for 10 minutes.

[0058] (MSC composition) In embodiments, the isolated MSCs can be formulated into a pharma- ceutically acceptable composition, for example, by using at least one pharma- ceutically acceptable carrier. In embodiments, a pharma-ceutically acceptable carrier generally refers to a carrier useful for preparing a pharmaceutical composition or formulation that is safe, non-toxic, and not biologically or otherwise undesirable, and includes carriers acceptable for human pharmaceutical use as well as veterinary use. Pharmaceutically acceptable carriers can include, for example, saline, phosphate buffered saline, Plasmalyte, Ringer's serum, Ringer's lactate serum, lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, alginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0059] The disclosed formulations include MSCs in combination with cytokines in the form of a composition, eg, a pharmaceutical composition, suitable for administration to a subject in need of treatment therewith.

[0060] The disclosed formulations may be "pre-filled" into an administration device, such as a syringe, prior to use.

[0061] The disclosed formulations may be provided as a kit. For example, the disclosed kit may include a pharma- ceutically acceptable carrier, an isolated population of mesenchymal stem cells, an isolated interferon, an isolated interleukin, and instructions for using the kit in a method for attenuating an immune response. The cell and stimulatory factor, e.g., cytokine, components of the kit may be administered separately or may be combined in vitro and then administered as a mixture. The kit may optionally include a means for administering the composition, e.g., by injection.

[0062] In an embodiment, pelleted hUC-MSCs are obtained, for example, at 1×10 6 Add 200uL of D-PBS to ensure hUC-MSCs are injected.- / - 1.3×10 inside 6 Cells at a concentration of 100 mM D-PBS - / - In an embodiment, the hUC-MSC / D-PBS solution (200 uL) is loaded into one U-100 BD Ultra-Fine Short Insulin Syringe (Beckton, Dickinson, and Company) for injection in mice, e.g., tail vein injection.

[0063] (MSC-based treatment) Disclosed embodiments may include administration of MSCs to treat various conditions and diseases. For example, vesicles derived from placental MSCs may be employed for therapeutic use. In embodiments, the stem cells may be autologous to the subject. If available, autologous stem cells may be beneficial to the subject as they reduce or eliminate the possibility of adverse immune responses, such as stem cell rejection or graft-versus-host disease. Autologous stem cells may be, for example, stem cells (e.g., MSCs) directly isolated from the subject or iPS cells produced from non-stem cells derived from the subject.

[0064] In some embodiments, allogeneic stem cells may be used when autologous stem cells are not available or are not indicated for a particular subject. In embodiments, the allogeneic stem cells are "matched" as closely as possible to the subject (e.g., via HLA genotype) to reduce the chance of rejection or graft-versus-host disease. In another embodiment, the stem cell donor is a first-degree relative (e.g., parent, sibling, or child) of the subject, which increases the chance of finding a closely matching donor. In yet another embodiment, the stem cell donor may be an extended relative of the subject. In some embodiments, the stem cell donor may be from the same racial or ethnic group as the subject. However, certain stem cells may be immune privileged and may be used allogeneically without the need for a match between donor and subject.

[0065] In an embodiment, the MSCs are used to treat a patient, for example, to treat a disease, condition, disorder, etc., such as liver disease, and symptoms thereof.

[0066] MSCs may be administered, e.g., injected, in any suitable manner, e.g., subcutaneously, intra-articularly, intralesional (tendon, ligament, disc), intravenously, intraperitoneally, or intramuscularly. In embodiments, administration may include, e.g., injection. For example, in embodiments, administration may include mixing or suspending MSCs with, e.g., plasma, HypoThermasol HTS-FRS, Cryostor (containing 0, 2, 5, and 10% DMSO as CSB, CS2, CS5, and CS10), human serum, human serum albumin, 0.7-0.9% isotonic saline, Plasmalyte, phosphate buffered saline (PBS), stem cell culture medium (e.g., Rooster Replenish CC / Rooster Nourish CC), exosome isolation medium (e.g., RoosterCollect-EV CC), Infuvite, lactated Ringer's solution, and the like. These solutions may be used alone or in combination with each other.

[0067] An appropriate MSC dosage is, for example, 1 x 10 3 cells, 2.5 x 10 3 cells, 5 x 10 3 cells, 1 x 10 4 cells, 2.5 x 10 4 cells, 5 x 10 4 cells, 1 x 10 5 cells, 2.5 x 10 5 cells, 5 x 10 5 cells, 1 x 10 6 cells, 2.5 x 10 6 cells, 5 x 10 6 cells, 1 x 10 7 cells, 2.5 x 10 7 cells, 5 x 10 7 cells, 1 x 10 8 cells, 2.5 x 10 8 cells, 5 x 10 8 cells, 1 x 10 9 cells, 2.5 x 10 9 cells, 5 x 10 9 cells, 1 x 10 10 cells, 2.5 x 10 10 cells, 5 x 10 10cells, 1 x 10 11 cells, 2.5 x 10 11 cells, 5 x 10 11 cells, 1 x 10 12 cells, 2.5 x 10 12 cells, 5 x 10 12 cells, 1 x 10 13 cells, 2.5 x 10 13 cells, 5 x 10 13 cells, 1 x 10 14 cells, 2.5 x 10 14 cells, 5 x 10 14 cells, 1 x 10 15 cells, 2.5 x 10 15 cells, 5 x 10 15 It may be a cell, or more, etc.

[0068] In embodiments, a suitable MSC dosage is, for example, 1×10 3 2.5 x 10 cells 3 Between cells, 5 × 10 3 From cells 1 x 10 4 Between cells, 2.5 × 10 4 5 × 10 cells 4 Between cells, 1 x 10 5 2.5 x 10 cells 5 Between cells, 5 × 10 5 From cells 1 x 10 6 Between cells, 2.5 × 10 6 Between cells, 5 × 10 6 From cells 1 x 10 7 Between cells, 2.5 × 10 7 5 × 10 cells 7 Between cells, 1 x 10 8 2.5 x 10 cells 8 Between cells, 5 × 10 8 From cells 1 x 10 9 Between cells, 2.5 × 10 9 5 × 10 cells 9 Between cells, 1 x 10 10 2.5 x 10 cells 10 Between cells, 5 × 10 10 From cells 1 x 10 11 Between cells, 2.5 × 10 11 5 × 10 cells 11 Between cells, 1 x 1012 2.5 x 10 cells 12 Between cells, 5 × 10 12 From cells 1 x 10 13 Between cells, 2.5 × 10 13 5 × 10 cells 13 Between cells, 1 x 10 14 2.5 x 10 cells 14 Between cells, 5 × 10 14 From cells 1 x 10 15 Between cells, 2.5 × 10 15 5 × 10 cells 15 It may be between cells, or more, etc.

