Improved stem cell composition

Abstract

To solve the problem in which the use of stem cells alone to promote angiogenesis remains limited because of insufficient expression of angiogenic factors in many types of stem cells.SOLUTION: The disclosure provides stem cells which express high levels of Angiopoietin-1 (Ang1) and methods for their production. Such stem cells may be used in a wide range of therapeutic applications.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to stem cells expressing high levels of angiopoietin-1 (Ang1) and methods for producing the same. Such stem cells may be used in a wide range of therapeutic applications, for example to promote vascularization and / or angiogenesis. [Background technology]

[0002] Angiopoietins are part of a family of vascular growth factors involved in early and postnatal angiogenesis. Ang1 promotes migration of endothelial cells and some non-endothelial cells, such as smooth muscle cells. Ang1 also induces endothelial cell sprouting and remodeling into renal tubules. Ang1 exerts a potent anti-inflammatory effect on endothelial cells, suppressing vascular endothelial growth factor (VEGF) inducing upregulation of E-selectin, ICAM-1, and VCAM-1, and inhibiting leukocyte adhesion and transmigration in response to VEGF and TNF-α (Kim et al. 2001a).

[0003] Many studies have shown that overexpression of Ang1, i.e., the addition of supplemental Ang1, has beneficial effects in alleviating ischemia and restoring function in several organs, including the limbs, brain, joints, kidneys, and most importantly, in the heart. Other beneficial effects include alleviating thrombosis (Kim et al. 2001b). Thus, Ang1 displays a number of important properties that suggest its usefulness as a therapeutic agent for cardiovascular disease.

[0004] Among the cascade of growth factors required for the development of a functional vasculature, Ang1 and VEGF play central roles, and therefore the use of Ang1 in combination with VEGF for therapeutic angiogenesis in the treatment of ischemic myocardium is also considered a very promising candidate.

[0005] Previously, it has been shown that combined administration of Ang1 and VEGF-A to myocardial infarction or infarct zone in test animals increases angiogenesis, reduces myocardial apoptosis, and leads to cardiomyocyte regeneration at the injection site, as well as improving vascular perfusion and cardiac function. Submaximal doses of Ang1 and VEGF in a ratio of approximately 20:1 potentiate these effects, making them more potent than when either factor is used alone (Chae et al. 2000). These results indicate that combined treatment with Ang1 and VEGF may be used to generate therapeutic angiogenesis.

[0006] Recently, stem cell therapy has emerged as one of the potential treatments for ischemic heart disease (Huang et al. 2011; Lijie et al. 2007). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Bongso et al. 1889, Improved quality of human embryos when co-cultured with human ampullary cells.Human Reprod., 4(6), 706-13. [Non-Patent Document 2] Chae et al. 2000, Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization.Arterioscler.Thromb.Vasc.Biol.20, 2573-2578. [Non-Patent Document 3] Eisenberg & Bader, 1996, Establishment of the mesodermal cell line QCE-6.A model system for cardiac cell differentiation.Circ Res., 78(2), 205-16. [Non-Patent Document 4] Gnecchi et al. 2008, Paracrine mechanisms in adult stem cell signaling and therapy.Circ Res., 103(11), 1204-1219. [Non-Patent Document 5] Huang et al. 2011, Transplantation of Sendai Viral Angiopoietin-1-Modified Mesenchymal Stem Cells for Ischemic Heart Disease.Targets in Gene Therapy, Prof. Yongping You (ed.), ISBN:978-953-307-540-2, InTech, DOI:10.5772 / 19590.Available from: http: / / www.intechopen.com / books / targets-in-gene-therapy / transplantation-of-sendai-viral-angiopoietin-1-modified-mesenchymal-stem-cells-for-ischemic-heart-di. [Non-Patent Document 6] Gronthos and Simmons 1995, The growth factor requirements of STRO-1-positive human bone marrow derived stromal precursors under serum-deprived conditions in vitro.Blood, 85, 929-940. [Non-Patent Document 7] Gronthos et al. (2003) Molecular and cellular characterization of highly purified stromal stem cells derived from human bone marrow.Journal of Cell Science 116:1827-1835 [Non-Patent Document 8] Kanatsu-Shinohara et al. 2003, Long-term proliferation in culture and germline transmission of mouse male germline stem cells, Biol.Reprod., 69(2), 612-616. [Non-Patent Document 9] Kim et al. 2001a, Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression.Circ Res., 89(6), 477-479. [Non-Patent Document 10] Kim et al. 2001b, Angiopoietin-1 negatively regulates expression and activity of tissue factor in endothelial cells.FASEB J., 16(1), 126-128. [Non-Patent Document 11] Lijie et al. 2007, Mesenchymal stem cells modified with angiopoietin-1 improve remodelling in a rat model of acute myocardial infarction.Biochem Biophys Res Commun., 357(3), 779 - 84. [Non-Patent Document 12] Matsui et al. 1992, Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture.Cell, 70(5):841 - 7. [Non-Patent Document 13] Mummery et al. 2002, Cardiomyocyte differentiation of mouse and human embryonic stem cells.J Anat., 200(3), 233 - 242. [Non-Patent Document 14] Passier R et al. 2005, Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.Stem Cells, 23(6), pg 772 - 80. [Non-Patent Document 15] Resnick et al. 1992, Long-term proliferation of mouse primordial germ cells in culture.Nature, 359(6395), 550 - 551. [Non-Patent Document 16] Reubinoff et al. 2000, Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol., 18(4), 399 - 404. [Non-Patent Document 17] Shamblott et al. 1998, Derivation of pluripotent stem cells from cultured human primordial germ cells, Proc. Natl. Acad. Sci. USA, 95(23), 13726 - 31. [Non-Patent Document 18] Shinohara et al. 2004, Generation of pluripotent stem cells from neonatal mouse testis, Cell, 119(7), 1001 -1012. [Non-Patent Document 19] Stanners et al., 1971, Two Types of Ribosome in Mouse-Hamster Hybrid Cells.Nat New Biol., 230, 52-54. [Non-Patent Document 20] Takebayashi et al. 2008, Experimental Medicine, 26(5) (Suppl.), 41 -46, YODOSHA (Tokyo, Japan). [Non-Patent Document 21] Thomson et al. 1995, Isolation of a primate embryonic stem cell line, Proc. Natl. Acad. Sci USA, 92(17), 7844 - 8. [Non-Patent Document 22] Thomson et al. 1996, Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts.Biol.Reprod., 55(254), 254 - 9. [Non-Patent Document 23] Thomson et al. 1998, Embryonic stem cell lines derived from human blastocysts.Science, 282, 1145-7. [Non-Patent Document 24] Vacanti, et al. 1991, Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation.Plast.Reconstr.Surg.88, 753-9. [Non-Patent Document 25] Yoshiaki et al. 2012, Human periodontal ligament fibroblasts are the optimal cell source for induced pluripotent stem cells.Histochemistry & Cell Biology, 137(6), 719-732. [Non-Patent Document 26] Zannettino, ACet al.1998, The Sialomucin CD164 (MGC-24v) Is an Adhesive Glycoprotein Expressed by Human Hematopoietic Progenitors and Bone Marrow Stromal Cells That Serves as a Potent Negative Regulator of Hematopoiesis.Blood 92:2613-2628. Summary of the Invention [Problem to be solved by the invention]

[0008] The use of stem cells alone to promote angiogenesis remains limited due to the poor expression of angiogenic factors in many types of stem cells.Genetic modification of stem cells, including the transfection of stem cells with nucleic acid molecules encoding Ang1, has been used to address this limitation.However, the use of genetically modified stem cells has drawbacks due to the technical complexity and potential undesirable effects caused by the genetic modification process. [Means for solving the problem]

[0009] The present disclosure is based on the unexpected production of a population of stem cells that express Ang 1 at high levels without the need for transfection of the cells with a nucleic acid that expresses Ang 1. In one example, this population of stem cells also expressed VEGF at low levels, and the ratio of Ang 1 to VEGF produced matched the ratio that Chae et al. (2000) showed to be particularly effective in enhancing angiogenesis.

