Compositions and methods for treating vascular diseases

Abstract

The present invention relates generally 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 critical limb ischemia, by administering meso-VPCs to a subject. TIFF2022545737000011.tif81153

Description

[Technical Field]

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

[0002] Field of Invention This invention relates to novel mesoderm-derived vascular progenitor cells (meso-VPCs) and methods for producing said meso-VPCs. This invention also relates to methods for treating vascular diseases such as ischemia using meso-VPCs. [Background technology]

[0003] Background of the Invention Vascular diseases affect the body's vascular network. More than 78 million Americans have hypertension, the most common form of vascular disease. In addition, peripheral artery disease (PAD) affects 12 to 15 million people in the United States, with many more undiagnosed cases.

[0004] Peripheral artery disease (PAD) is narrowing or blockage of the blood vessels that carry blood from the heart to other organs and tissues. This is primarily caused by the accumulation of fatty plaque in the arteries, known as atherosclerosis. PAD can occur in any blood vessel, but it is more common in the lower extremities than in the upper extremities.

[0005] Ischemia is a condition caused by peripheral arterial disease involving interruption of arterial blood supply to tissues, organs or limbs, which, if untreated, can lead to tissue death. This can be caused by embolism, thrombosis of atherosclerotic arteries or trauma. Venous problems such as venous outflow obstruction and low blood flow can cause acute arterial ischemia. Ischemia in the lower limbs can lead to lower limb pain or cramps (claudication) associated with activity, skin color changes, blisters or ulcers, and a feeling of fatigue in the lower limbs. Complete loss of circulation can lead to gangrene and limb loss.

[0006] The treatment of vascular diseases such as ischemia is limited. Most treatment methods involve invasive surgery, while some focus on preventing the progression of existing conditions. Therefore, in the art, improved treatments for vascular diseases such as ischemia are still needed. 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 a method for treating vascular diseases, such as severe ischemic limbs, using the meso-VPCs of the present invention.

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

[0009] In one embodiment, mesoderm cells are induced from pluripotent stem cells by culturing them in a culture medium containing one or more mesoderm-inducible 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-VPC is produced as a vasculonoid. In another embodiment, meso-VPC is dissociated into single cells.

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

[0012] In one embodiment, 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 approximately 3 to 5 days.

[0013] In one embodiment, the small molecule inhibitor of the transforming growth factor beta (TGF-β) type I receptor is SB431542. In another embodiment, one or more factors include VEGF165, FGF-2, BMP4, and SB431542. In one embodiment, 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 process of culturing mesoderm cells is carried out for approximately 3 to 7 days.

[0015] In one embodiment, the process of culturing mesoderm cells is carried out under normal oxygen concentration conditions of 5% CO2 and 20% O2.

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

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

[0018] In one aspect, the present invention provides a method for producing a population of meso-VPCs from pluripotent stem cells, comprising the steps of: (a) culturing pluripotent stem cell-derived mesoderm cells on the surface of the extracellular matrix 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 the surface of the extracellular matrix 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 small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of meso-VPCs.

[0019] In one embodiment, mesoderm cells are induced from pluripotent stem cells by culturing them in a culture medium containing one or more mesoderm-inducible 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 includes a step of dissociating a population of meso-VPCs into single cells.

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

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

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

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

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

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

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

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

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

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

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

[0032] In one embodiment, SB431542 is used at a concentration of approximately 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 culture in step (a) is carried out for approximately one day.

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

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

[0037] In one embodiment, the culture in step (b) is carried out under low oxygen concentration conditions of 5% CO2 and 5% O2.

[0038] In one embodiment, pluripotent stem cells are cultured under normal oxygen concentration 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, a population of meso-VPCs produced according to any of the methods of the present invention expresses at least one of the cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb.

[0042] In one embodiment, a population of meso-VPCs produced according to any of the methods of the present invention expresses the cell surface marker (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, a population of meso-VPCs produced according to any of the methods of the present invention shows limited detection or no detection of (a) one or more cell surface markers selected from the group consisting of CXCR7, CD45, and NG2, (b) CXCR7, CD45, and NG2, or (c) one or more cell surface markers selected from the group consisting of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2.

[0044] In one embodiment, a population of meso-VPCs produced according to any of the methods of the present invention 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.

[0045] In one embodiment, a population of meso-VPCs produced according to any of the methods of the present invention does not express, or expresses only limited, 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, a population of meso-VPCs produced according to any of the methods of the present invention expresses hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p.

[0047] In one embodiment, a population of meso-VPCs produced according to any of the methods of the present invention includes 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, a population of meso-VPCs produced according to any of the methods of the present invention includes at least one meso-VPC that exhibits limited expression 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 includes the step of producing vascular endothelial cells by differentiation of meso-VPC.

[0050] In one embodiment, differentiation occurs on the fibronectin coat surface.

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

[0052] In one aspect, the present invention provides a composition comprising a population of meso-VPCs produced by in vitro differentiation of pluripotent stem cell-derived mesoderm 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.

[0053] In one embodiment, a 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, a 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 the cell surface markers CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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 detection or no detection of (a) one or more cell surface markers selected from the group consisting of CXCR7, CD45, and NG2, (b) CXCR7, CD45, and NG2, or (c) one or more cell surface markers selected from the group consisting of 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 meso-VPC population includes meso-VPC vascularoids.

[0061] In one embodiment, a population of meso-VPCs includes a single meso-VPC cell.

[0062] In one embodiment, the present invention provides meso-VPC produced by in vitro differentiation of pluripotent stem cell-derived mesoderm cells, which is positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p.

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

[0064] In one embodiment, 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, and then these are differentiated into meso-VPCs.

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

[0070] In one aspect, the present invention provides a method for treating a vascular disease or vascular disorder in a subject, comprising the step of administering an effective amount of any one of the following to the subject: a composition comprising a population of the meso-VPCs of the present invention, or mesoderm-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, vascular disease or vascular disorder is selected from the group consisting of atherosclerosis, peripheral artery disease (PAD), carotid artery disease, venous disease, blood clots, aortic aneurysm, fibromuscular dysplasia, lymphedema, and vascular injury.

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

[0073] In one embodiment, peripheral artery disease is a severe limb ischemia.

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

[0075] In one embodiment, administration of a composition containing 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 containing a population of meso-VPCs, meso-VPCs, or a pharmaceutical composition promotes angiogenesis and / or angiogenesis in a subject.

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

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

[0079] In one embodiment, approximately 1 × 10 4 ~Approx. 1×10 13 Each meso-VPC is administered to the target individual.

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

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

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

[0083] In one embodiment, the osmotic activator is a salt.