[0069] In embodiments, a suitable MSC dosage is, for example, at least 1×10 3 cell 、 At least 2.5 x 10 3 Cells, at least 5 x 10 3 Cells, at least 1 x 10 4 cell 、 At least 2.5 x 10 4 Cells, at least 5 x 10 4 Cells, at least 1 x 10 5 Cells, at least 2.5 x 10 5 Cells, at least 5 x 10 5 Cells, at least 1 x 10 6 Cells, at least 2.5 x 10 6 Cells, at least 5 x 10 6 Cells, at least 1 x 10 7 Cells, at least 2.5 x 10 7 Cells, at least 5 x 10 7 Cells, at least 1 x 10 8 Cells, at least 2.5 x 10 8 Cells, at least 5 x 10 8 Cells, at least 1 x 10 9 Cells, at least 2.5 x 10 9 Cells, at least 5 x 10 9 Cells, at least 1 x 10 10 Cells, at least 2.5 x 10 10 Cells, at least 5 x 10 10 Cells, at least 1 x 10 11Cells, at least 2.5 x 10 11 Cells, at least 5 x 10 11 Cells, at least 1 x 10 12 Cells, at least 2.5 x 10 12 Cells, at least 5 x 10 12 Cells, at least 1 x 10 13 Cells, at least 2.5 x 10 13 Cells, at least 5 x 10 13 Cells, at least 1 x 10 14 Cells, at least 2.5 x 10 14 Cells, at least 5 x 10 14 Cells, at least 1 x 10 15 Cells, at least 2.5 x 10 15 Cells, at least 5 x 10 15 It may be a cell, or more, etc.

[0070] In embodiments, a suitable MSC dosage is, for example, at most 1×10 3 Cells, at most 2.5 × 10 3 Cells, at most 5 × 10 3 Cells, at most 1 × 10 4 Cells, at most 2.5 × 10 4 Cells, at most 5 × 10 4 Cells, at most 1 × 10 5 Cells, at most 2.5 × 10 5 Cells, at most 5 × 10 5 Cells, at most 1 × 10 6 Cells, at most 2.5 × 10 6 Cells, at most 5 × 10 6 Cells, at most 1 × 10 7 Cells, at most 2.5 × 10 7 Cells, at most 5 × 10 7 Cells, at most 1 × 10 8 Cells, at most 2.5 × 10 8 Cells, at most 5 × 10 8 Cells, at most 1 × 10 9 Cells, at most 2.5 × 10 9 Cells, at most 5 × 10 9 Cells, at most 1 × 10 10 Cells, at most 2.5 × 1010 Cells, at most 5 × 10 10 Cells, at most 1 × 10 11 Cells, at most 2.5 × 10 11 Cells, at most 5 × 10 11 Cells, at most 1 × 10 12 Cells, at most 2.5 × 10 12 Cells, at most 5 × 10 12 Cells, at most 1 × 10 13 Cells, at most 2.5 × 10 13 Cells, at most 5 × 10 13 Cells, at most 1 × 10 14 Cells, at most 2.5 × 10 14 Cells, at most 5 × 10 14 Cells, at most 1 × 10 15 Cells, at most 2.5 × 10 15 Cells, at most 5 × 10 15 It may be a cell, or more, etc.

[0071] In embodiments, the dose of each MSC injection is, for example, 5×10 6 Cells / kg to 5 x 10 7 The dosage may range between 100 and 200 cells / kg.

[0072] In embodiments, MSCs may be administered once, twice, three times, four times, five times, monthly, or every three months, six months, or yearly.

[0073] The disclosed methods may also involve co-administration of stem cells and a bioactive agent. By "co-administration" is intended administration prior to, simultaneously with (e.g., in the same formulation or in combination with a bioactive agent as a separate formulation), or subsequent to administration of the therapeutic composition described above. As used herein, a "bioactive agent" refers to any organic, inorganic, or biological agent that is biologically active or relevant. For example, a bioactive agent may be a protein (e.g., albumin), a polypeptide, a nucleic acid, a polysaccharide (e.g., heparin), an oligosaccharide, a monosaccharide, a disaccharide, an organic compound, an organometallic compound, or an inorganic compound. A bioactive agent may include a living or senescent cell, a bacterium, a virus, or a portion thereof. A bioactive agent may include a bioactive molecule, such as a hormone, a growth factor, a growth factor-producing virus, a growth factor inhibitor, a growth factor receptor, an anti-inflammatory agent, an antimetabolite, an integrin blocker, or a complete or partial functional sense or antisense gene (including siRNA). Bioactive agents may also include man-made particles or materials carrying biologically relevant or bioactive materials, such as nanoparticles that include a drug-bearing core and a coating on the core. Bioactive agents may also include drugs, such as chemical or biological compounds that may have a therapeutic effect on a biological organism. Non-limiting examples include, but are not limited to, growth factors, anti-rejection agents, anti-inflammatory agents, anti-infective agents (e.g., antibiotics and antivirals), and analgesics and analgesic combinations. Anti-inflammatory agents may serve as an additional agent to counteract the inflammatory aspects of the fibrotic process.

[0074] Combinations, mixtures, or other formulations of any of the aforementioned examples may be made and still be considered bioactive agents within the intended meaning of this specification. Aspects of the present disclosure directed to bioactive agents may include any or all of the aforementioned examples. In another embodiment, the bioactive agent may be a growth factor. A growth factor is any agent that promotes the proliferation, differentiation, and functionality of transplanted stem cells. Non-limiting examples of suitable growth factors may include, but are not limited to, leukemia inhibitory factor (LIF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), human growth hormone (hGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), interleukins, cytokines, and / or combinations thereof. The bioactive agent may be a blood-derived supplement containing a mixture of growth factors, such as platelet lysate.

[0075] In embodiments, the bioactive agent may include an immunosuppressant. An immunosuppressant is any agent that prevents, delays the onset of, or reduces the intensity of an undesirable immune response, such as the rejection of transplanted cells, tissues, or organs, or graft-versus-host disease. Preferred are immunosuppressants that suppress cellular immune responses against cells that are identified as non-self by the immune system. Examples of immunosuppressants include, but are not limited to, cyclosporine, cyclophosphamide, prednisone, dexamethasone, methotrexate, azathioprine, mycophenolate, thalidomide, FK-506, systemic steroids, as well as a wide variety of antibodies, receptor agonists, receptor antagonists, and other such agents known to those skilled in the art. In another embodiment, the bioactive agent is selected from the group consisting of, but not limited to, nintedanib, INT-767, emricasan, VBY-376, PF-04634817, EXC001, GM-CT-01, GCS-100, refanalin, SAR156597, tralokinumab, pomalidomide, STX-100, CC-930, simtuzumab, anti-miR-21, PRM-151, BOT191, palomid 529, IMD1041, serelaxin, PEG-relaxin, ANG-4011, FT011, pirfenidone, F351 (a perfenidone derivative), THR-184, CCX-140, FG-3019, avosentan, GKT137831, PF-00489791, pentoxifylline, fresolimumab, and LY2382770.

[0076] Disclosed therapeutic methods may involve frozen and thawed MSCs, for example, the cells may be frozen prior to or following activation and then thawed for subsequent use.

[0077] (Example) The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of representative embodiments, and should not be construed as limiting any of the embodiments described herein.

[0078] Example 1 Fah- / -, Rag2- / -, Il2rgc- / - (FRG) KO liver humanized mice were generated by crossing Fah- / - mice (RIKEN) and Rag2- / -, Il2rgc- / -.

[0079] Mice were irradiated using a 2 mm filter at 250 kV, 16 mA, and a 50 cm FSD, with a dose rate (cGy) of 150 cGy over a 10 cm × 10 cm area.

[0080] Human hematopoietic stem cells (HSCs) were prepared in sterile medium after collection from fetal liver donor cells (1 day after irradiation) at a concentration of 5 × 10 5 cells / mouse were injected intrahepatically into irradiated recipients.

[0081] Animals were fed autoclaved / irradiated diet and acidified autoclaved water with or without SMZ (7.8 ml of SMZ for every 250 ml of drinking water) on alternating weekly basis for the duration of the animals' postweaning lives.

[0082] Blood was collected from the facial vein of human stem cell xenografted mice.

