Treatment of immune disorders

Abstract

To increase production of anti-inflammatory cells in human subjects using genetically unmodified stem cells that express Ang1 at high levels without the need for transfection of the cells with a nucleic acid expressing Ang1.SOLUTION: The present disclosure relates to stem cells which express high levels of Angiopoietin-1 (Ang1), and uses thereof in inhibiting M1-type macrophage production and treating inflammatory disease such as diabetes.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to stem cells that express high levels of angiopoietin-1 (Ang1) and their use in treating inflammatory diseases such as diabetes by inhibiting the release of TNF-α and / or IL-6.

Background Art

[0002] Angiopoietin is part of a family of vascular growth factors involved in initial and postnatal angiogenesis. Ang1 promotes the migration of several non-endothelial cells such as endothelial cells and smooth muscle cells. Ang1 also induces the sprouting and reorganization of endothelial cells within the renal tubules. Ang1 exerts a strong anti-inflammatory effect on endothelial cells, suppresses vascular endothelial growth factor (VEGF) that induces upregulation of E-selectin, ICAM-1 and VCAM-1, and inhibits leukocyte adhesion and extravasation in response to VEGF and TNF-α (Kim et al., Circ Res., 89(6), 477-479, 2001).

[0003] Current treatment for the majority of type I diabetic patients is based on the regular subcutaneous injection of a mixture of rapid-acting and long-acting insulin formulations. Suspensions of soluble insulin particles of different sizes are administered to provide intermediate and long-acting components with a more sustained action profile to achieve a constant basal level of the hormone (Heine et al., Br Med J (Clin Res Ed) 290:204-205, 1985).

[0004] The drawback of this current treatment is that sustained-release formulations generally do not produce a smooth background level of insulin, resulting in either hyperglycemia or hypoglycemia. Hyperglycemia is a problem in that it can lead to further complications in diabetic patients. For example, chronic hyperglycemia leads to severe microvascular (retinopathy and nephropathy), macrovascular (stroke, myocardial infarction) and neurological complications. These severe complications can be prevented by normalizing blood glucose levels.

[0005] In recent years, stem cell-based technologies have emerged as a potential approach for treating diabetes. However, in addition to issues related to fundamental autoimmune diseases that may require lifelong immune regulation, these technologies have not yet been clearly established as viable treatment options.

[0006] Therefore, in patients suffering from diabetes and / or its related conditions or symptoms who require new treatment options, the therapeutic need remains unmet. The citation or identification of any document in this application does not admit that the document is available as prior art for the present disclosure.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0008] The inventors have found that genetically unmodified stem cells that express Ang1 at high levels can increase the production of anti-inflammatory cells in human subjects without the need to transfect the cells with nucleic acids that express Ang1.

[0009] The inventors have also found that it is possible to reduce TNF-alpha levels, reduce IL-6 levels and / or increase IL-10 levels in human subjects. These findings suggest that such genetically unmodified stem cells that express high levels of Ang1 may be suitable for treating inflammatory diseases such as diabetes and its related conditions and symptoms.

[0010] In fact, the inventors have found that it is possible to reduce HbA1c, reduce fasting insulin levels and / or increase adiponectin levels in human subjects suffering from diabetes, without the need to transfect cells with nucleic acids expressing Ang1, using genetically unmodified stem cells that express Ang1 at high levels.

[0011] In other examples, the inventors have also shown that it is possible to improve the symptoms of rheumatoid arthritis and diabetic nephropathy in human subjects, without the need to transfect cells with nucleic acids expressing Ang1, using genetically unmodified stem cells that express Ang1 at high levels.

[0012] Accordingly, in one example, the present disclosure provides a method of increasing the production and / or function of anti-inflammatory cells in a subject in need thereof, the method comprising administering to the subject a composition comprising genetically unmodified stem cells, wherein the genetically unmodified stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 6 cells.

[0013] In one example, the anti-inflammatory cells are Th2 cells, TReg cells or M2 macrophages.

[0014] In another example, the method increases the number of M2 macrophages in the subject.

[0015] In another example, the method increases the number of M2 macrophages in the subject to at least 10% of the total mononuclear cell population.

[0016] In another example, the method increases the number of M2 macrophages in the subject to at least 20% of the total mononuclear cell population.

[0017] In another example, the method increases the number of M2 macrophages in the subject to at least 40% of the total mononuclear cell population.

[0018] In another embodiment, the method increases the number of M2 macrophages in a subject to at least 80% of the total mononuclear cell population.

[0019] In another embodiment, the method increases the number of M2 macrophages in a subject to at least 90% of the total mononuclear cell population.

[0020] In another embodiment, the M2 macrophages are CD14+CD16+.

[0021] In another embodiment, the M2 macrophages are CD14+CD16+CD163+.

[0022] In another embodiment, the M2 macrophages are CD14+CD16+CD206+.

[0023] In another embodiment, the M2 macrophages are CD14+CD16+CD163+CD206+.

[0024] In another embodiment, the M2 macrophages are CD14++CD16+.

[0025] In another embodiment, the M2 macrophages are CD14++CD16+CD163+.

[0026] In another embodiment, the M2 macrophages are CD14++CD16+CD206+.

[0027] In another embodiment, the M2 macrophages are CD14++CD16+CD163+CD206+.

[0028] In another embodiment, the method promotes the polarization of macrophages from the M1 to the M2 phenotype.

[0029] In another embodiment, the method promotes the differentiation of pro-inflammatory helper T cells into Th2 cells or TReg cells.

[0030] In one embodiment, the inflammation-inducing helper T cells are Th17 cells.

[0031] In one embodiment, an increase in the production and / or function of anti-inflammatory cells in a subject results in · a decrease in IL-6 levels in the subject; · a decrease in TNF-alpha levels in the subject; and / or · an increase in IL-10 levels in the subject.

[0032] In one embodiment, the method increases the level of anti-inflammatory cytokines in a subject.

[0033] In one embodiment, the method increases the level of IL-10 in a subject.

[0034] In one embodiment, the method decreases the level of inflammation-inducing cytokines in a subject.

[0035] In one embodiment, the method decreases the level of any one of IL-6, TNF-alpha, and / or IL-17 in a subject.

[0036] In one embodiment, the method also inhibits the production and / or function of inflammation-inducing cells.

[0037] In one embodiment, the inflammation-inducing cells are Th17 cells or M1 macrophages.

[0038] In another embodiment, the present disclosure provides a method of inhibiting M1 macrophage polarization.

[0039] In another embodiment, the present disclosure provides a method of promoting the polarization of macrophages from the M1 to the M2 phenotype.

[0040] In another embodiment, the present disclosure provides a method of inhibiting cytokine release from M1 macrophages.

[0041] In another embodiment, the present disclosure provides a method in which the M1 macrophage-derived cytokines to be inhibited are TNF-alpha and / or IL-6.

[0042] In another embodiment, the present disclosure provides a method for treating an inflammatory disease, the method comprising administering to a subject a composition comprising genetically unmodified stem cells, wherein the genetically unmodified stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 6 cells.

[0043] In another embodiment, the inflammatory disease is diabetes or a diabetes-related condition or symptom selected from the group consisting of abnormal wound healing, symptoms of a heart attack, symptoms of a stroke, symptoms of peripheral vascular disease, amputation, symptoms of kidney disease, kidney failure, blindness, neuropathy, nephrosis, retinopathy, inflammation, impotence, or non-alcoholic steatohepatitis (NASH).

[0044] For example, the present disclosure provides a method for treating rheumatoid arthritis.

[0045] For example, the present disclosure provides a method for treating diabetic retinopathy.

[0046] In another embodiment, the method of the present disclosure comprises administering to a subject a composition comprising genetically unmodified stem cells, wherein the genetically unmodified stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 6 cells. In another embodiment, the stem cells express Ang1 in an amount of at least 0.5 μg / 10 6 cells. In another embodiment, the stem cells express Ang1 in an amount of at least 0.7 μg / 10 6 cells. In another embodiment, the stem cells express Ang1 in an amount of at least 1 μg / 10 6 cells.

[0047] In another embodiment, the stem cells are about 0.1 μg / 10 6Express VEGF in an amount less than the cells. In another embodiment, the stem cells are about 0.05 μg / 10 6 Express VEGF in an amount less than the cells. In another embodiment, the stem cells are about 0.04 μg / 10 6 Express VEGF in an amount less than the cells. In another embodiment, the stem cells are about 0.03 μg / 10 6 Express VEGF in an amount less than the cells. In another embodiment, the stem cells are about 0.02 μg / 10 6 Express VEGF in an amount less than the cells. In another embodiment, the stem cells are about 0.01 μg / 10 6 Express VEGF in an amount less than the cells.

[0048] In another embodiment, the stem cells express Ang1:VEGF at a ratio of at least about 2:1. In another embodiment, the stem cells express Ang1:VEGF at a ratio of at least about 10:1. In another embodiment, the stem cells express Ang1:VEGF at a ratio of at least about 20:1. In another embodiment, the stem cells express Ang1:VEGF at a ratio of at least about 30:1. In another embodiment, the stem cells express Ang1:VEGF at a ratio of at least about 50:1.

[0049] In another embodiment, the stem cells are mesenchymal stem cells. In another embodiment, the stem cells are mesenchymal progenitor cells. In another embodiment, the stem cells are induced pluripotent stem cells (iPS cells).

[0050] In another embodiment, the composition further comprises an acceptable pharmaceutical carrier.

[0051] In another embodiment, the composition is produced by culturing genetically unmodified stem cells according to the in vitro method described below.

[0052] In one embodiment, the stem cells can be obtained from any mammal. For example, the stem cells may be derived from primates, cows, sheep, horses, dogs, cats, or goats. In another embodiment, the stem cells are human stem cells.

[0053] In another embodiment, the inflammatory disease is type II diabetes.

[0054] In one embodiment, a method of treating type II diabetes may include administering to a subject from about 0.1×10 6 to about 3×10 6 stem cells per kg of body weight.

[0055] In one embodiment, a method of treating type II diabetes may include administering to a subject from about 0.3×10 6 to about 2×10 6 stem cells per kg of body weight.

[0056] In one embodiment, a method of treating type II diabetes may include administering to a subject from about 1×10 6 to about 2×10 6 stem cells per kg of body weight.

[0057] In one embodiment, a method of treating type II diabetes may include administering to a subject about 2×10 6 stem cells per kg of body weight.

[0058] In one embodiment, treatment of diabetes or a diabetes-related condition or symptom in a subject is indicated by any one of the following. · A decrease in HbA1c value (percentage of total hemoglobin); · A decrease in fasting insulin level; · A decrease in IL-6 level; · A decrease in TNF-α level; and / or · An increase in adiponectin level

[0059] In one embodiment, the subject has type II diabetes and the subject's glucose level is not well controlled.

[0060] In one embodiment, the subject's glucose level is not well controlled by metformin.

[0061] In one embodiment, the subject has a baseline HbA1c value of greater than 7.5%.

[0062] In one embodiment, the subject has a baseline HbA1c value of 8% or more.

[0063] In one embodiment, the inflammatory disease is rheumatoid arthritis.

[0064] In one embodiment, a method of treating rheumatoid arthritis may include administering to the subject from about 0.5×10 6 per kg to about 3.0×10 6 stem cells.

[0065] In one embodiment, a method of treating rheumatoid arthritis may include administering to the subject from about 1.0×10 6 per kg to about 2.0×10 6 stem cells.

[0066] In one embodiment, that rheumatoid arthritis has been treated is indicated by any one of the following. ·ACR20; ·ACR50 ·ACR70 ·A decrease in IL-6 levels; and / or ·A decrease in disease activity score

[0067] In another embodiment, the inflammatory disease is diabetic neuropathy.

[0068] In one embodiment, a method of treating rheumatoid arthritis may include administering to the subject from about 1.0×10 8 per kg to about 4.0×10 8 stem cells.