[0084] In one embodiment, the salt is sodium chloride. [Invention 1001] A method for producing a population of meso-VPCs (meso-VPCs) from pluripotent stem cells, A step of culturing pluripotent stem cell-derived mesoderm cells under non-adhesion or low-adhesion conditions in a culture 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 small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs). The method, including the method described above. [Invention 1002] The method of the present invention 1001, wherein mesodermal cells are induced from pluripotent stem cells by culturing pluripotent stem cells in a culture medium containing one or more mesoderm-inducible 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). [Invention 1003] The method according to the present invention 1001 or 1002, wherein meso-VPC is produced as a vasculonoid. [Invention 1004] The method of the present invention 1003, further comprising the step of dissociating meso-VPC in a vascularonoid into single cells. [Invention 1005] A method according to any one of the present invention 1002 to 1004, wherein the mesoderm-inducible growth factor comprises activin A, VEGF165, FGF-2, and BMP4. [Invention 1006] The method of the present invention 1005, wherein activin A is used at a concentration of approximately 5 to 15 ng / mL. [Invention 1007] The method of the present invention 1005, wherein VEGF165 is used at a concentration of approximately 5 to 25 ng / mL. [Invention 1008] The method of the present invention 1005, wherein FGF-2 is used at a concentration of approximately 5 to 25 ng / mL. [Invention 1009] The method of the present invention 1005, wherein BMP4 is used at a concentration of approximately 5-50 ng / mL. [Invention 1010] Any method according to 1002 to 1009 of the present invention, further comprising the step of removing activin A from the culture medium after culturing for approximately 24 hours. [Invention 1011] A method according to any one of the present invention 1002 to 1010, wherein pluripotent stem cells are cultured on the surface of the extracellular matrix. [Invention 1012] The method of the present invention 1011, wherein the extracellular matrix surface is a Matrigel coated surface. [Invention 1013] A method according to any of items 1002 to 1012 of the present invention, wherein pluripotent stem cells are cultured for approximately 3 to 5 days. [Invention 1014] A method according to any one of the present invention 1001 to 1013, wherein the small molecule inhibitor of the transforming growth factor beta (TGF-β) type I receptor is SB431542. [Invention 1015] A method according to any one of the present invention 1001 to 1014, wherein one or more factors include VEGF165, FGF-2, BMP4, and SB431542. [Invention 1016] Any method of the present invention 1001 to 1015, wherein one or more factors further comprise forskolin. [Invention 1017] The method of the present invention 1016, wherein forskolin is used at a concentration of approximately 2 to 10 μM. [Invention 1018] A method according to any of the present invention 1015 to 1017, wherein VEGF165 is used at a concentration of approximately 10 to 50 ng / mL. [Invention 1019] A method according to any of the present invention 1015 to 1017, wherein FGF-2 is used at a concentration of approximately 10 to 50 ng / mL. [Invention 1020] Any of the methods described in items 1015 to 1017 of this invention, wherein BMP4 is used at a concentration of approximately 10 to 50 ng / mL. [Invention 1021] Any method according to invention 1014 to 1020, wherein SB431542 is used at a concentration of approximately 5 to 20 μM. [Invention 1022] A method according to any of the present invention 1001 to 1021, wherein the step of culturing mesoderm cells is performed for approximately 3 to 7 days. [Invention 1023] The process of culturing mesoderm cells is performed using 5% CO2. 2 and 20% 2 Any of the methods described in 1001 to 1022 of this invention, performed under normal oxygen concentration conditions. [Invention 1024] Pluripotent stem cell culture is performed in 5% CO2 2 and 20% 2 Any of the methods described in 1002 to 1023 of this invention, performed under normal oxygen concentration conditions. [Invention 1025] Any method 1001 to 1024 of the present invention, wherein the non-adhesion or low-adhesion conditions are on an ultra-low-adhesion surface. [Invention 1026] A method for producing a population of meso-VPCs (meso-VPCs) from pluripotent stem cells, (a) A step of culturing pluripotent stem cell-derived mesodermal cells on the surface of the extracellular matrix in a culture 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) A step of culturing the cells produced in step (a) on the surface of the extracellular matrix 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 small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor, thereby producing a population of mesoderm-derived vascular progenitor cells. The method, including the method described above. [Invention 1027] The method of the present invention 1026, wherein mesodermal cells are induced from pluripotent stem cells by culturing pluripotent stem cells in a culture medium containing one or more mesoderm-inducible 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). [Invention 1028] The method of the present invention 1026 or 1027, further comprising the step of dissociating a population of meso-VPCs into single cells. [Invention 1029] The method of the present invention 1027 or 1028, wherein the mesoderm-inducible growth factor comprises activin A, VEGF165, FGF-2, and BMP4. [Invention 1030] The method of the present invention 1029, wherein activin A is used at a concentration of approximately 5 to 15 ng / mL. [Invention 1031] The method of the present invention 1029, wherein VEGF165 is used at a concentration of approximately 5 to 25 ng / mL. [Invention 1032] The method of the present invention 1029, wherein FGF-2 is used at a concentration of approximately 5 to 25 ng / mL. [Invention 1033] The method of the present invention 1029, wherein BMP4 is used at a concentration of approximately 5-50 ng / mL. [Invention 1034] Any method according to item 1029 to 1033 of the present invention, further comprising the step of removing activin A from the culture medium after culturing for approximately 24 hours. [Invention 1035] A method according to any of the present invention 1026 to 1034, wherein the extracellular matrix surface in step (a) is a collagen IV coated surface. [Invention 1036] A method according to any of items 1027 to 1035 of the present invention, wherein pluripotent stem cells are cultured for approximately 3 to 5 days. [Invention 1037] A method according to any one of the present invention 1026 to 1036, wherein one or more factors in step (a) include VEGF165, FGF-2, and BMP4. [Invention 1038] A method according to any one of the present invention 1026 to 1037, wherein the small molecule inhibitor of the transforming growth factor beta (TGF-β) type I receptor is SB431542. [Invention 1039] A method according to any one of the present invention 1026 to 1038, wherein one or more factors in step (b) include VEGF165, FGF-2, BMP4, and SB431542. [Invention 1040] A method according to any one of the present invention 1026 to 1039, wherein one or more factors in step (a) further comprises forskolin. [Invention 1041] Any method of the present invention 1026 to 1040, wherein one or more factors in step (b) further comprises forskolin. [Invention 1042] The method of the present invention 1040 or 1041, wherein forskolin is used at a concentration of approximately 2 to 10 μM. [Invention 1043] Any method according to invention 1037 to 1042, wherein VEGF165 is used at a concentration of approximately 10 to 50 ng / mL. [Invention 1044] Any method according to invention 1037 to 1042, wherein FGF-2 is used at a concentration of approximately 10 to 50 ng / mL. [Invention 1045] Any of the methods described in invention 1037 to 1042, wherein BMP4 is used at a concentration of approximately 10 to 50 ng / mL. [Invention 1046] The method of the present invention 1038 or 1039, wherein SB431542 is used at a concentration of approximately 5 to 20 μM. [Invention 1047] A method according to any one of the present invention 1026 to 1046, wherein the extracellular matrix surface in step (a) and step (b) is a collagen IV coated surface. [Invention 1048] A method according to any of the present invention 1026 to 1047, wherein the culture in step (a) is carried out for approximately one day. [Invention 1049] A method according to any of the present invention 1026 to 1048, wherein the culture in step (b) is carried out for approximately 4 to 7 days. [Invention 1050] The culture in step (a) is 5% CO 2 and 20% 2 Any of the methods described in items 1026 to 1049 of this invention, performed under normal oxygen concentration conditions. [Invention 1051] The culture in step (b) is 5% CO 2 and 5% 2 Any of the methods described in 1026 to 1050 of this invention, performed under low oxygen concentration conditions. [Invention 1052] Pluripotent stem cell culture is performed in 5% CO2 2 and 20% 2 Any method 1027 to 1051 of the present invention, performed under normal oxygen concentration conditions. [Invention 1053] A method according to any of the present invention 1001 to 1052, wherein the pluripotent stem cells are human embryonic stem cells. [Invention 1054] A method according to any one of the present invention 1001 to 1052, wherein the pluripotent stem cells are human induced pluripotent stem cells. [Invention 1055] A method according to any of the present invention 1001 to 1054, wherein a population of meso-VPCs expresses at least one of the cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb. [Invention 1056] The method of the present invention 1055, wherein a population of meso-VPCs expresses the cell surface markers (a) CD146, CD31 / PECAM1 and CD309 / KDR, or (b) CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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. [Invention 1057] The method according to any of items 1001 to 1056 of the present invention, wherein the population of meso-VPCs shows limited detection or no detection of (a) one or more cell surface markers selected from the group consisting of CXCR7, CD45, and NG2, (b) CXCR7, CD45, and NG2, or (c) one or more cell surface markers selected from the group consisting of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2. [Invention 1058] A method according to any of the Invention 1001 to 1057, wherein 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. [Invention 1059] A method according to any of the present invention 1001 to 1058, wherein a population of meso-VPCs exhibits limited expression 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. [Invention 1060] A method according to any of items 1001 to 1059 of the present invention, wherein a population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p. [Invention 1061] The method according to any one of the invention 1001 to 1060, wherein the population of meso-VPCs includes 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. [Invention 1062] The method of invention 1061, wherein the miRNA marker is mir483-5p. [Invention 1063] Any method of the present invention 1001 to 1062, wherein the population of meso-VPCs includes at least one meso-VPC that exhibits limited expression or does not express at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a. [Invention 1064] Any method of the present invention 1001 to 1063, further comprising the step of producing vascular endothelial cells by differentiation of meso-VPC. [Invention 1065] The method of the present invention 1064, wherein differentiation takes place on the fibronectin-coated surface. [Invention 1066] A composition comprising a group of meso-VPCs produced by any of the methods described in invention 1001 to 1063. [Invention 1067] A composition comprising a population of meso-VPCs produced by in vitro differentiation of pluripotent stem cell-derived mesoderm 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. [Invention 1068] The composition of Invention 1067, wherein 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. [Invention 1069] A composition according to any one of the invention 1067 to 1068, wherein a population of meso-VPCs expresses the cell surface markers CD146, CD31 / PECAM1, and CD309 / KDR. [Invention 1070] A composition according to any of the Invention 1067-1069, wherein a population of meso-VPCs expresses the cell surface markers CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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. [Invention 1071] A composition according to any of Invention 1067 to 1070, wherein the population of meso-VPCs exhibits limited detection or no detection of (a) one or more cell surface markers selected from the group consisting of CXCR7, CD45, and NG2, (b) CXCR7, CD45, and NG2, or (c) one or more cell surface markers selected from the group consisting of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, and NG2. [Invention 1072] A composition according to any one of the invention 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. [Invention 1073] A composition according to any one of the Invention 1067 to 1072, wherein the population of meso-VPCs exhibits limited expression 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. [Invention 1074] A composition according to any one of the invention 1067 to 1073, wherein the population of meso-VPCs expresses hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p. [Invention 1075] A composition according to any one of the present invention 1067 to 1074, wherein a group of meso-VPCs contains vascularoids of meso-VPCs. [Invention 1076] A composition according to any one of the present invention 1067 to 1074, wherein a population of meso-VPCs contains a single meso-VPC cell. [Invention 1077] A meso-VPC produced by in vitro differentiation of pluripotent stem cell-derived mesoderm cells, wherein the meso-VPC is positive for at least one miRNA marker selected from the group consisting of mir126, mir125a-5p, mir24, and mir483-5p. [Invention 1078] Meso-VPC according to Invention 1077, which is positive for the miRNA marker mir483-5p. [Invention 1079] A meso-VPC according to 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. [Invention 1080] A composition according to any of the invention items 1066 to 1079 or meso-VPC, wherein the pluripotent stem cells are human pluripotent stem cells. [Invention 1081] A composition of the present invention 1080 or meso-VPC, wherein the pluripotent stem cells are human embryonic stem cells (hESCs). [Invention 1082] The composition of the present invention 1080 or meso-VPC, wherein the pluripotent stem cells are human induced pluripotent stem cells (hiPSCs). [Invention 1083] A composition according to any of the present invention 1066 to 1082, or a meso-VPC, wherein pluripotent stem cells are first differentiated into mesodermal cells, and then these are differentiated into meso-VPCs. [Invention 1084] A pharmaceutical composition comprising any of the compositions described in invention 1066 to 1083 or meso-VPC. [Invention 1085] A method for treating vascular disease or vascular disorder in a subject, A step of administering an effective amount of any composition of Invention 1066 to 1083 or meso-VPCs derived from meso-VPCs or the pharmaceutical composition of Invention 1083 to a subject, thereby treating a vascular disease or vascular disorder in the subject. The method, including the method described above. [Invention 1086] The method of the present invention 1085, wherein the vascular disease or vascular disorder is selected from the group consisting of atherosclerosis, peripheral artery disease (PAD), carotid artery disease, venous disease, blood clots, aortic aneurysm, fibromuscular dysplasia, lymphedema, and vascular injury. [Invention 1087] The method of the present invention 1086, wherein the peripheral artery disease is selected from the group consisting of severe limb ischemia, enteroischemic syndrome, renal artery disease, popliteal artery entrapment syndrome, Raynaud's phenomenon, and Buerger's disease. [Invention 1088] The method of the present invention 1087, wherein peripheral artery disease is severe limb ischemia. [Invention 1089] A method according to any one of the present invention 1085 to 1088, wherein the composition, meso-VPC, or pharmaceutical composition is administered intramuscularly or systemically. [Invention 1090] A method of any of the invention 1085 to 1089, wherein administration of a composition, meso-VPC, or pharmaceutical composition increases blood flow in a subject. [Invention 1091] Any method of the present invention 1085 to 1090, wherein administration of a composition, meso-VPC, or pharmaceutical composition promotes angiogenesis and / or angiogenesis in a subject. [Invention 1092] A method according to any of items 1085 to 1091 of the present invention, wherein administration of a composition, meso-VPC, or pharmaceutical composition reduces the severity of ischemia in a subject. [Invention 1093] A method of any of invention 1085 to 1092, wherein administration of a composition, meso-VPC, or pharmaceutical composition reduces the necrotic area of ​​a limb in a subject. [Invention 1094] Approximately 1×10 4 ~Approx. 1×10 13 A method according to any of the present invention 1085 to 1093, wherein a meso-VPC is administered to the subject. [Invention 1095] Any method 1085 to 1094 of the present invention, wherein meso-VPC is administered as a pharmaceutical composition. [Invention 1096] Pharmaceutical compositions, (a) A buffering agent to maintain the solution at physiological pH, (b) at least 5% (w / v) glucose, and (c) Osmotic activators that maintain the solution at physiological osmotic pressure The method of the present invention 1095, including the method of the present invention. [Invention 1097] The method of the present invention 1096, wherein glucose is D-glucose (dextrose). [Invention 1098] The method of the present invention 1096, wherein the osmotic activator is a salt. [Invention 1099] The method of the present invention 1096, wherein the salt is sodium chloride. [Brief explanation of the drawing]

[0085] [Figure 1] This is a schematic diagram illustrating the process of in vitro differentiation of human pluripotent stem cells into mesodermal cells. [Figure 2A] This graph shows the 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] This graph shows that mesodermal cells differentiated from the human induced pluripotent stem cell line GMP1 selectively express or do not express cell surface markers for pluripotent cells, endodermal cells, ectoderm cells, and hematopoietic cells, thereby confirming differentiation into the mesodermal lineage. [Figure 3] This diagram illustrates the process of in vitro differentiation of human pluripotent stem cells into mesodermal cells (left), and the process of in vitro differentiation of mesodermal cells into mesoderm-derived vascular progenitor cells (meso-VPCs) using the Meso-3D-Vasculonoid VPC1 protocol (upper right) or the Meso-3D-Vasculonoid VPC2 protocol (lower right). [Figure 4] This diagram shows the process for in vitro differentiation of human pluripotent stem cells into mesodermal cells (left), and the process for in vitro differentiation of mesodermal cells into mesoderm-derived vascular progenitor cells (meso-VPCs) using the Meso-2D VPC2 protocol (upper right) or the Meso-2D VPC3 protocol (lower right). [Figure 5] This panel shows microscopic images illustrating the ability of meso-VPCs produced by the Meso-3D-Vasculonoid protocol to undergo further differentiation into the endothelial lineage. The top panel shows the morphology of meso-VPCs on day 5 before harvesting. The middle panel shows endothelial differentiation of meso-VPCs using fibronectin-coated plates and endothelial differentiation-promoting media. The bottom panel shows the capillary-like Matrigel network formed by meso-VPCs. [Figure 6A] This graph shows the expression of cell surface markers CD31 / PECAM1, CD309 / KDR, CXCR4 / CD184, CD43, CD146, and PDGFRb in meso-VPC produced using the Meso-3D-Vasculonoid-VPC1 protocol, Meso-3D-Vasculonoid-VPC2 protocol, Meso-2D-VPC2 protocol, or Meso-2D-VPC3 protocol. [Figure 6B] This heatmap shows the fraction of cells positive for selected cell surface markers among meso-VPCs and comparative hematopoietic endothelial cells (HE) or angioblasts (HB). Comparisons with undifferentiated pluripotent stem cells (J1 and GMP1) and human umbilical vein endothelial cells (HUVEC) are also shown. [Figure 6C] This is a principal component analysis (PCA) plot showing the expression profiles of vascular cell surface markers for meso-VPC produced by the Meso-3D-vascularronoid protocol or the Meso-2D protocol, hematopoietic endothelial cells (HE) for comparison, angioblasts (HB) for comparison, undifferentiated pluripotent stem cells (J1 and GMP1), or human umbilical vein endothelial cells (HUVEC). [Figure 7]This panel shows microscopic images illustrating the ability of meso-VPCs produced by the Meso-2D protocol to undergo further differentiation into 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 endothelial differentiation-promoting media. The bottom panel shows the capillary-like Matrigel network formed by meso-VPCs. [Figure 8] This graph shows the increase in blood flow in animals treated with meso-VPC, as described in Example 9. Specifically, animals are treated with sham surgery (1M), vehicle control (2M), J1-HDF Meso-2D VPC2 (3M), J-HDF Meso-3D vascularonoid VPC2 (4M), GMP1HDF Meso-2D VPC2 (5M), GMP1-HDF Meso-3D vascularonoid VPC2 (6M), or GMP1-HDF Meso-3D vascularonoid VPC1 (7M). [Figure 9] This graph shows the change in vascular density in animals treated with meso-VPC, as described in Example 9. Specifically, animals were treated with vehicle control (2M), J1-HDF Meso-2D VPC2 (3M TI1), J-HDF Meso-3D vascularonoid VPC2 (4M TI2), GMP1HDF Meso-2D VPC2 (5M TI3), GMP1-HDF Meso-3D vascularonoid VPC2 (6M TI4), or GMP1-HDF Meso-3D vascularonoid VPC1 (7M TI5). [Figure 10]This graph shows the quantitative results of CD34+ staining (an indicator of small capillary formation), total blood vessel count, and blood flow tests in animals treated with meso-VPC. Specifically, animals are treated with either simming (1M), vehicle control (2M), J1-HDF Meso-2D VPC2 (3M TI1), J-HDF Meso-3D vascularonoid VPC2 (4M TI2), GMP1HDF Meso-2D VPC2 (5M TI3), GMP1-HDF Meso-3D vascularonoid VPC2 (6M TI4), or GMP1-HDF Meso-3D vascularonoid VPC1 (7M TI5). [Figure 11] Each group of animals treated with meso-VPC showed a strong and statistically significant correlation between blood flow and mean capillary density, as measured by laser Doppler. [Figure 12]Figure 12A is a plot and graph showing the unique human miRNAs found in a population of J1-derived Meso-3D vasculonoid VPC2 cells from three replicates, including hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p, compared to a population of J1 cells and J1-derived HE cells. Figure 12A also shows the unique human miRNAs found in a population of J1-derived HE cells, including hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. "Expression" refers to normalized expression, where >0 is the expression level in all three replicates. Figure 12B is a graph showing the miRNA expression levels in a population of J1-derived Meso-3D vascularonoid VPC2 cells, which were previously analyzed using single cells. It 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 vascularonoid VPC2 cell population. Figure 12C is a graph showing that the J1-derived Meso-3D vascularonoid VPC2 cell population expresses hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, and hsa-miR-142-3p, while hsa-let-7e-3p, hsa-miR-99a-3p, and hsa-miR-133a-5p are either not expressed or expressed at low levels. Figure 12D is a graph showing that a population of J1-derived Meso-3D vasculonoid VPC2 cells expresses hsa-miR-483-5p and hsa-miR-483-3p. [Figure 13-1] This graph shows the expression of the most upregulated or most downregulated genes in J1-derived Meso-3D vasculonoid VPC2 cell samples compared to single J1 cells 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 is a low-magnification (10× objective) image of the extensive vascular network extending from the embedded aggregates of J1-derived Meso-3D vascularonoid VPC2 vascularonoids, stained with DAPI and UAE1 after 14 days. Figure 14B is a graph showing that when J1-derived Meso-3D vascularonoid VPC2 vascularonoids ("multicellular") or J1-derived Meso-3D vascularonoid VPC2 cells ("single cells") dissociated into single cells were cultured in vitro under CLI-mimicking conditions, under normal oxygen concentrations (20% O2) (left panel) or hypoxic concentrations (5% O2) (right panel), the vascularonoids showed better cell survival compared to J1-derived Meso-3D vascularonoid VPC2 cells that had been cryopreserved as single cells. Figure 14C is a graph showing statistically significant improvements in blood flow over the entire study, compared to the vehicle-treated group (GS2 medium only), after administration of J1-derived Meso-3D vascularonoid VPC2 single cells ("sc") or vascularonoid; Tukey's test following two-way ANOVA. [Figure 15]Figure 15A is a graph showing that animals treated with meso-3D vascularonoid VPC2 cells had better mean necrosis scores (left panel) and functional scores (right panel) at day 21 compared to HE cells and HB cells. Dunnett's test following one-way ANOVA. Mean ± sem. Figure 15B is a graph showing improved blood flow at day 63 in animals treated with meso-3D vascularonoid VPC2 cells, HE cells, and HB cells compared to the vehicle. *p<0.05 vs. vehicle. Mean ± sd. Tukey's test following two-way ANOVA. Figure 15C is a graph showing CD34+ vascular growth in the quadriceps muscle of animals treated with Meso-3D vascularonoid VPC2 cells, HE cells, and HB cells. *p<0.05 vs. vehicle. Mean ± sm. Fisher's LSD test without adjustment following two-way ANOVA. Figure 15D is a graph showing the improvement of the gastrocnemius muscle after administration of Meso-3D vascularonoid VPC2 cells, HE cells, or HB cells. *p<0.05 vs. vehicle. Mean ± sem. Uncorrected Fisher's LSD test following two-way ANOVA. [Figure 16A] This graph shows engrafted donor GMP1-Meso3D vasculonoid VPC2 cells at day 63 and day 180, stained with Ku80+, demonstrating long-term cell engraftment. [Figure 16B] This graph shows that meso-3D vasculonoid VPC2 cells engrafted on days 35 and 63, as indicated by Ku80+ staining. [Figure 16C] Fluorescence images of injected Meso3D vascularoid VPC2 63 days after HLI surgery in Balb / c nude mice, showing long-term graft survival (Ku80+), formation of human vascular structures (UEA1+ vessels), and promotion of paracrine host vascular growth (IB4+ and SMA+ vessels). [Modes for carrying out the invention]

