Conditioned medium and uses thereof

Abstract

The present disclosure relates to conditioned media and methods for producing the same, which may be particularly useful in providing therapeutic compositions.

Description

[Technical Field]

[0001] The present disclosure relates to conditioned media and methods for producing the same, which may be particularly useful in providing therapeutic compositions. [Background technology]

[0002] Multipotent mesenchymal stem cells (MLPSCs), such as multipotent mesenchymal stem cells (MSCs), have been proposed as attractive candidates for therapeutic applications due to their beneficial properties, such as high proliferation and differentiation potential and immunomodulatory / anti-inflammatory properties (Caplan AI (2007) J. Cell Physiol., 213, 341-347; Prockop DJ (2007) Clin Pharmacol Ther., 82, 241-243). To generate numbers suitable for therapeutic applications, it is often necessary to expand sparse populations of MSCs in vitro.

[0003] Despite various advances, in vitro expansion of MLPSCs, while important, still has limitations. Moreover, recent studies have demonstrated that MLPSCs may exert biological effects through secreted factors.

[0004] Thus, there remains an unmet need for compositions and methods for consistently producing therapeutically effective compositions comprising MLPSCs and / or factors derived therefrom. Summary of the Invention

[0005] The present inventors have unexpectedly demonstrated that culturing MLPSCs with the addition of neonatal serum results in conditioned medium with enhanced angiogenic potential. For example, neonatal bovine serum (NBCS) is commonly marketed as an equivalent / acceptable alternative to fetal bovine serum (FBS). However, the present inventors unexpectedly discovered that this is not the case, as the addition of NBCS induced increased levels of angiogenic markers, as described herein. Furthermore, conditioned medium obtained from MLPSCs cultured in NBCS-supplemented medium promoted angiogenesis. Analysis of neonatal serum used in culturing MLPSCs surprisingly revealed increased levels of cytokines, particularly for cytokines whose corresponding receptors are expressed by MLPSCs. These findings provide the basis for generating novel compositions by culturing and expanding MLPSCs with specific proinflammatory cytokines and / or neonatal serum.

[0006] Thus, in one aspect, the present invention relates to a composition comprising conditioned medium or extracellular vesicles obtained thereby, wherein the conditioned medium is obtained from a population of MLPSCs culture-expanded in a medium containing interferon (IFN)-γ and / or tumor necrosis factor (TNF)-α, and the level(s) of IFN-γ and / or TNF-α in the medium is less than 1 ng / ml. For example, the level of IFN-γ can be <500 pg / ml. In one example, the level of IFN-γ is <100 pg / ml. In one example, the level of TNF-α is <750 pg / ml. In another example, the level of TNF-α is <500 pg / ml. In one example, the levels of IFN-γ and TNF-α are both <500 pg / ml. In these examples, the conditioned medium can be characterized by increased levels of angiogenic marker(s), where the increased levels are determined compared to conditioned medium obtained from a control population.

[0007] In another aspect, the present disclosure relates to a composition comprising conditioned medium or extracellular vesicles obtained thereby, wherein the conditioned medium is characterized by increased levels of angiogenic marker(s), and the conditioned medium is IFN-γ and / or TNF-α, and / or - obtained from a population of MLPSCs culture-expanded in a culture medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, IP-10; Increased levels of angiogenic marker(s) are determined relative to conditioned medium obtained from a control population.

[0008] In one example, the angiogenic marker is increased levels of angiogenin compared to conditioned medium obtained from a control group.

[0009] In another example, a marker of angiogenesis is increased endothelial network formation compared to conditioned medium obtained from a control group.

[0010] In another example, a marker of angiogenesis is increased endothelial length compared to conditioned medium obtained from a control group.

[0011] In another example, the angiogenic marker is increased endothelial branch length compared to conditioned medium obtained from a control population.

[0012] In one example, the angiogenic marker is - increased levels of angiogenin compared to conditioned medium obtained from the control group, - increased endothelial network formation compared to conditioned medium obtained from the control group, - increased endothelial length compared to conditioned medium obtained from the control group, - one or more or all of the endothelial branch lengths are increased compared to conditioned medium obtained from a control population.

[0013] In one example, the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

[0014] In another example, the conditioned medium contains an angiogenin level greater than about 1200 pg / ml. In another example, the conditioned medium contains an SDF-1 level greater than about 3000 pg / ml. In another example, the conditioned medium contains a VEGF level greater than about 3200 pg / ml. In another example, the conditioned medium contains a VEGF level greater than about 0.12 mm 2 / mm 2 In another example, the conditioned medium induces the formation of an endothelial network of approximately 5 mm 2 / mm 2 In another example, the conditioned medium induces an endothelial network length of about 15 1 / mm 2 Induce ultra-endothelial branch length.

[0015] In another example, the culture medium contains three or more proinflammatory cytokines. In another example, the culture medium contains two or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In another example, the culture medium contains IL-6. In another example, the culture medium contains IL-8 and / or IL-17A.

[0016] In another example, the culture medium contains IFN-γ and TNF-α. In another example, the IFN-γ level is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml. In another example, the TNF-α level is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

[0017] In another example, the culture medium contains serum that includes pro-inflammatory cytokines.

[0018] In another example, the serum is a newborn mammal serum. For example, the newborn mammal serum can be newborn bovine serum. In one example, the serum is obtained within 21 days after birth.

[0019] In one example, the culture medium comprises: i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0020] In another example, the culture medium comprises at least 5% (v / v) newborn mammalian serum. In another example, the culture medium is serum-free.

[0021] In one example, the extracellular vesicles obtained from the conditioned medium are exosomes.

[0022] In another example, the present disclosure relates to conditioned medium and extracellular vesicles obtained thereby produced by culturing a population(s) of MLPSCs in neonatal serum. Accordingly, in one example, the present disclosure relates to a composition comprising conditioned medium or extracellular vesicles obtained thereby, wherein the conditioned medium is obtained from a population of MLPSCs culture-expanded in a medium containing neonatal mammalian serum. In one example, the serum is neonatal bovine serum. In one example, the neonatal serum is obtained within 21 days after birth.

[0023] In another example, the conditioned medium comprises an increased level of angiogenic marker(s) compared to conditioned medium obtained from a control population. In one example, the angiogenic marker is an increased level of angiogenin compared to conditioned medium obtained from a control group. In another example, the angiogenic marker is increased endothelial network formation compared to conditioned medium obtained from a control group. In another example, the angiogenic marker is increased endothelial length compared to conditioned medium obtained from a control group. In another example, the angiogenic marker is increased endothelial branch length compared to conditioned medium obtained from a control population. In one example, the angiogenic marker is - increased levels of angiogenin compared to conditioned medium obtained from the control group, - increased endothelial network formation compared to conditioned medium obtained from the control group, - increased endothelial length compared to conditioned medium obtained from the control group, - one or more or all of the endothelial branch lengths are increased compared to conditioned medium obtained from a control population.

[0024] In one example, the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the conditioned medium contains an angiogenin level greater than about 1200 pg / ml. In another example, the conditioned medium contains an SDF-1 level greater than about 3000 pg / ml. In another example, the conditioned medium contains a VEGF level greater than about 3200 pg / ml. In another example, the conditioned medium contains a VEGF level greater than about 0.12 mm 2 / mm 2 In another example, the conditioned medium induces the formation of an endothelial network of approximately 5 mm 2 / mm 2 In another example, the conditioned medium induces an endothelial network length of about 15 1 / mm 2 Induce ultra-endothelial branch length.

[0025] In another example, the newborn serum is serum from a newborn on postnatal day 1 to day 7. In one example, the concentration of the newborn serum is about 2% (v / v) to about 12% (v / v). In another example, the concentration of the newborn serum is about 5% (v / v).

[0026] In another example, the culture medium contains fetal serum and neonatal serum of the same species, and the ratio of the concentration of the fetal serum to the concentration of the neonatal serum is 1: 1 or less. For example, the concentration of the fetal serum and the concentration of the neonatal serum may be 5% (v / v), respectively.

[0027] In another example, the concentration in fetal serum will be lower than the concentration in newborn serum.

[0028] In another example, the extracellular vesicles obtained from the conditioned medium are exosomes.

[0029] In another example, the MLPSCs are human mesenchymal stem cells (hMSCs). + The population of pluripotent cells is culture expanded. [Brief explanation of the drawings]

[0030] [Figure 1] Assessment and comparison of serum cytokine levels in 1:1 FCS / NBCS (serum A), fetal bovine serum (serum B), and FBS from another source (serum C). [Figure 2] Quantitative measurement of in vitro angiogenesis induced by MLPSC-conditioned medium using the IncuCyte® 96-Well Kinetic Angiogenesis PrimeKit Assay. [Figure 3] 1 shows the results of a Luminex assay demonstrating increased production of angiogenin by MLPSCs cultured with or without neonatal serum. [Figure 4] Levels of angiogenic markers in cGMP lot MLPSC conditioned medium cultured with or without neonatal serum. DETAILED DESCRIPTION OF THE INVENTION

[0031] General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein are assumed to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular biology, stem cell biology, biochemistry).