[0083] 100 μl of blood per mouse was collected into a 1.5 mL sterile microcentrifuge tube containing 100 μl of 20 mM PBS-EDTA and placed on ice. PBMCs were resuspended in red blood cell lysis buffer (1x ACK lysis buffer) and incubated at room temperature (25°C) for 5 min.

[0084] Cells were centrifuged twice at 469×g and resuspended in 2% (vol / vol) FBS / PBS containing human CD45, mouse CD45 antibody, and 7-AAD mix. Human immune reconstituted (% human CD45 + cells / total CD45 + Cells) were examined using flow cytometry analysis.

[0085] Human umbilical cord mesenchymal stem cells (hUC-MSCs) were isolated from the perivascular Wharton's jelly region of human umbilical cord and the cells were characterized for surface marker expression by flow cytometry, trilineage mesodermal differentiation potential (adipocytes, osteocytes, and chondrocytes), indoleamine-2,3-dioxygenase (IDO) activity, sterility, endotoxin, and mycoplasma testing. hUC-MSCs were cultured and harvested according to the RB manufacturing protocol. 1.5 to 2 million hUC-MSCs were plated in 25 ml of RB complete medium in a T225 vented flask and cultured at 37°C, 5% CO2 for 48 hours.

[0086] After initial culture of hUC-MSCs, e.g., between 20 and 50 hours or between 36 and 38 hours after initial culture of hUC-MSCs, activation media consisting of human TNF-α, human IFNγ, and human IL-17 was added to each T225 flask containing hUC-MSCs at a final concentration of 2 ng / mL for each cytokine. Flasks can be cultured with the added activation media for an additional period of time, e.g., between 2 and 20 hours (e.g., between 10 and 12 hours), at 37° C., 5% CO2.

[0087] The cells were then harvested. The Rooster culture medium was removed and the cells were resuspended in 10 mL of D-PBS. - / - The flasks were washed with 10 mL of CTS-TrypLE and then the medium was removed. 10 mL of CTS-TrypLE was added to the flasks and incubated at 37°C for 5-6 minutes. Then 10 mL of medium was added to quench trypsin activity. The cell suspension was removed and the flasks were washed with 25 mL of D-PBS. - / - The cell suspension mixture was centrifuged at 280 x g for 10 minutes at 4°C.

[0088] The pelleted hUC-MSCs were diluted with 200uL of D-PBS to ensure that 1,000,000 hUC-MSCs were injected. - / - At a concentration of 1,300,000 cells in D-PBS - / -The hUC-MSC / D-PBS solution (200uL) was loaded into one U-100 BD Ultra-Fine Short Insulin Syringe for tail vein injection. Each mouse was injected with 5 doses of 1,000,000 cells / injection within 3 weeks, once on days 0, 2, and 5 in the first week, and once weekly thereafter for an additional 2 weeks.

[0089] (Cohort 1) Fifty mice were fed a modified high-fat Lieber-DeCarli (LD) liquid diet with alcohol (3.5% w / v) and high-fat or isocaloric dextrin for 4 weeks (75 to 103 days of age). During HFCD feeding, mice were given 53% aqueous ethanol by gavage only twice a week for a total of 8 doses at a dose of 4 g / kg ethanol. For the maltose control group, 1-month HFCD-fed mice were simultaneously given isocaloric dextrin-maltose by gavage only twice a week for a total of 8 doses. Thirty-one mice died before treatment and subsequent attempts to image with CT+ / -ultrasound elastography. After alcohol pretreatment and attempts to image with sedation, 19 humanized mice (10 males, 9 females) at 104 days of age survived to randomize and complete the cohort 1 study.

[0090] Mice were randomly assigned to placebo (PBS) (n=5) or nonactivated MSCs (n=14), and PBS or MSCs were administered according to protocol. Of the 14 treated mice, 8 received MSCs IV and IP, and 6 received IP only.

[0091] Group 1: 5 mice received vehicle (PBS) IP and IV.

[0092] Group 2: 8 mice received 1,000,000 non-activated mesenchymal stem cell therapeutic intravenously (IV) and intraperitoneally (IP).

[0093] Group 3: 6 mice received 1,000,000 non-activated mesenchymal stem cell therapeutic intraperitoneally (IP).

[0094] An overdose of alcohol was administered every other week for three weeks. Mice were given MSCs or PBS three times in the first week and twice each week for the next two weeks (eight times over three weeks).

[0095] (Cohort 2) After analysis of the initial data, the binge drinking period was shortened and no attempt was made to obtain computed tomography scans.An additional 33 humanized mice that underwent 3 weeks of binge alcohol pretreatment were divided into five groups.

[0096] Group 1: 5 mice received 1,000,000 non-activated mesenchymal cells IV and IP.

[0097] Group 2: 7 mice received vehicle (PBS) IV and IP.

[0098] Group 3: 7 mice received 1,000,000 activated mesenchymal cells IP.

[0099] Group 4: 7 mice received 1,000,000 activated mesenchymal cells IV.

[0100] Group 5: 7 mice received 1,000,000 activated mesenchymal cells IV and IP.

[0101] Cells / PBS were administered three times during the first week and once a week for an additional two weeks (FIG. 25A).

[0102] For both cohorts, blood samples were taken on the first day of treatment and on the day of death or euthanasia and were sent for measurement of ALT and AST levels.

[0103] For cohort 1, mice were observed to survive for up to 93 days after the initiation of MSC or PBS treatment. Surviving mice were euthanized by cardiac puncture and cervical dislocation at the end of the experiment. Liver tissues were fixed in 10% neutral buffered formalin for hematoxylin-eosin staining and histological evaluation of tumors.

[0104] For cohort 2, mice were observed surviving up to 25 days after initiation of MSC or PBS treatment. Surviving mice were euthanized in a similar manner 2 days after the last MSC treatment. Necropsy was performed on mice that died before the endpoint.

[0105] Blood collected from FRG-huHSC / Hep mice was used to quantify human leukocyte reconstitution at 4 weeks post-infection and at the euthanasia endpoint, 60 days (day 167) after initiation of MSC treatment. Blood was collected to measure the human engraftment efficacy of fetal liver cells.

[0106] After euthanasia, representative sections of liver tissue were fixed in 10% neutral buffered formalin and processed for histological evaluation. The extent of steatosis, necrosis, and fibrosis were quantified by blinded specimen analysis by examination of representative hematoxylin and eosin stained sections.

[0107] Steatosis was graded on a 4-point scale (0–3) with 0 being <5% steatosis, 1 being 5–33%, 2 being 34–66%, and 3 being >66%. Necrosis was also graded on a 4-point scale (0–3) with 0 being <5% necrosis, 1 being 5–10%, 2 being ≤20%, and 3 being >21%.

[0108] The log-rank (Mantel-Cox) test, Gehan-Breslow-Wilcoxon test, and chi-square test were used to calculate statistics for mouse survival studies. For mouse histological studies, ANOVA test or Student's T test were used.

[0109] Table 1 contains details of dosing, survival, pathology, and AST and ALT levels for all 52 mice that were randomized and completed the study. [Table 1]

[0110] Table 2 provides details of the mice with regard to sex, treatment, survival, AST and ALT levels, and histology. [Table 2]

[0111] Table 3 shows the primer sets used for qPCR. The primer sets were ordered from Integrated DNA technologies (IDT). [Table 3]

[0112] (Cohort 1) Four (out of five) control mice died on days 3, 5, 9, and 13 after randomization and first treatment. None of the 14 mice treated with non-activated MSCs died during the experiment. All surviving animals were sacrificed on day 93 after randomization.

