Treatment methods for vascular diseases

Abstract

The present invention provides methods for treating vascular disease using hemogenic endothelial cells (HE) derived from pluripotent stem cells in vitro. The present invention also provides compositions of HE and methods for producing HE.

Description

[Technical Field] 【0001】 Related applications This application claims the benefit of U.S. Provisional Application No. 62 / 892,712, filed on 28 August 2019 pursuant to 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety. 【0002】 Field of Invention The present invention relates to a method for treating vascular diseases using hematopoietic endothelial cells obtained by in vitro differentiation of pluripotent stem cells. [Background technology] 【0003】 background Cardiovascular disease is a type of illness that affects the heart or blood vessels, and it is the leading cause of death worldwide. In the United States alone, approximately 84 million people suffer from cardiovascular disease, and nearly one in three deaths are attributable to it. 【0004】 Pulmonary hypertension (PH) is a condition characterized by increased pressure in the main pulmonary arteries. A fatal form of PH is pulmonary artery hypertension (PAH), which typically leads to death within an average of 2.8 years of diagnosis. PAH is characterized by vasoconstriction and remodeling of the pulmonary blood vessels. While available standard treatments can improve quality of life and patient prognosis, they usually do not directly prevent the pathological remodeling process and may cause serious side effects. 【0005】 Peripheral artery disease (PAD) is the abnormal narrowing and occlusion of arteries other than those of the cerebral and coronary circulation. Critical limb ischemia (CLI) is a severe form of PAD that results in severe occlusion of arteries in the lower extremities. CLI is associated with limb loss, myocardial infarction, stroke, and death. Currently, there is no effective treatment for CLI. 【0006】 Coronary artery disease is the most common form of cardiovascular disease, caused by reduced blood flow and oxygen to the heart muscle due to atherosclerosis of the arteries of the heart. Patients with coronary artery disease often undergo balloon angioplasty or stenting to open the blocked arteries. Some patients undergo costly and high-risk coronary artery bypass surgery. 【0007】 Therefore, better methods are needed to treat and prevent vascular diseases. [Overview of the project] 【0008】 The present invention provides a method for treating vascular disease, comprising the step of administering a composition containing hematopoietic endothelial cells (HE) obtained by in vitro differentiation of pluripotent stem cells to a target. 【0009】 In some embodiments, vascular diseases include coronary artery disease (e.g., arteriosclerosis, atherosclerosis, and other diseases or injuries of arteries, arterioles and capillaries, or related conditions), myocardial infarction (e.g., acute myocardial infarction), organizing myocardial infarction, ischemic heart disease, arrhythmias, left ventricular dilation, embolism, heart failure, congestive heart failure, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditis, chronic coronary ischemia, dilated cardiomyopathy, restenosis, arrhythmias, angina, hypertension (e.g., pulmonary hypertension, glomerular hypertension, portal hypertension), myocardial hypertrophy, peripheral artery disease including severe limb ischemia, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cor pulmonale, dyscardial infarction, inflammatory heart disease, congenital heart disease, rheumatic heart disease, diabetic vascular disease, and endothelial lung injury. The group is selected from a selection of diseases (e.g., acute lung injury (ALI), acute respiratory distress syndrome (ARDS)). In one specific embodiment, the vascular disease is pulmonary hypertension. In another embodiment, the vascular disease is pulmonary artery hypertension. 【0010】 In any embodiment of the methods disclosed herein, the mean pulmonary (arterial) blood pressure is reduced in the subject. 【0011】 The present invention also provides a method for increasing blood flow in the pulmonary artery, comprising the step of administering to a subject a composition containing HE obtained by in vitro differentiation of pluripotent stem cells. In one embodiment, the subject has pulmonary hypertension. Specifically, in one embodiment, the subject has pulmonary artery hypertension. 