Methods for treating heart failure in subjects with persistent inflammation

Abstract

The present disclosure relates to cellular compositions with anti-inflammatory properties and their use in methods of treating and / or preventing advanced heart failure.

Description

[Technical Field]

[0001] The present disclosure relates to cellular compositions with anti-inflammatory properties and their use in methods of treating and / or preventing advanced heart failure. [Background technology]

[0002] Myocardial infarction (MI) remains one of the leading causes of mortality and morbidity in developed countries. A recent review of US Medicare records evaluated data on 350,509 patients over 65 years of age who were hospitalized with acute MI and survived to discharge after the event (Schuster et al. (2004) Physiol Heart Circa Physiol., 287(2):525-32). Within one year after the index event, 25.9% of MI patients died, and 50.5% were rehospitalized. In the month following MI, the likelihood of death was 21 times higher, and the likelihood of hospitalization was 12 times higher, compared with the overall Medicare age population.

[0003] Over the past decade, numerous clinical trials evaluating novel drug therapies have been conducted in patients with advanced heart failure (HF). Although progress has been made in reducing morbidity and mortality in HF patients, those with advanced disease continue to experience an unfavorable clinical course characterized by frequent hospitalizations and premature death.

[0004] Clearly, there is a need for techniques to treat or prevent progressive heart failure. Summary of the Invention

[0005] The present inventors have surprisingly recognized that mesenchymal progenitor or stem cells (MLPSCs) cultured in culture medium containing non-fetal serum are particularly effective in treating certain subjects with advanced heart failure, particularly in the context of subjects with persistent inflammation. One reason this is surprising is that newborn bovine serum (NBCS, non-fetal serum) is commonly marketed as an equivalent or acceptable alternative to fetal bovine serum (FBS). Indeed, the present inventors unexpectedly discovered that this was not the case, as NBCS supplementation improved patient outcomes. Analysis of the newborn 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 use of novel MLPSC compositions by culturing certain pro-inflammatory cytokines and / or newborn serum.

[0006] Thus, in one example, the present disclosure relates to a method of treating progressive heart failure in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs) or conditioned medium obtained therefrom, wherein the subject has persistent inflammation and the MLPSCs are culture-expanded in a cell culture medium containing at least one proinflammatory cytokine. In one example, the proinflammatory cytokine is selected from the group consisting of IL-1β, IL-6, TNF-α, IFN-γ, and / or IL-1RA. In one example, the MLPSCs are culture-expanded in a medium containing IFN-γ and / or TNF-α, and / or 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. In one example, the medium contains three or more proinflammatory cytokines. In one example, 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. 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 medium contains IFN-γ. In one example, the level of IFN-γ is <1 ng / ml. 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 one example, the medium contains TNF-α. In one example, the level of TNF-α is <1 ng / ml. In one example, the level of TNF-α is <750 pg / ml. In one example, the level of TNF-α is <400 pg / ml.

[0007] In one example, the proinflammatory cytokines are provided in non-fetal serum. Thus, in one example, the cell culture medium comprises non-fetal serum. In one example, the medium contains serum comprising the proinflammatory cytokines. In one example, the medium comprises non-fetal serum. In one example, the serum is newborn mammalian serum. In one example, the serum is newborn bovine serum (NBCS). In one example, the non-fetal serum is NBCS. In one example, the serum is obtained within 21 days after birth. In one example, the serum is obtained between the day of birth and 21 days after birth. In one example, the serum is obtained between the day of birth and 14 days after birth. In one example, the serum is obtained between the day of birth and 10 days after birth. In one example, the serum is obtained between the day of birth and 7 days after birth. In one example, the medium comprises at least 5% (v / v) newborn bovine serum (NBCS). In one example, the medium comprises 5% non-fetal serum. In one example, the medium comprises 5% non-fetal serum and 5% fetal serum. In these examples, the non-fetal serum is NBCS. In one example, the fetal serum is fetal bovine serum.

[0008] In one example, the medium is serum-free and / or xeno-free. In one example, the medium is xeno-free medium. In one example, the xeno-free medium includes human serum. In one example, the medium is serum-free.

[0009] 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.

[0010] In another example, the present disclosure relates to a method of treating progressive heart failure in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs) or conditioned medium obtained therefrom, wherein the MLPSCs have been culture-expanded in medium containing interferon (IFN)-γ and / or tumor necrosis factor (TNF)-α, and the level(s) of IFN-γ and / or TNF-α in the medium is <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.

[0011] In one example, the administered composition comprises a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs are culture-expanded in a medium containing 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.

[0012] Thus, in another embodiment, the administered composition comprises a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), the MLPSCs comprising: IFN-γ and / or TNF-α, and / or The cells are cultured and grown in a medium containing 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. In one example, the medium contains three or more proinflammatory cytokines. 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. 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 proinflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1, MIP-1α, MIP-1β, and IP-10.

[0013] In one example, the level of IFN-γ 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 another example, the level of TNF-α in the medium 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.

[0014] In one example, the medium contains serum containing proinflammatory cytokines. In one example, the serum is non-fetal serum. In one example, the serum is newborn mammalian serum. For example, the serum can be newborn bovine serum. In one example, the newborn serum is obtained within 21 days after birth.

[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 above 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 bovine serum. In another example, the medium is serum-free.

[0021] In another example, the present disclosure relates to a method for treating progressive heart failure in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded MLPSCs, wherein the MLPSCs have been culture-expanded in a cell culture medium comprising non-fetal serum.

[0022] In one example, the non-fetal serum is newborn bovine serum (NBCS). In one example, the NBCS is obtained within 21 days after the birth of the calf. For example, the NBCS is obtained from the day of birth to 21 days after birth of the calf. In another example, the NBCS is obtained from the day of birth to 14 days after birth of the calf. In another example, the NBCS is obtained from the day of birth to 10 days after birth of the calf. In another example, the NBCS is obtained from the day of birth to 7 days after birth of the calf. In one example, the NBCS is obtained after the calf has ingested colostrum.

[0023] In one example, a subject treated in accordance with the present disclosure has persistent inflammation.

[0024] In one example, persistent inflammation is characterized by elevated C-reactive protein (CRP). In one example, elevated CRP is characterized by a CRP of ≥ 2 mg / L. Thus, in one example, the subject's CRP level is ≥ 2 mg / L. In another example, the subject's CRP level is 2-5 mg / L, preferably 2-4 mg / L, more preferably 2-3 mg / L. In one example, the subject has persistent left ventricular dysfunction. In one example, the subject has an LVEF of less than about 45%. In one example, the subject has an LVEF of less than 40%. In one example, the subject has an LVEF of 30-35%. In one example, the subject has an LVEF of 30-35% or less. In one example, the subject has an LVEF less than 35%. In another example, the subject has an LVEF of more than 70 ml. In one example, the subject has an LVEF of 70 ml to 160 ml.

[0025] In one example, the subject has class II heart failure according to the New York Heart Association (NYHA) classification scale. In one example, the subject has myocardial ischemia and / or diabetes. In one example, the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level is >1000 pg / mL, or 1000 pg / mL to 2500 pg / mL. In one example, the subject has experienced a hospitalization due to heart failure within the past 9 months. In one example, the subject's heart failure is due to an ischemic event or a non-ischemic event. In one example, the heart failure is due to an ischemic event.

[0026] In one example, the disclosed method includes the steps of: i) selecting a subject with advanced heart failure for treatment, wherein the subject has microvascular disease and / or macrovascular disease; and ii) administering MLPSCs. For example, the method includes the steps of: i) selecting a subject with microvascular disease and / or macrovascular disease for treatment; and ii) administering MLPSCs. In these example embodiments, the subject has persistent inflammation. In one example, the method includes the steps of: i) selecting a subject with a CRP level of ≧2 mg / L for treatment; and ii) administering MLPSCs. In one example, the disclosed method includes the steps of: i) selecting a subject with advanced heart failure and a CRP level of ≧2 mg / L for treatment; and ii) administering MLPSCs. In one example, the subject's CRP level is 2-5 mg / L. In one example, the subject's CRP level is 2-4 mg / L. In one example, the subject's CRP level is 2-3 mg / L.

[0027] In one example, the composition is administered transendocardially and / or intravenously. In one example, the treatment methods disclosed herein involve administering 1x10 7 ~2x10 8 The method includes administering cells of the present invention.

[0028] In one example, the subject has a reduced risk of cardiac death after treatment. In one example, the reduced risk is compared to the risk of cardiac death in a subject not administered the MLPSCs.

[0029] In one example, treatment improves the subject's LVEF by at least 4 percentage points. In one example, treatment improves the subject's LVEF by at least 5 percentage points or at least 6 percentage points. In one example, treatment improves the subject's LVEF by 4-7 percentage points, or 5-7 percentage points. In one example, treatment improves the subject's LVEF by 5-7 percentage points, or 5-7 percentage points.

[0030] In one example, treatment improves the subject's LVESV by at least 17 ml. In one example, treatment improves the subject's LVESV by at least 20 ml. In one example, treatment improves the subject's LVESV by 15 ml to 30 ml.

[0031] In one example, treatment improves the subject's LVEDV by at least 15 ml. In one example, treatment improves the subject's LVEDV by 15 ml to 25 ml.

[0032] In one example, the subject has a reduced risk of ischemic MACE (MI or stroke) following treatment.

[0033] In one example, the subject has a left ventricular assist device (LVAD). In one example, the subject has heart failure due to an LVAD, ischemic event. In one example, the subject's IL-6 level is increased 60 days after LVAD implantation compared to baseline. In one example, the treatment reduces the subject's risk of all-cause mortality. In one example, the treatment reduces the subject's risk of all-cause mortality by 10% to 90%. In one example, the treatment reduces the subject's risk of all-cause mortality by more than 50%. In one example, the treatment reduces the subject's risk of all-cause mortality by 20 to 85%. In one example, the treatment reduces the subject's risk of all-cause mortality by approximately 80%. In one example, the risk reduction is compared to the risk of all-cause mortality in a subject not administered MLPSCs.

[0034] In one example, the MLPSCs are mesenchymal progenitor cells (MPCs). In one example, the MPCs are isolated from bone mononuclear cells with anti-STRO-3 antibodies before expansion in culture.

[0035] In one example, the MLPSCs are mesenchymal stem cells (MSCs).

[0036] In one example, the MLPSCs are allogeneic.

[0037] In one example, the cells are cryopreserved prior to administration.

[0038] In one example, the population of MLPSCs disclosed herein is administered in a composition. In one example, the composition further comprises Plasmalyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA). In another example, the composition further comprises a solution of Plasmalyte A (70%), DMSO (10%), and HSA (25%), wherein the HSA solution comprises 5% HSA and 15% buffer.

[0039] In one example, the composition comprises greater than 6.68×10 6 / mL viable cells.

[0040] In one example, the composition comprises allogeneic MPCs derived from human bone marrow that have been isolated from bone mononuclear cells with anti-STRO-3 antibodies, expanded in vitro in culture medium containing NBCS, and cryopreserved.

[0041] The inventors also surprisingly recognized that MLPSCs cultured in culture medium containing certain proinflammatory cytokines and / or non-fetal serum increased angiogenesis and increased levels of angiogenic markers. Thus, the inventors have arrived at criteria that can be used in one or more useful efficacy assays to establish the therapeutic efficacy of culture-expanded MLPSC populations (or the conditioned medium obtained thereby), particularly in the context of advanced heart failure.

[0042] Thus, in one example, the present disclosure provides a method for selecting a population of culture-expanded MLPSCs for use in treating progressive heart failure in a subject, the MLPSCs being culture-expanded in a cell culture medium containing at least one pro-inflammatory cytokine, the method comprising: (i) obtaining a population of MLPSCs; (ii) determining the level of one or more angiogenic markers in the MLPSC population, wherein the one or more angiogenic marker(s) are selected from the group consisting of levels of VEGF, angiogenin, SDF-1α expressed by the MLPSCs under culture conditions, and / or levels of endothelial network formation, endothelial network length, and endothelial branch length measured after treating an endothelial cell population with conditioned medium obtained from the MLPSCs; and (iii) selecting MLPSCs having increased level(s) of the one or more angiogenic markers for use in the treatment. In one example, the treatment is treatment of progressive heart failure. In one example, the MLPSCs are Increased levels of one or more of VEGF, angiogenin, and SDF-1α expressed by MLPSCs under culture conditions; and The endothelial cell population is selected for use in therapy based on an increase in one or more of the following levels: endothelial network formation, endothelial network length, and endothelial branch length measured after treatment of the endothelial cell population with conditioned medium obtained from MLPSCs.

[0043] In one example, the MLPSC-conditioned medium or soluble factors obtained therefrom are Increased levels of one or more of VEGF, angiogenin, and SDF-1α expressed by MLPSCs under culture conditions; and The endothelial cell population is selected for use in therapy based on an increase in one or more of the following levels: endothelial network formation, endothelial network length, and endothelial branch length measured after treatment of the endothelial cell population with conditioned medium obtained from MLPSCs.

[0044] In one example, the soluble factors obtained from the conditioned medium are exosomes.

[0045] In the example embodiments described above, the pro-inflammatory cytokine(s) is one or more of the above cytokine(s), or a combination thereof.

[0046] In the example embodiment described above, the pro-inflammatory cytokine(s) are provided in non-fetal serum. Thus, in one example, the cell culture medium comprises non-fetal serum, such as newborn bovine serum.

[0047] In another example, the present disclosure provides a method for determining the efficacy of a population of culture-expanded MLPSCs or conditioned medium obtained thereby, wherein the MLPSCs are culture-expanded in a cell culture medium containing at least one pro-inflammatory cytokine, the method comprising determining the level of one or more angiogenic markers in the MLPSC population, the one or more angiogenic markers being selected from the group consisting of levels of VEGF, angiogenin, and SDF-1α expressed by the MLPSCs under culture conditions, and / or levels of endothelial network formation, endothelial network length, and endothelial branch length measured after treating an endothelial cell population with conditioned medium obtained from the MLPSCs, wherein increased levels of the one or more angiogenic markers indicate biological activity or a therapeutic effect in treating progressive heart failure. In one example, increased levels of the one or more angiogenic markers indicate biological activity or a therapeutic effect in treating progressive heart failure.

[0048] In the example embodiments described above, the pro-inflammatory cytokines are provided in non-fetal serum. Thus, in one example, the cell culture medium comprises non-fetal serum, such as newborn bovine serum.

[0049] In one example, the increased level of one or more angiogenic markers is determined compared to a control population of MLPSCs.For example, the increased level of one or more angiogenic markers can be determined compared to a population of MLPSCs cultured and grown in a cell culture medium containing 10% fetal bovine serum.In one example, a suitable control group does not contain non-fetal bovine serum.

[0050] In one example, a population of MLPSCs used in the disclosed methods is culture-expanded in a culture medium containing non-fetal serum, cryopreserved, and thawed. In one example, the MLPSCs are culture-expanded, cryopreserved, and thawed, followed by evaluation of potency. In another example, the MLPSCs are culture-expanded, cryopreserved, and thawed twice, followed by evaluation of potency.

[0051] In one example, the level of VEGF is greater than about 3 ng / ml, preferably greater than about 3.45 ng / ml.

[0052] In one example, the level of angiogenin is greater than about 1000 pg / ml, preferably greater than about 1114 pg / ml.

[0053] In one example, the level of SDF-1α is greater than about 3000 ng / ml.

[0054] In one example, the formation of an endothelial network occurs within a range of approximately 0.1 mm. 2 / mm 2 More than 0.12 mm, preferably about 0.12 mm 2 / mm 2 It's super.

[0055] In one example, the length of the endothelial network is approximately 4 mm. 2 / mm 2 More than 5 mm, preferably about 5 mm 2 / mm 2 It's super.

[0056] In one example, the length of the endothelial branch is approximately 12 1 / mm 2 greater than, preferably about 15 1 / mm 2 It's super.

[0057] In one example, endothelial network formation, endothelial network length, and / or endothelial branch length are measured using an in vitro angiogenesis assay.

[0058] In one example, the method further comprises culturing and expanding the selected population of MLPSCs to provide a pharmaceutical composition.

[0059] In one example, the present disclosure provides a selected population of MLPSCs obtained by the methods disclosed herein. In one example, the present disclosure provides a cryopreserved cell intermediate comprising a population of culture-expanded MLPSCs selected according to the methods disclosed herein. In one example, the selected MLPSC population is used in a method for treating advanced heart failure.

[0060] The present inventors have also surprisingly recognized a novel population of MLPSCs that are characterized as having high angiogenic potential, as determined by the expression levels of one or more angiogenic markers.

[0061] Thus, in one example, the present disclosure provides a culture-expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein the population of MLPSCs is selected based on high angiogenic potential as determined by the level of angiogenin expressed by the MLPSCs under culture conditions. In one example, the MLPSCs are culture-expanded in a cell culture medium containing at least one pro-inflammatory cytokine as disclosed herein.

[0062] In one example, MLPSCs expressing increased levels of angiogenin compared to a control population are selected. In one example, the control population is a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, MLPSCs expressing angiogenin at a level greater than about 1200 pg / ml are selected. In one example, MLPSCs expressing angiogenin at a level greater than about 1100 pg / ml are selected. In one example, MLPSCs expressing angiogenin at a level greater than about 1000 pg / ml are selected. In one example, MLPSCs expressing angiogenin at a level greater than about 700 pg / ml are selected. In one example, the level of angiogenin is measured in conditioned medium obtained from the cultured and expanded cells. In one example, the level of angiogenin is measured in a cell lysate of the cultured and expanded cells.

[0063] In one example, the disclosure provides a cultured expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein the population of MLPSCs is measured after treating a population of endothelial cells with conditioned medium obtained from the MLPSCs. - Formation of the endothelial network, - the length of the endothelial network, or -Selected based on high angiogenic potential as determined by one or more levels of endothelial branch length.

[0064] In one example, MLPSCs are cultured and expanded in a cell culture medium comprising at least one pro-inflammatory cytokine disclosed herein.

[0065] In one example, MLPSCs that induce an increase in one or more levels of endothelial network formation, endothelial length, or endothelial branch length compared to a control population are selected. In one example, the control population is a population of MLPSCs cultured and grown in a cell culture medium containing 10% fetal bovine serum.