[0113] After 4 weeks of mesenchymal stem cell treatment (PrimeGen), the MSC-injected group (n=14) showed higher survival rates compared to the PBS control group (n=5) (Figure 1). Statistical significance was shown at p<0.0001 by Mantel-Cox test and Gehan-Breslow-Wilcoxon test.

[0114] The pathology was mixed, with variable degrees of steatosis, and only six animals showed necrosis rates anywhere between 5 and 10%. No fibrosis was observed in hematoxylin-eosin stained sections. In Table 1, all control mice (n=5) exhibited some degree of steatosis, and three exhibited some degree of intralobular inflammation. Of the 14 mice treated with MSCs, 11 did not exhibit steatosis or other significant findings, and three exhibited no steatosis and only minimal inflammation. Of note, all treated mice survived but were euthanized more than 2 months after the last injection, which may have been too late to see damage as surviving mice would have healed by that time. This was taken into account in the next set of experiments.

[0115] All animals exhibited elevated AST and ALT at the time of randomization, indicating the presence of liver injury. These levels were significantly reduced in MSC-treated animals by the time of sacrifice. However, these levels remained elevated in PBS-treated animals, including the lone survivor (Table 1, Figure 2).

[0116] (Cohort 2) Surviving mice were euthanized 25 days after the first treatment. Of the 5 non-activated MSC-treated mice, 60% survived. Of the 7 PBS (untreated mice), only one in seven or 14% survived. Of the 21 mice treated with activated MSC, 100% survived.

[0117] Six of seven PBS-treated mice (86%) died between days 5 and 19 after randomization and the first treatment. Two of five non-activated MSC-treated mice died on day 4 after randomization and the first treatment (Figure 3). All surviving mice were euthanized on day 2 after the last treatment.

[0118] Representative sections of the liver were fixed in 10% neutral buffered formalin, processed, and hematoxylin-eosin stained sections were obtained and examined to assess steatosis, inflammation, necrosis, and fibrosis. Of the seven placebo mice, three exhibited no significant pathological changes, three showed steatosis, and two showed 5-10% necrosis.

[0119] Of the seven mice that received activated mesenchymal stem cells both IP and IV, five had a grade of steatosis and two had no significant findings. None of the mice had necrosis or significant inflammation.

[0120] Of the seven mice that received activated mesenchymal stem cells IP, six developed steatosis and one had no significant findings. None of the mice developed necrosis or significant inflammation.

[0121] Of the seven mice that received activated mesenchymal cells IV, five exhibited steatosis, two had no notable findings, and one had necrosis.

[0122] Of the five mice treated with non-activated stem cells, two exhibited steatosis, three had no notable findings, and one had necrosis.

[0123] Hematoxylin and eosin staining did not reveal fibrosis in any group.

[0124] FIG. 4 shows some of the pathology findings at the time of death or euthanasia.

[0125] All three routes of activated MSC injection improved the mortality rate of alcoholic hepatitis, indicating that IP and / or IV can be used for MSC treatment.Pathological examination showed no significant differences among all groups, indicating that MSC treatment can have an impact on the systemic improvement of alcoholic hepatitis.

[0126] AST and ALT were taken at the start of treatment and at time of death, including those that were euthanized. All mice exhibited elevated enzymes at the time of randomization, indicative of liver injury. All PBS-treated mice, including one surviving mouse, exhibited elevated enzymes at time of death. All mice receiving non-activated or activated cells, including those that died (2 with non-activated cells), exhibited a significant decrease in enzymes at time of death. The most pronounced decrease was seen in mice receiving activated cells both IP and IV (Figure 5).

[0127] The MSC group exhibited a better survival rate than the PBS group, and the activated MSC group exhibited a better survival rate than the non-activated group, further demonstrating the role of MSCs in survival in this animal model and indicating that activated MSCs exhibit a better outcome.

[0128] AST and ALT were examined at the start of treatment and at the time of death including those sacrificed. All mice exhibited elevated enzymes at the time of randomization, indicative of liver damage. 100% of PBS-treated mice exhibited elevated enzymes at the time of death including one surviving mouse. All mice receiving non-activated or activated cells including those that died (2 with non-activated cells) showed a significant decrease in enzymes at the time of death. The most pronounced decrease was seen in mice receiving MSCs (p<0.0001) (Figure 4A,B). To determine the significance of elevated AST and ALT, a control group of mice was fed isocaloric dextrin-maltose by gavage only twice a week for 4 weeks without alcohol overdose. These mice had blood drawn for AST and ALT at the same time points as the mice receiving the alcohol overdose. Compared to the elevated laboratory values ​​for the test mice, ALT and AST levels ranged between 7U / L and 16U / L.

[0129] (The presence of the MSC lineage marker vimentin demonstrates human MSCs in the liver) To investigate the location of human MSCs, we stained the PBS, non-activated, and activated MSC groups with vimentin, a human-specific MSC lineage marker. Immunohistochemistry revealed vimentin expression only in activated MSC-treated mice (Figure 5A). To further analyze vimentin expression, we isolated RNA from all three groups of mice and performed quantitative polymerase chain reaction (PCR). Quantitative PCR revealed a statistically significant increase in vimentin expression in the activated MSC-treated group (n=3) compared to the PBS control group (n=3) (Figure 5B). These results demonstrate that the MSCs found in the liver of the activated MSC group are indeed human.

[0130] KI67 and myeloperoxidase cDNA levels indicate the importance of activated MSCs Ki-67, a marker of liver regeneration, has previously been shown to be elevated in patients with alcoholic liver disease, and myeloperoxidase (MPO), a neutrophil marker, has also been shown to be elevated in alcohol-treated mice.

[0131] To examine the efficacy of MSCs, we isolated RNA from all three groups of mice and performed quantitative PCR (Table S2). Ki-67 expression was significantly elevated in the activated MSC group compared to the PBS control group (Figure 5C). Conversely, MPO levels were significantly decreased in MSC-treated mice (Figure 5D). Taken together, these data sets indicate the importance of activated MSCs in alleviating alcohol-induced liver injury in these mice.

[0132] (Activated MSC-treated mice maintained human serum albumin levels after treatment) To investigate the amount of functional hepatocytes in the liver after treatment, we isolated RNA from all three groups of mice and measured human albumin levels relative to the mice. Before alcohol liver injury, we showed that the human mitochondrial DNA levels in our humanized FRG mice were between 60% and 70% (Figure S2). Quantitative PCR analysis revealed significantly higher human albumin levels in activated MSC-treated mice relative to the mice (Figure 5E). These data indicate the importance of activated MSCs in alleviating liver injury in our humanized mouse model.

[0133] (Receptor-interacting protein kinase 3 (RIPK3) immunofluorescence demonstrates the ability of MSCs to inhibit the necroptosis pathway) Receptor-interacting protein kinase 3 (RIPK3) has previously been shown to be a key molecule regulating necroptosis

[26] . To determine whether our MSC-treated mice expressed RIPK3, we stained paraffin-embedded liver tissues from PBS, non-activated MSC, and activated MSC groups. Confocal microscopy revealed elevated levels of RIPK3 in PBS-treated mice compared with the MSC-treated group (Figure 6A). Immunoreactivity scores of confocal images showed significantly lower RIPK3 levels in the activated MSC group compared with the PBS control group (Figure 6B). To confirm the expression of RIPK3 at the protein level, we performed Western blot analysis using protein lysates from livers of the activated and PBS-treated groups. Our results revealed that RIPK3 levels were decreased in the activated MSC group compared with the PBS control group (Figure 6C). Thus, our RIPK3 study indicates that activated MSCs inhibited necroptosis in this group of mice.

