Compositions and methods for treating vascular disease

Abstract

To provide a new method for producing mesoderm-derived vascular progenitor cells by in vitro differentiation of pluripotent stem cells. Further provided are methods of treating vascular disease, e.g., critical limb ischemia.SOLUTION: A method for producing a population of mesodermal-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, comprising culturing pluripotent stem cell-derived mesodermal cells under non-adherent or low-adherent conditions in a medium comprising one or more factors selected from the group consisting of vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), bone morphogenetic protein-4 (BMP4), and small-molecule inhibitors of transforming growth factor-beta (TGF - β) type I receptor, thereby producing a population of mesodermal-derived vascular progenitor cells. The method includes: SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 892,724, filed August 28, 2019, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to novel mesodermally derived vascular progenitor cells (meso-VPCs) and methods for producing said meso-VPCs. The present invention also relates to methods for treating vascular diseases, such as ischemia, using meso-VPCs. [Background technology]

[0003] Background of the Invention Vascular disease is a condition that affects the body's vascular network. Over 78 million Americans have hypertension, the most common form of vascular disease. In addition, peripheral arterial disease (PAD) affects 12 to 15 million people in the United States, with the number of undiagnosed cases being much higher.

[0004] Peripheral arterial disease (PAD) is the narrowing or blockage of blood vessels that carry blood from the heart to other organs and tissues. It is primarily caused by the buildup of fatty plaque in the arteries, called atherosclerosis. PAD can affect any blood vessel, but is more common in the lower extremities than the upper extremities.

[0005] Ischemia is a condition caused by peripheral arterial disease, which involves the interruption of arterial blood supply to tissues, organs, or limbs, which, if untreated, can lead to tissue death. It can be caused by embolism, atherosclerotic arterial thrombosis, or trauma. Venous problems, such as impaired venous outflow, and low blood flow can cause acute arterial ischemia. Ischemia in the lower limbs can lead to leg pain or cramping with activity (claudication), skin discoloration, sores or ulcers, and leg fatigue. Complete loss of circulation can lead to gangrene and limb loss.

[0006] Treatments for vascular diseases such as ischemia are limited. Most treatment methods involve invasive surgery, while others focus on preventing the progression of an existing condition. Thus, there remains a need in the art for improved treatments for vascular diseases such as ischemia. Summary of the Invention

[0007] The present invention relates to a novel method for producing mesoderm-derived vascular progenitor cells (meso-VPCs) by in vitro differentiation of pluripotent stem cells. The present invention further provides methods for treating vascular diseases, such as critical limb ischemia, using the meso-VPCs of the present invention.

[0008] Thus, in one aspect, the present invention provides a method for producing a population of mesodermal-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, comprising culturing mesodermal cells derived from pluripotent stem cells under non-adherent or low-adherent conditions in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesodermal-derived vascular progenitor cells (meso-VPCs).

[0009] In one embodiment, mesodermal cells are derived from pluripotent stem cells by culturing the pluripotent stem cells in a medium containing one or more mesoderm-inducing growth factors selected from the group consisting of activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4).

[0010] In one embodiment, meso-VPCs are produced as vasculonoids. In another embodiment, meso-VPCs are dissociated into single cells.

[0011] In one embodiment, the mesoderm-inducing growth factors include activin A, VEGF165, FGF-2, and BMP4. In one embodiment, activin A is used at a concentration of about 5 to 15 ng / mL. In one embodiment, VEGF165 is used at a concentration of about 5 to 25 ng / mL. In one embodiment, FGF-2 is used at a concentration of about 5 to 25 ng / mL. In one embodiment, BMP4 is used at a concentration of about 5 to 50 ng / mL. In one embodiment, the method further comprises the step of removing activin A from the culture medium after about 24 hours of culture.

[0012] In one embodiment, the pluripotent stem cells are cultured on an extracellular matrix surface. In one embodiment, the extracellular matrix surface is a Matrigel-coated surface. In one embodiment, the pluripotent stem cells are cultured for about 3 to about 5 days.

[0013] In one embodiment, the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542. In another embodiment, the one or more factors include VEGF165, FGF-2, BMP4, and SB431542. In one embodiment, the one or more factors further include forskolin. In one embodiment, forskolin is used at a concentration of about 2 to 10 μM. In one embodiment, VEGF165 is used at a concentration of about 10 to 50 ng / mL. In one embodiment, FGF-2 is used at a concentration of about 10 to 50 ng / mL. In one embodiment, BMP4 is used at a concentration of about 10 to 50 ng / mL. In one embodiment, SB431542 is used at a concentration of about 5 to 20 μM.

[0014] In one embodiment, the step of culturing the mesodermal cells is carried out for about 3 to about 7 days.

[0015] In one embodiment, the step of culturing the mesodermal cells is carried out under normoxic conditions of 5% CO2 and 20% O2.

[0016] In one embodiment, the pluripotent stem cells are cultured under normoxic conditions of 5% CO2 and 20% O2.

[0017] In one embodiment, the non-adherent or low-adherent conditions are on an ultra-low-adherent surface.

[0018] In one aspect, the present invention provides a method for producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, the method comprising the steps of: (a) culturing mesoderm cells derived from pluripotent stem cells on an extracellular matrix surface in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4); and (b) culturing the cells produced in step (a) on an extracellular matrix surface in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesoderm-derived vascular progenitor cells.

[0019] In one embodiment, mesodermal cells are derived from pluripotent stem cells by culturing the pluripotent stem cells in a medium containing one or more mesoderm-inducing growth factors selected from the group consisting of activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4).

[0020] In one embodiment, the method further comprises dissociating the population of meso-VPCs into single cells.

[0021] In one embodiment, the mesoderm-inducing growth factors include activin A, VEGF165, FGF-2, and BMP4. In one embodiment, activin A is used at a concentration of about 5 to 15 ng / mL. In one embodiment, VEGF165 is used at a concentration of about 5 to 25 ng / mL. In one embodiment, FGF-2 is used at a concentration of about 5 to 25 ng / mL. In one embodiment, BMP4 is used at a concentration of about 5 to 50 ng / mL. In one embodiment, the method further comprises the step of removing activin A from the culture medium after about 24 hours of culture.

[0022] In one embodiment, the extracellular matrix surface in step (a) is a collagen IV coated surface.

[0023] In one embodiment, the pluripotent stem cells are cultured for about 3 to about 5 days.

[0024] In one embodiment, the one or more factors in step (a) comprise VEGF165, FGF-2 and BMP4.

[0025] In one embodiment, the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542. In another embodiment, the one or more factors in step (b) include VEGF165, FGF-2, BMP4 and SB431542.

[0026] In one embodiment, the one or more factors in step (a) further comprise forskolin.

[0027] In one embodiment, the one or more factors in step (b) further comprise forskolin.

[0028] In one embodiment, forskolin is used at a concentration of about 2-10 μM.

[0029] In one embodiment, VEGF165 is used at a concentration of about 10 to 50 ng / mL.

[0030] In one embodiment, FGF-2 is used at a concentration of about 10 to 50 ng / mL.

[0031] In one embodiment, BMP4 is used at a concentration of about 10 to 50 ng / mL.

[0032] In one embodiment, SB431542 is used at a concentration of about 5-20 μM.

[0033] In one embodiment, the extracellular matrix surface in steps (a) and (b) is a collagen IV coated surface.

[0034] In one embodiment, the culturing in step (a) is carried out for about 1 day.

[0035] In one embodiment, the culture in step (b) is carried out for about 4 to about 7 days.

[0036] In one embodiment, the culturing in step (a) is carried out under normoxic conditions of 5% CO2 and 20% O2.

[0037] In one embodiment, the culturing in step (b) is carried out under hypoxic conditions of 5% CO 2 and 5% O 2 .

[0038] In one embodiment, the pluripotent stem cells are cultured under normoxic conditions of 5% CO2 and 20% O2.

[0039] In one embodiment, the pluripotent stem cells are human embryonic stem cells.

[0040] In one embodiment, the pluripotent stem cells are human induced pluripotent stem cells.

[0041] In one embodiment, the population of meso-VPCs produced according to any of the methods of the invention express at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb.

[0042] In one embodiment, the population of meso-VPCs produced according to any of the methods of the invention express cell surface markers (a) at least one of CD146, CD31 / PECAM1, and CD309 / KDR, or (b) CD31 / PECAM1, CD309 / KDR, CD146, and (i) CD144, CD34, CD184 / CXCR4, CD43, or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4.

[0043] In one embodiment, the population of meso-VPCs produced according to any of the methods of the invention exhibits limited or no detection of one or more of the cell surface markers selected from the group consisting of: (a) CXCR7, CD45, and NG2; (b) CXCR7, CD45, and NG2; or (c) CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2.

[0044] In one embodiment, the population of meso-VPCs produced according to any of the methods of the present invention express at least one miRNA marker selected from hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p.

[0045] In one embodiment, the population of meso-VPCs produced according to any of the methods of the present invention exhibits no or limited expression of at least one miRNA marker selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p.

[0046] In one embodiment, the population of meso-VPCs produced according to any of the methods of the invention expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p.

[0047] In one embodiment, the population of meso-VPCs produced according to any of the methods of the invention comprises at least one meso-VPC that is positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. In one embodiment, the miRNA marker is mir483-5p.

[0048] In one embodiment, the population of meso-VPCs produced according to any of the methods of the present invention comprises at least one meso-VPC that exhibits limited or no expression of at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a.

[0049] In one embodiment, the method of the present invention further comprises the step of producing vascular endothelial cells by differentiation of meso-VPCs.

[0050] In one embodiment, differentiation is performed on a fibronectin-coated surface.

[0051] In one aspect, the invention provides a composition comprising a population of meso-VPCs produced by any one of the methods of the invention.

[0052] In one aspect, the present invention provides a composition comprising a population of mesoderm-derived vascular progenitor cells (meso-VPCs) produced by in vitro differentiation of mesoderm cells derived from pluripotent stem cells, wherein the population of meso-VPCs express at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb.

[0053] In one embodiment, the composition comprising a population of meso-VPCs expresses at least two cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb.

[0054] In one embodiment, the composition comprising a population of meso-VPCs expresses the cell surface markers CD146, CD31 / PECAM1 and CD309 / KDR.

[0055] In one embodiment, a composition comprising a population of meso-VPCs expresses cell surface markers CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43, or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4.

[0056] In one embodiment, a composition comprising a population of meso-VPCs exhibits limited or no detection of one or more cell surface markers selected from the group consisting of: (a) CXCR7, CD45, and NG2; (b) CXCR7, CD45, and NG2; or (c) CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2.

[0057] In one embodiment, a composition comprising a population of meso-VPCs expresses at least one miRNA marker selected from hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p.

[0058] In one embodiment, a composition comprising a population of meso-VPCs exhibits limited or no expression of at least one miRNA marker selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p.

[0059] In one embodiment, a composition comprising a population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p.

[0060] In one embodiment, the population of meso-VPCs comprises vasculoid meso-VPCs.

[0061] In one embodiment, the population of meso-VPCs comprises single cells of meso-VPCs.

[0062] In one aspect, the present invention provides meso-VPCs produced by in vitro differentiation of mesodermal cells derived from pluripotent stem cells, wherein the meso-VPCs are positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p.

[0063] In one embodiment, the meso-VPC is positive for the miRNA marker mir483-5p.

[0064] In one embodiment, the meso-VPC is negative for at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p and mir133a.

[0065] In one embodiment, the pluripotent stem cells are human pluripotent stem cells.

[0066] In one embodiment, the pluripotent stem cells are human embryonic stem cells (hESCs).

[0067] In one embodiment, the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0068] In one embodiment, pluripotent stem cells are first differentiated into mesodermal cells, which are then differentiated into meso-VPCs.

[0069] In one aspect, the invention provides a pharmaceutical composition comprising a composition comprising a population of meso-VPCs of the invention, or comprising any one of the meso-VPCs of the invention.

[0070] In one aspect, the present invention provides a method for treating a vascular disease or vascular disorder in a subject, the method comprising administering to the subject an effective amount of a composition comprising a population of meso-VPCs of the present invention or any one of the mesodermally derived vascular progenitor cells (meso-VPCs) of the present invention, or any one of the pharmaceutical compositions of the present invention, thereby treating the vascular disease or vascular disorder in the subject.

[0071] In one embodiment, the vascular disease or disorder is selected from the group consisting of atherosclerosis, peripheral arterial disease (PAD), carotid artery disease, venous disease, blood clots, aortic aneurysms, fibromuscular dysplasia, lymphedema, and vascular injury.

[0072] In one embodiment, the peripheral arterial disease is selected from the group consisting of critical limb ischemia, intestinal ischemia syndrome, renal artery disease, popliteal entrapment syndrome, Raynaud's phenomenon, and Buerger's disease.

[0073] In one embodiment, the peripheral arterial disease is critical limb ischemia.

[0074] In one embodiment, the composition comprising a population of meso-VPCs, meso-VPCs, or pharmaceutical composition is administered intramuscularly or systemically.

[0075] In one embodiment, administration of a composition comprising a population of meso-VPCs, meso-VPCs, or a pharmaceutical composition increases blood flow in a subject.

[0076] In one embodiment, administration of a composition comprising a population of meso-VPCs, meso-VPCs, or pharmaceutical composition promotes angiogenesis and / or vasculogenesis in a subject.

[0077] In one embodiment, administration of a composition comprising a population of meso-VPCs, meso-VPCs, or pharmaceutical composition reduces ischemia severity in a subject.

[0078] In one embodiment, administration of a composition comprising a population of meso-VPCs, meso-VPCs, or pharmaceutical composition reduces the area of ​​necrosis in a limb in a subject.

[0079] In one embodiment, about 1 x 10 4 ~Approx. 1×10 13 meso-VPCs are administered to the subject.

[0080] In one embodiment, the meso-VPC is administered as a pharmaceutical composition.

[0081] In one embodiment, the pharmaceutical composition comprises (a) a buffering agent that maintains the solution at physiological pH, (b) at least 5% (w / v) glucose, and (c) an osmotically active agent that maintains the solution at physiological osmolality.

[0082] In one embodiment, the glucose is D-glucose (dextrose).

[0083] In one embodiment, the osmotically active agent is a salt.