[0066] In one example, approximately 0.12 mm 2 / mm 2 MLPSCs that induce the formation of an endothelial network of greater than about 0.11 mm are selected. 2 / mm 2 MLPSCs that induce the formation of an endothelial network of greater than about 0.10 mm are selected. 2 / mm 2 MLPSCs that induce the formation of an endothelial network of greater than about 0.14 mm are selected. 2 / mm 2 MLPSCs that induce the formation of a transendothelial network are selected.

[0067] In one example, approximately 5 mm 2 / mm 2 MLPSCs that induce an endothelial network length of greater than 4 mm are selected. 2 / mm 2 MLPSCs that induce an endothelial network length of greater than 5.5 mm are selected. 2 / mm2 MLPSCs that induce an endothelial network length of greater than 5.75 mm are selected. 2 / mm 2 MLPSCs that induce endothelial network lengths of up to 1000 μm are selected.

[0068] In one example, approximately 15 1 / mm 2 MLPSCs that induce endothelial branch lengths of greater than 14 1 / mm are selected. 2 MLPSCs that induce endothelial branch lengths of greater than about 10 1 / mm are selected. 2 MLPSCs that induce endothelial branch lengths of greater than 16 1 / mm are selected. 2 MLPSCs that induce endothelial branch lengths of greater than 1000 are selected.

[0069] The present disclosure also provides a method for producing a pharmaceutical product comprising a population of mesenchymal progenitor or stem cells (MLPSCs), the method comprising obtaining a determination of whether a test population of MLPSCs has a predetermined level of angiogenic potential under culture conditions, and treating at least a portion of the test population of MLPSCs as a pharmaceutical product, thereby producing the pharmaceutical product, if the test population of MLPSCs has at least the predetermined level of angiogenic potential under culture conditions, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of angiogenic potential under culture conditions, wherein the angiogenic potential is measured by a predetermined level of angiogenin measured under culture conditions.

[0070] In one example, the predetermined level of angiogenin is increased compared to a control group. For example, the control population is a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the predetermined level of angiogenin is greater than about 1200 pg / ml. In one example, the predetermined level of angiogenin is greater than about 1100 pg / ml. In one example, the predetermined level of angiogenin is greater than about 1000 pg / ml. In one example, the predetermined level of angiogenin is greater than about 700 pg / ml. In one example, the predetermined level of angiogenin is measured in conditioned medium obtained from a test population of MLPSCs. In one example, the predetermined level of angiogenin is measured in conditioned medium obtained from cultured expanded cells. In one example, the predetermined level of angiogenin is measured in a lysate of cultured expanded cells.

[0071] In one example, the disclosure provides a method of producing a pharmaceutical product comprising a population of mesenchymal progenitor or stem cells (MLPSCs), the method comprising obtaining a determination of whether a test population of MLPSCs has a predetermined level of angiogenic potential under culture conditions, and treating at least a portion of the test population of MLPSCs as a pharmaceutical product, thereby producing the pharmaceutical product, if the test population of MLPSCs has at least the predetermined level of angiogenic potential under culture conditions, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of angiogenic potential under culture conditions, wherein the angiogenic potential is -measured after treating endothelial cell populations with conditioned medium obtained from MLPSCs, o Formation of endothelial network, o The length of the endothelial network, and / or o As measured by one or more predetermined levels of endothelial branch length measured in an in vitro angiogenesis assay.

[0072] In one example, a predetermined level of one or more of endothelial network formation, endothelial length, or endothelial branch length is increased compared to a control population, for example, a population of MLPSCs cultured and grown in a cell culture medium containing 10% fetal bovine serum.

[0073] In one example, the predetermined level of endothelial network formation is about 0.12 mm 2 / mm 2 In one example, the predetermined level of endothelial network formation is about 0.11 mm. 2 / mm 2 In one example, the predetermined level of endothelial network formation is greater than about 0.10 mm. 2 / mm 2 In one example, the predetermined level of endothelial network formation is greater than about 0.14 mm. 2 / mm 2 It's super.

[0074] In one example, the predetermined level of endothelial network length is about 5 mm. 2 / mm 2 In one example, the predetermined level of endothelial network length is about 4 mm. 2 / mm 2 In one example, the predetermined level of endothelial network length is about 5.5 mm. 2 / mm 2 In one example, the predetermined level of endothelial network length is about 5.75 mm. 2 / mm 2 It's super.

[0075] In one example, the predetermined level of endothelial branch length is about 15 1 / mm 2 In one example, the predetermined level of endothelial branch length is greater than about 14 1 / mm 2 In one example, the predetermined level of endothelial branch length is greater than about 10 1 / mm 2 In one example, the predetermined level of endothelial branch length is greater than about 16 1 / mm 2 It's super.

[0076] In one example, according to the present disclosure, MLPSCs having high angiogenic potential can be cultured and expanded in a cell culture medium containing at least one pro-inflammatory cytokine. In one example, the MLPSCs are cultured and expanded in a medium containing 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, and IP-10. In one example, the medium contains three or more pro-inflammatory cytokines. In one example, 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. 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 medium contains IFN-γ. In one example, the level of IFN-γ is <1 ng / ml. 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 one example, the medium contains TNF-α. In one example, the level of TNF-α is <1 ng / ml. In one example, the level of TNF-α is <750 pg / ml. In one example, the level of TNF-α is <400 pg / ml.

[0077] In one example, the proinflammatory cytokines are provided in non-fetal serum. Thus, in one example, the cell culture medium comprises non-fetal serum. In one example, the medium contains serum comprising the proinflammatory cytokines. In one example, the medium comprises non-fetal serum. In one example, the serum is newborn mammalian serum. In one example, the serum is newborn bovine serum (NBCS). In one example, the non-fetal serum is NBCS. In one example, the serum is obtained within 21 days after birth. In one example, the serum is obtained between the day of birth and 21 days after birth. In one example, the serum is obtained between the day of birth and 14 days after birth. In one example, the serum is obtained between the day of birth and 10 days after birth. In one example, the serum is obtained between the day of birth and 7 days after birth. In one example, the medium comprises at least 5% (v / v) newborn bovine serum (NBCS). In one example, the medium comprises 5% non-fetal serum. In one example, the medium comprises 5% non-fetal serum and 5% fetal serum. In these examples, the non-fetal serum is NBCS.

[0078] In one example, the medium is serum-free and / or xeno-free. In one example, the medium is xeno-free medium. In one example, the xeno-free medium includes human serum. In one example, the medium is serum-free.

[0079] In one example, the proinflammatory cytokines are provided in non-fetal serum. Thus, in one example, MLPSCs are cultured and grown in a culture medium containing non-fetal serum. In one example, the non-fetal serum is newborn serum. In one example, the non-fetal serum is newborn mammalian serum. In one example, the newborn mammalian serum is newborn bovine serum (NBCS). In one example, MLPSCs are cultured and grown in a culture medium containing about 5% non-fetal serum. In one example, MLPSCs are cultured and grown in a culture medium containing about 5% to about 10% non-fetal serum. In one example, MLPSCs are cultured and grown in a culture medium containing about 5% non-fetal serum and about 5% fetal serum. In these examples, the non-fetal serum can be newborn mammalian serum, such as newborn bovine serum (NBCS). Thus, in one example, MLPSCs are cultured and grown in a culture medium containing about 5% NBCS. In one example, MLPSCs are cultured and grown in a culture medium containing less than 10% fetal bovine serum.

[0080] In one example, the proinflammatory cytokines are provided in a xeno-free medium. In one example, the proinflammatory cytokines are provided in a serum-free medium. Thus, in one example, the MLPSCs are cultured and expanded in the xeno-free medium. In one example, the xeno-free medium includes human serum. In one example, the xeno-free medium includes 3% human serum. In another example, the xeno-free medium is serum-free. Thus, in one example, the MLPSCs are cultured and expanded in the serum-free medium.

[0081] 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.

[0082] In one example, angiogenin levels, endothelial network formation, endothelial network length, and / or endothelial branch length indicate the biological activity or therapeutic effect of culture-expanded MLPSCs. [Brief explanation of the drawings]

[0083] [Figure 1-1] 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 1-2] 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 1-3] 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 1-4] 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 1-5] 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 2-1] Quantitative measurement of in vitro angiogenesis induced by MLPSC-conditioned medium using the IncuCyte® 96-Well Kinetic Angiogenesis PrimeKit Assay. [Figure 2-2] Quantitative measurement of in vitro angiogenesis induced by MLPSC-conditioned medium using the IncuCyte® 96-Well Kinetic Angiogenesis PrimeKit Assay. [Figure 2-3] 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] MPCs cultured in medium supplemented with neonatal serum significantly reduced 3-point MACE in all patients. [Figure 9] CV mortality in subjects with persistent inflammation (hsCRP ≥ 2) with MPC cultured with or without non-fetal serum. [Figure 10] Three-point composite MACE (MI, stroke, or CV death) in subjects with persistent inflammation (hsCRP ≥ 2) with MPC cultured with or without non-fetal serum. [Figure 11-1] MPCs cultured in medium supplemented with neonatal serum significantly reduced CV mortality (A) and TCE (B) in patients at highest risk (CRP>2mg / ml, NTpro-BNP>1000ng / ml). [Figure 11-2] MPCs cultured in medium supplemented with neonatal serum significantly reduced CV mortality (A) and TCE (B) in patients at highest risk (CRP>2mg / ml, NTpro-BNP>1000ng / ml). [Figure 12] (A) Plasma levels of the proinflammatory cytokine IL-6 in control LVAD patients: pathogenesis of ischemic and non-ischemic HFrEF. (B) Plasma levels of the proinflammatory cytokine IL-6 in LVAD patients: ischemic control group and ischemic LVAD patients receiving MPC. [Figure 13] All-cause mortality within 12 months in ischemic and non-ischemic LVAD patients. [Figure 14-1] All-cause mortality over 12 months in LVAD patients receiving "approved" Rexlemestrocel-L, "unapproved" Rexlemestrocel-L, and control patients. (A) All LVAD patients (ischemic and non-ischemic groups). (B) Ischemic LVAD patients. [Figure 14-2]All-cause mortality over 12 months in LVAD patients receiving "approved" Rexlemestrocel-L, "unapproved" Rexlemestrocel-L, and control patients. (A) All LVAD patients (ischemic and non-ischemic groups). (B) Ischemic LVAD patients. DETAILED DESCRIPTION OF THE INVENTION

[0084] General Techniques and Definitions Unless otherwise defined, all technical and scientific terms used herein should be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular biology, stem cell culture, immunology, and biochemistry).

[0085] 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 described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Messrs. (editors), and F.M.A.usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions), 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 It is described and explained throughout the literature in sources such as Wiley & Sons (including all current editions).

[0086] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0087] As used herein, the term "about" refers to + / - 10%, more preferably + / - 5% of the specified value, unless otherwise specified.

[0088] In one example, a sample (e.g., a blood sample) is obtained from a patient or subject, and the level of a substance in the sample is measured. For example, a blood sample can be obtained to measure the level of CRP in a subject.

[0089] "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, a subject treated according to the present disclosure can have elevated CRP. The term "elevated CRP" is used in the context of the present disclosure to refer to an increased CRP level compared to baseline CRP levels. In one example, a CRP level of 1 mg / L 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. In one example, persistent inflammation is characterized by a CRP level of ≧2 mg / L.

[0090] 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.

[0091] In the context of the present disclosure, the term "conditioned medium" refers to medium obtained from MLPSCs under culture conditions. Such medium contains the secretome of MLPSCs, proteins shed from the surface of MLPSCs, and other particles such as extracellular vesicles. The conditioned medium of the present disclosure contains extracellular vesicles, pro-angiogenic factors such as angiogenin, or secreted metabolites such as prostaglandin E2. The pro-angiogenic ability of the conditioned medium disclosed herein and / or factors obtained thereby can be confirmed, if desired, using one or more of the angiogenesis assays disclosed herein (e.g., endothelial network formation, endothelial length, endothelial branch length). In certain examples, the present disclosure relates to extracellular vesicles, such as exosomes, obtained from conditioned medium obtained from MLPSCs under culture conditions. In one example, the conditioned medium is obtained when MLPSCs are in the exponential growth phase. In one example, the conditioned medium is obtained at least 2-3 days after culture. In another example, the conditioned medium is obtained approximately 30-84 hours after culture.

[0092] In one example, the level of a particular marker, such as a pro-angiogenic factor(s), is determined under culture conditions. The term "culture conditions" is used to refer to the growth of cultured cells. In one example, culture conditions refer to a population of cells that are actively dividing. Such cells may, in one example, be in an exponential growth phase. Alternatively, such cells may be in a stationary phase.

[0093] In one example, in the context of measuring the level of IL2-RA inhibition, the culture conditions can include co-culturing the MLPSC population disclosed herein with a second cell population, such as a population containing peripheral blood mononuclear cells (PBMCs). In one example, the co-culturing includes culturing the MLPSC population disclosed herein with an activated PBMC population. For example, the PBMCs can be activated using anti-CD3 and anti-CD28 antibodies before co-culturing with the MLPSC population disclosed herein. In this example, the "culture conditions" can include co-culturing MLPSCs with T cells at a ratio of approximately 1 MLPSC:2 T cells. For example, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or 1 MLPSC:100 T cells, or less. In this example, the level of IL2-RA inhibition is determined after approximately 30 to 84 hours of cell culture under the culture conditions.

[0094] In one example, this level is expressed in terms of how much of a particular marker is released from the cells described herein under culture conditions.

[0095] In one example, the level is expressed in mg / L. For example, the level of CRP is expressed in mg / L. In one example, the level is expressed in ng / ml. For example, the level of VEGF can be expressed in ng / ml. In one example, the level of SDF-1α can be expressed in ng / ml. In one example, the level is expressed in pg / ml. For example, the level of NT-proBNP can be expressed in pg / ml. In one example, the level of angiogenin can be expressed in pg / ml.

[0096] In one example, the level of a particular marker is measured in a cell population (or in the supernatant obtained after cell culture) and divided by the number of cells in the population. In this example, the level is calculated as 10 6 It can be expressed in units per cell (eg, pg).

[0097] 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. One skilled in the art will understand that secreted markers can be measured by sampling the culture medium, while markers expressed on the cell surface can be measured by evaluating a sample of cell lysate. In one example, the sample is taken when the cells are in the exponential growth phase. In one example, the sample is taken after at least 2-3 days of culture. In another example, the sample is taken after approximately 30-84 hours of culture. In another example, the sample is taken when the cells are in the stationary phase.

[0098] In one example, a sample is taken from a co-culture of MLPSCs and activated PBMCs. In this example, the cell sample can be lysed and the level of a marker can be measured. For example, the level of IL2-RA can be determined. In this example, the level of IL2-RA can be determined using a variety of methods, such as an enzyme-linked immunosorbent assay (ELISA)-based method. In one example, ELISA is (i) adding sample dilutions to each well of a microplate pre-coated 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 IL2-RA in the sample; (iv) washing the microplate; (v) adding IL2-RA conjugate to the well; (vi) incubating the microplate for a time sufficient to allow the conjugate to specifically bind to the captured IL2-RA; (vii) washing the microplate; (viii) adding a substrate solution to the wells; (ix) incubating the microplate for a sufficient time to allow 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.

[0099] 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.

[0100] 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.

[0101] In one example, a method of manufacturing a medicament according to the present disclosure includes determining the level of one or more angiogenic markers under culture conditions.

[0102] Culturing and expanding cells from cryopreserved intermediates means thawing cryogenically frozen cells and culturing them in vitro under conditions suitable for cell growth.

[0103] In one example, the "level" of a particular marker is determined after the cells are cryopreserved and then returned to culture. For example, the level can be determined after the first cryopreservation of the cells. In another example, the level can be determined after the second cryopreservation of the cells. In one example, cells are isolated from a suitable stem cell source, such as bone marrow (e.g., using immunoselection against marker(s), such as STRO-1), and expanded in culture, thereby providing an intermediate cell population that is evaluated to determine the level of the particular marker. In this example, the level can be determined before or after cryopreservation. In one example, the level is determined after cryopreservation of the intermediate cell population. In another example, cells are expanded in culture from a cryopreserved intermediate, cryopreserved again, and then replated in culture, allowing the level of the particular marker to be measured under culture conditions.

[0104] As used herein, the terms "treating," "treat," "treatment," and "attenuating progression" include administering a population of mesenchymal stem cells or progenitor cells cultured according to the present disclosure and / or their progeny and / or soluble factors derived therefrom and / or extracellular vesicles derived therefrom to thereby reduce or eliminate at least one symptom of progressive heart failure, or, in the context of attenuating progression, delay the onset of progressive heart failure.

[0105] In one example, the present disclosure encompasses selecting certain subjects with advanced heart failure for treatment with the cell compositions disclosed herein. In one example, a subject with persistent inflammation is selected for treatment. In one example, persistent inflammation is determined based on CRP levels. For example, a subject with persistent inflammation has elevated CRP. In one example, a subject with a CRP level of ≧2 mg / L is selected for treatment. In one example, persistent inflammation is determined based on IL-6 levels. For example, a subject with persistent inflammation has elevated IL-6. In one example, a subject with persistently elevated IL-6 levels after LVAD implantation is selected for treatment. In another example, a subject with microvascular disease and / or macrovascular disease is selected for treatment. In one example, a subject with Class II heart failure is selected for treatment. In one example, a subject with a high risk of cardiac death is selected for treatment.

[0106] The term "subject" as used herein refers to a human subject. For example, the subject may be an adult. In another example, the subject may be a child. In another example, the subject may be an adolescent. Terms such as "subject," "patient," or "individual" are terms that can be used interchangeably in this disclosure, depending on the context. Subjects in need of treatment include those who already have progressive heart failure, as well as those who need to prevent, delay, or stop progressive heart failure.

[0107] 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.

[0108] 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).