[0086] Detailed description of the invention I. definition To make the present invention easier to understand, certain terms are first defined. Whenever a parameter value or range is stated, it should be noted that the values ​​and ranges between the stated values ​​are also part of the present invention.

[0087] In the following description, specific figures, materials, and components are given for illustrative purposes to ensure a full understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be carried out without these specific details. Where appropriate, well-known features may be omitted or simplified so as not to obscure the invention. Furthermore, where the specification refers to phrases such as “one aspect” or “a certain aspect,” it means that the specific features, structures, or characteristics described in relation to that aspect are included in at least one aspect of the invention. Where phrases such as “in one aspect” are used in different places in this specification, they do not necessarily all refer to the same aspect.

[0088] The articles “a” and “an” are used herein to refer to one or more (i.e., at least one) grammatical object of that article. For example, “an element” refers to one or more elements.

[0089] The terms “including” or “contain” are used herein in reference to compositions, methods and their constituent elements that are essential to the disclosure, but the inclusion of elements not specified, whether essential or not, is also permitted.

[0090] As used herein, “pluripotent cells,” “pluripotent stem cells,” and “PSCs” broadly refer to cells that, under appropriate conditions, remain undifferentiated, exhibit a stable (preferably normal) karyotype, and have the ability to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm), while also having the ability to proliferate in vitro for extended periods or virtually indefinitely. Typically, pluripotent cells (a) have the ability to induce teratomas when transplanted into immunodeficient (SCID) mice, (b) have the ability to differentiate into all three germ layer cell types (e.g., ectoderm, mesoderm, and endoderm), 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. Further exemplary pluripotent cells include, but are not limited to, those produced from embryonic stem cells, induced pluripotent (iPS) cells, embryo-derived cells, 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 transplanted into recipient oocytes). Exemplary pluripotent cells can be produced without destroying the embryo. For example, induced pluripotent cells can be produced from cells obtained without embryonic destruction. As a further example, pluripotent cells can be produced from biopsied blastomeres (which can be achieved without harming the remaining embryo). Optionally, any remaining embryos can be cryopreserved, cultured, and / or implanted in a suitable host. Pluripotent cells (whatever their source) can be genetically modified or otherwise altered.

[0091] As used herein, "embryonic" or "embryonic" broadly refers to a developing mass of cells that has not yet implanted in the uterine membrane of a maternal host. "Embryonic cells" are cells isolated from an embryo or cells 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) encompass pluripotent cells produced from embryonic cells (e.g., from cultured inner cell mass cells or cultured blastomeres). In many cases, such cells are serially passaged as cell lines or are serially passaged. 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 induction from embryos produced by any means (including by sexual or asexual means), 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 as embryonic stem cells, including when non-embryonic cells are used in the process. For example, ES cells can be embryonic stem cells induced from one or more blastomeres, as can be induced from the ICM of a blastocyst-stage embryo. Such embryonic stem cells can be produced by fertilization or generated from embryonic material through asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenesis. As further mentioned above, ES cells can be genetically modified or otherwise modified.

[0093] ES cells can be generated to be homozygous or heterozygous in one or more HLA genes, for example, by genetic engineering or screening for spontaneous loss of heterozygosity. Embryonic stem cells, regardless of their source or the specific method used to produce them, 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. Examples of embryonic stem cells that can be used in embodiments of the present invention include, but are not limited to, human ES cells ("hESCs" or "hES cells") such as CT2, MA01, MA09, ACT-4, No.3, J1, H1, H7, H9, H14, and ACT30 embryonic stem cells. Further exemplary cell lines include NED1, NED2, NED3, NED4, NED5, and NED7. See also the NIH Human Embryonic Stem Cell Registry. An example of a human embryonic stem cell line that can be used is the J1 cell line.

[0094] Examples of 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 and differentiation factor 3 (GDF3), REX1 (reduced expression 1), fibroblast growth factor 4 (FGF4), embryonic 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, germline nuclear factors, and stem cell factors (SCF or c-Kit ligands). In addition, 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 first be 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, e.g., human dermal fibroblasts (HDF). MEF cells or human feeder cells can be mitotically inactivated before seeding ESCs into co-culture by, for example, mitomycin C, gamma irradiation, or any other known method, so that MEFs do not proliferate in culture. Therefore, ESC cell cultures can be examined under a microscope, and colonies containing non-ESC cell morphology can be picked up 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 the ESCs have been harvested for seeding to form embryoid bodies.

[0096] Alternatively, hES cells may be cultured 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) under feeder-free conditions by any method known in the art, such as Klimanskaya et al., Lancet 365:1636-1641 (2005). Therefore, hES cells used in the methods described herein may be cultured in a feeder-free culture.

[0097] As used herein, “embryonic cells” (EDCs) broadly refer to pluripotent morula-derived cells, blastocyst-derived cells including inner cell masses, hyposhield or epiblastoid cells, or other pluripotent stem cells of early embryos including primitive endoderm, ectoderm and mesoderm, as well as their derivatives. “EDCs” also include blastomeres and aggregated single blastomeres or cell masses from embryos at various developmental stages, but do not include human embryonic stem cells that have been passaged as cell lines.

[0098] As used herein, “induced pluripotent stem cells,” “iPSCs,” or “iPS cells” refer to pluripotent stem cells generated by the 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 newly generated (by processes known in the art) before initiating differentiation into vascular progenitor cells (VPCs) or other cell types. The creation of iPS cells can be the first step in the production of differentiated cells. iPS cells can be specifically generated using material from a particular patient or compatible 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 autologous cells or histocompatible cells for the intended recipient. As further mentioned above (see "Pluripotent Cells"), pluripotent cells, including iPS cells, can be genetically modified or otherwise altered. An example human iPSC cell line that can be used is GMP1 cells.

[0099] As a further example, induced pluripotent stem cells can be generated by reprogramming somatic cells or other cells by exposing them to one or more reprogramming factors. For example, reprogramming factors can be expressed by cells in response to factors such as small molecules or microRNAs that promote or induce the expression of those genes, either 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 Reports, 10.1038 / srep00657, September 14, 2012. (These references are incorporated herein by reference in their entirety.) Reprogramming factors are provided from exogenous sources, for example by addition to culture media, 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, bioristic particle delivery systems (gene guns), and microinjection. In one embodiment, a combination of Oct4 (sometimes called Oct3 / 4), Sox2, c-Myc, and Klf4 can be used to reprogram somatic cells into pluripotent stem cells. In another embodiment, a combination of Oct-4, Sox2, Nanog, and Lin28 can be used to reprogram somatic cells into pluripotent stem cells.In another embodiment, somatic cells are reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. In yet another embodiment, somatic cells are reprogrammed by expressing Oct4, Sox2, MYC, Klf4, Nanog, and Lin28. In yet another embodiment, further 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 expressing 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 one embodiment, the somatic cells are fibroblasts, such as dermal fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblastic somatic cells. In one embodiment, 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 reprogramming factors can be induced by contacting somatic cells with at least one active agent that induces the expression of reprogramming factors, such as an organic small molecule active agent.

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

[0102] Once reprogramming factors are expressed in 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. The cells can be selected and subcultured, for example, based on ES morphology or based on the expression of selectable or detectable markers. The cells can be cultured to produce a culture of cells similar to 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 may have different expression profiles. Exemplary iPS cells may express Oct-4, alkaline phosphatase, SSEA3 surface antigen, SSEA4 surface antigen, TRA 1 60, and / or TRA 1 81.

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

[0104] As used herein, “mesoderm” refers to one of the three primary germ layers in the very early embryo of all belaterian animals. The mesoderm forms the mesenchyme, mesothelium, non-epithelial vascular cells, and coelocellular cells. Early mesoderm restraint arises from epithelial-mesenchymal transition, followed by the migration of designated mesodermal lineage cells inward as gastrulation progresses. Cells of the mesodermal lineage are destined to form the vascular and lymphoid systems, including angioblasts and multipotent mesenchymal stem cells capable of differentiating into several designated cell types. The mesoderm induces angiogenesis through the formation of the extraembryonic mesoderm and the subsequent 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 embodiment, cells of the mesodermal lineage are destined to become vascular precursor cells or vascular progenitor cells. In one embodiment, pluripotent stem cells, such as hESCs or iPSCs, such as hiPSCs, can be differentiated into mesoderm-lineage cells, such as mesoderm precursor cells. Therefore, the term “mesoderm” includes mesoderm-lineage cells derived from pluripotent stem cells, regardless of their maturity, and thus the term encompasses mesoderm cells at various maturity levels, 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, and INHB. Examples of markers 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 inhibitors, Wnt-3a, Wnt-8a, MESDC2, Nicalin, Brachyury, EOMES, FoxC1, FoxF1, Goosecoid, HAND1, MIXL1, Slug, Snail, TBX6, Twist-1, and Twist-2. In one embodiment, mesoderm cells are mesoderm 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. This includes the formation of mesoderm-derived endothelium. As used herein, "angiogenesis" refers to the formation of new blood vessels from existing ones. See, for example, 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 endothelial cells, smooth muscle cells, pericytes, and other cells capable of differentiating into blood vessel cell lineages. In one embodiment, the vascular progenitor cells are meso-VPCs (meso-VPCs).

[0108] As used herein, “meso-VPCs” refer to VPCs generated from mesoderm cells induced by in vitro differentiation of pluripotent stem cells, such as ESCs or iPSCs. Meso-VPCs may be identified by the expression of one or more cell surface markers, which are further described herein. In one embodiment, meso-VPCs are generated by the in vitro differentiation of pluripotent stem cells, such as ESCs or iPSCs, into mesoderm cells, which then differentiate into meso-VPCs.

[0109] meso-VPCs can be induced in vitro from both mouse and human PSCs. Meso-VPCs have the ability to differentiate into hematopoietic cell lineages and endothelial cell lineages, and may also have the ability to become smooth muscle cells. The meso-VPC population of the present invention may 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 1, 2, 3, 4, 5, 6, 7, or 8 of the markers listed above. In one embodiment, the meso-VPC population is positive for CD146, CD31 / PECAM1, and CD309 / KDR. In another embodiment, a population of meso-VPCs expresses CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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 one aspect, the meso-VPC population includes 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- The system 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 one embodiment, 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 meso-VPC population includes 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 one embodiment, the meso-VPC population expresses CD31 and KDR at higher levels than the HE cell population. In another embodiment, the meso-VPC population expresses CD146 at lower levels than the HE cell population. In yet another embodiment, the meso-VPC population expresses CD184 / CXCR4 at lower levels than the HE cell population.

[0110] In any embodiment, the meso-VPC population exhibits limited detection or no detection of one, two, or three of CXCR7, CD45, and NG2. In any embodiment, the meso-VPC population exhibits or no detection of all of CXCR7, CD45, and NG2. In any embodiment, the meso-VPC population exhibits 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 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 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 one embodiment, the meso-VPC of the present invention exhibits limited expression 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 aspect of the present invention, the population of meso-VPCs includes at least one meso-VPC that exhibits limited expression or does not 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 miRNA markers selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a.