[0084] In one embodiment, the salt is sodium chloride. [The present invention 1001] 1. A method for producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, comprising: Culturing mesodermal cells derived from pluripotent stem cells under non- or low-adherent conditions in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs). The method comprising: [The present invention 1002] 1001. The method of claim 1001, wherein mesodermal cells are derived from pluripotent stem cells by culturing the pluripotent stem cells in a medium comprising one or more mesoderm-inducing growth factors selected from the group consisting of activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and bone morphogenetic protein 4 (BMP4). [The present invention 1003] The method of any one of claims 1001 to 1002, wherein meso-VPC is produced as a vasculonoid. [The present invention 1004] The method of claim 1003, further comprising the step of dissociating meso-VPCs in the vasculoids into single cells. [The present invention 1005] The method of any of claims 1002 to 1004, wherein the mesoderm-inducing growth factors include activin A, VEGF165, FGF-2 and BMP4. [The present invention 1006] 1005. The method of claim 10, wherein activin A is used at a concentration of about 5 to 15 ng / mL. [The present invention 1007] The method of claim 1005, wherein VEGF165 is used at a concentration of about 5 to 25 ng / mL. [The present invention 1008] 1005. The method of claim 10, wherein FGF-2 is used at a concentration of about 5 to 25 ng / mL. [The present invention 1009] 1005. The method of claim 10, wherein BMP4 is used at a concentration of about 5 to 50 ng / mL. [The present invention 1010] The method of any of claims 1002 to 1009, further comprising the step of removing activin A from the culture medium after about 24 hours of culture. [The present invention 1011] The method of any of claims 1002 to 1010, wherein the pluripotent stem cells are cultured on an extracellular matrix surface. [The present invention 1012] The method of claim 1011, wherein the extracellular matrix surface is a Matrigel-coated surface. [The present invention 1013] The method of any one of claims 1002 to 1012, wherein the pluripotent stem cells are cultured for about 3 to about 5 days. [The present invention 1014] The method of any one of claims 1001 to 1013, wherein the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542. [The present invention 1015] The method of any of claims 1001 to 1014, wherein the one or more factors include VEGF165, FGF-2, BMP4 and SB431542. [The present invention 1016] 1016. The method of any of claims 1001 to 1015, wherein the one or more factors further comprise forskolin. [The present invention 1017] 1016. The method of claim 10, wherein forskolin is used at a concentration of about 2-10 μM. [The present invention 1018] 18. The method of any of claims 1015 to 1017, wherein VEGF165 is used at a concentration of about 10 to 50 ng / mL. [The present invention 1019] 18. The method of any of claims 1015 to 1017, wherein FGF-2 is used at a concentration of about 10 to 50 ng / mL. [The present invention 1020] Any of the methods of claims 1015 to 1017, wherein BMP4 is used at a concentration of about 10 to 50 ng / mL. [The present invention 1021] Any of the methods of claims 1014 to 1020, wherein SB431542 is used at a concentration of about 5 to 20 μM. [The present invention 1022] The method of any one of claims 1001 to 1021, wherein the step of culturing the mesodermal cells is carried out for about 3 to about 7 days. [The present invention 1023] The method of any one of claims 1001 to 1022, wherein the step of culturing the mesodermal cells is carried out under normoxic conditions of 5% CO2 and 20% O2. [The present invention 1024] The method of any one of claims 1002 to 1023, wherein the pluripotent stem cells are cultured under normoxic conditions of 5% CO2 and 20% O2. [The present invention 1025] The method of any one of claims 1001 to 1024, wherein the non-adhesion or low-adhesion conditions are on an ultra-low-adhesion surface. [The present invention 1026] 1. A method for producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, comprising: (a) culturing mesodermal cells derived from pluripotent stem cells on an extracellular matrix surface in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4); and (b) culturing the cells produced in step (a) on an extracellular matrix surface in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesodermally derived vascular progenitor cells. The method comprising: [The present invention 1027] 1026. The method of claim 1026, wherein mesodermal cells are derived from pluripotent stem cells by culturing the pluripotent stem cells in a medium comprising one or more mesoderm-inducing growth factors selected from the group consisting of activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4). [The present invention 1028] The method of any one of claims 1026 to 1027, further comprising the step of dissociating the population of meso-VPCs into single cells. [The present invention 1029] The method of any one of claims 1027 to 1028, wherein the mesoderm-inducing growth factors comprise activin A, VEGF165, FGF-2 and BMP4. [The present invention 1030] 1029. The method of claim 1029, wherein activin A is used at a concentration of about 5 to 15 ng / mL. [The present invention 1031] 1029. The method of claim 1029, wherein VEGF165 is used at a concentration of about 5 to 25 ng / mL. [The present invention 1032] 1029. The method of claim 1029, wherein FGF-2 is used at a concentration of about 5 to 25 ng / mL. [The present invention 1033] 1029. The method of claim 1029, wherein BMP4 is used at a concentration of about 5 to 50 ng / mL. [The present invention 1034] The method of any of claims 1029 to 1033, further comprising the step of removing activin A from the culture medium after about 24 hours of culture. [This invention 1035] The method according to any one of claims 1026 to 1034, wherein the extracellular matrix surface in the step (a) is a collagen IV-coated surface. [The present invention 1036] The method of any one of claims 1027 to 1035, wherein the pluripotent stem cells are cultured for about 3 to about 5 days. [This invention 1037] The method of any of claims 1026 to 1036, wherein the one or more factors in step (a) include VEGF165, FGF-2 and BMP4. [The present invention 1038] The method of any one of claims 1026 to 1037, wherein the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542. [This invention 1039] The method of any of claims 1026 to 1038, wherein the one or more factors in step (b) include VEGF165, FGF-2, BMP4 and SB431542. [The present invention 1040] 1039. The method of any of claims 1026 to 1039, wherein the one or more factors in step (a) further comprise forskolin. [The present invention 1041] 1040. The method of any of claims 1026 to 1040, wherein the one or more factors in step (b) further comprise forskolin. [The present invention 1042] 1042. The method of any one of claims 1040 to 1041, wherein forskolin is used at a concentration of about 2 to 10 μM. [This invention 1043] The method of any of claims 1037 to 1042, wherein VEGF165 is used at a concentration of about 10 to 50 ng / mL. [This invention 1044] 1043. The method of any of claims 1037 to 1042, wherein FGF-2 is used at a concentration of about 10 to 50 ng / mL. [This invention 1045] 1043. The method of any of claims 1037 to 1042, wherein BMP4 is used at a concentration of about 10 to 50 ng / mL. [The present invention 1046] The method of any one of claims 1038 to 1039, wherein SB431542 is used at a concentration of about 5 to 20 μM. [This invention 1047] The method of any one of claims 1026 to 1046, wherein the extracellular matrix surface in steps (a) and (b) is a collagen IV-coated surface. [This invention 1048] 1048. The method of any one of claims 1026 to 1047, wherein the culture in step (a) is carried out for about 1 day. [This invention 1049] 1049. The method of any one of claims 1026 to 1048, wherein the culture in step (b) is carried out for about 4 to about 7 days. [The present invention 1050] 1049. The method of any one of claims 1026 to 1049, wherein the culturing in step (a) is carried out under normoxic conditions of 5% CO2 and 20% O2. [This invention 1051] The method of any of claims 1026 to 1050, wherein the culture in step (b) is carried out under low oxygen concentration conditions of 5% CO2 and 5% O2. [This invention 1052] The method of any one of claims 1027 to 1051, wherein the pluripotent stem cells are cultured under normoxic conditions of 5% CO2 and 20% O2. [This invention 1053] The method of any one of claims 1001 to 1052, wherein the pluripotent stem cells are human embryonic stem cells. [This invention 1054] The method of any one of claims 1001 to 1052, wherein the pluripotent stem cells are human induced pluripotent stem cells. [This invention 1055] Any of the methods of claims 1001 to 1054, wherein the population of meso-VPCs expresses at least one of cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb. [This invention 1056] 1055. The method of claim 1055, wherein the population of meso-VPCs expresses cell surface markers (a) CD146, CD31 / PECAM1 and CD309 / KDR, or (b) CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43 or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CD184 / CXCFR4. [This invention 1057] Any of the methods of claims 1001 to 1056, wherein the population of meso-VPCs exhibits limited or no detection of one or more of the cell surface markers selected from the group consisting of: (a) CXCR7, CD45, and NG2, (b) CXCR7, CD45, and NG2, or (c) CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2. [This invention 1058] Any of the methods of claims 1001 to 1057, wherein the population of meso-VPCs expresses at least one miRNA marker selected from hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p. [This invention 1059] Any of the methods of claims 1001 to 1058, wherein the population of meso-VPCs exhibits limited or no expression of at least one miRNA marker selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [The present invention 1060] Any of the methods of claims 1001 to 1059, wherein the population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p. [The present invention 1061] Any of the methods of claims 1001 to 1060, wherein the population of meso-VPCs comprises at least one meso-VPC that is positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24 and mir483-5p. [The present invention 1062] The method of the present invention 1061, wherein the miRNA marker is mir483-5p. [The present invention 1063] Any of the methods of claims 1001 to 1062, wherein the population of meso-VPCs comprises at least one meso-VPC that exhibits limited or no expression of at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p and mir133a. [The present invention 1064] The method of any one of claims 1001 to 1063, further comprising the step of producing vascular endothelial cells by differentiation of meso-VPCs. [This invention 1065] The method of claim 1064, wherein differentiation is carried out on a fibronectin-coated surface. [The present invention 1066] A composition comprising a population of meso-VPCs produced by any of the methods of the present inventions 1001 to 1063. [This invention 1067] A composition comprising a population of mesoderm-derived vascular progenitor cells (meso-VPCs) produced by in vitro differentiation of mesoderm cells derived from pluripotent stem cells, wherein the population of meso-VPCs expresses at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb. [The present invention 1068] 1067. The composition of the present invention, wherein the population of meso-VPCs expresses at least two cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb. [This invention 1069] 1069. The composition of any of claims 1067 to 1068, wherein the population of meso-VPCs expresses the cell surface markers CD146, CD31 / PECAM1 and CD309 / KDR. [The present invention 1070] Any of the compositions of claims 1067 to 1069, wherein the population of meso-VPCs expresses the cell surface markers CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43 or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CD184 / CXCFR4. [This invention 1071] Any of the compositions of claims 1067 to 1070, wherein the population of meso-VPCs exhibits limited or no detection of one or more cell surface markers selected from the group consisting of: (a) CXCR7, CD45, and NG2; (b) CXCR7, CD45, and NG2; or (c) CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2. [This invention 1072] Any of the compositions of the present inventions 1067 to 1071, wherein the population of meso-VPCs expresses at least one miRNA marker selected from hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p and miR-199a-3p. [This invention 1073] Any of the compositions of claims 1067 to 1072, wherein the population of meso-VPCs exhibits limited or no expression of at least one miRNA marker selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [This invention 1074] Any of the compositions of claims 1067 to 1073, wherein the population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p. [This invention 1075] Any of the compositions of claims 1067 to 1074, wherein the population of meso-VPCs comprises meso-VPC vasculoids. [This invention 1076] Any of the compositions of claims 1067 to 1074, wherein the population of meso-VPCs comprises single meso-VPC cells. [This invention 1077] A meso-VPC produced by in vitro differentiation of mesodermal cells derived from pluripotent stem cells, said meso-VPC being positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24 and mir483-5p. [This invention 1078] meso-VPCs of the present invention 1077 positive for the miRNA marker mir483-5p. [This invention 1079] The meso-VPC of the present invention 1077 or 1078, which is negative for at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p and mir133a. [The present invention 1080] The composition or meso-VPC of any of claims 1066 to 1079, wherein the pluripotent stem cells are human pluripotent stem cells. [This invention 1081] The composition or meso-VPC of the present invention 1080, wherein the pluripotent stem cells are human embryonic stem cells (hESCs). [This invention 1082] The composition or meso-VPC of the present invention 1080, wherein the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs). [This invention 1083] The composition or meso-VPC of any of claims 1066 to 1082, wherein pluripotent stem cells are first differentiated into mesodermal cells, which are then differentiated into meso-VPCs. [This invention 1084] A pharmaceutical composition comprising any one of the compositions of the present inventions 1066 to 1083 or meso-VPC. [This invention 1085] 1. A method of treating a vascular disease or disorder in a subject, comprising: Administering an effective amount of any of the compositions of the present inventions 1066 to 1083, or mesoderm-derived vascular progenitor cells (meso-VPCs), or the pharmaceutical composition of the present invention 1083 to a subject, thereby treating a vascular disease or vascular disorder in the subject. The method comprising: [The present invention 1086] The method of claim 1085, wherein the vascular disease or vascular disorder is selected from the group consisting of atherosclerosis, peripheral arterial disease (PAD), carotid artery disease, venous disease, blood clots, aortic aneurysms, fibromuscular dysplasia, lymphedema, and vascular injury. [This invention 1087] 1086. The method of claim 1086, wherein the peripheral arterial disease is selected from the group consisting of critical limb ischemia, intestinal ischemia syndrome, renal artery disease, popliteal artery entrapment syndrome, Raynaud's phenomenon, and Buerger's disease. [This invention 1088] The method of claim 1087, wherein the peripheral arterial disease is critical limb ischemia. [This invention 1089] The method of any of claims 1085 to 1088, wherein the composition, meso-VPC or pharmaceutical composition is administered intramuscularly or systemically. [The present invention 1090] The method of any of claims 1085 to 1089, wherein administration of the composition, meso-VPC, or pharmaceutical composition increases blood flow in the subject. [This invention 1091] The method of any of claims 1085 to 1090, wherein administration of the composition, meso-VPC or pharmaceutical composition promotes angiogenesis and / or vasculogenesis in the subject. [This invention 1092] 20. The method of any of claims 1085 to 1091, wherein administering the composition, meso-VPC or pharmaceutical composition reduces ischemia severity in the subject. [This invention 1093] The method of any of claims 1085 to 1092, wherein administering the composition, meso-VPC or pharmaceutical composition reduces the area of ​​necrosis in the limb in the subject. [This invention 1094] Approximately 1×10 4 ~Approx. 1×10 13 The method of any of claims 1085 to 1093, wherein meso-VPCs are administered to the subject. [This invention 1095] The method of any of claims 1085 to 1094, wherein the meso-VPC is administered as a pharmaceutical composition. [This invention 1096] The pharmaceutical composition comprises: (a) a buffering agent that maintains the solution at physiological pH; (b) at least 5% (w / v) glucose, and (c) an osmotically active agent that maintains the solution at physiological osmolality The method of the present invention 1095, comprising: [This invention 1097] The method of claim 1096, wherein the glucose is D-glucose (dextrose). [This invention 1098] The method of claim 1096, wherein the osmotically active agent is a salt. [This invention 1099] 1096. The method of claim 1096, wherein the salt is sodium chloride. [Brief explanation of the drawings]

[0085] [Figure 1] 1 is a schematic representation of the process of in vitro differentiation of human pluripotent stem cells into mesodermal cells. [Figure 2A] Graph showing expression of cell surface markers KDR, CD56 / NCAM1, APLNR / APJ, GARP or CD13 in mesodermal cells differentiated from the human induced pluripotent stem cell line GMP1, confirming differentiation into the mesodermal lineage. [Figure 2B] Figure 1 shows the restricted or absent expression of pluripotent, endodermal, ectodermal, and blood vascular cell surface markers on mesodermal cells differentiated from the human induced pluripotent stem cell line GMP1, confirming differentiation into the mesodermal lineage. [Figure 3] Schematic representation of the process for in vitro differentiation of human pluripotent stem cells into mesodermal cells (left), and for in vitro differentiation of mesodermal cells into mesoderm-derived vascular progenitor cells (meso-VPCs) using the Meso-3D-Vasculonoid VPC1 protocol (top right) or the Meso-3D-Vasculonoid VPC2 protocol (bottom right). [Figure 4] Schematic diagram of the process for in vitro differentiation of human pluripotent stem cells into mesodermal cells (left), and for in vitro differentiation of mesodermal cells into mesoderm-derived vascular progenitor cells (meso-VPCs) using the Meso-2D VPC2 protocol (top right) or the Meso-2D VPC3 protocol (bottom right). [Figure 5] These panels show microscopic images demonstrating the ability of meso-VPCs produced by the Meso-3D-vasculoid protocol to undergo further differentiation toward the endothelial lineage. The top panel shows the morphology of meso-VPCs at day 5 before harvesting. The middle panel shows endothelial differentiation of meso-VPCs using fibronectin-coated plates and media that promotes endothelial differentiation. The bottom panel shows capillary-like Matrigel networks formed by meso-VPCs. [Figure 6A]Graph showing expression of cell surface markers CD31 / PECAM1, CD309 / KDR, CXCR4 / CD184, CD43, CD146, and PDGFRb in meso-VPCs produced using the Meso-3D-Vasculonoid-VPC1 protocol, the Meso-3D-Vasculonoid-VPC2 protocol, the Meso-2D-VPC2 protocol, or the Meso-2D-VPC3 protocol. [Figure 6B] Heatmap showing the fraction of cells positive for selected cell surface markers among meso-VPCs and comparison hemogenic endothelial cells (HE) or hemangioblasts (HB), as well as undifferentiated pluripotent stem cells (J1 and GMP1) and human umbilical vein endothelial cells (HUVEC). [Figure 6C] Principal component analysis (PCA) plots showing vascular cell surface marker expression profiles of meso-VPCs produced by the Meso-3D-vasculoid protocol or the Meso-2D protocol, control hemogenic endothelial cells (HE), control hemangioblasts (HB), undifferentiated pluripotent stem cells (J1 and GMP1), or human umbilical vein endothelial cells (HUVEC). [Figure 7] These panels show microscopic images demonstrating the ability of meso-VPCs produced by the Meso-2D protocol to undergo further differentiation toward the endothelial lineage. The top panel shows the morphology of meso-VPCs at day 7 before harvesting. The middle panel shows endothelial differentiation of meso-VPCs using fibronectin-coated plates and media that promotes endothelial differentiation. The bottom panel shows capillary-like Matrigel networks formed by meso-VPCs. [Figure 8]10 is a graph showing increased blood flow in animals treated with meso-VPC as described in Example 9. Specifically, animals were sham-operated (1M) or treated with vehicle control (2M), J1-HDF Meso-2D VPC2 (3M), J-HDF Meso-3D vasculonoid VPC2 (4M), GMP1HDF Meso-2D VPC2 (5M), GMP1-HDF Meso-3D vasculonoid VPC2 (6M), or GMP1-HDF Meso-3D vasculonoid VPC1 (7M). [Figure 9] 10 is a graph showing changes in vascular density in animals treated with meso-VPC as described in Example 9. Specifically, animals are treated with vehicle control (2M), J1-HDF Meso-2D VPC2 (3M TI1), J-HDF Meso-3D vasculonoid VPC2 (4M TI2), GMP1HDF Meso-2D VPC2 (5M TI3), GMP1-HDF Meso-3D vasculonoid VPC2 (6M TI4), or GMP1-HDF Meso-3D vasculonoid VPC1 (7M TI5). [Figure 10] Graphs show the quantitative results of CD34+ staining, an indicator of small capillary formation, total vessel count, and blood flow assays in animals treated with meso-VPC. Specifically, animals were sham-operated (1M) or treated with vehicle control (2M), J1-HDF Meso-2D VPC2 (3M TI1), J-HDF Meso-3D Vasculonoid VPC2 (4M TI2), GMP1HDF Meso-2D VPC2 (5M TI3), GMP1-HDF Meso-3D Vasculonoid VPC2 (6M TI4), or GMP1-HDF Meso-3D Vasculonoid VPC1 (7M TI5). [Figure 11] It shows a strong and statistically significant correlation between blood flow measured by laser Doppler and mean capillary density for each group of animals treated with meso-VPC. [Figure 12]Figure 12A shows plots and graphs showing unique human miRNAs found in the J1-derived Meso-3D vasculoid VPC2 cell population from three replicates, including hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p, compared to the J1 cell population and J1-derived HE cells. Figure 12A also shows unique human miRNAs found in the J1-derived HE cell population, including hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. "Expression" refers to normalized expression >0 in all three replicates. Figure 12B is a graph showing miRNA expression levels in populations of J1-derived Meso-3D vasculonoid VPC2 cells previously analyzed at single cells, demonstrating that hsa-miR-126-5p, hsa-miR-125a-5p, and hsa-miR-24-3p are expressed in both J1 and J1-derived Meso-3D vasculonoid VPC2 cell populations. Figure 12C is a graph showing that J1-derived Meso-3D vasculonoid VPC2 cell populations express hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, and hsa-miR-142-3p, but do not or only poorly express hsa-let-7e-3p, hsa-miR-99a-3p, and hsa-miR-133a-5p. Figure 12D is a graph showing that a population of J1-derived Meso-3D vasculoid VPC2 cells expresses hsa-miR-483-5p and hsa-miR-483-3p. [Figure 13-1] Graph showing expression of most up- or down-regulated genes in J1-derived Meso-3D vasculoid VPC2 cell samples compared to single J1 or single HUVEC cells in single-cell RNA-seq analysis. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14]Figure 14A shows a low-magnification (10x objective) image of an extensive vascular network extending from embedded aggregates of J1-derived Meso-3D vasculonoid VPC2 vasculonoids after 14 days, stained with DAPI and UAE1. Figure 14B shows that J1-derived Meso-3D vasculonoid VPC2 vasculonoids ("multicellular") or dissociated J1-derived Meso-3D vasculonoid VPC2 cells ("single cells") exhibited better cell survival than J1-derived Meso-3D vasculonoid VPC2 cells cryopreserved as single cells when cultured in vitro under CLI-mimicking conditions, normoxia (20% O2) (left panel) or hypoxia (5% O2) (right panel). Figure 14C is a graph showing statistically significant improvement in blood flow after administration of J1-derived Meso-3D vasculoid VPC2 single cells ("sc") or vasculoid compared to the vehicle-treated group (GS2 medium only) throughout the study; two-way ANOVA followed by Tukey's test. [Figure 15]Figure 15A is a graph showing that animals treated with meso-3D vasculoid VPC2 cells had better mean necrosis scores (left panel) and function scores (right panel) at day 21 compared to HE and HB cells. One-way ANOVA followed by Dunnett's test. Mean ± s.e.m. Figure 15B is a graph showing improved blood flow at day 63 in animals treated with meso-3D vasculoid VPC2 cells, HE cells, and HB cells compared to vehicle. *p<0.05 vs. vehicle. Mean ± s.d. Two-way ANOVA followed by Tukey's test. Figure 15C is a graph showing CD34+ blood vessel growth in the quadriceps muscle of animals treated with meso-3D vasculoid VPC2 cells, HE cells, and HB cells. *p<0.05 vs. vehicle. Mean ± s.e.m. Two-way ANOVA followed by Fisher's LSD test without correction. Figure 15D is a graph showing improvement in the gastrocnemius muscle after administration of Meso-3D vasculoid VPC2 cells, HE cells, or HB cells. *p<0.05 vs. vehicle. Mean±sem. Two-way ANOVA followed by Fisher's LSD test without correction. [Figure 16A] Graph showing engrafted donor GMP1-Meso3D vasculoid VPC2 cells by Ku80+ staining at days 63 and 180, demonstrating long-term engraftment of the cells. [Figure 16B] 10 is a graph showing meso-3D vasculoid VPC2 cell engraftment demonstrated by Ku80+ staining at days 35 and 63. [Figure 16C] Fluorescence images of injected Meso3D vasculoid VPC2 showing long-term engraftment (Ku80+), formation of human vasculature (UEA1+ vessels), and promotion of paracrine host vascular growth (IB4+ and SMA+ vessels) 63 days after HLI surgery in Balb / c nude mice. DETAILED DESCRIPTION OF THE INVENTION

[0086] Detailed Description of the Invention I. definition In order that the present invention may be more readily understood, certain terms are first defined. It should also be noted that whenever a value or range of values ​​for a parameter is recited, values ​​and ranges between the recited values ​​are also part of the present invention.

[0087] In the following description, for purposes of explanation, specific numbers, materials, and configurations are set forth to provide a thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, when the specification refers to a phrase such as "one embodiment" or "an embodiment," it means that the particular feature, structure, or characteristic described with respect to that embodiment is included in at least one embodiment of the present invention. The appearance of a phrase such as "in one embodiment" in various places throughout this specification does not necessarily all refer to the same embodiment.

[0088] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or to more than one element.

[0089] The terms "comprising" or "comprises" are used herein in reference to compositions, methods, and their respective components that are essential to the disclosure, but also allow for the inclusion of non-specified elements, whether essential or not.

[0090] As used herein, "pluripotent cells," "pluripotent stem cells," and "PSCs" broadly refer to cells capable of proliferating in vitro for extended periods or virtually indefinitely, while remaining undifferentiated under appropriate conditions, exhibiting a stable (preferably normal) karyotype, and possessing the ability to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm). Typically, pluripotent cells (a) possess the ability to induce teratomas when transplanted into immunodeficient (SCID) mice, (b) possess the ability to differentiate into cell types of all three germ layers (e.g., ectodermal, mesodermal, and endodermal cell types), and (c) express at least one hES cell marker (e.g., Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, NANOG, TRA 1 60, TRA 1 81, SOX2, REX1). Exemplary pluripotent cells may express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA 1 60, and / or TRA 1 81. Additional exemplary pluripotent cells include, but are not limited to, embryonic stem cells, induced pluripotent (iPS) cells, embryo-derived cells, pluripotent cells produced from embryonic germ (EG) cells (e.g., by culturing in the presence of FGF-2, LIF, and SCF), parthenogenetic ES cells, ES cells produced from cultured inner cell mass cells (ICM), ES cells produced from blastomeres, and ES cells produced by nuclear transfer (e.g., somatic cell nuclei transferred into recipient oocytes). Exemplary pluripotent cells may be produced without destroying embryos. For example, induced pluripotent cells may be produced from cells obtained without embryo destruction. As a further example, pluripotent cells may be produced from biopsied blastomeres (which can be accomplished without harming the remaining embryo). Optionally, the remaining embryos may be cryopreserved, cultured, and / or implanted into a suitable host. The pluripotent cells (whatever their source) may be genetically or otherwise modified.

