Pre-approved compositions and cell culture methods

Abstract

The present disclosure relates to MLPSC populations, and methods and cell culture media for producing the same, which may be particularly useful for facilitating the prequalification of mesenchymal progenitor or stem cells (MLPSCs).

Description

[Technical Field]

[0001] The present disclosure relates to MLPSC populations, and methods and cell culture media for producing the same, which may be particularly useful for facilitating the prequalification of mesenchymal progenitor or stem cells (MLPSCs). [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] Conventional media used for the isolation and expansion of MSCs consist of defined basal media (e.g., Dulbecco's modified Eagle's medium (DMEM) or alpha-modified minimal essential medium (α-MEM)) supplemented with fetal bovine serum (FBS) due to their high content of stimulatory growth factors. Although these media are generally reported to support multiple rounds of MLPSC proliferation, batch-to-batch variation in FBS may lead to inconsistencies in the therapeutic properties or potency of MLPSCs.

[0004] Thus, there remains an unmet need for compositions and methods for the consistent production of therapeutically effective MLPSCs in serum-containing cell culture. Summary of the Invention

[0005] The present inventors unexpectedly discovered that MLPSC cultures supplemented with neonatal serum exhibit a pre-adapted (also known as "pre-approved") anti-inflammatory state that enhances therapeutic efficacy, particularly in the setting of persistent inflammation. For example, neonatal bovine serum (NBCS) is commonly marketed as an equivalent or acceptable alternative to fetal bovine serum (FBS). However, the present inventors unexpectedly discovered that this is not the case, as, as described herein, the addition of NBCS elicited a pre-approved effect on cultured MLPSCs. Analysis of the neonatal serum used to culture MLPSCs with enhanced therapeutic efficacy surprisingly revealed increased levels of cytokines, particularly for cytokines whose corresponding receptors are expressed by MLPSCs. These findings support the generation of novel MLPSC compositions by culture expansion in certain pro-inflammatory cytokines and / or neonatal serum.

[0006] Thus, in one aspect, the present disclosure relates to a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs have been culture-expanded in medium containing interferon (IFN)-γ and / or tumor necrosis factor (TNF)-α, and wherein the level(s) of IFN-γ and / or TNF-α in the medium is <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.

[0007] In another aspect, the present disclosure relates to a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs have been cultured in a medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

[0008] Thus, in another aspect, the present disclosure provides a method for producing a medicament for a method of manufacturing a medicament for a medical device, comprising: (i) A composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), the MLPSCs comprising: IFN-γ and / or TNF-α, and / or - expanded in culture in a medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

[0009] In one example, the medium contains three or more pro-inflammatory cytokines.

[0010] In another example, the 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.

[0011] In another example, the medium contains IL-6. In another example, the medium contains IL-8 and / or IL-17A. In another example, the medium contains IFN-γ and TNF-α. For example, the medium may contain IFN-γ, 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.

[0012] In one example, the level of IFN-γ is <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 another example, the level of TNF-α is <1 ng / ml. For example, the level of TNF-α can be <750 pg / ml. In one example, the level of TNF-α is <400 pg / ml.

[0013] In another example, the medium contains serum containing proinflammatory cytokines. In one example, the serum is newborn mammal serum. For example, the serum can be newborn bovine serum. In one example, the newborn serum is obtained within 21 days after birth.

[0014] In one example, the compositions of the present disclosure are cryopreserved.

[0015] In one example, the medium is IFN-γ levels greater than 1 pg / ml, TNF-α levels greater than 2 pg / ml, IL-6 levels greater than 3 pg / ml, IL-8 levels above 500 pg / ml, IL-17A levels greater than 0.2 pg / ml, MCP-1 levels greater than 3 pg / ml, MIP-1-α levels above 0.5 pg / ml, MIP-1-β levels greater than 3 pg / ml, Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0016] In one example, the medium is IFN-γ levels between 1pg / ml and 1ng / ml; TNF-α levels greater than 2pg / ml to less than 1ng / ml, IL-6 levels greater than 3 pg / ml, IL-8 levels above 500 pg / ml, IL-17A levels greater than 0.2 pg / ml, MCP-1 levels greater than 3 pg / ml, MIP-1-α levels above 0.5 pg / ml, MIP-1-β levels greater than 3 pg / ml, Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0017] In another example, the medium may contain a level of IFN-γ between 1 pg / ml and <1 ng / ml, a level of TNF-α between >2 pg / ml and <1 ng / ml, and IFN-γ levels between 1pg / ml and 1ng / ml; TNF-α levels greater than 2pg / ml to less than 1ng / ml, IL-6 levels greater than 3 pg / ml, IL-8 levels above 500 pg / ml, IL-17A levels greater than 0.2 pg / ml, MCP-1 levels greater than 3 pg / ml, MIP-1-α levels above 0.5 pg / ml, MIP-1-β levels greater than 3 pg / ml, Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0018] In another example, the medium is IFN-γ levels greater than 10 pg / ml, TNF-α levels above 20 pg / ml, IL-6 levels above 30 pg / ml, IL-8 levels greater than 5,000 pg / ml, IL-17A levels greater than 2 pg / ml, MCP-1 levels above 30 pg / ml, MIP-1-α levels greater than 5 pg / ml, MIP-1-β levels above 30 pg / ml, · Characterized by the addition of serum containing one or more or all of the following levels of IP-10 greater than 5,000 pg / ml.

[0019] 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.

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

[0021] Thus, in another aspect, the present disclosure relates to a composition for the pre-approval of MLPSCs, comprising: (i) a human cell population enriched for MLPSCs; and (ii) a serum-containing cell culture medium, wherein the serum comprises one or more pro-inflammatory cytokines.

[0022] In another aspect, the present disclosure relates to a composition for pre-qualification and cryopreservation of MLPSCs, comprising (i) a human cell population enriched for MLPSCs, (ii) serum containing one or more pro-inflammatory cytokines, and (iii) a cryopreservation agent. In one example, the MLPSCs are cryopreserved at least twice.

[0023] In another aspect, the present invention relates to a cell culture medium suitable for the growth and pre-qualification of MLPSCs, wherein the cell culture medium comprises serum containing one or more pro-inflammatory cytokines.

[0024] In another aspect, the present disclosure relates to an in vitro method for prequalification of human mesenchymal progenitor or stem cells (MLPSCs), the method comprising: - IFN-γ and / or TNF-α, and / or one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, IP-10, and / or - Culturing MLPSCs in a medium containing newborn mammalian serum obtained within 21 days after birth.

[0025] For example, the method can include culturing MLPSCs in a medium containing IFN-γ and TNF-α. In one example, the level(s) of IFN-γ and / or TNF-α is <1 ng. In another example, the method can include culturing MLPSCs 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, and IP-10. In another example, the method can include culturing MLPSCs in a culture medium containing newborn mammalian serum obtained within 21 days after birth.

[0026] In one example, the method includes culturing MLPSCs in a medium containing 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.

[0027] In one example, the medium contains three or more proinflammatory cytokines. In this example, two or more of the proinflammatory cytokines may be selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. In one example, the medium contains IL-6. In one example, the medium contains IL-8 and / or IL-17A. In one example, the medium contains IFN-γ and TNF-α. In one example, the level of IFN-γ is <500 pg / ml. In one example, the level of IFN-γ is <100 pg / ml. In another example, the level of TNF-α is <750 pg / ml. In one example, the level of TNF-α is <400 pg / ml.

[0028] In one example, the medium contains serum containing proinflammatory cytokines. For example, the serum can be newborn serum, such as newborn bovine serum. In one example, the newborn serum is obtained within 21 days after birth.

[0029] 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.

[0030] 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.

[0031] In one example, the medium comprises at least 5% (v / v) newborn mammalian serum. In another example, the medium comprises 5% (v / v) newborn mammalian serum. In another example, the medium comprises 5% (v / v) newborn mammalian serum and 5% (v / v) fetal bovine serum. In another example, the medium is serum-free.

[0032] In one example, the MLPSCs in the composition express increased levels of angiogenic marker(s) compared to a control population. In one example, the MLPSCs in the composition have increased levels of angiogenin compared to a control population. In one example, conditioned medium from the MLPSCs induces increased levels of one or more of endothelial network formation, endothelial length, or endothelial branch length compared to a control population. In one example, the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

[0033] In one example, the MLPSCs express angiogenin levels greater than about 1200 pg / ml. In another example, the MLPSCs express SDF-1 levels greater than about 3000 pg / ml. In another example, the MLPSCs express VEGF levels greater than about 3200 pg / ml.