[0111] As used herein, "vascularonoid" refers to a colony-like aggregate of cells, such as meso-VPCs, formed, for example, during cell culture. In one embodiment, vascularonoids are formed by meso-VPCs produced using a 3D-vascularonoid differentiation platform.

[0112] As used herein, “treatment,” “therapeutic,” “procedure,” “to treat,” or “procedure” broadly means treating a disease, stopping or reducing the development of the disease or its clinical symptoms and / or alleviating the disease, causing a regression of the disease or its clinical symptoms. “Treatment,” “therapeutic,” “procedure,” or “procedure” encompasses prevention, prevention, treatment, care, correction, reduction, mitigation, and / or alleviation of the disease, signs and / or symptoms of the disease. “Treatment,” “therapeutic,” “procedure,” or “procedure” encompasses the reduction of signs and / or symptoms in patients who are experiencing persistent signs and / or symptoms of the disease. “Treatment,” “therapeutic,” “procedure,” or “procedure” encompasses “prevention” and “prevention.” Prevention includes preventing the disease from occurring in a patient after treatment of the disease, or reducing the incidence or severity of the disease in a patient. The terms "reduced" in relation to treatment, "therapeutic," "treating," "administering," or "administering" broadly refer to a clinically significant reduction of signs and / or symptoms. "Treatment," "therapeutic," "treating," "administering," or "administering" include treating relapsing or recurrent signs and / or symptoms. "Treatment," "therapeutic," "treating," "administering," or "administering" include, but are not limited to, preventing the appearance of signs and / or symptoms, as well as reducing or eliminating existing signs and / or symptoms. "Treatment," "therapeutic," "treating," "administering," or "administering" include treating chronic diseases ("maintenance") and treating acute diseases. For example, treatment includes treating or preventing relapses or recurrences of signs and / or symptoms. In one embodiment, treatment includes a clinically significant reduction of signs and / or symptoms of vascular disease, such as critical limb ischemia.

[0113] As used herein, "normalizing a pathological condition" refers to restoring abnormal structures and / or functions caused by a disease to a more normal state. Normalization implies that the progression of the pathological condition can be managed and improved by correcting the abnormalities in the structure and / or function of tissues, organs or cell types caused by the disease. For example, after treatment with the meso-VPCs of the present invention, abnormalities in limbs as a result of vascular diseases, such as critical ischemic limb, 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 diseases outside the heart can occur anywhere. The most common vascular diseases are stroke, peripheral artery disease (PAD), abdominal aortic aneurysm (AAA), carotid artery disease (CAD), arteriovenous malformation (AVM), critical ischemic limb (CLI), pulmonary embolism (blood clot), deep vein thrombosis (DVT), chronic venous insufficiency (CVI) and varicose veins. In one aspect, the vascular disease is peripheral artery disease (PAD). In one aspect, the vascular disease is an ischemic disease such as critical ischemic limb (CLI). In one aspect, 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 aspect, the vascular disease is a peripheral artery disease such as critical ischemic limb (CLI), intestinal ischemia syndrome, renal artery disease, popliteal artery 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 mesoderm-derived vascular progenitor cells (meso-VPCs) from pluripotent stem cells. The method includes the steps of producing mesoderm cells by culturing pluripotent stem cells in a medium containing one or more mesoderm-inducing growth factors, and culturing the mesoderm cells on a suitable surface in a medium containing one or more factors that direct the differentiation of the mesoderm cells into mesoderm-derived vascular progenitor cells (meso-VPCs). In some aspects, the method further includes the step of 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 described above. In one embodiment, pluripotent stem cells, such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), are cultured under feeder-free (FF) conditions and plated onto an extracellular matrix. In another embodiment, pluripotent stem cells are cultured under feeder culture conditions and plated onto an extracellular matrix.

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

[0118] In some embodiments, pluripotent stem cells are cultured in a medium suitable for supporting pluripotency, any such medium being known in the art. In some embodiments, the pluripotency-supporting medium is Nutristom®. In some embodiments, the pluripotency-supporting medium is TeSR®. In some embodiments, the pluripotency-supporting medium is StemFit®. In another embodiment, the pluripotency-supporting medium is Knockout® DMEM (Gibco), which may be supplemented with Knockout® serum substitute (Gibco), LIF, bFGF, or any other factor. Each of these exemplary media is known in the art and commercially available. In further embodiments, the pluripotency-supporting medium may be supplemented with bFGF or any other factor. In some embodiments, 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 some embodiments, the medium is serum-free. In another embodiment, the medium contains serum.

[0119] Pluripotent stem cells can be cultured, subcultured, or harvested in any suitable container known in the art. Exemplary tissue culture vessels include 15 cm tissue culture plates, 10 cm tissue culture plates, 3 cm 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, pluripotent stem cells are cultured in a 6-well tissue culture plate.

[0120] In some embodiments, after approximately 1, 2, 3, 4, 5, or 6 days of culture, the culture medium is changed to maintain optimal conditions for the pluripotent stem cells. The culture medium may be the same as the starting medium, or the medium may be adjusted according to the culture needs. In some embodiments, the pluripotent stem cells are divided and passaged after approximately 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, or when the cell culture reaches approximately 60–90% confluence. The culture medium may be the same as the starting medium, or the medium may be adjusted according to the culture needs. The cells may be divided and passaged at dilution ratios 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, pluripotent stem cells are passaged at a dilution ratio of 1:3.

[0121] In some embodiments, pluripotent stem cells can be cultured under normal oxygen concentration conditions of approximately 5% CO2 and approximately 20% O2, or under other known conditions suitable for the growth of pluripotent stem cells.

[0122] In some embodiments, pluripotent stem cells are cultured, passaged, or harvested in a culture medium under feeder-free conditions, in which no feeder cell layer is included in the culture. In some embodiments, pluripotent stem cells are cultured, passaged, or harvested in a culture medium under feeder culture conditions, in which a feeder cell layer, such as human dermal fibroblasts (HDF) 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, e.g., hESCs or hiPSCs, are cultured on a suitable surface, e.g., an extracellular matrix surface. In some embodiments, the extracellular matrix is ​​selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycans, enterin, collagen, collagen I, collagen IV, heparan sulfate, soluble preparations from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells, Matrigel, gelatin, and human basement membrane extracts. In one embodiment, the extracellular matrix may be of any mammalian origin, including human. 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 culture medium for approximately 1 to 24 hours to allow the cells to settle prior to induction of differentiation. To induce differentiation of pluripotent stem cells into mesodermal cells, the pluripotent stem cells are cultured in culture medium on a suitable surface, such as the extracellular matrix surface described above.

[0125] The culture medium for inducing the 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 hematopoietic culture and / or hematopoietic vasodilation, 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-inducible 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 this method is VEGF165. In one embodiment, the FGF used in this 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 approximately 1, 2, 3, 4, 5, 6, or 7 days. In one embodiment, the culture duration is approximately 4 days. In one embodiment, the culture medium is replaced after approximately 24 hours of culture with a culture medium that does not contain activin A.

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

[0127] Differentiation of pluripotent stem cells into mesodermal cells can occur under normal oxygen concentration conditions of approximately 5% CO2 and 20% O2, or under other known conditions suitable for the 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. Examples of tissue culture containers include, but are not limited to, 15 cm tissue culture plates, 10 cm tissue culture plates, 3 cm 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 a 10 cm tissue culture plate.

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

[0130] In one embodiment, 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 contain N-cadherins, 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 heterodimers, BMP-2 / BMP-7 heterodimers, BMP-2a, BMP-4, BMP-6, BMP-7, cryptic, FABP4 / A-FABP, FGF-5, GDF-1, GDF-3, INHBA, INHBB, and nodal In addition, one or more other mesodermal markers selected from the group consisting of 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 inhibitors, Wnt-3a, Wnt-8a, MESDC2, nicarin, brachiuri, EOMES, FoxC1, FoxF1, goosecoid, HAND1, MIXL1, slug, snail, TBX6, Twist-1, and Twist-2 may also be expressed.

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

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

[0134] The method for a 3D-vascularonoid differentiation platform is carried out by culturing mesodermal cells in a culture medium under non-adhesion or low-adhesion conditions, for example, on an ultra-low-adhesion 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 hematopoietic vascular culture and / or hematopoietic vasodilation, 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 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 the 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 the 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 culture duration is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. In one embodiment, the culture duration is approximately 5 days. In one embodiment, the culture medium is replaced approximately 2 days and 4 days after the start of differentiation.

[0135] VEGF, for example VEGF165, can be used at concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably at concentrations of approximately 10 ng / mL to approximately 100 ng / mL. In one embodiment, VEGF is used at concentrations of approximately 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, for example bFGF, can be used at concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably at concentrations of approximately 10 ng / mL to approximately 100 ng / mL. In one embodiment, FGF is used at concentrations of approximately 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 concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably at concentrations of approximately 10 ng / mL to approximately 100 ng / mL. In one embodiment, BMP4 is used at concentrations of approximately 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. A small molecule inhibitor of the transforming growth factor beta (TGF-β) type I receptor, such as SB431542, can be used at concentrations of approximately 0.1 μM to approximately 100 μM, or more preferably, approximately 1 μM to approximately 100 μM.In one aspect, the small molecule inhibitor of the transforming growth factor beta (TGF-β) type I receptor is 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 aspect, 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 aspect, 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 mesoderm cells into meso-VPCs using a 3D-basculonoid differentiation platform can be performed under normoxic conditions of about 5% CO2 and about 20% O2, or under other known conditions suitable for the differentiation of pluripotent stem cells.

[0138] Differentiation of mesoderm cells into meso-VPCs using the 3D-vascularonoid differentiation platform can be carried out in any suitable container known in the art. Examples of tissue culture containers include, but are not limited to, 15 cm tissue culture plates, 10 cm tissue culture plates, 3 cm 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, differentiation of mesoderm cells into meso-VPCs using the 3D-vascularonoid differentiation platform is carried out in a 10 cm tissue culture plate.

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

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

[0141] The 2D differentiation platform provides a method for in vitro differentiating 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 in culture medium 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, proteoglycans, enterin, collagen, collagen I, collagen IV, heparan sulfate, soluble preparations from EHS (Engelbreth-Holm-Swarm) mouse sarcoma cells, Matrigel, gelatin, and human basement membrane extracts. In one embodiment, the extracellular matrix may be derived from any mammalian origin, 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 a culture medium known in the art. In some embodiments, the culture medium may be any medium that supports hemovascular culture and / or hemovascular dilation, 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 inhibitors of the 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.

[0144] In one embodiment, a method for a 2D differentiation platform for differentiating mesodermal cells to obtain meso-VPC comprises two steps. First, the mesodermal cells are differentiated in a culture medium that supports differentiation. This culture medium may be a culture medium known in the art. In some embodiments, the culture medium may be any medium that supports hemovascular culture and / or hemovascular dilation, 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 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 approximately 12 hours to approximately 2 days. In one embodiment, the first step of the 2D differentiation platform for differentiating mesoderm cells into meso-VPCs is carried out for approximately 1 day.

[0145] VEGF, for example VEGF165, can be used at concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably 10 ng / mL to approximately 100 ng / mL. In one embodiment, VEGF is used at concentrations of approximately 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, for example bFGF, can be used at concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably 10 ng / mL to approximately 100 ng / mL. In one embodiment, FGF is used at concentrations of approximately 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 concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably at concentrations of approximately 10 ng / mL to approximately 100 ng / mL. In one embodiment, BMP4 is used at concentrations of approximately 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 concentrations of approximately 0.1 μM to approximately 10 μM. In one embodiment, forskolin is used at concentrations of approximately 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 was used at a concentration of approximately 50 ng / mL, FGF at a concentration of approximately 50 ng / mL, BMP4 at a concentration of approximately 25 ng / mL, and forskolin at a concentration of approximately 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 normal oxygen concentration conditions of approximately 5% CO2 and 20% O2, or under other known conditions suitable for mesodermal cell differentiation.

[0148] The second step of the 2D differentiation platform is to further differentiate the cells obtained in the first step into meso-VPCs in a culture medium that supports differentiation. In some embodiments, the culture medium may be any medium that supports hematopoietic vascular culture and / or hematopoietic vasodilation, 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 such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), bone morphogenetic protein 4 (BMP4), small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptor, and / or forskolin. In one embodiment, the culture medium contains VEGF165, bFGF, BMP4, and SB431542. In one embodiment, the culture medium contains 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 mesoderm cells into meso-VPCs is carried out for about 6 days. In one embodiment, the culture medium is replaced about 2 days and about 4 days after the start of the second step of the 2D differentiation platform.

[0149] VEGF, for example VEGF165, can be used at concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably 10 ng / mL to approximately 100 ng / mL. In one embodiment, VEGF is used at concentrations of approximately 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, for example bFGF, can be used at concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably 10 ng / mL to approximately 100 ng / mL. In one embodiment, FGF is used at concentrations of approximately 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 concentrations of approximately 1 ng / mL to approximately 100 ng / mL, or more preferably at concentrations of approximately 10 ng / mL to approximately 100 ng / mL. In one embodiment, BMP4 is used at concentrations of approximately 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. A small molecule inhibitor of the transforming growth factor beta (TGF-β) type I receptor, such as SB431542, can be used at concentrations of approximately 0.1 μM to approximately 100 μM, or more preferably, approximately 1 μM to approximately 100 μM.In one embodiment, small molecule inhibitors of transforming growth factor beta (TGF-β) type I receptors are used at concentrations of approximately 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 concentrations of approximately 0.1 μM to approximately 10 μM. In one embodiment, forskolin is used at concentrations of approximately 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 approximately 50 ng / mL, FGF at a concentration of approximately 50 ng / mL, BMP4 at a concentration of approximately 25 ng / mL, small molecule inhibitors at a concentration of approximately 10 μM, and forskolin at a concentration of approximately 2 μM.

