Method for treating inflammation using a cell composition

Abstract

The present disclosure relates to cell compositions with anti-inflammatory properties and their use in methods for treating inflammation.

Description

[Technical Field]

[0001] The present disclosure relates to cell compositions with anti-inflammatory properties and their use in methods for treating inflammation. [Background technology]

[0002] Inflammation is a host defense response to various stimuli, including foreign substances (e.g., microorganisms), trauma, malignant tumors, and toxins. It is a complex, multifaceted response broadly regulated by an increase in proinflammatory cytokines, which leads to the accumulation and activation of leukocytes. Excessive or persistent inflammation can cause or result in inflammatory diseases, such as diabetes, graft-versus-host disease, chronic pain, cancer, and infectious or post-infectious diseases.

[0003] Despite the wide variety of drugs on the market to treat inflammation, inflammatory diseases afflict millions of people worldwide each year, resulting in significant morbidity and mortality. Accordingly, there is a need in the art for improved treatments for inflammation and inflammatory diseases. Summary of the Invention

[0004] The present inventors have surprisingly found that mesenchymal progenitor or stem cells (MLPSCs) culture-expanded in a culture medium containing non-fetal serum are particularly effective in treating subjects with persistent inflammation.

[0005] Thus, in one example, the disclosure relates to a method of treating inflammation in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs are culture-expanded in a cell culture medium comprising at least one proinflammatory cytokine. In one example, the proinflammatory cytokine is selected from the group consisting of IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1RA. In one example, the proinflammatory cytokine is provided in non-fetal serum. Thus, in one example, the cell culture medium comprises non-fetal serum.

[0006] Thus, in one example, the present disclosure also provides a method of treating inflammation in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs have been culture-expanded in a cell culture medium comprising non-fetal serum.

[0007] In one example, the subject has persistent inflammation.

[0008] In one example, the non-fetal serum is newborn bovine serum (NBCS). In one example, the NBCS is obtained ≤30 days after the birth of the calf. In one example, the NBCS is obtained ≤21 days after the birth of the calf. In one example, the NBCS is obtained from the day of birth of the calf to 21 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 14 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 10 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 7 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 4 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 3 days after birth. In one example, the NBCS is obtained after the calf has ingested colostrum.

[0009] In one example, the non-fetal serum is characterized by the presence of one or more pro-inflammatory cytokines, hi one example, the non-fetal serum is characterized by the presence of any one or more of IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1RA.

[0010] In one example, the methods of the disclosure include treating an inflammatory disease in a subject.

[0011] In one example, the inflammatory disease is refractory to steroid immunosuppressants and / or biologic therapy.

[0012] In one example, the inflammatory disease is mediated by T cell activation and / or proliferation.

[0013] In one example, the inflammatory disease is refractory to steroid immunosuppressants and / or ruxolitinib. In one example, the inflammatory disease is refractory to steroid immunosuppressants and ruxolitinib. In one example, the inflammatory disease is GVHD, and the GVHD is refractory to steroid immunosuppressants and ruxolitinib.

[0014] In one example, the inflammatory disease is hyperinflammation. In one example, the hyperinflammation is caused by a coronavirus infection. For example, the coronavirus is SARS-CoV, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), or COVID-19.

[0015] In one example, the inflammatory disease is multisystem inflammatory syndrome (MIS). In one example, the inflammatory disease is acute respiratory distress syndrome (ARDS).

[0016] In one example, the inflammatory disease is selected from the group consisting of graft versus host disease (GvHD), pruritus, dermatitis, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's disease, myasthenia gravis, type I or type II diabetes, diabetic nephropathy, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, Sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory disease of the joints, skin or muscles, acute idiopathic inflammatory arthritis or chronic idiopathic inflammatory arthritis, myositis, demyelinating disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, interstitial nephritis, and chronic active hepatitis.

[0017] In one example, the inflammatory disease is diabetes or a condition or symptom associated with diabetes selected from the group consisting of abnormal wound healing, renal failure, blindness, neuropathy, nephropathy, retinopathy, inflammation, impotence, or non-alcoholic steatohepatitis (NASH). In one example, the inflammatory disease is type II diabetes. In one example, the inflammatory disease is rheumatoid arthritis. In one example, the inflammatory disease is diabetic nephropathy.

[0018] In one example, the inflammatory condition is pain. In one example, the pain is chronic pain. In one example, the pain can be lower back pain. In one example, the inflammatory condition is lower back pain associated with disc degeneration.

[0019] In one example, the MLPSCs are administered systemically. In one example, the MLPSCs are administered intravenously. In one example, the MLPSCs are administered in multiple doses. In one example, 1x10 7 ~2x10 8 of cells are administered.

[0020] In one example, the MLPSCs are mesenchymal progenitor cells (MPCs). In one example, MPCs are isolated from bone mononuclear cells using anti-STRO-3 antibodies prior to culture expansion.

[0021] In one example, the mesenchymal progenitor or stem cells are mesenchymal stem cells (MSCs).

[0022] In one example, the MLPSCs are allogeneic.

[0023] In one example, the cells are cryopreserved prior to administration. The cells are cryopreserved prior to being culture-expanded in cell culture medium containing non-fetal serum.

[0024] In one example, a population of MLPSCs is provided in a composition comprising Plasmalyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). In one example, the composition comprises a Plasmalyte A (70%), DMSO (10%), and HSA (25%) solution, where the HSA solution comprises 5% HSA and 15% buffer. In one example, the composition comprises 6.68x10 6 Contains more than 1000 viable cells / mL.

[0025] In one example, MLPSCs are culture-expanded in a cell factory. In one example, the cell factory is passively primed with CO2 before seeding the cells.

[0026] In one example, the method comprises: - the levels of VEGF, angiogenin, and / or SDF-1α expressed by MLPSCs under culture conditions, and / or selecting for use in the treatment culture-expanded MLPSCs having increased levels of one or more angiogenic markers selected from the group consisting of levels of endothelial network formation, endothelial network length, and endothelial branch length measured after treating the endothelial cell population with conditioned medium obtained from the MLPSCs.

[0027] In one example, an increase in the level of one or more angiogenic markers is determined compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. [Brief explanation of the drawings]

[0028] [Figure 1] Assessment and comparison of serum cytokine levels in 1:1 FCS / NBCS (serum A), fetal bovine serum (serum B), and FBS from another source (serum C). [Figure 2] Quantitative measurement of in vitro angiogenesis induced by MLPSC-conditioned medium using the IncuCyte® 96-Well Kinetic Angiogenesis PrimeKit Assay. [Figure 3] 1 shows the results of a Luminex assay demonstrating increased production of angiogenin by MLPSCs cultured with or without neonatal serum. [Figure 4] Levels of angiogenic markers in cGMP lot MLPSC conditioned medium cultured with or without neonatal serum. [Figure 5] Analysis of changes from baseline in echo parameters after 12 months - all subjects. [Figure 6] Analysis of change from baseline in echo parameters after 12 months - subjects with persistent inflammation (hsCRP ≥ 2). [Figure 7] Analysis of changes from baseline in echo parameters after 12 months - subjects without persistent inflammation (hsCRP<2). [Figure 8] CV mortality in subjects with persistent inflammation (hsCRP≧2) by MPC cultured with or without neonatal serum. [Figure 9] Three-point composite MACE (MI, stroke, or CV death) in subjects with persistent inflammation (hsCRP≧2) by MPC cultured with or without neonatal serum. [Figure 10] 3-point composite MACE (MI, stroke, or CV death) in subjects receiving MPC cultured with or without neonatal serum at all passages or at the final passage. The left panel represents data for all patients. The right panel represents data for patients with persistent inflammation (CRP>2 mg / ml). [Figure 11] Three-point composite MACE (left panel) and terminal cardiac events (TCE, right panel) in subjects with the most severe disease (NTpro-BNP>1000ng / ml, CRP>2mg / ml) treated with MPC cultured with or without neonatal serum at all or the final passage. [Figure 12] (A) Plasma levels of the proinflammatory cytokine IL-6 in control LVAD patients: pathogenesis of ischemic and non-ischemic HFrEF. (B) Plasma levels of the proinflammatory cytokine IL-6 in LVAD patients: ischemic control group and ischemic LVAD patients receiving MPC. [Figure 13] All-cause mortality within 12 months in ischemic and non-ischemic LVAD patients. [Figure 14] All-cause mortality over 12 months in LVAD patients receiving "approved" MPC formulations, "unapproved" MPC formulations, and control patients. (A) All LVAD patients (ischemic and non-ischemic groups). (B) Ischemic LVAD patients. DETAILED DESCRIPTION OF THE INVENTION

[0029] General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein are assumed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell therapy, molecular biology, stem cell culture, immunology, biochemistry).

[0030] Unless otherwise indicated, the cell culture techniques and assays utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are explained in such publications as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H.Means (editors), and F.M.A.usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0031] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0032] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0033] As used herein, the term "inflammation" in the context of the present disclosure refers to a physiological and / or pathophysiological state characterized by the presence of inflammatory biomarkers, such as proinflammatory cytokines and immune cell activation. As used herein, an "inflammatory disease" refers to any disease, disorder, or condition in which a subject experiences ongoing or persistent inflammation. "Inflammatory disease" according to the present disclosure also includes any disease, disorder, or condition secondary to an acute inflammatory episode.

[0034] "Persistent inflammation" is defined by elevated C-reactive protein levels. In one example, persistent inflammation is characterized by CRP levels of ≧2 mg / L. "C-reactive protein" or "CRP" is an inflammatory mediator. CRP levels are elevated during acute inflammatory flare-ups and normalize shortly after the inflammation subsides. Thus, CRP is a useful marker of persistent inflammation. In one example, a subject treated according to the present disclosure can have elevated CRP. The term "elevated CRP" is used in the context of the present disclosure to refer to an increased CRP level compared to baseline CRP levels. In one example, a CRP level of ≧1 mg / L is elevated. In another example, a CRP level of ≧1.5 mg / L is elevated. In another example, a CRP level of ≧2 mg / L is elevated. In another example, a CRP level of ≧4 mg / L is elevated.

[0035] In one example, persistent inflammation is persistent systemic inflammation.In this example, persistent inflammation is characterized by systemic biomarkers of inflammation, such as CRP.Therefore, in this example, the method of the present disclosure is directed to treating inflammation characterized by persistent systemic inflammation.In one example, the method of the present disclosure is used to treat inflammation associated with persistently elevated levels of IL-6.

[0036] As used herein, the term "angiogenic marker" refers to an indicator of angiogenesis. As used herein, "angiogenic marker" includes pro-angiogenic molecules such as VEGF, angiogenin, SDF-1α, etc. In another example, an angiogenic marker is a cellular indicator of angiogenesis, such as endothelial network formation, endothelial network length, and endothelial branch length. In this example, the cellular indicator of angiogenesis is determined in an in vitro angiogenesis assay, as disclosed herein.

[0037] The terms "level" and "amount" are used to define the amount of a particular substance in a sample from a subject or cell culture medium (or a sample therefrom). For example, a particular concentration, weight, percentage (e.g., v / v%), or ratio can be used to define the level of a particular substance in a sample. In one example, the level is expressed in terms of the extent to which a particular marker is expressed by the cells of the present disclosure under culture conditions. In one example, expression refers to cell surface expression. In another example, the level is expressed in terms of how much a particular marker is released from the cells described herein under culture conditions. In one example, a sample (e.g., a blood sample) is obtained from a patient or subject, and the level of the substance is measured in the sample to determine the level of the substance in the sample. For example, a blood sample can be obtained to measure the level of CRP in a subject.

[0038] In one example, the levels are expressed in mg / L. For example, the levels of CRP are expressed in mg / L. In one example, the levels are expressed in ng / ml. For example, the levels of VEGF can be expressed in ng / ml. In one example, the levels of SDF-1α can be expressed in ng / ml. In one example, the levels are expressed in pg / ml. In one example, the levels of angiogenin can be expressed in pg / ml.

[0039] In one example, the level of a particular marker is measured in a cell population (or in the supernatant obtained after cell culture) and divided by the number of cells in the population. In this example, the level is calculated as 10 6It can be expressed in units per cell (eg, pg).

[0040] In one example, the level of a particular marker in a cell culture medium is determined under culture conditions. The term "culture conditions" is used to refer to cells growing in culture. In one example, the culture conditions refer to a population of cells that are actively dividing. Such cells may, in one example, be in an exponential growth phase. In one example, the cells are in a stationary phase.

[0041] In one example, the culture conditions include co-culturing a population of MLPSCs disclosed herein with a second cell population, such as a population comprising peripheral blood mononuclear cells (PBMCs). In one example, the co-culturing includes culturing a population of MLPSCs disclosed herein and an activated population of PBMCs. For example, the PBMCs can be activated using anti-CD3 and anti-CD28 antibodies prior to co-culturing with the population of MLPSCs disclosed herein.

[0042] In one example, the "culture conditions" include co-culturing MLPSCs and T cells at a ratio of approximately 1 MLPSC:2 T cells. For example, the MLPSC:T cell ratio is 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or less. In this example, the level of IL2-RA inhibition is determined after approximately 30-84 hours of cell culture under the culture conditions.

[0043] In one example, the level of a particular marker can be determined by taking a sample of cell culture medium and measuring the level of the marker in the sample. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in a cell lysate. Those skilled in the art will understand that secreted markers are generally measured by sampling the culture medium, while markers expressed on the cell surface can generally be measured by evaluating a sample of cell lysate. In one example, a sample is taken when the cells are in the exponential growth phase. In one example, a sample is taken after at least two days of culture. In another example, a sample is taken after approximately 30-84 hours of culture. In one example, a sample is taken after culturing in cell culture medium containing non-fetal serum followed by culturing in cell culture medium containing 10% fetal serum for two to three days.

[0044] In one example, a sample is taken from a co-culture of MLPSCs and activated PBMCs. In this example, the cell sample can be lysed and the level of a marker can be measured. For example, the level of IL2-RA can be determined. In this example, the level of IL2-RA can be determined using a variety of methods, such as an enzyme-linked immunosorbent assay (ELISA)-based method. In one example, ELISA is (i) adding sample dilutions to each well of a microplate precoated with a monoclonal antibody specific for IL2-RA; (ii) adding the co-culture samples to wells of a microplate pre-coated with a monoclonal antibody specific for IL2-RA; (iii) incubating the microplate for a time sufficient to allow the monoclonal antibody specific for IL2-RA to specifically bind to any IL2-RA in the sample; (iv) washing the microplate; (v) adding IL2-RA complexes to the wells; (vi) incubating the microplate for a time sufficient to allow the complex to specifically bind to any captured IL2-RA; (vii) washing the microplate; (viii) adding a substrate solution to the wells; (ix) incubating the microplate for a time sufficient for color development; (x) adding a stop solution to the wells; (xi) reading the optical density on a microplate reader set at 450 nm with wavelength compensation set at 570 nm; (xii) determining the level of IL2-RA.

[0045] In another example, the level of IL2-RA is determined using fluorescence-activated cell sorting (FACS) with an appropriate antibody, such as anti-CD25. Additional antibodies can be used if it is necessary to distinguish between CD25+ cell types. While the above example refers to IL-2RA, it will be appreciated that similar methods can be used to determine the levels of other markers disclosed herein, such as angiogenin. In these examples, co-culture may not be required to determine the level. For example, the level of angiogenin can be measured in a population of MLPSCs under culture conditions.

[0046] Culturing and expanding cells from cryopreserved intermediates means thawing cryogenically frozen cells and culturing them in vitro under conditions suitable for cell growth.