[0032] Unless otherwise indicated, the cell culture techniques and assays utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are explained in such publications as J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G.lover and B.D. Hames (editors), and F.M.A.usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0033] The term "and / or", e.g., "X and / or Y", is understood to mean either "X and Y" or "X or Y", and is considered to explicitly endorse both meanings or either meaning.

[0034] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0035] The term "level" is used to define the amount of a particular substance present in a sample, cell culture medium, serum preparation, or composition of the present disclosure. For example, a particular concentration, weight, percentage (e.g., v / v%), or ratio can be used to define the level of a particular substance.

[0036] In the context of the present disclosure, the term "conditioned medium" refers to medium obtained from MLPSCs under culture conditions. Such medium contains the secretome of MLPSCs, proteins shed from the surface of MLPSCs, and other particles such as extracellular vesicles. The conditioned medium of the present disclosure contains extracellular vesicles, pro-angiogenic factors such as angiogenin, or secreted metabolites such as prostaglandin E2. The pro-angiogenic ability of the conditioned medium disclosed herein and / or factors obtained thereby can be confirmed, if desired, using one or more of the angiogenesis assays disclosed herein (e.g., endothelial network formation, endothelial length, endothelial branch length). In certain examples, the present disclosure relates to extracellular vesicles, such as exosomes, obtained from conditioned medium obtained from MLPSCs under culture conditions. In one example, the conditioned medium is obtained when MLPSCs are in the exponential growth phase. In one example, the conditioned medium is obtained at least 2-3 days after culture. In another example, the conditioned medium is obtained approximately 30-84 hours after culture.

[0037] In one example, the level of a particular marker, such as a pro-angiogenic factor(s), is determined under culture conditions. The term "culture conditions" is used to refer to the growth of cultured cells. In one example, culture conditions refer to a population of cells that are actively dividing. Such cells may, in one example, be in an exponential growth phase. Alternatively, such cells may be in a stationary phase.

[0038] In one example, in the context of measuring the level of IL2-RA inhibition, the culture conditions can include co-culturing the MLPSC population disclosed herein with a second cell population, such as a population containing peripheral blood mononuclear cells (PBMCs). In one example, the co-culturing includes culturing the MLPSC population disclosed herein with an activated PBMC population. For example, the PBMCs can be activated using anti-CD3 and anti-CD28 antibodies before co-culturing with the MLPSC population disclosed herein. In this example, the "culture conditions" can include co-culturing MLPSCs with T cells at a ratio of approximately 1 MLPSC:2 T cells. For example, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1 MLPSC:100 T cells, or less. In this example, the level of IL2-RA inhibition is determined after approximately 30 to 84 hours of cell culture under the culture conditions.

[0039] In another example, the level of a particular marker can be determined by taking a sample of conditioned medium and measuring the level of the marker in the sample. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in a cell lysate. Those skilled in the art will understand that secreted markers can be measured by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of cell lysate. In one example, the sample is taken when the cells are in the exponential growth phase. In one example, the sample is taken after culturing for at least 2-3 days. In another example, the sample is taken after culturing for approximately 30-84 hours. In one example, the sample is taken when the cells are in stationary phase.

[0040] In one example, a sample is taken from a co-culture of MLPSCs and activated PBMCs. In this example, the cell sample can be lysed and the level of a marker can be measured. For example, the level of IL2-RA can be determined. In this example, the level of IL2-RA can be determined using a variety of methods, such as an enzyme-linked immunosorbent assay (ELISA)-based method. In one example, ELISA is (i) adding sample dilutions to each well of a microplate precoated with a monoclonal antibody specific for IL2-RA; (ii) adding the co-culture samples to wells of a microplate pre-coated with a monoclonal antibody specific for IL2-RA; (iii) incubating the microplate for a time sufficient to allow the monoclonal antibody specific for IL2-RA to specifically bind to any IL2-RA in the sample; (iv) washing the microplate; (v) adding IL2-RA complexes to the wells; (vi) incubating the microplate for a time sufficient to allow the complex to specifically bind to any captured IL2-RA; (vii) washing the microplate; (viii) adding a substrate solution to the wells; (ix) incubating the microplate for a time sufficient for color development; (x) adding a stop solution to the wells; (xi) reading the optical density on a microplate reader set at 450 nm with wavelength compensation set at 570 nm; (xii) determining the level of IL2-RA.

[0041] In another example, the level of IL-2RA is determined using fluorescence-activated cell sorting (FACS) with an appropriate antibody, such as anti-CD25. Additional antibodies can be used if it is necessary to distinguish between CD25+ cell types. While the above example refers to IL-2RA, it will be appreciated that similar methods can be used to determine the levels of other markers disclosed herein, such as angiogenin. In these examples, co-culture may not be required to determine the levels.

[0042] In another example, the level is measured based on evaluation of conditioned medium (or a property thereof) obtained from a population of MLPSCs under culture conditions. For example, the conditioned medium can be obtained from a population of MLPSCs disclosed herein under culture conditions before being used in one or more of the angiogenesis assays disclosed below.

[0043] In the context of this disclosure, the term "sufficient" is used to define an amount that provides a particular concentration when dissolved in stem cell culture medium. A "sufficient amount" depends on the amount of culture medium required.

[0044] The term "angiogenic marker" as used herein refers to an indicator of angiogenesis. As used herein, "angiogenic marker" includes pro-angiogenic factors such as angiogenin, SDF-1α, and VEGF. In another example, an angiogenic marker is a cellular indicator of angiogenesis, such as endothelial network formation, endothelial network length, and endothelial branch length. In this example, the cellular indicator of angiogenesis is determined in an in vitro angiogenesis assay as disclosed herein. In one example, characterization of an angiogenic marker can be used to characterize the MLPSC populations disclosed herein (e.g., cryopreserved intermediates or pharmaceutical products disclosed herein).

[0045] In one example, the composition of the present disclosure comprises non-genetically modified MLPSCs. As used herein, the term "non-genetically modified" refers to cells that have not been modified by the introduction of a nucleic acid. For the avoidance of doubt, in the context of the present disclosure, MLPSCs into which a nucleic acid encoding a protein has been introduced are considered to be genetically modified.

[0046] As used herein, the term "sample" refers to an extract from a cell culture in which the level of a particular marker can be measured. A "sample" includes extracts and / or derivatives and / or fractions of a sample. In one example, a "sample" is a cell population, for example, a cell population under culture conditions. In one example, a sample is a supernatant obtained after cell culture, for example, a cell-conditioned medium. In these examples, a sample is any extract of a cell culture in which an angiogenic marker can be measured. In one example, a sample is contacted with another cell population to determine the level of an angiogenic marker.

[0047] Throughout this specification the word "comprises" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a specified element, integer, step, or group of elements, integers, or steps, but not the exclusion of other elements, integers, steps, or groups of elements, integers, or steps.

[0048] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter will be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0049] Those skilled in the art will understand that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all of the steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, as well as any combination of any two or more of the steps or features.

[0050] The present disclosure is not limited in scope by the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure as described herein.

[0051] Unless otherwise specified, examples disclosed herein are intended to apply mutatis mutandis to other examples.

[0052] Mesenchymal progenitor or stem cells (MLPSCs) As used herein, the term "mesenchymal progenitor or stem cell (MLPSC)" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining multipotency and to differentiate into many cell types of mesenchymal origin, such as osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, tendons, or non-mesodermal origin, such as hepatocytes, neurons, and epithelial cells. For the avoidance of doubt, "mesenchymal progenitor cells" refers to cells that can differentiate into mesenchymal cells, such as bone, cartilage, muscle, and adipocytes, as well as fibrous connective tissue.

[0053] The term "mesenchymal progenitor or stem cells" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursor cells, and their undifferentiated progeny.

[0054] Mesenchymal progenitor or stem cells can be autologous, allogeneic, xenogeneic, syngenic, or allogeneic. Autologous cells are isolated from the same individual into which they will be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngenic or allogeneic cells are isolated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0055] In one example, the mesenchymal progenitor or stem cells are allogeneic. In one example, the allogeneic mesenchymal progenitor or stem cells are expanded in culture and cryopreserved.

[0056] Mesenchymal progenitor or stem cells reside primarily in bone marrow, but have also been shown to reside in a variety of host tissues, including umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone, and dental pulp. They are also found in the skin, spleen, pancreas, brain, kidney, liver, heart, retina, hair follicles, intestine, lung, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis, and periosteum, and can differentiate into germline cells such as mesoderm and / or endoderm and / or ectoderm. Thus, mesenchymal progenitor or stem cells can differentiate into numerous cell types, including, but not limited to, adipose, bony, cartilaginous, elastic, muscular, and fibrous connective tissue. The specific lineage commitment and differentiation pathways these cells enter depend on various influences from mechanical and / or endogenous bioactive factors, such as growth factors, cytokines, and / or local microenvironmental conditions established by the host tissue.