【0012】 The present invention further provides a method for reducing blood pressure in a subject, comprising the step of administering to the subject a composition containing HE obtained by in vitro differentiation of pluripotent stem cells. In one embodiment, the subject has pulmonary hypertension. Specifically in one embodiment, the subject has pulmonary artery hypertension. In another embodiment, blood pressure is diastolic pressure. In yet another embodiment, blood pressure is systolic pressure. In yet another embodiment, blood pressure is mean pulmonary (arterial) blood pressure. Furthermore, by any of the methods of the present invention, blood pressure can be reduced by at least 20% in the subject. 【0013】 In one embodiment, the pluripotent stem cells disclosed herein are embryonic stem cells. In another embodiment, the pluripotent stem cells disclosed herein are induced pluripotent stem cells. 【0014】 In yet another embodiment, the HE disclosed herein is obtained by culturing pluripotent stem cells under adhesion conditions, in a differentiation medium, and in the absence of methylcellulose. In yet another embodiment, the HE disclosed herein is obtained by in vitro differentiation of pluripotent stem cells without embryoid body formation. 【0015】 In any of the methods provided herein, the subject may be human. In addition, the pluripotent stem cells disclosed herein may be human pluripotent stem cells. Furthermore, the HE disclosed herein may be human HE. 【0016】 HE disclosed herein may be positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In one embodiment, HE is positive for (i) miRNA-214, miRNA-199a-3p, and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In another embodiment, HE is positive for (i) miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. In one embodiment, HE is positive for miRNA-214. 【0017】 In any of these embodiments, the HE disclosed herein may be negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. In one embodiment, the HE is negative for miRNA-223 and miRNA-142-3p. In another embodiment, the HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. 【0018】 In one embodiment, HE is positive for miRNA-214, miRNA-199a-3p, and miRNA-335, and negative for miRNA-223 and miRNA-142-3p. 【0019】 In one embodiment, each of the HEs disclosed herein expresses at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD144, CD34, CXCR4, CD146, Tie2, CD140b, CD90, CD271, and CD105. In one embodiment, the HEs of the present invention express CD146, CXCR4, CD309 / KDR, CD90, and CD271. In another embodiment, the HEs of the present invention express CD146. In one embodiment, the HEs of the present invention express CD31 / PECAM1, CD309 / KDR, CD144, CD34, and CD105. 【0020】 In one embodiment, HE shows limited detection or no detection of at least one cell surface marker selected from the group consisting of CD34, CXCR7, CD43, and CD45. In another embodiment, HE shows limited detection or no detection of CXCR7, CD43, and CD45. In yet another embodiment, HE shows limited detection or no detection of CD43 and CD45. 【0021】 In one embodiment, the HE of the present invention is CD43(-), CD45(-), and / or CD146(+). In another embodiment, the HE expresses CD31, calponin (CNN1), and NG2, and therefore has the potential to differentiate into endothelial cells (CD31+), smooth muscle (calponin+), and / or pericytes (NG2+). 【0022】 In one aspect, CD144(VECAD)-expressing HE of the present invention is isolated from HE. In one aspect, the isolated CD144(VECAD)-expressing HE cells further express CD31 and / or CD309 / KDR(FLK-1). In another aspect, the isolated CD144(VECAD)-expressing HE cells further express at least one gene described in Tables 22 and 23. In another aspect, the isolated CD144(VECAD)-expressing HE cells further express at least one cell marker selected from the group consisting of PLVAP, GJA4, ESAM, EGFL7, KDR / VEGFR2, and ESAM. In one aspect, the isolated CD144(VECAD)-expressing HE cells further express at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA. In another aspect, the isolated CD144(VECAD)-expressing HE cells further express at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. In one aspect, the composition comprising CD144(VECAD)-expressing HE isolated from HE of the present invention is substantially free of CD144(VECAD)-negative HE. 