[0091] As used herein, "embryo" or "embryonic" refers broadly to a developing mass of cells that has not implanted into the uterine membrane of a maternal host. An "embryonic cell" is a cell isolated from or contained within an embryo. This includes blastomeres and aggregated blastomeres obtained as early as the two-cell stage.

[0092] "Embryonic stem cells" (ES cells or ESCs) include pluripotent cells produced from embryonic cells (e.g., from cultured inner cell mass cells or from cultured blastomeres). Often, such cells are or have been serially passaged as cell lines. Embryonic stem cells can be used as pluripotent stem cells in the process of producing mesodermal cells and meso-VPCs as described herein. For example, ES cells can be produced by methods known in the art, including derivation from embryos (including by sexual or asexual means) produced by any method, such as fertilization of egg cells with sperm or sperm DNA, nuclear transfer (including somatic cell nuclear transfer), or parthenogenesis. As a further example, cells produced by somatic cell nuclear transfer are also included in embryonic stem cells, even when non-embryonic cells are used in the process. For example, ES cells can be embryonic stem cells derived from one or more blastomeres, and can also be derived from the ICM of a blastocyst-stage embryo. Such embryonic stem cells can be generated from embryonic material produced by fertilization or by asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. As further described above, ES cells can be genetically or otherwise modified.

[0093] ES cells can be generated to be homozygous or heterozygous for one or more HLA genes, for example, by genetic manipulation or screening for spontaneous loss of heterozygosity. Regardless of their source or the particular method used to produce them, embryonic stem cells typically possess one or more of the following attributes: (i) the ability to differentiate into cells of all three germ layers, (ii) expression of at least Oct-4 and alkaline phosphatase, and (iii) the ability to produce teratomas when transplanted into immunocompromised animals. Embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, human embryonic stem cells ("hESCs" or "hES cells") such as CT2, MA01, MA09, ACT-4, No. 3, J1, H1, H7, H9, H14, and ACT30 embryonic stem cells. Additional exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also the NIH Human Embryonic Stem Cell Registry. An exemplary human embryonic stem cell line that can be used is J1 cells.

[0094] Exemplary human embryonic stem cell (hESC) markers include, but are not limited to, alkaline phosphatase, Oct-4, Nanog, stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), TRA-1-60, TRA-1-81, TRA-2-49 / 6E, Sox2, growth differentiation factor 3 (GDF3), REX1 (reduced expression 1), fibroblast growth factor 4 (FGF4), germ cell-specific antigen 1 (ESG1), DPPA2 (developmental pluripotency-associated 2), DPPA4, telomerase reverse transcriptase (hTERT), SALL4, E-cadherin, CD30 (Cluster designation 30), Cripto (TDGF-1), GCTM-2, Genesis, germ cell nuclear factor, and stem cell factor (SCF or c-Kit ligand). In addition, the embryonic stem cells may express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA 1 60 and / or TRA 1 81.

[0095] ESCs can be initially cultured in any culture medium known in the art that maintains the pluripotency of ESCs, with or without feeder cells, such as mouse embryonic feeder (MEF) cells or human feeder cells, such as human dermal fibroblasts (HDFs). The MEF cells or human feeder cells can be mitotically inactivated, for example, by mitomycin C, gamma irradiation, or any other known method, before seeding the ESCs into coculture, so that the MEFs do not proliferate in culture. Therefore, ESC cell cultures can be examined microscopically, and colonies containing non-ESC cell morphology can be picked and discarded, for example, using a stem cell cutting tool, laser ablation, or other means. Typically, no additional MEF cells or human feeder cells are used after harvesting ESCs for seeding to form embryoid bodies.

[0096] Alternatively, hES cells can be cultured under feeder-free conditions on a solid surface, such as an extracellular matrix (e.g., Matrigel®, laminin, or iMatrix-511, or any other extracellular matrix disclosed herein or known in the art), by any method known in the art, e.g., Klimanskaya et al., Lancet 365:1636-1641 (2005). Thus, hES cells used in the methods described herein can be cultured in feeder-free culture.

[0097] As used herein, "embryonic-derived cells" (EDCs) broadly refer to pluripotent morula-derived cells, blastocyst-derived cells, including cells of the inner cell mass, embryonic shield, or epiblast, or other pluripotent stem cells of the early embryo, including primitive endoderm, ectoderm, and mesoderm, and their derivatives. "EDCs" also include blastomeres, aggregated single blastomeres, or cell masses from embryos at various developmental stages, but do not include human embryonic stem cells passaged as cell lines.

[0098] As used herein, "induced pluripotent stem cells" or "iPSCs" or "iPS cells" refer to pluripotent stem cells generated by reprogramming of somatic cells. iPSCs can be generated by expressing or inducing the expression of a combination of factors ("reprogramming factors"). iPS cells can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. iPS cells can be obtained from cell banks. Alternatively, iPS cells can be generated de novo (by processes known in the art) before initiating differentiation into vascular progenitor cells (VPCs) or other cell types. iPS cell generation can be the first step in the production of differentiated cells. iPS cells can be specifically generated using material from a particular patient or matched donor for the purpose of generating histocompatible VPCs. iPS cells can be produced from cells that are substantially non-immunogenic in the intended recipient, for example, from cells autologous to the intended recipient or from cells that are histocompatible. As discussed further above (see "Pluripotent Cells"), pluripotent cells, including iPS cells, may be genetically or otherwise modified. An exemplary human iPSC cell line that may be used is GMP1 cells.

[0099] As a further example, induced pluripotent stem cells may be generated by reprogramming somatic or other cells by contacting the cells with one or more reprogramming factors. For example, reprogramming factors can be expressed by cells in response to factors such as small molecules, microRNAs, etc. that promote or induce expression of the gene, e.g., from exogenous nucleic acids added to the cell, or from endogenous genes (Suh and Blelloch, Development 138, 1653-1661 (2011); Miyoshi et al., Cell Stem Cell (2011), doi:10.1016 / j.stem.2011.05.001; Sancho-Martinez et al., Journal of Molecular Cell Biology (2011) 1-3; Anokye-Danso et al., Cell Stem Cell 8, 376-388, April 8, 2011; Orkin and Hochedlinger, Cell 145, 835-850, June 10, 2011; or Warren et al., Scientific (See, e.g., J. Cell. Reprogramming, 10.1038 / srep00657, September 14, 2012. Each of these references is incorporated herein by reference in its entirety.) Reprogramming factors can be provided from exogenous sources, for example, by adding them to culture medium, and can be introduced into cells by methods known in the art, such as coupling to cell entry peptides, protein or nucleic acid transfection agents, lipofection, electroporation, biolistic particle delivery systems (gene guns), microinjection, etc. In certain embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, and Klf4. In another embodiment, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, a combination of Oct-4, Sox2, Nanog, and Lin28.In another embodiment, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In another embodiment, somatic cells are reprogrammed by expressing Oct4, Sox2, MYC, Klf4, Nanog, and Lin28. In another embodiment, additional reprogramming factors are identified and used alone or in combination with one or more known reprogramming factors to reprogram somatic cells into pluripotent stem cells. iPS cells can typically be identified by the expression of the same markers as embryonic stem cells, although specific iPS cell lines may have different expression profiles.

[0100] Induced pluripotent stem cells can be produced by expressing or inducing the expression of one or more reprogramming factors in somatic cells. In some embodiments, the somatic cells are fibroblasts, such as dermal fibroblasts, synovial fibroblasts, or pulmonary fibroblasts, or non-fibroblastic somatic cells. In some embodiments, the somatic cells are reprogrammed by expressing at least one, two, three, four, or five of the above-mentioned reprogramming factors. In another embodiment, the expression of the reprogramming factors can be induced by contacting the somatic cells with at least one agent, such as a small organic molecule agent, that induces the expression of the reprogramming factors.

[0101] Somatic cells can also be reprogrammed using a combinatorial approach in which reprogramming factors are expressed (e.g., using viral vectors, plasmids, etc.) and expression of the reprogramming factors is induced (e.g., using small organic molecules). For example, reprogramming factors can be expressed in somatic cells by infection with a viral vector, such as a retroviral vector or a lentiviral vector. Reprogramming factors can also be expressed in somatic cells using a non-integrating vector, such as an episomal plasmid or mRNA. See, e.g., Yu et al., Science. 2009 May 8;324(5928):797-801, which is incorporated herein by reference in its entirety. When reprogramming factors are expressed using a non-integrating vector, the factors can be expressed in the cells using electroporation, transfection, or transformation of the somatic cells with the vector.

[0102] Once the reprogramming factors are expressed in the cells, the cells can be cultured by any method known in the art. Over time, cells with ES characteristics will appear in the culture dish. Cells can be selected and subcultured, for example, based on ES morphology or on the expression of selectable or detectable markers. Cells can be cultured to produce cultures of cells that resemble ES cells, which are putative iPS cells. iPS cells can typically be identified by the expression of the same markers as other embryonic stem cells, although specific iPS cell lines can differ in their expression profiles. Exemplary iPS cells can express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA1 60, and / or TRA1 81.

[0103] To confirm the pluripotency of iPS cells, cells can be tested by one or more pluripotency assays.For example, cells can be tested for the expression of ES cell markers, can be evaluated for the ability to produce teratomas when transplanted into SCID mice, and can be evaluated for the ability to differentiate into the cell types of all three germ layers.Once pluripotent iPS cells are obtained, they can be used to produce mesodermal cells and vascular progenitor cells, for example, mesodermal-derived vascular progenitor cells.

[0104] As used herein, "mesoderm" refers to one of the three primary germ layers in the very early embryos of all bilaterian animals. The mesoderm forms mesenchyme, mesothelium, non-epithelial vascular cells, and coelomocytes. Early mesodermal commitment arises from an epithelial-mesenchymal transition, followed by medial migration of specified mesodermal lineage cells as gastrulation progresses. Cells of the mesodermal lineage are fated to form the vascular and lymphatic systems, including hemangioblasts and multipotent mesenchymal stem cells capable of differentiating into multiple specified cell types. The mesoderm initiates angiogenesis through the formation of extraembryonic mesoderm followed by embryonic visceral mesoderm. Growth factors such as vascular endothelial growth factor (VEGF) and placental growth factor (PIGF or PGF) stimulate the growth and development of new blood vessels. In one aspect, cells of the mesodermal lineage are fated to become vascular precursor or progenitor cells. In one embodiment, pluripotent stem cells, e.g., hESCs or iPSCs, e.g., hiPSCs, can be differentiated into mesoderm-lineage-committed cells, e.g., mesoderm precursor cells. Thus, the term "mesoderm" also includes mesoderm-lineage-committed cells derived from pluripotent stem cells, regardless of the maturity of the cells, and thus encompasses mesoderm cells at various levels of maturity, including mesoderm precursor cells.

[0105] Exemplary mesoderm markers include CD309 / KDR, CD56 / NCAM1, APLNR / APJ, GARP, CD13, N-cadherin, activin A, activin AB, activin AC, activin B, activin C, BMP and other activin receptor activators, BMP and other activin receptor inhibitors, BMP-2, BMP-2 / BMP-4, BMP-2 / BMP-6 heterodimer, BMP-2 / BMP-7 heterodimer, BMP-2a, BMP-4, BMP-6, BMP-7, Cryptic, FABP4 / A-FABP, FGF-5, GDF-1, GDF-3, INHBA, INHB Examples of markers that may be used include, but are not limited to, B, Nodal, TGF-beta, TGF-beta 1, TGF-beta 1, 2, 3, TGF-beta 1.2, TGF-beta 1 / 1.2, TGF-beta 2, TGF-beta 2 / 1.2, TGF-beta 3, TGF-beta receptor inhibitor, Wnt-3a, Wnt-8a, MESDC2, Nicalin, Brachyury, EOMES, FoxC1, FoxF1, Goosecoid, HAND1, MIXL1, Slug, Snail, TBX6, Twist-1, and Twist-2. In one embodiment, the mesodermal cells are mesodermal precursor cells positive for one or more markers selected from CD309 / KDR, CD56 / NCAM1, APLNR / APJ, GARP, and CD13.

[0106] As used herein, "vasculogenesis" refers to the formation of new blood vessels. Vasculogenesis includes the formation of mesodermally derived endothelium. As used herein, "angiogenesis" refers to the formation of blood vessels from pre-existing vessels. See, e.g., Developmental Biology by Gilbert, Scott F. Sunderland (MA): Sinauer Associates, Inc.; c2000, and Molecular Biology of the Cell, 4th ed. Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter. New York and London: Garland Science; c2002.

[0107] As used herein, "vascular progenitor cells" (VPCs) refer to cells that have the potential to differentiate into endothelial cells, smooth muscle cells, pericytes, and other blood vascular cell lineages. In one embodiment, the vascular progenitor cells are mesodermally derived vascular progenitor cells (meso-VPCs).

[0108] As used herein, "mesodermal-derived vascular progenitor cells" (meso-VPCs) refer to VPCs generated from mesodermal cells induced by in vitro differentiation of pluripotent stem cells, e.g., ESCs or iPSCs. Meso-VPCs can be identified by the expression of one or more cell surface markers, as further described herein. In one embodiment, mesodermal-derived vascular progenitor cells are generated by in vitro differentiation of pluripotent stem cells, e.g., ESCs or iPSCs, into mesodermal cells, which then differentiate into meso-VPCs.

[0109] Meso-VPCs can be derived in vitro from both mouse and human PSCs. Meso-VPCs have the ability to differentiate into hematopoietic and endothelial cell lineages and may also have the ability to become smooth muscle cells. The meso-VPC population of the present invention can be positive for at least one marker, such as CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb. In one embodiment, the meso-VPC population is positive for one, two, three, four, five, six, seven, or eight of the above-listed markers. In one embodiment, the meso-VPC population is positive for CD146, CD31 / PECAM1, and CD309 / KDR. In another embodiment, the population of meso-VPCs express CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43, or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4. In some embodiments, the population of meso-VPCs comprises miR-3917, miR-450a-2-3p, miR-542-5p, miR-126-5p, miR-125a-5p, miR-24-3p, miR-let-7e-5p, miR-99a-5p, miR-223-5p, miR-142-3p, miR-483-5p, miR- The cell expresses at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen miRNA markers selected from miR-483-3p, miR214, miR335-3p, and miR-199a-3p.In one embodiment, a population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p. In some embodiments, a population of meso-VPCs is considered to express a particular marker if at least about 20% of the meso-VPCs in the composition express that marker. In one embodiment, the meso-VPCs of the present invention are positive for at least one, at least two, at least three, or at least four miRNA markers selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. In one embodiment, the miRNA marker is mir483-5p. In one embodiment, the population of meso-VPCs contains at least one meso-VPC that is positive for at least one, at least two, at least three, or at least four miRNA markers selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. In one embodiment, the miRNA marker is mir483-5p. In certain embodiments, the population of meso-VPCs express CD31 and KDR at higher levels than the population of HE cells. In another embodiment, the population of meso-VPCs express CD146 at lower levels than the population of HE cells. In yet another embodiment, the population of meso-VPCs express CD184 / CXCR4 at lower levels than the population of HE cells.

[0110] In any embodiment, the population of meso-VPCs exhibits limited detection or no detection of one, two, or three of CXCR7, CD45, and NG2. In any embodiment, the population of meso-VPCs exhibits limited detection or no detection of all of CXCR7, CD45, and NG2. In any embodiment, the population of meso-VPCs exhibits limited detection or no detection of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, or NG2. In some embodiments, a population of meso-VPCs exhibits limited or no expression of at least one, at least two, at least three, at least four, at least five, at least six, or at least seven miRNA markers selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In some embodiments, a population of meso-VPCs is considered to exhibit limited or no expression of a marker if less than about 20% of the meso-VPCs in the composition express that marker. In certain embodiments, the meso-VPCs of the present invention exhibit limited or no expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine miRNA markers selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a.In one embodiment of the present invention, the population of meso-VPCs comprises at least one meso-VPC that exhibits limited or no expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine miRNA markers selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a.

[0111] As used herein, "vasculonoids" refer to colony-like aggregates of cells, such as mesodermal-derived vascular progenitor cells (meso-VPCs), formed, for example, in cell culture. In one embodiment, vasculonoids are formed by meso-VPCs produced using a 3D-vasculonoid differentiation platform.

[0112] As used herein, "therapy," "therapeutic," "treating," "treat," or "treatment" broadly refers to treating a disease, halting or reducing the development of a disease or its clinical symptoms and / or alleviating the disease, causing regression of a disease or its clinical symptoms. "therapy," "therapeutic," "treating," "treat," or "treatment" includes prevention, prevention, treatment, cure, remediation, reduction, relief, and / or alleviating a disease, signs and / or symptoms of a disease. "therapy," "therapeutic," "treating," "treat," or "treatment" includes the reduction of signs and / or symptoms of a disease in a patient in whom signs and / or symptoms of the disease continue. "therapy," "therapeutic," "treating," "treat," or "treatment" encompasses "prophylaxis" and "prevention." Prevention includes preventing a disease from occurring after treatment of the disease in a patient or reducing the incidence or severity of a disease in a patient. The term "reduced" with respect to therapy, "therapeutic," "treating," "treat," or "treatment" broadly refers to a clinically significant reduction in signs and / or symptoms. "Therapy," "therapeutic," "treating," "treat," or "treatment" includes treating relapsing or recurring signs and / or symptoms. "Therapy," "therapeutic," "treating," "treat," or "treatment" includes, but is not limited to, preventing the appearance of signs and / or symptoms, as well as reducing existing signs and / or symptoms, eliminating existing signs and / or symptoms. "Therapy," "therapeutic," "treating," "treat," or "treatment" includes treating chronic diseases ("maintenance") and treating acute diseases. For example, treatment includes treating or preventing relapses or recurrence of signs and / or symptoms. In one embodiment, treatment includes a clinically significant reduction in signs and / or symptoms of vascular disease, such as critical limb ischemia.

[0113] As used herein, "normalizing a pathological condition" refers to restoring abnormal structure and / or function resulting from a disease to a more normal state. Normalization suggests that correcting the abnormal structure and / or function of a tissue, organ, or cell type resulting from a disease can control and improve the progression of the pathological condition. For example, after treatment with meso-VPCs of the present invention, limb abnormalities resulting from vascular disease, such as critical limb ischemia, can be improved, corrected, and / or reversed.

[0114] As used herein, "vascular disease" refers to any abnormal condition of blood vessels (arteries and veins). Vascular disease can occur anywhere outside the heart. The most common vascular diseases are stroke, peripheral artery disease (PAD), abdominal aortic aneurysm (AAA), carotid artery disease (CAD), arteriovenous malformation (AVM), critical limb ischemia (CLI), pulmonary embolism (blood clot), deep vein thrombosis (DVT), chronic venous insufficiency (CVI), and varicose veins. In one embodiment, the vascular disease is peripheral artery disease (PAD). In one embodiment, the vascular disease is an ischemic disease such as critical limb ischemia (CLI). In one embodiment, the vascular disease is atherosclerosis, peripheral artery disease (PAD), carotid artery disease, venous disease, blood clot, aortic aneurysm, fibromuscular dysplasia, lymphedema, or vascular injury. In one embodiment, the vascular disease is peripheral arterial disease such as critical limb ischemia (CLI), intestinal ischemia syndrome, renal artery disease, popliteal entrapment syndrome, Raynaud's phenomenon, or Buerger's disease.

[0115] II. In vitro generation of mesoderm-derived vascular progenitor cells (meso-VPCs) The present invention provides a method for producing mesodermal-derived vascular progenitor cells (meso-VPCs) from mesodermal cells derived from pluripotent stem cells. The method includes producing mesodermal cells by culturing the pluripotent stem cells in a medium containing one or more mesoderm-inducing growth factors, and culturing the mesodermal cells on a suitable surface in a medium containing one or more factors that direct the differentiation of the mesodermal cells into mesodermal-derived vascular progenitor cells (meso-VPCs). In some embodiments, the method further includes dissociating a plurality of meso-VPCs into single cells.

[0116] The pluripotent stem cells used in the present invention can be obtained and cultured by any of the methods presented above.In one embodiment, pluripotent stem cells, such as human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC), are cultured under feeder-free (FF) conditions and plated on extracellular matrix.In one embodiment, pluripotent stem cells are cultured under feeder culture conditions and plated on extracellular matrix.