[0034] In one example, conditioned medium from MLPSCs was approximately 0.12 mm 2 / mm 2 In one example, conditioned medium from MLPSCs induces the formation of an endothelial network of approximately 5 mm2 / mm 2 In one example, conditioned medium from MLPSCs induces an endothelial network length of approximately 15 1 / mm. 2 Induce ultra-endothelial branch length.

[0035] In a further aspect, the present disclosure relates to an in vitro method for the pre-qualification of MLPSCs, the method comprising culturing a human cell population enriched for MLPSCs in a serum-containing cell culture medium suitable for the maintenance and expansion of MLPSCs, wherein the serum comprises one or more pro-inflammatory cytokines. In one example, the present disclosure relates to a composition produced by the aforementioned exemplary method.

[0036] In another aspect, the present disclosure relates to a cryopreserved composition, comprising: (i) a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs are culture-expanded in newborn mammalian serum obtained within 21 days after birth; and (ii) a cryopreservative.

[0037] In some embodiments of any of the foregoing compositions, culture media, or methods, the one or more proinflammatory cytokines comprise IL-1β, IL-6, IFN-, TNF-α, or IL-1 receptor antagonist (IL-1RA). In some embodiments, the one or more proinflammatory cytokines comprise IL-1β, IL-6, IFN-, TNF-α, and IL-1RA.

[0038] In some embodiments of any of the foregoing compositions, culture media, or methods, the serum comprising one or more proinflammatory cytokines comprises newborn serum. In some preferred embodiments, the newborn serum is newborn bovine serum. In some embodiments, the newborn serum is from a newborn 1 day after birth to a 7 day after birth. In some embodiments, the newborn serum is from a newborn 1 day after birth. In some embodiments, the newborn serum is from a newborn 1 day after birth to a 20 day after birth.

[0039] In some embodiments of any of the aforementioned compositions, culture media, or methods, when the serum containing one or more inflammatory cytokines is neonatal serum, the concentration of the neonatal serum is about 2% (v / v) to about 12% (v / v). In some preferred embodiments, the concentration of the neonatal serum is about 5% (v / v). In other embodiments, the concentration of the neonatal serum is about 10% (v / v).

[0040] In some embodiments, the concentrations of cytokines in neonatal serum are as follows: (i) about 2 ng / ml to about 50 ng / ml for IL-1; (ii) IL-1β: about 2 ng / ml to about 50 ng / ml (iii) about 0.2 ng / ml to about 1.2 ng / ml for IL-6; (iv) about 0.1 ng / ml to about 0.2 ng / ml for IFN-γ; (v) about 6 ng / ml to about 33 ng / ml for IL-1ra, or (vi) about 0.1 ng / ml to about 0.7 ng / ml for TNF-α.

[0041] In some embodiments, the concentrations of cytokines in neonatal serum are as follows: (i) about 2 ng / ml to about 50 ng / ml for IL-1; (ii) IL-1β: about 2 ng / ml to about 50 ng / ml (iii) about 0.2 ng / ml to about 1.2 ng / ml for IL-6; (iv) about 0.1 ng / ml to about 0.2 ng / ml for IFN-γ; (v) about 6 ng / ml to about 33 ng / ml for IL-1ra, and (vi) about 0.2 ng / ml to about 0.7 ng / ml for TNF-α.

[0042] In some embodiments of any of the aforementioned compositions, culture media, or methods, fetal serum is included or used in addition to newborn serum. In some embodiments, when both fetal serum and newborn serum are included or used, the ratio of the concentration of fetal serum to the concentration of newborn serum is 1:1. In some embodiments, when the ratio of the concentrations of fetal serum to newborn serum is 1:1, the concentrations of fetal serum and newborn serum are 5% (v / v), respectively. In other examples, the concentration of fetal serum is lower than the concentration of newborn serum.

[0043] In some embodiments of any of the aforementioned compositions, culture media, or methods, neonatal serum is substantially the only source of exogenous pro-inflammatory cytokines.

[0044] In some embodiments of any of the aforementioned methods, the method also includes determining or having determined the level of one or more pro-inflammatory cytokines in the serum.

[0045] In some embodiments of any of the foregoing compositions, culture media, or methods, the MLPSCs are maintained in an undifferentiated state.

[0046] In some embodiments of any of the foregoing compositions, culture media, or methods, the MLPSCs are human mesenchymal stem cells (hMSCs). In other examples, the MLPSCs are culture-expanded from a population of STRO-1+ pluripotent cells.

[0047] In another aspect, the present disclosure relates to a medium for culturing MLPSCs, the medium comprising: IFN-γ and / or TNF-α, and / or - 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.

[0048] For example, the medium can contain IFN-γ and / or TNF-α, hi one example, the level of IFN-γ and / or TNF-α in the medium is <1 ng.

[0049] In one example, the medium contains three or more proinflammatory cytokines. In one example, the 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 one example, the medium contains IL-6. In another example, the medium contains IL-8 and / or IL-17A. In one example, the medium contains IFN-γ and TNF-α. In one example, the level of IFN-γ is <500 pg / ml. In one example, the level of IFN-γ is <100 pg / ml. In another example, the level of TNF-α is <750 pg / ml. In one example, the level of TNF-α is <400 pg / ml.

[0050] In one example, the medium contains serum containing proinflammatory cytokines. In one example, the serum is newborn mammalian serum, such as newborn bovine serum. In one example, the newborn serum is obtained within 21 days after birth.

[0051] 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.

[0052] In one example, the medium contains a level of IFN-γ greater than 1 pg / ml, a level of TNF-α greater than 2 pg / ml, and IL-6 levels greater than i.3 pg / ml; ii. IL-8 levels greater than 500 pg / ml; iii. IL-17A levels greater than 0.2 pg / ml; iv. MCP-1 levels greater than 3 pg / ml; v. MIP-1-α levels greater than 0.5 pg / ml; vi. MIP-1-β levels greater than 3 pg / ml; vii. Characterized by one or more or all of the following: IP-10 levels greater than 500 pg / ml.

[0053] In one example, the medium comprises at least 5% (v / v) newborn mammalian serum, hi another example, the medium comprises 5% (v / v) newborn mammalian serum.

[0054] In one example, the medium is serum-free. For example, the present disclosure encompasses a serum-free MLPSC culture medium, which 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-1-α, MIP-1-β, IP-10. [Brief explanation of the drawings]

[0055] [Figure 1] Assessment and comparison of serum cytokine levels in 1:1 FCS / NBCS (serum A), fetal bovine serum (serum B), and FBS from another source (serum C). [Figure 2] Quantitative measurement of in vitro angiogenesis induced by MLPSC-conditioned medium using the IncuCyte® 96-Well Kinetic Angiogenesis PrimeKit Assay. [Figure 3]1 shows the results of a Luminex assay demonstrating increased production of angiogenin by MLPSCs cultured with or without neonatal serum. [Figure 4] Levels of angiogenic markers in cGMP lot MLPSC conditioned medium cultured with or without neonatal serum. [Figure 5] Analysis of changes from baseline in echo parameters after 12 months - all subjects. [Figure 6] Analysis of change from baseline in echo parameters after 12 months - subjects with persistent inflammation (hsCRP ≥ 2). [Figure 7] Analysis of changes from baseline in echo parameters after 12 months - subjects without persistent inflammation (hsCRP<2). [Figure 8] CV mortality in subjects with persistent inflammation (hsCRP≧2) by MPC cultured with or without neonatal serum. [Figure 9] Three-point composite MACE (MI, stroke, or CV death) in subjects with persistent inflammation (hsCRP≧2) by MPC cultured with or without neonatal serum. [Figure 10] 3-point composite MACE (MI, stroke, or CV death) in subjects receiving MPC cultured with or without neonatal serum at all passages or at the final passage. The left panel represents data for all patients. The right panel represents data for patients with persistent inflammation (CRP>2 mg / ml). [Figure 11] Three-point composite MACE (left panel) and terminal cardiac events (TCE, right panel) in subjects with the most severe disease (NTpro-BNP>1000ng / ml, CRP>2mg / ml) treated with MPC cultured with or without neonatal serum at all or the final passage. DETAILED DESCRIPTION OF THE INVENTION

[0056] 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).

[0057] 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. Clover 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).