[0010] Therefore, the present invention provides a method for the treatment of a pulmonary artery disease comprising administering a pulmonary artery disease to a patient having a pulmonary artery disease, ... and 6 A composition is provided that includes a genetically unmodified stem cell that expresses Ang1 in a cell quantity.

[0011] In another embodiment, the composition has a concentration of at least 0.5 μg / 10 6 In another embodiment, the stem cells express Ang1 in an amount of at least 0.7 μg / 10 6 In another embodiment, the stem cells express Ang1 in an amount of at least 1 μg / 10 6 The amount of cells expressing Ang1.

[0012] In another embodiment, the stem cells are at about 0.05 μg / 10 6 In another embodiment, the stem cells express VEGF at levels less than about 0.03 μg / 10 6 In another embodiment, the stem cells express VEGF at levels less than about 0.02 μg / 10 6 They express VEGF in subcellular amounts.

[0013] In another embodiment, the ungenetically modified stem cells express Ang1:VEGF in a ratio of at least about 2:1. In another embodiment, the ungenetically modified stem cells express Ang1:VEGF in a ratio of at least about 10:1. In another embodiment, the stem cells express Ang1:VEGF in a ratio of at least about 20:1. In another embodiment, the stem cells express Ang1:VEGF in a ratio of at least about 30:1. In another embodiment, the stem cells express Ang1:VEGF in a ratio of at least about 50:1.

[0014] 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 derived from induced pluripotent stem cells (iPS cells).

[0015] In another embodiment, the composition further comprises an acceptable pharmaceutical carrier.

[0016] In another embodiment, the composition is produced by culturing non-genetically modified stem cells according to the methods described below.

[0017] The present disclosure also provides an in vitro method for inducing expression of Ang1 in stem cells, the method comprising culturing a population of stem cells in a cell culture medium comprising: Contains short-acting L-ascorbic acid derivatives but does not contain significant amounts of long-acting L-ascorbic acid derivatives; and / or · Supplemented with less than 10% (v / v) fetal bovine serum.

[0018] The present disclosure also provides an in vitro method for inducing expression of Ang1 in stem cells, the method comprising culturing a population of stem cells in a cell culture medium comprising: Contains short-acting L-ascorbic acid derivatives but does not contain significant amounts of long-acting L-ascorbic acid derivatives; and / or Supplemented with less than 10% (v / v) fetal bovine serum; and / or · Supplemented with non-fetal serum.

[0019] In one embodiment, the method further comprises measuring the level of Ang1 to determine that expression of Ang1 is induced. 6 In another embodiment, the expression of Ang1 is induced when the amount of stem cells expressing Ang1 is at least 0.7 μg / 10 6 In another embodiment, the expression of Ang1 is induced when the amount of stem cells expressing Ang1 is at least 1 μg / 10 6When Ang1 is expressed in the amount of cells, the expression of Ang1 is induced.

[0020] In one embodiment, the method selects for cells with induced expression of Ang1. In one embodiment, the method selects for cells with induced expression of Ang1. 6 In another embodiment, cells expressing Ang1 in an amount of at least 0.7 μg / 10 6 In another embodiment, cells expressing Ang1 in an amount of at least 1 μg / 10 6 Cells expressing Ang1 in any amount are selected.

[0021] In one embodiment, the method further comprises measuring the level of Ang1 to determine that expression of Ang1 is induced and selecting cells having induced expression of Ang1.

[0022] In one embodiment, the short-acting ascorbic acid derivative is L-ascorbate.

[0023] In one embodiment, the short-acting ascorbic acid derivative is L-ascorbic acid sodium salt.

[0024] In one example, the cell culture medium is supplemented with less than 10% (v / v) fetal calf serum (FCS).

[0025] In one example, the cell culture medium is supplemented with less than 8% (v / v) fetal calf serum (FCS).

[0026] In one example, the cell culture medium is supplemented with less than 7% (v / v) fetal calf serum (FCS).

[0027] In one example, the cell culture medium is supplemented with less than 6% (v / v) fetal calf serum (FCS).

[0028] In one example, the cell culture medium is supplemented with less than 5% (v / v) fetal bovine serum.

[0029] In one embodiment, the cell culture medium is supplemented with one or more stimulatory factors selected from the group consisting of 1α,25-dihydroxyvitamin D3 (1,25D), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor alpha (SDF-1α).

[0030] In one embodiment, the cell culture medium is supplemented with non-fetal serum.

[0031] In one embodiment, the cell culture medium is supplemented with non-fetal mammalian serum.

[0032] In one embodiment, the cell culture medium is supplemented with human non-fetal serum.

[0033] In one embodiment, the cell culture medium is supplemented with newborn serum.

[0034] In one embodiment, the cell culture medium is supplemented with newborn mammalian serum.

[0035] In one embodiment, the cell culture medium is supplemented with newborn calf serum (NBCS).

[0036] In one embodiment, the cell culture medium is supplemented with human newborn serum.

[0037] In one embodiment, the cell culture medium is supplemented with human newborn serum obtained from umbilical cord blood.

[0038] In one embodiment, the cell culture medium is supplemented with adult serum.

[0039] In one embodiment, the cell culture medium is supplemented with adult mammalian serum.

[0040] In one example, the cell culture medium is supplemented with adult bovine serum.

[0041] In one embodiment, the cell culture medium is supplemented with adult human serum.

[0042] In one embodiment, the cell culture medium is supplemented with human AB serum.

[0043] In another embodiment, the cell culture medium is supplemented with at least about 5% (v / v) NBCS.

[0044] In another embodiment, the cell culture medium is supplemented with at least about 2% (v / v) NBCS.

[0045] In one embodiment, the cell culture medium is supplemented with a mixture of NBCS and FCS, for example, the ratio of FCS to NBCS can be about 1:1.

[0046] In one embodiment, the cell culture medium is supplemented with at least about 5% (v / v) FCS and at least about 5% (v / v) NBCS.

[0047] In one embodiment, the cell culture medium is not supplemented with fetal serum.

[0048] The present invention provides a method for the treatment of a patient having a disease comprising obtaining at least one cell population(s) comprising stem cells from at least one donor, culturing the stem cells, determining the amount of Ang1 expressed by each stem cell of said at least one cell population, and measuring the amount of Ang1 expressed by each stem cell of at least 0.1 μg / 10 6 Also provided is a method for obtaining genetically unmodified stem cells suitable for use in promoting vascularization and / or angiogenesis, comprising selecting stem cells expressing Ang1 in a quantity of cells.

[0049] In one embodiment, the method further comprises determining the amount of VEGF expressed by each stem cell of the at least one cell population(s) and selecting stem cells expressing an Ang1:VEGF ratio of at least 2:1, or at least 10:1, or at least 20:1, or at least 30:1, or preferably at least 50:1.

[0050] The present disclosure also provides for the use of the compositions described herein to promote vascularization and / or angiogenesis.The present disclosure also provides for the use of the compositions described herein as antithrombotic.The present disclosure also provides for the use of the compositions described herein to treat conditions in which increased expression of Ang1 is desired.

[0051] The present disclosure also provides a method for promoting vascularization and / or angiogenesis in a subject, the method comprising administering to the subject a composition described herein.The present disclosure also provides a method for reducing thrombus formation in a subject, the method comprising administering to the subject a composition described herein.The present disclosure provides a method for treating a condition in a subject in which increased Ang1 expression is desired, the method comprising administering to the subject a composition described herein.

[0052] The present disclosure also provides for the use of the compositions described herein in the manufacture of a medicament for promoting vascularization and / or angiogenesis.The present disclosure also provides for the use of the compositions described herein in the manufacture of a medicament for reducing thrombus formation.The present disclosure also provides for the use of the compositions described herein in the manufacture of a medicament for treating a condition in which increased expression of Ang1 is desired.

[0053] The stem cells described in this disclosure can be obtained from any mammal. For example, the stem cells may be from a primate, cow, sheep, horse, dog, cat, or goat. In one embodiment, the stem cells are human stem cells.

[0054] In another embodiment, the present disclosure relates to a population of stem cells cultured according to or obtained by the methods of the present disclosure.