[0057] As used herein, the terms "enriched," "enriched," or variations thereof are used to describe a population of cells in which the proportion of one particular cell type or the number of several particular cell types is increased compared to a population of untreated cells (e.g., cells in their native environment). In one example, a population enriched for mesenchymal progenitor or stem cells contains 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% mesenchymal progenitor or stem cells. In this regard, the term "a population of cells enriched for mesenchymal progenitor or stem cells" is interpreted as providing explicit support for the term "a population of cells comprising X% mesenchymal progenitor or stem cells," where X% is a percentage as described herein. Mesenchymal progenitor or stem cells, in some instances, can form clonogenic colonies, e.g., CFU-F (fibroblasts) or a subset thereof (e.g., 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0058] In one example of the present disclosure, the mesenchymal progenitor or stem cells are mesenchymal stem cells (MSCs). The MSCs can be a homogenous composition or a mixed cell population enriched for MSCs. A homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers identified by unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium. In one example, the MSCs are allogeneic. In one example, the MSCs are cryopreserved. In one example, the MSCs are culture-expanded and cryopreserved.

[0059] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0060] Isolated or enriched mesenchymal progenitor or stem cells can be expanded in vitro by culture. Isolated or enriched mesenchymal progenitor or stem cells can be cryopreserved, thawed, and then expanded in vitro by culture.

[0061] In one example, isolated or enriched mesenchymal progenitor or stem cells are cultured in a culture medium (serum-free or serum-supplemented), such as alpha minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, at a density of 1 cm. 2 Seed cells at 50,000 viable cells per well and allow them to attach to the culture vessel overnight at 37 °C and 20% O. Then, replace and / or change the culture medium as needed and culture the cells for an additional 68-72 h at 37 °C and 5% O.

[0062] As will be appreciated by those skilled in the art, cultured mesenchymal progenitor or stem cells are phenotypically distinct from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal progenitor or stem cells are also biologically distinct from in vivo cells, having a higher proliferation rate than the majority of non-cycling (quiescent) cells in vivo.

[0063] In one example, a population of cells is enriched from a cell preparation containing STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., mesenchymal progenitor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (which may be STRO-1 bright). Thus, the designation that cells are STRO-1+ does not mean that the cells are selected solely by STRO-1 expression. In one example, cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+). For example, MPCs can be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0064] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. Although, in some instances, these terms provide support for selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or vascular tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues listed herein.

[0065] In one example, the cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0066] By "individually" it is meant that the present disclosure encompasses the listed markers or groups of markers separately, and that even if individual markers or groups of markers cannot be separately recited herein, the appended claims may define such markers or groups of markers separately and divisibly from one another.

[0067] "Collectively" means that the disclosure encompasses any number or combination of the listed markers or markers, and that notwithstanding that such number or combination of markers or markers may not be specifically recited herein, the appended claims may define such combination or subcombination separately and divisibly from any other combination of markers or markers.

[0068] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, this term encompasses liver isoform (LAP), bone isoform (BAP) and kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule capable of binding to STRO-3 antibody produced by the hybridoma cell line deposited with ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0069] Additionally, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0070] In one example, a significant proportion of STRO-1+ cells can differentiate into at least two different germ cell lineages. Non-limiting examples of lineages that STRO-1+ cells can commit include bone progenitors; hepatocyte precursors that are multipotent into bile duct epithelial cells and hepatocytes; neural-restricted cells that can generate glial precursors that develop into oligodendrocytes and astrocytes; neuronal precursors that develop into neurons; cardiac muscle and cardiomyocyte precursors; 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: retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular precursors, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocyte precursors.

[0071] In one example, mesenchymal precursor or stem cells are obtained from a single donor or multiple donors, and the donor samples or mesenchymal precursor or stem cells are then pooled and then expanded in culture.

[0072] The mesenchymal precursor or stem cells encompassed by the present disclosure can also be cryopreserved before administration to a subject, hi one example, the mesenchymal precursor or stem cells are expanded in culture and cryopreserved before administration to a subject.

[0073] In one example, the present disclosure encompasses mesenchymal progenitor or stem cells and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal progenitor or stem cells and conditioned medium obtained therefrom under culture conditions. In another example, the present disclosure encompasses mesenchymal progenitor or stem cells and extracellular vesicles isolated therefrom. For example, mesenchymal progenitor lineage cells or stem cells of the present disclosure can be cultured and grown for a period of time and under conditions suitable for secreting extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for therapeutic use. If desired, such extracellular vesicles can be characterized using one or more of the angiogenesis assays disclosed herein (e.g., endothelial network formation, endothelial length, endothelial branch length).

[0074] As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, but are typically less than 200 nm. They may contain proteins, nucleic acids, lipids, metabolites, or organelles from the cells that release them (e.g., mesenchymal stem cells; STRO-1+ cells).

[0075] As used herein, the term "exosome" refers to a type of extracellular vesicle that generally ranges in size from about 30 nm to about 150 nm and originates from the endosomal compartment of mammalian cells, from which it is transported and released to the plasma membrane. They contain nucleic acids (e.g., RNA, microRNA), proteins, lipids, and metabolites and function in intercellular communication by being secreted from one cell and taken up by other cells to carry their cargo.

[0076] As used herein, the term "pre-approved" or "approved" refers to a process by which MLPSCs achieve functional maturity, whereby pre-approved or approved MLPSCs, when administered to a subject, release significantly less inflammatory cytokines than non-pre-approved MLPSCs.

[0077] As used herein, the terms "enriched," "enriched," or variations thereof, are used to describe a cell population in which the percentage of a particular cell type or the percentage of the number of a particular cell type is increased when compared to an untreated cell population (e.g., cells in their native environment). In one example, a population enriched for STRO-1+ cells contains at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% STRO-1+ cells. In this regard, the term "cell population enriched for STRO-1+ cells" is construed as expressly supporting the term "a cell population comprising X% STRO-1+ cells," where X% is a percentage described herein. STRO-1+ cells, in some examples, can form clonogenic colonies; for example, CFU-F (fibroblasts) or a subset thereof (e.g., 50%, 60%, 70%, 80%, 90%, or 95%) can possess this activity.

[0078] In one example, a population of cells is enriched from a cell preparation that contains STRO-1+ cells in a selectable morphology. In this regard, the term "selectable morphology" is understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but need not be, STRO-1. For example, cells (e.g., mesenchymal progenitor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and STRO-1 bright (It may be that the cells are STRO-1+. Thus, the designation that a cell is STRO-1+ does not mean that the cell was selected by STRO-1 expression. In one example, the cell was selected based on at least STRO-3 expression, e.g., STRO-3+ (TNAP+).

[0079] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells can be selected, isolated, or enriched from a wide variety of sources. Although, in some instances, these terms support selection from any tissue containing STRO-1+ cells (e.g., mesenchymal progenitor cells), or angiogenic tissue, or tissue containing pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues described herein.

[0080] In one example, mesenchymal progenitor or stem cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0081] Use of the term "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 individually and separably from one another, even if the individual markers or groups of markers are not individually described herein.

[0082] Use of the term "collectively" means that the disclosure encompasses any number or combination of the listed markers or markers, and that even if such number or combination of markers or markers is not specifically recited herein, the appended claims may define such combination or subcombination as separate and distinct from other combinations of markers or markers.

[0083] In one example, STRO-1+ cells are bright (Synonym: STRO-1 bri ) In another example, STRO-1 bri STRO-1 cells dim cells or STRO-1 intermediateIn another example, the STRO-1bri cells are additionally enriched relative to TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), and / or CD146+. For example, the cells may be selected for and / or shown to express one or more of the aforementioned markers. In this regard, it is not necessary to specifically test cells shown to express a marker; rather, previously enriched or isolated cells can be tested and subsequently used, and it can be reasonably assumed that the isolated or enriched cells also express the same markers.

[0084] In one example, the mesenchymal precursor cells are perivascular mesenchymal precursor cells as defined in WO 2004 / 85630 and are characterized by the presence of the perivascular marker 3G5.

[0085] Cells that are "positive" for a given marker may express 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; this term refers to the intensity of the fluorescence or other marker used in the cell sorting process. The distinction between lo (or dim or dull) and bri is understood in the context of the marker used in the particular cell population being sorted. Cells that are "negative" for a given marker are not necessarily completely absent from the cell. This term means that the marker is expressed at a relatively very low level by the cell, producing a very low signal when detectably labeled, or is not detectable above background levels, such as those detected using an isotype control antibody.