[0058] 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.

[0059] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0060] "C-reactive protein" or "CRP" is an inflammatory mediator. CRP levels are elevated during acute inflammatory flare-ups and normalize quickly once the inflammation subsides. Thus, CRP is a useful marker of persistent inflammation. In one example, the term "persistent inflammation" is used to refer to a subject with elevated CRP. The term "elevated CRP," in the context of the present disclosure, is used to refer to an increased CRP level compared to baseline CRP levels. In one example, a CRP level of 1 mg / L or greater is elevated. In another example, a CRP level of 1.5 mg / L or greater is elevated. In another example, a CRP level of ≧2 mg / L is elevated. Thus, in one example, persistent inflammation is characterized by a CRP level of ≧2 mg / L.

[0061] N-terminal pro-B-type natriuretic peptide (NT-proBNP) is an inactive peptide released along with the active peptide hormone BNP when the heart wall is stretched or the heart is under pressure. In the context of the present disclosure, high-risk patients (e.g., patients at high risk of cardiovascular death) can be defined based on their NT-proBNP levels. In one example, the level is >1000 pg / mL or between 1000 pg / mL and 2500 pg / mL. In one example, these patients also have persistent inflammation. For example, these patients can have CRP levels of ≥2 mg / L.

[0062] 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.

[0063] In one example, the level of a particular marker is determined under culture conditions. The term "culture conditions" is used to refer to cells growing in culture. In one example, the culture conditions refer to a population of cells that are actively dividing. Such cells may, in one example, be in an exponential growth phase.

[0064] In one example, the culture conditions include co-culturing a population of MLPSCs disclosed herein with a second cell population, such as a population comprising peripheral blood mononuclear cells (PBMCs). In one example, the co-culturing includes culturing a population of MLPSCs disclosed herein and an activated population of PBMCs. For example, the PBMCs can be activated using anti-CD3 and anti-CD28 antibodies prior to co-culturing with the population of MLPSCs disclosed herein.

[0065] In one example, the "culture conditions" include co-culturing MLPSCs and T cells at a ratio of approximately 1 MLPSC:2 T cells. For example, the MLPSC:T cell ratio is 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100 or less. In this example, the level of IL2-RA inhibition is determined after approximately 30-84 hours of cell culture under the culture conditions.

[0066] In one example, the level of a particular marker can be determined by taking a sample of cell culture medium and measuring the level of the marker in the sample. In another example, the level of a particular marker can be determined by taking a sample of cells and measuring the level of the marker in a cell lysate. Those skilled in the art will understand that secreted markers 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, a sample is taken when the cells are in the exponential growth phase. In one example, a sample is taken after at least 2-3 days of culture. In another example, a sample is taken after approximately 30-84 hours of culture. 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 the 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 various methods, such as methods based on enzyme-linked immunosorbent assay (ELISA). 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.

[0067] In another example, the level of IL2-RA is determined using fluorescence-activated cell sorting (FACS) with an appropriate antibody, such as anti-CD25. Additional antibodies can be used if it is necessary to distinguish between CD25+ cell types. While the above example refers to IL-2RA, it will be appreciated that similar methods can be used to determine the levels of other markers disclosed herein, such as angiogenin. In these examples, co-culture may not be required to determine the level. For example, the level of angiogenin can be measured in a population of MLPSCs under culture conditions.

[0068] 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.

[0069] 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.

[0070] The term "angiogenic marker" as used herein refers to an indicator of angiogenesis. As used herein, "angiogenic marker" includes pro-angiogenic molecules such as VEGF, angiogenin, and SDF-1α. 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).

[0071] 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.

[0072] 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. "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. In another example, a sample is obtained from a patient (e.g., a blood sample). Such a sample can be used to determine the level of a marker such as CRP.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] Unless otherwise specified, examples disclosed herein are intended to apply mutatis mutandis to other examples.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] "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.

[0094] 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.

[0095] Additionally, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In one example, the present disclosure encompasses mesenchymal progenitor or stem cells, 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 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 use in therapy.

[0100] As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, but typically are less than 200 nm in size. They are released from the releasing cells (e.g., mesenchymal stem cells; STRO-1). + The fragment may contain proteins, nucleic acids, lipids, metabolites or organelles from the cell.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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+).

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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, STRO-1 bri The cells may further be one or more of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), and / or CD146+. For example, the cells may be selected for 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.

[0110] 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.

[0111] 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.

[0112] 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 Cells have a two-log increase in STRO-1 surface expression compared to "background," i.e., STRO-1-negative cells. dim cells and / or STRO-1 intermediate Cells have less than 2 logs of STRO-1 surface expression, typically about 1 log or less above "background."

[0113] 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.

[0114] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0115] 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 duct 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.

[0116] 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.

[0117] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs (e.g., remestemcel-L).

[0118] 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.

[0119] Culture-expanded MLPC In an example, culture-expanded MLPSCs of the present disclosure are characterized by expression of angiogenic marker(s). For example, culture-expanded MLPSC populations according to the present disclosure are characterized by increased levels of VEGF, angiogenin, and / or SDF-1α under culture conditions. In another example, MLPSC populations can be characterized based on evaluation of conditioned medium obtained from the MLPSC population under culture conditions. In one example, the conditioned medium increases the level of endothelial network formation, endothelial network length, and / or endothelial branch length in a population of endothelial cells when endothelial cells are treated with conditioned medium obtained from culture-expanded MLPSCs. In one example, the increase is determined compared to 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.

[0120] In one example, the expanded MLPSC population 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.

[0121] In one example, the MLPSCs have increased levels of angiogenin compared to a control population. In one example, the expanded MLPSC population 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 between about 1000 pg / ml and 1200 pg / ml. In one example, the angiogenin level is between about 1100 pg / ml and 1150 pg / ml. In one example, the angiogenin level is about 1114 pg / ml.

[0122] In one example, the expanded MLPSC population 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.

[0123] In one example, a culture-expanded MLPSC population is about 0.1 mm 2 / mm 2 The method is characterized by a conditioned medium that stimulates the formation of an endothelial network of greater than 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 2is.

[0124] In one example, a culture-expanded MLPSC population was grown to approximately 4 mm 2 / mm 2 The conditioned medium is characterized by stimulating an endothelial network length of greater than 4 mm. In one example, the endothelial network length 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, a culture-expanded MLPSC population has a density of about 12 1 / mm 2 The conditioned medium is characterized by stimulating endothelial branch lengths of greater than 12 mm. In one example, endothelial branch lengths are approximately 12 mm. 2 ~approximately 17 1 / mm 2 In one example, the length of the endothelial branch is about 15 1 / mm 2 is.

[0125] In one example, the culture-expanded MLPSC population is characterized by an increased level of one or more angiogenic markers compared to 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% compared to 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% 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, levels of angiogenic markers are increased compared to a population of MLPSCs cultured and expanded in cell culture medium without IFN-γ or TNF-α.

[0126] In one example, the expanded MLPSC population is characterized by an increased level of one or more angiogenic markers compared to an MLPSC population cultured and expanded in cell culture medium without neonatal 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% compared to a population of MLPSCs cultured and expanded in cell culture medium without neonatal serum. 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%.

[0127] In another example, the cultured and expanded MLPSCs of the present disclosure are characterized based on their therapeutic effect.For example, MLPSCs can be characterized based on their therapeutic effect in inflammatory diseases.In one example, MLPSCs are characterized by their therapeutic effect on heart failure.In another example, MLPSCs are characterized by their therapeutic effect in T cell-mediated diseases such as GVHD.

[0128] In another example, culture-expanded MLPSCs are characterized by their ability to inhibit IL-2RA expression by CD3 / CD28-activated PBMCs under culture conditions. In one example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 60% compared to a control group. In another example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 65% compared to a control group. In another example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by at least 70% compared to a control group. In another example, culture-expanded MLPSCs inhibit IL-2RA expression by CD3 / CD28-activated PBMCs by 60-70% compared to a control group.

[0129] "Culture-expanded" MLPSCs are distinguished from freshly isolated cells in that they are cultured and passaged (ie, subcultured) in cell culture medium.

[0130] 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+.

[0131] Thus, in one example, relevant cells are isolated and cultured for two passages to provide an intermediate MLPSC population. In certain examples, the intermediate MLPSC population is then cultured and expanded to produce a drug product (DP). For example, the DP composition of the present disclosure is produced by culturing cells from an intermediate cryopreserved MLPSC population, in other words, a cryopreserved intermediate. In one example, the intermediate cell population can be cultured for three passages (a total of five passages) to provide a DP.