[0134] (B cell lymphoma 2 (BCL2) is expressed in activated MSC-treated mice) B cell lymphoma 2 (BCL2) has been well studied as an anti-apoptotic molecule involved in the necroptosis and pyroptosis pathways. To determine whether BCL-2 was expressed in our alcohol-excessive FRG mice, we isolated protein lysates and performed Western blot analysis. Our results revealed BCL-2 expression in activated MSC-treated mice (Figure 6D). BCL-2 was slightly present in the non-activated MSC-treated group but was not expressed in PBS-treated mice. These results indicate the importance of activated MSC treatment in alleviating liver injury.

[0135] (The BCL-2 promoter is induced after addition of MSC-conditioned medium) Since BCL-2 has been shown to inhibit necroptosis and pyroptosis, we next performed luciferase reporter assays targeting signal transducer and activator of transcription 3 (STAT3) and cyclic adenosine monophosphate response element-binding protein (CREB1) in the BCL-2 promoter. Specifically, Huh7 cells were transfected with various BCL-2 promoter constructs and stimulated with either PlasmaLyte or MSC-conditioned medium. Our results showed that MSC-conditioned medium activated BCL-2 expression in STAT3- and CREB1-deleted BCL-2 constructs (Figure 6E). Interestingly, other BCL-2 promoter constructs showed minimal relative luciferase activity in both groups. This may be due to an AML-1 (acute myeloid leukemia-1) binding site (-1473 upstream of the TSS) that has previously been shown to be a repressor of BCL-2 expression.

[0136] (Treatment of gastrodermin D levels highlights the importance of activated MSCs in alleviating liver injury) We next examined the levels of gasdermin D (GSDMD) in PBS-, non-activated, and activated MSC-treated groups. GSDMD has been shown to be an important inflammatory response molecule. Western blots showed decreased expression of cleaved GSDMD in activated MSC-treated groups compared to both non-activated MSC and PBS control groups (Figure 6F). This result indicates the importance of activated MSCs in mitigating liver injury in our treated mice.

[0137] Transduction of sh-CD44 reduces the ability of MSCs to migrate to the liver. CD44 has previously been shown to be involved in cell trafficking by binding to its ligand hyaluronan. To track where MSCs go after alcohol-induced liver injury, we transduced activated MSCs with sh-CD44 lentivirus. Bioluminescence images revealed that a higher number of cells were present in the liver of sh-scramble-injected mice. In contrast, sh-CD44-injected mice exhibited a lower amount of luciferase expression (Figure 7A, B). These images indicate that CD44 influences the migration of MSCs to the liver after alcohol-induced liver injury.

[0138] MSCs have the ability to differentiate into different types of cells, migrate to the site of injury, and exhibit anti-inflammatory properties. When tissue damage or injury occurs in the body, MSCs migrate to the site of injury. Once MSCs arrive at this site of injury, they interact with various inflammatory cells and different types of stromal cells to initiate the regeneration process and repair the damaged area. Previous studies have shown that MSCs secrete different types of growth factors, cytokines, and adhesion molecules that affect the damaged tissue area and thus maintain a positive paracrine effect on the tissue repair process. Other studies have shown that MSCs can produce many different growth factors, such as vascular endothelial growth factor, hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor 1, and IL-6. The majority of these cytokine factors are upregulated by the activation of NF-κB, which is derived from the exposure of proinflammatory stimuli such as TNF-α, IFN-γ, IL-1β, lipopolysaccharide, and hypoxia. Several studies propose that MSCs are not naturally immunosuppressive but require activation for upregulation of their immunomodulatory properties. The most important MSC activating or priming factors are IFN-γ, TNF-α, IL-17, and IL-1β. After MSC activation, derived from these three proinflammatory cytokines, these proliferation cytokine factors are upregulated to promote tissue regeneration and repair by recruitment or stimulation of tissue progenitor cells, fibroblasts, and endothelial cells in the damaged tissue area or by production of anti-inflammatory cytokines. These activated MSCs can function to inhibit the proliferation of T helper cells and cytotoxic T cells through various pathways. Initiation of anti-inflammatory responses is triggered by regulatory T cell differentiation and activation of T helper type 2 cells. IL-6 can inhibit T cell activation and maturation of immature dendritic cells by reducing the expression of costimulatory molecules CD40, CD80, and CD86, by suppressing proinflammatory cytokines, and by upregulating anti-inflammatory cytokines such as IL-10. In previous studies using our original method, our activated MSCs had the ability to highly express IL-6 in vitro.We propose that the increased production of IL-6 in our activated MSCs may be responsible for the modulation of the inflammatory state in the acute alcoholic liver injury model, increasing survival and preventing apoptosis and pyrolysis. In future studies, we would like to analyze all of our potential pro- and anti-inflammatory treatment groups to better understand and propose potential pro- and anti-apoptotic pathways and mechanisms to explain why our activated MSCs increased survival in our model. Acute alcoholic hepatitis differs in many ways from chronic liver disease, the most important being its reversibility. Thus, the humanized mouse liver damaged by alcohol overdose presented an ideal model to test both non-activated and activated umbilical cord cells for their potential to increase survival and affect pathways of liver injury. Our first alcoholic hepatitis cohort included two groups (PBS control and non-activated MSC-treated). All non-activated MSC-treated mice survived, whereas the PBS-treated control group exhibited a 20% survival rate. Statistical significance was indicated at p<0.0001. In the second cohort, activated MSC-treated mice exhibited a 100% survival rate, non-activated MSC-treated mice exhibited a 60% survival rate, whereas, similar to cohort 1, the PBS control group exhibited a 14% survival rate.

[0139] Analysis of liver chemical composition before and after treatment with PBS or cells revealed a significant improvement in animals receiving MSCs compared to those receiving PBS. Furthermore, those receiving activated cells showed a more significant improvement compared to nonactivated cells. In cohort 1, PBS-treated mice had varying degrees of steatosis, whereas the 14 surviving mice treated with MSCs had no significant findings. We hypothesized that the lack of findings in surviving mice may have been due to the long observation time, which allowed the liver to heal, as evidenced by the significant decrease in liver chemical composition. In cohort 2, animals were sacrificed 2 days after the last treatment, with the majority of mice exhibiting varying degrees of steatosis as well as other signs of injury. Furthermore, we hypothesized that alcohol may have acted more systemically, as liver pathology could not explain the difference in survival. Publicly available RNA-seq datasets have demonstrated the importance of BCL-2 and CD44 in the pyroptosis and necroptosis pathways. Our findings with RIPK3, BCL-2, CD44, and GSDMD provide clues as to whether activated MSCs alleviated liver injury and inhibited necroptosis and pyroptosis (Figure 6G). In the future, we would like to perform chromatin immunoprecipitation / quantitative PCR and site-directed mutagenesis to see whether CREB1 and STAT3 actually activate the BCL-2 promoter. The results from our two cohort experiments show promising results as a treatment to combat alcoholic hepatitis. Although our mouse studies were limited in number, we look forward to a larger cohort of FRG mice and ultimately larger animal studies. It will be interesting to see how long-term high fat / cholesterol + alcohol overdose feeding fares with activated MSC treatment. In summary, activated MSC treatment is a strategically important strategy to improve the high mortality rate of patients with alcoholic hepatitis.