[0086] As used herein, the term "bright" or "bri" refers to a marker on the cell surface that generates a relatively high signal when detectably labeled. Without being limited by theory, it is believed 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, as determined by fluorescence-activated cell sorting (FACS) analysis, the expression of STRO-1 is higher in the bright cell surface than in the bright cell surface. bri Cells were identified as non-clear cells (STRO-1) when labeled with FITC-conjugated STRO-1 antibody. dull / dim ) produces a fluorescent signal greater than 0.1% of the most brightly labeled bone marrow mononuclear cells in the starting sample. In another example, the "bright" cells comprise 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 example, STRO-1 bright STRO-1 cells have a two-log increase in STRO-1 surface expression compared to "background," i.e., STRO-1-negative cells. intermediate Cells have less than 2 logs of STRO-1 surface expression, typically about 1 log or less above "background."

[0087] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue non-specific alkaline phosphatase. For example, this term encompasses the liver isoform (LAP), the bone isoform (BAP), and the kidney isoform (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule capable of binding to the STRO-3 antibody produced by the hybridoma cell line deposited with the ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0088] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0089] In one example, a significant proportion of STRO-1+ pluripotent cells can differentiate into at least two different germ cell lineages. Non-limiting examples of lineages into which pluripotent cells may commit include bone progenitor cells, hepatocyte progenitor cells that are pluripotent for bile duct epithelial cells and hepatocytes, neural-restricted cells that can generate glial progenitor cells that progress to oligodendrocytes and astrocytes, neuronal progenitor cells that progress to neurons, cardiac muscle and cardiac muscle cell progenitors, and glucose-responsive insulin-secreting pancreatic beta cell lines. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, chondrocytes, and progenitor cells such as retinal pigment epithelial cells, fibroblasts, skin cells such as keratinocytes, dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericytes, blood vessels, epithelium, glia, neurons, astrocytes, and oligodendrocyte cells.

[0090] In one embodiment of the present disclosure, the currently described mesenchymal progenitor or stem cells are MSCs. The MSCs can be homogenous compositions or mixed cell populations enriched for MSCs. Homogeneous MSC cell compositions can be obtained by culturing adherent bone marrow or periosteal cells, and MSCs can be identified by specific cell surface markers recognized by unique monoclonal antibodies. Methods for obtaining MSC-enriched cell populations are described, for example, in U.S. Patent No. 5,486,359. Alternative sources of MSCs include, but are not limited to, blood, skin, umbilical cord blood, muscle, fat, bone, and perichondrium.

[0091] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs (e.g., remestemcel-L).

[0092] As will be appreciated by those skilled in the art, cultured mesenchymal progenitor or stem cells are phenotypically distinct from in vivo cells. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultured mesenchymal progenitor or stem cells also differ biologically from in vivo cells, having a higher proliferation rate compared to the mostly non-cycling (quiescent) cells in vivo. Mesenchymal progenitor or stem cells cultured using the methods of the present disclosure can also be cryopreserved.

[0093] conditioned medium In one example, the conditioned medium or extracellular vesicles obtained thereby can be characterized by the expression of angiogenic marker(s). For example, the conditioned medium or extracellular vesicles obtained thereby can be characterized by increased levels of VEGF, angiogenin, and / or SDF-1α under culture conditions. In another example, the conditioned medium or extracellular vesicles obtained thereby can be characterized based on one or more functional criteria. In one example, when culture-expanded MLPSCs are treated with conditioned medium or extracellular vesicles obtained thereby, the conditioned medium or extracellular vesicles obtained thereby increase the level of endothelial network formation, endothelial network length, and / or endothelial branch length in a population of endothelial cells. In one example, the increase is determined by comparison with conditioned medium or extracellular vesicles obtained thereby from a control population of MLPSCs. In one example, the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

[0094] In one example, the conditioned medium is characterized by a VEGF level of greater than about 3 ng / ml. In one example, the VEGF level is about 3 ng / ml to 4 ng / ml. In one example, the VEGF level is greater than about 3.1 ng / ml. In one example, the VEGF level is greater than about 3.2 ng / ml. In one example, the VEGF level is greater than about 3.3 ng / ml. In one example, the VEGF level is greater than about 3.4 ng / ml. In one example, the VEGF level is greater than about 3.5 ng / ml. In one example, the VEGF level is about 3.2 to 3.6 ng / ml. In one example, the VEGF level is about 3.45 ng / ml.

[0095] In one example, the conditioned medium or extracellular vesicles obtained thereby contain an increased level of angiogenin compared to a control population. In one example, the conditioned medium is characterized by an angiogenin level greater than about 1000 pg / ml. In one example, the angiogenin level is greater than about 1100 pg / ml. In one example, the angiogenin level is about 1000 pg / ml to 1200 pg / ml. In one example, the angiogenin level is about 1100 pg / ml to 1150 pg / ml. In one example, the angiogenin level is about 1114 pg / ml.

[0096] In one example, the conditioned medium is characterized by a level of SDF-1α greater than about 3000 ng / ml. In one example, the level of SDF-1α is greater than about 3100 ng / ml. In one example, the level of SDF-1α is greater than about 3200 ng / ml. In one example, the level of SDF-1α is greater than about 3300 ng / ml. In one example, the level of SDF-1α is greater than about 3400 ng / ml. In one example, the level of SDF-1α is greater than about 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3400 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3300 ng / ml. In one example, the level of SDF-1α is about 3100 ng / ml to 3400 ng / ml.In one example, the level of SDF-1α is about 3100 ng / ml to 3300 ng / ml.

[0097] In one example, the conditioned medium is about 0.1 mm 2 / mm 2 In one example, the formation of an endothelial network is stimulated at a depth of about 0.1 mm. 2 / mm 2 ~0.2mm 2 / mm 2 In another example, the formation of an endothelial network is about 0.12 mm 2 / mm 2 is.

[0098] In one example, the conditioned medium is approximately 4 mm 2 / mm 2 In one example, the length of the endothelial network is approximately 4 mm. 2 / mm 2 ~about 6mm 2 / mm 2 In one example, the length of the endothelial network is about 5 mm. 2 / mm 2 In one example, the conditioned medium has a density of about 12 l / mm 2 Stimulates endothelial branch lengths of greater than 1 / 2 mm. In one example, the endothelial branch length is approximately 1 / 2 mm. 2 ~approximately 17 1 / mm2 In one example, the length of the endothelial branch is about 15 1 / mm 2 is.

[0099] In one example, the conditioned medium or extracellular vesicles obtained thereby are characterized by an increased level of one or more angiogenic markers compared to conditioned medium or extracellular vesicles obtained thereby from a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of the angiogenic marker is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In one example, the level of the angiogenic marker is increased by about 5% to about 60%. In one example, the level of the angiogenic marker is increased by about 5% to about 40%. In one example, the level of the angiogenic marker is increased by about 40%. In one example, the level of the angiogenic marker is increased by at least about 5%. In one example, the level of the angiogenic marker is increased by at least about 10%. In one example, the levels of angiogenic markers are increased compared to conditioned medium or extracellular vesicles obtained from a population of MLPSCs cultured and expanded in cell culture medium without IFN-γ or TNF-α.

[0100] In one example, the conditioned medium or extracellular vesicles obtained therefrom are characterized by increased levels of one or more angiogenic markers compared to conditioned medium or extracellular vesicles obtained therefrom from a population of MLPSCs cultured and expanded in neonatal serum-free cell culture medium. In one example, the level of the angiogenic marker is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In one example, the level of the angiogenic marker is increased by about 5% to about 60%. In one example, the level of the angiogenic marker is increased by about 5% to about 40%. In one example, the level of the angiogenic marker is increased by at least about 5%. In one example, the level of the angiogenic marker is increased by at least about 10%.

[0101] "Culture-expanded" MLPSCs are distinguished from freshly isolated cells in that they are cultured and passaged (ie, subcultured) in cell culture medium.

[0102] In one example, the freshly isolated cells are expanded in culture for about 1 or 2 passages to provide an intermediate population. In one example, the freshly isolated cells are expanded in culture for 2 passages to provide an intermediate population. In another example, the freshly isolated cells are expanded in culture for about 1-3 passages to provide an intermediate population. In one example, the freshly isolated cells are STRO-1+.

[0103] Thus, in one example, relevant cells are isolated and culture-expanded for two passages to provide an intermediate MLPSC population. In certain examples, the intermediate MLPSC population is then culture-expanded to produce a drug product (DP). In one example, conditioned medium or extracellular vesicles obtained thereby are generated by culturing cells from an intermediate cryopreserved MLPSC population, i.e., a cryopreserved intermediate. In one example, the intermediate cell population can be cultured for three passages (a total of five passages) to provide a DP. In another example, conditioned medium or extracellular vesicles obtained thereby are obtained from DP MLPSCs.

[0104] In one example, MLPSCs are culture-expanded for approximately 4 to 10 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs are culture-expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, or at least 10 passages, thereby providing the resulting conditioned medium or extracellular vesicles. For example, MLPSCs can be culture-expanded for at least 5 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be culture-expanded for at least 5 to 10 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be culture-expanded for at least 5 to 8 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be culture-expanded for at least 5 to 7 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be culture-expanded for more than 7 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In these instances, the MLPSCs can be culture-expanded prior to cryopreservation to provide an intermediate cryopreserved MLPSC population, which can then be further culture-expanded to provide the resulting conditioned medium or extracellular vesicles.