[0132] In one example, the MLPSCs have been culture-expanded for approximately 4 to 10 passages. In one example, the MLPSCs have been culture-expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, or at least 10 passages. For example, the MLPSCs can be culture-expanded for at least 5 passages. In one example, the MLPSCs can be culture-expanded for at least 5 to 10 passages. In one example, the MLPSCs can be culture-expanded for at least 5 to 8 passages. In one example, the MLPSCs can be culture-expanded for at least 5 to 7 passages. In one example, the MLPSCs can be culture-expanded for more than 7 passages. In these examples, the MLPSCs are culture-expanded before being cryopreserved to provide an intermediate cryopreserved MLPSC population, which can then be further culture-expanded.

[0133] In one example, a composition of the present disclosure includes MLPSCs culture-expanded from a cryopreserved intermediate. In one example, the cell culture culture-expanded from a cryopreserved intermediate is culture-expanded for at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 passages. For example, the MLPSCs can be culture-expanded for at least 3 passages. In one example, the MLPSCs can be culture-expanded for at least 3-10 passages. In one example, the MLPSCs can be culture-expanded for at least 3-8 passages. In one example, the MLPSCs can be culture-expanded for at least 3-7 passages. In one example, the culture of MLPSCs culture-expanded from a cryopreserved intermediate is culture-expanded in a medium disclosed herein (e.g., a medium containing newborn bovine serum).

[0134] In one example, MLPSCs can be obtained from a single donor or from multiple donors, where donor samples or MLPSCs are subsequently pooled and optionally culture-expanded. i. expanding viable cell numbers by passage expansion to provide a preparation of at least about 1 billion viable cells, the passage expansion comprising establishing a primary culture of isolated MLPSCs and then serially establishing a first non-primary (P1) culture of MLPSCs isolated from the previous culture; ii. Expanding the P1 culture of isolated MLPSCs by passage expansion into a second, non-primary (P2) culture of MLPSCs; and iii. Preparing and cryopreserving an in-process intermediate MLPSC preparation obtained from a P2 culture of MLPSCs, and optionally iv. Thawing a cryopreserved in-process intermediate MLPSC preparation and expanding the in-process intermediate MLPSC preparation by passage expansion.

[0135] 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.

[0136] 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.

[0137] In one example, the selected cell population is immediately expanded in culture, hi another example, the selected cell population is cryopreserved to allow for expansion in culture at a later stage.

[0138] In one example, the selected cell population is expanded in culture to provide a pharmaceutical composition, which in one example is characterized by certain criteria, such as the level of angiogenic markers.

[0139] In the context of the present disclosure, the level of angiogenic marker(s) can be assessed between steps iii and iv of the culture expansion process described above. For example, the level of angiogenic marker(s) can be determined under the culture conditions and / or from the conditioned medium after step iii. In one example, step iv is performed only if a desired level of angiogenic marker(s) is observed under the culture conditions and / or from the conditioned medium. In this example, a cell population is selected for culture expansion based on the level of angiogenic marker(s) under the culture conditions and / or from the conditioned medium.

[0140] In one example, the culture-expanded MLPSC population is expanded from an intermediate MLPSC population that has increased levels of one or more angiogenic markers compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

[0141] In one example, the level of an angiogenic marker(s) disclosed herein is considered to be increased if it is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum. In one example, the level of an angiogenic marker is increased by about 5% to about 60%. In one example, the level of an angiogenic marker is increased by about 5% to about 40%. In one example, the level of an angiogenic marker is increased by about 40%. In one example, the level of an angiogenic marker is increased by at least about 5%. In one example, the level of an angiogenic marker is increased by at least about 10%.

[0142] In one example, the culture-expanded MLPSC population is expanded from an intermediate MLPSC population that has increased levels of one or more angiogenic markers compared to a population of MLPSCs cultured and expanded in cell culture medium without neonatal serum. In one example, the level of an angiogenic marker is considered increased if it is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in cell culture medium without neonatal serum. In one example, the level of an angiogenic marker is increased by about 5% to about 60%. In one example, the level of an angiogenic marker is increased by about 5% to about 40%. In one example, the level of an angiogenic marker is increased by about 40%. In one example, the level of an angiogenic marker is increased by at least about 5%. In one example, the level of an angiogenic marker is increased by at least about 10%.

[0143] In one example, a culture-expanded MLPSC preparation is i. less than about 0.75% CD45+ cells; ii. at least about 95% CD105+ cells; iii. have an antigenic and activity profile that includes at least about 95% CD166+ cells.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] composition The compositions disclosed herein are useful for pre-licensing of MLPSCs, a multi-step process that results in the functional maturation of MSCs and promotes their therapeutic efficacy.

[0152] Thus, provided herein is a composition for pre-approval of MLPSCs, comprising a human cell population enriched for MLPSCs and serum containing one or more pro-inflammatory cytokines. In other embodiments disclosed herein, the composition comprises (i) a human cell population enriched for MLPSCs, (ii) serum containing one or more pro-inflammatory cytokines, and (iii) a cryopreservative. Suitable cryopreservatives include, but are not limited to, one or more of dimethyl sulfoxide (DMSO), trehalose, and albumin.

[0153] In other embodiments, provided herein is a cell culture medium for the expansion and pre-qualification of MLPSCs, the cell culture medium comprising serum containing one or more pro-inflammatory cytokines.

[0154] The cell culture medium of the present invention can contain optional components such as fatty acids or lipids, vitamins, cytokines, antioxidants, buffers, inorganic salts, and the like.

[0155] The cell culture medium used in the present disclosure contains all essential amino acids and may also contain non-essential amino acids. Generally, amino acids are classified as essential amino acids (Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, His) and non-essential amino acids (Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, Pro).

[0156] Those skilled in the art will understand that for optimal results, key nutrients must be available at appropriate levels to enhance cell growth, and the basal medium must be appropriate for the cell line of interest. For example, if depletion of this energy source proves to be limiting for cell growth, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or to supplement glucose (or other energy source) over the course of the culture.

[0157] The culture medium of the present disclosure can be prepared using a basal culture medium. In the context of the present disclosure, "basal culture medium" refers to an unsupplemented medium suitable for exposure to cells such as MSCs. The basal medium includes, for example, Eagle's Minimum Essential Medium (MEM), alpha-modified MEM, StemSpan™, and mixtures thereof, and is not particularly limited as long as it can be used to culture MLPSCs.

[0158] In some preferred embodiments of the compositions for preapproval disclosed herein, the composition comprises one or more proinflammatory cytokines selected from IL-1β, IL-6, IFN-γ, TNF-α, and IL-1 receptor antagonist (IL-1ra). In some embodiments, the composition comprises each of IL-1β, IL-6, IFN-γ, TNF-α, and IL-1 receptor antagonist (IL-1ra). In some embodiments, the composition is substantially free of proinflammatory cytokines other than IL-1β, IL-6, IFN-γ, TNF-α, and IL-1ra. In some embodiments, the composition comprises only one, two, three, four, or five proinflammatory cytokines, wherein the one to five proinflammatory cytokines are selected from the group consisting of IL-1β, IL-6, IFN-γ, TNF-α, and IL-1ra.

[0159] In some embodiments, the compositions disclosed herein contain a neonatal serum IL-1β concentration of about 2 ng / ml to about 50 ng / ml, e.g., 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml, 40 ng / ml, 45 ng / ml, or another concentration of about 2 ng / ml to about 50 ng / ml. In some embodiments, the neonatal serum IL-6 concentration is about 0.2 ng / ml to about 1.2 ng / ml, e.g., 0.4 ng / ml, 0.5 ng / ml, 0.6 ng / ml, 0.7 ng / ml, 0.8 ng / ml, 1.0 ng / ml, or another concentration of about 0.2 ng / ml to about 1.2 ng / ml. In some embodiments, the concentration of IFN-γ in suitable neonatal serum is about 0.1 ng / ml to about 0.2 ng / ml, e.g., 0.12 ng / ml, 0.14 ng / ml, 0.16 ng / ml, 0.18 ng / ml, or another concentration of about 0.1 ng / ml to about 0.2 ng / ml. In some embodiments, the concentration of IL-1ra in suitable neonatal serum is about 6 ng / ml to about 33 ng / ml, e.g., 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 12 ng / ml, 15 ng / ml, 20 ng / ml, 22 ng / ml, 25 ng / ml, 27 ng / ml, 30 ng / ml, or another concentration of about 6 ng / ml to about 33 ng / ml. In some embodiments, the concentration of TNF-α in suitable neonatal serum is about 0.1 ng / ml to about 0.7 ng / ml, e.g., 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 0.5 ng / ml, 0.6 ng / ml, or another concentration of about 0.2 ng / ml to about 0.7 ng / ml.