[0140] Example 2: Use of frozen / thawed MSCs After acute injury, the liver can either regenerate and recover or develop end-stage liver failure. The balance between recovery and failure can be influenced by several factors, including, non-exclusively, the extent of injury and the underlying liver disease. In a previously reported study, the group showed that activated umbilical cord mesenchymal stem cells (MSCs) administered to mice with humanized livers that had developed liver injury secondary to alcohol could significantly affect survival.

[0141] The primary objective of this study was to evaluate the safety and efficacy of different doses of frozen-thawed activated MSCs compared to placebo in treating acute alcohol-induced liver injury in humanized mouse livers. Secondary objectives included evaluation of m, liver chemical components, biomarkers, and pathology at different doses.

[0142] Sixty-two humanized mice fed a high-fat diet and binge alcohol for 24 days were randomized to receive injections of 1 million, 500,000, 250,000, 100,000, or 28,000 activated umbilical cord cells or vehicle (Plasmalyte) alone via the tail vein three times during the first week and once a week for an additional two weeks. AST and ALT were obtained at baseline, week 1, week 2, week 3, and / or at death. Mice were followed for survival, and surviving mice were euthanized at week 4. Liver pathology was assessed for all animals at the time of death. Time-to-event data were analyzed using Kaplan-Meier curves and log-rank tests or Wilcoxon rank tests, using the Sidak method for multiple comparison adjustment as appropriate. Histology for all mouse livers was reported at the time of death.

[0143] The highest dose of 1 million stem cells resulted in a statistically significant increase in survival compared to the placebo group (p=0.03). Histological findings correlated with survival, with 27 surviving animals exhibiting 1 to 2+ steatosis without necrosis, 23 of the 35 animals that died exhibited necrosis, and all but 3 of the remaining mice exhibited varying degrees of steatosis.

[0144] Treatment with high-dose freeze-thawed activated umbilical cord MSCs can result in improved survival and histology in mice bearing humanized livers and suffering from alcohol-induced liver injury.

[0145] The liver is the primary site of alcohol metabolism and has been described as the primary target of alcohol-induced injury. The spectrum of liver disease varies from steatosis, steatohepatitis, fibrosis, acute alcoholic hepatitis, and the development of advanced liver disease, including cirrhosis. Acute alcoholic hepatitis is an inflammatory disease of the liver associated with recent severe excessive drinking and is characterized by steatosis, ballooned hepatocytes, Mallory-Denk bodies, and intralobular inflammation with a prominent component of neutrophils. Outcome is variable, with high 30-day mortality rates reported as 30-50% for severe cases defined by discriminant functions. Treatment is initial supportive, with variable reports of efficacy with different treatment techniques. Criteria for transplantation can vary between centers, and the supply of organs is limited, making the demand for efficient treatment urgent.

[0146] The remarkable regenerative properties of the liver are influenced by many factors that can alter the balance between recovery and failure. The potential of MSCs to promote regeneration while reducing the inflammatory response to liver injury suggests that activated stem cells may offer some advantage in promoting regeneration and improving outcomes in acute liver failure. In previously published papers, our group demonstrated improved survival in mice bearing humanized livers subjected to liver injury due to excessive alcohol drinking using repeated injections of 1 million MSCs (Table). Furthermore, survival was significantly improved with activated umbilical cord MSCs compared to non-activated cells, and both cells were significantly better than placebo.

[0147] A new set of experiments was designed to compare different doses, compare these doses with placebo, and obtain additional histological and biochemical data to better determine the optimal dose, evaluate toxicity at different dose levels, and follow liver chemistry and histological findings.

[0148] In addition to our own previous studies, several animal models have demonstrated the ability of MSCs to ameliorate organ failure after liver injury, demonstrating improved viability, histology, liver chemistry, and inflammatory markers (Table 6).

[0149] Materials and Methods (Preparation of humanized mice) After obtaining IACUC consent, we utilized FRG KO liver-humanized mice, a process routinely performed in Dr. Keigo Machida's laboratory.

[0150] (Breeding of FRG mice) Our laboratory generated Fah- / -, Rag2- / -, Il2rgc- / - (FRG) mice by crossing Fah- / - mice (RIKEN) and Rag2- / -, Il2rgc- / - (Jackson Lab). These FRG mice are distinct from commercially available strains. Genotyping was performed according to the USC genotyping guidelines.

[0151] (IRRADIATION OF FRG NEWBORN) Irradiation was performed at 250 kV, 16 mA, 50 cm FSD with a 2 mm filter. Irradiation dose rate (cGy): 150 cGy over an area size of 10 cm x 10 cm. Mice were housed in a pathogen-free facility with microisolator cages and monitored to ensure the absence of acute illness.

[0152] (Transplantation Procedure) Human hematopoietic stem cells (HSCs) were collected from fetal liver (donor cells). Human HSCs were prepared in sterile medium and injected intrahepatically into irradiated recipients. We used 5 × 10 5 Cells / mouse were used, which were injected one day after the irradiation procedure.

[0153] (Feeding with sterile water) We fed the animals autoclaved / irradiated food and maintained them on acidified autoclaved water with or without SMZ (7.8 ml SMZ for every 250 ml drinking water) on a weekly rotation basis for the duration of the animals' lives after they were weaned at 3 weeks of age. Animals were monitored daily by investigators after irradiation and HSC transplantation for potential signs of complications (poor body condition / weight loss, rough coat, inactivity, hunched posture, death without signs of disease). Body weights were measured.

[0154] (In vivo blood study of humanized immune cells in FRG mice) To determine whether humanized immune cells were retained at sufficient levels within the bloodstream of FRG mice, blood was collected from the facial vein of human stem cell xenografted mice.

[0155] (FACS analysis using peripheral blood cells from FRG mice reconstituted with human HSCs) Approximately 100 μl of blood was collected per mouse into a 1.5 mL sterile microcentrifuge tube containing 100 μl of 20 mM PBS-EDTA and placed on ice. The PBMCs, bottom part (PBMCs), were resuspended in red blood cell lysis buffer (1x ACK lysis buffer) and incubated for 5 min at room temperature (25°C). Cells were centrifuged twice at 469 g and resuspended in 2% (vol / vol) FBS / PBS containing human CD45, mouse CD45 antibody, and 7-AAD mix. We used human immune reconstituted (% human CD45 + cells / total CD45 + cells), which was examined using flow cytometry analysis.

[0156] (Preparation of umbilical cord mesenchymal cells and activated cells) (Human umbilical cord MSC culture) With informed consent, human umbilical cord mesenchymal stromal cells (hUC-MSCs) were isolated from the perivascular Wharton's jelly region of the human umbilical cord and provided by RoosterBio (RoosterVial-hUC-XF, manufactured and sold by RoosterBio, Frederick, MD, and supported by core technology from Tissue Regeneration Therapeutics (TRT) and licensed technology from the following patent families; U.S. Patent Application No. 8,790,923; U.S. Patent Application No. 8,278,102; U.S. Patent Application No. 7,547,546; U.S. Patent Application No. 9,611,456; U.S. Patent Application No. 9,611,456; U.S. Patent Application No. 8,481,311; U.S. Patent Application No. 9,611,456). Purchased hUC-MSC vials were additionally fully characterized according to the International Society for Cell and Gene Therapy (ISCT) minimum standards (24), which were performed by RB. Furthermore, RoosterBio performed additional tests to characterize hUC-MSC for surface marker expression by flow cytometry, trilineage mesodermal differentiation potential (adipocytes, osteocytes, and chondrocytes), indoleamine-2,3-dioxygenase (IDO) activity, sterility, endotoxin, and mycoplasma testing (data not shown). hUC-MSC were cultured and harvested according to the RB manufacturing protocol. 1.5 to 2 million hUC-MSC were placed in a T225 vented flask (Corning or ThermoFisher) in 25 ml of RB complete medium RoosterNourish-MSC-XF (RoosterBio) and cultured for 48 h, incubated at 37°C, 5% CO2.