[0055] In another embodiment, the methods of the present disclosure are used in the manufacture of a medicament for promoting vascularization and / or angiogenesis.

[0056] In another embodiment, the methods of the disclosure are used in the manufacture of a medicament for treating a condition in which increased expression of Ang1 is desired. [Brief description of the drawings]

[0057] [Figure 1] MPC proliferation in Process A and Process B. Y-axis indicates cell number and X-axis indicates time in days. [Diagram 2] MPC doubling time for Process A and Process B. Cells were passaged from P3 to P5. [Diagram 3] Population doubling time (PDL) of MPCs from Process A and Process B. MPCs were expanded from P3 to P5. MPCs expanded using Process A achieved 8 PDL and MPCs expanded using Process B achieved 7.33 PDL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] [General Techniques and Definitions] Throughout this specification, unless otherwise indicated or required by context, a reference to a single step, composition of matter, group of steps or group of compositions of matter should be interpreted as encompassing a plurality (i.e. one or more) of that step, composition of matter, group of steps or group of compositions of matter.

[0059] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions and compounds referred to or shown herein, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0060] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure as described herein.

[0061] Unless specifically stated otherwise, any embodiment disclosed herein should be construed as applying mutatis mutandis to any other embodiment.

[0062] Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., of cell culture, molecular genetics, stem cell differentiation, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0063] Unless otherwise indicated, the stem cell, 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 throughout the literature in such sources as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), TA Brown (ed.), Essential Molecular Biology: A Practical Approach, volumes 1 and 2, IRL Press (1991), DM Glover and BD Hames (eds.), and FM 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 JE Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0064] The term "and / or" should be interpreted as explicitly supporting both meanings or either meaning, for example, "X and / or Y" should be understood to mean either "X and Y" or "X or Y."

[0065] As used herein, the term about means + / - 10%, more preferably + / - 5% of the specified value, unless stated to the contrary.

[0066] The definition of volume percent (v / v%) is [(volume of solute) / (volume of solution)] × 100%. Volume percent 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 5ml FCS for every 100 ml of cell culture medium.

[0067] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to be meant to include a stated element, integer or step, or group of elements, integers or steps, but not to exclude any other element, integer or step, or group of elements, integers or steps.

[0068] [Stem cells] As used herein, the term "stem cell" refers to a self-renewing cell that can give rise to phenotypically and genotypically identical daughters and at least one other ultimate cell type (e.g., a terminally differentiated cell). The term "stem cell" includes totipotent, pluripotent and multipotent cells as well as progenitor and / or precursor cells derived from their differentiation. Stem cells may be adult or embryonic stem cells.

[0069] As used herein, the term "totipotent cell" or "totipotential cell" refers to a cell that is capable of forming a complete embryo (eg, a blastula).

[0070] As used herein, the term "pluripotent cell" or "pluripotential cell" refers to a cell that has the full differentiation potential, i.e., the ability to develop into any of the approximately 260 cell types in the mammalian body. Pluripotent cells can self-renew and remain dormant or quiescent within a tissue.

[0071] "Multipotential cell" or "multipotent cell" refers to a cell that can give rise to multiple mature cell types. As used herein, the term encompasses adult progenitor cells and the multipotent progeny of these cells. Unlike pluripotent cells, multipotent cells do not have the ability to form all of the cell types.

[0072] As used herein, the term "mesenchymal progenitor or stem cell" refers to a cell that can differentiate into a mesenchymal cell type. For example, mesenchymal progenitor and precursor cells can differentiate into bone tissue, cartilage tissue, muscle tissue, and fat cells, and fibrous connective tissue.

[0073] In one embodiment, the stem cells of the present invention are STRO-1+ mesenchymal precursor cells.

[0074] 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 follicle, intestine, lung, lymph nodes, thymus, bone, ligaments, tendons, 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 particular lineage commitment and differentiation pathway that these cells enter are determined by various influences from mechanical influences and / or intrinsic 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 give rise to daughter cells, either stem cells or progenitor cells, at a time when they will irreversibly differentiate to give rise to phenotypic cells.

[0075] In one embodiment, STRO-1+ cells are enriched from a sample obtained from a subject, e.g., the subject to be treated or a related or unrelated subject (whether of the same or different species). The terms "enriched" and "enrichment" or variations thereof are used herein to describe a population of cells in which the proportion of one particular cell type or the proportion of a number of particular cell types is increased compared to an untreated population of cells (e.g., cells in their native environment). In one embodiment, an enriched population of STRO-1+ cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, 75%, 85%, 95%, or 99% STRO-1+ cells. In this context, the term "enriched population of STRO-1+ cells" will be construed as explicitly supporting the term "a population of cells comprising X% STRO-1+ cells" (where X% is a percentage as described herein). STRO-1+ cells may, in some instances, form clonogenic colonies, e.g., CFU-F (fibroblasts), or a subset thereof (e.g., 50% or 60% or 70% or 80% or 90% or 95%) may have this activity.

[0076] In one embodiment, the stem cells of the present invention are enriched from a cell preparation comprising STRO-1+ cells in a selectable format. In this context, 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. MPCs) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 will also express STRO-1 (and STRO-1 bright(It may also be that the cell is selected based on expression of STRO-1+). Thus, indicating that a cell is STRO-1+ does not imply that the cell has been selected due to expression of STRO-1. In one embodiment, the cell is selected based on at least expression of STRO-3, e.g., STRO-3+(TNAP+). In one embodiment, the cell is selected based on at least expression of STRO-4, e.g., STRO-4+.

[0077] Reference to the selection of a cell or population does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated or enriched from a wide variety of sources. Accordingly, in some instances, these terms provide support for selection from either a tissue containing STRO-1+ cells (e.g., MPCs), 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.

[0078] In one embodiment, the stem cells of the present invention express one or more markers 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.

[0079] "Individually" means that the disclosure encompasses each listed marker or group of markers individually, and that the appended claims may define each such marker or group of markers individually and separably from one another, even though each marker or group of markers may not be individually listed herein.

[0080] "Collectively" means that the disclosure encompasses any number or combination of the listed markers or peptides, and that notwithstanding that such numbers or combinations of markers or markers may not be specifically recited herein, the appended claims may define such combinations or sub-combinations individually and separably from any other combinations of markers or markers.

[0081] In one embodiment, STRO-1+ cells are bright (syn.STRO-1 bri In one embodiment, STRO-1 bri STRO-1 cells dim or STRO-1 intermediate are preferentially enriched relative to cells.

[0082] In one embodiment, STRO-1 bright The cells may further be one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β) and / or CD146+. For example, the cells may be selected for one or more of the aforementioned markers and / or may show expression of one or more of these markers. In this regard, cells shown to express a marker need not be specifically tested, and conversely, previously enriched or isolated cells may be tested and subsequently used. Also, it may be reasonably assumed that the isolated or enriched cells express the same markers.

[0083] In one embodiment, STRO-1 bright is isolated by immune selection. In one embodiment, STRO-1 brightThe cells are isolated by immunoselection of cells expressing TNAP. The term "TNAP" as used herein is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, the term encompasses the liver isoform (LAP), the bone isoform (BAP) and the kidney isoform (KAP). In one embodiment, TNAP is BAP. In one embodiment, TNAP as used herein refers to a molecule capable of binding to STRO-3 antibody produced by a hybridoma cell line based on the deposit under the provisions of the Budapest Treaty, accession number PTA-7282, deposited with the ATCC on December 19, 2005.

[0084] In one embodiment, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ mesenchymal stem cells (e.g., remestemcel-L).

[0085] In one embodiment, the mesenchymal precursor cells are perivascular mesenchymal precursor cells as defined in WO2004 / 85630. For example, the mesenchymal precursor cells are cells that express perivascular cell markers, for example, STRO-1+ or STRO-1b. right and / or 3G5+, etc. In one embodiment, the cells are or were formerly progeny of cells isolated from vascular tissue or an organ or portion thereof.