【0023】 Therefore, the present invention also provides a composition comprising HE obtained by in vitro differentiation of pluripotent stem cells disclosed herein. The present invention further provides a pharmaceutical composition comprising HE obtained by in vitro differentiation of pluripotent stem cells disclosed herein and a pharmaceutically acceptable carrier. [Invention 1001] A method for treating vascular disease in subjects suffering from or suspected of suffering from vascular disease, comprising the step of administering a composition containing hematopoietic endothelial cells (HE) obtained by in vitro differentiation of pluripotent stem cells to a subject. [Invention 1002] Vascular diseases include coronary artery disease (e.g., arteriosclerosis, atherosclerosis, and other diseases or injuries of arteries, arterioles and capillaries, or related conditions), myocardial infarction (e.g., acute myocardial infarction), organizing myocardial infarction, ischemic heart disease, arrhythmias, left ventricular diastole, embolism, heart failure, congestive heart failure, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditis, chronic coronary ischemia, dilated cardiomyopathy, restenosis, arrhythmias, angina, hypertension (e.g., pulmonary hypertension, glomerular hypertension, portal hypertension), myocardial hypertrophy, peripheral artery disease including severe limb ischemia, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cor pulmonale, dyscardial infarction, inflammatory heart disease, congenital heart disease, rheumatic heart disease, diabetic vascular disease, and endothelial lung injury. The method of the present invention 1001, selected from the group consisting of disease) (e.g., acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)). [Invention 1003] The method of the present invention 1001, wherein the vascular disease is pulmonary hypertension. [Invention 1004] The method of the present invention 1001, wherein the vascular disease is pulmonary artery hypertension. [Invention 1005] A method according to any of the present invention 1001 to 1004, wherein the mean pulmonary (arterial) blood pressure is reduced in the subject. [Invention 1006] A method for increasing pulmonary artery blood flow in a subject suffering from or suspected of suffering from a vascular disease, comprising the step of administering a composition containing HE obtained by in vitro differentiation of pluripotent stem cells to the subject. [Invention 1007] The method of the present invention 1006, wherein the subject has pulmonary hypertension. [Invention 1008] The method of the present invention 1006, wherein the subject has pulmonary artery hypertension. [Invention 1009] A method for reducing blood pressure in subjects suffering from or suspected of suffering from vascular disease, comprising the step of administering a composition containing HE obtained by in vitro differentiation of pluripotent stem cells to a subject. [Invention 1010] The method of the present invention 1009, wherein the subject has pulmonary hypertension. [Invention 1011] The method of the present invention 1009, wherein the subject has pulmonary artery hypertension. [Invention 1012] The method of the present invention 1009, wherein blood pressure is diastolic pressure. [Invention 1013] The method of the present invention 1009, wherein blood pressure is systolic pressure. [Invention 1014] The method of the present invention 1009, wherein the blood pressure is the mean pulmonary (arterial) blood pressure. [Invention 1015] A method according to any of the present invention 1009 to 1014, wherein blood pressure is reduced by at least 20% in the subject. [Invention 1016] The method according to any one of the invention 1001 to 1015, wherein HE is positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. [Invention 1017] The method of the present invention 1016, wherein HE is positive for (i) miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [Invention 1018] The method of the present invention 1016, wherein HE is positive for (i) miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [Invention 1019] The method of the present invention 1016, wherein HE is positive for miRNA-214. [Invention 1020] The method according to any one of the invention 1001 to 1019, wherein HE is negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. [Invention 1021] The method of the present invention 1020, wherein HE is negative for miRNA-223 and miRNA-142-3p. [Invention 1022] The method of the present invention 1020, wherein HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. [Invention 1023] A method according to any one of the present invention 1001 to 1022, wherein HE expresses at least one cell surface marker selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD144, CD34, CXCR4, CD146, Tie2, CD140b, CD90, CD271, and CD105. [Invention 1024] The method of the present invention 1023, wherein HE expresses CD146, CXCR4, CD309 / KDR, CD90, and CD271. [Invention 1025] The method of the present invention 1023, wherein HE expresses CD146. [Invention 1026] The method of invention 1023, wherein HE expresses CD144 (VECAD). [Invention 1027] The method of the present invention 1026, wherein HE expresses at least one cell marker selected from the group consisting of CD31, CD309 / KDR(FLK-1), PLVAP, GJA4, ESAM, EGFL7, KDR / VEGFR2, and ESAM. [Invention 