[0157] (Activation of hUC-MSCs) Thirty-six to 38 hours after initial culture of hUC-MSCs, triple activation consisting of human TNF-α (PeproTech), human IFNγ (PeproTech), and human IL-17 (PeproTech) was added to each T225 flask containing hUC-MSCs at a final concentration of 2 ng / mL for each cytokine (25). Each hUC-MSC flask was left to culture in the supplemented activation medium for an additional 10 to 12 hours at 37°C in a 5% CO2 incubator.

[0158] (Collection of hUC-MSCs) Cell harvesting from a T225 flask was performed as follows: Rooster medium was removed and 10 mL of D-PBS was added. - / - (Gibco), then 10 mL of CTS-TrypLE (Gibco) was added and incubated at 37°C for 5-6 min. Then 10 mL of Rooster medium was added to quench trypsin activity. The cell suspension was then removed and the T225 flask was rehydrated with 25 mL of D-PBS. - / - The whole cell suspension mixture was centrifuged at 280×g for 10 min at 4° C., and the supernatant was removed.

[0159] (Freezing of activated hUC-MSCs) Pelleted activated hUC-MSCs were resuspended in 1 ml of CS10 freezing medium (BioLife) at a concentration of 5 or 10 million cells / mL and aliquoted into 1.8 ml cryovials (Nunc). The aliquoted cell vials were then frozen using a Planer Kryo-550-16 Control Rate Freezer (Planer Limited). They were then transferred to a Vapor phase LN2 tank for storage until use.

[0160] (Shipping of hUC-MSC cells and syringe preparations) One frozen vial of activated hUC-MSCs was thawed using a ThawStar Automated Cell Thawing System (BioLife Solutions). Thawed frozen cells were gently resuspended in 7 ml of Rooster Complete Nutrient Medium. The frozen-thawed cell suspension tube was centrifuged at 280 × g for 10 min at 4 °C. The supernatant was removed and the pelleted activated hUC-MSCs were resuspended in 10 mL of Rooster Complete Nutrient Medium. Cell counts were performed using a NuceloCounter NC-200 Cell Counter (Chemometec). Cells were aliquoted into 1.8 mL vials for each dose at a concentration of approximately 1,300,000 cells / mL in Rooster Complete Nutrient Medium (this will ensure that 1,000,000 hUC-MSCs are injected into each subject). Individual activated hUC-MSC dose vials were then placed in and shipped using PrimeGen's proprietary validated 4 °C shipping box. When ready for use for a treatment group, each vial containing the activated hUC-MSC suspension was centrifuged at 280 x g for 10 minutes at 4°C. The supernatant was removed and the pelleted activated hUC-MSCs were resuspended in 200uL of Plasmalyte at a concentration of 1,300,000 cells. The hUC-MSC / Plasmalyte solution (200uL) was immediately loaded into one U-100 BD Ultra-Fine Short Insulin Syringe (Beckton, Dickinson, and Company) for tail vein injection in mice.

[0161] (Excessive drinking) Of 64 mice initiated into the binge drinking regimen, 62 survived and were randomized to the following treatment groups:

[0162] (Treatment group) The 62 surviving mice were randomized by sex into one of six groups:

[0163] Group 1: injected with 1 million activated MSCs.

[0164] Group 2: injected with 500,000 million activated MSCs.

[0165] Group 3: injected with 250,000 million activated MSCs.

[0166] Group 4: injected with 100,000 million activated MSCs.

[0167] Group 5: injected with 28,000 million activated MSCs.

[0168] Group 6: Injected with only the vehicle, Plasmalyte.

[0169] Mice were injected three times a week for the first week, then once a week for the remaining three weeks. Mice were injected via the tail vein. Each group had five male and five female mice, except for the control group, which had four females and six males. Two additional female mice were assigned, one each to groups 1 and 4.

[0170] Half of the mice began their first injection on day 0 after the binge, and half began their first injection on day 1 after the binge. The reason for splitting the initial injections into two days was to allow for the time required to draw blood, inject the animals, and maintain proper documentation. After the initial injection, each group received subsequent injections on days 3, 7, 14, and 21 after the initial injection, and were followed up until day 28 after the start of the initial injections.

[0171] Mice were examined during the follow-up period for posture, grooming, respiratory rate, body weight, and food consumption.

[0172] Blood was drawn for AST and ALT before each injection and at the time of death.

[0173] Necropsies were performed on mice that died before the endpoint. Mice with physical symptoms were monitored, and once their physical condition deteriorated to the euthanasia endpoint threshold, mice were euthanized according to USC IACUC guidelines.

[0174] (Pathological analysis and evaluation of liver and blood in humanized FRG mice) We collected blood from FRG-huHSC / Hep mice to measure human leukocyte reconstitution at 4 weeks post-infection and at the euthanasia endpoint, 60 days (day 167) after initiation of MSC treatment. This blood was collected to measure the human engraftment efficacy of fetal liver cells. Blood was collected at baseline and at death to measure AST and ALT.

[0175] After euthanasia, representative sections of liver tissue were fixed in 10% neutral buffered formalin and processed for histological evaluation.

[0176] (statistical design) Time-to-event data were analyzed using Kaplan-Meier curves and the log-rank test or Wilcoxon rank test, using the Sidak method for adjustment for multiple comparisons when appropriate.

[0177] Kaplan-Meier curves and Wilcoxon rank tests were used for data analysis between groups. Sidak-adjusted p-values ​​were reported for post-hoc comparisons of each treatment and control group with multiple comparison adjustment.

[0178] (result) (Group comparison) Tables 4A and 4B show age, sex, start date of injection, and baseline AST and ALT for all six cohorts. [Table 4]

[0179] [Table 5]

[0180] [Table 6]

[0181] Example 3: Treatment of liver disease A 50-year-old man has liver disease. He has been receiving 1.5 x 10 6 The mice were treated with activated MSCs. The activation treatment included exposure of the MSCs to interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-12 (IL-12) for 12 hours.

[0182] The patient's symptoms decrease after treatment.

[0183] Example 4: Treatment of liver disease A 40-year-old woman suffers from liver disease. She has been diagnosed with 1.2 × 10 7 The mice were treated with activated MSCs. The activation treatment included exposing the MSCs to interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17) for 10 hours.

[0184] The patient's symptoms decrease after treatment.

[0185] Example 5: Treatment of liver disease A 70-year-old woman suffers from liver disease. She received 1.8 × 10 6 The mice were treated with activated MSCs. The activation treatment included exposing the MSCs to interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17) for 6 hours.

[0186] The patient's symptoms decrease after treatment.

[0187] Example 6: Treatment of liver disease A 55-year-old man has liver disease. He is taking 2 × 10 6 The mice were treated with activated MSCs. The activation treatment included exposing the MSCs to interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17) for 10 hours.

[0188] The patient's symptoms decrease after treatment.

[0189] Example 7: Freezing / Thawing MSCs P5 cells were 1.86 × 10 6 Two injections of cells / flask were prepared. Excess cells and medium from the activation flask were frozen and stored. The remaining cells (terminal P5 cells) that had already been activated and frozen from a previous experiment were thawed and cultured in two flasks (1.86 × 10 6 One flask was left to grow for 48 hours, the other was reactivated after 38 hours and cultured for an additional 10 hours.