[0047] In one example, the "level" or "amount" of a particular marker is determined after the cells are cryopreserved and then returned to culture. For example, the level may be determined after the first cryopreservation of the cells. In another example, the level is determined after the second cryopreservation of the cells. In one example, cells are isolated from a suitable stem cell source, such as bone marrow (e.g., using immunoselection against marker(s), such as STRO-1), and expanded in culture, thereby providing an intermediate cell population that is evaluated to determine the level of the particular marker. In this example, the level may be determined before or after cryopreservation. In one example, the level is determined after cryopreservation of the intermediate cell population. In another example, cells are culture-expanded from a cryopreserved intermediate, cryopreserved again, and then replated in culture, allowing the level of the particular marker to be measured under culture conditions.

[0048] As used herein, the terms "treating," "treat," "treatment," and "reducing progression" include administering a population of MLPSCs cultured according to the present disclosure and / or their progeny and / or soluble factors derived therefrom and / or extracellular vesicles derived therefrom to thereby reduce or eliminate at least one symptom of progressive heart failure, or, in the context of reducing progression, delay the onset of inflammation.

[0049] In one example, treatment reduces the level of one or more inflammatory biomarkers, such as, for example, neutrophilia, lymphopenia, thrombocytopenia, hypoalbuminemia, elevated CRP, erythrocyte sedimentation rate (ESR), fibrinogen, D-dimer, ferritin, lactate dehydrogenase (LDH), interleukin 6 (IL-6), elevated procalcitonin, etc. In one example, treatment reduces white blood cell count, triglyceride levels, CRP, or IL-6 levels. In one example, treatment reduces the level of one or more pro-inflammatory cytokines.

[0050] The term "subject" as used herein refers to a human subject. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be an adolescent. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in this disclosure, depending on the context. Subjects in need of treatment include those who already suffer from an inflammatory disease, as well as those who need to prevent, delay, or stop inflammation.

[0051] In one example, the composition of the present disclosure comprises non-genetically modified MLPSCs. As used herein, the term "non-genetically modified" refers to cells that have not been modified by the introduction of a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, MLPSCs into which a nucleic acid encoding a protein has been introduced are considered to be genetically modified.

[0052] As used herein, the term "sample" refers to an extract from a subject or cell culture in which the level of a particular marker can be measured. "Sample" includes extracts and / or derivatives and / or fractions of a sample. In one example, a sample is an extract from a subject in which the level of CRP can be measured. In the present disclosure, any biological material can be used as the sample as long as it can be collected from a subject or cell culture and analyzed to determine the level of a marker disclosed herein (e.g., the level of CRP in a subject). In one example, the sample is a blood sample. For example, a blood sample can be obtained from a subject with an inflammatory disease such as GVHD.

[0053] In one example, the present disclosure encompasses selecting a population of culture-expanded MLPSCs with a particular potency for use in the therapeutic methods disclosed herein. As used herein, the term "potency" refers to the particular ability or capacity of MLPSCs to produce a predetermined outcome. In one example, the outcome is a therapeutic effect, such as a reduction in an inflammatory biomarker, as disclosed herein.

[0054] "Therapeutic effect" is used in the context of the present disclosure to refer to the MLPSCs and compositions disclosed herein that can treat, inhibit, and / or prevent a disease. For example, the therapeutically effective MLPSCs and compositions disclosed herein can treat, inhibit, and / or prevent an inflammatory disease.

[0055] "Biological activity," in the context of this disclosure, is used to define the MLPSCs and compositions disclosed herein based on a particular activity. In one example, the biological activity is pro-angiogenic activity and / or anti-inflammatory activity. In one example, the biological activity is the ability to increase angiogenesis in vitro. In one example, the biological activity is increased expression of one or more angiogenic markers. In one example, the biological activity is characterized by reduced inflammation. In one example, the biological activity is characterized by reduced T-cell activation and / or proliferation.

[0056] The term "clinically proven" (used alone or to modify the term "effective") means that efficacy has been proven by a clinical trial, and that the clinical trial meets the approval standards of the US Food and Drug Administration, EMEA, or corresponding national regulatory agency. For example, the clinical study may be an appropriately sized, randomized, double-blind study used to clinically prove the efficacy of a composition. In one example, a clinically proven effective amount is an amount shown by a clinical trial to meet a specified endpoint. In one example, the endpoint is prevention of death. In other words, the endpoint increases survival rate. For example, 100-day survival rate may increase when a therapeutic agent according to the present disclosure is administered.

[0057] Thus, the terms "clinically proven efficacy" and "clinically proven effect" may be used in the context of the present disclosure to refer to a dose, dosing regimen, treatment, or method disclosed herein. Efficacy can be measured based on a change in the course of a disease in response to administration of a composition disclosed herein. For example, a composition of the present disclosure is administered to a subject in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of the inflammatory disease. Various indicators reflecting the severity of the disease can be evaluated to determine whether the amount and duration of treatment are sufficient. Such indicators include, for example, clinically recognized indicators of disease severity or symptoms. In one example, the degree of improvement is determined by a physician, who can make this determination based on signs, symptoms, or other test results. In one example, a clinically proven effective amount improves patient survival. In another example, a clinically proven effective amount reduces the subject's risk of death. In another example, a clinically proven effective amount extends 100-day survival. In one example, the disclosed method administers a clinically proven effective amount of a composition disclosed herein.

[0058] Throughout this specification the word "comprises" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a specified element, integer, step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, steps, or groups of elements, integers, or steps.

[0059] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

[0060] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, as well as any combination of any two or more of the steps or features.

[0061] The present disclosure is not limited in scope by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0062] Unless otherwise specified, examples disclosed herein are intended to apply mutatis mutandis to other examples.

[0063] Mesenchymal progenitor or stem cells (MLPSCs) As used herein, the term "mesenchymal progenitor or stem cell (MLPSC)" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining multipotency and to differentiate into many cell types of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, tendon, or non-mesodermal origin, such as hepatocytes, neurons, and epithelial cells. For the avoidance of doubt, "mesenchymal progenitor cell" refers to a cell that can differentiate into mesenchymal cells, such as bone, cartilage, muscle, adipocytes, and fibrous connective tissue.

[0064] The term "MLPSC" includes both the parent cell and its undifferentiated progeny. The term also includes mesenchymal progenitor cells (MPCs), multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal progenitor cells, and their undifferentiated progeny.

[0065] MLPSCs can be autologous, allogeneic, xenogeneic, syngeneic, or isogenic. Autologous cells are isolated from the same individual to be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or isogenic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0066] In one example, the MLPSCs are allogeneic. In one example, the allogeneic MLPSCs are culture-expanded and cryopreserved.

[0067] While primarily present in bone marrow, MLPSCs have also been shown to reside in various host tissues, including umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, cancellous bone, and dental pulp. They also reside in skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestine, lung, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum. They can differentiate into germ cell lineages, such as mesoderm and / or endoderm and / or ectoderm. Thus, MLPSCs can differentiate into numerous cell types, including adipose, bone, cartilage, elastic, muscle, and fibrous connective tissue. The specific lineage commitment and differentiation pathways these cells follow depend on various influences from endogenous bioactive factors, such as mechanical influences and / or growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0068] As used herein, the terms "enriched," "enrichment," or variations thereof, are used to describe a cell population in which the percentage of a particular cell type or the percentage of the number of a particular cell type is increased when compared to an untreated cell population (e.g., cells in their native environment). In one example, a population enriched for MLPSCs contains at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% MLPSCs. In this regard, the term "a population of cells enriched for MLPSCs" is interpreted as expressly supporting the term "a population of cells containing X% MLPSCs," where X% is a percentage described herein. MLPSCs, in some examples, can form clonogenic colonies, e.g., CFU-Fs (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 70%, 90%, or 95%) can have this activity.

[0069] In one example of the present disclosure, the MLPSCs are mesenchymal stem cells (MSCs). The MSCs may be homogenous or may be a mixed cell population enriched for MSCs. A homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified by unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are culture-expanded and cryopreserved.

[0070] In another example, the MLPSCs are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0071] Isolated or enriched MLPSCs can be expanded in vitro by culture. Isolated or enriched MLPSCs can be cryopreserved, thawed, and then expanded in vitro by culture.

[0072] In one example, isolated or enriched MLPSCs are cultured in culture medium (serum-free or serum-supplemented) (e.g., alpha-minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine) at a density of 50,000 viable cells / cm. 2 Cells are seeded at 100°C and allowed to adhere to the culture vessel overnight at 37°C and 20% O. The culture medium is then replaced and / or changed as needed, and the cells are cultured for an additional 68-72 hours at 37°C and 5% O.

[0073] As will be appreciated by those skilled in the art, cultured MLPSCs are phenotypically distinct from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured MLPSCs also differ biologically from in vivo cells, having a higher proliferation rate compared to the mostly non-cycling (quiescent) cells in vivo.

[0074] In one example, a population of cells is enriched from a cell preparation containing STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may, but need not be STRO-1. For example, as described and / or exemplified herein, cells (e.g., mesenchymal progenitor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (which may be STRO-1 bright). Thus, the designation that cells are STRO-1+ does not mean that the cells are selected solely by STRO-1 expression. In one example, the cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0075] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. Although, in some instances, these terms provide support for selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or vascular tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues listed herein.

[0076] In one example, the cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0077] By "individually" it is meant that the present disclosure encompasses the listed markers or groups of markers separately, and that even if individual markers or groups of markers cannot be separately recited herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0078] "Collectively" means that the disclosure encompasses any number or combination of the listed markers or markers, and that notwithstanding that such number or combination of markers or markers may not be specifically recited herein, the appended claims may define such combination or subcombination separately and divisibly from any other combination of markers or markers.

[0079] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, this term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0080] Additionally, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0081] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell lineages. Non-limiting examples of lineages that STRO-1+ cells can commit include bone progenitor cells; hepatocyte precursors that are multipotent into bile duct epithelial cells and hepatocytes; neural-restricted cells that can generate glial cell precursors that develop into oligodendrocytes and astrocytes; neuronal precursors that develop into neurons; cardiac muscle and cardiomyocyte precursors; and glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, progenitor cells such as odontoblasts, dentin-forming cells, chondrocytes, retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocytes.

[0082] In one example, MLPSCs are obtained from a single donor or from multiple donors, where donor samples or MLPSCs are later pooled and culture-expanded.

[0083] The MLPSCs encompassed by the present disclosure can also be cryopreserved before being administered to a subject. In one example, the MLPSCs are culture-expanded and cryopreserved before being administered to a subject.

[0084] In one example, the present disclosure encompasses MLPSCs and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses MLPSCs and extracellular vesicles isolated therefrom. For example, mesenchymal progenitor or stem cells of the present disclosure can be culture-expanded for a period of time and under conditions suitable for secreting extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0085] As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, but typically are less than 200 nm in size. They are released from the releasing cells (e.g., mesenchymal stem cells; STRO-1). + The fragment may contain proteins, nucleic acids, lipids, metabolites or organelles from the cell.

[0086] As used herein, the term "exosome" refers to a type of extracellular vesicle that generally ranges in size from about 30 nm to about 150 nm and originates from the endosomal compartment of mammalian cells, from which they are transported to and released from the plasma membrane. They may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolic products and may function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0087] In one example, the composition of the present disclosure includes cells that induce new blood vessel formation in a target tissue. In one example, the target tissue is the heart. In another example, the cells secrete factors that protect at-risk or damaged myocardium. In one example, the at-risk or damaged myocardium is exposed to insufficient blood flow due to an ischemic event. In one example, the cells secrete factors that reduce apoptosis of cardiomyocytes.

[0088] The MLPSCs disclosed herein can be modified to inhibit cell lysis upon administration. Antigen modification induces immunological unresponsiveness or tolerance, thereby preventing the induction of effector steps of the immune response (e.g., generation of cytotoxic T cells, production of antibodies, etc.) that ultimately lead to the rejection of foreign cells in a normal immune response. Antigens that can be modified to achieve this goal include, for example, MHC class I antigens, MHC class II antigens, LFA-3, and ICAM-1. MLPSCs can also be genetically engineered to express proteins important for the differentiation and / or maintenance of striated musculoskeletal cells. Exemplary proteins include growth factors (TGF-β, insulin-like growth factor 1 (IGF-1), FGF), myogenic factors (e.g., myoD, myogenin, myogenic factor 5 (Myf5), myogenic regulatory factors (MRF)), transcription factors (e.g., GATA-4), cytokines (e.g., cardiotropin-1), members of the neuregulin family (e.g., neuregulin 1, 2, 3), and homeobox genes (e.g., Csx, tinman, NKx family).

[0089] Culture-expanded MLPC In some examples, the culture-expanded MLPSCs of the present disclosure are characterized based on their therapeutic effects. For example, the MLPSCs can be characterized based on their therapeutic effects in inflammatory diseases. In one example, the MLPSCs are characterized by their therapeutic effects on heart failure. In another example, the MLPSCs are characterized by their therapeutic effects in T cell-mediated diseases such as GVHD.

[0090] In another example, culture-expanded MLPSCs are characterized by their ability to inhibit IL-2RA expression by CD3 / CD28-activated PBMCs under culture conditions. In one example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 60% compared to a control group. In another example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 65% compared to a control group. In another example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 70% compared to a control group. In another example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by 60-70% compared to a control group.

[0091] "Culture-expanded" MLPSCs are distinguished from freshly isolated cells in that they are cultured and passaged (ie, subcultured) in cell culture medium.

[0092] In one example, the freshly isolated cells are expanded in culture for about 1 or 2 passages to provide an intermediate population. In one example, the freshly isolated cells are expanded in culture for 2 passages to provide an intermediate population. In another example, the freshly isolated cells are expanded in culture for about 1-3 passages to provide an intermediate population. In one example, the freshly isolated cells are STRO-1+.

[0093] Thus, in one example, relevant cells are isolated and cultured for two passages to provide an intermediate MLPSC population. In certain examples, the intermediate MLPSC population is then cultured and expanded to produce a drug product (DP). For example, the DP composition of the present disclosure is produced by culturing cells from an intermediate cryopreserved MLPSC population, in other words, a cryopreserved intermediate. In one example, the intermediate cell population can be cultured for three passages (a total of five passages) to provide a DP.

[0094] In one example, the MLPSCs have been culture-expanded for approximately 4 to 10 passages. In one example, the MLPSCs have been culture-expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, or at least 10 passages. For example, the MLPSCs can be culture-expanded for at least 5 passages. In one example, the MLPSCs can be culture-expanded for at least 5 to 10 passages. In one example, the MLPSCs can be culture-expanded for at least 5 to 8 passages. In one example, the MLPSCs can be culture-expanded for at least 5 to 7 passages. In one example, the MLPSCs can be culture-expanded for more than 7 passages. In these examples, the MLPSCs are culture-expanded before being cryopreserved to provide an intermediate cryopreserved MLPSC population, which can then be further culture-expanded.

[0095] In one example, a composition of the present disclosure includes MLPSCs culture-expanded from a cryopreserved intermediate. In one example, the cell culture culture-expanded from a cryopreserved intermediate is culture-expanded for at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 passages. For example, the MLPSCs can be culture-expanded for at least 3 passages. In one example, the MLPSCs can be culture-expanded for at least 3-10 passages. In one example, the MLPSCs can be culture-expanded for at least 3-8 passages. In one example, the MLPSCs can be culture-expanded for at least 3-7 passages. In one example, the culture of MLPSCs culture-expanded from a cryopreserved intermediate is culture-expanded in a medium disclosed herein (e.g., a medium containing newborn bovine serum).

[0096] In one example, MLPSCs can be obtained from a single donor or from multiple donors, where donor samples or MLPSCs are subsequently pooled and optionally culture-expanded. i. expanding viable cell numbers by passage expansion to provide a preparation of at least about 1 billion viable cells, the passage expansion comprising establishing a primary culture of isolated MLPSCs and then serially establishing a first non-primary (P1) culture of MLPSCs isolated from the previous culture; ii. Expanding the P1 culture of isolated MLPSCs by passage expansion into a second, non-primary (P2) culture of MLPSCs; and iii. Preparing and cryopreserving an in-process intermediate MLPSC preparation obtained from a P2 culture of MLPSCs, and optionally iv. Thawing a cryopreserved in-process intermediate MLPSC preparation and expanding the in-process intermediate MLPSC preparation by passage expansion.