[0109] The term "angiogenic potential" as used herein refers to the ability of an MLPSC population to express one or more angiogenic markers. In one example, angiogenic potential is determined by the ability to induce angiogenesis using an assay as disclosed herein. In one example, MLPSCs of the present disclosure have increased angiogenic potential. In another example, a conditioned medium of the present disclosure has increased angiogenic potential. In one example, MLPSCs with increased angiogenic potential are cultured and expanded according to the methods disclosed herein. For example, MLPSCs can be cultured and expanded in medium supplemented with proinflammatory cytokines as disclosed herein and / or non-fetal serum, such as newborn bovine serum. In one example, MLPSCs or conditioned medium of the present disclosure have increased angiogenic potential compared to MLPSCs cultured and expanded in medium containing 10% FCS.

[0110] As used herein, the term "sample" refers to an extract from a subject or cell culture in which the level of a particular marker can be measured. "Sample" includes extracts and / or derivatives and / or fractions of a sample. In one example, a sample is an extract from a subject in which the level of CRP can be measured. In the present disclosure, any biological material can be used as the sample as long as it can be collected from a subject or cell culture and analyzed to determine the level of a marker disclosed herein (e.g., the level of CRP in a subject). In one example, the sample is a blood sample. For example, a blood sample can be obtained from a subject with NYHA class II heart failure.

[0111] In one example, the "sample" is a cell population, e.g., a cell population under culture conditions. In one example, the sample is a supernatant obtained after cell culture, e.g., cell-conditioned medium. In these examples, the sample is any extract of the cell culture in which angiogenic markers can be measured. In one example, the sample is contacted with another cell population to determine the level of angiogenic markers.

[0112] In one example, the present disclosure encompasses selecting a population of culture-expanded MLPSCs with a particular potency for use in the therapeutic methods disclosed herein. As used herein, the term "potency" refers to the particular ability or capacity of MLPSCs to produce a predetermined result. In one example, the result is a therapeutic outcome, such as improved cardiac outcomes, as disclosed herein.

[0113] "Therapeutic effect" is used in the context of the present disclosure to refer to the MLPSCs and compositions disclosed herein that can treat, inhibit, and / or prevent a disease. For example, the therapeutically effective MLPSCs and compositions disclosed herein can treat, inhibit, and / or prevent progressive heart failure.

[0114] "Biological activity," in the context of the present disclosure, is used to define the MLPSCs and compositions disclosed herein based on a particular activity. In one example, the biological activity is pro-angiogenic activity and / or anti-inflammatory activity. In one example, the biological activity is the ability to increase angiogenesis in vitro. In one example, the biological activity is an increase in the expression of one or more angiogenic markers. In one example, the biological activity is angiogenic potential as determined by the level of one or more angiogenic markers. In one example, the biological activity is characterized by an improvement in clinical outcome(s) (e.g., survival rate) and / or parameter(s) (e.g., LVEF).

[0115] The term "clinically proven" (used alone or to modify the term "effective") means that efficacy has been proven by a clinical trial, and that the clinical trial meets the approval standards of the US Food and Drug Administration, EMEA, or corresponding national regulatory agency. For example, the clinical study may be an appropriately sized, randomized, double-blind study used to clinically prove the efficacy of a composition. In one example, a clinically proven effective amount is an amount shown by a clinical trial to meet a specified endpoint. In one example, the endpoint is prevention of death. In other words, the endpoint increases survival rate. For example, 100-day survival rate may increase when a therapeutic agent according to the present disclosure is administered.

[0116] Thus, the terms "clinically proven efficacy" and "clinically proven effect" may be used in the context of this disclosure to refer to a dose, dosing regimen, treatment, or method disclosed herein. Efficacy can be measured based on changes in the course of a disease in response to administration of a composition disclosed herein. For example, a composition of the present disclosure is administered to a subject in an amount and for a time sufficient to induce an improvement, preferably a sustained improvement, in at least one indicator reflecting the severity of cardiovascular disease. To determine whether the amount and time of treatment are sufficient, various indicators reflecting the severity of the disease can be evaluated. Such indicators include, for example, clinically recognized indicators of disease severity or symptoms. In one example, the degree of improvement is determined by a physician, who can make this determination based on signs, symptoms, or other test results (e.g., echocardiogram, LVEF, LVESV). In one example, a clinically proven effective amount improves patient survival. In another example, a clinically proven effective amount reduces a subject's risk of death. In another example, a clinically proven effective amount extends 100-day survival. In another example, the clinically proven effective amount increases LVEF. In one example, the disclosed method administers a clinically proven effective amount of a composition disclosed herein.

[0117] 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.

[0118] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, composition of matter, group of steps or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of those steps, compositions of matter, group of steps or group of compositions of matter.

[0119] 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.

[0120] 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.

[0121] Unless otherwise specified, examples disclosed herein are intended to apply mutatis mutandis to other examples.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs.

[0130] 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.

[0131] In one example, isolated or enriched mesenchymal progenitor or stem cells are cultured in culture medium (serum-free or serum-supplemented) (e.g., alpha-minimal essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine) at a density of 50,000 viable cells / cm. 2 Cells are seeded in a 100% CO2-independent manner and allowed to adhere to the culture vessel overnight at 37°C and 20% O2. As used herein, the terms "culture medium" and "culture medium" are used interchangeably. The culture medium is then replaced and / or changed as needed, and the cells are cultured for an additional 68-72 hours at 37°C and 5% O2.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] "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.

[0138] 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.

[0139] Additionally, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0140] 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 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 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.

[0141] 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.

[0142] 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.

[0143] In one example, the present disclosure encompasses mesenchymal progenitor or stem cells and their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal progenitor or stem cells and conditioned medium obtained thereby under culture conditions. In another example, the present disclosure encompasses mesenchymal progenitor or stem cells and extracellular vesicles isolated therefrom. For example, mesenchymal progenitor lineage cells or stem cells of the present disclosure can be cultured and grown for a period of time and under conditions suitable for secreting extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for therapeutic use. If desired, such extracellular vesicles can be characterized using one or more of the angiogenesis assays disclosed herein (e.g., endothelial network formation, endothelial length, endothelial branch length).

[0144] As used herein, the term "extracellular vesicles" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, but are typically less than 200 nm. They are released by the releasing cells (e.g., mesenchymal stem cells, STRO-1). + The fragments may contain proteins, nucleic acids, lipids, metabolites, or organelles from the cell.

[0145] 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.

[0146] As used herein, the term "pre-approved" or "approved" refers to the 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.

[0147] 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, e.g., CFU-F (fibroblasts), or a subset thereof (e.g., 50%, 60%, 70%, 80%, 90%, or 95%), can possess this activity.

[0148] In one example, a population of cells is enriched from a cell preparation that contains 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, 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 the cells are STRO-1+ does not mean that the cells were selected by STRO-1 expression. In one example, the cells were selected based on at least STRO-3 expression, e.g., STRO-3+ (TNAP+).

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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, cells shown to express a marker need not be specifically tested; 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.

[0154] 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.

[0155] 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.

[0156] 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 In cells, the "background" i.e. STRO-1 - STRO-1 surface expression is two logs higher than in 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."

[0157] 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.

[0158] Furthermore, in one example, STRO-1+ cells are capable of giving rise to clonogenic CFU-F.

[0159] 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.

[0160] 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.

[0161] In another example, the mesenchymal progenitor or stem cells are CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSCs (e.g., remestemcel-L).

[0162] 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.

[0163] Mesenchymal progenitor or stem cells cultured using the methods of the present disclosure can also be cryopreserved.

[0164] Culture-expanded MLPSCs and conditioned medium obtained from the same In an example, cultured expanded MLPSCs of the present disclosure and / or conditioned medium obtained therefrom are characterized by expression of angiogenic marker(s). For example, a cultured expanded MLPSC population according to the present disclosure and / or conditioned medium obtained therefrom can be characterized by increased levels of VEGF, angiogenin, and / or SDF-1α under culture conditions. In another example, an MLPSC population 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 the cultured 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 cultured and expanded in cell culture medium containing 10% fetal bovine serum.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 2 is.

[0169] In one example, a culture-expanded MLPSC population is 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 2The 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.

[0170] 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 cultured and expanded in cell culture medium containing 10% fetal serum (e.g., fetal bovine 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 containing 10% fetal serum (e.g., fetal bovine 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.

[0171] 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%.

[0172] 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.

[0173] 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.

[0174] "Culture-expanded" MLPSCs are distinguished from freshly isolated cells in that they have been cultured and passaged (ie, subcultured) in cell culture medium.

[0175] 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+.

[0176] 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.

[0177] 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.

[0178] In one example, a composition of the present disclosure includes MLPSCs cultured and expanded from a cryopreserved intermediate. In one example, the cell culture cultured and expanded from a cryopreserved intermediate is cultured and 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 cultured and expanded for at least 3 passages. In one example, the MLPSCs can be cultured and expanded for at least 3-10 passages. In one example, the MLPSCs can be cultured and expanded for at least 3-8 passages. In one example, the MLPSCs cultured and expanded from a cryopreserved intermediate is cultured and expanded in a medium disclosed herein (e.g., a medium containing newborn bovine serum).

[0179] In one example, MLPSCs can be obtained from a single donor or from multiple donors, where the donor samples or MLPSCs are later pooled and optionally culture expanded. i. expanding the viable cell number by serial propagation to provide a preparation of at least about 1 billion viable cells, wherein serial propagation comprises establishing a primary culture of isolated MLPSCs and then successively establishing a first non-primary (P1) culture of MLPSCs isolated from the previous culture; ii. Expanding the P1 culture of isolated MLPSCs into a second, non-primary (P2) culture of MLPSCs by sub-expansion; 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 sub-expansion.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] 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 bovine serum.

[0186] 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 cultured and expanded in a cell culture medium containing 10% fetal bovine 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%.

[0187] 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%.

[0188] In one example, a culture-expanded MLPSC preparation is i. less than about 0.75% CD45+ cells; ii. at least approximately 95% CD105+ cells; iii. have an antigenic and activity profile that includes at least about 95% CD166+ cells.

[0189] conditioned medium In one example, the conditioned medium or extracellular vesicles obtained therefrom can be characterized by the expression of angiogenic marker(s). For example, the conditioned medium or extracellular vesicles obtained therefrom can be characterized by increased levels of VEGF, angiogenin, and / or SDF-1α under culture conditions. In another example, the conditioned medium or extracellular vesicles obtained therefrom can be characterized based on one or more functional criteria. In one example, when culture-expanded MLPSCs are treated with conditioned medium or extracellular vesicles obtained therefrom, the conditioned medium or extracellular vesicles obtained therefrom increase the level of endothelial network formation, endothelial network length, and / or endothelial branch length in a population of endothelial cells. In one example, the increase is determined by comparison with conditioned medium or extracellular vesicles obtained therefrom from a control population of MLPSCs. In one example, the control population is a population of MLPSCs cultured and expanded in cell culture medium containing 10% fetal bovine serum.

[0190] In one example, the conditioned medium is characterized by a VEGF level of greater than about 3 ng / ml. In one example, the VEGF level is about 3 ng / ml to 4 ng / ml. In one example, the VEGF level is greater than about 3.1 ng / ml. In one example, the VEGF level is greater than about 3.2 ng / ml. In one example, the VEGF level is greater than about 3.3 ng / ml. In one example, the VEGF level is greater than about 3.4 ng / ml. In one example, the VEGF level is greater than about 3.5 ng / ml. In one example, the VEGF level is about 3.2 to 3.6 ng / ml. In one example, the VEGF level is about 3.45 ng / ml.

[0191] In one example, the conditioned medium or extracellular vesicles obtained thereby contain an increased level of angiogenin compared to a control population. In one example, the conditioned medium is characterized by an angiogenin level greater than about 1000 pg / ml. In one example, the angiogenin level is greater than about 1100 pg / ml. In one example, the angiogenin level is about 1000 pg / ml to 1200 pg / ml. In one example, the angiogenin level is about 1100 pg / ml to 1150 pg / ml. In one example, the angiogenin level is about 1114 pg / ml.

[0192] In one example, the conditioned medium is characterized by a level of SDF-1α greater than about 3000 ng / ml. In one example, the level of SDF-1α is greater than about 3100 ng / ml. In one example, the level of SDF-1α is greater than about 3200 ng / ml. In one example, the level of SDF-1α is greater than about 3300 ng / ml. In one example, the level of SDF-1α is greater than about 3400 ng / ml. In one example, the level of SDF-1α is greater than about 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3400 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3300 ng / ml. In one example, the level of SDF-1α is about 3100 ng / ml to 3400 ng / ml.In one example, the level of SDF-1α is about 3100 ng / ml to 3300 ng / ml.

[0193] In one example, the conditioned medium is about 0.1 mm 2 / mm 2 In one example, the formation of an endothelial network is stimulated at a depth of about 0.1 mm. 2 / mm 2 ~0.2mm 2 / mm 2 In another example, the formation of an endothelial network is about 0.12 mm 2 / mm 2 is.

[0194] In one example, the conditioned medium is approximately 4 mm 2 / mm 2 In one example, the length of the endothelial network is approximately 4 mm. 2 / mm 2 ~about 6mm 2 / mm 2 In one example, the length of the endothelial network is about 5 mm. 2 / mm 2 In one example, the conditioned medium has a density of about 12 l / mm 2 Stimulates endothelial branch lengths of greater than 1 / 2 mm. In one example, the endothelial branch length is approximately 1 / 2 mm. 2 ~approximately 17 1 / mm 2 In one example, the length of the endothelial branch is about 15 1 / mm 2 is.

[0195] In one example, the conditioned medium or extracellular vesicles obtained therefrom are characterized by increased levels of one or more angiogenic markers compared to conditioned medium or extracellular vesicles obtained therefrom from a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of the angiogenic marker is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In one example, the level of the angiogenic marker is increased by about 5% to about 60%. In one example, the level of the angiogenic marker is increased by about 5% to about 40%. In one example, the level of the angiogenic marker is increased by about 40%. In one example, the level of the angiogenic marker is increased by at least about 5%. In one example, the level of the angiogenic marker is increased by at least about 10%. In one example, the levels of angiogenic markers are increased compared to conditioned medium or extracellular vesicles obtained from a population of MLPSCs cultured and expanded in cell culture medium without IFN-γ or TNF-α.

[0196] In one example, the conditioned medium or extracellular vesicles obtained therefrom are characterized by increased levels of one or more angiogenic markers compared to conditioned medium or extracellular vesicles obtained therefrom from a population of MLPSCs cultured and expanded in neonatal serum-free cell culture medium. In one example, the level of the angiogenic marker is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70%. In one example, the level of the angiogenic marker is increased by about 5% to about 60%. In one example, the level of the angiogenic marker is increased by about 5% to about 40%. In one example, the level of the angiogenic marker is increased by at least about 5%. In one example, the level of the angiogenic marker is increased by at least about 10%.

[0197] In one example, freshly isolated cells are culture-expanded for about 1 or 2 passages to provide an intermediate population. In one example, freshly isolated cells are culture-expanded for 2 passages to provide an intermediate population. In another example, freshly isolated cells are culture-expanded for about 1 to 3 passages to provide an intermediate population. In one example, the freshly isolated cells are STRO-1+. In one example, the culture-expanded cells produce conditioned medium or extracellular vesicles obtained therefrom to provide a cryopreserved intermediate.

[0198] 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). In one example, conditioned medium or resulting extracellular vesicles are obtained from the DP MLPSCs.

[0199] In one example, MLPSCs are cultured and expanded for approximately 4 to 10 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs are cultured and expanded for at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, or at least 10 passages, thereby providing the resulting conditioned medium or extracellular vesicles. For example, MLPSCs can be cultured and expanded for at least 5 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be cultured and expanded for at least 5 to 10 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be cultured and expanded for at least 5 to 8 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be cultured and expanded for at least 5 to 7 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In one example, MLPSCs can be cultured and expanded for more than 7 passages, thereby providing the resulting conditioned medium or extracellular vesicles. In these instances, the MLPSCs can be culture-expanded prior to cryopreservation to provide an intermediate cryopreserved MLPSC population, which can then be further culture-expanded to provide the resulting conditioned medium or extracellular vesicles.

[0200] In one example, the conditioned medium or extracellular vesicles obtained thereby are obtained from MLPSCs cultured and expanded from a cryopreserved intermediate. In one example, the cell culture cultured and expanded from a cryopreserved intermediate is cultured and 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 cultured and expanded for at least 3 passages. In one example, the MLPSCs can be cultured and expanded for at least 3 to 10 passages. In one example, the MLPSCs can be cultured and expanded for at least 3 to 8 passages. In one example, the MLPSCs cultured and expanded from a cryopreserved intermediate is cultured and expanded in a medium disclosed herein (e.g., a medium containing newborn bovine serum).

[0201] In one example, MLPSCs can be obtained from a single donor or from multiple donors, where the donor samples or MLPSCs are later pooled and optionally culture expanded. i. expanding the viable cell number by serial propagation to provide a preparation of at least about 1 billion viable cells, wherein serial propagation comprises establishing a primary culture of isolated MLPSCs and then successively establishing a first non-primary (P1) culture of MLPSCs isolated from the previous culture; ii. Expanding the P1 culture of isolated MLPSCs into a second, non-primary (P2) culture of MLPSCs by sub-expansion; 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 sub-expansion.

[0202] 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.

[0203] 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.

[0204] In one example, the selected cell population is immediately expanded in culture, hi another example, the selected cell population is cryopreserved for expansion in culture at a later stage.

[0205] In one example, the conditioned medium obtained from the culture-expanded MLPSC population or the extracellular vesicles obtained thereby is i. less than about 0.75% CD45+ cells; ii. at least approximately 95% CD105+ cells; iii. have an antigenic and activity profile that includes at least about 95% CD166+ cells.

[0206] Common MLPSC culture and proliferation methods 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.

[0207] 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.

[0208] 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.

[0209] In one example, the MLPSCs of the present disclosure are cultured and expanded in 2D culture. For example, the MLPSCs of the present disclosure can be cultured and 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 cultured and expanded in 2D culture before being further expanded in 3D culture. In one example, the intermediate cell population of the present disclosure is not cultured and 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.

[0210] 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.

[0211] 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 types of bioreactors, such as stirred tanks, wave bags, and vertical wheels.