[0105] In one example, the conditioned medium or extracellular vesicles obtained thereby are obtained from MLPSCs cultured and expanded from a cryopreserved intermediate. In one example, the cell culture expanded from a cryopreserved intermediate is cultured and expanded for at least three, at least five, at least six, at least seven, at least eight, at least nine, or at least ten passages. For example, the MLPSCs can be cultured and expanded for at least three passages. In one example, the MLPSCs can be cultured and expanded for at least three to ten passages. In one example, the MLPSCs can be cultured and expanded for at least three to eight passages. In one example, the MLPSCs can be cultured and expanded for at least three to seven passages. In one example, the culture of MLPSCs cultured and expanded from a cryopreserved intermediate is cultured and expanded in a medium disclosed herein (e.g., a medium containing newborn bovine serum).

[0106] In one example, MLPSCs can be obtained from a single donor or from multiple donors, where the donor samples or MLPSCs are subsequently pooled and optionally culture expanded. i. expanding viable cell numbers by passage expansion to provide a preparation of at least about 1 billion viable cells, the passage expansion comprising establishing a primary culture of isolated MLPSCs and then serially establishing a first non-primary (P1) culture of MLPSCs isolated from the previous culture; ii. Expanding the P1 culture of isolated MLPSCs by passage expansion into a second, non-primary (P2) culture of MLPSCs; and iii. Preparing and cryopreserving an in-process intermediate MLPSC preparation obtained from a P2 culture of MLPSCs, and optionally iv. Thawing a cryopreserved in-process intermediate MLPSC preparation and expanding the in-process intermediate MLPSC preparation by passage expansion.

[0107] In one example, the method of the present disclosure includes selecting an intermediate population (e.g., a cryopreserved intermediate) for further culture expansion based on certain criteria, such as the level of one or more angiogenic markers. The selection process is not particularly limited, as long as it allows for the selection of a cell population characterized by relevant criteria, such as the level of angiogenic markers. In one example, the levels of angiogenic markers are evaluated for a series of intermediate MLPSC populations, and a population that exceeds a threshold level of angiogenic markers described herein is selected for further expansion.

[0108] It is noted that the selection process does not require immediate culture expansion. Rather, the "selected" population can be cryopreserved and culture expanded at a later stage. In one example, a portion of the intermediate cell population is culture expanded, and the remainder of the population is cryopreserved for culture expansion at a later stage.

[0109] In one example, the selected cell population is immediately culture expanded, hi another example, the selected cell population is cryopreserved for culture expansion at a later stage.

[0110] In one example, the conditioned medium obtained from the culture-expanded MLPSC population or the extracellular vesicles obtained thereby is i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. have an antigenic and activity profile that includes at least about 95% CD166+ cells.

[0111] The process of MLPSC isolation and in vitro expansion can be carried out using any equipment and cell processing methods known in the art. Various culture expansion embodiments of the present disclosure employ steps that require cell manipulation, such as seeding, feeding, dissociation of adherent cultures, or washing. Any step that manipulates cells can potentially injure the cells. While MLPSCs can generally tolerate some damage during preparation, it is preferable to manipulate cells using procedures and / or equipment that appropriately perform a given step(s) while minimizing damage to the cells.

[0112] In one example, MLPSCs are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a spinning membrane filter with inlet and outlet ports, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, as described, for example, in U.S. Pat. No. 6,251,295, incorporated herein by reference.

[0113] In one example, MLPSC compositions cultured according to the present disclosure are 95% homogeneous for being CD105-positive, CD166-positive, and CD45-negative, and in one example, this homogeneity persists through in vitro expansion, i.e., multiple population doublings.

[0114] In one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture. For example, the MLPSCs of the present disclosure can be culture-expanded in a cell factory. In certain examples, 3D culture of the intermediates disclosed herein can be performed, for example, using a bioreactor. In one example, the MLPSCs of the present disclosure are first culture-expanded in 2D culture before being further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure is not culture-expanded in 3D culture. In one example, the level of one or more angiogenic markers is assessed before subsequent culture expansion in a cell factory or 3D culture.

[0115] In one example, the MLPSCs of the present disclosure are culture-expanded from an intermediate population. In one example, the MLPSCs of the present disclosure are culture-expanded from an intermediate in a 2D culture before being seeded into a 3D culture.

[0116] In the context of both the intermediate population and the therapeutic composition expanded thereby, in one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture for at least three days before being seeded into a further culture system, such as a 3D culture in a cell factory or bioreactor. In one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture for at least four days before being seeded into a further culture system. In one example, the MLPSCs of the present disclosure are culture-expanded in 2D culture for three to five days before being seeded into a further culture system. In these examples, the 2D culture can be performed in a cell factory. Various cell factory products are commercially available (e.g., Thermofisher, Sigma, Corning). In one example, the cell factory has at least five layers. In one example, the cell factory has at least 10 layers. In one example, the cell factory has at least 20 layers. 3D culture can be performed in various bioreactor types, such as stirred tanks, wave bags, and vertical wheels.

[0117] In one example, CO2 is provided during the culture and growth of MLPSCs. In one example, MLPSCs are cultured and grown in less than 9% CO2. In one example, MLPSCs are cultured and grown in less than 8% CO2. In one example, MLPSCs are cultured and grown in 5% CO2. For example, MLPSCs can be cultured and grown in 5% (+ / - 2%) CO2. In one example, MLPSCs are cultured and grown using passive priming of CO2. For example, a cell factory can be passively primed with 5% CO2. Priming cell factories maintains CO2 tension between the cell factory and the incubator and stabilizes the pH level of the growth medium. Active priming involves actively passing CO2 gas through a bacterial vent air filter into each culture vessel (e.g., cell factory) for a specified time (e.g., approximately 10 minutes). However, active priming requires an open port to provide gas, potentially introducing contaminants into the culture. Passive priming involves placing the sealed culture system in an incubator with the appropriate CO2 concentration prior to cell seeding (e.g., approximately 12-72 hours). In one example, the cells disclosed herein are STRO-3+ before being culture-expanded to obtain an intermediate cell population.

[0118] MLPSC culture medium In one embodiment, the invention encompasses the culture expansion of MLPSCs in medium supplemented with pro-inflammatory cytokine(s). In one example, the culture medium includes IFN-γ and / or TNF-α. In one example, the medium includes IFN-γ. For example, the level of IFN-γ can be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the medium includes TNF-α. For example, the level of TNF-α can be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the medium includes IFN-γ and TNF-α, and both levels are less than 1 ng / ml.

[0119] In one example, the medium includes one or more pro-inflammatory cytokines that can bind to receptors on the surface of the MLPSCs.

[0120] In one example, the medium comprises one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the medium can comprise IL-8.

[0121] In one example, the medium comprises IFN-γ and / or TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0122] In one example, the medium is i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0123] In another example, the medium is i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 50 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: IP-10 levels greater than 5,000 pg / ml.

[0124] In one example, the medium contains IL-10. In another example, the medium contains IL-36RA. In another example, the medium contains IL-10 and IL-36RA. In one example, the level of IL-10 is greater than 0.3 pg / ml. For example, the level of IL-10 can be greater than 30 pg / ml. In one example, the level of IL-10 is greater than 400 pg / ml. In one example, the level of IL-36RA is greater than 50 pg / ml.

[0125] In one example, the medium is serum-free.

[0126] In one example, the medium is serum-free and supplemented with PDGF and FGF2. In one example, the medium is serum-free and supplemented with PDGF, FGF2, and EGF. In one example, the PDGF is PDGF-BB. In one example, the serum-free medium is supplemented with 10 ng / ml PDGF-BB, 5 ng / ml EGF, and 1 ng / ml FGF2.

[0127] In one example, the above cytokines can be provided at a concentration of <1 ng / ml each. For example, the medium can be characterized by one or more or all of the following, each provided at <1 ng / ml: IFN-γ, TNF-α, IL-6, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

[0128] method The disclosed cell culture methods, and use of the methods to generate the conditioned medium disclosed herein, involve culturing a cell population enriched for MLPSCs (e.g., human MSCs) in a cell culture medium suitable for the maintenance and proliferation of MLPSCs.

[0129] In one example, the culture medium is a composition or medium referenced above. In one example, the culture medium is supplemented with serum containing one or more pro-inflammatory cytokines described herein. In some preferred embodiments, the culture medium used is supplemented with neonatal serum. In some preferred embodiments, the culture medium used is supplemented with equal concentrations of fetal serum and neonatal serum, with the total serum concentration in the culture medium being about 10% (v / v). In some preferred embodiments, MLPSCs are pre-qualified in cell culture medium containing 5% (v / v) neonatal serum and 5% (v / v) fetal serum.