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

[0152] Two-step differentiation of mesoderm cells into meso-VPCs using a 2D differentiation platform can be carried out in any suitable container known in the art. Examples of tissue culture vessels include, but are not limited to, 15 cm tissue culture plates, 10 cm tissue culture plates, 3 cm 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, differentiation of mesoderm cells into meso-VPCs using a 2D differentiation platform is carried out in a T-75 tissue culture flask.

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

[0154] In one embodiment, meso-VPC produced by a 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 may be further sorted by methods known in the art, such as flow cytometry, to select cells having 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 meso-VPCs obtained by in vitro differentiation of mesoderm cells induced from pluripotent stem cells using the method disclosed herein. In one embodiment, pluripotent stem cells are first differentiated into mesoderm cells, and then these are 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, the meso-VPC population of the present invention expresses 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, the meso-VPC population expresses CD31 / PECAM1, CD309 / KDR, and CD146. In another embodiment, a population of meso-VPCs expresses CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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 one embodiment, 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.

[0158] In any embodiment, the population of meso-VPCs exhibits limited detection or no detection of one or more 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 one or more of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, or NG2. In some embodiments, the population of meso-VPCs is considered to exhibit limited expression 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-VPC 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-VPC in the composition of the present invention express CD31 / PECAM1, CD309 / KDR, and CD146. 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-VPC in the composition of the present invention express CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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, the expression of one or more of CXCR7, CD45, and NG2 is less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the meso-VPC in the composition of the present invention. In any embodiment, the expression of all of CXCR7, CD45, and NG2 is less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the meso-VPC in the composition of the present invention. In any embodiment, the expression of one or more of CD144, CD34, CD184 / CXCR4, CXCR7, CD43, CD45, PDGFRb, or NG2 is less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the meso-VPC in the composition of the present invention.

[0161] The meso-VPC of the present invention can be further characterized by a single-cell miRNA profile. In one embodiment, the meso-VPC of the present invention 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 any embodiment, the meso-VPC is 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-VPC is positive for mir126, mir125a-5p, mir24, and mir483-5p. In another embodiment, 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-VPC in the composition 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 of the present 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 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, the 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 markers selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a is less than approximately 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the meso-VPC in the composition. 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-VPC 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-VPC in the composition are positive for mir483-5p.

[0163] In one aspect, the meso-VPC population includes 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- The meso-VPC population 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, the meso-VPC population expresses hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p. In one embodiment, the meso-VPC population is considered to express a particular marker if at least about 20% of the meso-VPC 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 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-m The organism 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 present 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 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, h The organism 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 sa-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-VPC 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-VPC in the composition express hsa-miR-3917, hsa-miR-450a-2-3p, and hsa-miR-542-5p.

[0165] In one embodiment, the meso-VPC population 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, the meso-VPC population exhibits limited or no expression of hsa-let-7e-3p, hsa-miR-99a-3p, and hsa-miR-133a-5p. In one embodiment, a population of meso-VPCs may exhibit limited or no expression of hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In one embodiment, a population of meso-VPCs may exhibit limited or no expression of a marker if less than approximately 20% of the meso-VPCs in the composition express that marker.

[0166] 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 exhibits limited expression 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-VPC in the composition exhibits limited expression 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 features described above, the meso-VPC of the present invention possesses 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, the meso-VPC of the present invention possesses the potential to differentiate into vascular endothelial cells. Other vascular properties of the meso-VPC can be determined, for example, by Matrigel and AcLDL uptake assays.

[0168] In one embodiment, the meso-VPC of the present invention has vascular cell morphologies such as cobblestone-like endothelial morphology. Another method for characterizing the meso-VPC of the present invention is karyotype analysis to determine chromosomal integrity.

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

[0170] meso-VPC is approximately 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%, 0.01%, 0 This composition may contain 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% of pluripotent stem cells and mesoderm cells.

[0171] IV. Pharmaceutical composition 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 formulation, in which case the meso-VPCs can be formulated for administration in any convenient manner for use in medicine. Suitable carriers for the present disclosure include conventionally used carriers, such as water, saline solution, dextrose aqueous solution, lactose, Ringer's solution, buffer solution, hyaluronan, and glycol, which are exemplary liquid carriers, particularly for solution formulations (if isotonic).

[0172] Other exemplary carriers or excipients include, for example, 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 It is listed in 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 nonaqueous solutions selected from the group consisting of dispersions, suspensions, and emulsions, sterile powders which can optionally be reconstituted into sterile injectable solutions or dispersions immediately before use, antioxidants, buffers, bactericidal agents, solutes, or suspending and thickening agents.

[0174] Exemplary pharmaceutical compositions of the present invention may be any formulation suitable for use in the treatment of human patients, for example, patients suffering from vascular disease or vascular disorders. In one embodiment, a pharmaceutical composition comprising meso-VPC is formulated as an injectable, for example, suitable for intramuscular injection. A pharmaceutical composition comprising meso-VPC may be administered in a physiological pH buffer solution further containing an osmotic activator that maintains the solution at a physiological osmotic pressure. In one embodiment, a pharmaceutical composition comprising meso-VPC may be administered in a buffer solution containing at least 5% (w / v) glucose. In one embodiment, a pharmaceutical composition comprising meso-VPC may be administered in a buffer solution containing sodium chloride. Other reagents known in the art may also be used to formulate pharmaceutical compositions. In one embodiment, the buffer or solution used to formulate the pharmaceutical composition is sterilized before use.

[0175] The pharmaceutical compositions containing meso-VPC used in the methods described herein may be delivered as suspensions, gels, colloids, slurries, or mixtures. Alternatively, for delivery, cryopreserved meso-VPC may be resuspended in commercially available equilibrium salt solutions to obtain the desired osmotic pressure and concentration for administration by injection (e.g., bolus or intravenous). The pharmaceutical compositions containing meso-VPC may be mixed with a durable, inert matrix and delivered, for example, by one or more injections into a subject. The durable, inert matrix, such as a hydrogel—a natural or synthetic water-insoluble polymer—can serve as a scaffold for cell growth and expansion at the administration site. In one embodiment, the pharmaceutical composition containing meso-VPC is administered in a hyaluronane hydrogel. In another embodiment, the pharmaceutical composition containing meso-VPC is administered in a methylcellulose hydrogel. Other suitable substances known in the art that serve as durable, inert matrix scaffolds for cell growth and expansion may also be used in the methods described herein.

[0176] Pharmaceutical compositions containing meso-VPC can be delivered, for example, by one or more injections using a syringe. Alternatively, pharmaceutical compositions containing meso-VPC can be delivered by other suitable methods known in the art. Suitable delivery methods further 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 and provide an optimal environment for cell growth. Therefore, pharmaceutical compositions containing meso-VPC can also be formulated, for example, in the form of hydrogel tubes, hydrogel sheets, bioengineered patches made from 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 may also be used in the delivery of the pharmaceutical composition described herein.

[0177] A pharmaceutical composition typically must be sterile and stable under manufacturing and storage conditions. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it may be desirable to include in the composition an isotonic agent, such as sugar, polyalcohol, such as mannitol, sorbitol, or sodium chloride. Sustained absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption, such as monostearate and gelatin. Furthermore, soluble factors can be administered as depot formulations, for example as compositions containing slow-release polymers. The active compound can be prepared using carriers that protect the compound from rapid release, such as controlled-release formulations including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid-polyglycolic acid copolymers (PLG) can be used. Methods for preparing such formulations are numerous and patented or widely known to those skilled in the art.

[0178] One aspect of the present invention is at least 10 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 or 10 13This invention relates to a pharmaceutical composition suitable for use in mammalian patients, such as human patients, comprising meso-VPCs and a pharmaceutically acceptable carrier. The concentration of the meso-VPC pharmaceutical preparation for administration can be any effective amount and, for example, substantially free of PSCs. For example, the pharmaceutical composition may contain the number and types of meso-VPCs described herein. In a particular embodiment, the pharmaceutical composition of meso-VPC is for systemic administration to a host requiring it, approximately 1 × 10⁶ 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 approximately 1 x 10 12 ~Approx. 1×10 13 Approximately 1 × 10⁶ meso-VPCs are included for local administration to hosts that require them. 4 ~Approx. 1×10 5 , about 1×10 5 ~Approx. 1×10 6 , 1 x 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 x 10 12 ~Approx. 1×10 13 Includes individual meso-VPCs.

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

[0180] Vascular disease refers to any abnormal condition of blood vessels (arteries and veins). Extracardiac vascular disease can occur anywhere. 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 clots), deep vein thrombosis (DVT), chronic venous insufficiency (CVI), and varicose veins. In one embodiment, vascular disease is peripheral artery disease (PAD). In one embodiment, vascular disease is ischemic disease such as critical limb ischemia (CLI). In one embodiment, vascular disease is atherosclerosis, peripheral artery disease (PAD), carotid artery disease, venous disease, blood clots, aortic aneurysm, fibromuscular dysplasia, lymphedema, or vascular injury. In one aspect, vascular diseases include peripheral artery diseases such as critical limb ischemia (CLI), enteroischemic syndrome, renal artery disease, popliteal artery entrapment syndrome, Raynaud's phenomenon, or Buerger's disease.

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

[0182] The meso-VPC or pharmaceutical composition of the present invention may be administered systemically or topically. The meso-VPC or pharmaceutical composition may be administered using modalities known in the art, such as intravenous, intracranial, intramuscular, intraperitoneal injection, or other routes of administration, or local implantation, depending on the specific medical condition being treated, for example, but not limited to these. 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 is made from biocompatible fibers, biocompatible threads, biocompatible foams, aliphatic polyesters, poly(amino acids), copolymers (ether esters), polyoxalates, alkylenes, polyamides, tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamide esters, polyoxaesters containing amine groups, poly(anhydrides), polyphosphazenes, biopolymers; lactides, glycolides, epsilon-caprolactone, para-dioxanone, trimethylene carbonate homopolymers and copolymers; lactides, glycolides, epsilon-caprolactone, para -It can be manufactured using materials selected from dioxanone, trimethylene carbonate homopolymers and copolymers, fibrous collagen, non-fibrous collagen, pepsin-untreated collagen, other polymers, growth factors, extracellular matrix proteins, bio-related peptide fragments, hepatocyte growth factor, platelet-derived growth factor, platelet-rich plasma, insulin growth factor, growth and differentiation factors, vascular endothelial cell-derived growth factor, nicotinamide, glucagon-like peptides, tenascin C, laminin, anti-rejection agents, analgesics, antioxidants, anti-apoptotic agents, anti-inflammatory agents, and cell growth inhibitors combined with collagen.

[0184] Specific treatment regimens, routes of administration, and adjuvant therapies may be adjusted based on the specific disease state, the severity of the disease, and the patient's overall health status. Administration of meso-VPC or pharmaceutical compositions may be effective in reducing the severity of disease manifestation and / or preventing further deterioration of disease manifestation.

[0185] The treatment modality of the present invention may include the administration of a single dose of meso-VPC or a pharmaceutical composition. Alternatively, the treatment modality described herein may include a course of treatment in which meso-VPC or a pharmaceutical composition is administered multiple times over a period of time. An exemplary course of treatment may include weekly, bi-weekly, monthly, quarterly, twice annually, or once annual treatment. Alternatively, treatment may progress in stages, such that multiple doses are initially required (e.g., daily doses in the first week), followed by the need for less frequent administration.

[0186] In one embodiment, meso-VPC or the pharmaceutical composition is administered to the patient once or periodically over the patient's lifetime. In yet another embodiment of the present invention, meso-VPC or the 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 the pharmaceutical composition is administered using a device once, periodically over the patient's lifetime, or depending on the needs of that particular patient and the patient's condition being treated. Treatment regimens that change over time are also conceivable. For example, more frequent treatment (e.g., daily or weekly treatment) may be required initially. Over time, as the patient's condition improves, the frequency of required treatment may decrease, or even no further treatment may be needed.

[0187] In some embodiments, approximately 1 × 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 Approximately 540 million, 560 million, 580 million, 600 million, 620 million, 640 million, 660 million, 680 million, 700 million, 720 million, 740 million, 760 million, 780 million, 800 million, 820 million, 840 million, 860 million, 880 million, 900 million, 920 million, 940 million, 960 million, or 980 million meso-VPCs are administered to the target. In some embodiments, approximately 1 billion, 2 billion, 3 billion, 4 billion, or 5 billion or more meso-VPCs are administered. In some aspects, the number of meso-VPCs ranges from approximately 20 million to 4 billion, 40 million to 1 billion, 60 million to 750 million, 80 million to 400 million, 100 million to 350 million, and 175 million to 250 million.

[0188] The methods described herein may further include a step of monitoring the efficacy of the 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 angiogenesis, 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 necrotic area in a subject. Other physical and functional changes in a subject can also be measured and quantified to determine the efficacy of the treatment method for vascular disease.

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

[0190] In some embodiments, the present invention provides kits comprising the meso-VPC of the present invention together with reagents for selecting, culturing, expanding, maintaining, and / or transplanting the meso-VPC. Representative examples of cell selection kits, culture kits, expansion kits, 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 with respect to the expression of their gene products.