[0160] In some preferred embodiments, the compositions disclosed herein are substantially free of pro-inflammatory cytokines other than those present in the serum prior to inclusion in the composition, i.e., the serum used is substantially the only source of exogenous pro-inflammatory cytokines in the composition.

[0161] In some embodiments, the concentration of neonatal serum in the described compositions is about 2% (v / v) to about 12% (v / v), such as 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, or another concentration between about 2% (v / v) and about 12% (v / v). In some preferred embodiments, the concentration of neonatal serum is about 5% (v / v). In other preferred embodiments, the concentration of neonatal serum is about 10% (v / v).

[0162] In some embodiments, the compositions disclosed herein comprise both fetal serum and newborn serum. In some preferred embodiments, the ratio of fetal serum to newborn serum in the composition is 1:1. In some embodiments, the ratio of fetal serum to newborn serum is greater than 1:1, e.g., 3:1, 2.8:1, 2.5:1, 2.2:1, 2:1, 1.8:1, 1.5:1, 1.2:1, or a ratio of fetal serum to newborn serum of about 3:1 or less. In other embodiments, the ratio of fetal serum to newborn serum is less than 1:1, e.g., 1:3, 1:2.8, 1:2.5, 1:2.2, 1:1.8, 1:1.5, 1:1.2, or another ratio of fetal serum to newborn serum of about 1:3 or greater.

[0163] In some preferred embodiments, the total concentration of serum (including the proportions of fetal serum and neonatal serum) in the composition is about 10% (v / v).

[0164] In some embodiments, when the composition contains a cryopreservative, e.g., DMSO, the concentration of neonatal serum is about 3% (v / v) to about 20% (v / v), e.g., 4%, 6%, 7%, 7.5%, 8%, 9%, 10%, 12%, 15%, or another concentration of about 3% (v / v) to about 15% (v / v). In some embodiments, the cryopreservation composition has a 1:1 ratio of fetal serum to neonatal serum, e.g., 5% (v / v) each.

[0165] In some embodiments, the serum containing one or more proinflammatory cytokines used in the compositions disclosed herein is newborn serum. In some preferred embodiments, the newborn serum used is, for example, newborn calf serum, newborn lamb serum, or newborn horse foal serum. In some preferred embodiments, the newborn serum is newborn calf serum. In embodiments in which the compositions disclosed herein contain newborn serum, the newborn serum is from a newborn on about postnatal day 1 to about postnatal day 7, e.g., postnatal day 2, postnatal day 3, postnatal day 4, postnatal day 5, or postnatal day 6. For clarity, postnatal day 1 as used herein refers to the day of birth. In some embodiments, the newborn serum used in the compositions described herein can include a mixture of newborn sera obtained on different days after birth.

[0166] In some embodiments, the neonatal serum used is bovine, ovine, caprine, equine, or human serum. In some preferred embodiments, the neonatal serum is bovine. In some embodiments, when the composition contains fetal serum, the fetal serum is bovine, ovine, equine, or caprine. In some preferred embodiments, the fetal serum is fetal bovine serum.

[0167] In one example, a composition of the present disclosure includes IFN-γ and / or TNF-α (e.g., serum containing IFN-γ and TNF-α). For example, the level of IFN-γ may be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the level of TNF-α may be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the composition includes IFN-γ and TNF-α, both at levels less than 1 ng / ml. In this example embodiment, the IFN-γ and TNF-α are provided in serum.

[0168] In one example, the composition comprises one or more pro-inflammatory cytokines capable of binding to receptors on the surface of MLPSCs.

[0169] In one example, the serum contains one or more proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10. For example, the serum can contain IL-8.

[0170] In one example, the composition comprises IFN-γ and / or TNF-α and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, IP-10. In one example, the level of IFN-γ and / or TNF-α is less than 1 ng / ml.

[0171] In one example, the composition comprises: i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 5 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: a level of IP-10 greater than 5,000 pg / ml;

[0172] In certain instances, serum may be diluted from its original concentration in cell culture medium. In these instances, the level of cytokines in serum is reduced accordingly. For example, serum may be provided at 10% (v / v) in cell culture medium. In these instances, serum may be provided at 10% (v / v) in cell culture medium. i. IFN-γ levels above 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; MCP-1 levels greater than vi.3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. Levels of IP-10 greater than 500 pg / ml.

[0173] In one example, the serum is newborn serum and contains the above levels of cytokine(s). In one example, the composition of the present disclosure comprises a medium described below.

[0174] In other examples, the compositions of the present disclosure comprise the above population(s) of culture-expanded MLPSCs.

[0175] In some preferred embodiments, the MLPSCs are human mesenchymal stem cells. In other embodiments, the MLPSCs are STRO-1 + Pluripotent cells, or STRO-1 + This is a population of MLPSCs culture-expanded from a pluripotent cell population.

[0176] In a preferred embodiment, the MLPSCs are maintained in an undifferentiated state.

[0177] Culture medium In one embodiment, the present disclosure encompasses MLPSC culture medium supplemented with pro-inflammatory cytokine(s). In one example, the culture medium includes IFN-γ and / or TNF-α. In one example, the medium includes IFN-γ. For example, the level of IFN-γ can be less than 1 ng / ml. In one example, the level of IFN-γ is less than 500 pg / ml or less than 100 pg / ml. In one example, the medium includes TNF-α. For example, the level of TNF-α can be less than 1 ng / ml. In one example, the level of TNF-α is less than 750 pg / ml or less than 400 pg / ml. In one example, the medium includes IFN-γ and TNF-α, both at levels less than 1 ng / ml.

[0178] In one example, the medium includes one or more pro-inflammatory cytokines that can bind to receptors on the surface of the MLPSCs.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] In one example, the medium is i. IFN-γ levels above 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 50 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. Serum characterized by one or more or all of the following: IP-10 levels greater than 5,000 pg / ml.

[0183] 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.

[0184] In one example, the medium is serum-free.

[0185] 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.

[0186] 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.

[0187] method The present disclosure provides an in vitro method for pre-qualifying MLPSCs by culturing a human cell population enriched for MLPSCs (e.g., hMSCs) in a cell culture medium suitable for the maintenance and expansion of MLPSCs.

[0188] 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.

[0189] 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.

[0190] In some embodiments, the methods disclosed herein also include determining, or having determined, the ability of culture medium (e.g., culture medium supplemented with neonatal serum) to stimulate MLPSCs to promote angiogenesis in an in vitro assay (e.g., tube formation by human umbilical vein endothelial cells (HUVECs), and analysis of network length, network area, and branch point formation). In some embodiments, such assays involve collecting MLPSC-conditioned medium after culturing in neonatal serum-supplemented medium as disclosed herein and quantifying the effect of such conditioned medium in the angiogenesis assays described above or similar assays.

[0191] 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.

[0192] 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.

[0193] The disclosed methods and cell culture media promote stem cell proliferation and pre-qualification while maintaining MLPSCs in an undifferentiated state. MLPSCs are considered undifferentiated if they have not progressed to a specific lineage. As explained above, MLPSCs exhibit morphological characteristics that distinguish them from differentiated cells. Furthermore, undifferentiated MLPSCs express genes that can be used as markers to detect their differentiated state. Polypeptide products can also be used as markers to detect their differentiated state. Therefore, those skilled in the art can easily determine whether the disclosed methods maintain MLPSCs in an undifferentiated state using routine morphological, genetic, and / or proteomic analyses. Methods for monitoring / confirming cell proliferation are also known in the art and, in certain instances, can be as basic as periodic visual inspection of cell cultures to confirm an increase in cell number. Other methods may include the use of cell viability dyes and / or imaging and counting live cells using commercially available products.