[0069] In one embodiment, a method of treating rheumatoid arthritis may include administering to the subject from about 1.5×10 8 per kg to about 3.0×10 8 stem cells.

[0070] In one embodiment, the treatment of diabetic nephropathy is indicated by any one of the following. · Inhibition of the decrease in eGFR and / or mGFR; · Improvement of eGFR and / or mGFR; and / or · Decrease in IL-6 levels

[0071] In one embodiment, the baseline eGFR of the subject is greater than about 35 ml / min / 1.73m 2 .

[0072] In one embodiment, the baseline eGFR of the subject is greater than 30 ml / min / 1.73m 2 .

[0073] In one embodiment, the baseline eGFR of the subject is greater than 28 ml / min / 1.73m 2 .

[0074] In one embodiment, the baseline eGFR of the subject is greater than 25 ml / min / 1.73m 2 .

[0075] In one embodiment, the stem cells are administered systemically.

[0076] In one embodiment, the stem cells are administered intravenously.

[0077] In another embodiment, the present disclosure relates to the use of a composition comprising genetically unmodified stem cells in the manufacture of a medicament for treating an inflammatory disease, wherein the genetically unmodified stem cells express high levels of angiopoietin-1 (Ang1).

[0078] In another embodiment, the present disclosure relates to a composition comprising genetically unmodified stem cells for use in the treatment of an inflammatory disease, wherein the genetically unmodified stem cells express high levels of angiopoietin-1 (Ang1).

[0079] In another embodiment, the present disclosure relates to a composition comprising genetically unmodified stem cells that, when used to treat an inflammatory disease, express high levels of angiopoietin-1 (Ang1). BRIEF DESCRIPTION OF THE DRAWINGS

[0080]

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Mode for Carrying Out the Invention

[0081] General Techniques and Definitions Throughout this specification, unless otherwise specified or required by context, references to a single step, composition of matter, group of steps, or group of compositions of matter shall be construed to include pluralities (i.e., one or more) of such steps, compositions of matter, groups of steps, or groups of compositions of matter.

[0082] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It should be understood that the disclosure includes all such variations and modifications. The disclosure includes all steps, features, compositions, and compounds individually or collectively referenced or shown herein, and any combinations and all combinations or any two or more of the foregoing steps or features.

[0083] The present disclosure is not limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure described herein.

[0084] Unless otherwise specified, any example disclosed herein should be construed as applicable to any other example.

[0085] Unless otherwise defined, all technical and scientific terms used herein should be construed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0086] Unless otherwise specified, the stem cells, cell culture, and immunological techniques utilized in this disclosure are standard procedures well-known to those skilled in the art. Such techniques are described and explained through references such 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 Harbour Laboratory Press (1989), T.A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (eds), and F.M. Ausubel et al. (eds), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (eds) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (eds) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0087] The term "and / or" is to be understood as meaning either "X and Y" or "X or Y", for example, "X and / or Y", and is to be interpreted as explicitly supporting both meanings or either meaning.

[0088] As used herein, the term "about" means + / - 10% of the specified value, more preferably + / - 5%, unless otherwise stated.

[0089] The definition of volume % (v / v%) is [(volume of solute) / (volume of solution)] × 100%. Volume % is proportional to the volume of the solution. For example, supplementing a cell culture medium with 5% (v / v) FCS means that there is approximately 5 ml of FCS per 100 ml of the cell culture medium.

[0090] Throughout this specification, it will be understood that the word "comprise", or variations such as "comprises" or "comprising", does not mean to exclude any other element, integer or step, or group of elements, integers or steps, although it includes the stated element, integer or step, or group of elements, integers or steps.

[0091] Stem cell As used herein, the term "stem cell" means a self-renewing cell that can give rise to phenotypically and genotypically identical daughter and at least one other final cell type (e.g., a terminally differentiated cell). The term "stem cell" includes totipotent, pluripotent and multipotent cells, as well as progenitor cells and / or precursors derived from their differentiation. Stem cells can be adult or embryonic stem cells.

[0092] As used herein, the term "totipotent cell" or "totipotential cell" means a cell capable of forming a complete embryo (e.g., a blastocyst).

[0093] As used herein, the term "pluripotent cell" or "pluripotential cell" means a cell having complete differentiation potentiality, i.e., the ability to grow into any of approximately 260 cell types of the mammalian body. Pluripotent cells can self-renew and can remain quiescent or stationary within a tissue.

[0094] "Multipotential cell" or "multipotent cell" means a cell that can give rise to multiple mature cell types. As used herein, this expression includes adult progenitor cells and the multipotent progeny of these cells. Unlike pluripotent cells, multipotential cells do not have the ability to form all cell types.

[0095] As used herein, the term "mesenchymal lineage progenitor or stem cell" means a cell that can differentiate into mesenchymal cell types. For example, mesenchymal lineage progenitor cells and mesenchymal progenitor cells can differentiate into bone tissue, cartilage tissue, muscle tissue and adipocytes, and fibrous connective tissue.

[0096] In one example, the stem cells that express high levels of Ang1 are STRO-1+ mesenchymal progenitor cells.

[0097] STRO-1+ multipotent cells are cells found in bone marrow, blood, dental pulp, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestine, lung, lymph nodes, thymus, bone, ligament, tendon, skeletal muscle, dermis, and periosteum. Thus, STRO-1+ multipotent cells can differentiate into a number of cell types including, but not limited to, adipose tissue, bone tissue, cartilage tissue, elastic tissue and fibrous connective tissue. The specific lineage determination and the differentiation pathways into which these cells enter are determined by mechanical influences and / or various influences from endogenous bioactive factors such as growth factors, cytokines and / or local microenvironmental conditions established by the host tissue. In one embodiment, STRO-1+ multipotent cells are non-hematopoietic progenitor cells that divide to produce daughter cells that are either stem cells or progenitor cells at a timing that will irreversibly differentiate to produce phenotypic cells.

[0098] In one embodiment, STRO-1+ cells are enriched from a sample obtained from a subject, such as a subject to be treated or a related or unrelated subject (regardless of whether the same or different species). The terms "enriched", "enrichment" or variations thereof are used herein to describe a cell population in which the ratio of one particular cell type or the ratio of a number of particular cell types is increased compared to an untreated collection of cells (e.g., cells in their native environment). In one embodiment, the enriched population of STRO-1+ cells comprises at least about 0.1% or 0.5% or 1% or 2% or 5% or 10% or 15% or 20% or 25% or 30% or 50% or 75% or 85% or 95% or 99% STRO-1+ cells. In this regard, the term "enriched population of STRO-1+ cells" will be construed as explicitly supporting the term "population of cells comprising X% STRO-1+ cells" (where X% is the percentage described herein). STRO-1+ cells, in some instances, can form clonogenic colonies, such as CFU-F (fibroblasts), or a subset thereof (e.g., 50% or 60% or 70% or 80% or 90% or 95%) can have this activity.

[0099] In one embodiment, stem cells that express high levels of Ang1 are enriched from a cell preparation that contains STRO-1+ cells in a selectable format. In this regard, the term "selectable format" will be 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 can be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., MPC) 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 (and STRO-1 bright(It may also be the case). Therefore, showing that a cell is STRO-1+ does not mean that the cell has been selected by the expression of STRO-1. In one example, cells are selected based on the expression of at least STRO-3. For example, STRO-3+ (TNAP+), etc. In one example, cells are selected based on the expression of at least STRO-4. For example, STRO-4+, etc.

[0100] References to the selection of cells or populations thereof do not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. According to that, in some examples, these terms provide support for selection from either a tissue containing STRO-1+ cells (e.g., MPC), or a vascularized tissue, or a tissue containing pericytes (e.g., STRO-1+ pericytes), or from any one or more of the tissues described herein.

[0101] In one example, stem cells that express high levels of Ang1 express one or more markers that are individually or collectively selected from the group consisting of STRO-1+, TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, CC9, or any combination thereof.

[0102] "Individually" means that the present disclosure encompasses the listed markers or groups of markers individually, and that the appended claims may define such markers or groups of markers separately and divisibly from each other, even if the individual markers or groups of markers are not individually listed herein.

[0103] "Collectively" means that the present disclosure encompasses any number or combination of the recited markers or group of peptides, and that the appended claims may define such combination or sub - combinations individually and separately from any other combination of markers or group of markers, even if the number or combination of such markers or group of markers is not specifically recited herein.

[0104] In one embodiment, STRO - 1+ cells are STRO - 1 bright (syn.STRO - 1 bri ). In one embodiment, STRO - 1 bri cells are preferentially enriched compared to STRO - 1 dim or STRO - 1 intermediate cells.

[0105] In one embodiment, STRO - 1 bright cells are further one or more of TNAP +, VCAM - 1 +, THY - 1 +, STRO - 2 +, STRO - 4+(HSP - 90β) and / or CD146 +. For example, the cells are selected for one or more of the aforementioned markers and / or exhibit expression of one or more of these markers. In this regard, cells that have been shown to express a marker need not be specifically tested; conversely, previously enriched or isolated cells can be tested and then used. Also, isolated or enriched cells can reasonably be assumed to express the same markers.

[0106] In one embodiment, STRO - 1 bright is isolated by immunoselection. In one embodiment, STRO - 1 brightCells are isolated by the immune selection of cells expressing TNAP. As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses liver isoform (LAP), bone isoform (BAP), and kidney isoform (KAP). In one embodiment, TNAP is BAP. In one embodiment, TNAP as used herein means a molecule capable of binding to the STRO-3 antibody produced by a hybridoma cell line based on the deposit accession number PTA-7282 under the terms of the Budapest Treaty, deposited with the ATCC on December 19, 2005.

[0107] In one embodiment, the mesenchymal progenitor cells or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ mesenchymal stem cells (e.g., remestemcel-L).

[0108] In one embodiment, the mesenchymal progenitor cells are perivascular mesenchymal progenitor cells as defined in WO2004 / 85630. For example, the mesenchymal progenitor cells are cells that express markers of perivascular cells. For example, STRO-1+ or STRO-1 bright and / or 3G5+ etc. In one embodiment, the cells are the progeny of cells isolated from vascular tissue or their organs or parts, whether present or previously present.

[0109] When referring to cells that are "positive" for a given marker, depending on the degree to which the marker is present on the cell surface, it can be either at a low (lo or dim) or high (bright, bri) level of that marker, and the term relates to the fluorescence intensity or other markers used in the cell sorting process. The distinction between lo (or dim or dull) and bri will be understood in the context of the markers used for the particular cell population being sorted. When referring to cells that are "negative" for a given marker, it does not mean that the marker is not expressed at all by the cell. This term means that the marker is expressed at a relatively very low level by the cell, and when detectably labeled, generates a very low signal or is not detected above the background level. For example, the level is detected using an isotype control antibody.

[0110] As used herein, the term "bright" means a marker on the cell surface that generates a relatively high signal when detectably labeled. Without wishing to be limited by theory, it is proposed that "bright" cells express more of the target marker protein (e.g., the antigen recognized by STRO-1) than other cells in the sample. For example, STRO-1 bri cells are brighter than non-bright cells (STRO-1 dull / dim ) when labeled with FITC-conjugated STRO-1 antibody as measured by fluorescence-activated cell sorting (FACS) analysis, generating a larger fluorescence signal. In one example, "bright" cells constitute at least about 0.1% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In other examples, "bright" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the most brightly labeled bone marrow mononuclear cells contained in the starting sample. In one example, STRO-1 brightThe cells have STRO-1 surface expression that is 2 log magnitudes higher compared to the "background", i.e., cells that are STRO-1-. When compared, STRO-1 dim and / or STRO-1 intermediate cells have STRO-1 surface expression that is less than 2 log magnitudes, typically less than about 1 log or less than "background".