[0191] The present invention is further illustrated by the following examples, but these examples are not intended to be limiting. All references, patents, and patent application publications referenced throughout this application and the drawings are incorporated herein by reference. [Examples]

[0192] Example 1: Culture and differentiation of human pluripotent stem cells into mesodermal cells These studies used the proprietary human embryonic stem cell (hES) strain J1 and the human induced pluripotent stem cell (hiPS) strain GMP1. Cells were maintained in mTeSR1 complete medium (Stem Cell Technologies) at 37°C and normal oxygen concentration conditions of 5% CO2 + 20% O2 in 6-well tissue culture plates pre-coated with Matrigel (Corning) for feeder-free culture conditions (FF) or with Matrigel + human dermal fibroblasts (HDF) for feeder 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 the cells reached 60-70% confluence. For subculturing, 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. The cells were incubated at 37°C for 5–7 minutes, or until the edges of the colonies lifted off the plate. The medium containing the dispase or CDB was carefully aspirated and removed from the plate, and all residual enzyme or buffer was removed by gently washing the cells with DMEM-F12 (Gibco). Next, fresh mTeSR1 complete medium was used to collect the colonies from the plate, and they were scraped off with a disposable cell scraper using a strong wash, taking care not to create air bubbles, and then centrifuged 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 homogeneously mixed cell suspension was added to each well of a 6-well tissue culture plate containing 2 mL of mTeSR1 complete medium (either pre-coated with Matrigel for FF culture or Matrigel + HDF, as described above). Approximately 500,000 cells from the small cell clusters were evenly distributed to each well in the case of FF culture, and 250,000 cells from the small cell clusters were evenly distributed to each well in the case of HDF culture.Next, the cells were spread within the wells by moving them back and forth multiple times without swirling. The culture was checked daily for growth quality and morphology.

[0193] To differentiate pluripotent stem cells into mesodermal cells, 10cm tissue culture dishes (Corning) pre-coated with Matrigel were prepared by adding 5 mL of Matrigel / dish. After removing any unadhered Matrigel from each dish, 10 mL of mTeSR1 complete medium / 10cm dish was immediately added to prevent the Matrigel-coated surface from drying out completely. Approximately 1.5 million cells from small cell clusters of GMP1 cell cultures cultured in FF or HDF culture (or approximately 300,000 cells / 10cm dish from small cell clusters of J1 cell cultures cultured in HDF) were evenly distributed into each 10cm Matrigel pre-coated dish in 10 mL of TeSR1 complete medium. Next, the cells were spread throughout the dish by moving it back and forth several times without swirling, and then the plates were incubated for 24 hours (D-1) at 37°C under normal oxygen conditions of 5% CO2 + 20% O2 (Figure 1). At differentiation point D0, mTeSR1 complete medium was replaced with 12 mL / 10 cm dish of Stemline II medium (Sigma) containing a cocktail of mesoderm-inducible 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 differentiation point D1, 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 mesoderm 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 until day 4 at 37°C and normal oxygen concentration conditions of 5% CO2 + 20% O2 (Figure 1). Next, cells were harvested by dissociating them into single cells using the Stempro Acutase enzyme (Gibco).Cell characterization (by FACS and q-PCR analysis) confirmed the presence of mesodermal characteristics in the cells recovered on D4 (Figure 2A-B).

[0194] Example 2: Differentiation of human mesodermal cells into vascular progenitor cells (MESO-VPC) using a 3D-vasclonoid differentiation platform A novel 3D vascularonoid differentiation platform was developed by using an ultra-low adhesion tissue culture dish (Corning) and suspending the mesoderm cells obtained in Example 1 in VPC differentiation medium in the presence of factors that promote the appearance and expansion of vascular progenitor cells (Figure 3). One million unsorted D4 mesoderm cells were suspended in each well of an ultra-low adhesion 6-well plate and differentiated in VPC 3D differentiation medium (containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4, 10 μM SB431542, and either containing 2 μM forskolin ("Meso-3D vascularonoid VPC1" protocol) or forskolin-free ("Meso-3D vascularonoid VPC2" protocol), Stemline II medium) under normal oxygen concentrations (37°C, 5% CO2 and 20% O2). The respective culture media were changed to D2 and D4, and differentiation was completed on day 5. After 5 days of differentiation, MESO-VPCs from both protocols were harvested by dissociating them into single cells using the Stempro Acutase enzyme. The cells were then counted, viability was measured, and the cells were cryopreserved.

[0195] Example 3: Differentiation of human mesodermal cells into vascular progenitor cells (MESO-VPC) using a 2D differentiation platform. A novel 2D-based VPC differentiation platform was also developed by seeding mesoderm cells produced according to Example 1 onto adherent human extracellular matrix (collagen IV coated tissue culture dish). 1.2 million unsorted D4 mesoderm cells (from above) were seeded onto human collagen IV coated (5 mg / cm2) T-175 flasks (Corning) and differentiated in VPC 2D differentiation medium using two different differentiation protocols (Meso-2D VPC2 and Meso-2D VPC3) (Figure 4). The Meso-2D VPC2 protocol used Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, and 25 ng / mL BMP4 (40 mL / flask) for D0, and Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, and 25 ng / mL BMP4 + 10 μM SB431542 for D1 (45 mL / flask) to D7. The Meso-2D VPC3 protocol used Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4, and 2 μM forskolin for D0, and Stemline II medium containing 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4, and 2 μM forskolin + 10 μM SB431542 for D1 to D7. D0 cells were cultured under normal oxygen conditions (37°C, 5% CO2, and 20% O2). D1-D7 cells were cultured under hypoxic conditions (37°C, 5% CO2, and 5% O2), and the medium was changed to D3 (50 mL / flask) and D5 (60 mL / flask) for differentiation. After 7 days of differentiation, MESO-VPCs from the Meso-2D VPC2 and Meso-2D VPC3 protocols were recovered as single cells by enzymatic dissociation using the Stempro Acutase enzyme. Next, as described in Example 2, the cells were counted, viability was measured, and then they were cryopreserved.

[0196] Example 4: Matrigel / AcLDL assay Meso-VPCs from cryopreserved Examples 2-3 were rapidly thawed in a 37°C water bath (2-3 minutes). The cells were then transferred to a 15 mL conical tube with 10 mL of endothelial cell medium, i.e., EC medium (LifeLine Cell Technology's VascuLife® VEGF medium) and centrifuged at 300 × g for 5 minutes. 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 suspensions with concentrations of 10,000-20,000 live MESO-VPCs / mL were prepared using EC medium. Cells for Matrigel and AcLDL uptake assays were prepared by plating 3 mL / well of this cell suspension on fibronectin (FN) coated 6-well plates under normal oxygen 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 minutes. Once the plate was coated, cells were added to 250 μL of EC medium per well, totaling 5.0 × 10⁶ cells. 4 Cells were seeded at a specified density. After 2-3 hours 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 normal oxygen levels. After 24 hours of incubation, the AcLDL-containing medium was removed, the plates were washed three times with D-PBS, and 250 μL of fresh EC medium / well was added. Finally, micrographs of each well were taken at 4x magnification using a Keyence microscope.

[0198] Example 5: Flow Cytometry Assay The frozen vials of Meso-3D vasculonoid VPCs collected on day 5 and Meso-2D VPCs collected on day 7 (obtained in Examples 2-3 above) were thawed and prepared as single-cell suspensions in EC medium for cell counting. After cell counting, the 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 a rate of 5 μL / sample per 100 μL total volume. Cells were incubated with the antibodies on ice for 20-30 minutes. After incubation, cells were washed with 1 mL of FACS buffer to remove unbound antibodies. Next, the cells were centrifuged at 300 × g for 5 minutes, the supernatant was removed, and the cells were resuspended at a dilution of 1:1000 in 100 μL of fresh FACS buffer containing propidium iodide (PI, Sigma). PI was added to the cell suspension and used to eliminate dead cells during FACS analysis. A Sony SA3800 spectral analyzer was used for the analysis. Compensation was set up by using a positive control (HUVEC) and a negative control (undifferentiated J1 or GMP1 cells).

[0199] Example 6: Cells for comparison For comparison with the meso-VPC of the present invention, hematopoietic endothelial cells (HE) and angioblasts (HB) were generated from human embryonic stem cells (e.g., J1 hESCs) or human induced pluripotent stem cells (e.g., GMP-1 iPSCs) using the HE and HB protocols described above in, for example, 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 the PCT application claiming priority thereunder (all of these documents are incorporated herein by reference as is). Briefly speaking, single-cell aggregates were obtained by dissociating hESCs or iPSCs with Gibco® Cell Dissociation Buffer (CDB) to generate HE. 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 a 10 cm plate coated with collagen IV (Day 1). The plate was placed overnight in an incubator with normal oxygen pressure. The following day (Day 0), the mTeSR® 1 / Y-27632 medium was carefully 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 culture medium was aspirated and 10-12 mL of fresh BVF-M was added to each 10 cm plate. On day 4, the culture medium was aspirated again and 10-15 mL of fresh BVF-M was added to each 10 cm plate. On day 6, the cells were harvested for transplantation and / or further testing. The plates were washed by aspirating the culture medium from each plate, adding 10 mL of D-PBS (Gibco), and then aspirating the D-PBS.5 mL of StemPro Accutase (Gibco) was added to each 10 cm plate and incubated in a normal oxygen-pressure CO2 incubator (5% CO2 / 20% O2) for 3–5 minutes. Cells were pipetted 5 times with a 5 mL pipette, then approximately 5 times with a P1000 pipette. The cells were then filtered through a 30 μM cell strainer and transferred to collection tubes. 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 in collection tubes. 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, the cells were centrifuged (250 × g, 5 minutes) and placed in frozen medium (10% DMSO + heat-inactivated FBS) in 3 × 10⁶ units. 6 The cells were resuspended at a concentration of cells / mL. To prepare frozen stocks, the cell suspension was dispensed into 2 mL of FBS (Hyclone) and DMSO (Sigma) per cryovial (6 × 10⁻¹⁰⁻¹ 6 Cells / 2mL / vial).

[0200] To generate hemangioblasts (HBs), hESCs or iPSCs were dissociated with 4 mg / mL collagenase IV (Gibco) to obtain cell aggregates, which 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 in ultra-low adhesion surface 6-well plates (Corning) at a density of approximately 750,000 to 1,200,000 cells per well. Embryoid bodies were formed by incubating the plates in a CO2 incubator at normal oxygen pressure for 48 hours (days 0-2). Next, the medium and cells from each well were 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. Therefore, the final concentration of bFGF in the cell suspension was approximately 25 mg / mL. 4 mL of the cell suspension was plated into each well of a 6-well plate with an ultra-low adhesion surface, and the plate was placed in a CO2 incubator at normal oxygen pressure for a further 48 hours (days 2-4) to allow embryoid body formation to continue. On day 4, the embryoid bodies were collected in a 15 mL tube, centrifuged at 120-300 × g for 2 minutes, washed with D-PBS, and dissociated into single-cell suspension using StemPro Accutase (Gibco). Accutase was inactivated using FBS (Hyclone), and the single cells were passed through a cell strainer and centrifuged to approximately 1 × 10⁶ cells. 6 The cells were resuspended in Stemline II medium (Sigma) to a concentration of approximately 3 × 10⁶ 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 m / mL VEGF (Humanzyme), 50 ng / mL TPO (PeproTech), 30 ng / mL bFGF (Humanzyme)], replated onto ultra-low adhesion surface 10 cm dishes (Corning), and incubated in a normal oxygen pressure CO2 incubator for 7 days (days 4-11) to form angioblasts. On day 11, angioblasts were harvested for transplantation and / or further testing. Angioblasts were collected by diluting methylcellulose with D-PBS (Gibco). The cell mixture was centrifuged twice at 300×g for 15 minutes, resuspended in 30 mL of EGM2 BulletKit medium (Lonza) or StemlineII, counted the cells as described above, and frozen.

[0201] Example 7A: Cells with vascular precursor properties are generated by a 3D vascular ronoid differentiation platform. As described in Example 2, two different 3D differentiation protocols (Meso-3D vascularonoid VPC1 and Meso-3D vascularonoid VPC2) were used to generate meso-VPCs for 5 days under normal oxygen concentration conditions (37°C, 5% CO2, and 20% O2) (Figure 3). The seeded mesoderm cells remained viable and formed cell aggregates as early as day 1 (data omitted). These cell aggregates (hereinafter referred to as "vascularonoids") increased in size until day 5 (Figure 5, top panel), at which point they were harvested. The Meso-3D vascularonoid VPC1 protocol produced larger vascularonoid aggregates compared to the Meso-3D vascularonoid VPC2 protocol. After cell harvesting, the cells were replated onto FN-coated plates to determine the cells' ability to undergo further differentiation into endothelial lineages. As shown in the middle panel of Figure 5, when cells were cultured on an FN-coated plate in a medium that promotes endothelial differentiation, they acquired a cobblestone-like 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 showed higher tube formation ability compared to VPC1 cells (Figure 5, bottom panel).

[0202] In addition, FACS analysis of vascular markers revealed that both J1-derived and GMP1-derived Meso-3D vascular markers VPC1 and VPC2 cells exhibited 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 profiles differed from those observed in undifferentiated pluripotent stem cells (GMP1 and J1) or HUVEC cells, and in other PSC-derived cells (e.g., HB and HE) (Figures 6B-C). Chromosomal stability of these differentiated cells was assessed by G-banding karyotype analysis, and the cells exhibited a normal karyotype, indicating that differentiation of hES and hiPS cells using the Meso-3D vascular marker VPC1 and Meso-3D vascular marker VPC2 protocols does not alter chromosomal stability during differentiation (data omitted).

[0203] Example 7B: Cells with vascular precursor properties are generated by the 2D differentiation platform. As described in Example 3, meso-VPCs were generated from iPS cells (GMP1) and hES cells (J1) for a total of 7 days under normal and hypoxic culture conditions using two different 2D differentiation protocols (Meso-2D VPC2 and Meso-2D VPC3) (Figure 4). The seeded mesoderm cells adhered to the collagen IV coat surface, grew, and expanded into larger, denser cell colonies by day 7 (harvesting day) as 2D differentiated adherent cell cultures (Figure 7, top panel). The Meso-2D VPC2 protocol produced denser cell colonies (colonies were more "spiky" or "swirly") compared to the Meso-2D VPC3 protocol, using both J1-derived and GMP1-derived cells. After harvesting the cells on day 7, they were further cultured on FN-coated plates and exposed to endothelial culture medium. The cells exhibited typical vascular precursor properties, including a cobblestone-like endothelial morphology (Figure 7, middle panel) and the ability to form capillary-like networks on Matrigel and take up AcLDL (Figure 7, bottom panel), but were smaller in scale than meso-3D cells (comparison of the bottom panels of Figure 5 and Figure 7).