1028] The method of the present invention 1026 or 1027, wherein HE further expresses at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA. [Invention 1029] Any method of the present invention 1026 to 1028, wherein HE further expresses at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. [Invention 1030] The method of the present invention 1023, wherein HE expresses CD31 / PECAM1, CD309 / KDR, CD144, CD34, and CD105. [Invention 1031] Any method of the present invention 1001 to 1029, wherein HE shows limited detection or no detection of at least one cell surface marker selected from the group consisting of CD34, CXCR7, CD43, and CD45. [Invention 1032] A method according to any of items 1001 to 1031 of the present invention, wherein HE shows limited detection or no detection of CXCR7, CD43, and CD45. [Invention 1033] Any method of the present invention 1001 to 1031, wherein HE shows limited detection of CD43 and CD45, or does not show detection. [Invention 1034] A method according to any of the invention 1001 to 1033, wherein HE is CD43(-), CD45(-), and CD146(+). [Invention 1035] A method according to any of the present invention 1001 to 1034, wherein the pluripotent stem cells are embryonic stem cells. [Invention 1036] A method according to any of the present invention 1001 to 1034, wherein the pluripotent stem cells are induced pluripotent stem cells. [Invention 1037] The HE is obtained by culturing pluripotent stem cells under adhesion conditions, in a differentiation medium, and in the absence of methylcellulose, according to any method of the present invention 1001 to 1036. [Invention 1038] A method according to any of the present invention 1001 to 1037, wherein HE is obtained by in vitro differentiation of pluripotent stem cells without embryoid body formation. [Invention 1039] A method according to any of the present invention 1001 to 1038, wherein the subject is a human. [Invention 1040] A method according to any of the present invention 1001 to 1039, wherein the pluripotent stem cells are human pluripotent stem cells. [Invention 1041] Any method of the present invention 1001 to 1040, wherein HE is human HE. [Invention 1042] A composition comprising HE obtained by in vitro differentiation of pluripotent stem cells, wherein the HE is CD43(-), CD45(-), and CD146(+). [Invention 1043] A composition comprising HE obtained by in vitro differentiation of pluripotent stem cells, wherein the HE is positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. [Invention 1044] A composition of the present invention 1043, wherein HE is positive for (i) miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [Invention 1045] A composition of the present invention 1043, wherein HE is positive for (i) miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [Invention 1046] A composition of the present invention 1043, wherein HE is positive for miRNA-214. [Invention 1047] A composition according to any one of the invention 1042 to 1046, wherein HE is negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. [Invention 1048] The composition of Invention 1047, wherein HE is negative for miRNA-223 and miRNA-142-3p. [Invention 1049] The composition of Invention 1047, wherein HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. [Invention 1050] A composition according to any one of the inventions 1043 to 1049, wherein HE is CD43(-), CD45(-), and CD146(+). [Invention 1051] A composition comprising HE obtained by in vitro differentiation of pluripotent stem cells, wherein the HE expresses CD144(VECAD). [Invention 1052] The composition of Invention 1051, wherein HE further expresses at least one cell marker selected from the group consisting of CD31, CD309 / KDR(FLK-1), PLVAP, GJA4, ESAM, EGFL7, KDR / VEGFR2, and ESAM. [Invention 1053] The composition of the present invention 1051 or 1052, wherein HE further expresses at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA. [Invention 1054] A composition according to any one of the present invention 1051 to 1053, wherein HE further expresses at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. [Invention 1055] A composition of any of the present invention 1051 to 1054 that substantially lacks CD144 (VECAD) negative HE cells. [Invention 1056] A pharmaceutical composition comprising HE obtained by in vitro differentiation of pluripotent stem cells and a pharmaceutically acceptable carrier, wherein the HE is CD43(-), CD45(-), and CD146(+). [Invention 1057] A pharmaceutical composition comprising HE obtained by in vitro differentiation of pluripotent stem cells and a pharmaceutically acceptable carrier, wherein the HE is positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. [Invention 1058] A pharmaceutical composition