[0117] In some embodiments, the extracellular matrix is ​​selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, a soluble preparation from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells, Matrigel® (Corning), gelatin, and human basement membrane extract. In one embodiment, the extracellular matrix can be derived from any mammalian source, including humans. In one embodiment, the extracellular matrix surface for culturing pluripotent stem cells is a Matrigel-coated surface.

[0118] In some embodiments, the pluripotent stem cells are cultured in a medium suitable for supporting pluripotency, all of which are known in the art. In some embodiments, the medium for supporting pluripotency is Nutristem™. In some embodiments, the medium for supporting pluripotency is TeSR™. In some embodiments, the medium for supporting pluripotency is StemFit™. In another embodiment, the medium for supporting pluripotency is Knockout™ DMEM (Gibco), which may be supplemented with Knockout™ Serum Replacement (Gibco), LIF, bFGF, or any other factor. Each of these exemplary media is known in the art and commercially available. In further embodiments, the medium for supporting pluripotency may be supplemented with bFGF or any other factor. In one embodiment, bFGF may be supplemented at a low concentration (e.g., 4 ng / mL). In another embodiment, bFGF may be supplemented at a higher concentration (e.g., 100 ng / mL). In one embodiment, the medium is serum-free. In another embodiment, the medium contains serum.

[0119] Pluripotent stem cells can be cultured, subcultured or collected in any suitable container known in the art.Exemplary tissue culture containers include 15cm tissue culture plate, 10cm tissue culture plate, 3cm tissue culture plate, 6-well tissue culture plate, 12-well tissue culture plate, 24-well tissue culture plate, 48-well tissue culture plate, 96-well tissue culture plate, T-25 tissue culture flask, T-75 tissue culture flask.In one embodiment, pluripotent stem cells are cultured in 6-well tissue culture plate.

[0120] In some embodiments, after about 1, 2, 3, 4, 5, or 6 days of culture, a medium change is performed to maintain optimal conditions for the pluripotent stem cells. The same culture medium as the starting conditions can be used for the medium change, or the medium can be adjusted according to culture needs. In some embodiments, the pluripotent stem cells are split and passaged after about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, or when the cell culture reaches about 60-90% confluence. The same culture medium as the starting conditions can be used for cell passage, or the medium can be adjusted according to culture needs. Cells can be split and passaged at a dilution ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In one embodiment, the pluripotent stem cells are passaged at a dilution ratio of 1:3.

[0121] In some embodiments, pluripotent stem cells may be cultured under normoxic conditions of about 5% CO 2 and about 20% O 2 , or other known conditions suitable for the growth of pluripotent stem cells.

[0122] In some embodiments, the pluripotent stem cells are cultured, passaged, or recovered in a culture medium under feeder-free conditions in which a feeder cell layer is not included in the culture. In some embodiments, the pluripotent stem cells are cultured, passaged, or recovered in a culture medium under feeder culture conditions in which a feeder cell layer, such as human dermal fibroblasts (HDFs) or other cell types known to those skilled in the art, is included in the culture.

[0123] To produce mesodermal cells by in vitro differentiation of pluripotent stem cells, pluripotent stem cells, such as hESCs or hiPSCs, are cultured on a suitable surface, such as an extracellular matrix surface. In some embodiments, the extracellular matrix is ​​selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, a soluble preparation from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells, Matrigel, gelatin, and human basement membrane extract. In one embodiment, the extracellular matrix can be derived from any mammalian source, including humans. In one embodiment, the extracellular matrix surface for in vitro differentiation of pluripotent stem cells into mesodermal cells is a Matrigel-coated surface.

[0124] In one embodiment, pluripotent stem cells are plated and cultured in a culture medium for about 1 hour to about 24 hours to allow the cells to settle prior to inducing differentiation. To induce differentiation of the pluripotent stem cells into mesodermal cells, the pluripotent stem cells are cultured in a culture medium on a suitable surface, such as the extracellular matrix surface described above.

[0125] The culture medium for inducing differentiation of pluripotent stem cells into mesodermal cells may be any medium that supports differentiation and may be a culture medium known in the art. In some embodiments, the culture medium may be any medium that supports blood vessel culture and / or blood vessel expansion, including, but not limited to, Stemline® II (Sigma), StemSpan™ SFEMII (StemCell Technologies), StemSpan™ AFC (StemCell Technologies), Minimum Essential Medium (MEM) (Gibco), and αMEM. In some embodiments, the culture medium is serum-free. In another embodiment, the culture medium contains serum. The culture medium may further contain one or more mesoderm-inducing growth factors, such as activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4). In one embodiment, the VEGF used in the method is VEGF165. In one embodiment, the FGF used in the method is basic FGF (bFGF). In one embodiment, pluripotent stem cells are cultured in a culture medium containing activin A, VEGF165, bFGF and BMP4. In one embodiment, the culture duration is about 1, 2, 3, 4, 5, 6 or 7 days. In one embodiment, the culture duration is about 4 days. In one embodiment, the culture medium is changed after about 24 hours of culture and replaced with a culture medium that does not contain activin A.

[0126] VEGF, e.g., VEGF165, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 5 ng / mL to about 20 ng / mL. In one embodiment, VEGF is used at a concentration of about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, or about 20 ng / mL. Activin A can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 5 ng / mL to about 20 ng / mL. In one embodiment, activin A is used at a concentration of about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, or about 20 ng / mL. FGF, e.g., bFGF, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably about 5 ng / mL to about 20 ng / mL. In one embodiment, FGF is used at a concentration of about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, or about 20 ng / mL. BMP4 can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably about 5 ng / mL to about 35 ng / mL. In one embodiment, BMP4 is used at a concentration of about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, or about 35 ng / mL. In one embodiment, VEGF is used at a concentration of 10 ng / mL, activin A is used at a concentration of 10 ng / mL, FGF is used at a concentration of 10 ng / mL, and BMP4 is used at a concentration of 25 ng / mL.

[0127] Differentiation of pluripotent stem cells into mesodermal cells can be carried out under normoxic conditions of about 5% CO 2 and about 20% O 2 , or under other known conditions suitable for differentiation of pluripotent stem cells.

[0128] The differentiation of pluripotent stem cells into mesodermal cells can be carried out in any suitable container known in the art.Exemplary tissue culture containers include, but are not limited to, 15cm tissue culture plates, 10cm tissue culture plates, 3cm tissue culture plates, 6-well tissue culture plates, 12-well tissue culture plates, 24-well tissue culture plates, 48-well tissue culture plates, 96-well tissue culture plates, T-25 tissue culture flasks and T-75 tissue culture flasks.In one embodiment, the differentiation of pluripotent stem cells into mesodermal cells is carried out in 10cm tissue culture plates.

[0129] The mesodermal cells may be further dissociated into single cells for further use. In one aspect, mesodermal cells produced by in vitro differentiation of pluripotent stem cells are dissociated into single cells by enzymatic treatment.

[0130] In one embodiment, the mesodermal cells express at least one, at least two, at least three, at least four or at least five markers selected from the group including CD309 / KDR, CD56 / NCAM1, APLNR / APJ, GARP and CD13.

[0131] Mesodermal cells express N-cadherin, activin A, activin AB, activin AC, activin B, activin C, BMPs and other activin receptor activators, BMPs and other activin receptor inhibitors, BMP-2, BMP-2 / BMP-4, BMP-2 / BMP-6 heterodimer, BMP-2 / BMP-7 heterodimer, BMP-2a, BMP-4, BMP-6, BMP-7, cryptic, FABP4 / A-FABP, FGF-5, GDF-1, GDF-3, INHBA, INHBB, and nodal receptors. , TGF-beta, TGF-beta 1, TGF-beta 1,2,3, TGF-beta 1.2, TGF-beta 1 / 1.2, TGF-beta 2, TGF-beta 2 / 1.2, TGF-beta 3, TGF-beta receptor inhibitor, Wnt-3a, Wnt-8a, MESDC2, Nicalin, Brachyury, EOMES, FoxC1, FoxF1, Goosecoid, HAND1, MIXL1, Slug, Snail, TBX6, Twist-1 and Twist-2 may also be expressed.

[0132] The mesodermal cells produced by the methods of the present invention are further differentiated into mesoderm-derived vascular progenitor cells (meso-VPCs) using one of two platforms disclosed herein: a 3D-vasculoid differentiation platform or a 2D differentiation platform.

[0133] The 3D-vasculoid differentiation platform provides a method for the in vitro differentiation of mesodermal cells produced from pluripotent stem cells, such as hESCs or hiPSCs, into meso-VPCs.

[0134] The 3D-vasculoid differentiation platform method is carried out by culturing mesodermal cells in a culture medium under non-adherent or low-adherent conditions, for example, on an ultra-low attachment surface or in suspension culture. The culture medium may be any culture medium that supports differentiation and may be known in the art. In some embodiments, the culture medium may be any medium that supports blood vessel culture and / or blood vessel expansion, including, but not limited to, Stemline® II (Sigma), StemSpan™ SFEMII (StemCell Technologies), StemSpan™ AFC (StemCell Technologies), Minimum Essential Medium (MEM) (Gibco), and αMEM. In one embodiment, the culture medium is serum-free. In another embodiment, the culture medium contains serum. The culture medium may further contain one or more factors that induce differentiation of mesodermal cells into meso-VPCs, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, and forskolin. In one embodiment, the VEGF used in this method is VEGF165. In one embodiment, the FGF used in this method is basic FGF (bFGF). In one embodiment, the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542. In one embodiment, pluripotent stem cells are cultured in a culture medium containing VEGF165, bFGF, BMP4, and SB431542. In one embodiment, pluripotent stem cells are cultured in a culture medium containing VEGF165, bFGF, BMP4, SB431542, and forksolin. In one embodiment, the duration of culture is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In one embodiment, the duration of culture is about 5 days. In one embodiment, the culture medium is changed about 2 days and about 4 days after the initiation of differentiation.

[0135] VEGF, e.g., VEGF165, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, VEGF is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. FGF, e.g., bFGF, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, FGF is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. BMP4 can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, BMP4 is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. Small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor, such as SB431542, can be used at a concentration of about 0.1 μM to about 100 μM, or more preferably about 1 μM to about 100 μM.In one embodiment, small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor are used at a concentration of about 0.1 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or 100 μM. Forskolin can be used at a concentration of about 0.1 μM to about 10 μM. In one embodiment, forskolin is used at a concentration of about 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM.

[0136] In one embodiment, VEGF is used at a concentration of about 50 ng / mL, FGF is used at a concentration of about 50 ng / mL, BMP4 is used at a concentration of about 25 ng / mL, the small molecule inhibitor is used at a concentration of about 10 μM, and forskolin is used at a concentration of about 2 μM.

[0137] Differentiation of mesodermal cells into meso-VPCs using the 3D-vasculoid differentiation platform can be performed under normoxic conditions of approximately 5% CO2 and approximately 20% O2, or under other known conditions suitable for differentiation of pluripotent stem cells.

[0138] The differentiation of mesodermal cells into meso-VPCs using a 3D-vasculoid differentiation platform can be carried out in any suitable container known in the art. Exemplary tissue culture containers include, but are not limited to, 15cm tissue culture plates, 10cm tissue culture plates, 3cm tissue culture plates, 6-well tissue culture plates, 12-well tissue culture plates, 24-well tissue culture plates, 48-well tissue culture plates, 96-well tissue culture plates, T-25 tissue culture flasks, and T-75 tissue culture flasks. In one embodiment, the differentiation of mesodermal cells into meso-VPCs using a 3D-vasculoid differentiation platform is carried out in a 10cm tissue culture plate.

[0139] Differentiation of mesodermal cells into meso-VPCs using the 3D-vasculoid differentiation platform can be performed under non-adherent conditions or under low-adhesion conditions where cells minimally adhere to the culture substrate. In one embodiment, differentiation of mesodermal cells into meso-VPCs using the 3D-vasculoid differentiation platform is performed on an ultra-low attachment surface or in suspension culture.

[0140] In some embodiments, meso-VPCs produced by the 3D-vasculoid differentiation platform form vasculoids. As used herein, vasculoids refer to cell aggregates, e.g., colony-like aggregates, formed by vascular cell lineages, e.g., meso-VPCs. The morphology of vasculoids may vary depending on the method used to produce vascular cells. The present invention further provides a method for dissociating multiple cells in vasculoids to obtain single cells. In one embodiment, meso-VPCs produced by the 3D-vasculoid differentiation platform can be further dissociated into single cells. In one embodiment, multiple meso-VPCs in vasculoids are dissociated into single cells by enzymatic treatment.

[0141] The 2D differentiation platform provides a method for the in vitro differentiation of mesodermal cells produced from pluripotent stem cells, such as hESCs or hiPSCs, into meso-VPCs.

[0142] The 2D differentiation platform method is carried out by culturing mesodermal cells on a suitable surface, such as an extracellular matrix surface, in a culture medium. In some embodiments, the extracellular matrix is ​​selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, heparan sulfate, a soluble preparation from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells, Matrigel, gelatin, and human basement membrane extract. In one embodiment, the extracellular matrix can be derived from any mammalian source, including humans. In one embodiment, the extracellular matrix surface for in vitro differentiation of mesodermal cells is a collagen IV-coated surface.

[0143] The culture medium may be any medium that supports the differentiation of mesodermal cells and may be any culture medium known in the art. In some embodiments, the culture medium may be any medium that supports blood vessel culture and / or blood vessel expansion, including, but not limited to, Stemline® II (Sigma), StemSpan™ SFEMII (StemCell Technologies), StemSpan™ AFC (StemCell Technologies), Minimum Essential Medium (MEM) (Gibco), and αMEM. In some embodiments, the culture medium is serum-free. In another embodiment, the culture medium contains serum. The culture medium may further contain one or more factors that induce the differentiation of mesodermal cells into meso-VPCs. These factors are selected from vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, and forskolin. In one embodiment, the VEGF used in the method is VEGF165. In one embodiment, the FGF used in the method is basic FGF (bFGF). In one embodiment, the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542.

[0144] In one embodiment, the 2D differentiation platform method for differentiating mesodermal cells to obtain meso-VPCs comprises two steps. First, mesodermal cells are differentiated in a culture medium that supports differentiation. This culture medium can be any culture medium known in the art. In some embodiments, the culture medium can be any medium that supports blood vessel culture and / or blood vessel expansion, including, but not limited to, Stemline® II (Sigma), StemSpan™ SFEMII (StemCell Technologies), StemSpan™ AFC (StemCell Technologies), Minimum Essential Medium (MEM) (Gibco), and αMEM. In one embodiment, the culture medium is serum-free. In another embodiment, the culture medium contains serum. The culture medium can further contain one or more factors selected from vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and forskolin. In one embodiment, the culture medium contains VEGF165, bFGF, and BMP4. In one embodiment, the culture medium contains VEGF165, bFGF, BMP4, and forskolin. The culture in this step is carried out for about 12 hours to about 2 days. In one embodiment, the first step of the 2D differentiation platform for differentiating mesodermal cells into meso-VPCs is carried out for about 1 day.

[0145] VEGF, e.g., VEGF165, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of 10 ng / mL to about 100 ng / mL. In one embodiment, VEGF is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. FGF, e.g., bFGF, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, FGF is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. BMP4 can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, BMP4 is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. Forskolin can be used at a concentration of about 0.1 μM to about 10 μM. In one embodiment, forskolin is used at a concentration of about 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM.

[0146] In one embodiment, VEGF is used at a concentration of about 50 ng / mL, FGF is used at a concentration of about 50 ng / mL, BMP4 is used at a concentration of about 25 ng / mL, and forskolin is used at a concentration of about 2 μM.

[0147] The first step in the differentiation of mesodermal cells into meso-VPCs using a 2D differentiation platform can be carried out under normoxic conditions of about 5% CO2 and about 20% O2, or under other known conditions suitable for the differentiation of mesodermal cells.

[0148] The second step of the 2D differentiation platform involves further differentiating the cells obtained in the first step into meso-VPCs in a differentiation-supportive culture medium. In some embodiments, the culture medium can be any medium that supports blood vessel culture and / or blood vessel expansion, including, but not limited to, Stemline® II (Sigma), StemSpan™ SFEMII (StemCell Technologies), StemSpan™ AFC (StemCell Technologies), minimum essential medium (MEM) (Gibco), and αMEM. In some embodiments, the culture medium is serum-free. In another embodiment, the culture medium contains serum. The culture medium can further contain one or more factors, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, and / or forskolin. In one embodiment, the culture medium comprises VEGF165, bFGF, BMP4, and SB431542. In one embodiment, the culture medium comprises VEGF165, bFGF, BMP4, SB431542, and forskolin. The culture in this step is carried out for about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In one embodiment, the second step of the 2D differentiation platform for differentiating mesodermal cells into meso-VPCs is carried out for about 6 days. In one embodiment, the culture medium is changed about 2 days and about 4 days after the start of the second step of the 2D differentiation platform.

[0149] VEGF, e.g., VEGF165, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of 10 ng / mL to about 100 ng / mL. In one embodiment, VEGF is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. FGF, e.g., bFGF, can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, FGF is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. BMP4 can be used at a concentration of about 1 ng / mL to about 100 ng / mL, or more preferably at a concentration of about 10 ng / mL to about 100 ng / mL. In one embodiment, BMP4 is used at a concentration of about 1 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. Small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor, such as SB431542, can be used at a concentration of about 0.1 μM to about 100 μM, or more preferably about 1 μM to about 100 μM.In one embodiment, small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor are used at a concentration of about 0.1 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or 100 μM. Forskolin can be used at a concentration of about 0.1 μM to about 10 μM. In one embodiment, forskolin is used at a concentration of about 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM or 10 μM.

[0150] In one embodiment, VEGF is used at a concentration of about 50 ng / mL, FGF is used at a concentration of about 50 ng / mL, BMP4 is used at a concentration of about 25 ng / mL, the small molecule inhibitor is used at a concentration of about 10 μM, and forskolin is used at a concentration of about 2 μM.

[0151] The second step of differentiation of mesodermal cells into meso-VPCs using the 2D differentiation platform can be carried out under hypoxic conditions of approximately 5% CO2 and approximately 5% O2, or under other known conditions suitable for differentiation into vascular progenitor cells.

[0152] The two-stage differentiation of mesodermal cells into meso-VPCs using a 2D differentiation platform can be carried out in any suitable container known in the art.Exemplary tissue culture containers include, but are not limited to, 15cm tissue culture plates, 10cm tissue culture plates, 3cm tissue culture plates, 6-well tissue culture plates, 12-well tissue culture plates, 24-well tissue culture plates, 48-well tissue culture plates, 96-well tissue culture plates, T-25 tissue culture flasks, and T-75 tissue culture flasks.In one embodiment, the differentiation of mesodermal cells into meso-VPCs using a 2D differentiation platform is carried out in a T-75 tissue culture flask.

[0153] The differentiation of mesodermal cells into meso-VPCs using a 2D differentiation platform can be carried out on any suitable surface. In one embodiment, the differentiation of mesodermal cells into meso-VPCs using a 2D differentiation platform is carried out on an extracellular matrix surface. In one embodiment, the extracellular matrix surface is a collagen IV-coated surface.

[0154] In one embodiment, meso-VPCs produced by the 2D differentiation platform can be further dissociated into single cells by enzymatic treatment.

[0155] In some embodiments of the present invention, the mesodermal cells or meso-VPCs produced in each step can be further sorted by methods known in the art, such as flow cytometry, to select cells with a specific expression profile of molecular markers, such as cell surface markers or miRNA markers. Methods for characterizing cells produced by the methods of the present invention are further described below.

[0156] III. Characteristics and composition of meso-VPC The present invention provides mesoderm-derived vascular progenitor cells (meso-VPCs) obtained by in vitro differentiation of mesodermal cells induced from pluripotent stem cells using the methods disclosed herein. In one embodiment, pluripotent stem cells are first differentiated into mesodermal cells, which are then differentiated into meso-VPCs. The expression levels of certain phenotypic markers can be determined by any method known in the art, such as flow cytometry / fluorescence-activated cell sorting (FACS), single-cell mRNA profiling, or immunohistochemistry. The expression of certain genes can be determined by any method known in the art, such as RT-PCR and RNA-Seq.