[0212] In one example, CO2 is provided during the culture and growth of MLPSCs. In one example, MLPSCs are cultured and grown at less than 9% CO2. In one example, MLPSCs are cultured and grown at less than 8% CO2. In one example, MLPSCs are cultured and grown at 5% CO2. For example, MLPSCs are cultured and grown at 5% + / - 2% CO2. In one example, MLPSCs are cultured and grown by passive priming with 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 pumping CO2 gas into each culture vessel (e.g., cell factory) through a bacterial vent filter for a set period of time (e.g., approximately 10 minutes). However, active priming requires an open port to supply gas, potentially introducing contaminants into the culture. Passive priming refers to placing a closed culture system in an incubator with the appropriate CO2 concentration prior to cell seeding (e.g., approximately 12-72 hours prior). In one example, the cells disclosed herein are STRO-3+ before being culture-expanded to provide an intermediate cell population.

[0213] Cell culture method The compositions of the present disclosure can be prepared by growth in culture in medium containing one or more pro-inflammatory cytokines and / or a non-fetal serum as disclosed herein, such as newborn serum.

[0214] For example, MLPSC culture medium can be 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.

[0215] In one example, the medium includes one or more pro-inflammatory cytokines that can bind to receptors on the surface of the MLPSCs.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] In one example, the medium is serum-free.

[0222] 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.

[0223] 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.

[0224] In another example, the compositions of the present disclosure can be prepared by culturing and growing MLPSCs in culture medium supplemented with serum containing one or more of the pro-inflammatory cytokines described herein. In preferred embodiments, the culture medium is supplemented with non-fetal serum, such as neonatal serum. In some preferred embodiments, the culture medium is supplemented with equal concentrations of fetal serum and neonatal serum, with the total serum concentration in the culture medium being approximately 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.

[0225] In some embodiments, the methods for preparing MLPSCs disclosed herein include the additional step of determining or having determined the levels 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.

[0226] In some embodiments, the methods for preparing MLPSCs 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., analysis of tube formation by human umbilical vein endothelial cells (HUVECs), as well as 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.

[0227] In some embodiments, the methods for preparing MLPSCs disclosed herein also include determining or having determined the levels of one or more of angiogenin, angiopoietin (Ang1 / ANGPT1), SDF-1α, and VEGF in the conditioned medium described above.

[0228] 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.

[0229] The methods and cell culture media used to prepare MLPSCs of the present disclosure promote stem cell proliferation while maintaining the 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 a differentiated state. Polypeptide products can also be used as markers to detect a differentiated state. Therefore, those skilled in the art can easily determine whether the methods of the present disclosure 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.

[0230] 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.

[0231] The cell culture medium used for culture growth 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.

[0232] 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, if this energy source is found to be depleted and growth is limited, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or to add glucose (or other energy source) during the course of the culture. In one example, dissolved oxygen (DO) levels may also be controlled.

[0233] In the above example, the basal medium such as Alpha MEM or StemSpan™ can be supplemented with a reference amount of serum, and in certain cases, can also be supplemented with other additives. Further examples of suitable culture media for culturing stem cells are described, for example, in WO2016139340.

[0234] serum "Non-fetal serum" refers to serum obtained after birth. For example, the culture medium can be supplemented with mammalian non-fetal serum (e.g., bovine). In one example, the culture medium can be supplemented with animal non-fetal serum. In another example, the culture medium can be supplemented with human non-fetal serum.

[0235] In one example, the cell culture medium is supplemented with at least about 1% v / v, at least about 2% v / v, at least about 3% v / v, at least about 4% v / v, at least about 5% v / v, at least about 6% v / v, at least about 7% v / v, at least about 8% v / v, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, or at least about 25% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 15% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 1% v / v to about 10% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 5% v / v to about 10% v / v of non-fetal serum. In one example, the cell culture medium is supplemented with about 5% v / v of non-fetal serum.

[0236] In one example, the non-fetal serum contains at least one proinflammatory cytokine. Methods for detecting the presence of cytokines in cell culture medium and / or serum are known in the art, such as enzyme-linked immunosorbent assay (ELISA). In another example, the presence of cytokines in serum is detected by measuring cytokine mRNA, for example, by polymerase chain reaction (PCR) techniques such as reverse transcription PCR.

[0237] In one example, the non-fetal serum is a newborn serum, such as newborn bovine 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.

[0238] In one example, the newborn serum is obtained within 4 weeks after the birth of the animal. In one example, the newborn serum is obtained within 21 days after the birth of the animal. For example, the newborn serum is obtained within 21 days after the birth of the animal. In one example, the newborn serum is obtained from the day of birth to 21 days after the birth of the animal. In one example, the newborn serum is obtained from the day of birth to 14 days after the birth of the animal. In one example, the newborn serum is obtained from the day of birth to 10 days after the birth of the animal. In one example, the newborn serum is obtained from the day of birth to 7 days after the birth of the animal. In one example, the newborn serum is obtained from 6 hours to 72 hours after the birth. In one example, the newborn serum is obtained from 6 hours to 48 hours after the birth. In one example, the newborn serum is obtained from 6 hours to 24 hours after the birth. In one example, the newborn serum is obtained from 12 hours to 24 hours after the birth.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] In one example, the cell culture medium is fetal serum-free.

[0248] In one example, the cell culture medium does not contain FCS.

[0249] 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.

[0250] 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.

[0251] Ascorbic acid In one example, a short-acting ascorbic acid derivative is added to the cell culture medium. The term "short-acting" encompasses ascorbic acid derivatives that are approximately 80-90% oxidized after 24 hours of cell culture under culture conditions of neutral pH and 37°C. In one example, the short-acting L-ascorbic acid derivative is an L-ascorbate salt, such as L-ascorbic acid sodium salt. In one example, the cell culture medium may contain at least about 0.005 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.01 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.02 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium may contain at least about 0.03 g / L of the short-acting ascorbic acid derivative. For example, the cell culture medium may contain at least about 0.04 g / L of the short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.05 g / L of a short-acting ascorbic acid derivative. In another example, the cell culture medium can contain at least about 0.06 g / L of a short-acting ascorbic acid derivative.

[0252] 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.

[0253] 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.

[0254] In the above example, the basal medium such as Alpha MEM or StemSpan™ can be supplemented with a reference amount of serum, and in certain cases, can also be supplemented with other additives. Further examples of suitable culture media for culturing stem cells are described, for example, in WO2016139340.

[0255] Angiogenesis markers According to the present disclosure, in certain embodiments, MLPSCs cultured according to the methods disclosed herein have increased levels of one or more angiogenic markers. The inventors have surprisingly found that MLPSCs with increased levels of one or more angiogenic markers improve therapeutic efficacy in patients with advanced heart failure. Thus, in one example, the methods of the present disclosure relate to selecting cultured expanded MLPSCs for use in a therapy, such as the treatment of advanced heart failure. Such methods include determining the level(s) of the marker(s) disclosed herein and selecting MLPSCs with increased levels of one or more marker(s) for use in the therapy.

[0256] Angiogenesis is the physiological process by which new blood vessels are formed. Pathophysiological events such as ischemia and inflammation increase angiogenesis at the site of injury through the release of growth factors such as vascular endothelial growth factor (VEGF) and chemokines such as stromal cell-derived factor 1 (SDF-1). DF-1α is a pro-angiogenic protein known to play a role in the migration, recruitment, and retention of endothelial progenitor cells to the site of ischemic injury, contributing to neovascularization. VEGF is considered the most important regulator of angiogenesis in both health and disease. It is essential for embryonic vasculogenesis and angiogenesis, and is also a key mediator of angiogenesis in cancer and other diseases. VEGF stimulates angiogenesis by acting through its cognate receptor kinase family in endothelial cells. At the cellular level, binding of VEGF to its main receptor kinase insert domain-containing receptor (KDR) mimics a complex network of signaling pathways including activation of phospholipase C-γ, protein kinase C, Ca(2+), ERK (extracellular signal-regulated protein kinase), Akt, Src, focal adhesion kinase, and calcineurin pathways.

[0257] Angiogenin is another potent proangiogenic factor that regulates angiogenesis and cell proliferation by stimulating basement membrane degradation, endothelial cell permeability, migration, and the formation of tubular vasculature. Angiogenin induces angiogenesis by binding to actin on the surface of endothelial cells. Angiogenin is a member of the RNase A superfamily and is encoded by the human ANG gene (NCBI Gene ID: 283, GenBank: AAH62698.1). The structure, function, and expression pattern of angiogenin, along with detection methods, are known in the art (see, e.g., Tello-Montoliu et al. J Thromb Haemost. 2006;4(9):1864-74). Commercially available enzyme-linked immunosorbent assay (ELISA) kits can be used to detect the protein in body fluids, such as serum, plasma, cell-conditioned medium (e.g., cell-conditioned media), and urine, using various commercially available antibodies targeting human angiogenin. Antibody-based detection assays can also be used to measure angiogenin in tissue or cell lysates. Other approaches to measuring angiogenin use human cytokine protein array technologies such as the Luminex assay, which uses an antibody array to simultaneously detect angiogenin among multiple additional factors from a variety of sources.

[0258] The disclosed method includes measuring the level of a pro-angiogenic factor, such as VEGF, angiogenin, and / or SDF-1α, expressed by MLPSCs under culture conditions. In one example, MLPSCs can be cultured and grown in a culture medium according to the methods disclosed herein. Conditioned medium from the cultured MLPSCs is then isolated (i.e., a sample is obtained from the cell culture), and the amount of angiogenic marker expressed therein is measured. The level of angiogenic markers in the MLPSC-conditioned medium can be measured by standard protein detection and / or gene expression methods known in the art. In one example, the level of angiogenic markers is measured by enzyme-linked immunosorbent assay (ELISA). For example, conditioned medium from MLPSCs is obtained and then contacted with anti-VEGF antibody, anti-SDF-1α antibody, and / or anti-angiogenin antibody. The degree of antibody binding is used to quantify the level of angiogenic markers (e.g., ng / L) in the conditioned medium. In this example, the level of angiogenic marker in the conditioned medium is a measure of the level of angiogenic marker expressed or secreted by MLPSCs.

[0259] In one example, the level of angiogenic markers is measured by Western blot. In one example, the level of angiogenic markers is measured by Luminex assay. In one example, the level of angiogenic markers is measured by reverse transcription RT-PCR. For example, the level of angiogenin in the conditioned medium obtained from cultured MLPSCs is measured by cytokine protein array, such as Luminex assay.

[0260] In one example, MLPSCs are selected for therapeutic use if they have elevated vascular endothelial growth factor (VEGF) expression levels. In one example, the VEGF level is greater than about 3 ng / ml. In one example, the VEGF level is greater than about 3 ng / ml to 4 ng / ml. In one example, the VEGF level is greater than about 3.1 ng / ml. In one example, the VEGF level is greater than about 3.2 ng / ml. In one example, the VEGF level is greater than about 3.3 ng / ml. In one example, the VEGF level is greater than about 3.4 ng / ml. In one example, the VEGF level is greater than about 3.5 ng / ml. In one example, the VEGF level is between about 3 ng / ml and 4 ng / ml. In one example, the VEGF level is between about 3.2 to 3.6 ng / ml. In one example, the VEGF level is about 3.45 ng / ml.

[0261] In one example, MLPSCs are selected for therapeutic use if they exhibit an increased level of VEGF compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the VEGF level is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the VEGF level is increased by about 5% to about 60%. In one example, the VEGF level is increased by about 5% to about 40%. In one example, the VEGF level is increased by about 40%. In one example, the VEGF level is increased by at least about 5%. In one example, the VEGF level is increased by at least about 10%.

[0262] In one example, MLPSCs are selected for therapeutic use if they have elevated angiogenin expression levels. In one example, the angiogenin level is greater than about 1000 pg / ml. In one example, the angiogenin level is greater than about 1100 pg / ml. In one example, the angiogenin level is between about 1000 pg / ml and 1200 pg / ml. In one example, the angiogenin level is between about 1100 pg / ml and 1150 pg / ml. In one example, the angiogenin level is greater than about 1114 pg / ml. In one example, the angiogenin level is greater than about 1200 pg / ml.

[0263] In one example, MLPSCs are selected for therapeutic use if their angiogenin levels are increased compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the angiogenin levels are increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the angiogenin levels are increased by about 5% to about 60%. In one example, the angiogenin levels are increased by about 5% to about 40%. In one example, the angiogenin levels are increased by about 40%. In one example, the angiogenin levels are increased by at least about 5%. In one example, the angiogenin levels are increased by at least about 10%.

[0264] In one example, MLPSCs are selected for therapeutic use if they have elevated expression levels of stromal-derived factor 1 alpha (SDF-1α). In one example, the level of SDF-1α is greater than about 3000 ng / ml. In one example, the level of SDF-1α is greater than about 3100 ng / ml. In one example, the level of SDF-1α is greater than about 3200 ng / ml. In one example, the level of SDF-1α is greater than about 3300 ng / ml. In one example, the level of SDF-1α is greater than about 3400 ng / ml. In one example, the level of SDF-1α is greater than about 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3500 ng / ml. In one example, the level of SDF-1α is between about 3000 ng / ml and 3400 ng / ml. In one example, the level of SDF-1α is about 3000 ng / ml to 3300 ng / ml. In one example, the level of SDF-1α is about 3100 ng / ml to 3400 ng / ml. In one example, the level of SDF-1α is about 3100 ng / ml to 3300 ng / ml.

[0265] In one example, MLPSCs are selected for therapeutic use if they have an increased level of SDF-1α compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of SDF-1α is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of SDF-1α is increased by about 5% to about 60%. In one example, the level of SDF-1α is increased by about 5% to about 40%. In one example, the level of SDF-1α is increased by about 40%. In one example, the level of SDF-1α is increased by at least about 5%. In one example, the level of SDF-1α is increased by at least about 10%.

[0266] In another example, the angiogenic marker is increased angiogenesis. In this example, increased angiogenesis is measured by an in vitro angiogenesis assay, such as a quantitative live-cell imaging assay. Briefly, endothelial cell lines (e.g., human umbilical vein endothelial cells (HUVECs), human dermal fibroblasts, human saphenous vein endothelial cells (HSaVECs), human coronary artery endothelial cells (HCAECs), human aortic endothelial cells (HAECs), brain microvascular endothelial cells (BMECs), or any combination thereof) are fluorescently labeled and seeded onto culture plates. Next, the endothelial cells are simultaneously incubated with or without MLPSC-conditioned medium and imaged using a live-cell imaging system.

[0267] In this example, angiogenesis can be measured by various network morphometric parameters identified and calculated by image analysis software as a composite of various factors listed in Table 1 (Lam et al. Biomaterials 290. (2022) 121826). In one example, the live-cell imaging system is an IncuCyte® Live-Cell Analysis System. Live-cell imaging systems allow for fluorescent identification of cells and visualization of angiogenesis over time through time-lapse image acquisition. Images can be analyzed using a computer-based image analysis tool. In one example, the image analysis tool is the IncuCyte® Angiogenesis Analysis Software Module. The IncuCyte® Angiogenesis Analysis Software Module measures angiogenesis outputs such as endothelial network length, endothelial network area, and endothelial branch point formation. Those skilled in the art will recognize that other image analysis applications, such as Image J and CellProfiler, can also be used. Other examples of live imaging in vitro angiogenesis assays are described, for example, in Lam et al. Biomaterials 290. (2022). 121826. [Table 1-1] [Table 1-2]

[0268] In one example, angiogenesis is measured by the level of endothelial network formation, endothelial network length, and / or endothelial branch length. In one example, angiogenic potential is measured by the level of endothelial network formation, endothelial network length, and / or endothelial branch length. In one example, a population of endothelial cells is treated with conditioned medium obtained from MLPSCs, and then the level of endothelial network formation, endothelial network length, and / or endothelial branch length is measured. In one example, the level of endothelial network formation, endothelial network length, and / or endothelial branch length is calculated using an IncuCyte® angiogenesis analysis software module.

[0269] In another example, endothelial network formation, endothelial network length, and / or endothelial branch length may be calculated as a composite of one or more of the number of nodes, number of junctions, number of segments, number of meshes, average mesh size, total mesh area, number of endpoints, total branch length, and / or number of branches. As used herein, "network formation" refers to the network area in mm 2 / mm 2 Further examples of methods for calculating endothelial network formation, endothelial network length, and / or endothelial branch length are described, for example, in Lam et al. Biomaterials 290. (2022).

[0270] In one example, MLPSCs are selected for use in therapy if they increase one or more levels of endothelial network formation, endothelial network length, and / or endothelial branch length. In one example, endothelial network formation is increased by about 0.1 mm 2 / mm 2 In one example, the formation of an endothelial network is observed when the endothelial network is greater than about 0.1 mm 2 / mm 2 ~0.2mm 2 / mm 2In one example, the formation of an endothelial network is approximately 0.12 mm 2 / mm 2 In one example, the formation of an endothelial network is approximately 0.12 mm 2 / mm 2 In one example, the length of the endothelial network is about 4 mm. 2 / mm 2 In one example, the length of the endothelial network is about 4 mm. 2 / mm 2 ~about 6mm 2 / mm 2 In one example, the length of the endothelial network is about 5 mm. 2 / mm 2 In one example, the length of the endothelial network is about 5 mm. 2 / mm 2 In one example, the length of the endothelial branch is approximately 12 1 / mm 2 In one example, the length of the endothelial branch is approximately 121 / mm 2 ~approximately 17 1 / mm 2 In one example, the length of the endothelial branch is about 15 1 / mm 2 In one example, the length of the endothelial branch is about 15 1 / mm 2 It's super.

[0271] In one example, MLPSCs are selected for therapeutic use if they increase one or more levels of endothelial network formation, endothelial network length, and / or endothelial branch length compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of endothelial network formation is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of endothelial network formation is increased by about 5% to about 60%. In one example, the level of endothelial network formation is increased by about 5% to about 40%. In one example, the level of endothelial network formation is increased by about 40%. In one example, the level of endothelial network formation is increased by at least about 5%. In one example, the level of endothelial network formation is increased by at least about 10%.