[0086] When referring to cells that are "positive" for a given marker, it can be either low (lo or dim) or high (bright, bri) levels of that marker, depending on the extent to which the marker is present on the cell surface, and the term refers to the fluorescence intensity or other marker used in the cell sorting process. The distinction between lo (or dim or dull) and bri will be understood in the context of the marker 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, produces a very low signal or is not detected above background levels. For example, the level is detected using an isotype control antibody.

[0087] As used herein, the term "bright" refers to a marker on the cell surface that produces 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 The cells were non-bright cells (STRO-1 dull / dim ) yields a greater fluorescent signal when labeled with FITC-conjugated STRO-1 antibody as measured by fluorescence activated cell sorting (FACS) analysis. In one embodiment, the "bright" cells comprise at least about 0.1% of the most brightly labeled bone marrow mononuclear cells in the starting sample. In other embodiments, the "bright" cells comprise 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 in the starting sample. In one embodiment, the "bright" cells comprise 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 in the starting sample. brightThe cells have 2 log magnitude higher expression of STRO-1 surface expression compared to "background", i.e., cells that are STRO-1-. dim and / or STRO-1 intermediate The cells have STRO-1 surface expression of less than 2 log magnitude, and typically less than about 1 log or below "background."

[0088] In one embodiment, a significant proportion of STRO-1+ multilineage cells can differentiate into at least two different germline cell lines. Non-limiting examples of lineages to which multilineage cells can be committed include bone progenitor cells; hepatocyte progenitor cells that are multipotent for bile duct epithelial cells and hepatocytes; neural-restricted cells that can give rise to glial progenitor cells that progress to oligodendrocytes and astrocytes; neural progenitor cells that progress to neurons; cardiac muscle and cardiac muscle cell progenitors, glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells and chondrocytes, as well as the following progenitor cells: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal tubular epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes and oligodendrocytes.

[0089] In another embodiment, STRO-1+ cells are unable to give rise to hematopoietic cells in culture.

[0090] In one embodiment, the stem cells described herein are mesenchymal stem cells. Mesenchymal stem cells (MSCs) may be of homogenous composition or may be a mixed cell population enriched in MSCs. Homogeneous mesenchymal stem cell compositions may be obtained by culturing adherent bone marrow or periosteal cells, and mesenchymal stem cells may be identified by specific cell surface markers identified using specific monoclonal antibodies. Methods for obtaining cell populations 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.

[0091] The recognition, selection and purification of stem cells bearing the above-mentioned cell surface markers can be achieved by a number of different methods, such as by applying a binding agent for the marker of interest, followed by isolation of those cells that show either high levels of binding, or low levels of binding, or no binding at all.

[0092] For example, the binding agent can include an antibody, such as a monoclonal antibody or an antibody-based molecule.

[0093] Antibodies and other binding molecules can be used in a variety of techniques to select and purify stem cells expressing specific cell surface markers.

[0094] 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 solid matrices, fluorescence-activated cell sorting (FACS).

[0095] Stem cells of the invention expressing specific markers 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, e.g., Gronthos and Simmons 1995).

[0096] According to the present disclosure, isolated stem cells can be expanded in vitro by culturing. As will be appreciated by one of skill in the art, stem cells can be expanded in vitro by cryopreservation, thawing, and subsequent culturing. In one embodiment, stem cells are seeded in growth medium and allowed to attach to the culture vessel overnight at 37° C. and 20% O2. The growth medium is then replaced and the cells are cultured for an additional 68-72 hours at 37° C. and 5% O2.

[0097] In the examples, isolated stem cells are cultured at 50,000 cells / cm in serum-supplemented growth medium. 2 Cells are seeded at 4°C and allowed to attach to the culture vessel overnight at 37°C and 20% O2. The growth medium is then replaced with chondrocyte basal medium (Lonza, Walker CBM, MD) supplemented with 0.5% bovine serum albumin (BSA) and the cells are cultured for an additional 68–72 h at 37°C and 5% O2.

[0098] 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.

[0099] Cultured stem cells are phenotypically distinct from cells in vivo, for example, they can express CD44.

[0100] In one embodiment, cultured stem cells are biologically distinct from in vivo cells and have a faster renewal rate.

[0101] Cultured stem cells may be cryopreserved prior to administration to a subject, for example mesenchymal progenitor cells may be cryopreserved prior to administration to a subject.

[0102] [Genetically unmodified cells] As used herein, the term "genetically unmodified" refers to cells that have not been modified by transfection with a nucleic acid that expresses or encodes Ang1. For the avoidance of doubt, in the context of the present disclosure, stem cells transfected with a nucleic acid encoding Ang1 are considered to be genetically modified. In the context of the present disclosure, "genetically unmodified" cells express Ang1 to some degree without being transfected with a nucleic acid encoding native Ang1.

[0103] [Ang1 and / or VEGF expression] The stem cells of the present invention are genetically unmodified and have a concentration of at least 0.1 μg / 10 6 However, in various embodiments, the stem cells of the present invention express Ang1 at a concentration of at least 0.2 μg / 10 6 cells, 0.3μg / 10 6 cells, 0.4μg / 10 6 cells, 0.5μg / 10 6 cells, 0.6μg / 10 6 cells, 0.7μg / 10 6 cells, 0.8μg / 10 6 cells, 0.9μg / 10 6 cells, 1μg / 10 6 cells, 1.1μg / 10 6 cells, 1.2μg / 10 6 cells, 1.3μg / 10 6 cells, 1.4μg / 10 6 cells, 1.5μg / 10 6 It is envisaged that Ang1 may be expressed in amounts of cells.

[0104] In another embodiment, the genetically unmodified stem cells of the present invention are at about 0.05 μg / 10 6 In various embodiments, however, the stem cells of the present invention express VEGF at levels of less than about 0.05 μg / 10 6 cells, 0.04μg / 10 6 cells, 0.03μg / 10 6 cells, 0.02μg / 10 6 cells, 0.01μg / 10 6cells, 0.009μg / 10 6 cells, 0.008μg / 10 6 cells, 0.007μg / 10 6 cells, 0.006μg / 10 6 cells, 0.005μg / 10 6 cells, 0.004μg / 10 6 cells, 0.003μg / 10 6 cells, 0.002μg / 10 6 cells, 0.001μg / 10 6 It is envisioned that VEGF may be expressed in subcellular amounts.

[0105] The amount of cellular Ang1 and / or VEGF expressed in the composition or stem cell culture may be determined by methods well known to those of skill in the art. Such methods include, but are not limited to, quantitative assays, such as, for example, 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 the stem cells of the present invention.

[0106] In one embodiment, the level of Ang1 or VEGF expressed by the composition or stem cell culture is determined by an ELISA assay. In such an assay, cell lysates from stem cell cultures are added to the wells of an ELISA plate. The wells may be coated with a primary antibody, either monoclonal or polyclonal antibody(ies), against Ang1 or VEGF. The wells are then washed and then contacted with a secondary antibody, either monoclonal or polyclonal antibody(ies), against the primary antibody. The secondary antibody is conjugated to a suitable enzyme, such as horseradish peroxidase. The wells may then be incubated and washed after a period of incubation. The wells are then contacted with a suitable substrate for the enzyme conjugated to the secondary antibody, such as one or more chromogens. Chromogens that may be used include, but are not limited to, hydrogen peroxide and tetramethylbenzidine. After the substrate(s) are added, the wells are incubated for a suitable period of time. Once incubation is complete, a "stop" solution is added to the wells to stop the reaction of the substrate(s) with the enzyme. The optical density (OD) of the sample is measured and correlated with that of samples containing known amounts of Ang1 or VEGF to determine the amount of Ang1 or VEGF expressed by the stem cell culture being tested.

[0107] In another aspect, the genetically unaltered stem cells of the present disclosure express Ang1:VEGF in a ratio of at least about 2: 1. However, in various embodiments, it is contemplated that the stem cells of the present invention may express Ang1:VEGF in a ratio 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.

[0108] Methods for determining the expression ratio of Ang1:VEGF will be apparent to those skilled in the art. In an embodiment of the method for determining the expression ratio of Ang1 and VEGF, the expression levels of Ang1 and VEGF are quantified via quantitative ELISA as described above. In such an embodiment, after the levels of Ang1 and VEGF are quantified, 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.