[0157] In one embodiment, a population of meso-VPCs of the present invention express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb. In one embodiment, a population of meso-VPCs expresses CD31 / PECAM1, CD309 / KDR, and CD146. In another embodiment, the population of meso-VPCs express CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43, or PDGFRb, (ii) CD34, CD184 / CXCR4, and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4. In certain embodiments, a population of meso-VPCs is considered to express a particular marker if at least about 20% of the meso-VPCs in the composition express the marker.

[0158] In any embodiment, the population of meso-VPCs exhibits limited or no detection of one or more of CXCR7, CD45, and NG2. In any embodiment, the population of meso-VPCs exhibits limited or no detection of all of CXCR7, CD45, and NG2. In any embodiment, the population of meso-VPCs exhibits limited or no detection of one or more of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, or NG2. In some embodiments, a population of meso-VPCs is considered to exhibit limited or no expression of a marker if less than about 20% of the meso-VPCs in the composition express that marker.

[0159] In one embodiment, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPCs in the composition express at least one, at least two, at least three, at least four, at least five, at least six, at least seven or at least eight markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb. In one embodiment of the present invention, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPCs in the composition of the present invention express CD31 / PECAM1, CD309 / KDR and CD146. In one embodiment of the invention, at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPCs in a composition of the invention express CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43 or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4.

[0160] In any embodiment, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of meso-VPCs in the compositions of the invention express one or more of CXCR7, CD45, and NG2. In any embodiment, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of meso-VPCs in the compositions of the invention express all of CXCR7, CD45, and NG2. In any embodiment, less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of meso-VPCs in the compositions of the invention express one or more of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, or NG2.

[0161] The meso-VPCs of the present invention may be further characterized by single-cell miRNA profiles. In one embodiment, the meso-VPCs of the present invention are positive for at least one, at least two, at least three, or at least four miRNA markers selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. In any embodiment, the meso-VPCs are negative for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine markers selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a. In one embodiment, the meso-VPCs are positive for mir126, mir125a-5p, mir24, and mir483-5p. In another embodiment, the meso-VPC is positive for mir483-5p.

[0162] In one embodiment, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the meso-VPCs in the composition are positive for at least one, at least two, at least three, or at least four miRNA markers selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. In one embodiment of the invention, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the meso-VPCs in the composition are positive for at least one, at least two, at least three, or at least four markers selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. In any embodiment, less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the meso-VPCs in the composition express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine of the markers selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p and mir133a. In one embodiment of the present invention, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the meso-VPCs in the composition are positive for mir126, mir125a-5p, mir24, and mir483-5p. In another embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the meso-VPCs in the composition are positive for mir483-5p.

[0163] In one embodiment, the population of meso-VPCs comprises miR-3917, miR-450a-2-3p, miR-542-5p, miR-126-5p, miR-125a-5p, miR-24-3p, miR-let-7e-5p, miR-99a-5p, miR-223-5p, miR-142-3p, miR-483-5p, miR- The meso-VPCs express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen miRNA markers selected from miR-483-3p, miR214, miR335-3p, and miR-199a-3p. In one embodiment, the population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p. In some embodiments, a population of meso-VPCs is considered to express a particular marker if at least about 20% of the meso-VPCs in the composition express that marker.

[0164] In one embodiment, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPC in the composition is selected from the group consisting of miR-3917, miR-450a-2-3p, miR-542-5p, miR-126-5p, miR-125a-5p, miR-24-3p, miR-let-7e-5p, miR-99a-5p, miR-223-5p, miR-m The cell expresses at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen miRNA markers selected from iR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p. In one embodiment of the invention, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPC in the composition is selected from the group consisting of hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-161-5p, hsa-miR-162-5p, hsa-miR-163-5p, hsa-miR-164-5p, hsa-miR-165-5p, hsa-miR-166-5p, hsa-miR-167-5p, hsa-miR-168-5p, hsa-miR-169 ... The cells express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen miRNA markers selected from miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p.In one embodiment, about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the meso-VPCs in the composition express hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p. In one embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the meso-VPCs in the composition express hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p.

[0165] In one embodiment, a population of meso-VPCs exhibits limited or no expression of at least one, at least two, at least three, at least four, at least five, at least six, or at least seven miRNA markers selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In one embodiment, a population of meso-VPCs exhibits limited or no expression of hsa-let-7e-3p, hsa-miR-99a-3p, and hsa-miR-133a-5p. In some embodiments, a population of meso-VPCs exhibits limited or no expression of hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In some embodiments, a population of meso-VPCs is considered to exhibit limited or no expression of a marker if less than about 20% of the meso-VPCs in the composition express that marker.

[0166] In one embodiment, approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPCs in the composition exhibit limited or no expression of at least one, at least two or at least three miRNA markers selected from hsa-let-7e-3p, hsa-miR-99a-3p and hsa-miR-133a-5p. In one embodiment, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the meso-VPCs in the composition exhibit limited or no expression of at least one, at least two or at least three miRNA markers selected from hsa-let-7e-3p, hsa-miR-99a-3p and hsa-miR-133a-5p.

[0167] In addition to the above characteristics, meso-VPCs of the present invention possess other properties of vascular progenitor cells, such as the potential to differentiate into vascular cells, such as endothelial cells, smooth muscle cells, and hematopoietic cells. In one embodiment, meso-VPCs of the present invention possess the potential to differentiate into vascular endothelial cells. Other vascular cell properties of meso-VPCs can be determined, for example, by Matrigel and AcLDL uptake assays.

[0168] In one embodiment, the meso-VPCs of the present invention have vascular cell morphology, such as a cobblestone endothelial-like morphology. Another method for characterizing the meso-VPCs of the present invention includes karyotyping to determine chromosomal integrity.

[0169] In one embodiment, the meso-VPCs of the present invention are substantially purified with respect to pluripotent stem cells and mesodermal cells. In a further embodiment, the meso-VPCs of the present invention are substantially purified with respect to pluripotent stem cells and mesodermal cells such that the cells comprise at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% meso-VPCs. The pluripotent stem cells can be any pluripotent stem cell described herein.

[0170] meso-VPC is about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.0 ... The composition may comprise less than 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% pluripotent stem cells and mesoderm cells. The composition may lack pluripotent stem cells and mesoderm cells.

[0171] IV. Pharmaceutical compositions containing meso-VPC The present invention provides pharmaceutical compositions comprising any of the meso-VPCs described herein. Pharmaceutical compositions comprising the meso-VPCs of the present invention can be formulated with a pharmaceutically acceptable carrier. For example, the meso-VPCs of the present invention can be administered alone or as a component of a pharmaceutical preparation, in which case the meso-VPC can be formulated for administration in any convenient manner for use in medicine. Carriers suitable for the present disclosure include those conventionally used, e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronan, and glycols are exemplary liquid carriers, particularly for solutions (when isotonic).

[0172] Other exemplary carriers or excipients are described, for example, in Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; and Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0173] Pharmaceutical compositions containing meso-VPC can be formulated in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions selected from the group consisting of dispersions, suspensions, emulsions, sterile powders that are optionally reconstituted into sterile injectable solutions or dispersions immediately prior to use, antioxidants, buffers, bactericides, solutes, or suspending and thickening agents.

[0174] Exemplary pharmaceutical compositions of the present invention can be in any formulation suitable for use in treating human patients, e.g., patients suffering from vascular disease or disorders. In one embodiment, a pharmaceutical composition comprising meso-VPC is formulated as an injectable formulation, e.g., suitable for intramuscular injection. A pharmaceutical composition comprising meso-VPC can be administered in a buffer solution at physiological pH, further containing an osmotically active agent to maintain the solution at physiological osmolality. In one embodiment, a pharmaceutical composition comprising meso-VPC can be administered in a buffer containing at least 5% (w / v) glucose. In one embodiment, a pharmaceutical composition comprising meso-VPC can be administered in a buffer containing sodium chloride. Other reagents known in the art can also be used to formulate pharmaceutical compositions. In one embodiment, the buffer or solution used to formulate the pharmaceutical composition is sterilized prior to use.

[0175] Pharmaceutical compositions containing meso-VPC used in the methods described herein can be delivered as a suspension, gel, colloid, slurry, or mixture. Alternatively, cryopreserved meso-VPC can be resuspended in commercially available balanced salt solutions to achieve the desired osmolality and concentration for administration by injection (e.g., bolus or intravenous). Pharmaceutical compositions containing meso-VPC can be mixed with a durable, inert matrix and delivered, for example, by one or more injections into a subject. Durable, inert matrices, such as hydrogels—natural or synthetic water-insoluble polymers—can provide a scaffold for cell growth and expansion at the administration site. In one embodiment, pharmaceutical compositions containing meso-VPC are administered in hyaluronan hydrogels. In another embodiment, pharmaceutical compositions containing meso-VPC are administered in methylcellulose hydrogels. Other suitable materials known in the art that provide durable, inert matrix scaffolds for cell growth and expansion can also be used in the methods described herein.

[0176] Pharmaceutical compositions containing meso-VPC can be delivered by one or more injections, for example, using a syringe. Alternatively, pharmaceutical compositions containing meso-VPC can be delivered by other suitable methods known in the art. Suitable delivery methods also facilitate the growth and survival of meso-VPC and prevent cell loss at the administration site. In certain embodiments, suitable delivery methods help retain meso-VPC at the administration site, providing an optimal environment for cell growth. Thus, pharmaceutical compositions containing meso-VPC can also be formulated into, for example, hydrogel tubes, hydrogel sheets, bioengineered patches made of natural or artificial materials, or cell sheets that provide sufficient support for meso-VPC in the pharmaceutical composition. In one embodiment, pharmaceutical compositions containing meso-VPC are delivered in the form of hydrogel tubes. In one embodiment, pharmaceutical compositions containing meso-VPC are delivered in the form of hydrogel sheets. In one embodiment, pharmaceutical compositions containing meso-VPC are delivered in the form of bioengineered patches. In one embodiment, the pharmaceutical composition comprising meso-VPC is delivered in the form of a cell sheet. Any other suitable method known in the art can also be used to deliver the pharmaceutical compositions described herein.

[0177] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as monostearate salts and gelatin in the composition. Furthermore, soluble factors can be administered in sustained-release formulations, for example, in compositions with slow-release polymers. The active compounds can be prepared with carriers that protect the compound against rapid release, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.

[0178] One aspect of the present invention is to 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 or 10 13The present invention relates to pharmaceutical compositions suitable for use in mammalian patients, such as human patients, comprising meso-VPC and a pharmaceutically acceptable carrier. The concentration of meso-VPC for administration of the pharmaceutical preparation can be any effective amount, e.g., substantially free of PSCs. For example, the pharmaceutical composition can include the number and types of meso-VPC described herein. In certain embodiments, the pharmaceutical composition of meso-VPC is administered in a concentration of about 1 x 10 for systemic administration to a host in need thereof. 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 6 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 9 ~Approx. 1×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 12 or about 1 x 10 12 ~Approx. 1×10 13 Approximately 1 x 10 meso-VPCs for local administration to a host containing or requiring 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 6 , 1×10 6 ~Approx. 1×10 7 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 8 ~Approx. 1×10 9 , about 1×10 9 ~Approx. 1×10 10 , about 1×10 10 ~Approx. 1×10 11 , about 1×10 11 ~Approx. 1×10 12 , or approximately 1 × 10 12 ~Approx. 1×10 13 Includes meso-VPCs.

[0179] V. Methods for treating vascular disease The meso-VPCs and pharmaceutical compositions containing meso-VPCs described herein can be used in cell-based treatments. In particular, the present invention provides a method for treating vascular diseases, such as critical limb ischemia. The method comprises administering to a subject in need thereof an effective amount of meso-VPCs, wherein the meso-VPCs are obtained by in vitro differentiation of mesodermal cells derived from pluripotent stem cells. In one embodiment, the pluripotent stem cells are differentiated into mesodermal cells, which are then differentiated into meso-VPCs.

[0180] Vascular disease refers to any abnormal condition of blood vessels (arteries and veins). Vascular disease can occur anywhere outside the heart. The most common vascular diseases are stroke, peripheral artery disease (PAD), abdominal aortic aneurysm (AAA), carotid artery disease (CAD), arteriovenous malformation (AVM), critical limb ischemia (CLI), pulmonary embolism (blood clot), deep vein thrombosis (DVT), chronic venous insufficiency (CVI), and varicose veins. In one embodiment, the vascular disease is peripheral artery disease (PAD). In one embodiment, the vascular disease is an ischemic disease such as critical limb ischemia (CLI). In one embodiment, the vascular disease is atherosclerosis, peripheral artery disease (PAD), carotid artery disease, venous disease, blood clot, aortic aneurysm, fibromuscular dysplasia, lymphedema, or vascular injury. In one embodiment, the vascular disease is peripheral arterial disease such as critical limb ischemia (CLI), intestinal ischemia syndrome, renal artery disease, popliteal entrapment syndrome, Raynaud's phenomenon, or Buerger's disease.

[0181] The meso-VPC or pharmaceutical composition can be used to treat any vascular disease in a subject. In one embodiment, the meso-VPC or pharmaceutical composition is used to treat peripheral arterial disease. In one embodiment, the meso-VPC or pharmaceutical composition is used to treat peripheral arterial disease, including critical limb ischemia (CLI), intestinal ischemia syndrome, renal artery disease, popliteal artery entrapment syndrome, Raynaud's phenomenon, or Buerger's disease. In one embodiment, the meso-VPC or pharmaceutical composition is used to treat critical limb ischemia (CLI).

[0182] The meso-VPC or pharmaceutical composition of the present invention can be administered systemically or locally. The meso-VPC or pharmaceutical composition can be administered using modalities known in the art, such as, but not limited to, intravenous, intracranial, intramuscular, intraperitoneal injection, or other administration routes, or by local implantation, depending on the particular condition being treated. In one embodiment, the meso-VPC or pharmaceutical composition is administered intramuscularly.

[0183] The meso-VPC or pharmaceutical composition of the present invention may be administered by local implantation using a delivery device. The delivery device of the present invention is biocompatible and biodegradable. The delivery device of the present invention may be made of a biocompatible fiber, a biocompatible thread, a biocompatible foam, an aliphatic polyester, a poly(amino acid), a copoly(ether-ester), an alkylene polyoxalate, a polyamide, a tyrosine-derived polycarbonate, a poly(iminocarbonate), a polyorthoester, a polyoxaester, a polyamide ester, a polyoxaester containing an amine group, a poly(anhydride), a polyphosphazene, a biopolymer; a homopolymer and copolymer of lactide, glycolide, epsilon-caprolactone, para-dioxanone, and trimethylene carbonate; a copolymer of lactide, glycolide, epsilon-caprolactone, para-dioxanone, and trimethylene carbonate; or a copolymer of lactide, glycolide, epsilon-caprolactone, para-dioxanone, and trimethylene carbonate. -can be made using materials selected from homopolymers and copolymers of dioxanone, trimethylene carbonate, fibrillar collagen, non-fibrillar collagen, non-pepsinized collagen, other polymers, growth factors, extracellular matrix proteins, biorelevant peptide fragments, hepatocyte growth factor, platelet-derived growth factor, platelet-rich plasma, insulin growth factor, growth differentiation factor, vascular endothelial cell-derived growth factor, nicotinamide, glucagon-like peptide, tenascin-C, laminin, collagen in combination with anti-rejection agents, analgesics, antioxidants, anti-apoptotic agents, anti-inflammatory agents, and cell proliferation inhibitors.

[0184] The specific treatment regimen, route of administration, and adjuvant therapy may be adjusted based on the specific condition, the severity of the condition, and the overall health of the patient. Administration of meso-VPC or a pharmaceutical composition may be effective in reducing the severity of the condition and / or preventing further degeneration of the condition.

[0185] The treatment modalities of the present invention may involve the administration of a single dose of meso-VPC or pharmaceutical composition. Alternatively, the treatment modalities described herein may involve a course of therapy in which meso-VPC or pharmaceutical composition is administered multiple times over a period of time. Exemplary treatment courses may include weekly, biweekly, monthly, quarterly, semi-yearly, or annual treatment. Alternatively, treatment may progress in a stepwise manner, requiring multiple administrations initially (e.g., daily administration for the first week), followed by less frequent dosing.

[0186] In one embodiment, meso-VPC or a pharmaceutical composition is administered to a patient once or multiple times periodically over the patient's lifetime. In yet another embodiment of the present invention, meso-VPC or a pharmaceutical composition is administered once a year, once every 6 to 12 months, once every 3 to 6 months, once every 1 to 3 months, or once every 1 to 4 weeks. Alternatively, more frequent administration may be desirable for certain conditions or disorders. In one embodiment, meso-VPC or a pharmaceutical composition is administered using a device once, multiple times periodically over the patient's lifetime, or depending on the needs of the particular patient and the condition being treated. Treatment regimens that vary over time are also contemplated. For example, initially, more frequent treatments (e.g., daily or weekly treatments) may be required. Over time, as the patient's condition improves, less frequent treatments may be required, or even no further treatments may be necessary.

[0187] In some embodiments, about 1 x 10 4 , about 1×10 5 , about 1.5×10 5 , about 2×10 5 , about 5×105 , about 1×10 6 , about 5×10 6 , about 10 million, about 20 million, about 40 million, about 60 million, about 80 million, about 100 million, about 120 million, about 140 million, about 160 million, about 180 million, about 200 million, about 220 million, about 240 million, about 206 million 0 million, about 280 million, about 300 million, about 320 million, about 340 million, about 360 million, about 380 million, about 400 million, about 420 million, about 440 million, about 460 million, about 480 million, about 500 million, about 520 million , about 540 million, about 560 million, about 580 million, about 600 million, about 620 million, about 640 million, about 660 million, about 680 million, about 700 million, about 720 million, about 740 million, about 760 million, about 780 million, about 800 million, about 820 million, about 840 million, about 860 million, about 880 million, about 900 million, about 920 million, about 940 million, about 960 million, or about 980 million meso-VPCs are administered to a subject. In some embodiments, about 1 billion, about 2 billion, about 3 billion, about 4 billion, or about 5 billion or more meso-VPCs are administered. In some embodiments, the range of numbers of meso-VPCs is about 20 million to about 4 billion meso-VPCs, about 40 million to about 1 billion meso-VPCs, about 60 million to about 750 million meso-VPCs, about 80 million to about 400 million meso-VPCs, about 100 million to about 350 million meso-VPCs, and about 175 million to about 250 million meso-VPCs.

[0188] The methods described herein may further include monitoring the efficacy of treatment or prevention using methods known in the art. In one embodiment, administration of meso-VPC or a pharmaceutical composition increases blood flow in a subject. In one embodiment, administration of meso-VPC or a pharmaceutical composition promotes vascularization, such as angiogenesis and neovascularization, in a subject. In one embodiment, administration of meso-VPC or a pharmaceutical composition reduces the severity of ischemia in a subject. In one embodiment, administration of meso-VPC or a pharmaceutical composition reduces the area of ​​necrosis in a subject. Other physical and functional changes in a subject can also be measured and quantified to determine the efficacy of a vascular disease treatment method.

[0189] VI. kit In some embodiments, the present invention provides kits containing meso-VPC or pharmaceutical compositions of the present invention in one or more separate compartments. The kits may further contain additional components, such as gelling agents, emollients, surfactants, humectants, viscosity enhancers, or emulsifiers, in one or more compartments. The kits may optionally include instructions for formulating the meso-VPC or pharmaceutical composition for diagnostic or therapeutic applications. The kits may also include instructions for using the components individually or together in the treatment of vascular disorders and / or diseases. In one embodiment, the kits of the present invention include a syringe for injecting a pharmaceutical composition containing meso-VPC.

[0190] In some embodiments, the present invention provides kits comprising meso-VPCs of the present invention together with reagents for selecting, culturing, expanding, maintaining, and / or transplanting meso-VPCs. Representative examples of cell selection, culture, expansion, and transplantation kits are known in the art. Cells can also be enriched in a sample by using positive selection, negative selection, or a combination thereof for the expression of their gene products.