[0097] In one example, the method of the present disclosure includes selecting an intermediate population (e.g., a cryopreserved intermediate) for further culture expansion based on certain criteria, such as the level of one or more angiogenic markers. The selection process is not particularly limited, as long as it allows for the selection of a cell population characterized by relevant criteria, such as the level of angiogenic markers. In one example, the levels of angiogenic markers are evaluated for a series of intermediate MLPSC populations, and a population that exceeds a threshold level of angiogenic markers described herein is selected for further expansion.

[0098] It is noted that the selection process does not require immediate culture expansion. Rather, the "selected" population can be cryopreserved and culture expanded at a later stage. In one example, a portion of the intermediate cell population is culture expanded, and the remainder of the population is cryopreserved for culture expansion at a later stage.

[0099] In one example, the selected cell population is immediately expanded in culture, hi another example, the selected cell population is cryopreserved to allow for expansion in culture at a later stage.

[0100] In one example, the selected cell population is expanded in culture to provide a pharmaceutical composition, which in one example is characterized by certain criteria, such as the level of angiogenic markers.

[0101] In the context of the present disclosure, the level of angiogenic marker(s) can be assessed between steps iii and iv of the culture expansion process described above. For example, the level of angiogenic marker(s) can be determined under the culture conditions and / or from the conditioned medium after step iii. In one example, step iv is performed only if a desired level of angiogenic marker(s) is observed under the culture conditions and / or from the conditioned medium. In this example, a cell population is selected for culture expansion based on the level of angiogenic marker(s) under the culture conditions and / or from the conditioned medium.

[0102] In one example, the culture-expanded MLPSC population is expanded from an intermediate MLPSC population that has increased levels of one or more angiogenic markers compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

[0103] In one example, the level of an angiogenic marker(s) disclosed herein is considered to be increased if it is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of an angiogenic marker is increased by about 5% to about 60%. In one example, the level of an angiogenic marker is increased by about 5% to about 40%. In one example, the level of an angiogenic marker is increased by about 40%. In one example, the level of an angiogenic marker is increased by at least about 5%. In one example, the level of an angiogenic marker is increased by at least about 10%.

[0104] In one example, the culture-expanded MLPSC population is expanded from an intermediate MLPSC population that has increased levels of one or more angiogenic markers compared to a population of MLPSCs cultured and expanded in cell culture medium without neonatal serum. In one example, the level of an angiogenic marker is considered increased if it is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in cell culture medium without neonatal serum. In one example, the level of an angiogenic marker is increased by about 5% to about 60%. In one example, the level of an angiogenic marker is increased by about 5% to about 40%. In one example, the level of an angiogenic marker is increased by about 40%. In one example, the level of an angiogenic marker is increased by at least about 5%. In one example, the level of an angiogenic marker is increased by at least about 10%.

[0105] In one example, a culture-expanded MLPSC preparation is i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. have an antigenic and activity profile that includes at least about 95% CD166+ cells.

[0106] The process of MLPSC isolation and in vitro expansion can be carried out using any equipment and cell processing methods known in the art. Various culture expansion embodiments of the present disclosure employ steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing. Any step that manipulates cells can potentially injure the cells. While MLPSCs can generally tolerate some damage during preparation, it is preferable to manipulate cells using procedures and / or equipment that appropriately perform a given step(s) while minimizing damage to the cells.

[0107] In one example, MLPSCs are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a spinning membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, as described, for example, in U.S. Pat. No. 6,251,295, incorporated herein by reference.

[0108] In one example, MLPSC compositions cultured according to the present disclosure are 95% homogeneous for being CD105-positive, CD166-positive, and CD45-negative, and in one example, this homogeneity persists through in vitro expansion, i.e., multiple population doublings.

[0109] In one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture. For example, the MLPSCs of the present disclosure can be culture-expanded in a cell factory. In certain examples, 3D culture of the intermediates disclosed herein can be performed, for example, using a bioreactor. In one example, the MLPSCs of the present disclosure are first culture-expanded in 2D culture before being further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure is not culture-expanded in 3D culture. In one example, the level of one or more angiogenic markers is assessed before subsequent culture expansion in a cell factory or 3D culture.

[0110] In one example, the MLPSCs of the present disclosure are culture-expanded from an intermediate population. In one example, the MLPSCs of the present disclosure are culture-expanded from an intermediate in a 2D culture before being seeded into a 3D culture.

[0111] In the context of both the intermediate population and the therapeutic composition expanded thereby, in one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture for at least three days before being seeded into a further culture system, such as a 3D culture in a cell factory or bioreactor. In one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture for at least four days before being seeded into a further culture system. In one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture for three to five days before being seeded into a further culture system. In these examples, the 2D culture can be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma, Corning). In one example, the cell factory has at least five layers. In one example, the cell factory has at least 10 layers. In one example, the cell factory has at least 20 layers. 3D culture can be performed in various bioreactor types, such as stirred tanks, wave bags, and vertical wheels.

[0112] In one example, CO2 is provided during the culture and growth of MLPSCs. In one example, MLPSCs are cultured and grown in less than 9% CO2. In one example, MLPSCs are cultured and grown in less than 8% CO2. In one example, MLPSCs are cultured and grown in 5% CO2. For example, MLPSCs can be cultured and grown in 5% (+ / - 2%) CO2. In one example, MLPSCs are cultured and grown using passive priming of CO2. For example, a cell factory can be passively primed with 5% CO2.

[0113] Priming cell factories maintains CO2 tension between the cell factory and the incubator and stabilizes the pH level of the growth medium. Active priming involves actively passing CO2 gas through a bacterial vent air filter into each culture vessel (e.g., cell factory) for a specified time (e.g., approximately 10 minutes). However, active priming requires an open port to provide gas, potentially introducing contaminants into the culture. Passive priming involves placing the sealed culture system in an incubator with the appropriate CO2 concentration prior to cell seeding (e.g., approximately 12-72 hours). In one example, the cells disclosed herein are STRO-3+ before being culture-expanded to obtain an intermediate cell population.

[0114] composition The compositions disclosed herein are useful for pre-licensing of MLPSCs, a multi-step process that results in the functional maturation of MSCs and promotes their therapeutic efficacy.

[0115] Thus, provided herein is a composition for pre-approval of MLPSCs, comprising a human cell population enriched for MLPSCs and serum containing one or more pro-inflammatory cytokines. In other embodiments disclosed herein, the composition comprises (i) a human cell population enriched for MLPSCs, (ii) serum containing one or more pro-inflammatory cytokines, and (iii) a cryopreservative. Suitable cryopreservatives include, but are not limited to, one or more of dimethyl sulfoxide (DMSO), trehalose, and albumin.

[0116] In other embodiments, provided herein is a cell culture medium for the expansion and pre-qualification of MLPSCs, the cell culture medium comprising serum containing one or more pro-inflammatory cytokines.

[0117] The cell culture medium of the present invention can contain optional components such as fatty acids or lipids, vitamins, cytokines, antioxidants, buffers, inorganic salts, and the like.

[0118] The cell culture medium used in the present disclosure contains all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified into essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0119] Those skilled in the art will understand that for optimal results, key nutrients must be available at appropriate levels to enhance cell growth, and the basal medium must be appropriate for the cell line of interest. For example, if depletion of this energy source proves to be limiting for cell growth, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or to supplement glucose (or other energy source) over the course of the culture.

[0120] The culture medium of the present disclosure can be prepared using a basal culture medium. In the context of the present disclosure, "basal culture medium" refers to an unsupplemented medium suitable for exposure to cells such as MSCs. The basal medium includes, for example, Eagle's Minimum Essential Medium (MEM), alpha-modified MEM, StemSpan™, and mixtures thereof, and is not particularly limited as long as it can be used to culture MLPSCs.

[0121] In some preferred embodiments of the compositions for preapproval disclosed herein, the composition comprises one or more proinflammatory cytokines selected from IL-1β, IL-6, IFN-γ, TNF-α, and IL-1 receptor antagonist (IL-1ra). In some embodiments, the composition comprises each of IL-1β, IL-6, IFN-γ, TNF-α, and IL-1 receptor antagonist (IL-1ra). In some embodiments, the composition is substantially free of proinflammatory cytokines other than IL-1β, IL-6, IFN-γ, TNF-α, and IL-1ra. In some embodiments, the composition comprises only one, two, three, four, or five proinflammatory cytokines, wherein the one to five proinflammatory cytokines are selected from the group consisting of IL-1β, IL-6, IFN-γ, TNF-α, and IL-1ra.

[0122] In some embodiments, the compositions disclosed herein contain a neonatal serum IL-1β concentration of about 2 ng / ml to about 50 ng / ml, e.g., 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 40 ng / ml, 45 ng / ml, or another concentration of about 2 ng / ml to about 50 ng / ml. In some embodiments, the neonatal serum IL-6 concentration is about 0.2 ng / ml to about 1.2 ng / ml, e.g., 0.4 ng / ml, 0.5 ng / ml, 0.6 ng / ml, 0.7 ng / ml, 0.8 ng / ml, 1.0 ng / ml, or another concentration of about 0.2 ng / ml to about 1.2 ng / ml. In some embodiments, the concentration of IFN- in suitable neonatal serum is about 0.1 ng / ml to about 0.2 ng / ml, e.g., 0.12 ng / ml, 0.14 ng / ml, 0.16 ng / ml, 0.18 ng / ml, or another concentration of about 0.1 ng / ml to about 0.2 ng / ml. In some embodiments, the concentration of IL-1ra in suitable neonatal serum is about 6 ng / ml to about 33 ng / ml, e.g., 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 12 ng / ml, 15 ng / ml, 20 ng / ml, 22 ng / ml, 25 ng / ml, 27 ng / ml, 30 ng / ml, or another concentration of about 6 ng / ml to about 33 ng / ml. In some embodiments, the concentration of TNF-α in suitable neonatal serum is about 0.1 ng / ml to about 0.7 ng / ml, e.g., 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 0.5 ng / ml, 0.6 ng / ml, or another concentration of about 0.2 ng / ml to about 0.7 ng / ml.

[0123] In some preferred embodiments, the compositions disclosed herein are substantially free of pro-inflammatory cytokines other than those present in the serum prior to inclusion in the composition, i.e., the serum used is substantially the only source of exogenous pro-inflammatory cytokines in the composition.

[0124] In some embodiments, the concentration of neonatal serum in the described compositions is about 2% (v / v) to about 12% (v / v), such as 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or another concentration between about 2% (v / v) and about 12% (v / v). In some preferred embodiments, the concentration of neonatal serum is about 5% (v / v). In other preferred embodiments, the concentration of neonatal serum is about 10% (v / v).

[0125] In some embodiments, the compositions disclosed herein comprise both fetal serum and newborn serum. In some preferred embodiments, the ratio of fetal serum to newborn serum in the composition is 1:1. In some embodiments, the ratio of fetal serum to newborn serum is greater than 1:1, e.g., 3:1, 2.8:1, 2.5:1, 2.2:1, 2:1, 1.8:1, 1.5:1, 1.2:1, or a ratio of fetal serum to newborn serum of about 3:1 or less. In other embodiments, the ratio of fetal serum to newborn serum is less than 1:1, e.g., 1:3, 1:2.8, 1:2.5, 1:2.2, 1:1.8, 1:1.5, 1:1.2, or another ratio of fetal serum to newborn serum of about 1:3 or greater.

[0126] In some preferred embodiments, the total concentration of serum (including the proportions of fetal serum and neonatal serum) in the composition is about 10% (v / v).

[0127] In some embodiments, when the composition contains a cryopreservative, e.g., DMSO, the concentration of neonatal serum is about 3% (v / v) to about 20% (v / v), e.g., 4%, 6%, 7%, 7.5%, 8%, 9%, 10%, 12%, 15%, or another concentration of about 3% (v / v) to about 15% (v / v). In some embodiments, the cryopreservation composition has a 1:1 ratio of fetal serum to neonatal serum, e.g., 5% (v / v) each.

[0128] In some embodiments, the serum containing one or more proinflammatory cytokines used in the compositions disclosed herein is newborn serum. In some preferred embodiments, the newborn serum used is, for example, newborn calf serum, newborn lamb serum, or newborn horse foal serum. In some preferred embodiments, the newborn serum is newborn calf serum. In embodiments in which the compositions disclosed herein contain newborn serum, the newborn serum is from a newborn on about postnatal day 1 to about postnatal day 7, e.g., postnatal day 2, postnatal day 3, postnatal day 4, postnatal day 5, or postnatal day 6. For clarity, postnatal day 1 as used herein refers to the day of birth. In some embodiments, the newborn serum used in the compositions described herein can include a mixture of newborn sera obtained on different days after birth.

[0129] In some embodiments, the neonatal serum used is bovine, ovine, caprine, equine, or human serum. In some preferred embodiments, the neonatal serum is bovine. In some embodiments, when the composition contains fetal serum, the fetal serum is bovine, ovine, equine, or caprine. In some preferred embodiments, the fetal serum is fetal bovine serum.

[0130] In one example, a composition of the present disclosure includes IFN-γ and / or TNF-α (e.g., serum containing IFN-γ and TNF-α). For example, the level of IFN-γ may be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the level of TNF-α may be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the composition includes IFN-γ and TNF-α, both at levels less than 1 ng / ml. In this example embodiment, the IFN-γ and TNF-α are provided in serum.

[0131] In one example, the composition comprises one or more pro-inflammatory cytokines capable of binding to receptors on the surface of MLPSCs.

[0132] In one example, the serum contains one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the serum can contain IL-8.

[0133] In one example, the composition comprises IFN-γ and / or TNF-α and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0134] In one example, the composition comprises: i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 5 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: a level of IP-10 greater than 5,000 pg / ml.

[0135] In certain instances, serum may be diluted from its original concentration in cell culture medium. In these instances, the level of cytokines in serum is reduced accordingly. For example, serum may be provided at 10% (v / v) in cell culture medium. In these instances, serum may be provided at 10% (v / v) in cell culture medium. i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Levels of IP-10 greater than 500 pg / ml.

[0136] In one example, the serum is newborn serum and contains the above levels of cytokine(s). In one example, the composition of the present disclosure comprises a medium described below.

[0137] In other examples, the compositions of the present disclosure comprise the above population(s) of culture-expanded MLPSCs.

[0138] In some preferred embodiments, the MLPSCs are human mesenchymal stem cells. In other embodiments, the MLPSCs are STRO-1 + Pluripotent cells, or STRO-1 + This is a population of MLPSCs culture-expanded from a pluripotent cell population.

[0139] In a preferred embodiment, the MLPSCs are maintained in an undifferentiated state.

[0140] Culture medium In one embodiment, the present disclosure encompasses MLPSC culture medium supplemented with pro-inflammatory cytokine(s). In one example, the culture medium includes IFN-γ and / or TNF-α. In one example, the medium includes IFN-γ. For example, the level of IFN-γ can be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the medium includes TNF-α. For example, the level of TNF-α can be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the medium includes IFN-γ and TNF-α, both at levels less than 1 ng / ml.

[0141] In one example, the medium includes one or more pro-inflammatory cytokines that can bind to receptors on the surface of the MLPSCs.

[0142] In one example, the medium comprises one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the medium can comprise IL-8.

[0143] In one example, the medium comprises IFN-γ and / or TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0144] In one example, the medium is i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0145] In one example, the medium is i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 50 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: IP-10 levels greater than 5,000 pg / ml.