[0190] At 48 hours, both flasks were harvested and media from each condition was used for Qiagen Multi-Analyte ELISA Kit. [Table 7] [Table 8]

[0191] This shows growth and viability data for activated cells that are activated once (P5 cells), when activated P5 cells are frozen / thawed and cultured for an additional 48 hours (P6 cells), and when activated P5 cells are frozen / thawed and reactivated a second time and cultured for a total of 48 hours.

[0192] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties (such as molecular weight), reaction conditions, and the like, used in the specification and claims should be understood to be modified in all instances by the term "about". Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present invention. While not attempting to limit the application of the doctrine of equivalents to the scope of the claims, at the very least, each numerical parameter should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Moreover, any numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation found in the testing measurements to which they relate.

[0193] In the context of describing the present invention (particularly in the context of the appended claims), the singular term should be construed as including both the singular and the plural, unless otherwise specified and unless clearly contradicted by the context. The description of ranges of values ​​herein merely serves as a shorthand method of individually referring to each of the individual values ​​falling within the range. Unless otherwise specified herein, each of these individual values ​​is incorporated herein as if it were individually described herein. All of the methods described herein can be performed in any suitable order, unless otherwise specified herein and unless clearly contradicted by the context. The use of any examples and illustrative expressions (e.g., "such as") described herein is intended only to make the present invention more clearly understandable, and does not limit the scope of the invention described in the claims in any way different from these examples and illustrative expressions. Any expressions described in the present invention should not be construed as indicating elements that are essential to the practice of the invention but are not described in the claims.

[0194] Collections of alternative elements or embodiments of the invention described herein are not to be construed as limitations. Each collection member may be referenced or claimed individually or in combination with other collection members or other elements found herein. One or more elements of a collection may be added to or deleted from a collection for reasons of convenience and / or patentability. When such additions or deletions occur, the specification shall be deemed to include the modified collection and shall satisfy the written description requirements for all mark-brush groups used in the claims.

[0195] Certain embodiments herein are described, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will be apparent to those skilled in the art in light of the foregoing description. The inventors contemplate such variations as those skilled in the art would employ as appropriate, and intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0196] Certain embodiments described herein may be further limited in the claims by the use of the phrases "consisting of" or "consisting essentially of." When used in a claim as filed or amended, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to include those materials or steps specified, in addition to those that do not materially affect the basic and novel properties. Embodiments of the invention as so claimed are inherently or explicitly described and enabled in the specification.

[0197] Additionally, various references to patents and publications are made throughout this specification. Each of the above references and publications is hereby incorporated by reference in its entirety.

[0198] Finally, it is to be understood that the embodiments of the invention described herein are illustrative of the principles of the invention. Other modifications which may be employed are within the scope of the invention. Thus, by way of example, and not of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not limited to that precise nature shown and described.

[0199] [Note] [Appendix 1] A method for treating liver disease comprising administering MSCs to a patient in need of treatment for liver disease.

[0200] [Appendix 2] 2. The method of claim 1, wherein the MSCs comprise activated MSCs.

[0201] [Appendix 3] The method of claim 2, wherein the activated MSCs include MSCs activated with at least one of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

[0202] [Appendix 4] The method of claim 2, wherein the activated MSCs include MSCs activated with at least two of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

[0203] [Appendix 5] The method of claim 2, wherein the activated MSCs include MSCs activated with interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

[0204] [Appendix 6] 4. The method of claim 3, wherein the patient is a mammal.

[0205] [Appendix 7] The method of claim 4, wherein the mammal is a human.

[0206] [Appendix 8] 2. The method of claim 1, wherein the administration comprises at least one of subcutaneous, intra-articular, intralesional, intravenous, intraperitoneal, or intramuscular administration.

[0207] [Appendix 9] 10. The method of claim 9, wherein the MSCs are autologous.

[0208] [Appendix 10] The method of claim 8, wherein the MSCs are allogeneic.

[0209] [Appendix 11] 1 x 10 MSCs 3 From cells 1 x 10 12 11. The method of claim 9 or 10, wherein the cell is administered in a dose between 10 and 15 cells.

[0210] [Appendix 12] 12. The method of claim 11, wherein the dose comprises at least two administrations.

[0211] [Appendix 13] 13. The method of claim 12, wherein the dose comprises at least three administrations.

[0212] [Appendix 14] 14. The method of claim 13, wherein the dose comprises at least four administrations.

[0213] [Appendix 15] 15. The method of claim 14, wherein the dose comprises at least 5 administrations.

[0214] [Appendix 16] A method for activating cytokine-producing MSCs, comprising stimulating MSCs with at least one of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

[0215] [Appendix 17] A method for activating cytokine-producing MSCs, comprising stimulating MSCs with at least two of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

[0216] [Appendix 18] A method for activating cytokine-producing MSCs, comprising stimulating MSCs with interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

[0217] [Appendix 19] 19. The method of any one of claims 16 to 18, wherein the cytokines include at least one of IL-6, IL-17A, IFNγ, TNFα, TGFβ, MCP1, HGF, IL-8, TIMP-1, TIMP-2, VEGF, IDO, MIP-1b, and IL-10.

Claims

1. A pharmaceutical composition comprising MSCs for use in a method for treating liver disease comprising administering the MSCs to a patient in need of such treatment.

2. The pharmaceutical composition of claim 1 , wherein the MSCs comprise activated MSCs.

3. 3. The pharmaceutical composition of claim 2, wherein the activated MSCs comprise MSCs activated with at least one of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

4. 3. The pharmaceutical composition of claim 2, wherein the activated MSCs comprise MSCs activated with at least two of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

5. 3. The pharmaceutical composition of claim 2, wherein the activated MSCs include MSCs activated by interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

6. The method of claim 3, wherein the patient is a mammal.

7. The pharmaceutical composition of claim 6, wherein the mammal is a human.

8. 10. The pharmaceutical composition of claim 1, wherein the administration comprises at least one of subcutaneous, intraarticular, intralesional, intravenous, intraperitoneal, or intramuscular administration.

9. The pharmaceutical composition of claim 8 , wherein the MSCs are autologous.

10. The pharmaceutical composition of claim 8, wherein the MSCs are allogeneic.

11. The MSCs were 1×10 ３ 1 × 10 cells １２ The pharmaceutical composition of claim 9 or 10, wherein the composition is administered in an intercellular dose.

12. The pharmaceutical composition of claim 11 , wherein the dose comprises at least two administrations.

13. The pharmaceutical composition of claim 12, wherein the dose comprises at least three administrations.

14. The pharmaceutical composition of claim 13 , wherein the dose comprises at least four administrations.

15. The pharmaceutical composition of claim 14, wherein the dose comprises at least 5 administrations.

16. A method for activating cytokine-producing MSCs, comprising stimulating the MSCs with at least one of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

17. A method for activating cytokine-producing MSCs, comprising stimulating the MSCs with at least two of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

18. A method for activating cytokine-producing MSCs, comprising stimulating MSCs with interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukin-17 (IL-17).

19. 19. The method of any one of claims 16 to 18, wherein the cytokines comprise at least one of IL-6, IL-17A, IFNγ, TNFα, TGFβ, MCP1, HGF, IL-8, TIMP-1, TIMP-2, VEGF, IDO, MIP-1b, and IL-10.

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