[0191] The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. All references, patents and published patent applications mentioned throughout this application and the drawings are hereby incorporated by reference. [Example]

[0192] Example 1: Culture of human pluripotent stem cells and differentiation into mesodermal cells These studies used the proprietary human embryonic stem cell (hES) line J1 and the human induced pluripotent stem cell (hiPS) line GMP1. Cells were maintained in mTeSR1 complete medium (Stem Cell Technologies) at 37°C in normoxic conditions of 5% CO2 and 20% O2 in 6-well tissue culture plates precoated with Matrigel (Corning) for feeder-free culture conditions (FF) or Matrigel + human dermal fibroblasts (HDF) for feeder-free culture conditions (HDF) (Figure 1). Medium changes were performed on days 1, 2, and 3 after cell plating (day 0). Cells were passaged on day 4 or when they reached 60–70% confluence. For passaging, 1 mL / well of dispase (1 U / ml, STEMCELL Technologies) was used for FF-cultured human pluripotent stem cells, and 1 mL / well of cell dissociation buffer (CDB) (Gibco) was used for HDF-cultured human pluripotent stem cells. Cells were incubated at 37°C for 5–7 minutes, or until the edges of the colonies lifted from the plate. The medium containing dispase or CDB was carefully aspirated from the plate, and the cells were gently washed with DMEM-F12 (Gibco) to remove any residual enzyme or buffer. Colonies were then harvested from the plate using fresh mTeSR1 complete medium, using vigorous washing to avoid creating bubbles, using a disposable cell scraper, followed by centrifugation at 300 × g for 5 minutes at room temperature (RT) to obtain a cell pellet. After removing the supernatant, the cell pellet was resuspended in mTeSR1 complete medium, and 1 mL of this homogenously mixed cell suspension was added to each well of a 6-well tissue culture plate (pre-coated with Matrigel for FF culture or pre-coated with Matrigel + HDF as described above) containing 2 mL of mTeSR1 complete medium. Approximately 500,000 cells in small cell clumps for FF culture and 250,000 cells in small cell clumps for HDF culture were evenly distributed into each well.The cells were then spread into the wells by multiple side-to-side movements without swirling. Cultures were checked daily for growth quality and morphology.

[0193] For differentiation of pluripotent stem cells into mesodermal cells, Matrigel-precoated 10-cm tissue culture dishes (Corning) were prepared by adding 5 mL of Matrigel per dish. After removing unattached Matrigel from each dish, 10 mL of mTeSR1 complete medium per 10-cm dish was immediately added to prevent the Matrigel-coated surface from drying out. Approximately 1.5 million cells in small cell clumps from FF-cultured or HDF-cultured GMP1 cell cultures (or approximately 300,000 cells in small cell clumps from HDF-cultured J1 cell cultures per 10-cm dish) were evenly distributed in 10 mL of TeSR1 complete medium into each Matrigel-precoated 10-cm dish. The cells were then spread across the dish by multiple side-to-side movements without swirling, and the plates were then incubated for 24 hours (D-1) at 37°C under normoxic conditions of 5% CO2 and 20% O2 (Figure 1). At D0 of differentiation, mTeSR1 complete medium was replaced with 12 mL / 10 cm dish of Stemline II medium (Sigma) containing a cocktail of mesoderm-inducing growth factors Activin A (10 ng / mL; Humanzyme), FGF-2 (10 ng / mL; Humanzyme), VEGF165 (10 ng / mL, Humanzyme), and BMP4 (25 ng / mL, Humanzyme). At D1 of differentiation, Activin A was removed from the mesoderm cocktail, and the medium was replaced with 12 mL / dish of fresh Stemline II medium containing FGF-2 (10 ng / mL), VEGF165 (10 ng / mL), and BMP4 (25 ng / mL) to promote the emergence and expansion of mesodermal cells. The final medium change was performed on day 3 of differentiation by adding 15 mL / dish of fresh Stemline II medium containing FGF-2 (10 ng / mL), VEGF165 (10 ng / mL), and BMP4 (25 ng / mL). Culture was continued under normoxic conditions of 5% CO2 + 20% O2 at 37°C until day 4 (Figure 1). Cells were then harvested by dissociating them into single cells using Stempro Acutase enzyme (Gibco).Cell characterization (by FACS and q-PCR analysis) confirmed the presence of mesodermal features in cells collected on D4 (Figure 2A-B).

[0194] Example 2: Differentiation of human mesodermal cells into vascular progenitor cells (MESO-VPCs) using a 3D-vasclonoid differentiation platform A novel 3D vasculoid differentiation platform was developed by suspending mesodermal cells obtained in Example 1 in VPC differentiation medium in the presence of factors promoting the emergence and expansion of vascular progenitor cells using ultra-low attachment tissue culture dishes (Corning) (Figure 3). On D0, one million unsorted D4 mesodermal cells were suspended in each well of an ultra-low attachment 6-well plate and differentiated in VPC 3D differentiation medium (Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4, 10 μM SB431542, with or without 2 μM forskolin ("Meso-3D vasculoid VPC1" protocol) or without forskolin ("Meso-3D vasculoid VPC2" protocol)) under normoxia (37°C, 5% CO2, and 20% O2). The respective media were changed to D2 and D4, and differentiation culture was completed on day 5. After 5 days of differentiation, MESO-VPCs from both protocols were harvested by dissociation into single cells using Stempro Acutase enzyme. Cells were then counted, viability determined, and cryopreserved.

[0195] Example 3: Differentiation of human mesodermal cells into vascular progenitor cells (MESO-VPCs) using a 2D differentiation platform We also developed a novel 2D-based VPC differentiation platform by seeding mesodermal cells produced according to Example 1 onto an adherent human extracellular matrix (collagen IV-coated tissue culture dishes). On D0, 1.2 million unsorted D4 mesodermal cells (from above) were seeded onto human collagen IV-coated (5 mg / cm) T-175 flasks (Corning) and differentiated in VPC 2D differentiation medium using two different (Meso-2D VPC2 and Meso-2D VPC3) differentiation protocols (Figure 4). For the Meso-2D VPC2 protocol, Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, and 25 ng / mL BMP4 was used on Day 0 (40 mL / flask), and Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, and 25 ng / mL BMP4 + 10 μM SB431542 was used from Day 1 (45 mL / flask) to Day 7. For the Meso-2D VPC3 protocol, Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4, and 2 μM forskolin was used on Day 0. From Days 1 to 7, Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4, and 2 μM forskolin + 10 μM SB431542 was used. D0 cells were cultured under normoxic conditions (37°C, 5% CO2, and 20% O2). D1–D7 cells were cultured under hypoxic conditions (37°C, 5% CO2, and 5% O2), with medium changes performed on Days 3 (50 mL / flask) and 5 (60 mL / flask) of differentiation. After 7 days of differentiation, MESO-VPCs from the Meso-2D VPC2 and Meso-2D VPC3 protocols were harvested as single cells by enzymatic dissociation using Stempro Acutase enzyme, and then counted and viability determined as described in Example 2 before cryopreservation.

[0196] Example 4: Matrigel / AcLDL assay Cryopreserved meso-VPCs from Examples 2 and 3 were rapidly thawed (2–3 min) in a 37°C water bath. The cells were then transferred to a 15 mL conical tube with 10 mL of endothelial cell medium, i.e., EC medium (VascuLife® VEGF Medium, LifeLine Cell Technology), and centrifuged at 300 × g for 5 min. After centrifugation, the supernatant was removed, and the cells were resuspended in 1 mL of fresh EC medium for cell counting. Cells were counted using trypan blue and a Nexcelom Bioscience K2 Cellometer. A total of 18 mL of cell suspension was prepared using EC medium at a concentration of 10,000–20,000 viable MESO-VPCs / mL. Cells for Matrigel and AcLDL uptake assays were prepared by plating 3 mL / well of this cell suspension onto fibronectin (FN)-coated 6-well plates under normoxic conditions (37°C, 5% CO2, and 20% O2) for 3–4 days.

[0197] For the Matrigel / AcLDL assay, 250 μL of basement membrane Matrigel (Corning) was added to each well of a Nunc™ 4-well plate (Thermo Scientific), and the plate was incubated at RT for 30 min. Once coated, cells were plated at 5.0 × 10 cells per well in 250 μL of EC medium. 4 Cells were seeded at a density of 100 μL / well. After 2–3 h of plating, the medium was replaced with 250 μL of fresh EC medium containing AcLDL (Molecular Probes) (5 μL AcLDL + 245 μL EC medium). The plates were incubated overnight under normoxic conditions. After 24 h of incubation, the AcLDL-containing medium was removed, the plates were washed three times with D-PBS, and 250 μL of fresh EC medium was added per well. Finally, photomicrographs of each well were taken at 4x magnification using a Keyence microscope.

[0198] Example 5: Flow cytometry assay Cryopreserved vials of Meso-3D vasculoid VPCs harvested on day 5 and Meso-2D VPCs harvested on day 7 (obtained in Examples 2 and 3 above) were thawed and prepared as single-cell suspensions in EC medium for cell counting. After cell counting, cells were resuspended in FACS buffer (D-PBS containing 2% FBS). For surface marker antibody staining, aliquots of cells (100,000–200,000 cells / FACS assay sample) were prepared in 100 μL of FACS buffer. Anti-human CD31 / PECAM1 (BioLegend), CD34 (BD Biosciences), CD144 / VE-Cadh (BioLegend), CD309 / KDR (BioLegend), CD43 (BD ​​Biosciences), CD45 (BD Biosciences), CD184 / CXCR4 (BD Biosciences), CXCR7 (BioLegend), CD146 (BioLegend), NG2 (BD Biosciences), and PDGFRb (BioLegend) monoclonal antibodies were used at 5 μL per sample in a total volume of 100 μL. Cells were incubated with antibodies on ice for 20–30 min. After incubation, cells were washed with 1 mL of FACS buffer to remove unbound antibodies. The cells were then centrifuged at 300 × g for 5 minutes, the supernatant removed, and resuspended in 100 μL of fresh FACS buffer containing propidium iodide (PI, Sigma) at a dilution of 1:1000. PI was added to the cell suspension to exclude dead cells during FACS analysis. A Sony SA3800 spectral analyzer was used for analysis. Compensation was established by using a positive control (HUVEC) and a negative control (undifferentiated J1 or GMP1 cells).

[0199] Example 6: Comparative cells For comparison with the meso-VPCs of the present invention, hemogenic endothelial cells (HE) and hemangioblasts (HB) were generated from human embryonic stem cells (e.g., J1 hESCs) or human induced pluripotent stem cells (e.g., GMP-1 iPSCs) using HE and HB protocols described, for example, in U.S. Patent No. 9,938,500, U.S. Patent No. 9,410,123, WO 2013 / 082543, WO 2014 / 100779, U.S. Patent No. 9,993,503, and U.S. Provisional Application No. 62 / 892,712 (filed August 28, 2019) and its PCT applications claiming priority thereto, all of which are incorporated by reference in their entireties. Briefly, to generate HE, hESCs or iPSCs were dissociated with Gibco® Cell Dissociation Buffer (CDB) to obtain single-cell aggregates. Cells were resuspended at a final density of 400,000 cells / 10 mL in mTeSR™1 medium (STEMCELL Technologies) containing Y-27632 (Stemgent) at a final concentration of 10 μM. 10 mL of this cell suspension was transferred to collagen IV-coated 10 cm plates (day -1). The plates were placed in a normoxic incubator overnight. The following day (day 0), the mTeSR™1 / Y-27632 medium was gently removed from each 10 cm plate and replaced with 10 mL of BVF-M medium [Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma); 25 ng / mL BMP4 (Humanzyme); 50 ng / mL VEGF165 (Humanzyme); 50 ng / mL FGF2 (Humanzyme)]. The plates were incubated in a hypoxic chamber (5% CO2 / 5% O2) for 2 days. On day 2, the medium was aspirated, and 10-12 mL of fresh BVF-M was added to each 10 cm plate. On day 4, the medium was aspirated again, and 10-15 mL of fresh BVF-M was added to each 10 cm plate. On day 6, cells were harvested for transplantation and / or further testing. The medium was aspirated from each plate, and the plates were washed by adding 10 mL of D-PBS (Gibco) and aspirating the D-PBS.Five mL of StemPro Accutase (Gibco) was added to each 10 cm plate and incubated in a normoxic CO2 incubator (5% CO2 / 20% O2) for 3–5 minutes. The cells were pipetted five times with a 5 mL pipette, followed by approximately five times with a P1000 pipette. The cells were then strained through a 30 μM cell strainer and transferred to a collection tube. Each 10 cm plate was rinsed again with 10 mL of EGM-2 medium (Lonza) or Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma), and the cells were passed through a 30 μM cell strainer and collected into a collection tube. The tubes were centrifuged at 120–250 × g for 5 minutes. The cells were then resuspended in EGM-2 medium or Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma) and counted. After counting, cells were spun down (250 x g, 5 min) and placed in freezing medium (10% DMSO + heat-inactivated FBS) at 3 x 10 6 The cells were resuspended at a concentration of 6 × 10 cells / mL. To make frozen stocks, the cell suspension was dispensed into 2 mL of FBS (Hyclone) and DMSO (Sigma) per cryovial. 6 cells / 2mL / vial).

[0200] To generate hemangioblasts (HBs), hESCs or iPSCs were dissociated with 4 mg / mL collagenase IV (Gibco) to obtain cell clumps. They were then resuspended in BV-M medium [Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma), 25 ng / mL BMP4 (Humanzyme), 50 ng / mL VEGF165 (Humanzyme)] and plated at a density of approximately 750,000–1,200,000 cells per well in ultra-low attachment surface 6-well plates (Corning). Embryoid bodies were formed by incubating the plates in a normoxic CO2 incubator for 48 hours (days 0–2). The medium and cells in each well were then collected and centrifuged at 120–300 g for 3 minutes. Half of the supernatant was removed and replaced with 2 mL of BV-M containing 50 ng / mL bFGF. The final concentration of bFGF in the cell suspension was therefore approximately 25 mg / mL. Four mL of the cell suspension was plated into each well of an ultra-low attachment 6-well plate and placed in a normoxic CO2 incubator for an additional 48 hours (days 2–4) to allow for continued embryoid body formation. On day 4, embryoid bodies were collected into 15 mL tubes, centrifuged at 120–300 × g for 2 minutes, washed with D-PBS, and dissociated into a single-cell suspension using StemPro Accutase (Gibco). The Accutase was inactivated using FBS (Hyclone), and single cells were passed through a cell strainer, centrifuged, and dissociated to approximately 1 × 10 6 The cells were resuspended in Stemline II medium (Sigma) at approximately 3 × 10 cells / mL. 6Cells were mixed in 30 mL of Methocult BGM medium [Methocult™ SF H4536 (without EPO) (StemCell Technologies), penicillin / streptomycin (Gibco), ExCyte cell growth supplement (1:100) (Millipore), 50 ng / mL Flt3 ligand (PeproTech), 50 ng / mL VEGF (Humanzyme), 50 ng / mL TPO (PeproTech), 30 ng / mL bFGF (Humanzyme)], replated onto ultra-low attachment 10 cm dishes (Corning), and incubated in a normoxic CO2 incubator for 7 days (days 4–11) to allow hemangioblast formation. On day 11, hemangioblasts were harvested for transplantation and / or further testing. Hemangioblasts were collected by diluting the methylcellulose with D-PBS (Gibco). The cell mixture was centrifuged twice at 300 × g for 15 min, resuspended in 30 mL of EGM2 BulletKit medium (Lonza) or Stemline II, and cells were counted and frozen as described above.

[0201] Example 7A: 3D vasculoid differentiation platform generates cells with vascular progenitor properties As described in Example 2, two different 3D differentiation protocols (Meso-3D Vasculonoid VPC1 and Meso-3D Vasculonoid VPC2) were used to generate meso-VPCs under normoxic conditions (37°C, 5% CO2, and 20% O2) for 5 days (Figure 3). Seeded mesodermal cells remained viable and formed cell aggregates as early as day 1 (data not shown). These cell aggregates (hereafter referred to as "vasculonoids") increased in size until day 5 (Figure 5, top), at which point they were harvested. The Meso-3D Vasculonoid VPC1 protocol yielded larger vasculonoid aggregates compared to the Meso-3D Vasculonoid VPC2 protocol. After cell harvest, cells were replated on FN-coated plates to determine their ability to undergo further differentiation toward the endothelial lineage. As shown in Figure 5, middle panel, when cells were cultured on FN-coated plates in a medium that promotes endothelial differentiation, they acquired the cobblestone morphology characteristic of endothelial cells. Meso-3D VPCs also exhibited a robust ability to form capillary-like networks on Matrigel and showed AcLDL uptake (Figure 5, bottom panel). VPC2 cells exhibited enhanced tube-forming potential compared to VPC1 cells (Figure 5, bottom panel).

[0202] Additionally, FACS analysis of vascular markers revealed that both J1- and GMP1-derived Meso-3D vasculonoid VPC1 and VPC2 cells showed robust expression (>20%) of endothelial markers KDR, CD31, and endothelial / pericyte (CD146) (Figure 6A), as well as low expression of the hematopoietic marker CD43. Their broad vascular marker expression profile differed from that observed in undifferentiated pluripotent stem cells (GMP1 and J1) or HUVEC cells and other PSC-derived cells (e.g., HB and HE) (Figure 6B-C). The chromosomal stability of these differentiated cells was assessed by G-banding karyotyping, and the cells displayed normal karyotypes, indicating that differentiation of hES and hiPSCs using the Meso-3D vasculonoid VPC1 and Meso-3D vasculonoid VPC2 protocols did not alter chromosomal stability during differentiation (data not shown).

[0203] Example 7B: 2D differentiation platform generates cells with vascular progenitor properties As described in Example 3, meso-VPCs were generated from iPS cells (GMP1) and hES cells (J1) using two different 2D differentiation protocols (Meso-2D VPC2 and Meso-2D VPC3) under normoxic and hypoxic culture conditions for a total of 7 days (Figure 4). Seeded mesodermal cells attached to the collagen IV-coated surface, grew, and expanded into larger, more compact cell colonies by day 7 (harvesting day) in 2D differentiated adherent cell culture (Figure 7, top panel). The Meso-2D VPC2 protocol produced more compact cell colonies (the colonies were more "spiky" or "swirly") for both J1- and GMP1-derived cells compared with the Meso-2D VPC3 protocol. After cell harvesting on day 7, further culture on FN-coated plates and exposure to endothelial culture medium resulted in cells exhibiting typical vascular precursor properties, including a cobblestone endothelial-like morphology (Figure 7, middle panel) and the ability to form capillary-like networks on Matrigel and take up AcLDL (Figure 7, bottom panel), although at a smaller scale than meso-3D cells (compare Figure 5 and bottom panel of Figure 7).

[0204] Additionally, FACS analysis of vascular markers demonstrated that both J1- and GMP1-derived Meso-2D VPC1 and Meso-2D VPC2 cells exhibited high expression of CD146, robust expression of the endothelial markers KDR and CD31 (>20%), and detectable expression of PDGFRb (10–40%). This expression profile was distinct from that observed in undifferentiated pluripotent stem cells or HUVEC cells and other PSC-derived vascular progenitor cells (e.g., HB and HE) (Figures 6B and 6C). Compared with Meso-3D cells, Meso-2D VPCs expressed higher levels of CD146 and showed distinct expression of PDGFR, suggesting a greater propensity for pericyte differentiation (Figures 6A–B). Furthermore, unlike Meso-3D cells, Meso-2D VPCs did not express the blood markers CD45 or CD43.