[0146] In one example, the medium contains IL-10. In another example, the medium contains IL-36RA. In another example, the medium contains IL-10 and IL-36RA. In one example, the level of IL-10 is greater than 0.3 pg / ml. For example, the level of IL-10 can be greater than 30 pg / ml. In one example, the level of IL-10 is greater than 400 pg / ml. In one example, the level of IL-36RA is greater than 50 pg / ml.

[0147] In one example, the medium is serum-free.

[0148] In one example, the medium is serum-free and supplemented with PDGF and FGF2. In one example, the medium is serum-free and supplemented with PDGF, FGF2, and EGF. In one example, the PDGF is PDGF-BB. In one example, the serum-free medium is supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF2.

[0149] In one example, the above cytokines can be provided at a concentration of <1 ng / ml each. For example, the medium can be characterized by one or more or all of the following, each provided at <1 ng / ml: IFN-γ, TNF-α, IL-6, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

[0150] method The present disclosure provides an in vitro method for pre-qualifying MLPSCs by culturing a human cell population enriched for MLPSCs (e.g., hMSCs) in a cell culture medium suitable for the maintenance and expansion of MLPSCs.

[0151] In one example, the culture medium is the composition or medium referenced above. In one example, the culture medium is supplemented with serum containing one or more pro-inflammatory cytokines described herein. In some preferred embodiments, the culture medium used is supplemented with neonatal serum. In some preferred embodiments, the culture medium used is supplemented with equal concentrations of fetal serum and neonatal serum, with the total serum concentration in the culture medium being about 10% (v / v). In some preferred embodiments, MLPSCs are pre-qualified in cell culture medium containing 5% (v / v) neonatal serum and 5% (v / v) fetal serum.

[0152] In some embodiments, the MLPSC methods disclosed herein include the additional step of determining or having determined the level of one or more pro-inflammatory cytokines in the serum contained in the culture medium used for pre-qualification of the MLPSCs. Methods for determining cytokine levels are well known in the art, such as, for example, ELISA.

[0153] In some embodiments, the methods disclosed herein also include determining, or having determined, the ability of culture medium (e.g., culture medium supplemented with neonatal serum) to stimulate MLPSCs to promote angiogenesis in an in vitro assay (e.g., tube formation by human umbilical vein endothelial cells (HUVECs), and analysis of network length, network area, and branch point formation). In some embodiments, such assays involve collecting MLPSC-conditioned medium after culturing in neonatal serum-supplemented medium as disclosed herein and quantifying the effect of such conditioned medium in the angiogenesis assays described above or similar assays.

[0154] In some embodiments, the MLPSC methods disclosed herein also include determining or having determined levels of one or more of angiogenin, angiopoietin (Ang1 / ANGPT1), SDF-1α, and VEGF in the conditioned medium described above.

[0155] In some embodiments, when a first lot or batch of neonatal serum is used in a conditioned medium that promotes greater angiogenesis or release of angiogenic factors than a conditioned medium in which a second lot / batch of neonatal serum is used, it is concluded that using a first lot of neonatal serum for prequalification and culture expansion of MLPSCs will produce MLPSCs with relatively greater therapeutic potency, particularly for the treatment of conditions in which an angiogenic or anti-inflammatory therapeutic mode of action is useful.

[0156] The disclosed methods and cell culture media promote stem cell proliferation and pre-qualification while maintaining MLPSCs in an undifferentiated state. MLPSCs are considered undifferentiated if they have not progressed to a specific lineage. As explained above, MLPSCs exhibit morphological characteristics that distinguish them from differentiated cells. Furthermore, undifferentiated MLPSCs express genes that can be used as markers to detect their differentiated state. Polypeptide products can also be used as markers to detect their differentiated state. Therefore, those skilled in the art can easily determine whether the disclosed methods maintain MLPSCs in an undifferentiated state using routine morphological, genetic, and / or proteomic analyses. Methods for monitoring / confirming cell proliferation are also known in the art and, in certain instances, can be as basic as periodic visual inspection of cell cultures to confirm an increase in cell number. Other methods may include the use of cell viability dyes and / or imaging and counting live cells using commercially available products.

[0157] The MLPSCs disclosed herein can be cultured and expanded in a variety of suitable cell culture media, including neonatal serum. As used in the context of this disclosure, the term "medium" or "media" includes components of the environment surrounding cells. A medium contributes to and / or provides suitable conditions for cell growth. A medium can be solid, liquid, gaseous, or a mixture of these phases and materials. A medium can include liquid growth media as well as liquid media that do not support cell growth. An exemplary gaseous medium includes the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0158] In one example, the disclosed method involves culturing and expanding in cell culture medium containing one or more pro-inflammatory cytokines. In one example, the cell culture medium contains IFN-γ and / or TNF-α. In one example, the cell culture medium contains IFN-γ. For example, the level of IFN-γ can be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the cell culture medium contains TNF-α. For example, the level of TNF-α can be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the cell culture medium contains IFN-γ and TNF-α, both at levels less than 1 ng / ml.

[0159] In one example, the cell culture medium includes one or more pro-inflammatory cytokines that can bind to receptors on the surface of the MLPSCs.

[0160] In one example, the cell culture medium comprises one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the cell culture medium can comprise IL-8.

[0161] In one example, the cell culture medium comprises IFN-γ and / or TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0162] In one example, the cell culture medium comprises: i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0163] In another example, the medium is i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 50 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: IP-10 levels greater than 5,000 pg / ml.

[0164] In one example, the medium contains IL-10. In another example, the medium contains IL-36RA. In another example, the medium contains IL-10 and IL-36RA. In one example, the level of IL-10 is greater than 0.3 pg / ml. For example, the level of IL-10 can be greater than 30 pg / ml. In one example, the level of IL-10 is greater than 400 pg / ml. In one example, the level of IL-36RA is greater than 50 pg / ml.

[0165] In another example, the disclosed methods involve culture expansion in cell culture medium containing newborn serum. Various examples of suitable serum (and levels thereof) are disclosed herein.

[0166] In one example, the disclosed method includes selecting an intermediate population of cryopreserved MLPSCs for culture-expansion in a medium disclosed herein. In one example, a cryopreserved intermediate culture expanded in 10% fetal serum is selected for culture-expansion according to the methods disclosed herein. In one example, a cryopreserved intermediate culture expanded in neonatal serum and / or pro-inflammatory cytokines disclosed herein is selected for culture-expansion.

[0167] The cell culture medium used for culture expansion contains all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified as essential (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro) amino acids.

[0168] Those skilled in the art will understand that for optimal results, the basal medium must be appropriate for the cell line of interest. For example, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or add glucose (or other energy source) during the culture process if this energy source is found to be depleted and therefore limiting growth. In one example, dissolved oxygen (DO) levels may also be controlled.

[0169] serum "Non-fetal serum" refers to serum obtained after birth. For example, the culture medium can be supplemented with mammalian non-fetal serum (e.g., bovine). In one example, the culture medium can be supplemented with animal non-fetal serum. In another example, the culture medium can be supplemented with human non-fetal serum.

[0170] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 10% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 5% v / v of non-fetal serum.

[0171] In one example, the non-fetal serum contains at least one proinflammatory cytokine. Methods for detecting the presence of cytokines in cell culture medium and / or serum are known in the art, such as enzyme-linked immunosorbent assay (ELISA). In another example, the presence of cytokines in serum is detected by measuring cytokine mRNA, for example, by polymerase chain reaction (PCR) techniques such as reverse transcription PCR.

[0172] "Newborn serum" refers to serum obtained after birth. For example, the culture medium can be supplemented with mammalian newborn serum (e.g., bovine). In one example, the culture medium can be supplemented with animal newborn serum. In another example, the culture medium can be supplemented with human newborn serum.

[0173] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 10% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 5% v / v of newborn serum.

[0174] In one example, the neonatal serum contains at least one proinflammatory cytokine. As used herein, the term "proinflammatory cytokine" refers to a signaling molecule that promotes inflammation. In an example, the one or more cytokines are selected from the group including IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1ra.

[0175] In one example, the newborn serum contains IFN-γ. In another example, the newborn serum contains TNF-α. In another example, the newborn serum contains IFN-γ and TNF-α. In another example, the newborn serum contains one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the newborn serum can contain IL-8. In one example, the newborn serum contains IFN-γ and / or TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In another example, the newborn serum contains IFN-γ and TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In one example, the level of IFN-γ is less than 1 ng / ml. In one example, the level of TNF-α is less than 1 ng / ml. In one example, the levels of both IFN-γ and TNF-α are less than 1 ng / ml. For example, the level of IFN-γ can be less than 500 pg / ml or less than 100 pg / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml.

[0176] Methods for detecting the presence of cytokines in serum are known in the art, for example, enzyme-linked immunosorbent assay (ELISA). In another example, the presence of cytokines in serum is detected by measuring cytokine mRNA by polymerase chain reaction (PCR) techniques, such as reverse transcription PCR.

[0177] In one example, the newborn serum can be newborn bovine serum (NBCS). In one example, the NBCS is obtained from a newborn calf that has been fed colostrum. In one example, the NBCS has an elevated level of at least one inflammatory cytokine compared to NBCS obtained from a calf that has not been fed colostrum. In one example, the NBCS has an elevated level of at least one inflammatory cytokine compared to fetal serum such as FCS.

[0178] In one example, the NBCS is obtained within 4 weeks after the birth of the calf. In one example, the NBCS is obtained within 21 days after the birth of the calf. For example, the NBCS is obtained ≦21 days after the birth of the calf. In one example, the NBCS is obtained from the day of birth of the calf to 21 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 14 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 10 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 7 days after birth. In one example, the NBCS is obtained from 6 hours to 72 hours after birth. In one example, the NBCS is obtained from 6 hours to 48 hours after birth. In one example, the NBCS is obtained from 6 hours to 24 hours after birth. In one example, the NBCS is obtained from 12 hours to 24 hours after birth.

[0179] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of NBCS. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of NBCS. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of NBCS. In one example, the cell culture medium is supplemented with at least about 5% v / v NBCS.

[0180] In one example, the culture medium is also supplemented with fetal serum. In one example, the fetal serum is fetal calf serum (FCS). In the context of the present disclosure, the terms fetal calf serum (FCS) and fetal bovine serum (FBS) are considered to be interchangeable. In one example, the cell culture medium is supplemented with less than 10% v / v FCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS.

[0181] In one example, the cell culture medium is fetal serum-free.

[0182] In one example, the cell culture medium does not contain FCS.

[0183] In one example, the culture medium is supplemented with a mixture of FCS and NBCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS and about 5% v / v NBCS (i.e., a 1:1 FCS:NBCS ratio). In one example, the culture medium can be supplemented with a mixture of FCS and NBCS, where the FCS:NBCS ratio is at least about 0.4:1, at least about 0.5:1, at least about 0.6:1, at least about 0.7:1, at least about 0.8:1, at least about 0.9:1, at least about 1:1, at least about 1.5:1, or at least about 2:1. In one example, the FCS:NBCS ratio is about 0.5:1 to about 2:1. In one example, the FCS:NBCS ratio is about 0.8:1 to about 1.5:1. In one example, the FCS:NBCS ratio is about 0.8:1 to about 1.2:1. In one example, the ratio of FCS:NBCS is about 1:1.

[0184] In one example, the mixture of FCS and NBCS can comprise at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise from about 1% v / v to about 15% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 2% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 5% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 8% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 10% v / v of the cell culture medium, but in this example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, or at least about 9% v / v, but less than 10% v / v, of FCS. In one example, the cell culture medium is supplemented with about 1% v / v to about 9% v / v of FCS. In one example, the cell culture medium is supplemented with about 3% v / v to about 8% v / v FCS, in one example, about 3% v / v to about 6% v / v FCS, and in one example, about 5% v / v FCS.

[0185] In one example, the non-fetal serum is adult serum. Thus, in one example, the culture medium is supplemented with adult serum. In one example, the culture medium is supplemented with adult bovine serum. In one example, the culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of adult serum.

[0186] In another example, the culture medium is supplemented with human adult serum. The cell culture medium can be supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, or at least about 9% v / v of human adult serum. In one example, the cell culture medium can be supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, or at least about 9% v / v of human AB serum. In one example, the cell culture medium is supplemented with at least about 3% human AB serum.

[0187] In another example, the culture medium is supplemented with neonatal serum (e.g., NBCS) and adult serum. In one example, the culture medium is supplemented with adult bovine serum. In one example, the culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of adult serum.

[0188] In another example, the culture medium is supplemented with human adult serum. The cell culture medium can be supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, or at least about 9% v / v of human adult serum. In one example, the cell culture medium can be supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, or at least about 9% v / v of human AB serum. In one example, the cell culture medium is supplemented with at least about 3% human AB serum.

[0189] Ascorbic acid In one example, a short-acting ascorbic acid derivative is added to the cell culture medium. The term "short-acting" encompasses ascorbic acid derivatives that are approximately 80-90% oxidized after 24 hours of cell culture under culture conditions of neutral pH and 37°C. In one example, the short-acting L-ascorbic acid derivative is an L-ascorbate salt, such as L-ascorbic acid sodium salt. In one example, the cell culture medium may contain at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.01 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.02 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.03 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.04 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.05 g / L of a short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.06 g / L of a short-acting ascorbic acid derivative.

[0190] In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not a substantial amount of a long-acting ascorbic acid derivative. For example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.04 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.03 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.02 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.01 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.005 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not a long-acting ascorbic acid derivative, hi another example, the cell culture medium contains L-ascorbic acid sodium salt but does not contain a substantial amount of L-ascorbic acid-2-phosphate.

[0191] Other additives In one example, the cell culture medium contains human-derived additives. For example, human serum and human platelet cell lysate can be added to the cell culture medium. In another example, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), 1α,25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor 1α (SDF-1α). In another embodiment, the cells can be cultured in the presence of at least one cytokine in an amount sufficient to maintain cell growth. In another embodiment, the cells can be cultured in the presence of heparin or a derivative thereof.

[0192] In the above example, the basal medium such as Alpha MEM or StemSpan™ can be supplemented with a reference amount of serum, and in certain cases, can also be supplemented with other additives. Further examples of suitable culture media for culturing stem cells are described, for example, in WO2016139340.

[0193] Angiogenesis markers According to the present disclosure, in certain embodiments, MLPSCs cultured according to the methods disclosed herein have increased levels of one or more angiogenic markers. In one example, the methods of the present disclosure relate to selecting culture-expanded MLPSCs for use in a therapy, such as treating inflammation. Such methods include determining the level(s) of the marker(s) disclosed herein and selecting MLPSCs for use in the therapy that have increased levels of one or more marker(s).

[0194] In certain examples, the disclosed methods include measuring the levels of pro-angiogenic factors, such as VEGF, angiogenin, and / or SDF-1α, expressed by MLPSCs under culture conditions. In one example, MLPSCs can be culture-expanded in culture medium according to the methods disclosed herein. Conditioned medium from the cultured MLPSCs is then isolated (i.e., a sample is obtained from the cell culture), and the amount of expressed angiogenic markers therein is measured. The level of angiogenic markers in MLPSC-conditioned medium can be measured by standard protein detection and / or gene expression methods known in the art. In one example, the level of angiogenic markers is measured by enzyme-linked immunosorbent assay (ELISA). For example, conditioned medium from MLPSCs is obtained and then contacted with anti-VEGF antibody, anti-SDF-1α antibody, and / or anti-angiogenin antibody. The degree of antibody binding is used to quantify the level (e.g., ng / L) of the angiogenic marker in the conditioned medium. In this example, the level of angiogenic marker in the conditioned medium is a measure of the level of angiogenic marker expressed or secreted by MLPSCs.