[0111] In one example, a significant proportion of STRO-1+ multipotent cells can differentiate into at least two different germ cell lineages. Non-limiting examples of lineages into which multipotent cells can be committed include: osteoprogenitor cells; hepatocyte progenitor cells that are pluripotent with respect to cholangiocytes and hepatocytes; neural restricted cells that can give rise to glial cell progenitors that progress into oligodendrocytes and astrocytes; neural progenitor cells that progress into neurons; progenitor cells of cardiomyocytes and cardiomyocytes, glucose-responsive insulin-secreting pancreatic β cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, and the following progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth muscle cells and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle, skeletal muscle, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes and oligodendrocytes.

[0112] In another example, STRO-1+ cells cannot give rise to hematopoietic cells upon culturing.

[0113] In one embodiment, the stem cells described herein are mesenchymal stem cells. The mesenchymal stem cells (MSCs) may be a homogeneous composition or a mixed cell population enriched in MSCs. A homogeneous mesenchymal stem cell composition may be obtained by culturing adherent bone marrow or periosteal cells, or the mesenchymal stem cells may be identified by specific cell surface markers identified using a proprietary monoclonal antibody. Methods for obtaining a cell population enriched in mesenchymal stem cells are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of mesenchymal stem cells include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0114] Recognition, selection, and purification of stem cells bearing the above-described cell surface markers can be accomplished by a number of different methods. For example, an agent that binds to the marker of interest is applied, followed by separation of those cells that exhibit either high-level binding, or low-level binding, or no binding.

[0115] For example, the binding agent can include an antibody such as a monoclonal antibody or an antibody-based molecule.

[0116] Antibodies and other binding molecules can be used in a variety of techniques to select and purify stem cells that express a particular cell surface marker.

[0117] Techniques for selection and purification may include, but are not limited to, magnetic separation using antibody-coated magnetic beads, affinity chromatography, and "panning" with antibodies bound to a solid matrix, fluorescence-activated cell sorting (FACS).

[0118] Stem cells that express a specific marker and a high level of Ang1 may be selected or purified from a cell population via positive immunoselection. For example, mesenchymal progenitor cells can be isolated and enriched from a cell population based on cell surface expression of the STRO-1 antibody (see, for example, Gronthos and Simmons 1995).

[0119] According to the present disclosure, isolated stem cells can be expanded in vitro by culturing. As will be understood by those skilled in the art, stem cells can be expanded in vitro by cryopreservation, thawing, and subsequent culturing. In one example, the stem cells are seeded in a growth medium and allowed to adhere to a culture vessel overnight at 37 °C and 20% O2. Thereafter, the growth medium is replaced and the cells are cultured for an additional 68-72 hours at 37 °C and 5% O2.

[0120] In one example, the isolated stem cells are seeded at 50,000 cells / cm 2 in a growth medium supplemented with serum and allowed to adhere to a culture vessel overnight at 37 °C and 20% O2. Thereafter, the growth medium is replaced with a cartilage basal medium (CBM; Lonza, Walkersville, MD) supplemented with 0.5% bovine serum albumin (BSA), and the cells are cultured for an additional 68-72 hours at 37 °C and 5% O2.

[0121] Various other methods of primary stem cell culture are well known in the art. For example, primary stem cell culture can be performed using the method described in Gronthos and Simmons 1995.

[0122] Cultured stem cells are phenotypically different from cells in vivo. For example, they can express CD44.

[0123] In one embodiment, cultured stem cells are biologically different from cells in vivo and have a faster regeneration rate.

[0124] Cultured stem cells may be cryopreserved prior to administration to a subject. For example, stem cells that express high levels of Ang1 are cryopreserved prior to administration to a subject.

[0125] Cells that have not been genetically modified As used herein, the term "not genetically modified" refers to cells that have not been modified by transfection with a nucleic acid that expresses or encodes Ang1. By way of clarification to avoid misunderstanding, stem cells transfected with a nucleic acid encoding Ang1 in the context of the present disclosure are considered to be genetically modified. In the context of the present disclosure, "not genetically modified" cells express Ang1 endogenously to some extent.

[0126] Expression of Ang1 and / or VEGF Stem cells that express high levels of Ang1 are not genetically modified and express Ang1 at a level of at least 0.1 μg / 10 6 cells. However, in various embodiments, stem cells that express high levels of Ang1 express Ang1 at a level of at least 0.2 μg / 10 6 cells, 0.3 μg / 10 6 cells, 0.4 μg / 10 6 cells, 0.5 μg / 10 6 cells, 0.6 μg / 10 6 cells, 0.7 μg / 10 6 cells, 0.8 μg / 10 6 cells, 0.9 μg / 10 6 cells, 1 μg / 10 6 cells, 1.1 μg / 10 6 cells, 1.2 μg / 10 6 cells, 1.3 μg / 10 6 cells, 1.4 μg / 10 6 cells, 1.5 μg / 10 6 cells and are expected to be able to express Ang1 at these levels.

[0127] In one example, stem cells that express high levels of Ang1 express Ang1 at a level of at least 0.2 μg / 10 6 cells to about 1.5 μg / 10 6 cells.

[0128] In one example, stem cells that express high levels of Ang1 express Ang1 at a level of at least 0.3 μg / 10 6 cells to about 1.4 μg / 10 6Express Ang1 in terms of the amount of cells.

[0129] In one embodiment, stem cells that express high levels of Ang1 express Ang1 in an amount of at least 0.4 μg / 10 6 cells to about 1.3 μg / 10 6 cells.

[0130] In one embodiment, stem cells that express high levels of Ang1 express Ang1 in an amount of at least 0.5 μg / 10 6 cells to about 1.2 μg / 10 6 cells.

[0131] In one embodiment, stem cells that express high levels of Ang1 express Ang1 in an amount of at least 0.55 μg / 10 6 cells to about 1.1 μg / 10 6 cells.

[0132] In one embodiment, stem cells that express high levels of Ang1 express Ang1 in an amount of at least 0.6 μg / 10 6 cells to about 1.0 μg / 10 6 cells.

[0133] In one embodiment, stem cells that express high levels of Ang1 express Ang1 in an amount of at least 0.65 μg / 10 6 cells to about 0.9 μg / 10 6 cells.

[0134] In one embodiment, stem cells that express high levels of Ang1 express Ang1 in an amount of at least 0.7 μg / 10 6 cells to about 0.8 μg / 10 6 cells.

[0135] In another aspect, genetically unmodified stem cells that express high levels of Ang1 express VEGF in an amount less than about 0.01 μg / 10 6 cells.

[0136] In another aspect, genetically unmodified stem cells that express high levels of Ang1 express VEGF in an amount of less than about 0.05 μg / 10 6 cells. However, in various embodiments, stem cells that express high levels of Ang1 express VEGF in an amount of about 0.05 μg / 10 6 cells, 0.04 μg / 10 6 cells, 0.03 μg / 10 6 cells, 0.02 μg / 10 6 cells, 0.01 μg / 10 6 cells, 0.009 μg / 10 6 cells, 0.008 μg / 10 6 cells, 0.007 μg / 10 6 cells, 0.006 μg / 10 6 cells, 0.005 μg / 10 6 cells, 0.004 μg / 10 6 cells, 0.003 μg / 10 6 cells, 0.002 μg / 10 6 cells, 0.001 μg / 10 6 cells or less. It is contemplated that stem cells that express high levels of Ang1 may express VEGF in an amount of less than about 0.05 μg / 10

[0137] In one example, stem cells that express high levels of Ang1 express VEGF in an amount of at least 0.001 μg / 10 6 cells to about 0.1 μg / 10 6 cells.

[0138] In one example, stem cells that express high levels of Ang1 express VEGF in an amount of at least 0.0025 μg / 10 6 cells to about 0.09 μg / 10 6 cells.

[0139] In one example, stem cells that express high levels of Ang1 express VEGF in an amount of at least 0.0075 μg / 10 6 cells to about 0.08 μg / 10 6 cells.

[0140] In one example, stem cells that express high levels of Ang1 express VEGF in an amount of at least 0.01 μg / 10 6Cells ~ 0.07 μg / 10 6 Express VEGF in an amount of cells.

[0141] In one example, stem cells that express high levels of Ang1 are at least 0.02 μg / 10 6 Cells ~ about 0.06 μg / 10 6 Express VEGF in an amount of cells.

[0142] In one example, stem cells that express high levels of Ang1 are at least 0.02 μg / 10 6 Cells ~ about 0.05 μg / 10 6 Express VEGF in an amount of cells.

[0143] The amount of Ang1 and / or VEGF in the cells expressed in the composition or in the culture of the stem cells may be determined by methods well known to those skilled in the art. Such methods include, but are not limited to, quantitative assays such as quantitative ELISA assays. However, it should be understood that the scope of the present disclosure is not limited to any particular method for determining the amount or level of Ang1 or VEGF expressed in stem cells that express high levels of Ang1.

[0144] In one embodiment, the levels of Ang1 or VEGF expressed by culturing the composition or stem cells are determined by an ELISA assay. In such an assay, cell lysates from the culturing of stem cells are added to the wells of an ELISA plate. The wells may be coated with a primary antibody, either monoclonal or polyclonal (plural possible), against Ang1 or VEGF. Then, after washing the wells, the wells are contacted with a secondary antibody, either monoclonal or polyclonal (plural possible), against the primary antibody. The secondary antibody is conjugated to a suitable enzyme, such as horseradish peroxidase, for example. The wells may then be incubated and washed after a certain period of incubation. The wells are then contacted with a substrate suitable for the enzyme conjugated to the secondary antibody, such as one or more chromogens. Chromogens that can be used include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After adding the substrate(s), the wells are incubated for an appropriate period of time. Once the incubation is complete, a "stop" solution is added to the wells to stop the reaction of the substrate(s) and the enzyme. The optical density (OD) of the sample is then measured. The optical density of the sample correlates with the optical density of a sample containing a known amount of Ang1 or VEGF to determine the amount of Ang1 or VEGF expressed by the culturing of the stem cells being tested.

[0145] In another aspect, genetically unmodified stem cells that express high levels of Ang1 express Ang1:VEGF at a ratio of at least about 2:1. However, in various embodiments, it is contemplated that stem cells that express high levels of Ang1 may express Ang1:VEGF at ratios of at least about 10:1, 15:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 50:1.

[0146] Methods for determining the expression ratio of Ang1:VEGF will be apparent to those skilled in the art. In an example of a method for determining the ratio of Ang1 and VEGF expression, as described above, the expression levels of Ang1 and VEGF are quantified via quantitative ELISA. In such an example, after quantifying the levels of Ang1 and VEGF, the ratio based on the quantified levels of Ang1 and VEGF can be expressed as follows. (Level of Ang1 / Level of VEGF) = Ratio of Ang1:VEGF.

[0147] Cell composition In one embodiment of the present disclosure, the stem cells are administered in the form of a composition. In one embodiment, such a composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0148] The terms "carrier" and "excipient" refer to a composition of substances conventionally used in the art to facilitate storage, administration, and / or the biological activity of the active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition, Mac Publishing Company (1980)). The carrier can also reduce any undesirable side effects of the active compound. A suitable carrier is, for example, stable and cannot react with other components in the carrier, for example. In one embodiment, the carrier does not cause significant local or systemic adverse effects to the recipient at the dosage and concentration used for treatment.

[0149] Suitable carriers for the present disclosure include those conventionally used. For example, water, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffer solutions, hyaluronic acid, and glycols are exemplary liquid carriers, especially for solutions (in the case of isotonicity). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerin, propylene glycol, water, ethanol, and the like.

[0150] In another embodiment, the carrier is, for example, a culture composition in which cells are growing or suspended. For example, the culture composition does not induce any harmful effects in the subject to which it is administered.

[0151] Exemplary carriers and excipients do not adversely affect the viability and / or the ability of the cells to reduce, prevent or delay metabolic syndrome and / or obesity.