[0205] Example 8: Single-cell miRNA profiling Further analysis using single-cell qRT-PCR to assess the expression levels of 96 microRNAs associated with pluripotency or vascular cell identity was performed as described below. TaqMan gene expression assays (Applied Biosystems) were ordered for 96 human miRNAs. A 10x assay was prepared by mixing 25 μL of 20x TaqMan assay with 25 μL of 2x Assay Loading Reagent (Fluidigm) for a final stock volume of 50 μL. Aliquots of cells (frozen or freshly harvested) ranging from 66,000 to 250,000 cells / mL were prepared. Cells were incubated with LIVE / DEAD staining solution (LIVE / DEAD Viability / Cytotoxicity Kit) for 10 minutes at room temperature. Cells were then washed, suspended in medium, and filtered through a 40 μm filter. Cell counts were performed using a cellometer to obtain viability and cell concentration. A cell mixture was prepared by mixing cells (60 μL) with suspension reagent (40 μL) (Fluidigm) at a 3:2 ratio. Six μL of the cell suspension mixture was loaded onto a primed C1 Single-Cell Autoprep IFC microfluidic chip for medium cells (10–17 μm) or large cells (17–25 μm). The chip was then processed on a Fluidigm C1 instrument using the "STA:Cell Load (1782x / 1783x / 1784x)" script. This process captured one cell in each of the 96 capture chambers. The chip was then transferred to a Keyence microscope, and each chamber was scanned to score the number of single-cell captures, cell live / dead status, and captured doublets / cell aggregates.For cell lysis, reverse transcription, and preamplification in C1, Harvest reagent, Lysis final mix, RT final mix, and Preamp mix were added to designated wells of the C1 chip according to the manufacturer's protocol. The IFC was then placed in the C1 chip and the "STA:miRNA Preamp (1782x / 1783x / 1784x)" script was used. cDNA recovery was programmed to finish the following morning. The cDNA was transferred from each chamber of the C1 chip to a fresh 96-well plate preloaded with 12.5 μL of C1 DNA Dilution Reagent. Tube controls, including no template and positive controls, were prepared for each experiment according to the manufacturer's instructions. The preamplified cDNA samples were analyzed by qPCR using a 96.96 Dynamic Array™ IFC and a BioMark™ HD system. The process of IFC priming in the JUNO instrument, followed by loading of the cDNA sample mix and 10x assay, was performed according to the manufacturer's protocol. The IFC was then placed in the Biomark™ HD system and the "GE96x96 miRNA Standard" script was used. PCR was performed using the Real-Time PCR Analysis Software (v1.pcl). Data analysis was performed using the real-time PCR analysis software provided by Fluidigm. Dead cells, duplicates, etc. were removed from the analysis, and the Linear Derivative Baseline and User Detector Ct Threshold based methods were used for analysis. Data were displayed in a heatmap diagram and exported as a CSV file. Next, an "FSO" file was obtained by performing "Outlier Identification" analysis using "R" software, and then the "Automatic Analysis" instructions were followed.

[0206] result (Table 1) miRNA profile TIFF2026001090000002.tif97164

[0207] As shown in Table 1, MESO-VPCs (3D or 2D) were negative for pluripotent stem cell miRNA markers (mir376, mir302a, mir302b, and mir302c) and positive for endothelial miRNA markers such as mir126, mir125a-5p, and mir24, which are expressed in HUVECs. Nevertheless, MESO-VPCs were negative for HUVEC-specific miRNAs (mirLet7-e, mir223, and mir99a). Finally, MESO-VPCs showed unique expression of miRNA 483-5P and were negative for HB- and HE-specific miRNAs mir142-3p and 133a, respectively.

[0208] Example 9: In vivo testing in a hindlimb ischemia model Peripheral arterial disease (PAD) is a form of peripheral vascular disease (PVD) characterized by partial or complete blockage of blood flow to the limbs, usually the lower limbs, resulting in impaired blood flow and hypoxia in tissues. PAD progresses to the stage of critical limb ischemia (CLI), which is accompanied by skin ulceration, gangrene, and unavoidable amputation. Animal models of hindlimb ischemia have been used to evaluate various therapeutic approaches. In this study, we used a stable critical ischemia model (Ishikane et al. (2008) Stem Cells, 16:2625-2633) to evaluate the efficacy of meso-VPCs and demonstrate improved blood flow restoration and signs of donor cell integration in the ischemic limb. Induction of hindlimb ischemia in mice involves two ligatures at the proximal ends of the iliac and femoral arteries and subsequent transection between the two ligatures. This procedure results in impaired blood flow and subsequent severe ischemic injury.

[0209] seed Mice were 6-8 weeks old at the start of the study, with minimum and maximum body weights within ±20% of the group mean body weight. nu (Charles River Laboratories).

[0210] Test Article Test Item 1 = J1-HDF Meso-2D VPC2 prepared according to Example 3

[0211] Test Item 2 = J1-HDF Meso-3D Vasculonoid VPC2 prepared according to Example 2

[0212] Test Item 3 = GMP-1-HDF Meso-2D VPC2 prepared according to Example 3

[0213] Test Item 4 = GMP1-HDF Meso-3D Vasculonoid VPC2 prepared according to Example 2

[0214] Test Item 5 = GMP1-HDF Meso-3D Vasculonoid VPC1 prepared according to Example 2

[0215] Vehicle (negative control) GS2 (cell-free medium described in WO 2017 / 031312, incorporated herein by reference in its entirety) [552.2 mL of GS2: 0.9% Sodium Chloride Wash USP (Baxter Healthcare or Hospira) (408.6 mL); 5% Dextrose / 0.9% Sodium Chloride Injection USP (Baxter or Braun) (33.2 mL) and BSS Wash Solution (Alcon) (110.4 mL)].

[0216] Study Design and Timeline The study was conducted according to the following study design (Table 2) and timeline (Table 3).

[0217] (Table 2) Study design TIFF2026001090000003.tif96164IM=Local intramuscular injection into the ischemic limb

[0218] (Table 3) Timeline TIFF2026001090000004.tif84164

[0219] Experimental procedure Morbidity and mortality observations Animals were continuously monitored during the day of surgery and twice daily thereafter (once daily on weekends).

[0220] body weight Body weights were recorded before treatment and weekly thereafter.

[0221] HLI surgery Under anesthesia and analgesia, the mice were placed in a supine position.

[0222] On the day of surgery (day 0), an incision was made in the skin of the right hind limb at the groin. The femoral artery was ligated twice with 6-0 silk suture and transected between the two ligatures. The wound was closed with 5-0 Vicryl absorbable suture, and the mice were allowed to recover.

[0223] Test item administration procedure Immediately after surgery on day 0, each animal received two intramuscular injections, one proximal and one distal to the surgical wound. Animals received 50 μl at each site, for a total of 100 μl per mouse. The total volume per mouse was 1 M cells / mouse.

[0224] Blood flow measurement procedure Blood flow in both hind limbs of each mouse was measured with a non-contact Peri-Med laser Doppler before surgery, immediately after surgery, and immediately before treatment for inclusion criteria (only animals with at least a 30% reduction in blood flow compared to the intact hind limb were included), as well as on post-surgery days 7, 14, 21, 28, and 35. Blood flow measurements were expressed as the ratio of flow in the ischemic limb to that in the normal limb after surgery and the ratio of flow in the right limb to that in the left limb.

[0225] Vascular Imaging Procedures Vascular imaging in both lower limbs (femoral and tibial regions) of three mice per group at three time points (7, 21, and 35 days after surgery) was performed using an RSOM Explorer P50 (i-Thera Medical) imaging system. The RSOM (Raster Scanning Optoacoustic Mesoscopy) Explorer P50 operates with 532 nm nanosecond laser pulses and a spherically focused 50 MHz detector. An 80-second acquisition time allowed imaging with a 5 × 5 mm field of view, a 3 mm depth, and 40 μm / 10 μm axial / lateral resolution.

[0226] Macroscopic assessment procedure for ischemic severity Macroscopic assessment of the ischemic limb was performed weekly starting from day 7 after surgery by using a morphological grade for the necrotic area according to Table 4 (see Goto et al. Tokai J. Exp. Clin. Med. 2006. 31:128-132).

[0227] Table 4. Morphological grade of necrotic areas TIFF2026001090000005.tif31138

[0228] In vivo assessment procedure for limb function Semiquantitative assessment of ischemic limb disability was performed weekly starting from day 7 after surgery using the scale in Table 5 below (see Stabile et al. Circulation. 2003. 108:205-210).

[0229] Table 5: Limb function assessment TIFF2026001090000006.tif26128

[0230] In cases of partial or complete limb amputation, limb function was graded as "not applicable" or "N / A." In such cases, blood flow measurements were not included in the statistical analysis.

[0231] Animal sacrifice and tissue fixation Mice were sacrificed on day 36. Gastrocnemius muscles were collected from both hind limbs, fixed in formalin, and embedded in paraffin (five animals per group). Muscles from three animals per group were embedded in OCT, frozen, and stored for further transportation. Embedded muscle samples were sectioned, stained with H&E+IHC isolectin B4-HRP conjugate, and evaluated by a pathologist. IHC was performed with a human-specific antibody (Stem121) for the presence of human cells in the tissue. ICH staining for CD34 and vascular density assessment were performed.

[0232] result death Fourteen animals died during the study, including one during surgery. Thirteen were found dead in their cages within 11 days of HLI surgery. Mice 19, 40, 99, 100, and 101 were in group 1M; 97 was in group 2M; 69, 72, 88, and 89 were in group 4M; 38 and 50 were in group 6M; and 28 was in group 7. Twenty mice were euthanized for humane reasons by lower limb amputation (mouse numbers 58 and 98 in group 2M; mouse numbers 61, 63, 64, 90, 91, 92, 93, and 95 in group 3M; mouse number 81 in group 4M; mouse number 21 in group 5M; mouse numbers 36, 37, 47, and 53 in group 6M; and mouse numbers 29, 30, 32, and 34 in group 7M). All surviving animals at each time point were evaluated at that time point.

[0233] body weight Body weight was monitored until study day 35. After surgery, body weight decreased during the first week but began to recover during the second week, with almost complete recovery by the final week. All animal groups recovered in parallel. Two-way ANOVA followed by Bonferroni post-hoc comparisons performed using GraphPad Prism 5 software revealed no statistically significant differences in body weight among all groups.

[0234] blood flow measurement Blood flow was assessed prior to test item treatment, and significant changes were observed afterward in all animals that underwent surgery. Significant improvements in blood flow were observed throughout the study in all treatment groups (3-7M) compared to the vehicle-treated group (2M). This improvement was statistically significant (two-way ANOVA followed by Bonferroni multiple comparisons) for the operated right limb from day 21 in the 3M group and from days 28 to 35 in the other treatment groups (Figure 8).

[0235] Vascular Imaging Vascular imaging in both lower limbs (femur and tibia) of three mice per group at three time points (7, 21, and 35 days after surgery) was measured using an RSOM Explorer P50 (i-Thera Medical) imaging system.

[0236] Several analytical methods were used to evaluate possible increases in small vessel density in the ischemic hindlimb. Finally, the integration of the highest 100 sections was used to more reliably assess vascular vascularization. Results were presented as a summary at day 35 as a percentage of day 7. To clarify the data, the average of all groups was presented compared to the increase or decrease from the vehicle group. Throughout the study, improvements in small vessel density were observed in the treatment groups (3, 4, 6, and 7M) compared to the vehicle-treated group (2M) (Figure 9).

[0237] Macroscopic assessment of ischemic severity The ischemic limbs were macroscopically assessed by a graded morphological scale for necrotic areas from day 7 to day 35. Paw amputations were observed in animals of all groups, with the least frequent in groups 4M and 5M (see Tables 6 and 7).

[0238] Table 6. Incidence of mice with limb necrosis scores of 0, 1, and 2 on day 7 TIFF2026001090000007.tif46161

[0239] Table 7. Incidence of mice with limb necrosis scores of 0, 1, and 2 at day 35 TIFF2026001090000008.tif46161

[0240] Limb function assessment Semiquantitative assessment of ischemic limb impairment was performed from day 7 to day 35 using a graded functional scale. Spontaneous improvement in limb function was found in all animal groups. Nevertheless, animals treated with test items in groups 4M and 5M showed better functional improvement than the vehicle-treated (2M) control group (see Tables 8 and 9).

[0241] Table 8. Incidence of mice with limb function scores of 0, 1, 2, and 3 on day 7 TIFF2026001090000009.tif61156

[0242] Table 9. Incidence of mice with limb function scores of 0, 1, 2, and 3 at day 35 TIFF2026001090000010.tif61156

[0243] Histological examination results All slides were stained with H&E and Masson's Trichrome stains and examined by a single pathologist. This evaluation was performed as a semiquantitative analysis (see grades below). CD34 + High-resolution histological images were transferred for quantitative image analysis.

[0244] Muscle atrophy grade: 0 = no atrophy at all. 1 = Very mild atrophy (up to 10% of muscle fibers) 2 = Mild atrophy (>10% and <25% of muscle fibers) 3 = Moderate atrophy (>25% and <75% of muscle fibers) 4 = Severe atrophy (>75% and <100% of muscle fibers)

[0245] Inflammation (macrophages and satellite cells) grade: 0 = no inflammatory infiltrate at all. 1 = mild cellular infiltration, increasing to 10 cells per × 20 HPF 2 = moderate cellular infiltration, an increase of 10–20 cells per × 20 HPF 3 = Severe cellular infiltration, with an increase of 20–50 cells per × 20 HPF 4 = very severe cellular infiltration with an increase of >50 cells per ×20 HPF

[0246] Moderate to severe atrophy of muscle fibers was observed in all animal groups. Degenerative adipose changes in muscle cells and increases in satellite cells and macrophages were observed. In some cases, there was a marked increase in fibrous tissue and lymphocytic infiltration. A few animals also showed some dystrophic mineralization. Groups 2M and 3M generally showed more severe changes compared to groups 4M, 5M, and 6M. Group 7M showed intermediate changes.

[0247] Immunohistochemistry and capillary density analysis Stained sections were evaluated and photographed using a fluorescence microscope (E600, Nikon, Tokyo, Japan) equipped with a Plan Fluor objective connected to a CCD camera (DMX1200F, Nikon). Cy3 exhibits bright red fluorescence: Ex(max): 543 nm; Em(max): 570 nm. Fluorescein dextran, on the other hand, exhibits strong green fluorescence (Ex(max): 488 nm; Em(max): 530 nM). Digital images were collected and analyzed using Image Pro+ software. Two sections of muscle samples were taken from the same area of ​​five animals in groups 1M and 7M. The area of ​​blood vessels was measured. The density was expressed as the average number of capillaries per field. Total vessels refers to all vessels in the measured area. The number of CD-34-positive capillaries was higher in all treatment groups compared to the control group 2M on day 36 of the study. Since CD-34 positive staining is considered an indicator of small capillary formation, the results obtained support the improved blood flow observed in the cell-treated animals. There was a strong, statistically significant correlation between blood flow and capillary density measured by laser Doppler (see Figures 10 and 11).

[0248] Consideration IM administration of the test item to the ischemic limb showed some improvement in limb function, blood flow (monitored by laser Doppler), RSOM imaging, and quantitative vascular histology, primarily in treatment groups 4M and 5M. Treatment restored blood perfusion (to 78% of its normal value in the best group, 4M) by the end of the study (day 36) in all treatment groups compared with the vehicle-treated group. This blood perfusion recovery correlated well with the results of RSOM imaging analysis and immunohistochemistry for capillary density in the operated hind limb. The rating of each group showed that 4M was the best, followed by 6M and 7M. STEM121 staining of paraffin-embedded gastrocnemius slides failed to demonstrate human stem cells, although they were visualized in the quadriceps muscle near the injection site.

[0249] Example 10: Bulk small RNA-seq analysis of Meso-3D vasculoid VPC2 cells Meso-3D vasculoid VPC2 cells were generated as described in Example 2. Pellets of approximately 1-2 million cells were lysed, and the isolated RNA was sequenced, bioinformatically aligned, and small RNA expression was analyzed for the known human transcriptome (approximately 2,000 miRNAs). Figure 12A shows the unique human miRNAs found in populations of J1-derived Meso-3D vasculoid VPC2 cells from three replicates, including hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p, compared to populations of J1 cells and J1-derived HE cells. Figure 12A also shows the unique human miRNAs found in the J1-derived HE cell population, including hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p.

[0250] Additionally, bulk small RNA-seq analysis revealed that miR214 was highly expressed in both J1-derived HE and meso 3D vasculoid VPC2 cells, miR335-5p was highly expressed in J1 and J1-derived HE cells, whereas miR335-3p was highly expressed in J1-derived HE and meso 3D vasculoid VPC2 cells. Similarly, miR199a-3p was highly expressed in both J1-derived HE and meso 3D vasculoid VPC2 cells (data not shown).

[0251] Figure 12B shows the miRNA expression levels in the J1-derived Meso-3D vasculoid VPC2 cell population, previously analyzed in single cells. Figure 12B shows that hsa-miR-126-5p, hsa-miR-125a-5p, and hsa-miR-24-3p are expressed in both the J1 cell population and the J1-derived Meso-3D vasculoid VPC2 cell population. Figure 12C shows that the J1-derived Meso-3D vasculoid VPC2 cell population expresses hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, and hsa-miR-142-3p, but does not or only poorly express hsa-let-7e-3p, hsa-miR-99a-3p, and hsa-miR-133a-5p. Furthermore, Figure 12D shows that a population of J1-derived Meso-3D vasculoid VPC2 cells expresses hsa-miR-483-5p and hsa-miR-483-3p.

[0252] Example 11: Single-cell RNA-seq analysis of Meso-3D vasculoid VPC2 cells Single-cell RNA-seq analysis was also performed on J1-derived Meso-3D vasculoid VPC2 cells generated according to Example 2. Approximately 3,700–8,000 single cells for each cell type (J1 cells, J1-derived Meso-3D vasculoid VPC2 cells, and HUVECs) were captured, processed, and analyzed for single-cell sequencing using the 10X Genomics (Pleasanton, CA) platform and its Cell Ranger analysis pipeline. Further data QC and analysis were performed using the R package Seurat (Butler et al., Nature Biotechnology 36:411–420 (2018); Stuart et al., Cell 177:1888–1902 (2019)). One such analysis was an integrated analysis of J1 cells, J1-derived Meso-3D vasculoid VPC2 cells, and HUVECs to identify the top differentially expressed genes among these three samples. This analysis followed the guidelines described in Stuart et al., Cell 177:1888-1902 (2019) and https: / / satijalab.org / seurat / v3.0 / pancreas_integration_label_transfer.html. Only genes expressed in ≥10 cells and cells with at least 200 detected genes were retained. Figure 13 shows the expression of the most up- or down-regulated genes in J1-derived Meso-3D vasculoid VPC2 cell samples compared to single J1 or single HUVEC cells.

[0253] Example 12: Vasculonoids exhibit increased cell viability in vitro and are effective in vivo Vasculonoids of J1-derived Meso-3D vasculoid VPC2 cells were generated according to Example 2, except that the cells were cryopreserved without dissociation into single cells so that the cells maintained their aggregated morphology.

[0254] Approximately 150 undissociated Meso-3D vasculoid VPC2 (equivalent to approximately 1,500,000 dissociated single cells) were mixed with collagen I and growth factor-reduced Matrigel at a 1:1 ratio in four wells of a 96-well plate. The gel was allowed to solidify at 37°C for 30 minutes before being overlaid with 50 μl of complete VascuLife® basal medium (Lifeline® Cell Technology, Frederick, MD) supplemented with 20 ng / mL FGF, 25 ng / mL BMP4, 45 ng / mL VEGF, and 10 μM SB431542. The vasculoids were cultured for 14 days. The gels were fixed with 4% PFA, permeabilized with 0.05% Triton-X for up to 4 hours, and stained overnight with rhodamine-conjugated Ulex europaeus I (UEA1), a human-specific endothelial cell marker. Gels were washed extensively and counterstained with the nuclear marker DAPI. Gels were imaged using a Leica SP8 confocal microscope. Figure 14A shows at low magnification (10x objective) an extensive vascular network extending from embedded aggregates of J1-derived Meso-3D vasculoid VPC2 vasculoids after 14 days.

[0255] Dissociated (i.e., "single cell") or undissociated (i.e., vasculoid or "plural") Meso-3D vasculoid VPC2 cells were then seeded in 100 μl of medium into tissue-culture-treated 96-well plates (approximately 14,000 single cells per well) or ultra-low-attachment 96-well plates (approximately 70 multicellular cells per well, i.e., vasculoid) in 100 μl of medium. To test CLI-mimicking conditions (i.e., hyperglycemia and / or hypoxia), cells were cultured with complete VascuLife® Basal Medium (Lifeline® Cell Technology, Frederick, MD) containing 5.5 mM D-glucose as a control or complete VascuLife® Basal Medium with a high glucose concentration (30 mM) under normoxic (20% O2) or hypoxic (5% O2) conditions for 72 hours. After 72 hours, relative cell viability was measured by incubating each well with 100 μl of CellTiter-Glo® reagent (Promega, Madison, WI) for 45 minutes according to the manufacturer's instructions. Luminescence was measured as a readout of cell viability for both single and multicell cultures under each oxygen condition and normalized to the 5.5 mM control. Figure 14B shows that when these vasculonoids were thawed as described above and cultured in vitro under normoxia (20% O2) or hypoxia (5% O2) in CLI-mimicking conditions, the vasculonoids exhibited better cell viability than J1-derived Meso-3D vasculonoid VPC2 cells cryopreserved as single cells.