[0195] In one example, the level of the angiogenic marker is measured by Western blot. In one example, the level of the angiogenic marker is measured by Luminex assay. In one example, the level of the angiogenic marker is measured by reverse transcription RT-PCR.

[0196] In one example, MLPSCs are selected for use in therapy if the expression level of vascular endothelial growth factor (VEGF) is elevated. In one example, the VEGF level is greater than about 3 ng / ml. In one example, the VEGF level is greater than about 3 ng / ml to 4 ng / ml. In one example, the VEGF level is greater than about 3.1 ng / ml. In one example, the VEGF level is greater than about 3.2 ng / ml. In one example, the VEGF level is greater than about 3.3 ng / ml. In one example, the VEGF level is greater than about 3.4 ng / ml. In one example, the VEGF level is greater than about 3.5 ng / ml. In one example, the VEGF level is between about 3 ng / ml and 4 ng / ml. In one example, the VEGF level is between about 3.2 to 3.6 ng / ml. In one example, the VEGF level is about 3.45 ng / ml.

[0197] In one example, MLPSCs are selected for therapeutic use if they exhibit an increased level of VEGF compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the VEGF level is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the VEGF level is increased by about 5% to about 60%. In one example, the VEGF level is increased by about 5% to about 40%. In one example, the VEGF level is increased by about 40%. In one example, the VEGF level is increased by at least about 5%. In one example, the VEGF level is increased by at least about 10%.

[0198] In one example, MLPSCs are selected for therapeutic use if they have elevated angiogenin expression levels. In one example, the angiogenin level is greater than about 1000 pg / ml. In one example, the angiogenin level is greater than about 1100 pg / ml. In one example, the angiogenin level is between about 1000 pg / ml and 1200 pg / ml. In one example, the angiogenin level is between about 1100 pg / ml and 1150 pg / ml. In one example, the angiogenin level is greater than about 1114 pg / ml.

[0199] In one example, MLPSCs are selected for therapeutic use if their angiogenin levels are increased compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the angiogenin levels are increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the angiogenin levels are increased by about 5% to about 60%. In one example, the angiogenin levels are increased by about 5% to about 40%. In one example, the angiogenin levels are increased by about 40%. In one example, the angiogenin levels are increased by at least about 5%. In one example, the angiogenin levels are increased by at least about 10%.

[0200] In one example, MLPSCs are selected for therapeutic use if they have elevated expression levels of stromal-derived factor 1 alpha (SDF-1α). In one example, the level of SDF-1α is greater than about 3000 ng / ml. In one example, the level of SDF-1α is greater than about 3100 ng / ml. In one example, the level of SDF-1α is greater than about 3200 ng / ml. In one example, the level of SDF-1α is greater than about 3300 ng / ml. In one example, the level of SDF-1α is greater than about 3400 ng / ml. In one example, the level of SDF-1α is greater than about 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3400 ng / ml. In one example, the level of SDF-1α is about 3000 ng / ml to 3300 ng / ml. In one example, the level of SDF-1α is about 3100 ng / ml to 3400 ng / ml. In one example, the level of SDF-1α is about 3100 ng / ml to 3300 ng / ml.

[0201] In one example, MLPSCs are selected for therapeutic use if they exhibit an increased level of SDF-1α compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of SDF-1α is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of SDF-1α is increased by about 5% to about 60%. In one example, the level of SDF-1α is increased by about 5% to about 40%. In one example, the level of SDF-1α is increased by about 40%. In one example, the level of SDF-1α is increased by at least about 5%. In one example, the level of SDF-1α is increased by at least about 10%.

[0202] In another example, the angiogenic marker is increased angiogenesis. In this example, increased angiogenesis is measured by an in vitro angiogenesis assay, such as a quantitative live-cell imaging assay. Briefly, endothelial cell lines (e.g., human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts, human saphenous vein endothelial cells (HSaVECs), human coronary artery endothelial cells (HCAECs), human aortic endothelial cells (HAECs), brain microvascular endothelial cells (BMECs), or any combination thereof) are fluorescently labeled and seeded onto culture plates. Next, the endothelial cells are simultaneously incubated with or without MLPSC-conditioned medium and imaged using a live-cell imaging system.

[0203] In this example, angiogenesis can be measured by various network morphometric parameters identified and calculated by image analysis software as a composite of various factors listed in Table 1 (Lam et al. Biomaterials 290. (2022) 121826). In one example, the live-cell imaging system is an IncuCyte® Live-Cell Analysis System. Live-cell imaging systems allow for fluorescent identification of cells and visualization of angiogenesis over time through time-lapse image acquisition. Images can be analyzed using a computer-based image analysis tool. In one example, the image analysis tool is the IncuCyte® Angiogenesis Analysis Software Module. The IncuCyte® Angiogenesis Analysis Software Module measures angiogenesis outputs such as endothelial network length, endothelial network area, and endothelial branch point formation. Those skilled in the art will recognize that other image analysis applications, such as Image J and CellProfiler, can also be used. Other examples of live imaging in vitro angiogenesis assays are described, for example, in Lam et al. Biomaterials 290. (2022). 121826. [Table 1-1] [Table 1-2]

[0204] In one example, angiogenesis is measured by the level of endothelial network formation, endothelial network length, and / or endothelial branch length. In one example, the level of endothelial network formation, endothelial network length, and / or endothelial branch length is calculated by an IncuCyte® Angiogenesis Analysis software module.

[0205] In another example, the endothelial network formation, endothelial network length, and / or endothelial branch length may be calculated as a composite of one or more of the number of nodes, number of junctions, number of segments, number of meshes, average mesh size, total mesh area, number of endpoints, total branch length, and / or number of branches. Further examples of methods for calculating endothelial network formation, endothelial network length, and / or endothelial branch length are described, for example, in Lam et al. Biomaterials 290. (2022).

[0206] In one example, MLPSCs are selected for use in a treatment if they increase one or more levels of endothelial network formation, endothelial network length, and / or endothelial branch length. In one example, endothelial network formation is increased by about 0.1 mm 2 / mm 2 In one example, the formation of an endothelial network is observed when the endothelial network is greater than about 0.1 mm 2 / mm 2 ~0.2mm 2 / mm 2 In one example, the formation of an endothelial network is approximately 0.12 mm 2 / mm 2 In one example, the length of the endothelial network is about 4 mm. 2 / mm 2 In one example, the length of the endothelial network is about 4 mm. 2 / mm 2 ~about 6mm 2 / mm 2In one example, the length of the endothelial network is about 5 mm. 2 / mm 2 In one example, the length of the endothelial branch is about 12 1 / mm 2 In one example, the length of the endothelial branch is approximately 12 1 / mm 2 ~approximately 17 1 / mm 2 In one example, the length of the endothelial branch is about 15 1 / mm 2 is.

[0207] In one example, MLPSCs are selected for use in a treatment if they increase one or more levels of endothelial network formation, endothelial network length, and / or endothelial branch length compared to a population of MLPSCs culture-expanded in a cell culture medium containing 10% fetal serum. In one example, the level of endothelial network formation is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in a cell culture medium containing 10% fetal serum. In one example, the level of endothelial network formation is increased by about 5% to about 60%. In one example, the level of endothelial network formation is increased by about 5% to about 40%. In one example, the level of endothelial network formation is increased by about 40%. In one example, the level of endothelial network formation is increased by at least about 5%. In one example, the level of endothelial network formation is increased by at least about 10%.

[0208] In one example, MLPSCs are selected if the level of endothelial network length is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of endothelial network length is increased by about 5% to about 60%. In one example, the level of endothelial network length is increased by about 5% to about 40%. In one example, the level of endothelial network length is increased by about 40%. In one example, the level of endothelial network length is increased by at least about 5%. In one example, the level of endothelial network length is increased by at least about 10%. In one example, MLPSCs are selected if the level of endothelial branch length is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of endothelial branch length is increased by about 5% to about 60%. In one example, the level of endothelial branch length is increased by about 5% to about 40%. In one example, the level of endothelial branch length is increased by about 40%. In one example, the level of endothelial branch length is increased by at least about 5%. In one example, the level of endothelial branch length is increased by at least about 10%.

[0209] composition The MLPSCs disclosed herein can be culture expanded from a cryopreserved intermediate to produce a preparation comprising at least one therapeutic dose.

[0210] In one example, a composition of the present disclosure comprises about 150 million cells.

[0211] In one example, the compositions of the present disclosure include a pharmaceutically acceptable carrier and / or excipient. The terms "carrier" and "excipient" refer to a composition of matter conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also reduce undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other ingredients in the carrier. In one example, a carrier does not cause significant local or systemic adverse effects in a recipient at dosages and concentrations used for therapy.

[0212] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffers, hyaluronan, and glycols are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0213] In another example, the carrier is, for example, a medium composition in which cells are grown or suspended. Such a medium composition does not induce any adverse effects in the subject to which it is administered. Exemplary carriers and excipients do not adversely affect cell viability and / or the ability of the cells to treat or prevent disease.

[0214] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at an appropriate pH, thereby exerting biological activity; for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it interacts minimally with the cells and factors, allowing for rapid release of the cells and factors; in such cases, the compositions of the present disclosure can be formulated as a liquid for direct application to the bloodstream or to tissues or areas surrounding or adjacent to tissues, e.g., by injection.

[0215] The composition of the present disclosure can be cryopreserved. Cryopreservation of MLPSCs can be carried out using slow cooling or "rapid" freezing protocols known in the art. Cryopreservation methods preferably maintain the phenotype, cell surface markers, and proliferation rate of cryopreserved cells comparable to those of unfrozen cells.

[0216] The cryopreservation composition may contain a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0217] Cryopreservation solutions can include a sterile, nonpyrogenic, isotonic solution, such as PlasmaLyte ATM. 100 mL of PlasmaLyte ATM contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H11NaO7); 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O), 37 mg of potassium chloride, USP (KCl), and 30 mg of magnesium chloride, USP (MgCl2·6H2O). No antimicrobial agents are included. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0218] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0219] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are typically added to cryopreservation solutions. Ideally, cryoprotectants should be non-toxic to cells and patients, non-antigenic, and chemically inert, provide high post-thaw survival rates, and allow for irrigation-free transplantation. However, DMSO, the most commonly used cryoprotectant, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0220] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes Plasma-Lyte A (70%), DMSO (10%), and HSA (25%) solution, where the HSA solution includes 5% HSA and 15% buffer.

[0221] In one example, the cryopreservation solution may further comprise one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0222] The cryopreserved composition can be thawed and administered directly to a subject or added to another solution containing, for example, hyaluronic acid. Alternatively, the cryopreserved composition can be thawed prior to administration and the MLPSCs resuspended in another carrier.

[0223] The compositions described herein may be administered alone or as a mixture with other cells. Different types of cells may be mixed with the disclosed compositions immediately prior to or immediately before administration, or may be co-cultured together for a period of time prior to administration.

[0224] In one example, the composition comprises an effective amount, or a therapeutically or prophylactically effective amount, of MLPSCs and / or their progeny and / or soluble factors derived therefrom. For example, the composition comprises about 1x10 5 ~approx. 1x10 9 of stem cells, or approximately 1.25x10 3 ~Approx. 1.25x10 7 / kg (80 kg subject) of stem cells. The exact amount of cells administered will depend on a variety of factors, including the age, weight, and sex of the subject, and the extent and severity of the disorder being treated.

[0225] Regardless of the number of cells provided in the composition, in one example, 50 x 10 6 ~200×10 7 In another example, 60×10 cells are administered. 6 ~200×10 6 cells or 75 x 10 6 ~150×10 6 In one example, 75 x 10 cells are administered. 6 In another example, 150 x 10 cells are administered. 6 cells are administered.

[0226] In one example, the composition contains 5.00 x 10 6 In another example, the composition contains more than 5.50 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.00 x 10 viable cells / mL. 6In another example, the composition contains more than 6.50 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.68 x 10 viable cells / mL. 6 Contains more than 1000 viable cells / mL.

[0227] In one example, the MLPSCs comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the cell population of the composition.

[0228] In one example, the composition may be packaged in a suitable container, optionally accompanied by instructions for a desired purpose.

[0229] The compositions of the present disclosure may be administered systemically, such as by intravenous administration. In one example, the compositions are administered transendocardially.

[0230] In one example, a composition of the present disclosure comprises a "clinically proven effective" amount of MLPSCs. In one example, a composition of the present disclosure comprises a "clinically proven effective" amount of MSCs. In one example, a composition of the present disclosure comprises a "clinically proven effective" amount of MPCs. In one example, a "clinically proven effective" amount of MLPSCs is administered as a total dose. The term "total dose," in the context of this disclosure, is used to refer to the total number of cells received by a subject treated according to this disclosure. In one example, the total dose consists of one administration of cells. In another example, the total dose consists of two administrations of cells. In another example, the total dose consists of three administrations of cells. In another example, the total dose consists of four or more administrations of cells. For example, the total dose can consist of two to four administrations of cells.

[0231] Treatment method The disclosed methods relate to treating inflammation in a subject by administering to the subject a composition comprising a population of MLPSCs as disclosed herein. In one example, the therapeutic methods disclosed herein reduce T cell activation and / or proliferation. In another example, the therapeutic methods disclosed herein improve patient survival.

[0232] As used herein, the terms "inflammation" and "inflammatory disease" should be interpreted to encompass any disease with an inflammatory component, such as autoimmune disease, pain, infection, cardiovascular disease, cancer, inflammatory lung disease, inflammatory bowel disease, trauma, and / or neurodegenerative disease. Examples of inflammatory diseases include, but are not limited to, graft-versus-host disease (GvHD), pruritus, dermatitis, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's disease, myasthenia gravis, type I or type II diabetes, diabetic nephropathy, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, Sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory diseases of the joints, skin, or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease (COPD), interstitial lung disease, interstitial nephritis, and chronic active hepatitis.

[0233] In one example, the inflammatory disease is refractory to first-line therapy. In one example, the inflammatory disease is refractory to steroid immunosuppressive therapy. For example, the inflammatory disease is refractory to systemic steroids. In one example, the steroid is a corticosteroid. In another example, the steroid is a glucocorticoid. In another example, the steroid is prednisone. In one example, the inflammatory disease is refractory to biological therapy. For example, the inflammatory disease is refractory to etanercept, infliximab, ruxolitinib, antithymocyte globulin, mycophenolic acid, alemtuzumab, basiliximab, or tocilizumab. In one example, the inflammatory disease is refractory to extracorporeal photopheresis. In one example, the inflammatory disease is refractory to steroid immunosuppressive therapy. In one example, the inflammatory disease is refractory to steroid therapy and second-line therapy. In one example, the second-line therapy is ruxolitinib.

[0234] In one example, the inflammatory disease is an autoimmune disease, hi one example, the inflammatory disease is rheumatoid arthritis.

[0235] In one example, the inflammatory disease is a cardiovascular disease. In one example, the inflammatory disease is caused by thrombosis, such as pulmonary embolism or venous or arterial thrombosis. As used herein, the term "thrombosis" refers to the formation of a blood clot or clot. In one example, thrombosis is referred to as "arterial thrombosis," in which a blood clot forms in an artery. Such blood clots are particularly dangerous to a subject because they can obstruct blood flow to major organs such as the heart or brain. As used herein, the term "pulmonary embolism" refers to a condition in which an artery in the lung is blocked by material that has traveled from elsewhere in the body through the bloodstream.

[0236] In one example, the inflammatory disease is associated with abnormal T cell proliferation. In one example, the inflammatory disease is mediated by T cell activation.

[0237] Graft-versus-host disease In one example, the inflammatory disease is GvHD. GvHD is an immune disease that is a major factor limiting the success and availability of allogeneic bone marrow or stem cell transplants. In one example, the inflammatory disease is acute GvHD. Acute GvHD typically develops within 100 days after bone marrow or stem cell transplantation. In one example, the inflammatory disease is chronic GvHD. Chronic GvHD typically appears later than acute GvHD (>100 days after transplantation) and has some characteristics of an autoimmune disease. It may develop de novo after resolution of aGvHD or as an extension of aGvHD. Chronic GvHD can cause a variety of debilitating symptoms, including widespread skin rashes, painful mouth sores, shortness of breath, and pain in the limbs and joints.