[0152] In one embodiment, the carrier or excipient provides a buffering action, for example, to maintain cells and / or soluble factors at an appropriate pH, thereby exerting a biological activity. For example, the carrier or excipient is phosphate buffered saline (PBS). PBS is an attractive carrier or excipient because, when the compositions of the present disclosure may be manufactured as a liquid for direct application (e.g., by injection) into the bloodstream, or into or around or adjacent to a tissue, PBS interacts minimally with the cells and factors and allows for rapid release of the cells and factors.

[0153] The stem cells and / or their progeny cells can also be incorporated into or encapsulated within a scaffold that degrades into a product that is recipient-compatible and not harmful to the recipient. These scaffolds provide support and protection for the cells transplanted into the recipient subject. Natural and / or synthetic biodegradable scaffolds are examples of such scaffolds.

[0154] A variety of different scaffolds may be successfully used in the practice of the present invention. Exemplary scaffolds include, but are not limited to, biological, degradable scaffolds. Natural biodegradable scaffolds include scaffolds of collagen, fibronectin, and laminin. Suitable synthetic materials for scaffolds for cell transplantation should be able to support extensive cell growth and cell function. Such scaffolds may be absorbable. Suitable scaffolds include polyglycolic acid scaffolds, such as those described in Vacanti et al. J.P. Surg. 23:3-9 1988; Cima et al. Biotechnol. Bioeng. 38:145 1991; Vacanti et al. Plast. Reconstr. Surg. 88:753-9 1991, or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

[0155] In another embodiment, the cells may be administered in a gel scaffold (such as Gelfoam from Upjohn Company).

[0156] The cell compositions described herein may be administered alone or as a mixture with other cells. Different types of cells may be mixed with the compositions of the present disclosure immediately before or shortly before administration, or may be co-cultured together for a certain period of time before administration.

[0157] In one embodiment, the composition contains an effective amount, or a therapeutically or prophylactically effective amount, of cells. For example, the composition contains from about 1×10 5 stem cells / kg with a high Ang1 level to about 1×10 7 stem cells / kg with a high Ang1 level, or from about 1×10 6 stem cells / kg with a high Ang1 level to about 5×10 6 stem cells / kg.

[0158] The exact dose of stem cells to be administered is determined according to various factors including, but not limited to, the age, weight and gender of the patient, the disease or disorder being treated, and its degree and severity.

[0159] In one embodiment, a low dose of cells is administered to a subject. Exemplary dosages are from about 0.1×10 4 to about 0.5×10 6 cells per kg, for example, from about 0.1×10 5 to about 0.5×10 6 cells per kg, for example, from about 0.5×10 5 to about 0.5×10 6 cells per kg, for example, from about 0.1×10 6 to about 0.5×10 6 cells per kg, for example, from about 0.2×10 6 cells or 0.3×10 6 cells or 0.4×10 6 cells per kg.

[0160] For example, dosages of about 0.1×10 6 , about 0.2×10 6 , about 0.3×10 6 , about 0.4×10 6 , about 0.5×10 6 cells per kg can be administered to a subject.

[0161] In other embodiments, dosages of about 0.6×10 6 , about 0.7×10 6 , about 0.8×10 6 , about 0.9×10 6 , about 1.0×10 6 , about 1.1×10 6 , about 1.2×10 6 , about 1.3×10 6 , about 1.4×10 6 cells per kg can be administered to a subject.

[0162] In one embodiment, a high dose of cells is administered to a subject. Exemplary dosages include at least about 1.5×10 6 cells / kg. For example, high dosages are from about 1.5×10 6 to about 6×10 6 cells / kg, for example, about 1.5×10 6~ about 5×10 6 cells / kg, for example, about 1.5×10 6 ~ about 4×10 6 cells / kg, for example, about 1.5×10 6 ~ about 3×10 6 cells / kg. For example, a high dose contains about 1.5×10 6 cells or about 2×10 6 cells / kg. For example, a high dose contains about 1.5×10 6 cells / kg. For example, a high dose contains about 2×10 6 cells / kg. For example, a high dose contains about 3×10 6 cells / kg.

[0163] In other embodiments, about 1.5×10 6 , about 1.6×10 6 , about 1.7×10 6 , about 1.8×10 6 , about 1.9×10 6 , about 2.0×10 6 , about 2.1×10 6 , about 2.2×10 6 , about 2.3×10 6 , about 2.4×10 6 , about 2.5×10 6 , about 2.6×10 6 , about 2.7×10 6 , about 2.8×10 6 , about 2.9×10 6 , about 3.0×10 6 , about 3.1×10 6 , about 3.2×10 6 , about 3.3×10 6 , about 3.4×10 6 , about 3.5×10 6 , about 3.6×10 6 , about 3.7×10 6 , about 3.8×10 6 , about 3.9×10 6 , about 4.0×10 6 cells are administered to the subject.

[0164] In other embodiments, about 1.0×10 8, about 1.1×10 8 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , about 2.5×10 8 , about 2.6×10 8 , about 2.7×10 8 , about 2.8×10 8 , about 2.9×10 8 cells, about 3.0×10 8 , about 3.1×10 8 , about 3.2×10 8 , about 3.3×10 8 , about 3.4×10 8 , about 3.5×10 8 , about 3.6×10 8 , about 3.7×10 8 , about 3.8×10 8 , about 3.9×10 8 , about 4.0×10 8 cells are administered to the subject.

[0165] Mesenchymal progenitor or stem cells can account for at least about 5% of the cell population of the composition. In other embodiments, mesenchymal progenitor or stem cells can account for 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% of the cell population of the composition.

[0166] Stem cells expressing CD44 can account for at least about 5% of the cell population of the composition. In other embodiments, stem cells expressing CD44 can account for 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%, at least about 99%, at least about 100% of the cell population of the composition.

[0167] In some embodiments, the cells are contained within a chamber where it is impossible for the cells to exit into the subject's circulation, but factors secreted by the cells can enter the circulation. In such a method, soluble factors may be administered to the subject by enabling the cells to secrete the factors into the subject's circulation. Such chambers may be implanted simultaneously at a site of the subject to increase the local level of the soluble factor.

[0168] Stem cells can be administered systemically, for example, by intravenous, intra-arterial, or intraperitoneal administration.

[0169] Mesenchymal progenitor or stem cells can also be administered by nasal, intramuscular, intra-articular, or intracardiac administration.

[0170] For example, mesenchymal progenitor or stem cells can be administered directly to a painful or swollen joint.

[0171] In another embodiment, stem cells expressing high levels of Ang1 are administered by intracoronary injection. For example, mesenchymal progenitor or stem cells can be administered to the left anterior descending (LAD) artery.

[0172] In another embodiment, stem cells expressing high levels of Ang1 are administered by intrarenal injection.

[0173] In one embodiment, stem cells that express high levels of Ang1 can be administered in a single dose.

[0174] In some embodiments, stem cells that express high levels of Ang1 can be administered in multiple doses. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 doses.

[0175] Compositions containing stem cells that express high levels of Ang1 may be cryopreserved. Cryopreservation of stem cells can be performed using slow cooling methods or "rapid" freezing procedures well known in the art. In one embodiment, the cryopreservation method maintains a similar phenotype, cell surface markers, and growth rate of cryopreserved cells compared to non-frozen cells.

[0176] Cryopreserved compositions may contain a cryopreservation solution. The pH of the cryopreservation solution is typically about 6.5 - 8.

[0177] In one embodiment, the pH of the cryopreservation solution is about 7.4.

[0178] The cryopreservation solution may contain, for example, sterile, non-pyrogenic, isotonic Plasmalyte A®. 100 mL of Plasmalyte A® contains 526 mg of sodium chloride, USP (NaCl), 502 mg of sodium gluconate (C6H 11 NaO7), 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O), 37 mg of potassium chloride, USP (KCI), and 30 mg of magnesium chloride, USP (MgCl2·6H2O). It does not contain antibacterial agents. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5 - 8.0).

[0179] To promote freezing, cryoprotectants such as dimethyl sulfoxide (DMSO), for example, are usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic for cells and patients, chemically inert, non-antigenic, provide a high survival rate after thawing, and enable transplantation without washing. 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 the cryopreservation solution.

[0180] The cryopreservation solution can contain one or more of DMSO, hydroxyethyl starch, human serum components, and other protein extenders. In one example, the cryopreservation solution contains about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further contain one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0181] The cryopreserved composition can also be thawed and administered directly to a subject. Alternatively, the cryopreserved composition can be thawed and resuspended in an alternative solution prior to administration of the mesenchymal precursor or stem cells.

[0182] Stem cells expressing high levels of Ang1 are administered to an animal in an amount effective to treat a disease or disorder of the animal. The animal can be a mammal, and the mammal can be a primate including humans and non-human primates.

[0183] In one example, stem cells expressing high levels of Ang1 are administered to a human.

[0184] In one example, stem cells expressing high levels of Ang1 are administered to a human.

[0185] In one example, stem cells expressing high levels of Ang1 are administered to a subject suffering from diabetes.

[0186] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from type II diabetes.

[0187] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from type II diabetes and having a baseline HbA1c value of greater than about 7%. For example, stem cells expressing high levels of Ang1 can be administered to a subject suffering from type II diabetes and having a baseline HbA1c value of about 7.1%, about 7.2%, about 7.3%, about 7.4%, about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%, about 8.7%, about 8.8%, about 8.9%, about 9.0% or greater.

[0188] In one embodiment, stem cells expressing high levels of Ang1 can be administered to a subject suffering from type II diabetes and having a baseline HbA1c value of about 8.0% or greater.

[0189] Methods for determining the HbA1c value (percentage of total hemoglobin) will be apparent to those skilled in the art. Examples of methods used to determine the HbA1c value (percentage of total hemoglobin) include high performance liquid chromatography (HPLC) or immunoassay. Those skilled in the art will also recognize that the HbA1c value can be expressed in other values, such as mmol / mol.

[0190] In one embodiment, the HbA1c value of the subject is determined by HPLC.

[0191] In one embodiment, the HbA1c value of the subject is determined by immunoassay.

[0192] In one embodiment, stem cells expressing high levels of Ang1 are administered to a patient suffering from type II diabetes, and the glucose level of the subject is not well controlled.

[0193] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from type II diabetes, and the subject's glucose levels are not adequately controlled by metformin or metformin and another oral therapeutic agent.

[0194] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from chronic kidney disease.

[0195] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from diabetic nephropathy.

[0196] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from type II diabetes and chronic kidney disease.

[0197] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from type II diabetes and diabetic nephropathy.

[0198] Estimated glomerular filtration rate (eGFR) is used to examine and detect initial kidney damage and monitor kidney status.

[0199] In one embodiment, stem cells expressing high levels of Ang1 are administered to subjects with a baseline eGFR greater than about 35 ml / min / 1.73m 2 , about 34 ml / min / 1.73m 2 , about 44 ml / min / 1.73m 2 , about 32 ml / min / 1.73m 2 , about 31 ml / min / 1.73m 2 , about 30 ml / min / 1.73m 2 , about 29 ml / min / 1.73m 2 , about 28 ml / min / 1.73m 2 , about 27 ml / min / 1.73m 2 , about 26 ml / min / 1.73m 2 , about 25 ml / min / 1.73m 2 .

[0200] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject having a renal function stage of 3A, 3B, 4, or 5.

[0201] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject having a renal function stage of 3B.

[0202] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject.

[0203] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject having a baseline eGFR of 30 ml / min / 1.73m 2 or greater.

[0204] Methods for estimating GFR will be apparent to those skilled in the art and are exemplified below. eGFR can be calculated using creatinine and / or cystatin C levels.