[0272] In one example, MLPSCs are selected if the level of endothelial network length is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of endothelial network length is increased by about 5% to about 60%. In one example, the level of endothelial network length is increased by about 5% to about 40%. In one example, the level of endothelial network length is increased by about 40%. In one example, the level of endothelial network length is increased by at least about 5%. In one example, the level of endothelial network length is increased by at least about 10%. In one example, MLPSCs are selected if the level of endothelial branch length is increased by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% compared to a population of MLPSCs cultured and expanded in a cell culture medium containing 10% fetal bovine serum. In one example, the level of endothelial branch length is increased by about 5% to about 60%. In one example, the level of endothelial branch length is increased by about 5% to about 40%. In one example, the level of endothelial branch length is increased by about 40%. In one example, the level of endothelial branch length is increased by at least about 5%. In one example, the level of endothelial branch length is increased by at least about 10%.

[0273] Efficacy test The present disclosure provides potency assays for identifying cells and conditioned medium having biological activity or therapeutic effects. The potency assays are based on and determine the increase of one or more angiogenic markers disclosed herein. Thus, in one example, the present disclosure relates to a method for determining the potency of a population of mesenchymal progenitor or stem cells (MLPSCs) cultured and expanded in a cell culture medium containing non-fetal serum. In this example, the method includes determining the level of one or more angiogenic markers in the population of MLPSCs. In one example, the method is applied to determine the potency of a conditioned medium.

[0274] In one example, the one or more angiogenic markers are selected from the group consisting of levels of VEGF, angiogenin, and SDF-1α expressed by MLPSCs under culture conditions, and / or levels of endothelial network formation, endothelial network length, and endothelial branch length measured after treating a population of endothelial cells with conditioned medium obtained from MLPSCs. In one example, increased levels of the one or more angiogenic markers indicate biological activity or a therapeutic effect. In one example, the efficacy assay is based on determining an increase in the levels of VEGF, angiogenin, and / or SDF-1α expressed by MLPSCs under culture conditions. In one example, the efficacy assay is based on determining an increase in the levels of at least two of VEGF, angiogenin, and SDF-1α expressed by MLPSCs under culture conditions. In another example, the efficacy assay is based on determining an increase in VEGF, angiogenin, and SDF-1α expressed by MLPSCs under culture conditions. In these examples, VEGF, angiogenin, and SDF-1α may be measured by ELISA or Luminex assay. In another example, the efficacy assay is based on determining an increase in endothelial network formation, endothelial network length, and / or endothelial branch length measured after treating a population of endothelial cells with conditioned medium obtained from a population of MLPSCs. In one example, the efficacy assay is based on determining an increase in at least two of endothelial network formation, endothelial network length, and endothelial branch length measured after treating a population of endothelial cells with conditioned medium obtained from a population of MLPSCs. The efficacy assay is based on determining an increase in endothelial network formation, endothelial network length, and endothelial branch length measured after treating a population of endothelial cells with conditioned medium obtained from a population of MLPSCs.

[0275] composition The MLPSCs disclosed herein can be culture expanded from a cryopreserved intermediate to produce a preparation comprising at least one therapeutic dose.

[0276] In one example, a composition of the present disclosure comprises about 150 million cells.

[0277] In one example, the compositions of the present disclosure include a pharmaceutically acceptable carrier and / or excipient. The terms "carrier" and "excipient" refer to a composition of matter conventionally used in the art to facilitate the storage, administration, and / or biological activity of an active compound (see, e.g., Remington's Pharmaceutical Sciences, 16th Ed., Mac Publishing Company (1980)). A carrier may also reduce undesirable side effects of an active compound. A suitable carrier is, for example, stable and, for example, incapable of reacting with other ingredients in the carrier. In one example, a carrier does not cause significant local or systemic adverse effects in a recipient at dosages and concentrations used for therapy.

[0278] Suitable carriers for the present disclosure include those conventionally used, for example, water, saline, aqueous dextrose, lactose, Ringer's solution, buffers, hyaluronan, and glycols are exemplary liquid carriers, particularly for solutions (when isotonic). Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

[0279] In another example, the carrier is, for example, a medium composition in which cells are grown or suspended. Such a medium composition does not induce any adverse effects in the subject to which it is administered. Exemplary carriers and excipients do not adversely affect cell viability and / or the ability of the cells to treat or prevent disease.

[0280] In one example, the carrier or excipient provides buffering activity to maintain the cells and / or soluble factors at an appropriate pH, thereby exerting biological activity; for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it interacts minimally with the cells and factors, allowing for rapid release of the cells and factors; in such cases, the compositions of the present disclosure can be formulated as a liquid for direct application to the bloodstream or to tissues or areas surrounding or adjacent to tissues, e.g., by injection.

[0281] The composition of the present disclosure can be cryopreserved. Cryopreservation of MLPSCs can be carried out using slow cooling or "rapid" freezing protocols known in the art. Cryopreservation methods preferably maintain the phenotype, cell surface markers, and proliferation rate of cryopreserved cells comparable to those of unfrozen cells.

[0282] The cryopreservation composition may contain a cryopreservation solution, the pH of which is typically 6.5 to 8, preferably 7.4.

[0283] Cryopreservation solutions can include a sterile, nonpyrogenic, isotonic solution, such as PlasmaLyte ATM. 100 mL of PlasmaLyte ATM contains 526 mg of sodium chloride, USP (NaCl); 502 mg of sodium gluconate (C6H11NaO7); 368 mg of sodium acetate trihydrate, USP (C2H3NaO2·3H2O), 37 mg of potassium chloride, USP (KCl), and 30 mg of magnesium chloride, USP (MgCl2·6H2O). No antimicrobial agents are included. The pH is adjusted with sodium hydroxide. The pH is 7.4 (6.5-8.0).

[0284] The cryopreservation solution may include Profreeze™. The cryopreservation solution may additionally or alternatively include culture medium, such as αMEM.

[0285] To facilitate freezing, cryoprotectants, such as dimethyl sulfoxide (DMSO), are typically added to cryopreservation solutions. Ideally, cryoprotectants should be non-toxic to cells and patients, non-antigenic, and chemically inert, provide high post-thaw survival rates, and allow for irrigation-free transplantation. However, DMSO, the most commonly used cryoprotectant, exhibits some cytotoxicity. Hydroxyethyl starch (HES) can be used as an alternative or in combination with DMSO to reduce the cytotoxicity of cryopreservation solutions.

[0286] The cryopreservation solution may include one or more of DMSO, hydroxyethyl starch, human serum components, and other protein bulking agents. In one example, the cryopreservation solution includes Plasma-Lyte A (70%), DMSO (10%), and HSA (25%) solution, where the HSA solution includes 5% HSA and 15% buffer.

[0287] In one example, the cryopreservation solution may further comprise one or more of methylcellulose, polyvinylpyrrolidone (PVP), and trehalose.

[0288] The cryopreserved composition can be thawed and administered directly to a subject or added to another solution containing, for example, hyaluronic acid. Alternatively, the cryopreserved composition can be thawed prior to administration and the MLPSCs resuspended in another carrier.

[0289] The compositions described herein may be administered alone or as a mixture with other cells. Different types of cells may be mixed with the disclosed compositions immediately prior to or immediately before administration, or may be co-cultured together for a period of time prior to administration.

[0290] In one example, the composition comprises an effective amount, or a therapeutically or prophylactically effective amount, of MLPSCs and / or their progeny and / or soluble factors derived therefrom. For example, the composition comprises about 1x10 5 ~approx. 1x10 9 of stem cells, or approximately 1.25x10 3~Approx. 1.25x10 7 / kg (80 kg subject) of stem cells. The exact amount of cells administered will depend on a variety of factors, including the age, weight, and sex of the subject, and the extent and severity of the disorder being treated.

[0291] Regardless of the number of cells provided in the composition, in one example, 50 x 10 6 ~200×10 7 In another example, 60x10 cells are administered. 6 ~200x10 6 or 75x10 6 ~150x10 6 In one example, 75 x 10 cells are administered. 6 In another example, 150x10 cells are administered. 6 of cells are administered.

[0292] In one example, the composition is 5.00x10 6 In another example, the composition contains more than 5.50 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.00 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.50 x 10 viable cells / mL. 6 In another example, the composition contains more than 6.68 x 10 viable cells / mL. 6 Contains more than 1000 viable cells / mL.

[0293] In one example, the MLPSCs comprise at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the cell population of the composition.

[0294] In one example, the composition may be packaged in a suitable container, optionally accompanied by instructions for a desired purpose.

[0295] The compositions of the present disclosure may be administered systemically, such as by intravenous administration. In one example, the compositions are administered transendocardially.

[0296] In one example, a composition of the present disclosure comprises a "clinically proven effective" amount of MLPSCs. In one example, a composition of the present disclosure comprises a "clinically proven effective" amount of MSCs. In one example, a composition of the present disclosure comprises a "clinically proven effective" amount of MPCs.

[0297] In one example, a "clinically proven effective" amount of MLPSCs is administered as a total dose. The term "total dose," in the context of this disclosure, is used to refer to the total number of cells received by a subject treated according to this disclosure. In one example, the total dose consists of one administration of cells. In another example, the total dose consists of two administrations of cells. In another example, the total dose consists of three administrations of cells. In another example, the total dose consists of four or more administrations of cells. For example, the total dose can consist of two to four administrations of cells.

[0298] Pharmaceuticals and their manufacturing methods The inventors also surprisingly confirmed that MLPSCs cultured in the culture medium according to the methods disclosed herein exhibit / express increased levels of angiogenic markers. MLPSCs with increased expression levels of angiogenic markers, such as angiogenin, promote increased angiogenesis, as determined by increased endothelial network formation, endothelial network length, or endothelial branch length. Thus, the inventors have arrived at a novel population of culture-expanded MLPSCs and a conditioned medium that can be selected for their high angiogenic potential.

[0299] As used herein, "high angiogenic potential" refers to MLPSCs and conditioned medium obtained thereby that promote angiogenesis. As described above, increased angiogenesis can be determined by an increase in one or more of endothelial network formation, endothelial network length, or endothelial branch length. As identified by the present inventors, MLPSCs that increase angiogenesis express one or more angiogenic markers at specific levels. Thus, in one example, MLPSCs with high angiogenic potential according to the present disclosure express specific levels of angiogenin measured under culture conditions and / or induce one or more of endothelial network formation, endothelial network length, or endothelial branch length measured after treating a population of endothelial cells with conditioned medium obtained from the MLPSCs. In one example, MLPSCs with high angiogenic potential and / or conditioned medium obtained thereby have improved therapeutic effects and / or biological activities.

[0300] The level of angiogenic marker(s) can be measured according to the methods disclosed herein. For example, the level of angiogenin in MLPSC-conditioned medium can be measured by standard protein detection and / or gene expression methods known in the art. In one example, the level of angiogenin is measured by enzyme-linked immunosorbent assay (ELISA). In one example, the level of angiogenin is measured by Luminex assay. Endothelial network formation, endothelial network length, and / or endothelial branch length can be measured in an in vitro angiogenesis assay as described above.

[0301] In one example, the present disclosure provides a culture-expanded population of mesenchymal progenitor or stem cells (MLPSCs), where the population of MLPSCs is selected based on high angiogenic potential as determined by the level of angiogenin expressed by the MLPSCs under culture conditions. In one example, the MLPSCs are culture-expanded in a cell culture medium supplemented with at least one proinflammatory cytokine disclosed herein and / or a non-fetal serum, such as newborn bovine serum.

[0302] In one example, the present disclosure provides a culture-expanded population of MLPSCs, wherein the population of MLPSCs is selected based on high angiogenic potential as determined by one or more levels of endothelial network formation, endothelial network length, or endothelial branch length measured after treating a population of endothelial cells with conditioned medium obtained from the MLPSCs. In one example, the MLPSCs are culture-expanded in a cell culture medium containing at least one pro-inflammatory cytokine.

[0303] In another example, the present disclosure provides a conditioned medium selected based on high angiogenic potential as determined by one or more levels of endothelial network formation, endothelial network length, or endothelial branch length measured after treating an endothelial cell population with the conditioned medium. In one example, the conditioned medium is obtained by culturing and expanding a population of MLPSCs according to the methods disclosed herein.

[0304] In one example, a cultured expanded population and / or conditioned medium of MLPSCs selected based on their high angiogenic potential is selected for administration. In one example, such a population or conditioned medium can be referred to as a pharmaceutical composition or drug product (DP). In one example, the drug product comprises a composition disclosed herein. In one example, the drug product comprises MLPSCs. For example, a DP can be administered to 2x10 6 In one example, the culture expanded population is an intermediate population disclosed herein.

[0305] The present inventors have further identified a method for producing a pharmaceutical product by selecting a population of MLPSCs with high angiogenic potential. Thus, in one example, the present disclosure provides a method for producing a pharmaceutical product comprising a population of MLPSCs, the method comprising obtaining a determination of whether a test population of MLPSCs has a predetermined level of angiogenic potential under culture conditions, and treating at least a portion of the test population of MLPSCs as a pharmaceutical product if the test population of MLPSCs has at least the predetermined level of angiogenic potential under culture conditions, thereby producing the pharmaceutical product, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has angiogenic potential below the predetermined level under culture conditions, wherein the angiogenic potential is measured by a predetermined level of angiogenin measured under culture conditions.

[0306] In one example, the disclosure provides a method of producing a pharmaceutical product comprising a population of MLPSCs, the method comprising obtaining a determination of whether a test population of MLPSCs has a predetermined level of angiogenic potential under culture conditions, and treating at least a portion of the test population of MLPSCs as a pharmaceutical product, thereby producing the pharmaceutical product, if the test population of MLPSCs has at least the predetermined level of angiogenic potential under culture conditions, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has less than the predetermined level of angiogenic potential under culture conditions, wherein the angiogenic potential is -measured after treating endothelial cell populations with conditioned medium obtained from MLPSCs, o Formation of endothelial network, o The length of the endothelial network, and / or o As measured by one or more predetermined levels of endothelial branch length measured in an in vitro angiogenesis assay.

[0307] In one example, the test population is obtained from a 3D-cultured MLPSC population. For example, the MLPSCs may be in bioreactor culture. In one example, the test population is obtained from a cryopreserved population of MLPSCs. In one example, the test population is representative of a larger MLPSC population, such as a plurality of cryopreserved MLPSC populations. In one example, the plurality of cryopreserved MLPSC populations are culture-expanded from the same MLPSC intermediate population. In one example, the manufacturing method is applied to conditioned medium obtained from MLPSCs.

[0308] As used herein, the term "predetermined level" refers to a level of an angiogenic marker that indicates high angiogenic potential. In one example, the predetermined level is a level of angiogenin that indicates high angiogenic potential. In one example, the predetermined level is a level of angiogenin greater than about 1200 pg / ml. In one example, the predetermined level of angiogenin is an increase over a control population of MLPSCs cultured and grown in a cell culture medium containing 10% fetal bovine serum.

[0309] In one example, the predetermined level is a level of endothelial network formation that exhibits high angiogenic potential. In one example, the predetermined level of endothelial network formation is about 0.12 mm 2 / mm 2 In one example, the predetermined level of endothelial network formation is the increase relative to a control population of MLPSCs cultured and grown in cell culture medium containing 10% fetal bovine serum.

[0310] In one example, the predetermined level is a level of endothelial network length that exhibits high angiogenic potential. In one example, the predetermined level of endothelial network length is about 5 mm. 2 / mm 2 In one example, the predetermined level of endothelial network length is the increase relative to a control population of MLPSCs cultured and grown in cell culture medium containing 10% fetal bovine serum.

[0311] In one example, the predetermined level is a level of endothelial branch length that indicates high angiogenic potential. In one example, the predetermined level of endothelial branch length is about 15 1 / mm2 In one example, the predetermined level of endothelial branch length is the increase relative to a control population of MLPSCs cultured and grown in cell culture medium containing 10% fetal bovine serum.

[0312] In one example, the predetermined level is a level of angiogenin, endothelial network formation, endothelial network length, and / or endothelial branch length indicative of high angiogenic potential according to the parameters shown in Table A.

[0313] In one example, the predetermined level is a clinically proven effective predetermined level. In one example, the level is clinically proven to be effective in treating heart failure. In another example, the predetermined level is predetermined by a regulatory agency, such as the US Food and Drug Administration (FDA). In one example, reaching the predetermined level improves survival rates of heart failure patients. In one example, the predetermined level is a "baseline level of angiogenin." In one example, the base line level of angiogenin is the level of angiogenin in an FDA-approved MLPSC population (e.g., an FDA MLPSC population approved for treating heart failure). In one example, the base line level of angiogenin provides a criterion for selecting a cell population according to the present disclosure. In one example, the predetermined level is a base line level of one or more of endothelial network formation, endothelial network length, or endothelial branch length. In one example, the reference level of one or more of endothelial network formation, endothelial network length, or endothelial branch length is the level of one or more of endothelial network formation, endothelial network length, or endothelial branch length of an FDA-approved MLPSC population (e.g., an FDA MLPSC population approved for the treatment of heart failure). In one example, the reference level of one or more of endothelial network formation, endothelial network length, or endothelial branch length provides a basis for selecting a cell population according to the present disclosure.

[0314] In one example, the present disclosure provides a method of producing an MSC medicine, such method including a first step of providing (e.g., culturing (e.g., small-scale or large-scale cell culture) or manufacturing) or acquiring (e.g., from a third party (including a contractually related third party or a contractually unrelated (e.g., independent) third party) a test MLPSC population (e.g., a sample of the test MLPSC population); a second step of obtaining (e.g., detecting, measuring, receiving, or acquiring) a value of at least one MLPSC parameter listed in Table A for the test MLPSC population; and determining whether at least a portion of the test MLPSC population (e.g., a manufacturing lot, culture, or run) meets the reference standard listed in Table A for the parameter. and a third step of processing a portion, an entire manufacturing lot, culture, or run, or multiple manufacturing lots, cultures, or runs) as an MLPSC product (e.g., in a form or packaging intended for administration as described herein below, optionally cryopreserved), thereby producing the MSC product. In one example, the value(s) include parameter number 1. In another example, the value(s) include parameter number 2. In another example, the value(s) include parameter number 3. In another example, the value(s) include parameter number 4. In another example, the value(s) include parameter numbers 1 and 2. In another example, the value(s) include parameter numbers 1 and 3. In another example, the value(s) include parameter numbers 1, 2, and 4.