[0238] In one example, the inflammatory disease is severe GvHD. Severe GvHD can be acute or chronic.

[0239] The severity of GVHD can also be classified by the pattern of organ dysfunction and clinical performance status. Multi-organ dysfunction can include skin rash, liver dysfunction, and / or gastrointestinal (GI) dysfunction. In one example, a subject suffers from GVHD with multi-organ dysfunction. Severe GvHD can be assessed, for example, by the IBMTR Severity Index for Acute GvHD (Rowlings et al., (1997) British Journal of Haematology; 97, 855-864), Glucksberg Clinical Stage and Grade for Acute GvHD (Glucksberg et al., (1974) Transplantation; 18, 295-304), and / or the MAGIC Algorithm Probability (MAP) (Major-Monfried et al., (2018) Blood; 131(25):2846-2855; Hartwell et al., (2017) JCI Insight.; 2(3):e89798).

[0240] In one example, severe GVHD is classified according to the Glucksberg scale (Glucksberg et al., 1974; Thomas et al., 1975) (Table 2). For example, a subject can suffer from Grade II GvHD or Grade III / IV GvHD according to the Glucksberg scale. In one example, a subject suffers from Grade II GvHD. In another example, a subject suffers from Grade III / IV GVHD.

[0241] In another example, severe GVHD is classified according to the IBMTR Severity Index (Table 3) (Rowlings et al., 1997). In one example, the subject has Grade B, Grade C, or Grade D GvHD according to the IBMTR severity scale.

[0242] In another example, the subject has Minnesota high-risk GVHD. Minnesota high-risk acute GvHD is defined as GvHD of either cutaneous stage 4; lower gastrointestinal (GI) stage 3-4 or hepatic stage 3-4; or cutaneous stage 3+ and lower GI stage 2-4 or hepatic stage 2-4 (MacMillan et al., 2015). In each of these examples, the subject may also have a high MAP score. For example, the subject may have a MAP score of ≥ 0.29. [Table 2] [Table 3]

[0243] In one example, severe GvHD manifests in the subject's stomach and / or intestines as damage to these organs. In this example, various sites of inflammation can occur along the stomach and / or intestines, and in certain instances, it may be preferable to administer treatment directly to one or more of these sites of inflammation. In one example, severe GvHD is associated with inflammatory bowel disease (IBD). For example, GVHD can be associated with Crohn's disease. Thus, in one example, the inflammatory disease is inflammatory bowel disease. In one example, the inflammatory disease is Crohn's disease.

[0244] In one example, the treatment of GVHD induces a partial response after initiation of treatment. In one example, the partial response is induced 28 days after initiation of treatment. In one example, the partial response is - At least 1 point reduction in skin %BSA score, - A decrease of at least 1 point in the oral score, - A decrease of at least 1 point in visual acuity score, - A decrease of at least 1 point in skin feature score, - A decrease of at least 1 point in the gastrointestinal score, - A decrease of at least 1 point in liver score, - At least 1 point reduction in pulmonary symptom score, - A decrease of at least 1 point in pulmonary FEV1 score, - At least 1 point reduction in joint and fascia score, - Characterized by one or more or all of the following: a decrease of at least 1 point in the reproductive tract score.

[0245] In another example, a partial response is: - At least 1 point reduction in skin %BSA score, - A decrease of at least 1 point in the oral score, - Characterized by one or more or all of the following: a decrease of at least 1 point in visual acuity score.

[0246] In one example, the treatment for GVHD induces a complete response after initiation of treatment. In one example, the complete response is induced 28 days after initiation of treatment.

[0247] Excessive inflammation In one example, the inflammatory disease is hyperinflammation. The term "hyperinflammation" is used in the context of the present disclosure to refer to a severe and ongoing inflammatory process in the body. For example, hyperinflammation may refer to a severe and ongoing inflammatory process in the airways and / or lungs, kidneys, or liver. As such, hyperinflammation may affect multiple organs and their vasculature in the body. In one example, hyperinflammation is associated with cytokine storm or cytokine release syndrome (CRS). In one example, cytokine storm or CRS involves the massive release of inflammatory cytokines such as IL-6.

[0248] In one example, hyperinflammation leads to secondary (or acquired) hemophagocytic lymphohistiocytosis (sHLH). Thus, in one example, the methods of the present disclosure encompass treatment of hemophagocytic lymphohistiocytosis (HLH).

[0249] In another example, excessive inflammation is associated with elevated CRP, PCT, IL-6, and / or ferritin. For example, ferritin can be greater than 2000 mg / dl. In another example, ferritin can be greater than 2500 mg / dl. In one example, excessive inflammation is associated with bacterial infection. In one example, a subject treated according to the present disclosure has elevated circulating CRP levels. For example, a subject treated according to the present disclosure can have a circulating CRP level greater than 100 mg / dl. In another example, a subject treated has a circulating CRP level greater than 120 mg / dl. In another example, a subject treated has a circulating CRP level greater than 150 mg / dl. In another example, a subject treated has a circulating CRP level between 90 mg / dl and 300 mg / dl.

[0250] In other examples, hyperinflammation is associated with elevated triglycerides or decreased fibrinogen. For example, a subject treated according to the present disclosure can have a triglyceride level of >1.5 mmol / L. In another example, a subject treated has a triglyceride level of >2, >3, or >4 mmol / L. In another example, a subject treated has a triglyceride level of 1.5-5 mmol / L. In another example, a subject treated has a fibrinogen level of 2.5 g / L or less. In another example, a subject treated has a fibrinogen level less than 2.5 g / L.

[0251] In one example, hyperinflammation can lead to multisystem inflammatory syndrome (MIS). For example, hyperinflammation can lead to pediatric MIS (MIS-C).

[0252] In one example, the hyperinflammation is caused by a viral infection. For example, the hyperinflammation can be caused by rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, the hyperinflammation can be caused by a coronavirus. For example, the coronavirus can be SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), or COVID-19. In one example, the hyperinflammation is caused by Epstein-Barr virus (EBV) or herpes simplex virus (HSV).

[0253] multisystem inflammatory syndrome In one example, the inflammatory disease is multisystem inflammatory syndrome (MIS). In one example, the subject is a child with MIS. For example, a subject with MIS is between 1 month and 18 years old. In one example, the subject suffers from acute heart failure. Heart failure (HF) is generally a clinical syndrome characterized by a series of symptoms (dyspnea, orthopnea, leg swelling) and signs (elevated jugular venous pressure, pulmonary congestion). Acute heart failure is broadly defined as a condition in which new or worsening signs and symptoms of heart failure appear rapidly. In one example, the subject has a reduced left ventricular ejection fraction. For example, the subject's LVEF may be less than 45%. In one example, the LVEF is less than 40%. In another example, the LVEF is less than 30%.

[0254] In one example, a subject with MIS meets the following criteria, which are one or more of the following: - Hypotension or shock (cardiac or vascular) - Features of severe cardiac disease, including but not limited to myocarditis, pericarditis, or valvulitis, markedly elevated troponin / proBNP, or coronary artery abnormalities. - Other severe end-organ impairment, including but not limited to neurological or renal disease (except in the case of severe respiratory disease alone); or Two or more of the following are true: -Maculopapular rash - Bilateral non-purulent conjunctivitis - Mucocutaneous inflammatory signs (mouth, hands, or feet) - Acute gastrointestinal symptoms (diarrhea, vomiting, or abdominal pain).

[0255] In one example, a subject who meets the above MIS criteria has fever (body temperature of 38 degrees or higher). In another example, a patient with MIS has elevated inflammatory biomarkers. For example, a subject may exhibit one or more of the following: neutrophilia, lymphopenia, thrombocytopenia, hypoalbuminemia, elevated CRP, erythrocyte sedimentation rate (ESR), fibrinogen, D-dimer, ferritin, lactate dehydrogenase (LDH), interleukin-6 (IL-6), elevated procalcitonin. In one example, a subject has two or more of the following: neutrophilia, lymphopenia, thrombocytopenia, hypoalbuminemia, elevated CRP, ESR, fibrinogen, D-dimer, ferritin, LDH, IL-6, elevated procalcitonin.

[0256] In one example, the subject suffers from one or more of myocarditis, pericarditis, or valvulitis. In one example, the subject suffers from viral myocarditis, pericarditis, or valvulitis. For example, the subject may have viral myocarditis.

[0257] In one example, MIS occurs secondary to infection with SARS-CoV, MERS-CoV, or COVID-19.

[0258] inflammatory lung disease In one example, the inflammatory disease is an inflammatory lung disease. The term "inflammatory lung disease" as used in the context of this disclosure refers to a disease caused by an ongoing inflammatory process in the airways and / or lungs. For example, COPD is an inflammatory lung disease that affects both the airways and lung tissue. It can manifest as a combination of chronic obstructive bronchitis and emphysema, the former resulting from chronic inflammation of the bronchi and the latter resulting from destruction of the alveoli. Other examples of inflammatory lung diseases include acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), pulmonary arterial hypertension (PAH), asthma, cystic fibrosis, pneumonia, and interstitial lung disease.

[0259] In one example, the inflammatory lung disease is caused by a viral infection. For example, the inflammatory lung disease can be caused by rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, the inflammatory lung disease can be caused by coronavirus. For example, the coronavirus can be severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or COVID-19. In one example, the inflammatory lung disease is characterized by a combination of the above symptoms. For example, the inflammatory lung disease can include chronic obstructive pulmonary disease (COPD) and acute respiratory distress syndrome (ARDS).

[0260] In one example, the inflammatory lung disease is acute respiratory distress syndrome (ARDS). The term "acute respiratory distress syndrome (ARDS)" is a type of respiratory failure characterized by widespread inflammation of the lungs, oxygen deficiency, non-compliance or "stiff" lungs. The disorder is generally associated with capillary endothelial injury and diffuse alveolar injury.

[0261] The severity of ARDS can be diagnosed according to the PaO2 / FiO2 ratio. For example, the severity of ARDS is diagnosed as follows (mild: 26.6 kPa < PaO2 / FiO2 ≤ 39.9 kPa, moderate: 13.3 kPa < PaO2 / FiO2 ≤ 26.6 kPa, severe: PaO2 / FiO2 ≤ 13.3 kPa). In one example, the severity of ARDS can be diagnosed according to the Berlin definition summarized in Table 4 below.

Table 4

[0262] In another example, the severity of ARDS is diagnosed as follows. Mild (PaO2 / FiO2 200 - 300 mmHg), moderate (PaO2 / FiO2 100 - 200 mmHg), severe (PaO2 / FiO2 less than 100 mmHg).

[0263] In one example, the ARDS is mild ARDS. In another example, the ARDS is moderate ARDS. In another example, the ARDS is severe ARDS. In another example, the ARDS is moderate or severe ARDS. In one example, a patient with moderate to severe ARDS has a circulating CRP level greater than 4 mg / L. In another example, the ARDS is moderate, severe, or very severe ARDS. In one example, the patient with ARDS is 65 years of age or older. In one example, the patient with ARDS is less than 65 years of age.

[0264] In one example, ARDS is caused by a viral infection. For example, ARDS can be caused by rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, ARDS can be caused by a coronavirus. For example, the coronavirus can be SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), or COVID-19. In one example, ARDS is caused by Epstein-Barr virus (EBV) or herpes simplex virus (HSV).

[0265] In another example, ARDS is caused by thrombosis. In another example, ARDS is caused by embolism. In one example, ARDS is caused by pulmonary embolism.

[0266] In another example, ARDS occurs secondary to hemophagocytic lymphohistiocytosis (HLH). HLH is a life-threatening disease characterized by hyperinflammation of lymphocytes and macrophages. HLH can be caused by viral infections such as EBV, CMV, and HHV. Therefore, in one example, HLH is secondary or acquired HLH. For example, HLH can occur secondary to a viral infection, leading to the development of ARDS in a patient.

[0267] In one example, the inflammatory disease is COPD. In one example, the COPD is mild COPD, moderate COPD, severe COPD, or very severe COPD. In one example, the severity of COPD is determined based on the Global Initiative for Obstructive Lung Disease (GOLD) criteria for COPD (see, for example, Rabe et al., (2007) Respir Crit Care Med., 176:532-555). In this example, a patient with COPD may have an FEV1 / FVC ratio of <0.70. In one example, for a patient with an FEV1 / FVC ratio of <0.70, the following is used to diagnose the severity of COPD: -GOLD 1-Mild: FEV1 ≥ 80% predicted; -GOLD 2 - Moderate: 50% ≤ FEV1 < 80% predicted; -GOLD 3-Severe: 30% ≤ FEV1 < 50% predicted, -GOLD 4-Very severe: FEV1<30% predicted.

[0268] Viral infection In one example, the inflammatory disease is caused by a viral infection, such as a rhinovirus, influenza virus, respiratory syncytial virus (RSV), or coronavirus. In one example, the viral infection is caused by a coronavirus. In one example, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), or COVID-19.

[0269] diabetes In one example, the inflammatory disease is diabetes mellitus. Diabetes mellitus (DM) is a diagnostic term for a group of disorders in which abnormal carbohydrate (e.g., glucose) homeostasis or metabolism results in elevated blood glucose levels. These disorders include several interrelated metabolic, vascular, and neurological components. The various components of DM are caused by endocrine and / or exocrine pancreatic function. For example, the metabolic component is generally characterized by hyperglycemia and includes alterations in carbohydrate, fat, and protein metabolism caused by absent or significantly reduced secretion of hormones, particularly insulin (i.e., endocrine function), and / or ineffective insulin action. At the exocrine level, the pancreas produces various enzymes involved in food digestion. For example, the pancreas produces amylase, but in DM, insufficient secretion of this enzyme results in the inability to digest carbohydrates, leading to exocrine pancreatic insufficiency, malnutrition, and weight loss.

[0270] The vascular component of DM includes vascular abnormalities, leading to cardiovascular, retinal, and renal complications. Abnormalities in the peripheral and autonomic nervous systems are also components of DM. Vascular complications can also result in impaired wound healing, necrosis, and gangrene. In one example, the inflammatory disease is a diabetes-related condition or symptom selected from the group consisting of impaired wound healing, renal failure, blindness, neuropathy, nephropathy, retinopathy, inflammation, impotence, or nonalcoholic steatohepatitis (NASH). In one example, the inflammatory disease is diabetic nephropathy.

[0271] There are several types of diabetes, including type I (also called insulin-dependent diabetes mellitus or IDDM), type II (also called non-insulin-dependent diabetes mellitus or NIDDM), gestational diabetes, and prediabetes (or disorders of glucose metabolism). In one example, the inflammatory disease is type I diabetes. In one example, the inflammatory disease is type II diabetes.

[0272] pain In one example, the inflammatory disease is pain, which can be a chronic condition associated with inflammation, pain, and has a significant impact on disability and quality of life.

[0273] In one example, the pain is chronic pain. In one example, the pain is lower back pain. For example, lower back pain is associated with intervertebral disc degeneration. In one example, lower back pain is associated with the intervertebral disc (IVD). The IVD provides flexibility and mechanical stability to the spine during axial compression, flexion, and extension. The IVD is composed of several specialized connective tissues: (i) hyaline cartilage of the cartilaginous endplates (CEPs) covering the surfaces of the vertebrae (vertebral bodies) above and below the disc; (ii) the fibrocartilaginous annulus fibrosus (AF) encasing the nucleus pulposus (NP); and (iii) the central gelatinous nucleus pulposus (NP). The IVD can degenerate due to aging or trauma. Common IVD lesions that cause chronic pain include i) rim lesions, which are transverse defects near where the AF attaches to the bone at the vertebral body edge; (ii) concentric (circumferential) tears, in which the annular lamellae separate from one another; and (iii) radial tears, which occur due to the propagation of a tear that begins within the NP. These lesions can cause a local inflammatory response and exacerbate the pain experienced by the individual.