[0204] In addition, FACS analysis of vascular markers revealed that Meso-2D VPC1 and Meso-2D VPC2 cells derived from J1 and GMP1 cells both exhibited high expression of CD146, robust expression (>20%) of the endothelial markers KDR and CD31, and detectable (10-40%) expression of PDGFRb. This expression profile differed 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 to Meso-3D cells, Meso-2D VPCs expressed higher levels of CD146 and exhibited specific PDGFR expression, suggesting a greater tendency towards pericyte differentiation (Figures 6A-B). Furthermore, unlike Meso-3D cells, Meso-2D VPCs did not express either the blood markers CD45 or CD43.

[0205] Example 8: Single-cell miRNA profile Further analysis using single-cell qRT-PCR analysis was performed to evaluate the expression levels of 96 microRNAs associated with pluripotency or vascular cell identity, as described below. TaqMan gene expression assays (Applied Biosystems) were ordered for 96 human miRNAs. A 10× assay was prepared for a final stock volume of 50 μL by mixing 25 μL of 20× Taqman assay with 25 μL of 2× Assay Loading Reagent (Fluidigm). 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) at room temperature for 10 minutes. Cells were then washed, suspended in culture medium, and filtered through a 40 μm filter. Cell counts were performed using a serometer to obtain viability and cell concentration. A cell mixture was prepared by mixing 60 μL of cells with 40 μL of suspension reagent (Fluidigm) in a 3:2 ratio. 6 μL of this cell suspension mixture was loaded onto a primed Single-Cell Autoprep IFC microfluidic chip for medium-sized cells (10–17 μm) or large cells (17–25 μm), and the chip was then processed using a Fluidigm C1 instrument with 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, the viability / death status of the cells, and the captured doublets / cell aggregates.For cell lysis, reverse transcription, and pre-amplification at C1, the harvest reagent, lysis final mix, RT final mix, and preamp mix were added to the designated wells of the C1 tip according to the manufacturer's protocol. Next, the IFC was placed in C1 and the "STA: miRNA Preamp (1782x / 1783x / 1784x) script" was used. cDNA recovery was programmed to be completed the following morning. The cDNA was transferred from each chamber of the C1 tip to a fresh 96-well plate preloaded with 12.5 μL of C1 DNA diluent. Tube controls, such as template-less controls and positive controls, were prepared for each experiment according to the manufacturer's instructions. Pre-amplified cDNA samples were analyzed by qPCR using the 96.96 Dynamic Array® IFC and BioMark® HD system. IFC priming in the JUNO instrument, followed by loading of the cDNA sample mix and 10× assay, was performed according to the manufacturer's protocol. Next, the IFC was placed in the BioMark® HD system and the protocol "GE96x96 miRNA Standard PCR was performed using v1.pcl). Data analysis was performed using real-time PCR analysis software provided by Fluidigm. Dead cells and duplicates were removed from the analysis, and linear derivative baseline and user detector Ct threshold based methods were used. The data was displayed as a heatmap and exported as a CSV file. Next, an "Outlier Identification" analysis was performed using "R" software to obtain an "FSO" file, and then the "Automatic Analysis" instructions were followed.

[0206] result (Table 1) miRNA profiles TIFF0007894203000001.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 expressed in HUVECs, such as mir126, mir125a-5p, and mir24. Nevertheless, MESO-VPCs were negative for HUVEC-specific miRNAs (mirLet7-e, mir223, and mir99a). Finally, MESO-VPCs showed specific expression of miRNA 483-5P and were negative for mir142-3p and 133a, which are specific miRNAs for HB and HE, respectively.

[0208] Example 9: In vivo study in a hindlimb ischemia model Peripheral artery disease (PAD) is a form of peripheral vascular disease (PVD) characterized by partial or complete occlusion of the limbs, usually the lower limbs, resulting in impaired blood flow and tissue hypoxia. As PAD progresses, it reaches the stage of critical limb ischemia (CLI), accompanied by skin ulceration, gangrene, and unavoidable amputation. Hindlimb ischemia animal models have been used to evaluate various therapeutic approaches. In this study, a stable critical ischemia model (Ishikane et al. (2008) Stem Cells, 16:2625-2633) was used to evaluate the efficacy of meso-VPC and demonstrate improved blood flow recovery and signs of donor cell integration in the ischemic limb. Induction of hindlimb ischemia in mice involves two ligations of the proximal ends of the iliac and femoral arteries, as well as transection between these two ligations. This surgery causes impaired blood flow and subsequent severe ischemic injury.

[0209] seed The mice used 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. (Mice / Balb / cOlaHsd-Foxn1) nu (Charles River Laboratories).

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

[0211] Test item 2 = J1-HDF Meso-3D vascularonoid 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 as described in WO 2017 / 031312, which is incorporated herein in its entirety by reference) [552.2 mL of GS2: 0.9% sodium chloride washing USP (Baxter Healthcare or Hospira) (408.6 mL); 5% dextrose / 0.9% sodium chloride injection USP (Baxter or Braun) (33.2 mL); and BSS washing solution (Alcon) (110.4 mL)].

[0216] Test design and timeline The test was conducted according to the following test design (Table 2) and timeline (Table 3).

[0217] (Table 2) Study Design TIFF0007894203000002.tif96164IM=Local intramuscular injection into ischemic limb

[0218] (Table 3) Timeline TIFF0007894203000003.tif84164

[0219] Experimental Procedure Observation of illness and death The animals were continuously monitored twice a day (once a day on weekends) during and after the surgery.

[0220] body weight Weight was recorded once a week before and after the procedure.

[0221] HLI surgery The mice were placed on their backs under anesthesia and analgesia.

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

[0223] Test item administration procedure Immediately after surgery on day 0, intramuscular injections were administered to two locations on the proximal and distal sides of the surgical wound in each animal. 50 μl was injected into each site, for a total of 100 μl per mouse. The total amount per mouse was 1M cells / mouse.

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

[0225] Vascular imaging procedure Vascular imaging of both lower extremities (thigh and tibia) was measured in three mice from each group at three time points (7, 21, and 35 days after surgery) using the RSOM Explorer P50'' (i-Thera Medical) imaging system. The RSOM (Raster Scanning Optoacoustic Mesoscopy) Explorer P50 operates with a 532 nm nanosecond laser pulse and a spherically focused 50 MHz detector. An 80-second acquisition time enabled imaging with a 5 × 5 mm field of view, a depth of 3 mm, and a distance / azimuth resolution of 40 μm / 10 μm.

[0226] Macroscopic assessment procedure for ischemia severity Macroscopic evaluation of the ischemic limb was performed weekly, starting on the 7th postoperative day, using morphological grading 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 area TIFF0007894203000004.tif31138

[0228] Procedure for in vivo assessment of limb function A semi-quantitative assessment of limb use impairment was performed weekly, starting on postoperative day 7, using the scale shown in Table 5 below (see Stabile et al. Circulation. 2003. 108:205-210).

[0229] (Table 5) Evaluation of limb function TIFF0007894203000005.tif26128

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

[0231] Animal slaughter and tissue fixation Mice were sacrificed on day 36. Gastrocnemius muscle was collected from both hind limbs, fixed in formalin, and embedded in paraffin (5 mice per group). Muscle from 3 animals per group was OCT embedded, frozen, and stocked for further transport. The embedded muscle samples were sectioned, stained with H&E+IHC isolectin B4-HRP conjugate, and evaluated by a pathologist. IHC was performed for the presence of human cells in the tissue using a human-specific antibody (Stem121). ICH staining and vascular density assessment were performed for CD34.

[0232] result death Fourteen animals died during the experiment. One of them died during surgery. Thirteen animals were found dead in their respective cages within 11 days after HLI surgery. Of these, mice 19, 40, 99, 100, and 101 were in Group 1M, mouse 97 in Group 2M, mice 69, 72, 88, and 89 in Group 4M, mice 38 and 50 in Group 6M, and mouse 28 in Group 7. Twenty mice were euthanized for humane reasons by amputating their lower limbs (mouse numbers 58 and 98 in Group 2M, mice 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, mice numbers 36, 37, 47 and 53 in Group 6M, and mice numbers 29, 30, 32 and 34 in Group 7M). All surviving animals at each time point were evaluated at that time.

[0233] body weight Body weight was monitored until day 35 of the study. Weight decreased during the first week after surgery, but began to recover during the second week and was almost fully recovered by the final week. All animal groups recovered in parallel. Two-way ANOVA and subsequent Bonferroni post-hoc comparisons using GraphPad Prism 5 software did not reveal any statistically significant differences in body weight among the groups.

[0234] blood flow measurement Prior to the treatment of the test items, blood flow was evaluated, and significant changes were observed in all animals that underwent surgery. Throughout the study, a significant improvement in blood flow was observed in all treatment groups (3-7M) compared to the vehicle treatment group (2M). This improvement was statistically significant in the surgically treated right limb from day 21 in the 3M group and from day 28 to day 35 in the other treatment groups (two-way ANOVA followed by Bonferroni multiple comparisons) (Figure 8).

[0235] Vascular imaging Vascular imaging of both lower extremities (thigh and tibia) in three mice from each group at three time points (7, 21, and 35 days after surgery) was measured using the RSOM Explorer P50 (i-Thera Medical) imaging system.

[0236] Several analytical methods were used to assess the possible increase in small vessel density in the ischemic hind limb. Finally, to observe angiogenesis, the integration of the highest 100 sections was used for greater certainty. Results are presented as a summary at day 35 as a percentage at day 7. To clarify the data, the mean of all groups compared to the increase or decrease from the vehicle group is presented. Throughout the study, improvements in small vessel density were observed in the treatment groups (3, 4, 6, and 7M) compared to the vehicle treatment group (2M) (Figure 9).

[0237] Macroscopic assessment of ischemia severity Ischemic limbs were macroscopically assessed from day 7 to day 35 using a graded morphological scale for necrotic areas. Foot amputation was observed in all animal groups, with the lowest incidence 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 TIFF0007894203000006.tif46161

[0239] (Table 7) Incidence of mice with limb necrosis scores of 0, 1, and 2 on day 35 TIFF0007894203000007.tif46161

[0240] Evaluation of limb function A semi-quantitative assessment of ischemic limb use impairment was performed from day 7 to day 35 using a graded functional scale. Spontaneous improvement in limb function was observed in all animal groups. Nevertheless, animals treated with the test item in groups 4M and 5M showed better functional improvement compared to 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 TIFF0007894203000008.tif61156

[0242] (Table 9) Incidence of mice with limb function scores of 0, 1, 2, and 3 on day 35 TIFF0007894203000009.tif61156

[0243] Histological examination results All slides were stained with H&E and Masson's trichrome and examined by a single pathologist. This evaluation was performed as a semi-quantitative analysis (see grade below). CD34 + High-resolution tissue 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 (macrophage and satellite cell) grade: 0 = No inflammatory infiltration whatsoever. 1 = × 20 Mild cell infiltration, which is an increase of up to 10 cells per HPF. 2 = × 20, moderate cell infiltration is an increase of 10-20 cells per HPF. 3 = × 20, which is a high degree of cell infiltration, representing an increase of 20-50 cells per HPF. 4 = × 20 HPF is an extremely high degree of cell infiltration, which is an increase of >50 cells per HPF.

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

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

[0248] Consideration Immunohistochemical (IM) administration of the test item to ischemic limbs resulted in some improvement in limb function, blood flow (monitored by laser Doppler), RSOM imaging, and quantitative histological examination of blood vessels, primarily in treatment groups 4M and 5M. Treatment resulted in recovery of hemoperfusion (up to 78% of normal levels in the best-performing group, 4M) by the end of the study (day 36) in all treatment groups compared to the vehicle-treated group. This hemoperfusion recovery correlated well with the results of RSOM imaging analysis and immunohistochemical results regarding capillary density in the surgically treated hind limbs. Ratings of each group indicated that 4M was the best, followed closely by 6M and 7M. STEM121 staining of gastrocnemius paraffin-embedded slides did not reveal 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 vasculonoid VPC2 cells Meso-3D vasculonoid VPC2 cells were generated according to Example 2. A pellet of approximately 1 to 2 million cells was lysed, isolated RNAs were sequenced, and bioinformatics was used to analyze small RNA expression for known human transcriptomes (approximately 2000 miRNAs). Figure 12A shows the unique human miRNAs found in the J1-derived Meso-3D vasculonoid 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 the J1-derived HE cell population. 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] In addition, bulk small RNA-seq analysis revealed that miR214 was highly expressed in both J1-derived HE cells and meso 3D vascularonoid VPC2 cells, while miR335-5p was highly expressed in J1 cells and J1-derived HE cells, while miR335-3p was highly expressed in both J1-derived HE cells and meso 3D vascularonoid VPC2 cells. Similarly, miR199a-3p was highly expressed in both J1-derived HE cells and meso 3D vascularonoid VPC2 cells (data omitted).

[0251] Figure 12B shows the miRNA expression levels in a population of J1-derived Meso-3D vascularonoid VPC2 cells, which were 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 vascularonoid VPC2 cell population. Figure 12C shows that the J1-derived Meso-3D vascularonoid VPC2 cell population expresses hsa-let-7e-5p, hsa-miR-99a-5p, hsa-miR-223-5p, and hsa-miR-142-3p, while hsa-let-7e-3p, hsa-miR-99a-3p, and hsa-miR-133a-5p are either not expressed or expressed at low levels. Furthermore, Figure 12D shows that the population of J1-derived Meso-3D vascularonoid VPC2 cells expresses hsa-miR-483-5p and hsa-miR-483-3p.