[0205] For example, eGFR can be calculated using the following MDRD equation. GFR (mL / min / 1.73m 2 ) = 175 × (S cr ) -1.154 × (age) -0.203 × (0.742 for females) × (1.212 for African Americans)

[0206] In other embodiments, eGFR may be calculated using the CKD-EPI equation (Levey et al., Ann Intern. Med. 150(9), 604-12, 2009). GFR = 141 × min(S cr / κ, 1) α × max(S cr / κ, 1) -1.209 × 0.993 年齢 × 1.018 [for females] × 1.159 [for blacks] Wherein, S cr is serum creatinine (mg / dL), κ is 0.7 for females and 0.9 for males, α is -0.329 for females and -0.411 for males, min represents the minimum value of S cr / κ or 1, max represents the maximum value of S cr / κ or 1.

[0207] Also, eGFR can be continuously calculated and monitored over time to determine whether eGFR is decreasing or improving. By comparing eGFR between a control group and a treatment group, it can be specified whether the decrease in eGFR is inhibited in the treatment group.

[0208] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from arthritis.

[0209] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from rheumatoid arthritis.

[0210] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from rheumatoid arthritis classified as an inadequate anti-TNFα responder.

[0211] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from rheumatoid arthritis in whom biological therapy for rheumatoid arthritis has not been successful.

[0212] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from rheumatoid arthritis in whom two biological therapies for rheumatoid arthritis have not been successful.

[0213] In one embodiment, stem cells expressing high levels of Ang1 are administered to a subject suffering from rheumatoid arthritis in whom three biological therapies for rheumatoid arthritis have not been successful.

[0214] Inhibition of TNF-alpha, IL-6, IL-17 TNF-alpha, IL-6, and IL-17 are chemical messengers known as cytokines. These molecules are released from cells in response to various signals. In the context of the present invention, the term "inhibit" or "inhibiting" refers to a measurable reduction or suppression of the level of a substance such as a protein (e.g., a cytokine) or a process (e.g., cell differentiation or cell polarization).

[0215] Thus, in one embodiment, administration of a cell composition comprising stem cells that express high levels of Ang1 is envisioned to reduce the measurable levels of TNF-alpha, IL-17, and / or IL-6 in a subject. In this embodiment, the release of TNF-alpha, IL-17, and / or IL-6 from the cells is inhibited.

[0216] In one embodiment, the present disclosure relates to a method of monitoring a subject's response to administration of stem cells that express high levels of Ang1. In this embodiment, after administration of the stem cells that express high levels of Ang1, the levels of inflammatory and / or anti-inflammatory markers such as cytokines can be monitored over a period of time.

[0217] In one embodiment, the present disclosure relates to a method of monitoring a subject's response after administration of stem cells that express high levels of Ang1, the method comprising evaluating the levels of inflammatory markers and / or anti-inflammatory marker levels in a sample such as a whole blood sample obtained from a subject administered with stem cells that express high levels of Ang1, and determining whether the subject has responded to the administration of the stem cells that express high levels of Ang1 based on the inflammatory and / or anti-inflammatory marker levels.

[0218] In one embodiment, an increase in anti-inflammatory marker levels and / or a decrease in inflammatory marker levels indicates that the subject has responded to the administration of the stem cells.

[0219] In one embodiment, the inflammatory marker and / or anti-inflammatory marker is a cell or cell population.

[0220] In one embodiment, an increase in the number of anti-inflammatory cells and / or a decrease in the number of inflammatory cells indicates that the subject has responded to the administration of stem cells.

[0221] In one embodiment, the anti-inflammatory cells are Th2 cells, Treg cells, and / or M2 macrophages.

[0222] In one embodiment, the inflammatory cells are Th17 cells and / or M1 macrophages.

[0223] A variety of assays that can be used to determine whether the number of inflammatory cells has decreased and / or the number of anti-inflammatory cells has increased are available to those skilled in the art.

[0224] For example, techniques based on flow cytometry, such as fluorescence-activated cell sorting (FACS), can be used to evaluate the expression of cell surface markers on cell populations isolated from whole blood samples.

[0225] In one example, CD14+ monocytes can be purified from whole blood samples obtained from subjects administered stem cells that express high levels of Ang1. The monocytes can be evaluated for the expression of CD16, CD163, and CD206 to identify the ratio of M1 and M2 macrophages. Multiple samples can be evaluated over time to determine whether the number of M1 macrophages has decreased or is decreasing and / or whether the level of M2 macrophages has increased or is increasing. In one embodiment, the number of M1 and / or M2 macrophages is evaluated in comparison to the number of M1 and / or M2 macrophages in a sample obtained from the subject before administration of the stem cells (e.g., a baseline or pre-treatment reference sample).

[0226] In another embodiment, the inflammatory marker and / or anti-inflammatory marker to be evaluated is a cytokine.

[0227] In one embodiment, the inflammatory marker is TNF-alpha, IL-17, and / or IL-6.

[0228] In one embodiment, the anti-inflammatory marker is IL-10.

[0229] A variety of assays capable of determining whether TNF-alpha, IL-17, and / or IL-6 levels have decreased or IL-10 levels have increased are available to those skilled in the art. In one example, the enriched levels of TNF-alpha, IL-17, IL-10, and / or IL-6 can be identified using spectrophotometric techniques such as the Immulite chemiluminescent immunoassay. In another example, the enriched levels of TNF-alpha, IL-17, IL-10, and / or IL-6 can be measured using a Luminex platform using a commercially available kit (Millipore).

[0230] In one embodiment, administration of a cell composition comprising stem cells that express high levels of Ang1 inhibits the release of TNF-alpha and / or IL-6 by macrophages. A variety of assays capable of determining whether the release of TNF-alpha and / or IL-6 from macrophages has decreased are available to those skilled in the art. For example, macrophages are cultured in vitro and exposed to either a composition comprising stem cells that express high levels of Ang1 or a suitable control. After a period of time, TNF-alpha and / or IL-6 release can then be evaluated using the exemplary methods described above. The TNF-alpha and / or IL-6 levels in the cells exposed to the cell composition comprising stem cells that express high levels of Ang1 can then be compared to the TNF-alpha and / or IL-6 levels in control cells to determine whether the TNF-alpha and / or IL-6 levels have decreased.

[0231] Regarding macrophages, two different polarization states have been identified: the classically activated (M1) macrophage phenotype (i.e., "M1 macrophages") and the alternatively activated (M2) macrophage phenotype (i.e., "M2 macrophages") (Gordon and Taylor., Nat. Rev. Immunol. 5:953 - 964, 2005; Mantovani et al., Trends Immunol. 23:549 - 555, 2002). M1 macrophages have a "pro - inflammatory" cytokine profile (e.g., TNF - alpha, IL - 6, IL - 1 - beta, IL - 12, IL - 23). In contrast, M2 macrophages have an "anti - inflammatory" cytokine profile (e.g., IL - 10). In one embodiment, administration of a cell composition containing stem cells that express high levels of Ang1 is envisioned to inhibit the release of cytokines by M1 macrophages. In another embodiment, administration of a cell composition containing stem cells that express high levels of Ang1 is envisioned to inhibit the release of TNF - alpha and / or IL - 6 by M1 macrophages. Various assays are available to those skilled in the art to determine whether the release of TNF - alpha, IL - 6, and / or other cytokines by M1 macrophages has decreased. For example, M1 macrophages are used in the in vitro assay described above. In this example, CD14+ mononuclear cells can be purified from a whole blood sample by immunoselection.

[0232] Increased production and / or function of anti - inflammatory cells In one embodiment, the present disclosure relates to a method of increasing the production and / or function of anti - inflammatory cells in a subject by administering stem cells that express high levels of Ang1.

[0233] The term "anti-inflammatory cell" is used in the context of the present disclosure to refer to cells that induce or mediate an anti-inflammatory response in a subject. "Anti-inflammatory cells" can act directly on a cell population or target to induce an anti-inflammatory response. Alternatively, anti-inflammatory cells encompassed by the present disclosure can express or secrete factors such as cytokines that act on a specific cell population or target to induce an anti-inflammatory response.

[0234] Examples of anti-inflammatory cells include Th2 cells, Treg cells, and M2 macrophages.

[0235] In one embodiment, the present disclosure relates to a method of increasing the number of Th2 cells, Tregs, and / or M2 macrophages in a subject by administering stem cells that express high levels of Ang1.

[0236] In one embodiment, the present disclosure relates to a method of increasing the number of M2 macrophages in a subject by administering stem cells that express high levels of Ang1.

[0237] In one embodiment, the method of the present disclosure increases the number of M2 macrophages in a subject to at least about 6%, at least about 7%, at least about 8%, at least about 9% of the total mononuclear cell population.

[0238] In one embodiment, the method of the present disclosure increases the number of M2 macrophages in a subject to at least about 10% of the total mononuclear cell population.

[0239] In one embodiment, the method of the present disclosure increases the number of M2 macrophages in a subject to 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% of the total mononuclear cell population.

[0240] One of ordinary skill in the art could readily identify the percentage of M2 macrophages relative to a total mononuclear cell population of interest using a variety of methods known in the art. For example, M2 macrophages can be identified based on the expression of other markers, such as CD14 and CD16, as well as CD163 and CD206.

[0241] In this example, a whole blood sample can be obtained from a subject, and cells can be immunoselected by FACS based on the expression of CD14. The CD14+ cells can then be evaluated for the expression of CD16, CD163, and CD206. The ratio of CD14+CD16+CD163+CD206+ can be calculated relative to the total CD14+ cell population in the sample.

[0242] In one example, an increase in the production and / or function of anti-inflammatory cells in a subject results in · a decrease in IL-6 levels in the subject; · a decrease in TNF-alpha levels in the subject; and / or · an increase in IL-10 levels in the subject.

[0243] In another example, the methods of the disclosure are methods of promoting the polarization of macrophages from an M1 to an M2 phenotype.

[0244] For example, the disclosure provides a method of promoting the polarization of M1 macrophages to M2 macrophages in a subject in need thereof, the method comprising administering to the subject a composition comprising non-genetically modified stem cells, the non-genetically modified stem cells expressing angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 6 cells.

[0245] In this example, the production or increase in the number of CD14+CD16+ cells, CD14++CD16+ cells, CD14+CD16+CD163+ cells, CD14++CD16+CD163+ cells, CD14+CD16+CD206+ cells, CD14++CD16+CD206+ cells, CD14+CD16+CD163+CD206+ cells, CD14++CD16+CD163+CD206+ cells, CD14+CD163+ cells, CD14++CD163+ cells, CD14+CD206+ cells, CD14++CD206+ cells, CD14+CD163+206+ cells, CD14++CD163+CD206+ cells, the decrease in the IL-6 level, the decrease in the TNF-alpha level, and / or the increase in the IL-10 level may indicate that the polarization of M1 macrophages to M2 macrophages has been promoted.

[0246] Conversely, in another example, the present disclosure provides a method of inhibiting the polarization of M2 macrophages to M1 macrophages.

[0247] In another example, the present disclosure provides a method of inhibiting the production and / or function of M1 macrophages in a subject in need thereof, the method comprising administering to the subject a composition comprising genetically unmodified stem cells that express high levels of angiopoietin-1 (Ang1).

[0248] Treatment method The present disclosure relates to a method of treating an inflammatory disease.

[0249] As used herein, the term "inflammatory disease" includes, but is not limited to, pruritus, skin inflammation, psoriasis, multiple sclerosis, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, type I or 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 joints, skin or muscles, acute or chronic idiopathic inflammatory arthritis, myositis, demyelinating diseases, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis and chronic active hepatitis.

[0250] In one embodiment, the inflammatory disease treated by the methods of the present disclosure is diabetes. In another embodiment, the inflammatory disease is a condition or symptom associated with diabetes. In this embodiment, the symptoms that can be treated include abnormal wound healing, symptoms associated with heart attacks such as chest pain, symptoms associated with stroke, peripheral vascular disease, amputations, kidney disease, renal failure, blindness, neuropathy, inflammation, sexual dysfunction or non-alcoholic steatohepatitis (NASH).

[0251] For example, the inflammatory disease treated by the methods of the present disclosure is rheumatoid arthritis.