[0315] In one example, such a method includes a second step of obtaining values ​​for any combination of two or more MLPSC parameters listed in Table A, and a third step of treating at least a portion of the test MLPSC population as an MLPSC pharmaceutical if the values ​​for any combination of two or more MLPSC parameters satisfy the corresponding reference standards for the parameters listed in Table A. [Table 2]

[0316] A population of MLPSCs with high angiogenic potential can be cultured and expanded in a cell culture medium containing at least one pro-inflammatory cytokine according to the methods disclosed herein. For example, MLPSCs are cultured and expanded in a cell culture medium containing non-fetal serum, such as newborn serum (e.g., newborn bovine serum). In one example, MLPSCs are cultured and expanded in a culture medium containing about 5% non-fetal serum and about 5% fetal serum. In one example, MLPSCs are cultured and expanded in a medium containing less than 10% fetal bovine serum. In one example, MLPSCs are cultured and expanded in a xeno-free culture medium supplemented with one or more pro-inflammatory cytokines disclosed herein. As used herein, "xeno-free" refers to a culture medium that contains only human-derived components and does not contain components derived from animals other than humans. In one example, the xeno-free culture medium contains human serum. In one example, the xeno-free culture medium is serum-free.

[0317] Treatment method The disclosed methods relate to treating progressive heart failure in a subject and include administering to the subject a composition comprising the MLPSC population disclosed herein and / or conditioned medium obtained thereby. Thus, in one example, the disclosed methods include administering culture-expanded MLPSCs. In another example, the disclosed methods include administering conditioned medium or soluble factors obtained thereby.

[0318] Cardiomyopathy is a disease of the myocardium that makes it difficult for the heart to pump blood to the rest of the body. Heart failure can occur when the heart cannot pump enough to maintain blood flow to meet the body's demands. Cardiomyopathy can occur after either an ischemic or non-ischemic event. One cause of ischemic heart failure is systolic dysfunction following a myocardial infarction (MI). An MI occurs when blood does not flow properly to a part of the heart. The lack of blood supply leads to localized myocardial necrosis, called myocardial infarction. The infarcted heart cannot pump enough blood to maintain blood flow to meet the body's needs, triggering various pathophysiological responses and ultimately leading to heart failure. Non-ischemic cardiomyopathy is not associated with known coronary artery disease. One example is dilated cardiomyopathy (DCM), a condition in which the left ventricle, the heart's main pumping chamber, becomes enlarged, dilated, and weakened, reducing the heart's ability to pump blood.

[0319] When the heart is no longer able to pump sufficiently to maintain blood flow to meet the body's demands, a series of compensatory mechanisms are initiated, contributing to the mitigation of the decline in cardiac output and helping to maintain sufficient blood pressure to perfuse vital organs. As a result, patients with heart failure may not progress over time. However, the compensatory mechanisms eventually fail to compensate for the damaged heart, resulting in a progressive decline in cardiac output referred to as "progressive heart failure." In the context of this disclosure, the terms chronic heart failure, congestive heart failure, congestive cardiac failure, systolic dysfunction, and progressive heart failure can be used interchangeably with "progressive heart failure."

[0320] The method of the present disclosure can be used to treat progressive heart failure in a specific group of MI patients.The subjects who need treatment include not only those who already have progressive heart failure, but also those who should be prevented, delayed or stopped from progressive heart failure.In these examples, subject can have the progressive heart failure of II or III according to NYHA.For example, subject can have the progressive heart failure of II degree.

[0321] In a first example, the present disclosure relates to the treatment of a subject defined based on the New York Heart Association (NYHA) classification scale. In one example, the subject's advanced heart failure is less than grade III. In one example, the subject has grade II heart failure. In one example, the NYHA classification is assigned based on the subject's symptoms. For example, the NYHA classification can be assigned based on the following table: [Table 3]

[0322] In one example, the subject's heart failure is caused by an ischemic event. In one example, the subject's heart failure is caused by myocardial infarction (MI). For example, the subject can be an MI subject. The term "myocardial infarction (MI) subject" is used to define a subject who has had myocardial infarction. In one example, the subject's heart failure is caused by non-ischemic cardiomyopathy.

[0323] In one example, the present disclosure relates to treating patients with advanced heart failure and persistent inflammation. "Persistent inflammation" is defined by elevated C-reactive protein levels. In one example, persistent inflammation is characterized by a CRP level of ≧2 mg / L. Thus, in this example, the present disclosure relates to treating subjects with advanced heart failure with a CRP level of ≧2 mg / L. In one example, these subjects may have advanced heart failure of Class II or Class III according to the NYHA classification. In another example, these subjects have microvascular disease and / or macrovascular disease. For example, these subjects may have ischemia and / or diabetes. Thus, in one example, the subject may have advanced heart failure, a CRP level of ≧2 mg / L, advanced stage II or III heart failure, and microvascular disease and / or macrovascular disease. In another example, the subject may have advanced heart failure, a CRP level of ≧2 mg / L, advanced stage II or III heart failure, and ischemia and / or diabetes. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or greater, advanced class II or III heart failure, and ischemia. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or greater, advanced class II or III heart failure, and diabetes. In another example, the subject may have advanced heart failure, a CRP level of 2 mg / L or greater, advanced class II heart failure, and ischemia. In another example, the subject may have advanced heart failure, a CRP level of ≥ 2 mg / L, advanced class II heart failure, and diabetes.

[0324] In a third example, the present disclosure relates to treating patients with advanced heart failure and microvascular and / or macrovascular disease. "Microvascular disease" (sometimes called small artery disease or small vessel disease) is a heart disease that affects the walls and intima of small coronary vessels that branch off from larger coronary arteries. In coronary MVD, the coronary vessels of the heart do not necessarily have plaque, but rather may have damage to the inner walls of the vessels, which can lead to spasms and reduced blood flow to the heart muscle. In one example, microvascular disease is "myocardial ischemia," a condition characterized by obstruction of blood flow to the myocardium (myocardium) due to partial or complete blockage of a coronary artery. Examples of myocardial ischemia include ischemic heart failure, angina pectoris, and stroke. "Macrovascular disease" is characterized by the process of atherosclerosis, which leads to narrowing of the arterial walls of the coronary vasculature. Atherosclerosis is believed to result from chronic inflammation in the coronary vasculature and damage to the arterial wall(s). Diabetes dramatically accelerates atherosclerosis by causing inflammation and slowing blood flow, and thus represents an example of macrovascular disease. In one example, diabetes is type I diabetes or type II diabetes. In one example, diabetes is type II diabetes.

[0325] Thus, in one example, the subject may have progressive heart failure and microvascular disease and / or macrovascular disease. In another example, the subject may have progressive heart failure and ischemia and / or diabetes. In another example, the subject may have progressive heart failure and ischemia. In another example, the subject may have progressive heart failure and diabetes. In these examples, the subject may also have persistent inflammation. For example, the subject may have progressive heart failure, a CRP level of ≧2 mg / L, and microvascular disease and / or macrovascular disease. In another example, the subject may have progressive heart failure, a CRP level of ≧2 mg / L, and ischemia and / or diabetes. In another example, the subject may have progressive heart failure, a CRP level of ≧2 mg / L, and ischemia. In another example, the subject may have progressive heart failure, a CRP level of ≧2 mg / L, and ischemia. In another example, the subject may have progressive heart failure, a CRP level of ≧2 mg / L, and diabetes.

[0326] The subjects in the first, second, and third examples above can be further characterized as follows: In one example, a subject treated according to the present disclosure has an initial CRP level of 2 mg / L or greater. For example, the subject may have stage II or III heart failure and an initial CRP level of 2 mg / L or greater. In another example, the subject may have stage II heart failure and an initial CRP level of 2 mg / L or greater. In one example, a subject treated according to the present disclosure has an initial CRP level of less than 5 mg / L. In another example, the subject has an initial CRP level of less than 4 mg / L. In another example, the subject has an initial CRP level of 2-6 mg / L. In another example, the subject has an initial CRP level of 3-6 mg / L. In another example, the subject has an initial CRP level of 4-5 mg / L.

[0327] There are various assays available for measuring CRP levels, such as antibody-based immunoassays. For example, CRP levels can be measured in blood samples using an enzyme-linked immunosorbent (ELISA) assay. In one example, a blood sample is obtained from a patient and purified before contacting with an anti-CRP antibody. The degree of antibody binding is used to quantify the level of CRP in the blood sample (e.g., mg / L).

[0328] B-type natriuretic peptide (BNP) is a hormone produced by the heart. N-terminal (NT) prohormone BNP (NT-proBNP) is an inactive prohormone released from the same molecule that produces BNP. Both BNP and NT-proBNP are released in response to changes in pressure within the heart. These changes can be associated with heart failure and other cardiac problems. Levels increase when heart failure develops or worsens, and decrease when heart failure stabilizes. Therefore, BNP is an effective marker of heart failure progression. In one example, the subject's NT-proBNP level before administering a composition of the present disclosure is less than 2500 pg / ml. In another example, the subject's NT-proBNP level before administering a composition of the present disclosure is less than 2400 pg / ml. In another example, the subject's NT-proBNP level before administering a composition of the present disclosure is less than 2000 pg / ml. In another example, the subject's NT-proBNP level before administration of a composition of the present disclosure is less than 1900 pg / ml. In another example, the subject's NT-proBNP level before administration of a composition of the present disclosure is between 2200 pg / ml and 1000 pg / ml. In another example, the subject's NT-proBNP level before administration of a composition of the present disclosure is between 2200 pg / ml and 1100 pg / ml. In another example, the subject's NT-proBNP level before administration of a composition of the present disclosure is between 2100 pg / ml and 1200 pg / ml. In another example, the subject's NT-proBNP level before administration of a composition of the present disclosure is between 2000 pg / ml and 1500 pg / ml.

[0329] In one example, the subject has a level of CRP above 2 mg / ml and NT-proBNP above 1000 ng / ml.

[0330] In another example, the subject has experienced a hospitalization due to heart failure within the past 12 months prior to administration of a composition disclosed herein. In another example, prior to administration of a composition disclosed herein, the subject has had a heart failure hospitalization event over the past 9 months. In another example, prior to administration of a composition disclosed herein, the subject has had a heart failure hospitalization event over the past 6-12 months. In one example, a heart failure hospitalization indicates worsening signs and symptoms of heart failure. In another example, a heart failure hospitalization event is an ischemic event. In another example, a heart failure hospitalization event is a non-ischemic event.

[0331] In another example, the subject can walk at least 320 meters in 6 minutes before administering a composition of the present disclosure. In another example, the subject can walk at least 330 meters in 6 minutes before administering a composition of the present disclosure. In another example, the subject can walk at least 340 meters in 6 minutes before administering a composition of the present disclosure. In another example, the subject can walk at least 350 meters in 6 minutes before administering a composition of the present disclosure.

[0332] In one example, the subject may have persistent left ventricular dysfunction. Left ventricular dysfunction is characterized by a decrease in myocardial contractility. Decreased myocardial contractility in the left ventricle results in a decrease in left ventricular ejection fraction (LVEF). Therefore, LVEF provides one way to determine left ventricular dysfunction. Another parameter of left ventricular function is left ventricular end-systolic volume (LVESV), which is a measure of the adequacy of cardiac ejection related to systolic function. Another parameter of left ventricular function is left ventricular end-diastolic function (LVEDV), which is a measure of the adequacy of ventricular filling during diastole (i.e., the blood volume in the ventricle just before systole). LVEF and LVESV are often used together to evaluate left ventricular systolic function and characterize persistent left ventricular dysfunction.

[0333] LVEF, LVESV, and LVEDV can be measured by several methods known in the art, such as echocardiogram (e.g., 2-dimensional echocardiogram), single-photon emission computed tomography (SPECT), cardiac magnetic resonance imaging (cMRI), or multi-gated acquisition scan.

[0334] In one example, a subject with an LVEF of less than 45% has left ventricular dysfunction. In another example, a subject with an LVEF of less than about 44%, 43%, 42%, or 41% has left ventricular dysfunction. In another example, a subject with an LVEF of less than about 40% has left ventricular dysfunction. In another example, a subject with an LVEF of less than about 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30% has left ventricular dysfunction.

[0335] In the context of this disclosure, the term "persistent left ventricular dysfunction" is used to define left ventricular dysfunction that persists over a period of time or series of measurements. For example, "persistent left ventricular dysfunction" can include left ventricular dysfunction that persists for about 1 day to about 14 days or longer.

[0336] In one example, the subject has an LVEF of less than 45%, in another example, the subject has an LVEF of less than 40%, or in other examples, the subject has an LVEF of less than 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30%.

[0337] In one example, the subject's LVESV is greater than 70 ml. In another example, the subject's LVESV is greater than 100 ml. In another example, the subject's LVESV is greater than 130 ml. In another example, the subject's LVESV is between 70 ml and 160 ml. In these examples, the subject may also have an LVEF as described above. For example, the subject may have an LVESV of greater than 70 ml and an LVEF of less than 45%.

[0338] In one example, the subject's heart failure results from an ischemic event or a non-ischemic event. In one example, the subject's heart failure results from an ischemic event as disclosed below.

[0339] The methods of the present disclosure relate to treating the progressive decline in cardiac output that is characteristic of advanced heart failure. Accordingly, "treating" and "treatment" in the context of this disclosure refer to both therapeutic treatment and prophylactic or preventative measures.

[0340] In one example, the treatment comprises administering a composition of the present disclosure. In one example, the method of the present disclosure reduces or inhibits the progression of advanced heart failure. In one example, the treatment improves the subject's left ventricular function. In one example, the treatment improves the subject's LVEF. In one example, the treatment improves the subject's LVEF by at least 1 percentage point (i.e., a subject with an LVEF of 35% before treatment improves their LVEF to 36% after treatment). In one example, the treatment improves the subject's LVEF by 2-10 percentage points, or 5-7 percentage points. In one example, the treatment improves the subject's LVEF by 4-7 percentage points, or 5-7 percentage points. In one example, the treatment improves the subject's LVEF by 5-7 percentage points, or 5-7 percentage points.

[0341] In one example, the treatment improves the subject's LVESV. In one example, the treatment improves the subject's LVEDV by at least 15 ml. In one example, the treatment improves the subject's LVESV by at least 17 ml. In one example, the treatment improves the subject's LVESV by at least 20 ml. In one example, the treatment improves the subject's LVESV by 15 ml to 30 ml. In one example, the treatment improves the subject's LVESV by 15 ml to 30 ml.

[0342] In one example, treatment improves the subject's LVEDV. In one example, treatment improves the subject's LVEDV by at least 15 ml. In one example, treatment improves the subject's LVEDV by 15 ml to 25 ml.

[0343] In one example, the treatment inhibits the subject's progression to NYHA Class III advanced heart failure. In another example, the treatment reduces the risk of cardiac death. In one example, the reduced risk of cardiac death is compared to the risk of cardiac death in a patient with NYHA Class III advanced heart failure. In one example, the reduced risk of cardiac death is compared to the risk of cardiac death in a subject not receiving MLPSCs. In one example, the reduced risk of cardiac death is compared to the risk of cardiac death in a subject with NYHA Class III advanced heart failure not receiving MLPSCs. In one example, the treatment reduces the risk of cardiac death by at least 20%. In one example, the treatment reduces the risk of cardiac death by at least 30%. In one example, the treatment reduces the risk of cardiac death by at least 40%. In one example, the treatment reduces the risk of cardiac death by at least 50%. In one example, the treatment reduces the risk of cardiac death by 35% to 45%. In one example, the treatment reduces the risk of cardiac death by 40% to 45%.

[0344] In another example, after treatment, the risk of ischemic MACE (MI or stroke) is reduced. In one example, the risk of ischemic MACE (MI or stroke) is reduced by at least 50% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 55% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 60% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 65% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 70% compared to baseline. In another example, the risk of ischemic MACE (MI or stroke) is reduced by at least 50%-70% compared to baseline.

[0345] In another example, the risk of 3-point MACE (cardiac death / MI / stroke) is reduced after treatment. In the context of this disclosure, "3-point MACE" is used to refer to a combination of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke (cardiac death / MI / stroke), defined as a composite of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke (cardiac death / MI / stroke). In one example, the risk of 3-point MACE is reduced by at least 30% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 40% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 45% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 50% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 55% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 30%-50% compared to baseline. In another example, the risk of 3-point MACE is reduced by at least 45%-55% compared to baseline.

[0346] In one example, the risk reduction is a 3-year risk reduction. In another example, the risk reduction is a 5-year risk reduction. In these examples, the risk of an ischemic event is reduced over a period of time.

[0347] In one example, the treatment increases the survival rate of the patient. In one example, the treatment increases the probability that the subject will survive for at least 1000 days after the start of treatment. In another example, the treatment increases the probability that the subject will survive for at least 2000 days after the start of treatment. In one example, the increased probability is determined compared to a subject not treated with the composition of the present disclosure. In one example, the improved probability is determined compared to a subject with grade III heart failure.

[0348] In one example, treatment reduces the likelihood or risk of heart failure-related major adverse cardiovascular events (HF-MACE), defined as a composite of cardiac-related death or resuscitated cardiac death, or non-fatal decompensated heart failure events. In one example, the likelihood or risk of HF-MACE is reduced for at least 6 months, at least 12 months, at least 24 months, or at least 36 months after administration of a composition disclosed herein. In one example, treatment reduces the likelihood or risk of all-cause mortality.

[0349] The present inventors have also surprisingly discovered that heart failure patients with left ventricular assist devices (LVADs) and experiencing heart failure due to an ischemic event also exhibit persistent inflammation. As disclosed herein, MLPSC compositions according to the present disclosure are particularly effective in treating heart failure patients with persistent inflammation.

[0350] An LVAD is a mechanical circulatory assist device that can be implanted in patients with end-stage heart failure. Patients can undergo LVAD implantation as bridge-to-transplant (BTT) therapy or as definitive therapy (DT) for subjects who are not eligible for transplant. Those skilled in the art will be aware of various LVAD models, including, but not limited to, HeartMate I, HeartMate II, HeartMate 3, and HeartWare.