[0109] [Cell composition] In one embodiment of the invention, the stem cells are administered in the form of a composition, hi one embodiment, such a composition comprises a pharma- ceutically acceptable carrier and / or excipient.

[0110] The terms "carrier" and "excipient" refer to a composition of matter conventionally used in the art to store, administer, and / or facilitate the biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th ed., Mac Publishing Company (1980)). A carrier may also reduce any undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other components in the carrier. In one embodiment, a carrier causes no significant local or systemic adverse effects in a recipient at the dosages and concentrations used therapeutically.

[0111] Suitable carriers for the present disclosure include those that are conventionally used.For example, physiological saline, aqueous dextrose, lactose, Ringer's solution, buffer, hyaluronic acid and glycols are exemplary liquid carriers, especially for liquids (when isotonic).Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerin, propylene glycol, water, ethanol, etc.

[0112] In another example, the carrier is, for example, a media composition that the cells are grown or suspended in. For example, the media composition does not induce any adverse effects in the subject to which it is administered.

[0113] Exemplary carriers and excipients do not adversely affect cell viability and / or the ability of the cells to reduce, prevent or delay metabolic syndrome and / or obesity.

[0114] In one embodiment, the carrier or excipient provides buffering, e.g., to maintain the cells and / or soluble factors at an appropriate pH, thereby exerting biological activity. For example, the carrier or excipient is phosphate buffered saline (PBS). PBS is an attractive carrier or excipient because it interacts minimally with the cells and factors, allowing for rapid release of the cells and factors, such that the compositions of the invention may be prepared as a liquid for direct application (e.g., by injection) into the bloodstream, or into tissues or into areas surrounding or adjacent to tissues.

[0115] Stem cells and / or their progeny may also be incorporated or embedded in scaffolds that are recipient-compatible and degrade into products that are not harmful to the recipient. These scaffolds provide support and protection for the cells that are transplanted into the recipient subject. Natural and / or synthetic biodegradable scaffolds are examples of such scaffolds.

[0116] 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 collagen, fibronectin and laminin scaffolds. Suitable synthetic materials for cell transplantation scaffolds should be able to support a wide range of cell growth and cell functions. Such scaffolds may be resorbable. 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 acids.

[0117] In another embodiment, the cells may be administered in a gel-like scaffold (such as Gelfoam from the Upjohn Company).

[0118] 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 invention just before or shortly before administration, or may be co-cultured together for a period of time before administration.

[0119] In one embodiment, the composition comprises an effective amount, or a therapeutically or prophylactically effective amount, of cells. For example, the composition comprises about 1×10 5 Approximately 1×10 stem cell cells / kg with high Ang1 levels 7 stem cells / kg, or approximately 1 x 10 6 Stem cells / kg ~ approx. 5 x 10 6 The exact amount of cells to be administered will depend on a variety of factors, including the age, weight and sex of the patient, as well as the extent and severity of the disorder being treated.

[0120] In one embodiment, a low dose of cells is administered to a subject. An exemplary dose is about 0.1×10 cells per kg. 4 ~about 0.5×10 6 Between 10 and 10 cells, for example, about 0.1 × 10 cells per kg 5 ~about 0.5×10 6 Between 10 and 10 cells, for example, about 0.5 × 10 cells per kg 5 ~about 0.5×10 6 Between 10 and 10 cells, for example, about 0.1 × 10 cells per kg 6 ~about 0.5×10 6 Between 10 and 10 cells, for example, about 0.2 × 10 cells per kg 6 pcs or 0.3×10 6 pcs or 0.4×10 6 Contains cells.

[0121] In one embodiment, a high dose of cells is administered to a subject. An exemplary dose is at least about 1.5×10 6 For example, a high dose would be approximately 1.5 x 10 cells / kg. 6 ~Approx. 6×10 6 Between 1.5 x 10 cells / kg, for example, about 1.5 x 10 6 ~Approx. 5×10 6 Between 1.5 x 10 cells / kg, for example, about 1.5 x 10 6 ~Approx. 4×10 6 Between 1.5 x 10 cells / kg, for example, about 1.5 x 10 6 ~Approx. 3×10 6 For example, a high dose would include approximately 1.5 x 10 cells / kg. 6 pcs or approx. 2 x 10 6 Contains cells / kg.

[0122] Other exemplary doses include at least about 1×10 6 For example, the dose may be about 1.0×10 6 ~Approx. 1×10 10 Between 1.1 x 10 cells, for example, 6 ~Approx. 1×10 9 Between 1.2 x 10 cells, for example, 6 ~Approx. 1×10 8 Between 1.3 x 10 cells, for example,6 ~Approx. 1×10 7 Between 1.4 x 10 cells, for example, 6 ~Approx. 9×10 6 Between 10 and 20 cells, for example, about 1.5 x 10 6 ~about 8x10 6 Between 1.6 x 10 cells, for example, 6 ~Approx. 7×10 6 Between 1.7 x 10 cells, for example, 6 ~Approx. 6×10 6 Between 1.8 x 10 cells, for example, 6 ~about 5x10 6 Between 1.9 x 10 cells, for example, 6 ~Approx. 4×10 6 Between 10 and 20 cells, for example, about 2 x 10 6 ~about 3x10 6 Includes cells between individuals.

[0123] In one embodiment, the dose is about 5×10 5 ~about 2×10 7 Between 10 and 20 cells, for example, about 6 × 10 6 ~ approx. 1.8 x 10 cells 7 For example, the dose may be about 6×10 6 pcs or approx. 1.8 x 10 7 Contains cells.

[0124] Mesenchymal precursors or stem cells comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the cell population of the composition.

[0125] In some embodiments, the cells are contained within a chamber that does not allow the cells to exit into the circulation of a subject, but allows factors secreted by the cells to enter the circulation. In such methods, a soluble factor may be administered to a subject by allowing the cells to secrete the factor into the circulation of the subject. Such a chamber may be simultaneously implanted at a site in the subject to increase local levels of the soluble factor, for example, in or near the heart.

[0126] The stem cells of the present disclosure are administered to an animal in an amount effective to treat the animal's disease or disorder. The animal may be a mammal, and the mammal may be a primate, including humans and non-human primates. The stem cells may be administered systemically, such as, for example, by intravenous, intraarterial, or intraperitoneal administration. The exact dose of stem cells administered depends on a variety of factors, including, but not limited to, the age, weight, and sex of the patient, the disease(s) or disorder(s) being treated, and their extent and severity.

[0127] Compositions comprising stem cells of the present disclosure may be cryopreserved. Cryopreservation of stem cells can be accomplished using slow cooling methods or "rapid" freezing procedures well known in the art. Preferably, the cryopreservation method maintains a similar phenotype, cell surface markers, and growth rate of the cryopreserved cells compared to non-frozen cells.

[0128] The cryopreserved composition may contain a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0129] The cryopreservation medium may, for example, comprise Plasmalyte A®, a sterile, non-pyrogenic, isotonic solution. 100 mL of Plasmalyte A® contains 526 mg of Sodium Chloride, USP (NaCl), 502 mg of Sodium Gluconate (C6H 11NaO7), 368 mg Sodium Acetate Trihydrate, USP (C2H3NaO2·3H2O), 37 mg Potassium Chloride, USP (KCI), and 30 mg Magnesium Chloride, USP (MgCl2·6H2O). Contains no antimicrobial agents. pH is adjusted with sodium hydroxide. pH is 7.4 (6.5-8.0).

[0130] To facilitate freezing, a cryoprotectant, such as dimethyl sulfoxide (DMSO), is usually added to the cryopreservation solution. Ideally, the cryoprotectant should be non-toxic for the cells and the patient, chemically inert, non-antigenic, provide high survival rates after thawing, and allow 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.

[0131] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one embodiment, the cryopreservation solution includes about 5% human serum albumin (HSA) and about 10% DMSO. The cryopreservation solution may further include one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0132] The cryopreserved composition may be thawed and administered directly to a subject, or the cryopreserved composition may be thawed and the mesenchymal precursors or stem cells resuspended in a replacement solution prior to administration.