[0194] The MLPSCs disclosed herein can be cultured and expanded in a variety of suitable cell culture media, including neonatal serum. As used in the context of this disclosure, the term "medium" or "media" includes components of the environment surrounding cells. A medium contributes to and / or provides suitable conditions for cell growth. A medium can be solid, liquid, gaseous, or a mixture of these phases and materials. A medium can include liquid growth media as well as liquid media that do not support cell growth. An exemplary gaseous medium includes the gas phase to which cells growing on a Petri dish or other solid or semi-solid support are exposed.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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. Levels of IP-10 greater than 500 pg / ml.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] In one example, the disclosed method includes selecting an intermediate population of cryopreserved MLPSCs for culture-expansion in a medium disclosed herein. In one example, a cryopreserved intermediate culture expanded in 10% fetal serum is selected for culture-expansion according to the methods disclosed herein. In one example, a cryopreserved intermediate culture expanded in neonatal serum and / or pro-inflammatory cytokines disclosed herein is selected for culture-expansion.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] In one example, the cell culture medium is fetal serum-free.

[0216] In one example, the cell culture medium does not contain FCS.

[0217] 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.

[0218] 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.

[0219] Ascorbic acid In one example, a cell culture medium is supplemented with a short-acting ascorbic acid derivative. 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] This application claims priority to US 63 / 386,876, filed December 9, 2022, and US 63 / 507,009, filed June 8, 2023, the disclosures of which are incorporated herein by reference.

[0225] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0226] 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]

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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).

[0231] Eagle's αMEM medium, suitable for culturing primary stem cells, can be obtained from a variety of sources, including Life Technologies and Sigma.

[0232] 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.

[0233] MLPSCs were cultured in a medium containing 10% fetal bovine serum (serum B), L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10 -7 Eagle's α-MEM medium supplemented with 0.1 mM of phosphate (0.01 mM) and / or inorganic phosphate (3 mM) was used.

[0234] 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, serum A). NBCS meets the specifications of standard fetal bovine serum but is 100% bovine serum obtained from animals less than 20 days old.

[0235] 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]

[0236] Example 4: Expansion of MLPSCs in medium supplemented with neonatal serum promotes angiogenesis To characterize the novel MLPSC populations obtained by culturing and expanding them in medium supplemented with neonatal serum and / or proinflammatory cytokines (and to identify the mechanism behind the observed increase in therapeutic efficacy described in Example 5), the angiogenic potential of MPCs cultured under different conditions was assessed.

[0237] Cell culture: MPCs were cultured in 5% NBCS / 5% FCS (Serum A) or 10% FCS (Serum B) to generate MPC-conditioned medium. To control for donor variability, MPCs were obtained from the same donor and cultured under different conditions. In some experiments, MPCs belonging to the same donor but cultured during different manufacturing expansions are indicated by different "lot" numbers.

[0238] 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.

[0239] Angiogenesis Efficacy Assay: In vitro angiogenesis was measured using a kinetic, quantitative 96-well co-culture angiogenesis model. Lentiviral-transduced human umbilical vein endothelial cells (HUVECs) expressing CytoLight Green (a GFP variant) were co-cultured with normal human dermal fibroblasts (NHDFs), seeded into 96-well plates, and simultaneously incubated and imaged using the IncuCyte® Live-Cell Analysis System. This system allowed for the determination of the angiogenesis potential of HUVECs (CytoLight Green). + Fluorescence identification of cells is now possible, and time-lapse image acquisition allows visualization of tube formation over time. The resulting images are analyzed using an integrated angiogenesis algorithm to measure network length, network area, and branch point formation, quantifying the stage and extent of angiogenesis throughout the assay.

[0240] Results: Conditioned medium from MPCs cultured in medium supplemented with neonatal bovine serum (NBCS) was found to increase angiogenesis. As shown in Figure 2, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS increased the network area (Figure 2A), network length (Figure 2B), and branch points (Figure 2C) in the coculture angiogenesis model. Furthermore, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS contained higher levels of VEGF compared to conditioned medium from cells cultured in 10% FCS (Figure 2A). Angiogenin levels were also increased in conditioned medium from MPCs cultured in 5% NBCS / 5% FCS compared to 10% FCS (Figure 3). Figure 4 shows further analysis of the levels of the angiogenic factors SDF-1α, VEGF, and Ang1 (ANGPT1) present in MPCs cultured in 10% FCS ("Serum B Medium") or 5% FCS / 5% NBCS ("Serum A Medium"). These data show that both VEGF and SDF-1α are elevated in neonatal serum medium-cultured MPCs.

[0241] Considering the data provided in Example 1, these data indicate that culture expansion of MLPSCs in medium supplemented with neonatal serum and / or pro-inflammatory cytokines results in a novel cell population with enhanced angiogenic potential. This enhanced potential can be characterized in various ways, as desired, e.g., to define the novel cell population we have identified, which can be, for example, The ability of conditioned medium obtained from MLPSCs to increase network area, network length, and / or branch points upon contact with HUVECs; · Including levels of angiogenin, VEGF, and / or SDF-1 in conditioned medium.

[0242] Example 5. MLPSCs cultured in medium supplemented with neonatal serum improve therapeutic outcomes in persistent inflammation High-risk heart failure with low ejection fraction (HFrEF), NYHA class II / III, is a clinical model of persistent inflammation. HFrEF patients are characterized by cardiac and systemic inflammation, as determined by elevated inflammatory biomarkers. MPCs cultured under different serum conditions were administered to HFrEF patients in the following clinical studies.

[0243] In patients with HFrEF, cardiac macrophages produce high levels of proinflammatory cytokines (IL-6, IL-1, and TNF-α), which induce endothelial dysfunction and cardiomyocyte apoptosis. Plasma concentrations of C-reactive protein (CRP), measured with a high-sensitivity CRP (hsCRP) assay, reflect acute-phase reactants produced in the liver in response to the high levels of proinflammatory cytokines (IL-6, IL-1, and TNF-α) produced by cardiac macrophages. Therefore, plasma concentrations of hsCRP (<2 mg / L vs. >2 mg / L) are representative systemic measures reflecting low or high levels of inflammation in the heart. In a subsequent study, patients with HFrEF were classified as having persistent inflammation if their hsCRP plasma concentrations were >2 mg / L.

[0244] Study Details: Eligible NYHA class II / III patients were enrolled in a double-blind, randomized, sham-controlled, parallel-group efficacy and safety study (DREAM HF-1) of allogeneic mesenchymal progenitor cells (Rexlemestrocel-L) for chronic heart failure due to left ventricular systolic dysfunction (ischemic or non-ischemic). Patients with HFrEF received (1) MPCs cultured in 10% fetal serum (n=37), (2) MPCs cultured in the presence of neonatal bovine serum (5% FCS / 5% NBCS, n=153), or (3) a sham control (i.e., no MPCs, n=241). As evidenced by serum analysis described in Example 1, cells cultured in medium supplemented with neonatal serum effectively cultivated a medium with elevated levels of proinflammatory cytokines. Cells were administered via a single intracardiac injection. Left ventricular systolic function in HFrEF was measured by echocardiographic (ECHO) parameters, including left ventricular ejection fraction (LVEF, %), left ventricular end-systolic volume (LVESV, mL), and left ventricular end-diastolic volume (LVEDV, mL), at baseline and 12 months after treatment. Plasma concentrations of CRP were measured to determine baseline levels of inflammation.

[0245] Results: MPCs cultured in the presence of neonatal bovine serum (5% FCS / 5% NBCS) were found to improve left ventricular (LV) systolic function in patients with HFrEF after 12 months. Specifically, neonatal serum-cultured MPCs significantly increased LVEF and decreased LVESV compared with sham controls (p = 0.0398 and 0.0426, respectively) (Figure 5).

[0246] HFrEF patients were then characterized based on whether their plasma hsCRP levels were <2 mg / L (normal baseline systemic inflammation) or >2 mg / L (elevated baseline systemic inflammation). Importantly, when HFrEF patients were classified according to their baseline systemic inflammation status (CRP >2), the effect of treatment with MPCs cultured in the presence of newborn calf serum (5% FCS / 5% NBCS) on LV systolic function recovery was more pronounced. In contrast, MPCs cultured in 10% FBS did not induce a significant effect (Figure 6). Compared to sham controls, newborn serum-cultured MPCs significantly increased LVEF% by a mean (LS mean) of 2.46 and reduced LVESV by a mean of 8.99 mL (p = 0.0033 and 0.0264, respectively). In contrast, MPCs cultured in 10% fetal serum or 5% / FCS / 5% NBCS showed improved LV systolic function in HFrEF patients without elevated baseline inflammation (HFrEF patients with CRP < 2) (Figure 7).