[0252] For example, the inflammatory disease treated by the methods of the present disclosure is diabetic retinopathy.

[0253] In one embodiment, the present disclosure relates to a method for treating diabetes.

[0254] In one embodiment, the present disclosure relates to a method for treating type II diabetes.

[0255] In one embodiment, the present disclosure relates to a method for treating diabetic nephropathy.

[0256] In one embodiment, the present disclosure relates to a method for treating rheumatoid arthritis.

[0257] As used herein, the terms "treat", "treatment" or "treating" should be understood to mean administering a therapeutically effective amount of cells. In the context of the present disclosure, the term "therapeutically effective amount of cells" refers to an amount of cells effective to prevent, ameliorate or treat an inflammatory disease or disorder. Such an effective amount will generally result in an improvement in the signs, symptoms and / or other indicators of the inflammatory disease or disorder. For example, in the case of diabetes, an effective amount of cells can result in a decrease in HbA1c levels, a decrease in cytokine levels such as IL-6 and / or TNF-α, a decrease in fasting insulin and / or an increase in adiponectin levels.

[0258] For example, in the case of rheumatoid arthritis, an effective amount of cells can result in the achievement of ACR20, ACR50 and / or ACR70, a decrease in cytokine levels such as IL-6 and / or a decrease in disease activity score.

[0259] For example, in the case of diabetic nephropathy, an effective amount of cells can result in inhibition of the decrease in eGFR or mGFR, improvement of eGFR or mGFR and / or a decrease in cytokine levels such as IL-6.

[0260] In the context of diabetes or its related conditions or symptoms, various conventional clinical assays that can be used to identify a decrease in HbA1c levels, a decrease in fasting insulin and / or an increase in adiponectin levels are available to those skilled in the art. For example, generally, after obtaining a blood sample from a subject, it is subjected to an immunoassay to detect HbA1c, insulin and adiponectin levels.

[0261] Cell culture method In one embodiment, a method of generating stem cells that express high levels of Ang1 includes culturing a population of stem cells in a cell culture medium that contains a short-acting L-ascorbic acid derivative but does not contain a substantial amount of a long-acting L-ascorbic acid derivative and / or is supplemented with less than 10% (v / v) fetal bovine serum.

[0262] The terms "media" or "medium" as used in connection with cell culture include the components of the environment surrounding the cells. The medium is assumed to contribute to and / or provide conditions sufficient to induce the expression of Ang1. The medium can be a solid, liquid, gas, or a mixture of phases and substances. The medium can include a liquid growth medium and a liquid medium that does not maintain cell growth. The medium also includes gelatinous media such as agar, agarose, gelatin, and collagen matrices. An exemplary gas medium includes the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed. The term "medium" also refers to a substance intended for use in cell culture even if it has not yet come into contact with cells.

[0263] The culture medium used in the method for producing stem cells that express high levels of Ang1 can be prepared using, as a basal medium, a medium used for culturing stem cells. The basal medium includes, for example, Eagle's Minimum Essential (MEM) medium, α-modified MEM medium, and mixed culture media thereof, and is not particularly limited as long as it can be used for culturing stem cells.

[0264] Furthermore, the culture medium can include components such as fatty acids or lipids, vitamins, growth factors, cytokines, antioxidants, buffers, and inorganic salts.

[0265] Cell culture media can include all essential amino acids and may include non-essential amino acids. Generally, amino acids are classified into essential amino acids (threonine, methionine, valine, leucine, isoleucine, phenylalanine, tryptophan, lysine, histidine) and non-essential amino acids (glycine, alanine, serine, cysteine, glutamine, asparagine, aspartic acid, tyrosine, arginine, proline).

[0266] Ascorbic acid is an essential supplement for the growth and differentiation of various cells in culture. Certain ascorbic acid derivatives are now understood to be "short-acting" because they are not stable in solution, particularly under normal cell culture conditions of neutral pH and 37°C. These short-acting derivatives rapidly oxidize to oxalic acid or threonic acid. In a culture medium (pH 7) at 37°C, the level of the short-acting ascorbic acid derivative decreases by approximately 80 - 90% after 24 hours due to oxidation. Therefore, short-acting ascorbic acid derivatives are replaced by more stable "long-acting" ascorbic acid derivatives in the conventional cell culture of various cell types.

[0267] In the context of the present disclosure, the term "short-acting" includes ascorbic acid derivatives that are oxidized by approximately 80 - 90% after 24 hours in cell culture under culture conditions of neutral pH and 37°C. In one example, a short-acting L-ascorbic acid derivative is L-ascorbate. For example, in the context of the present disclosure, sodium L-ascorbate is a "short-acting" ascorbic acid derivative.

[0268] In contrast, the term "long-acting" includes ascorbic acid derivatives that are not oxidized by approximately 80 - 90% after 24 hours in cell culture under culture conditions of neutral pH and 37°C. In one example, in the context of the present disclosure, L-ascorbic acid 2-phosphate is a "long-acting" ascorbic acid derivative. Other examples of long-acting ascorbic acid derivatives include ascorbyl tetrahexyldecyl, magnesium ascorbyl phosphate, and 2-O-α-D-glucopyranosyl-L-ascorbic acid.

[0269] In one embodiment, the culture medium used in the method of producing stem cells that express high levels of Ang1 is supplemented with a short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another embodiment, 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 embodiment, 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 embodiment, the cell culture medium may contain at least about 0.05 g / L of the short-acting ascorbic acid derivative. In another embodiment, the cell culture medium may contain at least about 0.06 g / L of the short-acting ascorbic acid derivative. In one example of this embodiment, the cell culture medium is supplemented with the sodium salt of L-ascorbic acid.

[0270] In another embodiment, the cell culture medium contains a short-acting ascorbic acid derivative but does not contain a significant amount of a long-acting ascorbic acid derivative. For example, the cell culture medium may contain a short-acting ascorbic acid derivative, but is 0.04 g / L or less of a long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may contain a short-acting ascorbic acid derivative, but is 0.03 g / L or less of a long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may contain a short-acting ascorbic acid derivative, but is 0.02 g / L or less of a long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may contain a short-acting ascorbic acid derivative, but is 0.01 g / L or less of a long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may contain a short-acting ascorbic acid derivative, but is 0.005 g / L or less of a long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may contain a short-acting ascorbic acid derivative, but may not contain a long-acting ascorbic acid derivative.

[0271] In another embodiment, the cell culture medium contains sodium L-ascorbate but does not contain a significant amount of L-ascorbic acid-2-phosphate.

[0272] The cell culture medium used in the method for producing stem cells that express high levels of Ang1 can be a serum-containing medium or a serum-free medium.

[0273] The culture medium may or may not contain a serum replacement. The serum replacement can be, for example, albumin (e.g., lipid-rich albumin), transferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thiol glycerol, or a serum equivalent appropriately containing them. Such a serum replacement can be prepared, for example, by the method described in International Patent Application No. 93 / 30679, or a commercially available product can also be used.

[0274] In one embodiment, the cell culture medium used in the method for producing stem cells that express high levels of Ang1 is supplemented with at least about 9% (v / v), at least about 8% (v / v), at least about 7% (v / v), at least about 6% (v / v), at least about 5% (v / v), at least about 4% (v / v), at least about 3% (v / v), at least about 2% (v / v), at least about 1% (v / v) FCS. Also, the terms fetal calf serum (FCS) and fetal bovine serum (FBS) are assumed to be used interchangeably in the context of the present invention.

[0275] In one embodiment, the cell culture medium is supplemented with non-fetal serum. The medium may 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), 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%, at least about 25% (v / v) non-fetal serum.

[0276] For example, the culture medium can be supplemented with non-fetal serum from a mammal.

[0277] For example, the culture medium can be supplemented with non-fetal serum from a human.

[0278] For example, the culture medium can be supplemented with neonatal serum. The 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), 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%, at least about 25% (v / v) of neonatal serum.

[0279] In one embodiment, the cell culture medium is supplemented with mammalian neonatal serum.

[0280] For example, the culture medium can be supplemented with newborn calf serum (NBCS). The 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), 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%, at least about 25% (v / v) of NBCS.

[0281] In one embodiment, the cell culture medium is supplemented with human neonatal serum.

[0282] For 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), at least about 9% (v / v) of human neonatal serum. For example, human neonatal serum can be obtained from "umbilical cord blood".

[0283] In one embodiment, the culture medium is supplemented with adult serum. It is contemplated that the 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), 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%, at least about 25% (v / v) of adult serum.

[0284] In one embodiment, the cell culture medium is supplemented with mammalian adult serum.

[0285] For 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), 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%, at least about 25% (v / v) of mammalian adult serum.

[0286] For 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), 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%, at least about 25% (v / v) adult bovine serum.

[0287] In one embodiment, the cell culture medium is supplemented with human adult serum.

[0288] For 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), at least about 9% (v / v) human adult serum.

[0289] For 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), at least about 9% (v / v) human AB serum.

[0290] In one example, the cell culture medium is supplemented with at least about 3% human AB serum.

[0291] In one embodiment, the culture medium is supplemented with a mixture of FCS and NBCS.

[0292] For example, the culture medium can be supplemented with a mixture of FCS and NBCS such that the ratio of FCS:NBCS 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, at least about 2:1.

[0293] For example, a mixture of FCS and NBCS is envisioned to contain 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%, at least about 25% (v / v) of cell culture medium. However, in this example, the cell culture medium is supplemented with less than 10% (v / v) FCS and 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% (v / v) of NBCS.

[0294] In one embodiment, the cell culture medium is serum-free FCS.

[0295] In one embodiment, the cell culture medium is fetal bovine serum-free.

[0296] In one embodiment, the cell culture medium is supplemented with non-fetal bovine serum.

[0297] In one embodiment, the cell culture medium is fetal bovine serum-free and is supplemented with non-fetal bovine serum.

[0298] In another embodiment, the cell culture medium is supplemented with one or more stimulatory factors selected from the group consisting of lα,25-dihydroxyvitamin D3 (1,25D), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor lα (SDF-lα). In another embodiment, the cells can be cultured in the presence of at least one cytokine in an amount sufficient to support cell growth.

[0299] In another embodiment, the cells are cultured in the presence of platelet cytolysate in an amount sufficient to support cell growth. For example, the cells can be cultured with cytolysate of human platelets in an amount sufficient to support cell growth.

[0300] In one example, the cells are cultured with human AB serum and cytolysate of human platelets in an amount sufficient to support cell growth.

[0301] Also, those skilled in the art can generate stem cells that express high levels of Ang1 using the methods exemplified below.

[0302] Analyzing the therapeutic / preventive potential of cells Methods for determining the ability of stem cells that express high levels of Ang1 to treat, prevent, or delay the onset or progression of a disease will be apparent to those skilled in the art. For example, the stem cells can be evaluated for their ability to increase the level of Ang1.

[0303] In one example, genetically unmodified stem cells that express Ang1 at a level of at least 0.1 μg / 10 6 cells are tested for their ability to increase Ang1 expression in vitro and / or in vivo. In these examples, the cells or tissues are evaluated for the development of Ang1 expression after administration of stem cells that express high levels of Ang1.

[0304] The present disclosure also provides the following methods for identifying or isolating cells for treating, preventing or delaying a disease, which will be apparent to those skilled in the art from the above. (i) Administer stem cells expressing high levels of Ang1 to a subject suffering from a related disease and evaluate the symptoms of the subject's disease. (ii) Compare the symptoms of the disorder of the subject in (i) with the symptoms or activities of the disease of a control subject suffering from the disease but not administered stem cells, and an improvement in the symptoms of the test subject compared to the control subject indicates that the stem cells treat the disease. The cells may be any of the cells described in the present disclosure according to any embodiment.

Example

[0305] Example 1: STRO-3 + Immunoselection of MPCs by cell selection Bone marrow (BM) is collected from healthy normal adult volunteers (20 - 35 years old). Briefly, 40 ml of BM is aspirated from the posterior iliac crest into tubes containing lithium-heparin anticoagulant.