[0133] [Cell culture method] The compositions of the present disclosure can be produced by a variety of cell culture methods.

[0134] Therefore, the present invention also provides an in vitro method for inducing Ang1 expression in stem cells.Surprisingly, the present inventors have identified cell culture medium conditions that induce Ang1 expression in stem cells.These conditions are also found to reduce VEGF expression and induce a high ANG1:VEGF ratio.

[0135] For example, these conditions may include culturing a population of stem cells in a cell culture medium comprising: i) contains a short-acting L-ascorbic acid derivative but does not contain a significant amount of a long-acting L-ascorbic acid derivative; and / or ii) Fetal bovine serum less than 10% (v / v).

[0136] In another embodiment, these conditions include culturing the population of stem cells in a cell culture medium comprising: i) Contains a short-acting L-ascorbic acid derivative but does not contain an appreciable amount of a long-acting L-ascorbic acid derivative. ii) Fetal bovine serum less than 10% (v / v); and / or iii) Non-fetal serum.

[0137] Thus, in one embodiment, the present disclosure relates to an in vitro method for inducing expression of Ang1 in stem cells, the method comprising culturing a population of stem cells in a cell culture medium comprising a short-acting L-ascorbic acid derivative but not containing an equivalent amount of a long-acting L-ascorbic acid derivative, and / or supplemented with less than 10% (v / v) fetal bovine serum.

[0138] In another embodiment, the present disclosure relates to an in vitro method for inducing expression of Ang1 in stem cells, the method comprising: culturing a population of stem cells in a cell culture medium, the cell culture medium comprising a short-acting L-ascorbic acid derivative but not comprising an equivalent amount of a long-acting L-ascorbic acid derivative, and / or supplemented with non-fetal serum.

[0139] In another embodiment, the disclosure relates to an in vitro method for inducing expression of Ang1 in stem cells, the method comprising: culturing a population of stem cells in a cell culture medium comprising a sufficient amount of human adult serum (e.g., human AB serum) to support the growth of the cells and non-fetal serum in the form of a cell lysate of human platelets.

[0140] The term "media" or "medium" as used in connection with cell culture includes components of the environment surrounding the cells. It is envisioned that the medium contributes to and / or provides conditions sufficient to induce expression of Ang1 expression. The medium may be solid, liquid, gaseous, or a mixture of phases and substances. Media can include liquid growth media and liquid media that do not support cell growth. Media also include gelatinous media such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed. The term "media" also refers to a substance intended for use in cell culture, even if it is not yet in contact with the cells.

[0141] The culture medium used in the method of the present disclosure can be prepared using a medium used for culturing stem cells as a basal medium. The basal medium includes, for example, Eagle's minimum essential (MEM) medium, α modified MEM medium, and mixed culture medium thereof, and is not particularly limited as long as it can be used for culturing stem cells.

[0142] In addition, the culture medium of the present invention can contain optional components such as fatty acids or lipids, vitamins, growth factors, cytokines, antioxidants, buffering agents, inorganic salts, and the like.

[0143] The cell culture medium used in the present disclosure includes all essential and optionally non-essential amino acids, which are generally classified as essential (threonine, methionine, valine, leucine, isoleucine, phenylalanine, tryptophan, lysine, histidine) and non-essential (glycine, alanine, serine, cysteine, glutamine, asparagine, aspartic acid, tyrosine, arginine, proline).

[0144] [Ascorbic acid] Ascorbic acid is an essential supplement for the growth and differentiation of various cells in culture. It is now understood that certain ascorbic acid derivatives are "short-acting" because they are not stable in solution, especially under normal cell culture conditions at neutral pH and 37°C. These short-acting derivatives are rapidly oxidized to oxalic acid or threonic acid. In culture medium at 37°C (pH 7), the levels of the short-acting ascorbic acid derivatives are reduced by approximately 80-90% after 24 hours due to oxidation. Thus, the short-acting ascorbic acid derivatives are replaced by the more stable "long-acting" ascorbic acid derivatives in conventional cell cultures of various cell types.

[0145] In the context of the present disclosure, the term "short-acting" includes ascorbic acid derivatives that are approximately 80-90% oxidized after 24 hours of cell culture under culture conditions of neutral pH and 37° C. In one embodiment, the short-acting L-ascorbic acid derivative is an L-ascorbate salt. For example, in the context of the present disclosure, sodium L-ascorbate salt is a "short-acting" ascorbic acid derivative.

[0146] In contrast, the term "long-acting" includes ascorbic acid derivatives that are not oxidized by about 80-90% after 24 hours in cell culture under culture conditions of neutral pH and 37°C. In one embodiment, 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 tetrahexyldecyl ascorbate, magnesium ascorbyl phosphate, and 2-O-α-D-glucopyranosyl-L-ascorbic acid.

[0147] The inventors have surprisingly found that replacement of a long-acting ascorbic acid derivative with a short-acting derivative can induce expression of Ang1 in stem cells. Thus, in an embodiment of the present disclosure, the cell culture medium is supplemented with a short-acting ascorbic acid derivative. For example, the cell culture medium may comprise at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another embodiment, the cell culture medium may comprise at least about 0.01 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may comprise at least about 0.02 g / L of the short-acting ascorbic acid derivative. In another embodiment, the cell culture medium may comprise at least about 0.03 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may comprise at least about 0.04 g / L of the short-acting ascorbic acid derivative. In another embodiment, the cell culture medium may comprise at least about 0.05 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may include at least about 0.06 g / L of a 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.

[0148] In another embodiment, the cell culture medium includes a short-acting ascorbic acid derivative but does not include a significant amount of a long-acting ascorbic acid derivative. For example, the cell culture medium may include a short-acting ascorbic acid derivative but not more than 0.04 g / L of the long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may include a short-acting ascorbic acid derivative but not more than 0.03 g / L of the long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may include a short-acting ascorbic acid derivative but not more than 0.02 g / L of the long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may include a short-acting ascorbic acid derivative but not more than 0.01 g / L of the long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may include a short-acting ascorbic acid derivative but not more than 0.005 g / L of the long-acting ascorbic acid derivative. In another embodiment, the cell culture medium may include a short-acting ascorbic acid derivative, but may not include a long-acting ascorbic acid derivative.

[0149] In another embodiment, the cell culture medium contains L-ascorbic acid sodium salt but does not contain an equivalent amount of L-ascorbic acid-2-phospahte.

[0150] [serum] The medium used in the culture method of the present disclosure can be a serum-containing medium or a serum-free medium.

[0151] The culture medium of the present disclosure may or may not contain a serum replacement. The serum replacement may be, for example, albumin (e.g., lipid-rich albumin), transferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thiolglycerol, or a serum equivalent that appropriately contains the same. Such serum replacement may be prepared, for example, by the method described in International Patent Application No. 93 / 30679, or may be commercially available.

[0152] Traditionally, stem cells are maintained in cell culture using medium supplemented with at least about 10-15% (v / v) serum, typically fetal calf serum (FCS). However, the present inventors have found that expression of Ang1 can also be induced by culturing a population of stem cells in cell culture medium supplemented with less than 10% (v / v) FCS. In one embodiment, the population of stem cells is cultured in cell culture medium 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. It is also envisioned that the terms fetal calf serum (FCS) and fetal bovine serum (FBS) may be used interchangeably in the context of the present invention.

[0153] In one embodiment, the cell culture medium is supplemented with non-fetal serum. It is contemplated that 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) of non-fetal serum.

[0154] For example, the culture medium can be supplemented with non-fetal mammalian serum.

[0155] For example, the culture medium can be supplemented with human non-fetal serum.

[0156] For example, the culture medium can be supplemented with neonatal serum. It is contemplated that 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) of neonatal serum.

[0157] In one embodiment, the cell culture medium is supplemented with neonatal mammalian serum.

[0158] For example, the culture medium can be supplemented with newborn bovine serum (NBCS). It is contemplated that 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%, or at least about 25% (v / v) of NBCS.

[0159] In one embodiment, the cell culture medium is supplemented with human newborn serum.