[0130] In some embodiments, the MLPSC methods disclosed herein include the additional step of determining or having determined the level of one or more pro-inflammatory cytokines in the serum contained in the culture medium used for pre-qualification of the MLPSCs. Methods for determining cytokine levels are well known in the art, such as, for example, ELISA.

[0131] In some embodiments, the MLPSC methods disclosed herein also include determining, or having determined, the ability of culture medium (e.g., culture medium supplemented with neonatal serum) to stimulate MLPSCs to produce conditioned medium that promotes angiogenesis in an in vitro assay (e.g., tube formation by human umbilical vein endothelial cells (HUVECs), and analysis of network length, network area, and branch point formation). In some embodiments, such assays involve collecting MLPSC-conditioned medium after culturing in neonatal serum-supplemented medium as disclosed herein and quantifying the effect of such conditioned medium in an angiogenesis assay described above or a similar assay.

[0132] In some embodiments, the MLPSC methods disclosed herein also include determining or having determined levels of one or more of angiogenin, angiopoietin (Ang1 / ANGPT1), SDF-1α, and VEGF in the conditioned medium described above.

[0133] In some embodiments, when a first lot or batch of neonatal serum is used in a conditioned medium that promotes greater angiogenesis or release of angiogenic factors than a conditioned medium in which a second lot / batch of neonatal serum is used, it is concluded that using a first lot of neonatal serum for prequalification and culture expansion of MLPSCs will produce MLPSCs with relatively greater therapeutic potency, particularly for the treatment of conditions in which an angiogenic or anti-inflammatory therapeutic mode of action is useful.

[0134] In certain instances, the disclosed methods and cell culture media maintain MLPSCs in an undifferentiated state. MLPSCs are considered undifferentiated if they have not progressed along a specific lineage. As explained above, MLPSCs exhibit morphological characteristics that distinguish them from differentiated cells. Furthermore, undifferentiated MLPSCs express genes that can be used as markers to detect a differentiated state. Polypeptide products can also be used as markers to detect a differentiated state. Therefore, those skilled in the art can easily determine whether the disclosed methods maintain MLPSCs in an undifferentiated state using routine morphological, genetic, and / or proteomic analyses. Methods for monitoring / confirming cell proliferation are also known in the art and, in certain instances, can be as basic as periodic visual inspection of cell cultures to confirm an increase in cell number. Other methods can include the use of cell viability dyes and / or imaging and counting live cells using commercially available products.

[0135] The terms "medium" or "media" as used in the context of this disclosure include the components of the environment surrounding cells. Media contribute to and / or provide suitable conditions for growing cells. Media can be solid, liquid, gaseous, or a mixture of these phases and materials. Media can include liquid growth media as well as liquid media that do not support cell growth. Exemplary gaseous media include the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0136] In one example, the disclosed method involves culturing and expanding in cell culture medium containing one or more pro-inflammatory cytokines. In one example, the cell culture medium contains IFN-γ and / or TNF-α. In one example, the cell culture medium contains IFN-γ. For example, the level of IFN-γ can be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the cell culture medium contains TNF-α. For example, the level of TNF-α can be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the cell culture medium contains IFN-γ and TNF-α, both at levels less than 1 ng / ml.

[0137] In one example, the cell culture medium includes one or more pro-inflammatory cytokines that can bind to receptors on the surface of the MLPSCs.

[0138] In one example, the cell culture medium comprises one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the cell culture medium can comprise IL-8.

[0139] In one example, the cell culture medium comprises IFN-γ and / or TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0140] In one example, the cell culture medium comprises: i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0141] In another example, the medium is i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 50 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: IP-10 levels greater than 5,000 pg / ml.

[0142] In one example, the medium contains IL-10. In another example, the medium contains IL-36RA. In another example, the medium contains IL-10 and IL-36RA. In one example, the level of IL-10 is greater than 0.3 pg / ml. For example, the level of IL-10 can be greater than 30 pg / ml. In one example, the level of IL-10 is greater than 400 pg / ml. In one example, the level of IL-36RA is greater than 50 pg / ml.

[0143] In another example, the disclosed methods involve culture expansion in cell culture medium containing newborn serum. Various examples of suitable serum (and levels thereof) are disclosed herein.

[0144] The cell culture medium used for culture expansion contains all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified as essential (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro) amino acids.

[0145] Those skilled in the art will understand that for optimal results, the basal medium must be appropriate for the cell line of interest. For example, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or add glucose (or other energy source) during the culture process if this energy source is found to be depleted and therefore limiting growth. In one example, dissolved oxygen (DO) levels may also be controlled.

[0146] serum "Newborn serum" refers to serum obtained after birth. For example, the culture medium can be supplemented with mammalian newborn serum (e.g., bovine). In one example, the culture medium can be supplemented with animal newborn serum. In another example, the culture medium can be supplemented with human newborn serum.

[0147] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 10% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of newborn serum. In one example, the cell culture medium is supplemented with about 5% v / v of newborn serum.

[0148] In one example, the neonatal serum contains at least one proinflammatory cytokine. As used herein, the term "proinflammatory cytokine" refers to a signaling molecule that promotes inflammation. In an example, the one or more cytokines are selected from the group including IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1ra.

[0149] In one example, the newborn serum contains IFN-γ. In another example, the newborn serum contains TNF-α. In another example, the newborn serum contains IFN-γ and TNF-α. In another example, the newborn serum contains one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the newborn serum can contain IL-8. In one example, the newborn serum contains IFN-γ and / or TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In another example, the newborn serum contains IFN-γ and TNF-α, and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10. In one example, the level of IFN-γ is less than 1 ng / ml. In one example, the level of TNF-α is less than 1 ng / ml. In one example, the levels of both IFN-γ and TNF-α are less than 1 ng / ml. For example, the level of IFN-γ can be less than 500 pg / ml or less than 100 pg / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml.

[0150] Methods for detecting the presence of cytokines in serum are known in the art, for example, enzyme-linked immunosorbent assay (ELISA). In another example, the presence of cytokines in serum is detected by measuring cytokine mRNA by polymerase chain reaction (PCR) techniques, such as reverse transcription PCR.

[0151] In one example, the newborn serum can be newborn bovine serum (NBCS). In one example, the NBCS is obtained from a newborn calf that has been fed colostrum. In one example, the NBCS has an elevated level of at least one inflammatory cytokine compared to NBCS obtained from a calf that has not been fed colostrum. In one example, the NBCS has an elevated level of at least one inflammatory cytokine compared to fetal serum such as FCS.

[0152] In one example, the NBCS is obtained within 4 weeks after the birth of the calf. In one example, the NBCS is obtained within 21 days after the birth of the calf. For example, the NBCS is obtained ≦21 days after the birth of the calf. In one example, the NBCS is obtained from the day of birth of the calf to 21 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 14 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 10 days after birth. In one example, the NBCS is obtained from the day of birth of the calf to 7 days after birth. In one example, the NBCS is obtained from 6 hours to 72 hours after birth. In one example, the NBCS is obtained from 6 hours to 48 hours after birth. In one example, the NBCS is obtained from 6 hours to 24 hours after birth. In one example, the NBCS is obtained from 12 hours to 24 hours after birth.

[0153] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of NBCS. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of NBCS. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of NBCS. In one example, the cell culture medium is supplemented with at least about 5% v / v NBCS.

[0154] In one example, the culture medium is also supplemented with fetal serum. In one example, the fetal serum is fetal calf serum (FCS). In the context of the present disclosure, the terms fetal calf serum (FCS) and fetal bovine serum (FBS) are considered to be interchangeable. In one example, the cell culture medium is supplemented with less than 10% v / v FCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS.

[0155] In one example, the cell culture medium is fetal serum-free.

[0156] In one example, the cell culture medium does not contain FCS.

[0157] In one example, the culture medium is supplemented with a mixture of FCS and NBCS. In one example, the cell culture medium is supplemented with about 5% v / v FCS and about 5% v / v NBCS (i.e., a 1:1 FCS:NBCS ratio). In one example, the culture medium can be supplemented with a mixture of FCS and NBCS, where the FCS:NBCS ratio is at least about 0.4:1, at least about 0.5:1, at least about 0.6:1, at least about 0.7:1, at least about 0.8:1, at least about 0.9:1, at least about 1:1, at least about 1.5:1, or at least about 2:1. In one example, the FCS:NBCS ratio is about 0.5:1 to about 2:1. In one example, the FCS:NBCS ratio is about 0.8:1 to about 1.5:1. In one example, the FCS:NBCS ratio is about 0.8:1 to about 1.2:1. In one example, the ratio of FCS:NBCS is about 1:1.

[0158] In one example, the mixture of FCS and NBCS can comprise at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise from about 1% v / v to about 15% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 2% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 5% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 8% v / v to about 12% v / v of the cell culture medium. In one example, the mixture of FCS and NBCS can comprise about 10% v / v of the cell culture medium, but in this example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, or at least about 9% v / v, but less than 10% v / v, of FCS. In one example, the cell culture medium is supplemented with about 1% v / v to about 9% v / v of FCS. In one example, the cell culture medium is supplemented with about 3% v / v to about 8% v / v FCS, in one example, about 3% v / v to about 6% v / v FCS, and in one example, about 5% v / v FCS.