[0247] MPCs cultured in medium supplemented with neonatal serum have also been shown to reduce other cardiac-related outcomes in HFrEF patients with CRP > 2, including a 43% reduction in the risk of cardiovascular death (Figure 8) and a 54% reduction in the incidence of 3-point MACE (cardiovascular death / MI / stroke) (Figure 9). These data indicate that MPCs cultured in medium supplemented with neonatal serum improve therapeutic efficacy in the context of persistent inflammation.

[0248] Further analysis of clinical responses surprisingly revealed the importance of culture-expanding MLPSCs in medium supplemented with neonatal serum and / or pro-inflammatory cytokines during the final passage(s) before administration. MLPSCs cultured in medium supplemented with neonatal serum and / or pro-inflammatory cytokines during the final passage reduced the three-point MACE (MI, stroke, or CV death) in patients, regardless of whether the MLPSCs were culture-expanded with FBS during the initial passages. Remarkably, this reduction in three-point MACE was observed in all patients, regardless of their inflammatory status (Figure 10). Subgroup analysis of patients revealed that the reduction in three-point MACE was observed in patients with persistent inflammation (CRP > 2 mg / ml, Figure 10) and those at highest risk for end-stage cardiac events (defined as total cardiac event (TCE), cardiovascular death, heart transplant, or left ventricular assist device (LVAD) implantation) in this study. Patients at highest risk for TCE were defined as CRP > 2 mg / ml and NT-proBNP > 1000 ng / ml (Figure 11).

[0249] In contrast, an increase in adverse events (3-point MACE) was observed in all patients who received MLPSCs culture-expanded in medium without neonatal serum and / or proinflammatory cytokines at the final passage before treatment. The increase in 3-point MACE was observed regardless of whether the MLPSCs were cultured in medium supplemented with neonatal serum and / or proinflammatory cytokines during the initial passage.

[0250] Summary: MPCs expanded in medium supplemented with newborn bovine serum (NBCS) and / or pro-inflammatory cytokines: Left ventricular systolic dysfunction improved in patients with HFrEF with inflammation, as measured by the LS mean change in LVEF and LVESV at 12 months. A 43% reduction in cardiovascular death in high-risk patients with NYHA class II / III HFrEF and inflammation A 54% reduction in long-term 3-point MACE in high-risk NYHA class II / III patients with HFrEF and inflammation.

[0251] Taken together with the results of Examples 1 and 4, these human study data demonstrate that the addition of neonatal serum and / or pro-inflammatory cytokines to cell culture media results in cell populations with distinct functional properties, at least in terms of their ability to elicit a therapeutic effect in an inflammatory environment.

[0252] These human trial data also demonstrate the importance of adding neonatal serum and / or proinflammatory cytokines to the culture medium at the final passage(s), e.g., during culture expansion of intermediate cell populations into pharmaceutical products. Thus, our findings provide the basis for at least two approaches to providing MLPSC populations with improved therapeutic efficacy upon administration to patients. Expanding MLPSCs into an intermediate population in neonatal serum and / or pro-inflammatory cytokines to provide a cryopreserved intermediate population, and expanding the cryopreserved intermediate population in neonatal serum and / or pro-inflammatory cytokines to provide a pharmaceutical product. Expanding MLPSCs in fetal serum to an intermediate population to provide a cryopreserved intermediate population, and expanding the cryopreserved intermediate population in neonatal serum and / or pro-inflammatory cytokines to provide a drug product.

[0253] Without wishing to be bound by any particular theory, the above data suggest that culturing MPCs in medium supplemented with neonatal serum and / or proinflammatory cytokines enables them to respond more effectively to an inflammatory environment. Furthermore, when combined with the results described in Example 4, these data suggest a potential mechanism by which MPCs cultured in medium supplemented with neonatal serum and / or proinflammatory cytokines may exhibit improved therapeutic efficacy, i.e., enhanced angiogenesis and increased production of the proangiogenic growth factors VEGF, SDF-1α, and angiogenin. Thus, these data provide the basis for a method for selecting cells with sufficient efficacy for treating inflammatory diseases. Notably, the data demonstrate threshold levels exceeding 3.45 ng / mL of VEGF, 3000 ng / mL of SDF-1α, or 1114 pg / mL of angiogenin, indicating therapeutic efficacy and enhanced biological activity of MPCs at concentrations above these levels. For example, cells can be cultured according to the methods disclosed herein, and conditioned medium can be collected and measured in angiogenesis assays and / or for VEGF and angiogenin levels. Cells producing VEGF / angiogenin above a threshold are considered therapeutic and biologically active. Similarly, cells with an area of ​​>0.12 mm 2 / mm 2 , the network length is 5 mm 2 / mm 2 , and / or bifurcation >15 mm 2 / mm 2 Cells that produce conditioned medium that promotes angiogenesis, as determined by their ability to stimulate angiogenesis, are also believed to be therapeutically effective and biologically active in treating inflammatory diseases.

[0254] The above-mentioned angiogenic markers may serve as relevant criteria (in addition to clinical response criteria demonstrated in heart failure) for the characterization of novel MLPSCs generated by culture in cell culture medium containing neonatal serum and / or proinflammatory cytokines. Example 6: MLPSCs cultured in medium supplemented with neonatal serum are effective in treating GVHD Ten GvHD patients received 2x10 MPCs culture-expanded in NBCS containing pro-inflammatory cytokines (Examples 2 and 4) once a week. 6 The doses were administered intravenously. Patient responses are summarized in Table 2. Eighty percent of GvHD patients who received MPC cultured in NBCS responded to treatment. One patient achieved a complete response, seven patients achieved a partial response, and two patients died. [Table 2]

[0255] Taken together with at least the results of Example 6, our findings support the broad application of pre-licensed MLPSCs (e.g., MPCs cultured in the presence of non-fetal serum, particularly neonatal serum, and / or in the presence of pro-inflammatory cytokines) for the treatment of any disease or disorder characterized by elevated inflammation, particularly diseases characterized by persistent inflammation such as heart failure, or T-cell mediated diseases such as GvHD.

[0256] Example 7. Medium analysis and summary of findings Based on the data described in Example 1, increased cytokine levels in the culture medium used to expand the MLPSC population were characterized by increased one or more angiogenic marker(s) (Example 4) and increased therapeutic efficacy in both heart failure (Example 5) and GvHD (Example 6) patients. The correlation between increased levels of pro-inflammatory cytokines in the culture medium and therapeutic efficacy in distinct disease indications associated with inflammation suggests a pre-approved effect for MLPSCs.

[0257] Surprisingly, the MLPSCs described herein appear to be pre-qualified by culturing with proinflammatory cytokines, even though these cytokines are present at very low levels (e.g., pg / ml levels). This is surprising because it was not previously thought that the presence of proinflammatory cytokines, particularly TNF-α and IFN-γ, at pg / ml levels could have such dramatic effects (e.g., increased angiogenic potential, increased therapeutic efficacy in disease indications such as heart failure and GvHD). While not wishing to be bound by any particular theory, the data provided by the inventors surprisingly suggest synergistic and / or more than additive effects of cytokines in the context of MLPSC culture expansion. For example, the present data show that providing culture medium containing TNF-α and IFN-γ at concentrations <1 ng / ml can have profound effects on MLPSCs cultured and expanded therein, and these effects can be characterized based on the levels of various angiogenic markers and / or clinical efficacy in patients.

[0258] Our findings therefore represent a significant advance in the art by providing a method for preparing novel MLPSC populations that can lead to improved therapeutic effects, particularly in the context of inflammation. These results not only suggest that culture expansion in media supplemented with proinflammatory cytokines can provide improved MLPSC populations, but also demonstrate that culture expansion in media supplemented with neonatal serum can provide relevant proinflammatory cytokines. Our findings therefore support the criteria for culture expansion of MLPSCs in serum- and serum-free media.

[0259] Example 8. Isolation and expansion of MLPSCs MLPSCs can be isolated using techniques such as STRO-3+ immunoselection of MPCs or density gradient separation of MSCs.

[0260] 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.

[0261] 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).