[0256] To test in vivo efficacy, hindlimb ischemia was induced as detailed in Example 9, and then GMP1-Meso3D VPCs were injected into the quadriceps muscles of Balb / c nude mice (n = 15 per group) either as single cells (Meso3D sc, 1 million total single cells per animal) or as undissociated multicellular or vasculonoids (Meso3D vasculonoids, 25,000 per animal, approximately equivalent to 1 million total single cells per animal). Blood flow was assessed by laser Doppler perfusion imaging (LDPI) immediately after surgery and weekly thereafter for up to 64 days. Figure 14C shows a statistically significant improvement in blood flow after administration of single cells or vasculonoids compared to the vehicle-treated group (GS2 medium only) throughout the study; two-way ANOVA followed by Tukey's test.

[0257] Example 13: Long-term effects of Meso-3D vasculoid VPC2 cells in the HLI model Meso-3D vasculoid VPC2 cells were generated (as dissociated single cells) as described in Example 2 and administered to the HLI animal model described in Example 9 to observe long-term effects. In these studies, following HLI surgery, mice were injected with 1 million GMP1-derived cells (GMP1 Meso3D vasculoid VPC2, GMP1-HE, and GMP1-HB) in GS2 medium or GS2 medium alone (vehicle) into the right quadriceps muscle (n = 12–19 mice / group). Limb necrosis and limb function were scored as described in Example 9. For some cell types, more than one lot of cells produced in independent differentiation experiments was used, resulting in increased animal numbers when combining data from more than one lot of the same cell type. Data are the mean ± sem averaged over two independent replicates. *p<0.05 vs. vehicle control (GS2 medium) by one-way ANOVA followed by Dunnett's test.

[0258] Figure 15A shows that animals treated with meso-3D vasculoid VPC2 cells had better mean necrosis and functional scores compared to HE and HB cells at day 21. Figure 15B shows improved blood flow at day 63 in animals treated with meso-3D vasculoid VPC2 cells, HE cells, and HB cells compared to vehicle. CD34 blood vessel growth in the quadriceps (Figure 15C) and gastrocnemius (Figure 15D) muscles showed improvement with all three cell types, with HB cells showing better growth around day 35. However, by day 63, all three cell types promoted growth similarly, with meso-3D vasculoid VPC2 cells appearing to promote growth slightly better in the gastrocnemius than HE and HB.

[0259] Meso-3D vasculoid VPC2 cells also demonstrated long-term engraftment beyond 63 days after treatment (Figure 16A). In this study, 1 million cells per mouse in GS2 medium or GS2 medium alone (vehicle) were injected into the right quadriceps muscle after HLI surgery (vehicle = 18 mice, GMP1-Meso3D Lot No. 1 = 19 mice, GMP1-Meso3D Lot No. 2 = 18 mice, GMP1-Meso3D Lot No. 3 = 19 mice, GMP1-HE = 19 mice, and J1-HE = 19 mice). The injection site was then marked with a tattoo. On days 14, 35, 63, and 180, quadriceps muscles were collected from the operated right hind limb, fixed in PFA, embedded in paraffin, and stained for the human-specific marker Ku80. Two images (20x magnification) per mouse were analyzed. Each group had at least n=3, except for J1-HE, which had only n=1 at day 14. Data represent mean ± sem by a blinded independent histopathologist using the following semiquantitative scale: 0 = no positive Ku-80 cells; 1 = <5 positive Ku-80 cells; 2 = >5 and <15 positive Ku-80 cells; 3 = >15 and <50 positive Ku-80 cells; 4 = >50 positive Ku-80 cells. Figure 16A shows engrafted donor GMP1-Meso3D vasculoid VPC2 cells at days 63 and 180, demonstrating long-term cell engraftment. However, GMP-1-derived HE appeared to exhibit better engraftment at day 180.

[0260] In the second study (Figure 16B), 1 million cells per animal in GS2 medium were injected into the right quadriceps muscle after HLI surgery (GMP1-Meso3D vasculoid VPC2 cells = 16 mice, GMP1-HE Lot No. 1 = 16 mice, GMP1-HE Lot No. 2 = 17 mice, GMP1-HB Lot No. 1 = 16 mice, GMP1-HB Lot No. 2 = 16 mice). The injection site was then marked by tattoo. On days 14, 35, and 63, quadriceps muscles were collected from the operated right hind limb, fixed in PFA, embedded in paraffin, and stained for the human-specific marker Ku80. Two images (20x magnification) per animal were analyzed using an Olympus BX60 light microscope, with at least two per group. Ku80+ cells were quantified by a blinded independent histopathologist. Data represent the mean ± sem. Figure 16B shows that meso-3D vasculoid VPC2 cells showed engraftment by days 35 and 63, although one lot of GMP-1-derived HE showed better engraftment at day 63.

[0261] In another study (Figure 16C), 1 million cells per mouse in GS2 medium were injected into the right quadriceps muscle after HLI surgery (GMP1-Meso3D vasculoid VPC2 cells = 24–25 mice from two lots). On day 63, quadriceps muscles were collected from the operated right hind limb, fixed in PFA, and embedded in paraffin. Sections were then stained with either isolectin-B4 (a marker for mouse endothelial cells) and Ulex europaeus I (UEA1, a marker for human endothelial cells) or Ku80 (a pan-human-specific marker), UEA1, and smooth muscle α-actin (SMA, a marker for both mouse and human smooth muscle). DAPI was used to counterlabel nuclei. Figure 16C shows fluorescent images of injected Meso3D vasculoid VPC2 63 days after HLI surgery in Balb / c nude mice, demonstrating long-term engraftment (Ku80+), formation of human vasculature (UEA1+ vessels), and promotion of paracrine host vascular growth (IB4+ and SMA+ vessels).

[0262] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims. The contents of all references, patents and published patent applications mentioned throughout this application are hereby incorporated by reference.

Claims

1. 1. A method for producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, comprising: Culturing mesodermal cells derived from pluripotent stem cells under non- or low-adherent conditions in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs). The method comprising:

2. The method of claim 1, wherein the mesodermal cells are derived from the pluripotent stem cells by culturing the pluripotent stem cells in a medium comprising one or more mesoderm-inducing growth factors selected from the group consisting of activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4).

3. 3. The method of claim 1 or 2, wherein the meso-VPC is produced as a vasculonoid.

4. The method of claim 3, further comprising dissociating meso-VPCs in the vasculoids into single cells.

5. The method of any one of claims 2 to 4, wherein the mesoderm-inducing growth factors include activin A, VEGF165, FGF-2 and BMP4.

6. The method of claim 5, wherein activin A is used at a concentration of about 5 to 15 ng / mL.

7. 6. The method of claim 5, wherein VEGF165 is used at a concentration of about 5 to 25 ng / mL.

8. The method of claim 5, wherein FGF-2 is used at a concentration of about 5 to 25 ng / mL.

9. The method of claim 5, wherein BMP4 is used at a concentration of about 5 to 50 ng / mL.

10. The method of any one of claims 2 to 9, further comprising removing activin A from the culture medium after about 24 hours of culture.

11. The method of any one of claims 2 to 10, wherein the pluripotent stem cells are cultured on an extracellular matrix surface.

12. 12. The method of claim 11, wherein the extracellular matrix surface is a Matrigel-coated surface.

13. The method of any one of claims 2 to 12, wherein the pluripotent stem cells are cultured for about 3 days to about 5 days.

14. 14. The method of any one of claims 1 to 13, wherein the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542.

15. The method of any one of claims 1 to 14, wherein the one or more factors include VEGF165, FGF-2, BMP4 and SB431542.

16. 16. The method of any one of claims 1 to 15, wherein the one or more factors further comprise forskolin.

17. 17. The method of claim 16, wherein forskolin is used at a concentration of about 2 to 10 μM.

18. The method of any one of claims 15 to 17, wherein VEGF165 is used at a concentration of about 10 to 50 ng / mL.

19. The method of any one of claims 15 to 17, wherein FGF-2 is used at a concentration of about 10 to 50 ng / mL.

20. The method of any one of claims 15 to 17, wherein BMP4 is used at a concentration of about 10 to 50 ng / mL.

21. The method of any one of claims 14 to 20, wherein SB431542 is used at a concentration of about 5 to 20 μM.

22. 22. The method of any one of claims 1 to 21, wherein the step of culturing the mesodermal cells is carried out for about 3 days to about 7 days.

23. The process of culturing mesodermal cells is carried out at 5% CO 2 and 20% O 2 23. The method of any one of claims 1 to 22, wherein the method is carried out under normoxic conditions.

24. Pluripotent stem cells were cultured under 5% CO 2 and 20% O 2 The method of any one of claims 2 to 23, wherein the method is carried out under normoxic conditions.

25. 25. The method of any one of claims 1 to 24, wherein the non-adhesion or low-adhesion conditions are on an ultra-low attachment surface.

26. 1. A method for producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells, comprising: (a) culturing mesodermal cells derived from pluripotent stem cells on an extracellular matrix surface in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4); and (b) culturing the cells produced in step (a) on an extracellular matrix surface in a medium containing one or more factors selected from the group consisting of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), and a small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesodermally derived vascular progenitor cells. The method comprising:

27. 27. The method of claim 26, wherein the mesodermal cells are derived from the pluripotent stem cells by culturing the pluripotent stem cells in a medium comprising one or more mesoderm-inducing growth factors selected from the group consisting of activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4).

28. The method of claim 26 or 27, further comprising dissociating the population of meso-VPCs into single cells.

29. The method of claim 27 or 28, wherein the mesoderm-inducing growth factors include activin A, VEGF165, FGF-2 and BMP4.

30. 30. The method of claim 29, wherein activin A is used at a concentration of about 5 to 15 ng / mL.

31. 30. The method of claim 29, wherein VEGF165 is used at a concentration of about 5 to 25 ng / mL.

32. 30. The method of claim 29, wherein FGF-2 is used at a concentration of about 5 to 25 ng / mL.

33. 30. The method of claim 29, wherein BMP4 is used at a concentration of about 5 to 50 ng / mL.

34. The method of any one of claims 29 to 33, further comprising removing activin A from the culture medium after about 24 hours of culture.

35. The method of any one of claims 26 to 34, wherein the extracellular matrix surface in step (a) is a collagen IV-coated surface.

36. The method of any one of claims 27 to 35, wherein the pluripotent stem cells are cultured for about 3 days to about 5 days.

37. The method of any one of claims 26 to 36, wherein the one or more factors in step (a) include VEGF165, FGF-2 and BMP4.

38. 38. The method of any one of claims 26 to 37, wherein the small molecule inhibitor of transforming growth factor beta (TGF-β) type I receptor is SB431542.

39. The method of any one of claims 26 to 38, wherein the one or more factors in step (b) include VEGF165, FGF-2, BMP4 and SB431542.

40. 40. The method of any one of claims 26 to 39, wherein the one or more factors in step (a) further comprise forskolin.

41. 41. The method of any one of claims 26 to 40, wherein the one or more factors in step (b) further comprise forskolin.

42. 42. The method of claim 40 or 41, wherein forskolin is used at a concentration of about 2 to 10 μM.

43. The method of any one of claims 37 to 42, wherein VEGF165 is used at a concentration of about 10 to 50 ng / mL.

44. The method of any one of claims 37 to 42, wherein FGF-2 is used at a concentration of about 10 to 50 ng / mL.

45. The method of any one of claims 37 to 42, wherein BMP4 is used at a concentration of about 10 to 50 ng / mL.

46. 40. The method of claim 38 or 39, wherein SB431542 is used at a concentration of about 5 to 20 μM.

47. The method of any one of claims 26 to 46, wherein the extracellular matrix surface in steps (a) and (b) is a collagen IV-coated surface.

48. The method of any one of claims 26 to 47, wherein the culturing in step (a) is carried out for about 1 day.

49. The method of any one of claims 26 to 48, wherein the culturing in step (b) is carried out for about 4 days to about 7 days.

50. The culture in step (a) is performed at 5% CO 2 and 20% O 2 50. The method of any one of claims 26 to 49, wherein the method is carried out under normoxic conditions.

51. The culture in step (b) is performed at 5% CO 2 and 5% O 2 The method of any one of claims 26 to 50, wherein the method is carried out under low oxygen concentration conditions.

52. Pluripotent stem cells were cultured under 5% CO 2 and 20% O 2 The method of any one of claims 27 to 51, wherein the method is carried out under normoxic conditions.

53. The method of any one of claims 1 to 52, wherein the pluripotent stem cells are human embryonic stem cells.

54. The method of any one of claims 1 to 52, wherein the pluripotent stem cells are human induced pluripotent stem cells.

55. The method of any one of claims 1 to 54, wherein the population of meso-VPCs expresses at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb.

56. 56. The method of claim 55, wherein the population of meso-VPCs expresses cell surface markers (a) CD146, CD31 / PECAM1 and CD309 / KDR, or (b) CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43 or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4.

57. 57. The method of any one of claims 1-56, wherein the population of meso-VPCs exhibits limited or no detection of one or more of the cell surface markers selected from the group consisting of: (a) CXCR7, CD45, and NG2; (b) CXCR7, CD45, and NG2; or (c) CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2.

58. 58. The method of any one of claims 1-57, wherein the population of meso-VPCs express at least one miRNA marker selected from hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p.

59. 59. The method of any one of claims 1 to 58, wherein the population of meso-VPCs exhibits limited or no expression of at least one miRNA marker selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p.

60. The method of any one of claims 1 to 59, wherein the population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p.

61. The method of any one of claims 1 to 60, wherein the population of meso-VPCs comprises at least one meso-VPC that is positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24 and mir483-5p.

62. The method of claim 61, wherein the miRNA marker is mir483-5p.

63. The method of any one of claims 1 to 62, wherein the population of meso-VPCs comprises at least one meso-VPC that exhibits limited or no expression of at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p and mir133a.

64. The method of any one of claims 1 to 63, further comprising the step of producing vascular endothelial cells by differentiation of meso-VPCs.

65. 65. The method of claim 64, wherein the differentiation is performed on a fibronectin-coated surface.

66. 64. A composition comprising a population of meso-VPCs produced by the method of any one of claims 1 to 63.

67. A composition comprising a population of mesoderm-derived vascular progenitor cells (meso-VPCs) produced by in vitro differentiation of mesoderm cells derived from pluripotent stem cells, wherein the population of meso-VPCs expresses at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb.

68. The composition of claim 67, wherein the population of meso-VPCs expresses at least two cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146 and PDGFRb.

69. The composition of any one of claims 67 to 68, wherein the population of meso-VPCs expresses the cell surface markers CD146, CD31 / PECAM1 and CD309 / KDR.

70. 70. The composition of any one of claims 67-69, wherein the population of meso-VPCs express the cell surface markers CD31 / PECAM1, CD309 / KDR, CD146, and at least one of (i) CD144, CD34, CD184 / CXCR4, CD43 or PDGFRb, (ii) CD34, CD184 / CXCR4 and PDGFRb, (iii) CD184 / CXCR4, (iv) PDGFRb, (v) CD144 and CD184 / CXCR4, (vi) CD184 / CXCR4 and CD43, or (vii) CC184 / CXCFR4.

71. The composition of any one of claims 67-70, wherein the population of meso-VPCs exhibits limited or no detection of one or more cell surface markers selected from the group consisting of: (a) CXCR7, CD45, and NG2; (b) CXCR7, CD45, and NG2; or (c) CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2.

72. 72. The composition of any one of claims 67-71, wherein the population of meso-VPCs express at least one miRNA marker selected from hsa-miR-3917, hsa-miR-450a-2-3p, hsa-miR-542-5p, hsa-miR-126-5p, hsa-miR-125a-5p, hsa-miR-24-3p, hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, hsa-miR-142-3p, hsa-miR-483-5p, hsa-miR-483-3p, miR214, miR335-3p, and miR-199a-3p.

73. 73. The composition of any one of claims 67 to 72, wherein the population of meso-VPCs exhibits limited or no expression of at least one miRNA marker selected from hsa-let-7e-3p, hsa-miR-99a-3p, hsa-miR-133a-5p, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p.

74. The composition of any one of claims 67 to 73, wherein the population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p.

75. The composition of any one of claims 67-74, wherein the population of meso-VPCs comprises meso-VPC vasculoids.

76. The composition of any one of claims 67-74, wherein the population of meso-VPCs comprises a single cell of meso-VPCs.

77. A meso-VPC produced by in vitro differentiation of mesodermal cells derived from pluripotent stem cells, said meso-VPC being positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24 and mir483-5p.

78. The meso-VPC of claim 77, which is positive for the miRNA marker mir483-5p.

79. The meso-VPC of claim 77 or 78, which is negative for at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p and mir133a.

80. 80. The composition or meso-VPC of any one of claims 66-79, wherein the pluripotent stem cells are human pluripotent stem cells.

81. The composition or meso-VPC of claim 80, wherein the pluripotent stem cells are human embryonic stem cells (hESCs).

82. The composition or meso-VPC of claim 80, wherein the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

83. The composition or meso-VPC of any one of claims 66-82, wherein the pluripotent stem cells are first differentiated into mesodermal cells, which are then differentiated into meso-VPCs.

84. 84. A pharmaceutical composition comprising the composition of any one of claims 66-83 or meso-VPC.

85. 1. A method of treating a vascular disease or disorder in a subject, comprising: Administering to a subject an effective amount of the composition or mesodermally derived vascular progenitor cells (meso-VPCs) of any one of claims 66-83 or the pharmaceutical composition of claim 83, thereby treating a vascular disease or disorder in the subject. The method comprising:

86. 86. The method of claim 85, wherein the vascular disease or disorder is selected from the group consisting of atherosclerosis, peripheral arterial disease (PAD), carotid artery disease, venous disease, blood clots, aortic aneurysms, fibromuscular dysplasia, lymphedema, and vascular injury.

87. 87. The method of claim 86, wherein the peripheral arterial disease is selected from the group consisting of critical limb ischemia, intestinal ischemia syndrome, renal artery disease, popliteal artery entrapment syndrome, Raynaud's phenomenon, and Buerger's disease.

88. 88. The method of claim 87, wherein the peripheral arterial disease is critical limb ischemia.

89. The method of any one of claims 85-88, wherein the composition, meso-VPC or pharmaceutical composition is administered intramuscularly or systemically.

90. 90. The method of any one of claims 85-89, wherein administering the composition, meso-VPC, or pharmaceutical composition increases blood flow in the subject.

91. 91. The method of any one of claims 85-90, wherein administering the composition, meso-VPC or pharmaceutical composition promotes angiogenesis and / or vasculogenesis in the subject.

92. 92. The method of any one of claims 85-91, wherein administering the composition, meso-VPC or pharmaceutical composition reduces ischemia severity in the subject.

93. 93. The method of any one of claims 85-92, wherein administering the composition, meso-VPC, or pharmaceutical composition reduces the area of ​​necrosis in the limb in the subject.

94. Approximately 1×10 4 ~Approx. 1×10 13 The method of any one of claims 85-93, wherein meso-VPCs are administered to the subject.

95. The method of any one of claims 85-94, wherein the meso-VPC is administered as a pharmaceutical composition.

96. The pharmaceutical composition comprises: (a) a buffering agent that maintains the solution at physiological pH; (b) at least 5% (w / v) glucose, and (c) an osmotically active agent that maintains the solution at physiological osmolality 96. The method of claim 95, comprising:

97. 97. The method of claim 96, wherein the glucose is D-glucose (dextrose).

98. 97. The method of claim 96, wherein the osmotically active agent is a salt.

99. 97. The method of claim 96, wherein the salt is sodium chloride.

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