[0274] In one example, the pain is associated with a disc that has not experienced a significant reduction in disc height compared to the height of adjacent healthy discs. In another example, the cause of the lower back pain is non-radicular. In another example, the pain is associated with one or more of a herniated disc with up to 3 mm of protrusion, nerve proliferation into the disc, or disc inflammation. In one example, the nerve proliferation or inflammation is in the disc space, the nucleus pulposus of the disc, or the annulus fibrosus.

[0275] In one example, the pain is axial pain. The term "axial pain" is used in the context of this disclosure to refer to localized pain confined to a particular location or area (e.g., neck pain or leg pain). In one example, axial pain confirmed by MRI is due to nerve root compression.

[0276] Treatment target selection In one example, the methods of the present disclosure relate to methods of selecting a subject with an inflammatory disease, such as a T cell-mediated inflammatory disease, for treatment with a stem cell composition according to the present disclosure.

[0277] In another example, the method includes: i) selecting a subject with an inflammatory disease disclosed herein; and ii) administering to the subject a composition comprising MLPSCs, wherein the MLPSCs are culture-expanded in a cell culture medium containing non-fetal serum. In one example, the subject suffers from persistent inflammation characterized by circulating CRP levels. In one example, the subject suffers from GvHD. [Example]

[0278] Example 1, Serum Analysis Mesenchymal progenitor cell lineages or stem cell populations were culture-expanded in 5% FCS / 5% NBCS (serum A) or 10% fetal bovine serum (serum B). These MLPSCs were used in Examples 4-6.

[0279] Cytokine levels were assessed in 5% FCS / 5% NBCS (Serum A) and 10% fetal bovine serum (Serum B). To provide an external control, cytokine levels were also assessed in FBS from a different source (Serum C). In both cases, cytokine concentrations were assessed in pure serum.

[0280] Surprisingly, serum specimens containing neonatal bovine serum had higher levels of pro-inflammatory cytokines (Figure 1). Particularly noteworthy was the increase in pro-inflammatory cytokines known to bind to receptors expressed on the surface of MLPSCs, including interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukins. For example, the following was observed in serum preparations containing neonatal bovine serum compared to fetal bovine serum: At least a two-fold increase in IFNγ At least a 13-fold increase in TNFα, At least an 8-fold increase in IL-6, At least a two-fold increase in IL-8, At least a two-fold increase in IL-17A.

[0281] Example 2: MLPSC composition obtained using a culture medium containing fetal serum Alpha modifications of Eagle's minimum essential medium (MEM) with Earle's balanced salts, commonly referred to as Eagle's α-MEM, contain non-essential amino acids, sodium pyruvate, and additional vitamins. These modifications were first described for growing hybrid mouse and hamster cells (Stanners et al. 1971).

[0282] Eagle's αMEM medium, suitable for culturing primary stem cells, can be obtained from a variety of sources, including Life Technologies and Sigma.

[0283] Detailed methods for establishing primary stem cell cultures, including the necessary growth factors used in the exemplified process, are described in Gronthos and Simmons 1995.

[0284] MLPSCs were cultured in a medium containing 10% fetal bovine serum (serum B), L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10 -7 Eagle's α-MEM medium supplemented with 0.1 mM of phosphate (0.01 mM) and / or inorganic phosphate (3 mM) was used.

[0285] Example 3. Approved culture medium (non-fetal serum culture medium) For MLPSC culture medium containing neonatal serum, the serum component of Eagle's αMEM culture medium described in Example 2 was modified by adding 5% (v / v) neonatal serum (the differences between fetal serum medium and neonatal serum medium are shown in Table 5). The neonatal serum used was neonatal bovine serum (NBCS, serum A). NBCS meets the specifications of standard fetal bovine serum but is 100% bovine serum obtained from animals less than 20 days old.

[0286] NBCS was obtained from a commercial supplier and is marketed as an FCS substitute that is very similar to FCS, can be used interchangeably, and is expected to exert similar effects on cell lines. [Table 5]

[0287] Example 4: Expansion of MLPSCs in medium supplemented with neonatal serum promotes angiogenesis To characterize the novel MLPSC populations obtained by culturing and expanding them in medium supplemented with neonatal serum and / or proinflammatory cytokines (and to identify the mechanism behind the observed increase in therapeutic efficacy described in Example 5), the angiogenic potential of MPCs cultured under different conditions was assessed.

[0288] Cell culture: MPCs were cultured in 5% NBCS / 5% FCS (Serum A) or 10% FCS (Serum B) to generate MPC-conditioned medium. To control for donor variability, MPCs were obtained from the same donor and cultured under different conditions. In some experiments, MPCs belonging to the same donor but cultured during different manufacturing expansions are indicated by different "lot" numbers.

[0289] Conditioned medium was obtained by separating cells from conditioned medium. Briefly, cryopreserved MPCs were thawed and cultured at 50,000 / cm in either αMEM and 10% FBS or 5% NBCS / 5% FCS. 2 Cells were seeded at 1×. After incubation at 37°C and 5% CO for 72 hours, conditioned medium (CM) was collected. VEGF, SDF-1, and angiogenin levels in CM were measured using Luminex (R&D Systems). CM was concentrated using a 3k protein concentration filtration column (Amicon® Ultra-15) and reconstituted at 1× or 0.25× in assay medium.

[0290] Angiogenesis Efficacy Assay: In vitro angiogenesis was measured using a kinetic, quantitative 96-well co-culture angiogenesis model. Lentiviral-transduced human umbilical vein endothelial cells (HUVECs) expressing CytoLight Green (a GFP variant) were co-cultured with normal human dermal fibroblasts (NHDFs), seeded into 96-well plates, and simultaneously incubated and imaged using the IncuCyte® Live-Cell Analysis System. This system allowed for the determination of the angiogenesis potential of HUVECs (CytoLight Green). + Fluorescence identification of cells is now possible, and time-lapse image acquisition allows visualization of tube formation over time. The resulting images are analyzed using an integrated angiogenesis algorithm to measure network length, network area, and branch point formation, quantifying the stage and extent of angiogenesis throughout the assay.

[0291] Results: Conditioned medium from MPCs cultured in medium supplemented with neonatal bovine serum (NBCS) was found to increase angiogenesis. As shown in Figure 2, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS increased the network area (Figure 2A), network length (Figure 2B), and branch points (Figure 2C) in the coculture angiogenesis model. Furthermore, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS contained higher levels of VEGF compared to conditioned medium from cells cultured in 10% FCS (Figure 2A). Angiogenin levels were also increased in conditioned medium from MPCs cultured in 5% NBCS / 5% FCS compared to 10% FCS (Figure 3). Figure 4 shows further analysis of the levels of the angiogenic factors SDF-1α, VEGF, and Ang1 (ANGPT1) present in MPCs cultured in 10% FCS ("Serum B Medium") or 5% FCS / 5% NBCS ("Serum A Medium"). These data show that both VEGF and SDF-1α are elevated in neonatal serum medium-cultured MPCs.

[0292] Considering the data provided in Example 1, these data indicate that culture expansion of MLPSCs in medium supplemented with neonatal serum and / or pro-inflammatory cytokines results in a novel cell population with enhanced angiogenic potential. This enhanced potential can be characterized in various ways, as desired, e.g., to define the novel cell population we have identified, which can be, for example, The ability of conditioned medium obtained from MLPSCs to increase network area, network length, and / or branch points upon contact with HUVECs; · Including levels of angiogenin, VEGF, and / or SDF-1 in conditioned medium.

[0293] Example 5. MLPSCs cultured in medium supplemented with neonatal serum improve therapeutic outcomes in persistent inflammation High-risk heart failure with low ejection fraction (HFrEF), NYHA class II / III, is a clinical model of persistent inflammation. HFrEF patients are characterized by cardiac and systemic inflammation, as determined by elevated inflammatory biomarkers. MPCs cultured under different serum conditions were administered to HFrEF patients in the following clinical studies.

[0294] In patients with HFrEF, cardiac macrophages produce high levels of proinflammatory cytokines (IL-6, IL-1, and TNF-α), which induce endothelial dysfunction and cardiomyocyte apoptosis. Plasma concentrations of C-reactive protein (CRP), measured with a high-sensitivity CRP (hsCRP) assay, reflect acute-phase reactants produced in the liver in response to the high levels of proinflammatory cytokines (IL-6, IL-1, and TNF-α) produced by cardiac macrophages. Therefore, plasma concentrations of hsCRP (<2 mg / L vs. >2 mg / L) are representative systemic measures reflecting low or high levels of inflammation in the heart. In a subsequent study, patients with HFrEF were classified as having persistent inflammation if their hsCRP plasma concentrations were >2 mg / L.

[0295] Study Details: Eligible NYHA class II / III patients were enrolled in a double-blind, randomized, sham-controlled, parallel-group efficacy and safety study (DREAM HF-1) of allogeneic mesenchymal progenitor cells (Rexlemestrocel-L) for chronic heart failure due to left ventricular systolic dysfunction (ischemic or non-ischemic). Patients with HFrEF received (1) MPCs cultured in 10% fetal serum (n=37), (2) MPCs cultured in the presence of neonatal bovine serum (5% FCS / 5% NBCS, n=153), or (3) a sham control (i.e., no MPCs, n=241). As evidenced by serum analysis described in Example 1, cells cultured in medium supplemented with neonatal serum effectively cultivated a medium with elevated levels of proinflammatory cytokines. Cells were administered via a single intracardiac injection. Left ventricular systolic function in HFrEF was measured by echocardiographic (ECHO) parameters, including left ventricular ejection fraction (LVEF, %), left ventricular end-systolic volume (LVESV, mL), and left ventricular end-diastolic volume (LVEDV, mL), at baseline and 12 months after treatment. Plasma concentrations of CRP were measured to determine baseline levels of inflammation.

[0296] Results: MPCs cultured in the presence of neonatal bovine serum (5% FCS / 5% NBCS) were found to improve left ventricular (LV) systolic function in patients with HFrEF after 12 months. Specifically, neonatal serum-cultured MPCs significantly increased LVEF and decreased LVESV compared with sham controls (p = 0.0398 and 0.0426, respectively) (Figure 5).

[0297] HFrEF patients were then characterized based on whether their plasma hsCRP levels were <2 mg / L (normal baseline systemic inflammation) or >2 mg / L (elevated baseline systemic inflammation). Importantly, when HFrEF patients were classified according to their baseline systemic inflammation status (CRP >2), the effect of treatment with MPCs cultured in the presence of newborn calf serum (5% FCS / 5% NBCS) on LV systolic function recovery was more pronounced. In contrast, MPCs cultured in 10% FBS did not induce a significant effect (Figure 6). Compared to sham controls, newborn serum-cultured MPCs significantly increased LVEF% by a mean (LS mean) of 2.46 and reduced LVESV by a mean of 8.99 mL (p = 0.0033 and 0.0264, respectively). In contrast, MPCs cultured in 10% fetal serum or 5% / FCS / 5% NBCS showed improved LV systolic function in HFrEF patients without elevated baseline inflammation (HFrEF patients with CRP < 2) (Figure 7).

[0298] MPCs cultured in medium supplemented with neonatal serum have also been shown to reduce other cardiac-related outcomes in HFrEF patients with CRP > 2, including a 43% reduction in the risk of cardiovascular death (Figure 8) and a 54% reduction in the incidence of 3-point MACE (cardiovascular death / MI / stroke) (Figure 9). These data indicate that MPCs cultured in medium supplemented with neonatal serum improve therapeutic efficacy in the context of persistent inflammation.

[0299] Further analysis of clinical responses surprisingly revealed the importance of culture-expanding MLPSCs in medium supplemented with neonatal serum and / or pro-inflammatory cytokines during the final passage(s) before administration. MLPSCs cultured in medium supplemented with neonatal serum and / or pro-inflammatory cytokines during the final passage reduced the three-point MACE (MI, stroke, or CV death) in patients, regardless of whether the MLPSCs were culture-expanded with FBS during the initial passages. Remarkably, this reduction in three-point MACE was observed in all patients, regardless of their inflammatory status (Figure 10). Subgroup analysis of patients revealed that the reduction in three-point MACE was observed in patients with persistent inflammation (CRP > 2 mg / ml, Figure 10) and those at highest risk for end-stage cardiac events (defined as total cardiac event (TCE), cardiovascular death, heart transplant, or left ventricular assist device (LVAD) implantation) in this study. Patients at highest risk for TCE were defined as CRP > 2 mg / ml and NT-proBNP > 1000 ng / ml (Figure 11).

[0300] In contrast, an increase in adverse events (3-point MACE) was observed in all patients who received MLPSCs culture-expanded in medium without neonatal serum and / or proinflammatory cytokines at the final passage before treatment. The increase in 3-point MACE was observed regardless of whether the MLPSCs were cultured in medium supplemented with neonatal serum and / or proinflammatory cytokines during the initial passage.

[0301] Summary: MPCs expanded in medium supplemented with newborn bovine serum (NBCS) and / or pro-inflammatory cytokines: Left ventricular systolic dysfunction improved in patients with HFrEF with inflammation, as measured by the LS mean change in LVEF and LVESV at 12 months. A 43% reduction in cardiovascular death in high-risk patients with NYHA class II / III HFrEF and inflammation A 54% reduction in long-term 3-point MACE in high-risk NYHA class II / III patients with HFrEF and inflammation.

[0302] Taken together with the results of Examples 1 and 4, these human study data demonstrate that the addition of neonatal serum and / or pro-inflammatory cytokines to cell culture media results in cell populations with distinct functional properties, at least in terms of their ability to elicit a therapeutic effect in an inflammatory environment.

[0303] These human trial data also demonstrate the importance of adding neonatal serum and / or proinflammatory cytokines to the culture medium at the final passage(s), e.g., during culture expansion of intermediate cell populations into pharmaceutical products. Thus, our findings provide the basis for at least two approaches to providing MLPSC populations with improved therapeutic efficacy upon administration to patients. Expanding MLPSCs into an intermediate population in neonatal serum and / or pro-inflammatory cytokines to provide a cryopreserved intermediate population, and expanding the cryopreserved intermediate population in neonatal serum and / or pro-inflammatory cytokines to provide a pharmaceutical product. Expanding MLPSCs in fetal serum to an intermediate population to provide a cryopreserved intermediate population, and expanding the cryopreserved intermediate population in neonatal serum and / or pro-inflammatory cytokines to provide a drug product.

[0304] Without wishing to be bound by any particular theory, the above data suggest that culturing MPCs in medium supplemented with neonatal serum and / or proinflammatory cytokines enables them to respond more effectively to an inflammatory environment. Furthermore, when combined with the results described in Example 4, these data suggest a potential mechanism by which MPCs cultured in medium supplemented with neonatal serum and / or proinflammatory cytokines may exhibit improved therapeutic efficacy, i.e., enhanced angiogenesis and increased production of the proangiogenic growth factors VEGF, SDF-1α, and angiogenin. Thus, these data provide the basis for a method for selecting cells with sufficient efficacy for treating inflammatory diseases. Notably, the data demonstrate threshold levels exceeding 3.45 ng / mL of VEGF, 3000 ng / mL of SDF-1α, or 1114 pg / mL of angiogenin, indicating therapeutic efficacy and enhanced biological activity of MPCs at concentrations above these levels. For example, cells can be cultured according to the methods disclosed herein, and conditioned medium can be collected and measured in angiogenesis assays and / or for VEGF and angiogenin levels. Cells producing VEGF / angiogenin above a threshold are considered therapeutic and biologically active. Similarly, cells with an area of ​​>0.12 mm 2 / mm 2 , the network length is 5 mm 2 / mm 2 , and / or bifurcation >15 mm 2 / mm 2 Cells that produce conditioned medium that promotes angiogenesis, as determined by their ability to stimulate angiogenesis, are also believed to be therapeutically effective and biologically active in treating inflammatory diseases.