[0351] Therefore, in another example, the heart failure patient with persistent inflammation treated according to the method of the present disclosure has an LVAD. For example, the subject can have an LVAD and heart failure caused by an ischemic event. In these examples, the patient with heart failure caused by an ischemic event and an LVAD shows persistent inflammation, which can be characterized based on the serum level of specific biomarker(s) as needed.

[0352] For example, in the context of LVAD patients, persistent inflammation can be characterized based on plasma levels of IL-6. Thus, LVAD patients treated according to the present disclosure may have increased IL-6 levels compared to baseline. In one example, the subject's IL-6 level is increased compared to baseline at least 30 days after LVAD implantation. In one example, the subject's IL-6 level is increased compared to baseline at least 60 days after LVAD implantation. In one example, the subject's IL-6 level is increased compared to baseline between 30 and 365 days after LVAD implantation.

[0353] In one example, LVAD patients who develop heart failure due to an ischemic event have a higher risk of all-cause mortality compared to LVAD-implanted patients who develop heart failure due to a non-ischemic event. As used herein, "all-cause mortality" (also referred to as "total mortality") is a measure of the total number of deaths from all causes. In one example, a subject's risk of all-cause mortality is approximately 30% higher than a subject implanted with an LVAD who develops heart failure due to a non-ischemic event. In one example, treating an LVAD subject with MLPSCs in accordance with the present disclosure reduces the subject's risk of all-cause mortality. In one example, the subject's risk of all-cause mortality is reduced by 10% to 90%. In one example, the subject's risk of all-cause mortality is reduced by more than about 50%. In one example, the subject's risk of all-cause mortality is reduced by more than about 80%. In one example, the subject's risk of all-cause mortality is reduced by about 80%.

[0354] Ischemic events In one example, the present disclosure relates to a method for reducing the risk or incidence of an ischemic event in a subject, particularly a subject with cardiomyopathy. In one example, the present disclosure relates to a method for reducing the risk or incidence of an ischemic event in a subject with cardiomyopathy and elevated CRP. In one example, the risk or incidence is reduced compared to a subject not administered a composition of the present disclosure. For example, the risk or incidence can be reduced compared to an untreated subject. In one example, the ischemic event is caused by the formation of an occlusion. In one example, the occlusion is an arterial occlusion. In one example, the ischemic event is the formation of a cerebrovascular occlusion. In another example, the ischemic event is the formation of a cardiac occlusion. For example, the occlusion can occur in a coronary artery.

[0355] Examples of ischemic events caused by the formation of a blockage include heart attack and stroke. Thus, in one example, the present disclosure relates to a method of reducing the risk or incidence of heart attack or stroke in a subject with cardiomyopathy.

[0356] The risk or incidence of ischemic events in subjects with cardiomyopathy is reduced by administering a cell therapy, such as a composition of the present disclosure.

[0357] In one example, the ischemic event is non-fatal. In one example, the ischemic event is fatal, and in this example, the method of the present disclosure reduces the risk of cardiac death due to the ischemic event. Thus, in one example, the method of the present disclosure encompasses a method of reducing the risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering to the subject a composition comprising MLPSCs. In one example, the subject: - Class II heart failure - Microvascular and / or macrovascular disease, - Have one or more or all of the following persistent inflammation:

[0358] For example, the disclosure encompasses a method of reducing the risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering to a subject with class II heart failure a composition comprising MLPSCs.

[0359] In another example, the disclosure includes a method for reducing the risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering a composition comprising MLPSCs to the subject, wherein the subject has microvascular disease and / or macrovascular disease.

[0360] In another example, the disclosure encompasses a method of reducing the risk of cardiac death or a non-fatal ischemic event in a subject, the method comprising administering to the subject a composition comprising MLPSCs, the subject having persistent inflammation.

[0361] In one example, the subject has non-ischemic cardiomyopathy. For example, the subject's cardiomyopathy may be caused by an enlarged left ventricle (dilated cardiomyopathy). In another example, the cardiomyopathy is caused by a viral infection.

[0362] In another example, the subject has grade II or III heart failure according to the New York Heart Association (NYHA) classification scale.

[0363] In another example, the subject's N-terminal pro-B-type natriuretic peptide (NT-proBNP) level prior to administration of the cells is between 1000 pg / ml and 2000 pg / ml. In another example, the subject has elevated C-reactive protein (CRP) levels. In another example, the subject's CRP level is 1.5 mg / L or greater. In another example, the subject's CRP level is 2 mg / L or greater. In another example, the subject's CRP level is between 1 and 5 mg / L. In another example, the subject's CRP level is between 3 and 5 mg / L.

[0364] In one example, the cells are administered transendocardially.

[0365] In one example, the risk reduction is a 3-year risk reduction. In another example, the risk reduction is a 5-year risk reduction. In these examples, the risk of an ischemic event is reduced over a period of time.

[0366] Selection of treatment targets In one example, the method of the present disclosure relates to a method of selecting a subject at an elevated risk of persistent inflammation and / or cardiac death for treatment with a stem cell composition according to the present disclosure. In one example, a CRP level of ≧2 mg / L indicates persistent inflammation and an elevated risk of cardiac death, myocardial infarction, or stroke. In one example, the CRP level is measured after an ischemic event. In one example, the ischemic event is a myocardial infarction.

[0367] Thus, in one example, the present disclosure relates to a method for treating progressive heart failure, the method comprising the steps of: i) selecting a subject for treatment having a CRP level of ≧2 mg / L; and ii) administering to the subject a composition comprising MLPSCs, wherein the MLPSCs are cultured and expanded in a cell culture medium comprising non-fetal serum, and the subject has persistent inflammation.

[0368] In another example, the method includes the steps of: i) selecting a subject with progressive heart failure for treatment, wherein the subject has microvascular disease and / or macrovascular disease; and ii) administering to the subject a composition comprising MLPSCs cultured and expanded in a cell culture medium comprising non-fetal serum, wherein the subject has persistent inflammation.

[0369] In another example, the method includes the steps of: i) selecting a subject with advanced heart failure for treatment, wherein the subject is implanted with an LVAD; and ii) administering to the subject a composition comprising MLPSCs cultured and expanded in a cell culture medium containing non-fetal serum, wherein the subject has persistent inflammation.

[0370] In another example, the method includes steps of: i) selecting a subject with advanced heart failure for treatment, wherein the subject has a CRP level of >2 mg / ml and an NTpro-BNP level of >1000 ng / ml; and ii) administering to the subject a composition comprising MLPSCs cultured and expanded in a cell culture medium containing non-fetal serum, wherein the subject has persistent inflammation.

[0371] In another example, the method includes the steps of: i) selecting for treatment a subject with advanced heart failure, wherein the subject has microvascular disease and / or macrovascular disease; and ii) administering to the subject a conditioned medium or a population of exosomes obtained thereby, wherein the conditioned medium is obtained by culturing and expanding a population of MLPSCs in a cell culture medium containing non-fetal serum, and the subject has persistent inflammation.

[0372] In another example, the method includes the steps of: i) selecting a subject for treatment having advanced heart failure, wherein the subject is implanted with an LVAD; and ii) administering to the subject a conditioned medium or a population of exosomes obtained thereby, wherein the conditioned medium is obtained by culturing and expanding a population of MLPSCs in a cell culture medium containing non-fetal serum, and the subject has persistent inflammation.

[0373] In another example, the method includes the steps of: i) selecting a subject with advanced heart failure for treatment, wherein the subject has a CRP level >2 mg / ml and an NTpro-BNP level >1000 ng / ml; and ii) administering to the subject a conditioned medium or a population of exosomes obtained thereby, wherein the conditioned medium is obtained by culturing and expanding a population of MLPSCs in a cell culture medium containing non-fetal serum. [Example]

[0374] 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 (also known as fetal calf serum) (Serum B). These MLPSCs were used in Examples 4-7.

[0375] 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.

[0376] 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.

[0377] Example 2: MLPSC composition obtained using culture medium supplemented with 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).

[0378] Eagle's αMEM medium, suitable for culturing primary stem cells, can be obtained from a variety of sources, including Life Technologies and Sigma.

[0379] 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.

[0380] 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.

[0381] 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 2). The neonatal serum used was neonatal bovine serum (NBCS). NBCS meets the specifications of standard fetal bovine serum but is 100% bovine serum obtained from animals less than 20 days old.

[0382] 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 4]

[0383] Example 4: Culture and proliferation of MLPSCs in a 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.

[0384] 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.

[0385] 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.

[0386] 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 cultured with normal human dermal fibroblasts (NHDFs), seeded into 96-well plates, and simultaneously incubated and imaged using an IncuCyte® live cell analysis system. This system allowed for the determination of angiogenesis by 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.

[0387] Results: We found that conditioned medium from MPCs cultured in medium supplemented with neonatal bovine serum (NEBS) increased angiogenesis. As shown in Figure 2, conditioned medium from MPCs cultured in 5% NBCS / 5% FCS increased network area (Figure 2A), network length (Figure 2B), and branch points (Figure 2C) in the coculture angiogenesis model. Conditioned medium from MPCs cultured in 5% NBCS / 5% FCS also increased angiogenin levels compared to 10% FCS (Figure 3). Figure 4 shows further analysis of the levels of 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 indicate that both VEGF and SDF-1α are elevated in neonatal serum-cultured MPCs.

[0388] Considering the data provided in Example 1, these data indicate that the expansion of MLPSCs in culture 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, for example, 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.

[0389] Without wishing to be bound by any particular theory, these data suggest a potential mechanism by which MPCs cultured in neonatal serum may improve therapeutic efficacy: promoting angiogenesis and increasing production of the pro-angiogenic growth factors VEGF, SDF-1α, and angiogenin. Furthermore, these data provide the basis for methods of selecting cells with sufficient efficacy for treating inflammatory diseases. Notably, the data demonstrate threshold levels of approximately 3.45 ng / mL VEGF, 3000 ng / mL SDF-1α, or 1114 pg / mL angiogenin, indicating that concentrations above these amounts are therapeutically effective and demonstrate increased biological activity of MPCs. For example, cells can be cultured according to the methods disclosed herein, and conditioned medium can be harvested and assayed in angiogenesis assays and / or for VEGF and angiogenin levels. Cells producing VEGF / angiogenin levels above the threshold are considered therapeutically effective and biologically active. Similarly, cells with a network area of ​​approximately >0.12 mm2 can be cultured in vitro. 2 / mm 2 , network length >5mm 2 / mm 2 , and / or branch points >15 1 / mm 2 Cells that produce conditioned medium that promotes angiogenesis, as determined by their ability to stimulate angiogenesis, are considered therapeutically potent / biologically active for the treatment of diseases with an inflammatory component, such as heart failure in patients with persistent inflammation.

[0390] Example 5. Selection of MPC populations with high angiogenic potential Introduction: The results of Example 4 demonstrate that MPCs with high angiogenic potential can be selected based on (i) the levels of growth factors expressed in MPC-conditioned medium, and / or (ii) the levels of network area, network length, and branch length induced in endothelial cells treated with conditioned medium obtained from MPCs.

[0391] Methods: MPCs were cultured in 10% FCS, 5% NBCS / 5% FCS, or xeno-free medium to generate MPC-conditioned medium (CM). In Example 4, angiogenin, VEGF, and Ang1 levels in CM were measured using Luminex (R&D Systems). Endothelial network area, network length, and branch length were measured using the angiogenesis assay described in Example 4 above and shown in Figure 2.

[0392] Results: Figure 3 and Table 3 show that MPCs cultured in both 5% NBCS / 5% FCS and xeno-free medium had elevated angiogenin levels. The mean angiogenin level for MPCs cultured in 10% FCS was approximately 695 pg / ml, whereas the mean angiogenin levels for MPCs cultured in 5% NBCS / 5% FCS and xeno-free medium were approximately 2010 pg / ml and 1372 pg / ml, respectively (total mean = 1691 pg / ml). This represents an approximately two-fold increase in angiogenin levels compared to MPCs cultured in 10% FCS. [Table 5]

[0393] Figure 2 shows that conditioned medium from the same cells expressing high levels of angiogenin also induces increases in endothelial cell network area, network length, and branch length. Conditioned medium from MPCs (cultured in 5% NBCS / 5% FCS) with mean angiogenin levels of approximately 2010 pg / ml increased network formation (measured by network area) (Figure 2A), network length (Figure 2B), and branch points (Figure 2C), as measured in the angiogenesis assay described in Example 4. Network area, network length, and branch points are increased compared to MPCs cultured in 10% FBS (mean angiogenin level approximately 695 pg / ml). Cells cultured in xeno-free medium (mean angiogenin level approximately 1372 pg / ml) also showed increased network area (Figure 6A), network length (Figure 6B), and branch length (Figure 6C) compared to MPCs cultured in 10% FCS (mean angiogenin level approximately 695 pg / ml). The estimated raw values ​​for network area, network length, and branch length are shown in Table 4. [Table 6-1] [Table 6-2]

[0394] Without wishing to be bound by any particular theory, these data indicate that angiogenin levels can be used to identify MPCs with high angiogenic potential. For example, MPCs expressing angiogenin levels above a threshold of approximately 1200 pg / ml have high angiogenic potential, as demonstrated by their ability to induce increases in network area, network length, and / or branch length in angiogenesis assays. Thus, the data provide a basis for obtaining novel compositions by selecting MPCs based on their high angiogenic potential as determined by angiogenin levels.

[0395] These data also provide a basis for obtaining novel compositions by selecting MPCs based on their high angiogenic potential as determined by one or more levels of endothelial network formation, endothelial network length, or endothelial branch length measured after treating an endothelial cell population with conditioned medium obtained from MPCs. For example, if conditioned medium collected from MPCs (i) produces endothelial cells with an angiogenic potential of approximately 0.12 mm 2 / mm 2 (ii) formation of a network of approximately 5 mm 2 / mm 2 and / or (iii) a network length of about 15 1 / mm 2 If MPCs can induce a threshold level of branch length above 100 μm, it indicates that they have high angiogenic potential.

[0396] Our findings also show that cells cultured in non-fetal serum or xeno-free medium produce cells with enhanced angiogenic potential (i.e., increased angiogenin, network formation, network length, and / or branch length) compared to cells cultured in medium containing 10% FCS. Given that 10% FCS is the amount of serum typically used in standard cell culture conditions, cells cultured in 10% FCS are considered an appropriate control for determining angiogenic potential.

[0397] Therefore, our findings also provide a basis for selecting cells with high angiogenic potential, as determined relative to MLPSCs cultured and grown in control medium containing 10% FCS. Furthermore, our findings support the broad application of high-potency MPC populations, i.e., populations of MPCs cultured and grown in cell culture medium containing at least one pro-inflammatory cytokine and selected on the basis of their high angiogenic potential.

[0398] Example 6. Evaluation of MPCs cultured with non-fetal and fetal serum: Therapeutic effects 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.

[0399] 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.

[0400] 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). HFrEF patients received (1) MPCs cultured in 10% fetal serum (n=37), (2) MPCs cultured in medium supplemented with newborn 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 newborn 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.

[0401] Results: MPCs cultured in medium supplemented with neonatal calf 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).

[0402] 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 medium supplemented with newborn calf serum (5% FCS / 5% NBCS) on LV systolic function recovery was more pronounced. In contrast, no significant improvement was observed with MPCs cultured in 10% FBS (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). The effect of MPCs cultured in 10% fetal bovine serum or 5% / FCS / 5% NBCS on LV systolic function in HFrEF patients without elevated baseline inflammation (HFrEF patients with CRP < 2) is shown in Figure 7 .

[0403] MPCs cultured in medium supplemented with neonatal serum reduced the incidence of the three-point MACE (cardiovascular death, MI, or stroke) in all patients (Figure 8). MPCs cultured in medium supplemented with neonatal serum also reduced other cardiac-related outcomes in HFrEF patients with CRP > 2, reducing the risk of cardiovascular death by 43% (Figure 9) and the incidence of the three-point MACE (cardiovascular death, MI, or stroke) by 54% (Figure 10). Notably, MPCs cultured in medium supplemented with neonatal bovine serum (5% FCS / 5% NBCS) significantly reduced CV death (Figure 11A) and TCE (Figure 11B) in the highest-risk patients (CRP > 2 mg / ml, NTpro-BNP > 1000 ng / ml).

[0404] These data indicate that MPCs cultured in medium supplemented with neonatal serum improve therapeutic efficacy, particularly in the context of persistent inflammation.

[0405] Summary: MPCs grown in media 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.

[0406] 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.

[0407] Without wishing to be bound by any particular theory, these data suggest that culturing in NBCS, particularly NBCS obtained from animals less than 20 days old, prequalifies MPCs to effectively respond to an inflammatory environment, and that this ability to respond is influenced by (or related to) increased levels of angiogenin. Indeed, angiogenin levels may be a useful assay for measuring the efficacy of treatments for conditions involving persistent inflammation, such as heart failure. While our findings demonstrate prequalification using non-fetal serum, it is likely that one or more pro-inflammatory cytokines in non-fetal serum facilitate prequalification. Therefore, our findings are not limited to the use of non-fetal serum, but rather extend to the culture and expansion of MLPSCs in cell culture medium containing at least one pro-inflammatory cytokine.

[0408] Our findings support the broad application of pre-approved MPCs (e.g., MPCs cultured in cell culture medium containing at least one pro-inflammatory cytokine, e.g., MPCs using culture medium containing non-fetal serum, particularly neonatal serum) for the treatment of any disease or disorder with elevated baseline levels of inflammation, particularly diseases characterized by persistent inflammation such as heart failure.

[0409] Example 7, Treatment of HFrEF patient population with approved MPLSCs. Left ventricular assist devices (LVADs) were implanted in patients with end-stage heart failure with reduced ejection fraction (HFrEF). HFrEF patients receiving LVADs were classified as having either ischemic or non-ischemic heart failure. LVAD patients in the treatment group received MPCs cultured in medium supplemented with neonatal serum or in medium containing 10% fetal bovine serum (FBS). Control patients did not receive stem cell therapy.