[0160] 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 newborn serum. For example, human newborn serum is obtained from "umbilical cord blood".

[0161] In one embodiment, the culture medium is supplemented with adult serum. It is contemplated that 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) of adult serum.

[0162] In one embodiment, the cell culture medium is supplemented with adult mammalian serum.

[0163] 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 adult mammalian serum.

[0164] 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.

[0165] In one embodiment, the cell culture medium is supplemented with adult human serum.

[0166] 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.

[0167] 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 AB serum.

[0168] In one embodiment, the cell culture medium is supplemented with at least about 3% human AB serum.

[0169] In one embodiment, the culture medium is supplemented with a mixture of FCS and NBCS.

[0170] 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.

[0171] For example, it is contemplated that the mixture of FCS and NBCS may comprise at least about 1% (v / v), at least about 2% (v / v), at least about 3% (v / v), at least about 4% (v / v), at least about 5% (v / v), at least about 6% (v / v), at least about 7% (v / v), at least about 8% (v / v), at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25% (v / v) of the cell culture medium. However, in this embodiment, the cell culture medium is supplemented with at least about 1% (v / v), at least about 2% (v / v), at least about 3% (v / v), at least about 4% (v / v), at least about 5% (v / v), at least about 6% (v / v), at least about 7% (v / v), at least about 8% (v / v), at least about 9% (v / v), but less than 10% (v / v) FCS.

[0172] In one embodiment, the cell culture medium is FCS serum-free.

[0173] In one embodiment, the cell culture medium is fetal serum free.

[0174] In one embodiment, the cell culture medium is supplemented with non-fetal serum.

[0175] In one embodiment, the culture medium is fetal serum free and supplemented with non-fetal serum.

[0176] [Stimulating factor] In another embodiment, the cell culture medium is supplemented with one or more stimulatory factors selected from the group consisting of 1α,25-dihydroxyvitamin D3 (1,25D), platelet-derived growth factor (PDGF) such as PDGF-BB, tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), stromal-derived factor alpha (SDF-1α), and EGF.

[0177] In another embodiment, the cells can be cultured in the presence of at least one cytokine in an amount sufficient to support proliferation of the cells.

[0178] In another embodiment, the cells are cultured in the presence of a platelet cell lysate in an amount sufficient to support the growth of the cells. For example, the cells can be cultured with a human platelet cell lysate in an amount sufficient to support the growth of the cells.

[0179] In one embodiment, the cells are cultured with human AB serum and human platelet cell lysate in an amount sufficient to support the growth of the cells.

[0180] [Analyzing the therapeutic / prophylactic potential of cells] Methods for determining the ability of the cells of the present disclosure to treat or prevent or delay the onset or progression of a disease will be apparent to one of skill in the art. For example, the stem cells of the present invention can be evaluated for their ability to increase the levels of Ang1.

[0181] In one embodiment, at least 0.1 μg / 10 6 Unmodified stem cells expressing Ang1 in cellular quantities are tested for their ability to increase levels of Ang1 in cardiac tissue in vitro and / or in vivo, in these examples, Ang1 levels are assessed in cell culture medium or tissue following administration of stem cells as described herein.

[0182] It will be apparent to one of skill in the art from the above that the present disclosure also provides the following methods for identifying or isolating cells for treating, preventing or delaying disease. (i) Cells are administered to a test subject suffering from the relevant disease and the subject is assessed for symptoms of the disease. (ii) comparing symptoms of the disorder in the subject of (i) with symptoms of the disease or activity in a control subject suffering from the disease but not administered the cells, where an improvement in the symptoms in the test subject compared to the control subject indicates that the stem cells treat the disease. The cells may be any cell described in this disclosure, according to any embodiment.

[0183] It will be understood by those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive. EXAMPLES

[0184] 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 a lithium-heparin anticoagulant-containing tube.

[0185] BMMNCs are prepared by density gradient separation using Lymphoprep™ (Nycomed Pharma, Oslo, Norway) as previously described (Zannettino et al. 1998). After centrifugation at 400×g and 4° C. for 30 min, the pale yellow layer is removed with a transfer pipette and washed three times in “HHF” consisting of Hanks’ Balanced Salt Solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0186] Next, STRO-3 +(or TNAP + ) cells were isolated by magnetic activated cell sorting as previously described (Gronthos et al. 2003; Gronthos and Simmons 1995). Briefly, approximately 1-3 × 10 8 BMMNCs are incubated on ice for 20 min in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells are incubated on ice for 1 h with 200 μl of STRO-3 mAb solution at 10 μg / ml in blocking buffer. The cells are then washed twice in HHF by centrifugation at 400×g. Goat anti-mouse γ-biotin (Southern Biotechnology Associates, Birmingham, UK) diluted 1 / 50 in HHF buffer is added and the cells are incubated on ice for 1 h. The cells are then incubated in MACS buffer (Ca2+, supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide) as above. 2+ and Mn 2+ The cells are washed twice in PBS (without HCl) and resuspended in a final volume of 0.9 ml MACS buffer.

[0187] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) are added to the cell suspension and incubated on ice for 15 min. The cell suspension is washed twice with 0.5 ml of MACS buffer, resuspended, and then loaded onto a mini MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3mAb (deposited at the American Type Culture Collection (ATCC) on December 19, 2005 under accession number PTA-7282; see International Patent Application No. 2006 / 108229). After adding a further 1 ml of MACS buffer, the column is removed from the magnet and TNAP +Cells are isolated by positive pressure. An aliquot of cells from each fraction can be stained with streptavidin-FITC and purity assessed by flow cytometry.

[0188] MPCs isolated in this way express STRO-1 bright It is MPC.

[0189] Example 2: Culture Initiation - Process A Alpha modifications of Eagle's minimum essential medium (MEM) with Earle's balanced salts, commonly referred to as Eagle's alpha MEM, contain nonessential amino acids, sodium pyruvate, and supplemented vitamins. These modifications were first described for use in growing hybrid mouse and hamster cells (Stanners et al. 1971).

[0190] Eagle's αMEM medium, suitable for culturing primary stem cells, can be obtained from a variety of sources, such as Life Technologies and Sigma.

[0191] Detailed methods for establishing primary stem cell cultures containing the necessary growth factors used in the exemplary process are described in Gronthos and Simmons 1995.

[0192] In process A, Eagle's αMEM medium supplemented with 10% fetal bovine serum, L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10−7 M), and / or inorganic phosphate (3 mM) was used to culture the stem cells.

[0193] Example 3: Modified Culture Medium - Process B In Process B, the Eagle's αMEM medium used in Process A was modified (modified αMEM) with: -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).

[0194] [Table 1]

[0195] Example 4: Cell Culture

[0196] Mesenchymal precursor cells (MPCs) are obtained from a single donor and preserved following cryopreservation.

[0197] Generally, cell culture involves the following steps.

[0198] Thaw cryopreserved MPCs and inoculate them at 10,000 cells / cm. 2 and grown at 37°C with 20% O2 until 90% confluence in either starting medium (process A, n = 3) or modified culture medium (process B, n = 3).

[0199] To generate conditioned medium, growth medium was added to 200 μl medium / cm 2 The medium was replaced with EBM-2 basal medium (Lonza) supplemented with FCS at a volume of 100 μg / ml. The cells were cultured for an additional 3 days, after which the medium was harvested and centrifuged to remove any cells, and the resulting supernatant was collected and stored at -80°C.

[0200] Growth factor concentrations were measured using the Luminex platform using commercially available kits (Millipore).

[0201] Following cell culture, MPC proliferation dynamics were assessed (see Figures 1-3). No significant changes in cell proliferation, MPC doubling time or population doubling time were observed following cell culture processes A and B.

[0202] MPCs are also characterized in terms of expression levels of the cell markers STRO-1, CC9 and STRO-4 as well as the pro-angiogenic growth factors Ang1 and VEGF.

[0203] Levels of STRO-1, CC9 and STRO-4 were comparable in MPCs after cell culture processes A and B.