[0305] The above-mentioned angiogenic markers may serve as relevant criteria (in addition to clinical response criteria demonstrated in heart failure) for the characterization of novel MLPSCs generated by culture in cell culture medium containing neonatal serum and / or proinflammatory cytokines. Example 6: MLPSCs cultured in medium supplemented with neonatal serum are effective in treating GVHD Ten GvHD patients received 2x10 MPCs culture-expanded in NBCS containing pro-inflammatory cytokines (Examples 2 and 4) once a week. 6 The doses were administered intravenously. Patient responses are summarized in Table 6. Eighty percent of GvHD patients who received MPC cultured in NBCS responded to treatment. One patient achieved a complete response, seven patients achieved a partial response, and two patients died. [Table 6]

[0306] Taken together with at least the results of Example 6, our findings support the broad application of pre-licensed MLPSCs (e.g., MPCs cultured in the presence of non-fetal serum, particularly neonatal serum, and / or in the presence of pro-inflammatory cytokines) for the treatment of any disease or disorder characterized by elevated inflammation, particularly diseases characterized by persistent inflammation such as heart failure, or T-cell mediated diseases such as GvHD.

[0307] Example 7, Treatment of HFrEF patient population with approved MPLSCs. Left ventricular assist devices (LVADs) were implanted in patients with end-stage heart failure with reduced ejection fraction (HFrEF). HFrEF patients receiving LVADs were classified as having either ischemic or non-ischemic heart failure. LVAD patients in the treatment group received MPCs cultured in medium supplemented with neonatal serum or in medium containing 10% fetal bovine serum (FBS). Control patients did not receive stem cell therapy.

[0308] Inflammation was assessed based on serum IL-6 levels. Before LVAD implantation, IL-6 levels were similarly elevated in both ischemic and non-ischemic heart failure control patients (Figure 12). From postoperative day 1 to 30, a similar surgery-related increase in IL-6 was observed in both the control ischemic and non-ischemic groups (Figure 12). From postoperative day 30 to 365, IL-6 levels in the non-ischemic group decreased to below levels observed before LVAD implantation. However, in patients in the ischemic group, IL-6 levels remained similar to pre-LVAD implantation levels and were significantly higher than those in the non-ischemic group (Figure 12). Furthermore, the ischemic control group had a significantly higher risk of all-cause mortality within 12 months after LVAD implantation compared with the non-ischemic control group (Figure 13).

[0309] These data suggest that LVAD implantation reduces the inflammatory process associated with end-stage HFrEF in patients with nonischemic heart failure but not in patients with ischemic heart failure. Furthermore, LVAD patients with ischemic end-stage HFrEF represent a distinct subgroup of patients with persistent inflammation and a higher risk of all-cause mortality.

[0310] Figure 12B shows that not only did administration of MPCs cultured in medium supplemented with neonatal serum reduce IL-6 levels, but that IL-6 levels also decreased over time to levels consistent with non-ischemic controls.

[0311] Figures 14 and 15 show the overall mortality rate 12.5 months after LVAD implantation in patients receiving MPCs cultured in medium supplemented with neonatal bovine serum, MPCs cultured in medium containing 10% FBS, and control patients who did not receive cell therapy. Surprisingly, ischemic LVAD patients receiving MPCs cultured in medium supplemented with neonatal bovine serum had a significantly reduced overall mortality rate compared to both the control group and patients receiving unapproved MPCs (Figure 14B). Specifically, MPCs cultured in medium supplemented with neonatal bovine serum reduced overall mortality in ischemic LVAD patients by 83% compared to the ischemic control group.

[0312] Our findings provide further evidence (in addition to the studies in Example 5) that MPCs culture-expanded in medium supplemented with at least one pro-inflammatory cytokine and / or newborn bovine serum are particularly effective in treating inflammation, particularly diseases characterized by persistent inflammation such as heart failure.

[0313] Example 8. Culture medium analysis and summary of findings Based on the data described in Example 1, increased cytokine levels in the culture medium used to expand the MLPSC population were characterized by increased levels of one or more angiogenic markers (Example 4) and increased therapeutic efficacy in both heart failure (Example 5) and GvHD (Example 6) patients. The correlation between increased levels of pro-inflammatory cytokines in the culture medium and therapeutic efficacy in distinct inflammation-related disease indications suggests a preapproval effect for MLPSCs. This is further supported by the reduction in all-cause mortality observed in ischemic LVAD patients (i.e., patients characterized by persistently elevated levels of IL-6, Example 7).

[0314] Surprisingly, the MLPSCs described herein appear to be pre-qualified by culturing with proinflammatory cytokines, even though these cytokines are present at very low levels (e.g., pg / ml levels). This is surprising because it was not previously thought that the presence of proinflammatory cytokines, particularly TNF-α and IFN-γ, at pg / ml levels could have such dramatic effects (e.g., increased angiogenic potential, increased therapeutic efficacy in disease indications such as heart failure and GvHD). While not wishing to be bound by any particular theory, the data provided by the inventors surprisingly suggest synergistic and / or more than additive effects of cytokines in the context of MLPSC culture expansion. For example, the present data show that providing culture medium containing TNF-α and IFN-γ at concentrations <1 ng / ml can have profound effects on MLPSCs cultured and expanded therein, and these effects can be characterized based on the levels of various angiogenic markers and / or clinical efficacy in patients.

[0315] Our findings therefore represent a significant advance in the art by providing a method for preparing novel MLPSC populations that can lead to improved therapeutic effects, particularly in the context of inflammation. These results not only suggest that culture expansion in media supplemented with proinflammatory cytokines can provide improved MLPSC populations, but also demonstrate that culture expansion in media supplemented with neonatal serum can provide relevant proinflammatory cytokines. Our findings therefore support the standard for culture expansion of MLPSCs in serum- and serum-free media.

[0316] Example 9. Isolation and expansion of MLPSCs MLPSCs can be isolated using techniques such as STRO-3+ immunoselection of MPCs or density gradient separation of MSCs.

[0317] Typically, for bone marrow-derived MLPCs, bone marrow (BM) is collected from healthy adult volunteers (20–35 years old). Briefly, 40 ml of BM is aspirated from the posterior iliac crest and placed into a tube containing lithium heparin anticoagulant.

[0318] BMMNCs were prepared by density gradient separation using Lymphoprep (Nycomed Pharma, Oslo, Norway) as previously described (Zannettino et al. 1998). After centrifugation at 400 × g for 30 minutes at 4°C, the buffy layer was removed with a transfer pipette and washed three times with "HHF," which consisted of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0319] In conjunction with immunoselection, STRO-3+ (or TNAP+) cells are isolated by magnetic-activated cell sorting as previously described (Gronthos et al. 2003; Gronthos and Simmons 1995). Briefly, approximately 1-3 x 108 BMMNCs are incubated on ice for 20 minutes in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells are then incubated on ice for 1 hour with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer. The cells are then centrifuged at 400 x g and washed twice with HHF. A 1 / 50 dilution of goat anti-mouse biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer is added, and the cells are incubated on ice for 1 hour. Cells were washed twice with MACS buffer (Ca2+- and Mn2+-free PBS supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide) as described above and resuspended in a final volume of 0.9 ml of MACS buffer.

[0320] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice, resuspended in 0.5 ml of MACS buffer, and then loaded onto a mini-MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3 mAb (Deposited with the American Type Culture Collection (ATCC) on December 19, 2005, under accession number PTA-7282 - see International Publication No. WO 2006 / 108229). After the addition of an additional 1 ml of MACS buffer, the column was removed from the magnet and TNAP+ cells were isolated under positive pressure. A portion of the cells from each fraction could be stained with streptavidin-FITC and their purity assessed by flow cytometry.

[0321] Alternatively, MSCs can be grown from BMMNCs using plastic adherence techniques. For example, bone marrow mononuclear cells are isolated using Ficoll-Hypaque and plated into two T175 flasks with 50 ml of culture expansion medium containing alpha-modified MEM (αMEM) supplemented with gentamicin, glutamine (2 mM), and 10% (v / v) fetal bovine serum (FBS).

[0322] Cells are cultured at 37 °C, 5% CO for 2-3 days, at which point non-adherent cells are removed and the remaining adherent cells are continuously cultured until cell confluence reaches 70% or greater (7-10 days), after which the cells are trypsinized and replaced into six T175 flasks containing Expansion Medium.

[0323] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the present disclosure as set forth in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0324] This application claims priority to US 63 / 386,876, filed December 9, 2022, US 63 / 386,883, filed December 9, 2022, and US 63 / 507,009, filed June 8, 2023, the disclosures of which are incorporated herein by reference.

[0325] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0326] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure and is not to be construed as an admission that any or all of these matters form part of the prior art or were general knowledge in the art relevant to the present disclosure as existing prior to the priority date of each claim of this application.

Claims

1. 1. A method of treating an inflammatory disease in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), the MLPSCs having been culture-expanded in a cell culture medium comprising at least one pro-inflammatory cytokine.

2. The MLPSC comprises: IFN-γ and / or TNF-α, and / or The method of claim 1, wherein the cells are cultured and expanded in a medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

3. The method of claim 1 or 2, wherein the medium contains three or more pro-inflammatory cytokines.

4. 3. The method of claim 1, wherein the medium contains two or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

5. The method according to any one of claims 1 to 4, wherein the medium contains IL-6.

6. The method according to any one of claims 1 to 5, wherein the medium contains IL-8 and / or IL-17A.

7. The method according to any one of claims 1 to 6, wherein the medium contains IFN-γ and TNF-α.

8. The method of any one of claims 1 to 7, wherein the level of IFN-γ is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml.

9. The method of any one of claims 1 to 8, wherein the level of TNF-α is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

10. The method according to any one of claims 1 to 9, wherein the medium contains serum containing the pro-inflammatory cytokines.

11. The method of claim 10 , wherein the medium comprises non-fetal serum.

12. The method of claim 10 or 11, wherein the serum is newborn mammalian serum.

13. The method according to any one of claims 10 to 12, wherein the serum is newborn bovine serum (NBCS).

14. The method of any one of claims 10 to 13, wherein the serum is obtained within 21 days after birth.

15. 15. The method of any one of claims 10 to 14, wherein the serum is obtained between the day of birth and postnatal day 21, between the day of birth and postnatal day 14, between the day of birth and postnatal day 10, or between the day of birth and postnatal day 7.

16. The method of any one of claims 10 to 15, wherein the serum is obtained between the day of birth and 10 days after birth.

17. The medium is i. IFN-γ levels greater than 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; vi. MCP-1 levels greater than 3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. A level of IP-10 greater than 500 pg / ml.

18. The method according to any one of claims 10 to 17, wherein the culture medium comprises at least 5% (v / v) newborn bovine serum.

19. The method of any one of claims 2 to 9 or 17, wherein the medium is serum-free and / or xeno-free.

20. The method of any one of claims 10 to 18, wherein the culture medium comprises 5% non-fetal serum.

21. The method according to any one of claims 10 to 18, wherein the culture medium comprises 5% non-fetal serum and 5% fetal serum.

22. 22. The method of claim 20 or 21, wherein the non-fetal serum is NBCS.

23. The method of any one of claims 1 to 22, wherein the inflammatory disease is mediated by T cell activation.

24. 24. The method of claim 23, wherein the inflammatory disease is refractory to steroid immunosuppressants and / or ruxolitinib.

25. The method of any one of claims 1 to 22, wherein the inflammatory disorder is hyperinflammation.

26. 26. The method of claim 25, wherein the hyperinflammation is caused by a coronavirus infection.

27. 27. The method of claim 26, wherein the coronavirus is SARS-CoV, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), or COVID-19.

28. The method of any one of claims 1 to 22, wherein the inflammatory disease is multisystem inflammatory syndrome (MIS) or acute respiratory distress syndrome (ARDS).

29. 23. The method of any one of claims 1 to 22, wherein the inflammatory disease is selected from the group consisting of graft-versus-host disease (GvHD), pruritus, dermatitis, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's disease, myasthenia gravis, type I or type II diabetes, diabetic nephropathy, asthma, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, seborrheic dermatitis, Sjogren's syndrome, keratoconjunctivitis, uveitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, inflammatory diseases of the joints, skin, or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease (COPD), interstitial lung disease, interstitial nephritis, and chronic active hepatitis.

30. 23. The method of any one of claims 1 to 22, wherein the inflammatory disease is diabetes or a diabetes-related condition or symptom selected from the group consisting of wound healing abnormalities, renal failure, blindness, neuropathy, nephropathy, retinopathy, inflammation, impotence, or non-alcoholic steatohepatitis (NASH).

31. The method of any one of claims 1 to 22, wherein the inflammatory disease is type II diabetes, rheumatoid arthritis, or diabetic nephropathy.

32. The method of any one of claims 1 to 22, wherein the inflammatory disorder is pain.

33. 33. The method of claim 32, wherein the pain is chronic pain.

34. 33. The method of claim 32, wherein the pain is lower back pain, preferably the lower back pain is associated with intervertebral disc degeneration.

35. The method of any one of claims 1 to 34, wherein the MLPCs are administered systemically.

36. 36. The method of any one of claims 1 to 35, wherein the MLPSCs are administered intravenously.

37. The method of any one of claims 1 to 36, wherein the MLPSCs are administered in multiple doses.

38. 38. The method of any one of claims 1 to 37, wherein the mesenchymal precursor or stem cells are mesenchymal precursor cells (MPCs).

39. 39. The method of claim 38, wherein the MPCs are isolated from bone mononuclear cells using an anti-STRO-3 antibody prior to culture expansion.

40. 37. The method of any one of claims 1 to 36, wherein the mesenchymal progenitor or stem cells are mesenchymal stem cells (MSCs).

41. 41. The method of any one of claims 1 to 40, wherein the MLPSCs are allogeneic.

42. 42. The method of any one of claims 1 to 41, wherein the cells are cryopreserved prior to administration.

43. 43. The method of any one of claims 1 to 42, wherein the cells are cryopreserved before being culture-expanded in cell culture medium containing non-fetal serum.

44. 1 x 10 ７ ~2 x 10 ８ 44. The method of any one of claims 1 to 43, comprising administering cells of

45. 45. The method of any one of claims 1 to 44, wherein the population of MLPSCs is provided in a composition comprising Plasmalyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA).

46. 46. ​​The method of claim 45, wherein the composition comprises a Plasmalyte A (70%), DMSO (10%), HSA (25%) solution, wherein the HSA solution comprises 5% HSA and 15% buffer.

47. The composition is 6.68 x 10 ６ 47. The method of claim 45 or claim 46, wherein the total amount of viable cells in the blood comprises more than 1000 cells / mL.

48. The method of any one of claims 1 to 47, wherein the MLPSCs are culture-expanded in a cell factory.

49. The cell factory is inoculated with CO before seeding the cells. ２ 49. The method of claim 48, wherein the antibody is passively primed with

50. The method comprises: - the levels of VEGF, angiogenin, and / or SDF-1α expressed by said MLPSCs under culture conditions, and / or - selecting for therapeutic use culture-expanded MLPSCs having increased levels of one or more angiogenic markers selected from the group consisting of levels of endothelial network formation, endothelial network length, and endothelial branch length measured after treating an endothelial cell population with conditioned medium obtained from said MLPSCs.

51. 51. The method of claim 50, wherein an increased level of one or more angiogenic markers is determined compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

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