[0410] Inflammation was assessed based on serum IL-6 levels. Before LVAD implantation, IL-6 levels were similarly elevated in both ischemic and non-ischemic heart failure control patients (Figure 12). From postoperative day 1 to 30, a similar surgery-related increase in IL-6 was observed in both the control ischemic and non-ischemic groups (Figure 12). From postoperative day 30 to 365, IL-6 levels in the non-ischemic group decreased to below levels observed before LVAD implantation. However, in patients in the ischemic group, IL-6 levels remained similar to pre-LVAD implantation levels and were significantly higher than those in the non-ischemic group (Figure 12). Furthermore, the ischemic control group had a significantly higher risk of all-cause mortality within 12 months after LVAD implantation compared with the non-ischemic control group (Figure 13).

[0411] These data suggest that LVAD implantation reduces the inflammatory process associated with end-stage HFrEF in patients with nonischemic heart failure but not in patients with ischemic heart failure. Furthermore, LVAD patients with ischemic end-stage HFrEF represent a distinct subgroup of patients with persistent inflammation and a higher risk of all-cause mortality.

[0412] Figure 12B shows that not only did administration of MPCs cultured in medium supplemented with neonatal serum reduce IL-6 levels, but that IL-6 levels also decreased over time to levels consistent with non-ischemic controls.

[0413] Figures 14 and 15 show the overall mortality rate 12.5 months after LVAD implantation in patients receiving MPCs cultured in medium supplemented with neonatal bovine serum, MPCs cultured in medium containing 10% FBS, and control patients who did not receive cell therapy. Surprisingly, ischemic LVAD patients receiving MPCs cultured in medium supplemented with neonatal bovine serum had a significantly reduced overall mortality rate compared to both the control group and patients receiving unapproved MPCs (Figure 14B). Specifically, MPCs cultured in medium supplemented with neonatal bovine serum reduced overall mortality in ischemic LVAD patients by 83% compared to the ischemic control group.

[0414] Our findings provide further evidence (in addition to the studies in Example 6) that MPCs cultured and expanded in medium supplemented with at least one pro-inflammatory cytokine and / or newborn bovine serum are particularly effective in treating diseases characterized by persistent inflammation, such as heart failure. They also provide a basis for selecting patients (e.g., ischemic LVAD patients) who will be particularly responsive to treatment with approved MPCs based on their level of persistent inflammation.

[0415] Example 8. Culture medium analysis and summary of findings Based on the data described in Example 1, increased cytokine levels in the culture medium used to expand the MLPSC population were characterized by increased one or more angiogenic marker(s) and increased therapeutic efficacy in patients with heart failure. 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.

[0416] 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.

[0417] Our findings therefore represent a significant advance in the art by providing a method for preparing novel MLPSC populations that can lead to improved therapeutic effects, particularly in the context of inflammation. These results not only suggest that culture expansion in media supplemented with proinflammatory cytokines can provide improved MLPSC populations, but also demonstrate that culture expansion in media supplemented with neonatal serum can provide relevant proinflammatory cytokines. Our findings therefore support the standard for culture expansion of MLPSCs in serum- and serum-free media.

[0418] Example 9. Isolation and expansion of MLPSCs MLPSCs can be isolated using techniques such as STRO-3+ immunoselection of MPCs or density gradient separation of MSCs.

[0419] Typically, for bone marrow-derived MLPSCs, 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.

[0420] 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).

[0421] In conjunction with immunoselection, STRO-3+ (or TNAP+) cells are isolated by magnetic-activated cell sorting as previously described (Gronthos et al. 2003; Gronthos and Simmons 1995). Briefly, approximately 1-3 x 108 BMMNCs are incubated on ice for 20 minutes in blocking buffer consisting of 10% (v / v) normal rabbit serum in HHF. The cells are then incubated on ice for 1 hour with 200 μl of a 10 μg / ml solution of STRO-3 mAb in blocking buffer. The cells are then centrifuged at 400 x g and washed twice in HHF. A 1 / 50 dilution of goat anti-mouse biotin (Southern Biotechnology Associates, Birmingham, UK) in HHF buffer is added, and the cells are incubated on ice for 1 hour. Cells were washed twice with MACS buffer (Ca2+- and Mn2+-free PBS supplemented with 1% BSA, 5 mM EDTA, and 0.01% sodium azide) as described above and resuspended in a final volume of 0.9 ml of MACS buffer.

[0422] 100 μl of streptavidin microbeads (Miltenyi Biotec; Bergisch Gladbach, Germany) were added to the cell suspension and incubated on ice for 15 minutes. The cell suspension was washed twice, resuspended in 0.5 ml of MACS buffer, and then loaded onto a mini-MACS column (MS Columns, Miltenyi Biotec) and washed three times with 0.5 ml of MACS buffer to recover cells that did not bind to STRO-3 mAb (Deposited with the American Type Culture Collection (ATCC) on December 19, 2005, under accession number PTA-7282 - see International Publication No. WO 2006 / 108229). After the addition of an additional 1 ml of MACS buffer, the column was removed from the magnet and TNAP+ cells were isolated under positive pressure. A portion of the cells from each fraction could be stained with streptavidin-FITC and their purity assessed by flow cytometry.

[0423] Alternatively, MSCs can be grown from BMMNCs using plastic adhesion techniques. For example, bone marrow mononuclear cells are isolated using Ficoll-Hypaque and placed into two T175 flasks at 50 ml per flask of culture growth medium containing alpha-modified MEM (αMEM) supplemented with gentamicin, glutamine (2 mM), and 10% (v / v) fetal bovine serum (FBS).

[0424] 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 growth medium.

[0425] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the present disclosure as set forth in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0426] This application claims priority to US 63 / 386,878 filed December 9, 2022, US 63 / 386,876 filed December 9, 2022, US 63 / 486,792 filed February 24, 2023, US 63 / 507,009 filed June 8, 2023, US 63 / 507,013 filed June 8, 2023, and US 63 / 513,777 filed July 14, 2023, the disclosures of which are incorporated herein by reference.

[0427] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0428] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure and is not to be construed as an admission that any or all of these matters form part of the prior art or were general knowledge in the art relevant to the present disclosure as existing prior to the priority date of each claim of this application.

Claims

1. 1. A method of treating progressive heart failure in a subject, the method comprising administering to the subject a composition comprising a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs) or conditioned medium obtained therefrom, wherein the subject has persistent inflammation and the MLPSCs have been culture-expanded in cell culture medium comprising at least one pro-inflammatory cytokine.

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

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

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

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

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

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

8. The method of any one of claims 2 to 7, wherein the level of IFN-γ is <1 ng / ml, preferably <500 pg / ml, more preferably <100 pg / ml.

9. The method of any one of claims 2 to 8, wherein the level of TNF-α is <1 ng / ml, preferably <750 pg / ml, more preferably <400 pg / ml.

10. The method of any one of claims 1-9, wherein the culture medium contains serum containing the pro-inflammatory cytokine(s).

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

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

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

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

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

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

17. The medium is i. IFN-γ levels greater than 1 pg / ml; ii. TNF-α levels greater than 2 pg / ml; iii. IL-6 levels greater than 3 pg / ml; iv. IL-8 levels greater than 500 pg / ml; v. IL-17A levels greater than 0.2 pg / ml; vi. MCP-1 levels greater than 3 pg / ml; vii. MIP-1-α levels greater than 0.5 pg / ml; viii. MIP-1-β levels greater than 3 pg / ml; ix. IP-10 levels greater than 500 pg / ml 17. The method according to any one of claims 1 to 16, characterized by one or more or all of the following:

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

19. 18. The method of any one of claims 1-9 or 17, wherein the medium is serum-free and / or xeno-free.

20. The method of any one of claims 10-19, wherein the culture medium contains 5% non-fetal serum.

21. The method of any one of claims 10-19, wherein the culture medium comprises 5% non-fetal serum and 5% fetal serum.

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

23. 18. The method of any one of claims 1-9 or 17, wherein the medium is a xeno-free medium.

24. 24. The method of claim 23, wherein the xeno-free medium comprises human serum.

25. 18. The method of any one of claims 1-9 or 17, wherein the medium is serum-free.

26. The method of any one of claims 1-25, wherein the subject has myocardial ischemia and / or diabetes.

27. 26. The method of any one of claims 1-25, wherein the subject's level of CRP is ≧2 mg / L.

28. The method of any one of claims 1-27, wherein the subject has a LVEF of less than about 45%, preferably less than 40%, preferably 30-35%.

29. 29. The method of any one of claims 1-28, wherein the subject has a LVESV of greater than 70 ml.

30. 30. The method of any one of claims 1-29, wherein the subject has a LVESV of between 70 ml and 160 ml.

31. 31. The method of any one of claims 1-30, wherein the subject has Class II heart failure according to the New York Heart Association (NYHA) classification scale.

32. 32. The method of any one of claims 1-31, comprising the steps of: i) selecting a subject with microvascular and / or macrovascular disease for treatment; and ii) administering said MLPSCs.

33. 33. The method of any one of claims 1-32, comprising the steps of: i) selecting a subject for treatment with a CRP level of > 2 mg / L; and ii) administering to said MLPSCs.

34. the subject's level of N-terminal pro-B-type natriuretic peptide (NT-proBNP) is >1000 pg / mL, or - 1000pg / ml to 2500pg / ml 34. The method of any one of claims 1 to 33, wherein

35. 35. The method of any one of claims 1-34, wherein the subject has a C-reactive protein (CRP) level of 2-5 mg / L, preferably 2-4 mg / L, more preferably 2-3 mg / L.

36. 36. The method of any one of claims 1-35, wherein the subject has experienced a hospitalization event due to heart failure within the past 9 months.

37. The method of any one of claims 1-36, wherein the subject has persistent left ventricular dysfunction.

38. 38. The method of any one of claims 1-37, wherein the subject's heart failure is due to an ischemic event or a non-ischemic event.

39. 39. The method of any one of claims 1-38, wherein the subject has a reduced risk of cardiac death following treatment.

40. 40. The method of any one of claims 1-39, wherein treatment improves LVEF in the subject by at least 4 percentage points.

41. 41. The method of any one of claims 1-40, wherein treatment improves the subject's LVEF by at least 5 percentage points or at least 6 percentage points.

42. 42. The method of any one of claims 1-41, wherein treatment improves the subject's LVEF by 4 to 7 percentage points, 5 to 7 percentage points.

43. With treatment, - the subject's LVESV improves by at least 17 ml; - the subject's LVESV is improved by at least 20 ml; - the subject's LVESV improves by 15 ml to 30 ml; or The method of any one of claims 1-42, wherein the subject's LVEDV is improved by at least 15 ml, preferably between 15 ml and 25 ml.

44. 44. The method of claim 43, wherein the reduced risk is compared to the risk of cardiac death in a subject not administered MLPSCs.

45. 45. The method of any one of claims 1-44, wherein the subject has a reduced risk of ischemic MACE (MI or stroke) following treatment.

46. The method of any one of claims 1-45, wherein the subject has a left ventricular assist device (LVAD).

47. 47. The method of claim 46, wherein the subject's level of IL-6 is increased compared to baseline at least 60 days after LVAD implantation.

48. 48. The method of claim 46 or 47, wherein treatment reduces the subject's risk of all-cause mortality.

49. 49. The method of any one of claims 46-48, wherein treatment reduces the subject's risk of all-cause mortality by between 10% and 90%, more than 50%, between 20% and 85%, preferably about 80%.

50. The method of any one of claims 1-49, wherein the composition is administered transendocardially and / or intravenously.

51. 51. The method of any one of claims 1-50, wherein the MLPSCs are mesenchymal progenitor cells (MPCs).

52. 52. The method of claim 51, wherein said MPCs are isolated from bone mononuclear cells with anti-STRO-3 antibodies prior to expansion in culture.

53. 53. The method of any one of claims 1-52, wherein the MLPSCs are mesenchymal stem cells (MSCs).

54. 54. The method of any one of claims 1-53, wherein the cells are allogeneic.

55. 55. The method of any one of claims 1-54, wherein the cells are cryopreserved prior to administration.

56. 1 x 10 ７ ~2 x 10 ８ 56. The method of any one of claims 1-55, comprising administering cells of

57. 57. The method of any one of claims 1-56, wherein the composition further comprises Plasmalyte A, dimethyl sulfoxide (DMSO), and human serum albumin (HSA).

58. 58. The method of any one of claims 1-57, wherein the composition further comprises a Plasmalyte A (70%), DMSO (10%), HSA (25%) solution, wherein the HSA solution comprises 5% HSA and 15% buffer.

59. The composition is 6.68 x 10 ６ 59. The method of any one of claims 1-58, comprising more than 100% viable cells / mL.

60. 60. The method of any one of claims 1-59, wherein the composition comprises human bone marrow-derived allogeneic MPCs isolated from bone mononuclear cells with anti-STRO-3 antibodies, expanded in vitro in culture medium containing NBCS, and cryopreserved.

61. 1. A method for selecting a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs) for use in treating advanced heart failure in a subject, wherein the MLPSCs are culture-expanded in a cell culture medium comprising at least one pro-inflammatory cytokine, the method comprising: (i) obtaining a population of MLPSCs; (ii) determining the level of one or more angiogenic markers in the MLPSC population, wherein the one or more angiogenic markers are: - the levels of VEGF, angiogenin, SDF-1α expressed by said MLPSCs under culture conditions, and / or - the level of endothelial network formation, endothelial network length, and endothelial branch length measured after treating an endothelial cell population with conditioned medium obtained from the MLPSCs; (iii) selecting the MLPSCs having increased level(s) of the one or more angiogenic markers for use in therapy.

62. A cultured expanded population of mesenchymal progenitor or stem cells (MLPSCs), wherein the population of MLPSCs is selected based on high angiogenic potential as determined by the level of angiogenin expressed by the MLPSCs under culture conditions.

63. A cultured expanded population of mesenchymal progenitor or stem cells (MLPSCs), the population of MLPSCs being measured after treating a population of endothelial cells with conditioned medium obtained from the MLPSCs. - Formation of the endothelial network, - the length of the endothelial network, or - The culture expansion population is selected based on high angiogenic potential as determined by one or more levels of endothelial branch length.

64. 63. The culture expanded population of claim 62, wherein MLPSCs expressing increased levels of angiogenin compared to a control population are selected.

65. 64. The cultured expanded population of claim 63, wherein MLPSCs are selected that induce an increase in one or more levels of endothelial network formation, endothelial length, or endothelial branch length compared to a control population.

66. 66. The culture-expanded population of claim 64 or 65, wherein the control population is a population of MLPSCs culture-expanded in cell culture medium containing 10% fetal bovine serum.

67. - expressing angiogenin levels greater than about 1200 pg / ml; - approx. 0.12 mm ２ / mm ２ Inducing the formation of a transendothelial network, -About 5mm ２ / mm ２ Inducing ultrathin endothelial network length -approximately 151 / mm ２ 67. The culture expanded population of any one of claims 62-66, wherein MLPSCs are selected that are characterized by one or more of: inducing endothelial branch lengths of greater than 1000 nm;

68. 1. A method for producing a pharmaceutical product comprising a population of mesenchymal progenitor or stem cells (MLPSCs), the method comprising: obtaining a determination of whether a test population of MLPSCs has a predetermined level of angiogenic potential under culture conditions; and if the test population of MLPSCs has at least the predetermined level of angiogenic potential under culture conditions, treating at least a portion of the test population of MLPSCs as a pharmaceutical product, thereby producing the pharmaceutical product; or if the population of MLPSCs has angiogenic potential under culture conditions less than the predetermined level, discarding at least a portion of the test population of MLPSCs, wherein the angiogenic potential is measured by a predetermined level of angiogenin measured under culture conditions.

69. 1. A method of producing a medicament comprising a population of mesenchymal progenitor or stem cells (MLPSCs), the method comprising obtaining a determination of whether a test population of MLPSCs has a predetermined level of angiogenic potential under culture conditions, and treating at least a portion of the test population of MLPSCs as a medicament, thereby producing the medicament, if the test population of MLPSCs has at least the predetermined level of angiogenic potential under culture conditions, or discarding at least a portion of the test population of MLPSCs if the population of MLPSCs has angiogenic potential below the predetermined level under culture conditions, wherein the angiogenic potential is - measured after treating an endothelial cell population with conditioned medium obtained from said MLPSCs; o Formation of endothelial network, o the length of the endothelial network, and / or Endothelial branch length measured in an in vitro angiogenesis assay The method, wherein the level is measured by a predetermined level of one or more of the following:

70. 69. The method of claim 68, wherein the predetermined level of angiogenin is measured in conditioned medium obtained from a test population of MLPSCs.

71. The predetermined level is as follows: - Endothelial network formation is approximately 0.12 mm ２ / mm ２ is larger than - The length of the endothelial network is approximately 5 ２ / mm ２ is larger than - The length of the endothelial branches is approximately 15 1 / mm ２ 70. The method of claim 69, wherein the

72. The MLPSC comprises: IFN-γ and / or TNF-α, and / or - the culture-expanded population of any one of claims 62-67, or the method of any one of claims 68-71, which has been cultured and expanded in a cell 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.

73. 72. The culture expanded population or method of any one of claims 62-71, wherein the medium contains serum containing pro-inflammatory cytokines.

74. 74. The culture expanded population or method of claim 73, wherein said serum is newborn bovine serum (NBCS).

75. 75. The culture expanded population or method of claim 74, wherein said serum is obtained within 21 days of birth.

76. 1. A method for determining the potency of a population of culture-expanded mesenchymal progenitor or stem cells (MLPSCs), wherein the MLPSCs are culture-expanded in a cell culture medium comprising at least one pro-inflammatory cytokine, the method comprising determining the level of one or more angiogenic markers in the population of MLPSCs, wherein the one or more angiogenic markers are - the levels of VEGF, angiogenin, SDF-1α expressed by said MLPSCs under culture conditions, and / or - the level of endothelial network formation, endothelial network length, and endothelial branch length measured after treating the endothelial cell population with conditioned medium obtained from the MLPSCs. is selected from the group consisting of The method, wherein increased levels of the one or more angiogenic markers are indicative of biological activity or a therapeutic effect.

77. 77. The method of claim 76, wherein the cell culture medium comprises non-fetal serum, preferably newborn bovine serum (NCBS).

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