[0306] Bone marrow mononuclear cells (BMMNCs) are prepared by density gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo, Norway) as previously described (Zannettino et al., Blood, 92:2613 - 2628, 1998). After centrifugation at 400×g for 30 minutes at 4°C, the pale yellow layer is removed with a whole pipette and washed three times in "HHF" consisting of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0307] Subsequently, STRO-3 + (or TNAP +) The cells were isolated by magnetic-activated cell sorting as previously described (Gronthos et al., Journal of Cell Science 116:1827-1835, 2003; Gronthos and Simmons, Blood, 85, 929-940, 1995). Briefly, approximately 1-3×10 8 individual BMMNCs were incubated on ice for 20 minutes in a blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells were incubated on ice for 1 hour with a 10 μg / ml STRO-3 mAb solution (200 μl) in the blocking buffer. Subsequently, the cells were washed twice in HHF by centrifugation at 400×g. Goat anti-mouse γ-biotin diluted 1 / 50 in HHF buffer (Southern Biotechnology Associates, Birmingham, UK) was added and the cells were incubated on ice for 1 hour. The cells were washed twice as above in MACS buffer (PBS supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide, without Ca 2+ and Mn 2+ and resuspended in 0.9 ml of MACS buffer at a final volume.

[0308] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) was added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice with 0.5 ml of MACS buffer, resuspended, loaded onto a mini MACS column (MS Columns, Miltenyi Biotec), and washed three times with 0.5 ml of MACS buffer to recover the cells that did not bind to STRO-3 mAb (deposited with the American Type Culture Collection (ATCC) under the accession number PTA-7282 on December 19, 2005; see International Patent Application No. 2006 / 108229). After adding an additional 1 ml of MACS buffer, the column was removed from the magnet and TNAP +Isolate cells under positive pressure. Cell aliquots from each fraction can be stained with streptavidin-FITC and the purity can be evaluated by flow cytometry.

[0309] The MPCs isolated in this way are STRO-1 bright MPCs.

[0310] Example 2: Starting Medium - Process A The alpha modification of Eagle's Minimum Essential Medium (MEM) with Earle's balanced salts, commonly referred to as Eagle's αMEM, contains non-essential amino acids, sodium pyruvate, and added vitamins. These modifications were first described for use in growing mouse and hamster hybrid cells (Stanners et al., Nat New Biol., 230, 52 - 54, 1971).

[0311] Eagle's αMEM medium suitable for culturing primary stem cells can be obtained from various sources such as Life Technologies and Sigma.

[0312] A detailed method for establishing a primary stem cell culture containing the necessary growth factors used in the exemplary process is described in Gronthos and Simmons, Blood, 85, 929 - 940, 1995. In Process A, Eagle's αMEM medium supplemented with 10% fetal bovine serum, L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10 - 7M), and / or inorganic phosphate (3 mM) was used to culture the stem cells.

[0313] Example 3: Modified Culture Medium - Process B In Process B, the Eagle's αMEM medium used in Process A was modified (modified αMEM) as follows. · Replace the long-acting ascorbic acid derivative L-ascorbic acid-2-phosphate with the short-acting ascorbic acid derivative sodium L-ascorbate (50 mg / L). ·Reduce FCS from 10% (v / v) to 5% (v / v). ·Supplement with non-fetal serum (5% v / v).

[0314]

Table 1

[0315] Example 4: Cell culture Mesenchymal progenitor cells (MPCs) were obtained from a single donor and stored using the following cryopreservation method.

[0316] Generally, cell culture included the following steps.

[0317] The cryopreserved MPCs were thawed and seeded at 10,000 cells / cm 2 and grown to 90% confluence in either starting medium (Process A, n = 3) or modified culture medium (Process B, n = 3) at 20% O2, 37 °C.

[0318] To generate the conditioning medium, the growth medium was exchanged with EBM-2 basal medium (Lonza) supplemented with FCS at a rate of 200 μl of medium / cm 2 The cells were cultured for an additional 3 days, after which the medium was harvested, centrifuged to remove any cells, the resulting supernatant was collected, and stored at -80 °C.

[0319] The concentration of growth factors was measured using a Luminex platform with a commercially available kit (Millipore).

[0320] Following cell culture, MPC growth dynamics were evaluated (see Figures 1 - 3). No significant changes in cell growth, MPC doubling time, or population doubling time were observed after cell culture processes A and B.

[0321] MPCs are also characterized in terms of the expression levels of cell markers for STRO-1, CC9, and STRO-4, as well as the pro-angiogenic growth factors Ang1 and VEGF.

[0322] The levels of STRO-1, CC9, and STRO-4 were equivalent to those of MPCs after cell culture processes A and B. However, culture process B · increased the Ang1 level, · decreased the VEGF level, · provided an Ang1:VEGF ratio that was consistent with the Ang1:VEGF ratio previously shown to be particularly effective in enhancing angiogenesis. The measured values of the Ang1 and VEGF levels (μg / 10 6 cells) in the conditioned media of MPCs cultured in process A or B are shown in Table 2.

[0323]

Table 2

[0324] Example 5: Modified Culture Conditions - Processes C and D To control the exchange of the long-acting ascorbic acid derivative, L-ascorbic acid-2-phosphate, with the short-acting ascorbic acid derivative, sodium L-ascorbate, MPCs from three different donors were continuously propagated with growth factors such as PDGF and EGF in α-MEM + 10% FCS + 50 mg / L sodium L-ascorbate (process C) or α-MEM + 3% human AB serum + 50 mg / L sodium L-ascorbate (process D).

[0325] The levels of Ang1 and VEGF were evaluated after cell culture in processes C and D. The levels of Ang1 and VEGF (ug / 10 6 cells) in the conditioned media of MPCs cultured in process C or D are shown in Table 3.

[0326] Compared to process C, culture process D · increased the Ang1 level, · decreased the VEGF level, · increased the VEGF:Ang1 ratio. Compared with Process A, Processes C and D result in a progressive increase in the expression level of Ang1. This suggests that the presence of a short-acting ascorbic acid derivative and non-fetal serum independently brings about an increase in the expression of Ang1 and exerts a synergistic effect of further increasing the expression of Ang1.

[0327]

Table 3

[0328] Example 6: Polarization of Inflammatory M1 Monocytes into M2 Phenotype CD14+ monocytes were immunoselected from whole blood. The monocyte population was characterized based on CD16 expression. 5.2% of the cells exhibited the phenotypic characteristics of M2 macrophages (CD14 + CD16 + ). (Figure 4)

[0329] CD14+ monocytes were co-cultured with MPCs expressing high levels of Ang1 for 7 days. The MPCs were obtained from two different donors (#023 and #009).

[0330] Three days after co-culture, the cells were evaluated for the following. · CD14, CD16 expression; and · Secretion of TNF-α in response to lipopolysaccharide (LPS). LPS was added at 1 ng / ml for 24 hours (the last 5 hours with + transduction inhibitor).

[0331] Co-culture resulted in the following. · Generation of macrophages showing the phenotypic characteristics of M2 macrophages (CD14 + CD16 + CD163 + CD206 + ). (Figure 4) · Inhibition of LPS-induced secretion of TNFα by macrophages. (Figure 5)

[0332] After 7 days of co - culture, the cells were evaluated for the secretion of IL - 10 in response to LPS. LPS was added at 1 ng / ml for 24 hours (the last 5 hours with + transduction inhibitor). IL - 10 expression was analyzed by intracellular flow cytometry. IL - 10 production by macrophages was enhanced by co - culture with MPCs in the presence of LPS (Figure 6).

[0333] These data indicate that MPCs expressing high levels of Ang1 promote the production of M2 - type macrophages (Figure 7).

[0334] Stem cells expressing high levels of Ang1 were cultured with increasing amounts of either IL - 1β alone or in combination with TNF - α, and the effect of these cytokines on PGE2 secretion was evaluated.

[0335] An additive effect of IL - 1β and TNF - α on PGE2 expression was observed (Figure 8).

[0336] PGE2 promotes the differentiation of Th17 cells into Th2 cells and TReg cells, as well as the polarization of M1 - type macrophages into M2 - type macrophages (Figure 7). High levels of IL - 1 and TNF - α have been observed in subjects with inflammatory diseases such as type II diabetes.

[0337] Therefore, the increased secretion of PGE2 from stem cells expressing high levels of Ang1 in response to TNFα and IL1β suggests that stem cells expressing high levels of Ang1 can increase the production of anti - inflammatory cells in subjects with inflammatory diseases. In particular, stem cells expressing high levels of Ang1 can · increase M2 - type macrophage production by promoting the polarization of M1 - type macrophages into M2 - type macrophages; and / or · increase the production of Th2 and Treg by promoting the differentiation of Th17 cells.

[0338] Example 7: Sheep model of collagen - induced arthritis Stem cells expressing high levels of Ang1 were administered to a murine model of rheumatoid arthritis (Thorpe et al., Clinical & Exp. Rheumatology, 10:143-150 (1992)). This model is characterized by inflammatory manifestations of rheumatoid arthritis in both the systemic and joint compartments.

[0339] 150 million cryopreserved ovine MPCs were administered intravenously via the jugular vein.

[0340] IL-17 and IL-10 levels were evaluated over a 2-week period. Changes in the levels of pro-inflammatory and anti-inflammatory cytokines are shown in (Figure 9).

[0341] At the establishment of late-stage disease (day 42), stem cells expressing high levels of Ang1 were also administered. Administration of stem cells resulted in a decrease in the levels of inflammatory cytokines in the joints (Figure 10).

[0342] Example 8: Stem cell administration in diabetes Human subjects with type 2 diabetes that was not adequately controlled by metformin or metformin and another oral therapy were given a single intravenous injection of three doses of mesenchymal progenitor cells (MPC) and compared to a placebo control. Changes in baseline HbA1c, IL-6, TNF-alpha, fasting insulin, adiponectin, osteocalcin, and hsCRP were evaluated over 12 weeks.

[0343] Subjects were divided into three cohorts (Figure 11). Cohort 1: MPC dose 1 (300,000 cells / kg) [n = 15] or placebo [n = 5] Cohort 2: MPC dose 1 (1 million cells / kg) [n = 15] or placebo [n = 5] Cohort 3: MPC dose 1 (2 million cells / kg) [n = 15] or placebo [n = 5]

[0344] In the subjects treated with MPC, a slight decrease in HbA1c was observed compared with the placebo control subjects, in whom there was a slight increase (Table 4). A greater decrease in HbA1c was observed in cohort 2 compared with placebo at 8 weeks (Table 4). A tendency for a greater decrease in HbA1c was observed in subjects with a baseline HbA1c value of 8% or higher (Figure 15). Eight (17.8%) of the 45 subjects achieved the target HbA1c (<7.0%) at 12 weeks (Figure 16). Improvement tendencies in fasting insulin and adiponectin levels (Figure 12) were observed in the MPC-treated subjects compared with the placebo control subjects. Decreases in TNF-alpha and IL-6 levels were observed in the MPC-treated subjects compared with the placebo control subjects, and the most prominent decrease was observed in cohort 3 (Figure 13). In cohort 3, the changes from baseline in TNF-alpha (Figure 13) and IL-6 (Figure 14) were -0.26 pg / ml and -0.47 pg / ml, respectively (Figure 13).

[0345] [Table 4]

[0346] Example 9: Stem cell administration in rheumatoid arthritis Human subjects with rheumatoid arthritis classified as inadequate anti-TNFα responders or human subjects in whom up to two other biological agents had failed were injected with a single intravenous injection of two doses of mesenchymal progenitor cells (MPC) and compared with a placebo control.

[0347] The 48 subjects included in the study had +RF / anti-CCP; >4 swollen / tender joints; ESR / CRP >ULN.