[0262] In conjunction with immunoselection, STRO-3+ (or TNAP+) cells are isolated by magnetic-activated cell sorting as previously described (Gronthos et al. 2003; Gronthos and Simmons 1995). Briefly, approximately 1-3 x 108 BMMNCs are incubated on ice for 20 minutes in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells are then incubated on ice for 1 hour with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer. The cells are then centrifuged at 400 x g and washed twice with HHF. A 1 / 50 dilution of goat anti-mouse biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer is added, and the cells are incubated on ice for 1 hour. Cells were washed twice with MACS buffer (Ca2+- and Mn2+-free PBS supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide) as described above and resuspended in a final volume of 0.9 ml of MACS buffer.

[0263] 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 was stained with streptavidin-FITC and purity assessed by flow cytometry.

[0264] 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).

[0265] 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: (i) a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), said MLPSCs comprising: IFN-γ and / or TNF-α, and / or - The composition is cultured and expanded in a medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

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

3. 3. The composition according to claim 1 or claim 2, wherein the medium contains two or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

4. The composition according to any one of claims 1 to 3, wherein the medium contains IL-6.

5. The composition according to any one of claims 1 to 4, wherein the medium contains IL-8 and / or IL-17A.

6. The composition according to any one of claims 1 to 5, wherein the medium contains IFN-γ and TNF-α.

7. The composition of any one of claims 1 to 6, wherein the level of IFN-γ is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml.

8. The composition of any one of claims 1 to 7, wherein the level of TNF-α is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

9. The composition according to any one of claims 1 to 8, wherein the medium contains serum containing the pro-inflammatory cytokines.

10. The composition of claim 9, wherein the serum is newborn mammalian serum.

11. The composition of claim 9, wherein the serum is newborn bovine serum.

12. 12. The composition of claim 10 or claim 11, wherein the serum is obtained within 21 days of birth.

13. The medium is i. IFN-γ levels greater than 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; vi. MCP-1 levels greater than 3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. A composition according to any one of claims 1 to 12, characterized by one or more or all of the following: a level of IP-10 greater than 500 pg / ml.

14. The composition according to any one of claims 1 to 12, wherein the culture medium comprises at least 5% (v / v) newborn mammalian serum.

15. The composition of any one of claims 1 to 6 or 9, wherein the medium is serum-free.

16. The composition of any one of claims 1 to 15, wherein the MLPSCs express increased levels of angiogenin compared to a control population.

17. 17. The composition of any one of claims 1 to 16, wherein the MLPSCs induce increased levels of one or more of endothelial network formation, endothelial length, or endothelial branch length compared to a control population.

18. 18. The composition of claim 16 or claim 17, wherein the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal serum.

19. The composition of any one of claims 1 to 18, wherein the MLPSCs express angiogenin levels greater than about 1200 pg / ml.

20. The composition of any one of claims 1 to 19, wherein the MLPSCs express a level of SDF-1 greater than about 3000 pg / ml.

21. The composition of any one of claims 1 to 20, wherein the MLPSCs express a level of VEGF greater than about 3200 pg / ml.

22. The conditioned medium from the MLPSCs was approximately 0.12 mm ２ / mm ２ The composition according to any one of claims 1 to 21, which induces the formation of a transendothelial network.

23. The conditioned medium from the MLPSCs was collected at approximately 5 mm ２ / mm ２ 23. The composition of any one of claims 1 to 22, which induces an endothelial network length of greater than 1000 ng / cm.

24. The conditioned medium from the MLPSCs was approximately 15 l / mm ２ 24. The composition of any one of claims 1 to 23, which induces endothelial branch lengths of greater than 1000kJ / cm.

25. 1. A cryopreserved composition comprising: (i) a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs have been culture-expanded in a medium containing newborn mammalian serum obtained within 21 days of birth; and (ii) a cryopreservation agent.

26. 26. The composition of claim 25, wherein the MLPSCs are cryopreserved at least twice.

27. 1. An in vitro method for prequalification of human mesenchymal progenitor or stem cells (MLPSCs), said method comprising: i. one or more pro-inflammatory cytokines selected from the group consisting of IFN-γ and / or TNF-α, and / or IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, IP-10, and / or ii. The method, comprising culturing the MLPSCs in a medium containing newborn mammalian serum obtained within 21 days after birth.

28. 28. The method of claim 27, wherein the medium contains three or more pro-inflammatory cytokines.

29. 29. The method of claim 27 or 28, 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.

30. The method according to any one of claims 27 to 29, wherein the medium contains IL-6.

31. The method according to any one of claims 27 to 30, wherein the medium contains IL-8 and / or IL-17A.

32. The method according to any one of claims 27 to 31, wherein the medium contains IFN-γ and TNF-α.

33. The method of any one of claims 27 to 32, wherein the culture medium contains serum containing the pro-inflammatory cytokines.

34. 34. The method of claim 33, wherein the serum is newborn mammalian serum.

35. 35. The method of claim 34, wherein the serum is newborn bovine serum.

36. 36. The method of claim 34 or claim 35, wherein the serum is obtained within 21 days of birth.

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

38. 38. The method of any one of claims 27 to 37, wherein the culture medium comprises at least 5% (v / v) newborn mammalian serum.

39. 38. The method of any one of claims 27 to 32 or 37, wherein the medium is serum-free.

40. A composition produced by the method of any one of claims 27 to 39.

41. The composition or method of any one of claims 7 to 40, wherein the newborn serum is serum from a newborn between 1 day after birth and 7 days after birth.

42. 41. The composition or method of any one of claims 7 to 40, wherein the concentration of serum is from about 2% (v / v) to about 12% (v / v).

43. 41. The composition or method of any one of claims 7 to 40, wherein the concentration of the serum is about 5% (v / v).

44. 41. The composition or method of any one of claims 7 to 40, wherein the concentration of serum is about 10% (v / v).

45. 45. The composition or method of any one of claims 7 to 44, wherein the culture medium comprises fetal serum and newborn serum of the same species, and the ratio of the concentration of the fetal serum to the concentration of the newborn serum is 1:1 or less.

46. 46. ​​The composition or method of claim 45, wherein the concentration of the fetal serum and the concentration of the newborn serum are each 5% (v / v).

47. 46. ​​The composition or method of claim 45, wherein the concentration of the fetal serum is less than the concentration of the newborn serum.

48. 48. The composition or method of any one of claims 1 to 47, wherein the MLPSCs are maintained in an undifferentiated state.

49. 49. The composition or method of any one of claims 1 to 48, wherein the MLPSCs are human mesenchymal stem cells (hMSCs).

50. 50. The composition or method of any one of claims 1 to 49, wherein the MLPSCs are culture-expanded from a population of STRO-1+ pluripotent cells.

51. A medium for culturing MLPSCs, the medium comprising: IFN-γ and / or TNF-α, and - the medium containing one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

52. 52. The medium of claim 51, wherein the medium contains three or more pro-inflammatory cytokines.

53. The medium of claim 51 or claim 52, wherein the medium contains two or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1-α, MIP-1-β, and IP-10.

54. The medium according to any one of claims 51 to 53, wherein the medium contains IL-6.

55. The medium according to any one of claims 51 to 54, wherein the medium contains IL-8 and / or IL-17A.

56. The medium according to any one of claims 51 to 55, wherein the medium contains IFN-γ and TNF-α.

57. The medium according to any one of claims 51 to 56, wherein the medium contains serum containing the pro-inflammatory cytokine.

58. 58. The medium of claim 57, wherein the serum is newborn mammalian serum.

59. 58. The medium of claim 57, wherein the serum is newborn bovine serum.

60. 60. The medium of claim 58 or claim 59, wherein the serum is obtained within 21 days of birth.

61. The serum is i. IFN-γ levels greater than 10 pg / ml; ii. TNF-α levels greater than 20 pg / ml; iii. IL-6 levels greater than 30 pg / ml; iv. IL-8 levels greater than 5,000 pg / ml; v. IL-17A levels greater than 2 pg / ml; vi. MCP-1 levels greater than 30 pg / ml; vii. MIP-1-α levels greater than 5 pg / ml; viii. MIP-1-β levels greater than 30 pg / ml; ix. A medium according to any one of claims 57 to 60, characterized by one or more or all of the following: a level of IP-10 greater than 5,000 pg / ml.

62. 62. The medium of any one of claims 57 to 61, wherein the medium comprises at least 5% (v / v) newborn mammalian serum.

63. 62. The medium of any one of claims 57 to 61, wherein the medium comprises 5% (v / v) newborn mammalian serum.

64. The medium according to any one of claims 57 to 56, wherein the medium is serum-free.

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