[0159] Ascorbic acid In one example, a short-acting ascorbic acid derivative is added to the cell culture medium. The term "short-acting" encompasses ascorbic acid derivatives that are approximately 80-90% oxidized after 24 hours of cell culture under culture conditions of neutral pH and 37°C. In one example, the short-acting L-ascorbic acid derivative is an L-ascorbate salt, such as L-ascorbic acid sodium salt. In one example, the cell culture medium may contain at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.01 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.02 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.03 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.04 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.05 g / L of a short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.06 g / L of a short-acting ascorbic acid derivative.

[0160] In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not a substantial amount of a long-acting ascorbic acid derivative. For example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.04 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.03 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.02 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.01 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not more than 0.005 g / L of a long-acting ascorbic acid derivative. In another example, the cell culture medium may contain a short-acting ascorbic acid derivative but not a long-acting ascorbic acid derivative, hi another example, the cell culture medium contains L-ascorbic acid sodium salt but does not contain a substantial amount of L-ascorbic acid-2-phosphate.

[0161] Other additives In one example, the cell culture medium contains human-derived additives. For example, human serum and human platelet cell lysate can be added to the cell culture medium. In another example, additional factors can be added to the cell culture medium. For example, the cell culture medium can be supplemented with one or more stimulatory factors selected from the group consisting of platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), 1α,25-dihydroxyvitamin D3 (1,25D), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and stromal-derived factor 1α (SDF-1α). In another embodiment, the cells can be cultured in the presence of at least one cytokine in an amount sufficient to maintain cell growth. In another embodiment, the cells can be cultured in the presence of heparin or a derivative thereof.

[0162] In the above example, the basal medium such as AlphaMEM or StemSpan™ can be supplemented with a reference amount of serum, and in certain cases, can also be supplemented with other additives. Further examples of suitable culture media for culturing stem cells are described, for example, in WO2016139340.

[0163] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0164] This application claims priority to US 63 / 386,876 filed December 9, 2022 and US 63 / 507,013 filed June 8, 2023, the disclosures of which are incorporated herein by reference.

[0165] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0166] 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 is not to be construed as an admission that any or all of these matters form part of the prior art or were general knowledge in the art relevant to the present invention as existing prior to the priority date of each claim of this application. [Example]

[0167] Example 1, Serum Analysis Mesenchymal progenitor cell lineages or stem cell populations were culture-expanded in 5% FCS / 5% NBCS (serum A) or 10% fetal bovine serum (serum B). These MLPSCs were used in Examples 4-6.

[0168] Cytokine levels were assessed in 5% FCS / 5% NBCS (Serum A) and 10% fetal bovine serum (Serum B). To provide an external control, cytokine levels were also assessed in FBS from a different source (Serum C). In both cases, cytokine concentrations were assessed in pure serum.

[0169] Surprisingly, serum specimens containing neonatal bovine serum had higher levels of pro-inflammatory cytokines (Figure 1). Particularly noteworthy was the increase in pro-inflammatory cytokines known to bind to receptors expressed on the surface of MLPSCs, including interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukins. For example, the following was observed in serum preparations containing neonatal bovine serum compared to fetal bovine serum: At least a two-fold increase in IFNγ At least a 13-fold increase in TNFα, At least an 8-fold increase in IL-6, At least a two-fold increase in IL-8, At least a two-fold increase in IL-17A.

[0170] Example 2: MLPSC composition obtained using a culture medium containing fetal serum Alpha modifications of Eagle's minimum essential medium (MEM) with Earle's balanced salts, commonly referred to as Eagle's α-MEM, contain non-essential amino acids, sodium pyruvate, and additional vitamins. These modifications were first described for growing hybrid mouse and hamster cells (Stanners et al. 1971).

[0171] Eagle's αMEM medium, suitable for culturing primary stem cells, can be obtained from a variety of sources, including Life Technologies and Sigma.

[0172] Detailed methods for establishing primary stem cell cultures, including the necessary growth factors used in the exemplified process, are described in Gronthos and Simmons 1995.

[0173] MLPSCs were cultured in Eagle's α-MEM medium supplemented with 10% fetal bovine serum, L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10−7 M), and / or inorganic phosphate (3 mM).

[0174] Example 3: MLPSC composition obtained using a culture medium containing neonatal serum For MLPSC culture medium containing neonatal serum, the serum component of Eagle's αMEM culture medium described in Example 2 was modified by adding 5% (v / v) neonatal serum (the differences between fetal serum medium and neonatal serum medium are shown in Table 1). The neonatal serum used was neonatal bovine serum (NBCS). NBCS meets the specifications of standard fetal bovine serum but is 100% bovine serum obtained from animals less than 20 days old.

[0175] NBCS was obtained from a commercial supplier and is marketed as an FCS substitute that is very similar to FCS, can be used interchangeably, and is expected to exert similar effects on cell lines. [Table 1]

[0176] Example 4: Expansion of MLPSCs in culture medium supplemented with neonatal serum promotes angiogenesis To characterize novel MLPSC populations obtained by expansion in culture medium supplemented with neonatal serum and / or proinflammatory cytokines, we evaluated the angiogenic potential of conditioned medium obtained from MLPSCs cultured under different conditions.

[0177] Cell culture: MPCs were cultured in either 10% FCS or 5% NBCS / 5% FCS to generate MPC-conditioned medium. To control for donor variability, MPCs were obtained from the same donor and cultured under different conditions. In some experiments, MPCs belonging to the same donor but cultured during different manufacturing expansions are indicated by different "lot" numbers.

[0178] Conditioned medium was obtained by separating cells from conditioned medium. Briefly, cryopreserved MPCs were thawed and cultured at 50,000 / cm in either αMEM and 10% FBS or 5% NBCS / 5% FCS. 2 Cells were seeded at 1×. After incubation at 37°C and 5% CO for 72 hours, conditioned medium (CM) was collected. VEGF, SDF-1, and angiogenin levels in CM were measured using Luminex (R&D Systems). CM was concentrated using a 3k protein concentration filtration column (Amicon® Ultra-15) and reconstituted at 1× or 0.25× in assay medium.

[0179] Angiogenesis Efficacy Assay: In vitro angiogenesis was measured using a kinetic, quantitative 96-well co-culture angiogenesis model. Lentiviral-transduced human umbilical vein endothelial cells (HUVECs) expressing CytoLight Green (a GFP variant) were co-cultured with normal human dermal fibroblasts (NHDFs), seeded into 96-well plates, and simultaneously incubated and imaged using the IncuCyte® Live-Cell Analysis System. This system allowed for the determination of the angiogenesis potential of HUVECs (CytoLight Green). + Fluorescence identification of cells is enabled, and time-lapse image acquisition allows visualization of tube formation over time. The acquired images are analyzed using an integrated angiogenesis algorithm to measure network length, network area, and branch point formation to quantify the stage and extent of angiogenesis throughout the assay.

[0180] Results: Surprisingly, we found that conditioned medium from MPCs cultured in medium supplemented with neonatal bovine serum (NEBS) increased angiogenesis. As shown in Figure 2, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS increased network area (Figure 2A), network length (Figure 2B), and branch points (Figure 2C) in the coculture angiogenesis model. Furthermore, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS contained higher levels of VEGF compared to conditioned medium from cells cultured in 10% FCS (Figure 2A). Angiogenin levels were also increased in conditioned medium from MPCs cultured in 5% NBCS / 5% FCS compared to 10% FCS (Figure 3). Figure 4 shows further analysis of the levels of the angiogenic factors SDF-1α, VEGF, and Ang1 (ANGPT1) present in MPCs cultured in 10% FCS ("Serum B Medium") or 5% FCS / 5% NBCS ("Serum A Medium"). These data show that both VEGF and SDF-1α are elevated in MPCs cultured in neonatal serum medium. These data show that both VEGF and SDF-1α are elevated in MPCs cultured in neonatal serum medium.

[0181] Considering the data provided in Example 1, these data indicate that culture expansion of MLPSCs in medium supplemented with neonatal serum and / or pro-inflammatory cytokines results in a novel conditioned medium with enhanced angiogenic potential. This enhanced potential can be characterized in various ways, as desired, for example, by defining the novel conditioned medium identified by the inventors, which can be, for example, The ability of conditioned medium obtained from MLPSCs to increase network area, network length, and / or branch points upon contact with HUVECs; · Including levels of angiogenin, VEGF, and / or SDF-1 in conditioned medium.

[0182] Example 5. Isolation and expansion of MLPSCs MLPSCs can be isolated using techniques such as STRO-3+ immunoselection of MPCs or density gradient separation of MSCs.