[0204] However, the cultivation process B is - Increases Ang1 levels, - Reduces VEGF levels, - Provides a ratio of Ang1:VEGF that is consistent with Ang1:VEGF ratios previously shown to be particularly effective in enhancing angiogenesis.

[0205] Ang1 and VEGF levels (μg / 10 6 The measured values ​​for the number of cells are shown in Table 2.

[0206] [Table 2]

[0207] Example 5: Modified Culture Conditions - Processes C and D To control for 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 serially propagated 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) with growth factors such as PDGF and EGF.

[0208] Ang1 and VEGF levels were assessed following cell culture in processes C and D. Ang1 and VEGF levels (μg / ml) in conditioned medium of MPCs cultured in process C or D are shown in Table 3.

[0209] Compared to process C, cultivation process D has the following advantages: - Increases Ang1 levels, - Reduces VEGF levels, - Increases the VEGF:Ang1 ratio.

[0210] Compared to Process A, Processes C and D lead to a progressive increase in the expression levels of Ang1, suggesting that the short-acting ascorbic acid derivative and the presence of non-fetal serum each independently lead to increased expression of Ang1 and exert a synergistic effect to further increase Ang1 expression.

[0211] [Table 3]

[0212] It will be appreciated by those skilled in the art that numerous variations and / or modifications may be made to the present disclosure as illustrated in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0213] This disclosure claims priority to AU2014901247, filed April 7, 2014, the disclosure of which is incorporated herein by reference.

[0214] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0215] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and should not be understood as an admission that any or all of such matter existed prior to the priority date of each claim of this application and therefore forms part of the prior art base or is common general knowledge in the field relevant to the present invention.

Claims

1. 1. A composition comprising genetically unmodified stem cells, the genetically unmodified stem cells being at least 0.1 μg / 10 ６ A composition comprising an amount of cells expressing angiopoietin-1 (Ang1).

2. The stem cells are at least 0.5 μg / 10 ６ The composition of claim 1 , wherein the composition expresses Ang1 in a cellular amount.

3. The stem cells are at least 0.7 μg / 10 ６ The composition of claim 1 , wherein the composition expresses Ang1 in a cellular amount.

4. The stem cells are at least 1 μg / 10 ６ The composition of claim 1 , wherein the composition expresses Ang1 in a cellular amount.

5. The stem cells are about 0.05 μg / 10 ６ The composition according to any one of claims 1 to 4, which expresses vascular endothelial growth factor (VEGF) in amounts less than that of cells.

6. The stem cells are about 0.05 μg / 10 ６ The composition according to any one of claims 1 to 5, which expresses VEGF in amounts less than that of cells.

7. The stem cells are about 0.03 μg / 10 ６ The composition according to any one of claims 1 to 5, which expresses VEGF in amounts less than that of cells.

8. The composition of any one of claims 1 to 7, wherein the stem cells express Ang1:VEGF in a ratio of at least 2:

1.

9. The composition of any one of claims 1 to 7, wherein the stem cells express Ang1:VEGF in a ratio of at least 10:

1.

10. The composition of any one of claims 1 to 7, wherein the stem cells express Ang1:VEGF in a ratio of at least 20:

1.

11. The composition of any one of claims 1 to 7, wherein the stem cells express Ang1:VEGF in a ratio of at least 30:

1.

12. The composition of any one of claims 1 to 7, wherein the stem cells express Ang1:VEGF in a ratio of at least 50:

1.

13. The composition according to any one of claims 1 to 12, wherein the stem cells are mesenchymal stem cells.

14. The composition of claim 13 , wherein the stem cells are mesenchymal progenitor cells.

15. The composition of any one of claims 1 to 14, further comprising a pharma- ceutically acceptable carrier.

16. 1. An in vitro method for inducing expression of Ang1 in stem cells, the method comprising culturing a population of stem cells in a cell culture medium, the cell culture medium comprising: contains a short-acting L-ascorbic acid derivative but does not contain a significant amount of a long-acting L-ascorbic acid derivative; and / or Supplemented with less than 10% (v / v) fetal calf serum (FCS), and / or The cell culture medium is supplemented with non-fetal serum.

17. The method of claim 16, further comprising measuring the level of Ang1 to determine that expression of Ang1 is induced.

18. The method of claim 16 or 17, further comprising selecting cells with induced expression of Ang1.

19. The method according to any one of claims 16 to 18, wherein the short-acting L-ascorbic acid derivative is L-ascorbate.

20. 20. The method of claim 19, wherein the short-acting L-ascorbate salt is L-ascorbic acid sodium salt.

21. The method according to any one of claims 16 to 20, wherein the cell culture medium is supplemented with at least about 5% (v / v) FCS.

22. The cell culture medium is a medium containing 1α,25-dihydroxyvitamin D ３ 22. The method of any one of claims 16 to 21, wherein the stimulatory factor is supplemented with one or more stimulatory factors selected from the group consisting of (1,25D), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor lα (SDF-lα).

23. The method of any one of claims 16 to 22, wherein the cell culture medium is supplemented with non-fetal serum.

24. 24. The method of claim 23, wherein the non-fetal serum is newborn serum.

25. 25. The method of claim 24, wherein the newborn serum is newborn bovine serum (NBCS).

26. 25. The method of claim 24, wherein the newborn serum is human serum.

27. 24. The method of claim 23, wherein the non-fetal serum is adult serum.

28. The method of claim 23, wherein the adult serum is human serum.

29. 26. The method of any one of claims 16 to 25, wherein the cell culture medium comprises NBCS at a level of at least about 5% (v / v).

30. 26. The method of any one of claims 16 to 25, wherein the cell culture medium comprises NBCS at a level of at least about 2% (v / v).

31. The method according to any one of claims 16 to 25, wherein the cell culture medium is supplemented with a mixture of NBCS and FCS.

32. 32. The method of claim 31 , wherein the ratio of FCS to NBCS is about 1:

1.

33. 33. The method according to claim 31 or 32, wherein the culture medium is supplemented with 5% (v / v) FCS and 5% (v / v) NBCS.

34. 1. A method for obtaining genetically unmodified stem cells suitable for use in promoting vascularization and / or angiogenesis, comprising: Obtaining at least one cell population comprising stem cells from at least one donor; Culturing the stem cells; determining the amount of Ang1 expressed by the stem cells of each of the at least one cell population; At least 0.1 μg / 10 ６ and selecting cells expressing Ang1 in the amount of cells.

35. determining the amount of VEGF expressed by the stem cells of each of the at least one cell population(s); 35. The method of claim 34, further comprising selecting stem cells that express Ang1:VEGF in a ratio of at least 10:

1.

36. 36. The method of claim 35, wherein the stem cells express Ang1:VEGF in a selected ratio of at least 20:

1.

37. 36. The method of claim 35, wherein the stem cells expressing Ang1:VEGF in a ratio of at least 30:1 are selected.

38. 36. The method of claim 35, wherein the stem cells expressing Ang1:VEGF in a ratio of at least 50:1 are selected.

39. Use of a composition according to any one of claims 1 to 15 for promoting vascularisation and / or angiogenesis.

40. Use of a composition according to any one of claims 1 to 15 for treating a condition in which increased Ang1 expression is desired.

41. A method for promoting vascularization and / or angiogenesis in a subject, comprising administering to the subject a composition according to any one of claims 1 to 15.

42. A method for treating a condition in a subject in which additional Ang1 expression is desired, comprising administering to the subject a composition according to any one of claims 1 to 15.

43. A stem cell population cultured by the method according to any one of claims 16 to 33 or obtained by the method according to any one of claims 34 to 38.

44. The composition according to any one of claims 1 to 15, wherein said stem cells are produced by culturing a population of stem cells according to a method according to any one of claims 16 to 33, or obtained by a method according to any one of claims 34 to 38.

45. Use of a composition according to any one of claims 1 to 15 or a method according to any one of claims 16 to 38 in the manufacture of a medicament for promoting vascularization and / or angiogenesis.

46. Use of a composition according to any one of claims 1 to 15 or a method according to any one of claims 16 to 38 in the manufacture of a medicament for treating a condition in which increased Ang1 expression is desired.

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