[0252] Example 11: Single-cell RNA-seq analysis of Meso-3D vascularonoid VPC2 cells Single-cell RNA-seq analysis was also performed on J1-derived Meso-3D vascularonoid VPC2 cells generated according to Example 2. Approximately 3,700–8,000 single cells were captured, processed, and analyzed for single-cell sequencing for each cell type (J1 cells, J1-derived Meso-3D vascularonoid VPC2 cells, and HUVEC) using the 10X Genomics (Pleasanton, California) platform and its Cell Ranger analysis pipeline. Further data QC and data 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 involved performing an integrated analysis of J1 cells, J1-derived Meso-3D vasculonoid VPC2 cells, and HUVEC cells 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. The analysis retained only genes expressed in cells with ≥10 cells and at least 200 detected genes. Figure 13 shows the expression of the most upregulated or most downregulated genes in the J1-derived Meso-3D vasculonoid VPC2 cell sample compared to a single J1 cell or a single HUVEC cell.

[0253] Example 12: Vasculonoids increased in vitro cell viability and demonstrated efficacy in vivo. Vasculonoids from J1-derived Meso-3D vasculonoid VPC2 cells were generated according to Example 2. However, the cells were cryopreserved without dissociating into single cells in order to maintain their aggregated morphology.

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

[0255] Next, dissociated (i.e., "single") or undissociated (i.e., vascularonoid or "plural") Meso-3D vascularonoid VPC2 cells were seeded in 100 μl of culture medium into tissue culture-treated 96-well plates (approximately 14,000 single cells per well) or ultra-low adhesion 96-well plates (approximately 70 vascularonoid cells per well, equivalent to approximately 14,000 single cells per well). To test CLI-mimicking conditions (i.e., hyperglycemia and / or hypoxia), cells were cultured for 72 hours under normal oxygen conditions (20% O2) or hypoxic conditions (5% O2) with either complete VascuLife® basal medium containing 5.5 mM D-glucose as a control (Lifeline® Cell Technology, Frederick, Maryland) or complete VascuLife® basal medium with a high glucose concentration (30 mM). After 72 hours, relative cell viability was measured by incubating each well with 100 µl of CellTiter-Glo® reagent (Promega, Madison, Wisconsin) for 45 minutes as directed by the manufacturer. For both single-cell and multicellular cells under each oxygen condition, luminescence was measured as a readout of cell viability and normalized to a 5.5 mM control. Figure 14B shows that these vasculonoids exhibited better cell viability when cultured in vitro under CLI-mimicking conditions at normal oxygen concentrations (20% O2) or hypoxic concentrations (5% O2) after thawing as described above, compared to J1-derived Meso-3D vasculonoid VPC2 cells that had been cryopreserved as single cells.

[0256] To test in vivo efficacy, after inducing hindlimb ischemia as detailed in Example 9, GMP1-Meso3D VPC was injected into the quadriceps muscles of Balb / c nude mice (n=15 in each group) as single cells (Meso3D sc, 1 million total single cells per mouse) or as undissociated multicellular vascularoids (Meso3D vascularoids, 25,000 per mouse, roughly equivalent to 1 million total single cells per mouse). Blood flow was assessed by laser Doppler perfusion imaging (LDPI) immediately after surgery and weekly thereafter until day 64. Figure 14C shows statistically significant improvements in blood flow over the entire study compared to the vehicle-treated group (GS2 medium only) after administration of single cells or vascularoids; Tukey's test following two-way ANOVA.

[0257] Example 13: Long-term effects of Meso-3D vascularonoid VPC2 cells in an HLI model Meso-3D vascularonoid VPC2 cells were generated (as dissociated single cells) according to Example 2 and administered to the HLI animal model described in Example 9, and the long-term effects were observed. In these studies, after HLI surgery, 1 million GMP1-derived cells per mouse (GMP1 Meso3D vascularonoid VPC2, GMP1-HE, and GMP1-HB) were injected into the right quadriceps muscle in GS2 medium, or GS2 medium alone (vehicle) was injected (n=12-19 mice / group). Limb necrosis and limb function were scored as described in Example 9. For some cell types, more than two lots of cells produced in independent differentiation experiments were used, so the number of animals increased when data from more than two lots of the same cell type were combined. Data are mean ± sem, averaged from two independent repeated studies. *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 vascularonoid VPC2 cells had better mean necrosis and mean functional scores at day 21 compared to HE and HB cells. Figure 15B shows the improvement in blood flow at day 63 compared to the vehicle in animals treated with meso-3D vascularonoid VPC2 cells, HE cells, and HB cells. CD34 vascular growth in the quadriceps (Figure 15C) and gastrocnemius (Figure 15D) showed improvement with all three cell types, with HB showing better growth around day 35. However, by day 63, all three cell types promoted growth similarly, and meso-3D vascularonoid VPC2 cells appeared to promote growth slightly better in the gastrocnemius than HE and HB.

[0259] Meso-3D vasculonoid VPC2 cells also showed long-term engraftment exceeding 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 number 1 = 19 mice, GMP1-Meso3D lot number 2 = 18 mice, GMP1-Meso3D lot number 3 = 19 mice, GMP1-HE = 19 mice, and J1-HE = 19 mice). The injection sites were then marked with tattoos. On days 14, 35, 63, and 180, quadriceps muscles were collected from the surgically treated right hind limb, fixed with PFA, embedded in paraffin, and stained for the human-specific marker Ku80. Two images (20x magnification) were analyzed per mouse. Each group had at least n=3, except for J1-HE on day 14, where there was only n=1. Data are represented mean ± sem by blinded independent histopathologists using the following semi-quantitative scale: 0 = no positive Ku-80 cells; 1 = < 5 positive Ku-80 cells present; 2 = > 5 and < 15 positive Ku-80 cells present; 3 = > 15 and < 50 positive Ku-80 cells present; 4 = > 50 positive Ku-80 cells present. Figure 16A shows engrafted donor GMP1-Meso3D vasculonoid VPC2 cells at days 63 and 180, which demonstrate long-term cell engraftment. However, GMP-1 derived HE appeared to show better engraftment at day 180.

[0260] In the second trial (Figure 16B), 1 million cells per mouse in GS2 medium were injected into the right quadriceps muscle after HLI surgery (GMP1-Meso3D vasculonoid VPC2 cells = 16 mice, GMP1-HE lot number 1 = 16 mice, GMP1-HE lot number 2 = 17 mice, GMP1-HB lot number 1 = 16 mice, GMP1-HB lot number 2 = 16 mice). The injection sites were then marked with tattoos. On days 14, 35, and 63, quadriceps muscles were collected from the surgically treated right hind limb, fixed with PFA, embedded in paraffin, and stained for the human-specific marker Ku80. For each group, at least n=2 mice were analyzed, with two images (20x magnification) taken using an Olympus BX60 optical microscope. Ku80+ cells were quantified by blinded independent histopathologists. Data represent mean ± sem. Figure 16B shows that meso-3D vasculonoid VPC2 cells engrafted by day 35 and day 63. However, one lot of GMP-1-derived HE showed better engraftment at day 63.

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

[0262] Equal portions A person skilled in the art will recognize, or at best verify, by routine experimentation, numerous equivalents of specific embodiments of the invention described herein. Such equivalents are intended to be included in the claims below. All references, patents, and published patent applications referred to throughout this application are incorporated herein by reference.

Claims

1. A method for producing a population of meso-VPCs (meso-VPCs) from pluripotent stem cells, (a) To produce mesoderm cells, in a mesoderm differentiation medium containing activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4), on the extracellular matrix surface, 5% CO2 2 and 20% 2 The process involves culturing pluripotent stem cells for 3 to 5 days under normal oxygen concentration conditions, and (b) In meso-VPC differentiation medium containing VEGF, FGF, BMP4 and transforming growth factor beta (TGF-β) type I receptor inhibitors, under non-adhesion conditions at 5% CO2 2 and 20% 2 A process of culturing pluripotent stem cell-derived mesoderm cells for 3 to 7 days under normal oxygen concentration conditions, thereby producing a population of mesoderm-derived vascular progenitor cells (meso-VPCs). The method, including the method described above.

2. I) The mesoderm differentiation medium contains 5–15 ng / mL activin A, 5–25 ng / mL VEGF, 5–25 ng / mL FGF and 5–50 ng / mL BMP4, and / or II) Meso-VPC is produced as a vasculonoid, and / or III) The meso-VPC is produced as a vascularonoid, and the method further includes a step of dissociating the meso-VPC in the vascularonoid into single cells. The method according to claim 1.

3. I) The mesoderm differentiation medium contains 10 ng / mL activin A, 10 ng / mL VEGF165, 10 ng / mL FGF-2 and 25 ng / mL BMP4, and / or II) The extracellular matrix surface is a Matrigel® coated surface. The method according to claim 1 or 2.

4. I) The meso-VPC differentiation medium contains 10–50 ng / mL VEGF, 10–50 ng / mL FGF, 10–50 ng / mL BMP4 and 5–20 μM of a transforming growth factor beta (TGF-β) type I receptor inhibitor, and / or II) The inhibitor of the transforming growth factor beta (TGF-β) type I receptor is SB431542, and / or III) The meso-VPC differentiation medium contains 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4 and 10 μM SB431542, and / or IV) The meso-VPC differentiation medium further contains forskolin, and / or V) The meso-VPC differentiation medium further contains forskolin, and forskolin is used at a concentration of 2–10 μM. The method according to any one of claims 1 to 3.

5. A method for producing a population of meso-VPCs (meso-VPCs) from pluripotent stem cells, (a) To produce mesoderm cells, in a mesoderm differentiation medium containing activin A, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and bone morphogenetic protein 4 (BMP4), on the extracellular matrix surface, 5% CO2 2 and 20% 2 A process of culturing pluripotent stem cells for 3 to 5 days under normal oxygen concentration conditions; (b) In a first medium containing VEGF, FGF, and BMP4, on the surface of the extracellular matrix, 5% CO 2 and 20% 2 A step of culturing pluripotent stem cell-derived mesodermal cells for one day under normal oxygen concentration conditions; and (c) On the extracellular matrix surface, in a second medium containing inhibitors of VEGF, FGF, BMP4, and transforming growth factor beta (TGF-β) type I receptor, at 5% CO 2 and 5% O 2 culturing the cells produced in step (b) for 4 to 7 days under low oxygen concentration conditions, thereby producing a population of mesoderm-derived vascular progenitor cells, step The method, including the method described above.

6. I) The mesoderm differentiation medium contains 5–15 ng / mL activin A, 5–25 ng / mL VEGF, 5–25 ng / mL FGF and 5–50 ng / mL BMP4, and / or II) The method further comprises a step of dissociating a population of meso-VPCs into single cells, and / or III) The mesoderm differentiation medium contains 10 ng / mL activin A, 10 ng / mL VEGF165, 10 ng / mL FGF-2 and 25 ng / mL BMP4, and / or IV) The extracellular matrix surface in step (a) is a Matrigel® coated surface, and / or V) The first medium contains 10–50 ng / mL VEGF, 10–50 ng / mL FGF, and / or 10–50 ng / mL BMP4. VI) The first medium contains 50 ng / mL VEGF165, 50 ng / mL FGF-2 and 25 ng / mL BMP4, and / or VII) The second medium contains 10–50 ng / mL VEGF, 10–50 ng / mL FGF, 10–50 ng / mL BMP4 and 5–20 μM of a transforming growth factor beta (TGF-β) type I receptor inhibitor, and / or VIII) The inhibitor of the transforming growth factor beta (TGF-β) type I receptor is SB431542, and / or IX) The second medium contains 50 ng / mL VEGF165, 50 ng / mL FGF-2, 25 ng / mL BMP4 and 10 μM SB431542, and / or X) The first medium and / or the second medium further contain forskolin, and / or XI) The first medium and / or the second medium further contain forskolin, and forskolin is used at a concentration of 2–10 μM, and / or XII) The extracellular matrix surface in steps (b) and (c) is a collagen IV coated surface. The method according to claim 5.

7. I) The pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells, and / or II) The meso-VPC population expresses at least one of the cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD43, CD144, CD34, CD184 / CXCR4, CD146, and PDGFRb, and / or III) The population of meso-VPCs expresses the cell surface marker (a) CD146, CD31 / PECAM1, and CD309 / KDR, or (b) CD31 / PECAM1, CD309 / KDR, CD146, and (i) at least one of 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, and / or IV) Less than 20% of the meso-VPC population express (a) one or more cell surface markers selected from the group consisting of CXCR7, CD45, and NG2, or (b) CXCR7, CD45, and NG2, and / or V) The meso-VPC population 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, and / or VI) Less than 20% of the meso-VPC population express 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, and / or VII) The meso-VPC population expresses hsa-miR-3917, hsa-miR-450a-2-3p and hsa-miR-542-5p, and / or VIII) The population of meso-VPCs includes 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, and / or IX) The population of meso-VPCs includes meso-VPCs that are positive for mir483-5p, and / or X) Less than 20% of the meso-VPC population express at least one miRNA marker selected from the group consisting of mir367, mir302a, mir302b, mir302c, mirLet7-e, mir223, mir99a, mir142-3p, and mir133a, and / or XI) The method further comprises a step of producing vascular endothelial cells by differentiation of meso-VPCs, and / or XII) The method further includes a step of producing vascular endothelial cells by differentiation of meso-VPCs on the fibronectin coat surface, The method according to any one of claims 1 to 6.

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