[0348] The subjects were divided into two cohorts. Cohort 1: MPC dose (1 million cells / kg) [n = 16] or placebo [n = 8] Cohort 2: MPC dose (2 million cells / kg) [n = 16] or placebo [n = 8]

[0349] The evaluation endpoints at 3 months after injection included the following. ·Levels of TNFα; IL-6 (Figure 17), IL-17; RANKL; MMP-1, 3, 9; TIMP-1, 2, 4 and osteocalcin. Levels were also evaluated at 0, 1, 2, 4, 6, 8 and 10 weeks. ·ACR20 (Figure 18) / 50 (Figure 19) / 70 (Figure 20); ·ACR individual assets (Figure 21; Figure 22); ·Remission (disease activity score (DAS28(CRP)) < 2.6) (Figure 23); ·Disease activity score (DAS28); ESR / CRP; HAQ-DI (Figure 24); SF-36 (remission DAS28(CRP) < 2.6); change from baseline for response DAS28(CRP) < 3.2 (Figure 23); ·X-rays of hands / wrists at 6 and 12 months

[0350] There were no serious adverse events (SAEs) related to the treatment associated with cohort 1. Early and sustained efficacy was observed over 3 months.

[0351] Over 1 - 12 weeks, in cohort 1 compared to placebo, the IL-6 level decreased from baseline (Figure 17).

[0352] The data supports the suggestion of a potential remission rate of approximately 20% that appears to be higher than other biological agents (Figure 23).

[0353] In the primary endpoint at 12 weeks for cohort 1, there is a consistent trend of improved response over placebo.

[0354] The interim analysis during follow-up shows the persistence of the responder effect over time.

[0355] Cohort 2 is undergoing testing with more single doses.

[0356] Example 10: Administration of Stem Cells in Diabetic Nephropathy Human subjects with type 2 diabetes and moderate to severe chronic kidney disease, and who were on a stable regimen of ACEi or ARB therapy for diabetic nephropathy, were given a single intravenous injection of two doses of mesenchymal progenitor cells (MPCs) and compared to a placebo control.

[0357] The subjects were divided into two cohorts. Cohort 1: MPC1 dose (150 million cells / kg) [n = 10] or placebo [n = 5] Cohort 2: MPC1 dose (300 million cells / kg) [n = 10] or placebo [n = 5]

[0358] Evaluation endpoints at 3 and 6 months post-injection: · Measured GFR (mGFR[99Tc DTPA]) (Figure 25), estimated GFR (eGFR[MDRD]) (Figure 25), IL-6 (Figure 26); and levels of serum creatinine; · Correlation between IL-6 and serum creatinine levels (Figure 27)

[0359] During the initial 24-week study period, a trend towards prevention or improvement of renal function as measured by both measured GFR and estimated GFR was observed in MPC-treated subjects compared to placebo. · The treatment effect was similar at both MPC doses. · In subjects with baseline eGFR > 30 ml / min / 1.73m 2 the treatment effect with MPCs was more pronounced (Figure 28). · A more pronounced treatment effect with MPCs in subjects with baseline IL-6 levels above the median; · A significant correlation between baseline IL-6 levels and MPC-related improvement in serum creatinine (Figure 27); · There was a dose-dependent change in serum IL-6 levels at 12 weeks in the MPC group vs placebo (Figure 26).

[0360] Those skilled in the art will understand that numerous variations and / or modifications can be made to the present disclosure without departing from the spirit or scope of the disclosure as broadly described, as shown in the specific embodiments. Accordingly, the present embodiments should be considered as illustrative in all respects and not as restrictive.

[0361] This application claims the priority of AU2014902194 filed on June 10, 2014 and AU2014902257 filed on June 13, 2014, and incorporates these disclosures herein by reference.

[0362] All publications discussed and / or referenced herein are hereby incorporated by reference in their entirety.

[0363] The discussion of documents, acts, materials, devices, articles, etc. contained herein is for the purpose of providing context only for the present invention. It should not be construed as an admission that any or all of these matters formed part of the prior art base or were common general knowledge in the relevant field of the present invention as of the priority date of each claim of this application.

Claims

1. A method for increasing the production and / or function of anti-inflammatory cells in a subject in need thereof, said method comprising administering to said subject a composition comprising genetically unmodified stem cells, said genetically unmodified stem cells expressing angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 ６ cells, said method.

2. The method according to claim 1, wherein the anti-inflammatory cells are Th2 cells, TReg cells or M2 macrophages.

3. The method according to claim 1 or 2, wherein in the subject, the number of M2 macrophages is increased.

4. The method according to any one of claims 1 to 3, wherein in the subject, the number of M2 macrophages is increased to at least 10% of the total mononuclear cell population.

5. The method according to any one of claims 1 to 3, wherein in the subject, the number of M2 macrophages is increased to at least 20% of the total mononuclear cell population.

6. The method according to any one of claims 1 to 3, wherein in the subject, the number of M2 macrophages is increased to at least 40% of the total mononuclear cell population.

7. The method according to any one of claims 1 to 3, wherein in the subject, the number of M2 macrophages is increased to at least 80% of the total mononuclear cell population.

8. The method according to any one of claims 2 to 7, wherein the M2 macrophages are CD14++CD16+.

9. The method according to any one of claims 1 to 9, which promotes the polarization of macrophages from the M1 to the M2 phenotype.

10. The method according to any one of claims 1 to 9, which promotes the differentiation of pro-inflammatory helper T cells into Th2 cells or TReg cells.

11. The method according to claim 10, wherein the pro-inflammatory helper T cells are Th17 cells.

12. The increase in the production and / or function of the anti-inflammatory cells in the subject is - a decrease in the IL-6 level in the subject; - a decrease in the TNF-alpha level in the subject; and / or - an increase in the IL-10 level in the subject resulting in the method according to any one of claims 1 to 11.

13. The method according to any one of claims 1 to 12, wherein in the subject, the level of anti-inflammatory cytokines is increased.

14. The method according to any one of claims 1 to 13, wherein in the subject, the level of IL-10 is increased.

15. The method according to any one of claims 1 to 14, wherein in the subject, the level of pro-inflammatory cytokines is decreased.

16. The method according to any one of claims 1 to 15, wherein in the subject, the level of any one of IL-6, TNF-alpha and / or IL-17 is decreased.

17. The method according to any one of claims 1 to 16, which also inhibits the production and / or function of inflammation-inducing cells.

18. The method according to claim 17, wherein the inflammation-inducing cells are Th17 cells or M1 macrophages.

19. A method of treating a target inflammatory disease, the method comprising administering to the target a composition comprising genetically unmodified stem cells, wherein the genetically unmodified stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 ６ cells, said method.

20. The method according to any one of claims 1 to 19, wherein the genetically unmodified stem cells express Ang1 in an amount of at least 0.5 μg / 10 ６ cells.

21. The method according to any one of claims 1 to 19, wherein the genetically unmodified stem cells express Ang1 in an amount of at least 0.7 μg / 10 ６ cells.

22. The method according to any one of claims 1 to 19, wherein the genetically unmodified stem cells express vascular endothelial growth factor (VEGF) in an amount of less than about 0.05 μg / 10 ６ cells.

23. The method according to any one of claims 1 to 22, wherein the non-genetically modified stem cells express Ang1:VEGF at a ratio of at least about 20:

1.

24. The method according to any one of claims 1 to 23, wherein the non-genetically modified stem cells are mesenchymal progenitor cells.

25. The method according to claim 24, wherein the stem cells are mesenchymal stem cells.

26. The inflammatory disease is diabetes or a diabetes-related condition or symptom selected from the group consisting of abnormal wound healing, symptoms of a heart attack, symptoms of a stroke, symptoms of peripheral vascular disease, amputation, symptoms of kidney disease, renal failure, blindness, neuropathy, nephrosis, retinopathy, inflammation, impotence, or non-alcoholic steatohepatitis (NASH). The method according to any one of claims 19 to 25.

27. The method according to any one of claims 19 to 26, wherein the inflammatory disease is type II diabetes.

28. Approximately 0.1×10 per 1 kg ６ to approximately 3×10 ６ The method according to any one of claims 1 to 27, comprising administering to the subject from ６ to ６ stem cells per 1 kg

29. About 0.3×10 ６ to about 2×10 ６ administering stem cells to the subject, the method according to any one of claims 1 to 27.

30. About 1×10 per kg ６ to about 2×10 ６ The method according to any one of claims 1 to 27, comprising administering to the subject from about 1×10 to about 2×10 stem cells per kg of body weight.

31. administering to the subject about 2×10 ６ stem cells per kg, the method according to any one of claims 1 to 27.

32. The method according to any one of claims 20 to 25, wherein the treatment of the diabetes or diabetes-related condition or symptom in the subject is indicated by any one of the following: ・ A decrease in the HbA1c value (percentage of total hemoglobin); ・ A decrease in the fasting insulin level; ・ A decrease in the IL-6 level; ・ A decrease in the TNF-α level; and / or ・ An increase in the adiponectin level

33. The method according to any one of claims 26 to 32, wherein the glucose level of the subject is not adequately controlled by metformin.

34. The method according to any one of claims 26 to 33, wherein the subject has a baseline HbA1c value of more than 7.5%.

35. The method according to any one of claims 26 to 34, wherein the subject has a baseline HbA1c value of 8% or more.

36. The method according to any one of claims 26 to 31, wherein the inflammatory disease is rheumatoid arthritis.

37. About 0.5×10 ６ to about 3.0×10 ６ The method according to any one of claims 26 to 31 and 36, comprising administering to the subject from

38. About 1.0×10 ６ to about 2.0×10 ６ The method according to any one of claims 26 to 31 and 36, comprising administering to the subject from

39. The method according to any one of claims 26 to 31 and 36 to 38, wherein the treatment of the rheumatoid arthritis is indicated by any one of the following: - ACR20; - ACR50; - ACR70; - A decrease in the IL-6 level; and / or - A decrease in the disease activity score

40. The method according to any one of claims 26 to 31, wherein the inflammatory disease is diabetic nephropathy.

41. About 1.0 × 10 ８ cells to about 4.0 × 10 ８ cells of stem cells to the subject, the method according to any one of claims 26 to 31 and 40.

42. About 1.5 × 10 ８ cells to about 3.0 × 10 ８ cells, the method according to any one of claims 26 to 31 and 40, comprising administering the stem cells to the subject.

43. The method according to any one of claims 26 to 31 and 40 to 42, wherein the treatment of the diabetic nephropathy is indicated by any one of the following: - Inhibition of the decrease in eGFR and / or mGFR; - Improvement of eGFR and / or mGFR; and / or - A decrease in the IL-6 level

44. The eGFR of the baseline of the subject is greater than about 35 ml / min / 1.73 m ２ The method according to any one of claims 26 to 31 or 40 to 43.

45. The eGFR of the baseline of the subject is greater than 30 ml / min / 1.73 m ２ The method according to any one of claims 26 to 31 or 40 to 44.

46. The eGFR of the baseline of the subject is greater than 28 ml / min / 1.73 m ２ The method according to any one of claims 26 to 31 or 40 to 44.

47. The eGFR of the baseline of the subject is greater than 25 ml / min / 1.73 m ２ The method according to any one of claims 26 to 31 or 40 to 44.

48. The method according to any one of claims 1 to 47, wherein the stem cells are administered systemically.

49. The method according to any one of claims 1 to 48, wherein the stem cells are administered intravenously.

50. The method according to any one of claims 1 to 49, wherein the stem cells are administered in multiple doses.

51. A composition comprising genetically unmodified stem cells for use in the treatment of inflammatory diseases, wherein the genetically unmodified stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 ６ cells, said composition.

52. Use of a composition comprising non-genetically modified stem cells in the manufacture of a medicament for treating an inflammatory disorder, wherein the non-genetically modified stem cells express angiopoietin-1 (Ang1) in an amount of at least 0.1 μg / 10 ６ cells, said use.

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