[0183] Typically, for bone marrow-derived MLPCs, bone marrow (BM) is collected from healthy adult volunteers (20–35 years old). Briefly, 40 ml of BM is aspirated from the posterior iliac crest and placed into a tube containing lithium heparin anticoagulant.

[0184] BMMNCs were prepared by density gradient separation using Lymphoprep (Nycomed Pharma, Oslo, Norway) as previously described (Zannettino et al. 1998). After centrifugation at 400 × g for 30 minutes at 4°C, the buffy layer was removed with a transfer pipette and washed three times with "HHF," which consisted of Hank's balanced salt solution (HBSS; Life Technologies, Gaithersburg, MD) containing 5% fetal calf serum (FCS, CSL Limited, Victoria, Australia).

[0185] In conjunction with immunoselection, STRO-3+ (or TNAP+) cells are isolated by magnetic-activated cell sorting as previously described (Gronthos et al. 2003; Gronthos and Simmons 1995). Briefly, approximately 1-3 x 108 BMMNCs are incubated on ice for 20 minutes in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells are then incubated on ice for 1 hour with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer. The cells are then centrifuged at 400 x g and washed twice in HHF. A 1 / 50 dilution of goat anti-mouse biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer is added, and the cells are incubated on ice for 1 hour. Cells were washed twice with MACS buffer (Ca2+- and Mn2+-free PBS supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide) as described above and resuspended in a final volume of 0.9 ml of MACS buffer.

[0186] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice, resuspended in 0.5 ml of MACS buffer, and then loaded onto a mini-MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3 mAb (Deposited with the American Type Culture Collection (ATCC) on December 19, 2005, under accession number PTA-7282 - see International Publication No. WO 2006 / 108229). After the addition of an additional 1 ml of MACS buffer, the column was removed from the magnet and TNAP+ cells were isolated under positive pressure. A portion of the cells from each fraction could be stained with streptavidin-FITC and their purity assessed by flow cytometry.

[0187] Alternatively, MSCs can be grown from BMMNCs using plastic adherence techniques. For example, bone marrow mononuclear cells are isolated using Ficoll-Hypaque and plated into two T175 flasks with 50 ml of culture expansion medium containing alpha-modified MEM (αMEM) supplemented with gentamicin, glutamine (2 mM), and 10% (v / v) fetal bovine serum (FBS).

[0188] Cells are cultured at 37°C, 5% CO for 2-3 days, at which point non-adherent cells are removed and the remaining adherent cells are continuously cultured until cell confluence reaches 70% or greater (7-10 days), after which the cells are trypsinized and replaced into six T175 flasks containing Expansion Medium.

Claims

1. 1. A composition comprising conditioned medium or extracellular vesicles obtained thereby, wherein the conditioned medium is characterized by increased levels of angiogenic marker(s), the conditioned medium comprising: IFN-γ and / or TNF-α, and / or - The composition is obtained from a population of MLPSCs culture-expanded in a culture medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, IP-10, wherein the increased levels of angiogenic marker(s) are determined relative to conditioned medium obtained from a control population.

2. The angiogenesis marker is i. increased levels of angiogenin compared to conditioned medium obtained from a control group; ii. increased levels of endothelial network formation compared to conditioned medium obtained from a control group; iii. increased endothelial length compared to conditioned medium obtained from a control group; iv. The composition of claim 1, wherein one or more or all of the following are increased endothelial branch length compared to conditioned medium obtained from a control population.

3. 3. The composition of claim 1 or claim 2, wherein the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

4. The composition of any one of claims 1 to 3, wherein the conditioned medium contains an angiogenin level greater than about 1200 pg / ml.

5. The composition of any one of claims 1 to 4, wherein the conditioned medium contains a level of SDF-1 greater than about 3000 pg / ml.

6. The composition of any one of claims 1 to 5, wherein the conditioned medium contains a level of VEGF greater than about 3200 pg / ml.

7. The conditioned medium is about 0.12 mm ２ / mm ２ The composition according to any one of claims 1 to 6, which induces the formation of a transendothelial network.

8. The conditioned medium is about 5 mm ２ / mm ２ The composition of any one of claims 1 to 7, which induces an endothelial network length of greater than 1000 ng / cm.

9. The conditioned medium has a density of about 15 l / mm ２ 9. The composition of claim 1, wherein the composition induces an endothelial branch length of greater than 100 μg / ml.

10. The composition of any one of claims 1 to 9, wherein the culture medium contains three or more pro-inflammatory cytokines.

11. The composition of any one of claims 1 to 10, wherein the culture medium contains two or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

12. The composition according to any one of claims 1 to 11, wherein the culture medium contains IL-6.

13. The composition according to any one of claims 1 to 12, wherein the culture medium contains IL-8 and / or IL-17A.

14. The composition according to any one of claims 1 to 13, wherein the culture medium contains IFN-γ and TNF-α.

15. The composition of any one of claims 1 to 14, wherein the level of IFN-γ is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml.

16. The composition of any one of claims 1 to 15, wherein the level of TNF-α is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

17. The composition of any one of claims 1 to 16, wherein the culture medium contains serum containing pro-inflammatory cytokines.

18. 18. The composition of claim 17, wherein the serum is newborn mammalian serum, preferably newborn bovine serum.

19. 18. The composition of claim 17, wherein the serum is obtained within 21 days after birth.

20. The culture medium comprises: i. IFN-γ levels greater than 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; vi. MCP-1 levels greater than 3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. A composition according to any one of claims 1 to 19, characterized by one or more or all of the following: a level of IP-10 greater than 500 pg / ml.

21. The composition of any one of claims 1 to 20, wherein the culture medium comprises at least 5% (v / v) newborn mammalian serum.

22. The composition of any one of claims 1 to 16 or 20, wherein the medium is serum-free.

23. The composition of any one of claims 1 to 22, wherein the extracellular vesicles obtained from the conditioned medium are exosomes.

24. A composition comprising conditioned medium or extracellular vesicles obtained thereby, wherein the conditioned medium is obtained from a population of MLPSCs culture-expanded in a medium containing newborn mammalian serum.

25. 25. The composition of claim 24, wherein the serum is newborn bovine serum.

26. 26. The composition of claim 24 or claim 25, wherein the serum is obtained within 21 days of birth.

27. 27. The composition of any one of claims 24 to 26, wherein the conditioned medium comprises increased levels of angiogenic marker(s) compared to conditioned medium obtained from a control population.

28. The angiogenesis marker is i. increased levels of angiogenin compared to conditioned medium obtained from a control group; ii. increased endothelial network formation compared to conditioned medium obtained from a control group; iii. increased endothelial length compared to conditioned medium obtained from a control group; iv. The composition of claim 27, wherein one or more or all of the following are increased endothelial branch length compared to conditioned medium obtained from a control population.

29. 29. The composition of claim 27 or claim 28, wherein the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

30. 30. The composition of any one of claims 24 to 29, wherein the conditioned medium contains an angiogenin level greater than about 1200 pg / ml.

31. The composition of any one of claims 24 to 30, wherein the conditioned medium contains a level of SDF-1 greater than about 3000 pg / ml.

32. 32. The composition of any one of claims 24 to 31, wherein the conditioned medium contains a level of VEGF greater than about 3200 pg / ml.

33. The conditioned medium is about 0.12 mm ２ / mm ２ The composition according to any one of claims 24 to 32, which induces the formation of a transendothelial network.

34. The conditioned medium is about 5 mm ２ / mm ２ 34. The composition of any one of claims 24 to 33, which induces an endothelial network length of greater than 1000 ng / cm.

35. The conditioned medium has a density of about 15 l / mm ２ 35. The composition of any one of claims 24 to 34, which induces an endothelial branch length of greater than 100 μg / ml.

36. The composition according to any one of claims 18 to 35, wherein the newborn serum is a newborn serum from a newborn on day 1 to day 7 after birth.

37. 37. The composition of any one of claims 18 to 36, wherein the concentration of the newborn serum is from about 2% (v / v) to about 12% (v / v).

38. The composition of any one of claims 18 to 36, wherein the concentration of the newborn serum is about 5% (v / v).

39. 37. The composition of any one of claims 18 to 36, wherein the culture medium comprises fetal serum and neonatal serum of the same species, and the ratio of the concentration of the fetal serum to the concentration of the neonatal serum is 1:1 or less.

40. 40. The composition of claim 39, wherein the concentration of the fetal serum and the concentration of the newborn serum are each 5% (v / v).

41. 40. The composition of claim 39, wherein the concentration of the fetal serum is less than the concentration of the newborn serum.

42. The composition of any one of claims 24 to 41, wherein the extracellular vesicles obtained from the conditioned medium are exosomes.

43. The composition of any one of claims 1 to 42, wherein the MLPSCs are human mesenchymal stem cells (hMSCs).

44. 44. The composition of any one of claims 1 to 43, wherein the MLPSCs are culture-expanded from a population of STRO-1+ pluripotent cells.

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