of the present invention 1057, wherein HE is positive for (i) miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [Invention 1059] A pharmaceutical composition of Invention 1057, wherein HE is positive for (i) miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, and / or (ii) hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p. [Invention 1060] A pharmaceutical composition of the present invention 1057, wherein HE is positive for miRNA-214. [Invention 1061] A pharmaceutical composition according to any of the present invention 1057 to 1060, wherein HE is negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. [Invention 1062] A pharmaceutical composition of the present invention 1061, wherein HE is negative for miRNA-223 and miRNA-142-3p. [Invention 1063] A pharmaceutical composition of the present invention 1061, wherein the HE test is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. [Invention 1064] A pharmaceutical composition according to any of invention 1057 to 1063, wherein HE is CD43(-), CD45(-), and CD146(+). [Invention 1065] A pharmaceutical composition comprising HE obtained by in vitro differentiation of pluripotent stem cells and a pharmaceutically acceptable carrier, wherein the HE expresses CD144(VECAD), CD31, and CD309 / KDR(FLK-1). [Invention 1066] The pharmaceutical composition of Invention 1065, wherein HE further expresses at least one cell marker selected from the group consisting of CD31, CD309 / KDR(FLK-1), PLVAP, GJA4, ESAM, EGFL7, KDR / VEGFR2, and ESAM. [Invention 1067] A pharmaceutically acceptable composition according to Invention 1065 or 1066, wherein HE further expresses at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA. [Invention 1068] A pharmaceutically acceptable composition according to any one of the invention 1065 to 1067, wherein HE further expresses at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. [Invention 1069] A pharmaceutical composition according to any of items 1065 to 1068 of the present invention, substantially lacking CD144 (VECAD) negative HE cells. [Invention 1070] Any method according to items 1026 to 1041 of the present invention, wherein HE expresses (i) CD144 (VECAD) and (ii) CD31 and / or CD309 / KDR (FLK-1). [Invention 1071] A method according to any one of Invention 1026-1041 or Invention 1070, wherein HE expresses at least one gene listed in Tables 22 and 23. [Invention 1072] A composition according to any one of the present inventions 1051 to 1054, wherein HE expresses (i) CD144 (VECAD) and (ii) CD31 and / or CD309 / KDR (FLK-1). [Invention 1073] A composition according to any one of Invention 1051-1054 or Invention 1072, wherein HE expresses at least one gene listed in Tables 22 and 23. [Invention 1074] A pharmaceutically acceptable composition according to any of the inventions 1065 to 1069, wherein HE expresses (i) CD144 (VECAD) and (ii) CD31 and / or CD309 / KDR (FLK-1). [Invention 1075] A pharmaceutical composition according to any of Invention 1065-1069 or Invention 1074, wherein HE expresses at least one gene listed in Tables 22 and 23. [Brief explanation of the drawing] 【0024】 [Figure 1] This is an overview of an exemplary method for producing HE. [Figure 2] This is an overview of an exemplary method for producing hemangioblasts (HB). [Figure 3] This bar graph shows the intracellular expression of PDGFRa, HAND1, FOXF1, APLNR, and PECAM / CD31 during the differentiation process from ES cells (day -1). The time points for the study were day -1 (ES cells), day 2 (D2), day 4 (D4), and day 6 (D6). [Figure 4A]This graph shows the expression levels of CD31, CD43, CD34, KDR, CXCR4, CD144, CD146, and CD105 in J1-HE cells (red bars on the left) and GMP1-HE cells (blue bars on the right) obtained on day 6 of the differentiation process. [Figure 4B-1] The graphs show the levels of CD31, VECAD, CD34, FLK1 (KDR), CD105, CD146, CD43, CXCR4, CD140b (PDGFRb), and NG2 in J1-HE and GMP1-HE cells obtained on day 6 of the differentiation process. Red indicates stained cells, and gray indicates unstained control cells. [Figure 4B-2] This is a continuation of Figure 4B-1. [Figure 5-1] The graphs show the J1-HE and GMP1-HE populations gated to CD31-positive (red) and CD31-negative (blue) cells, as well as their respective FLK1 / CD309, CD144 / VECAD, CD34, CD105, and CD43 expression levels. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 6] The bottom panel shows representative images of GMP-1-derived HE cells stained with CD31, NG2, or CNN1 antibodies. The top panel uses HUVEC cells for comparison. [Figure 7] This is a TSNE plot of miRNAs from HUVEC cells, J1 hESCs, J1-HE, or J1-HB. [Figure 8] This shows the effects of HB(VPC1) and HE(VPC2) on the survival rate of sugen-hypoxia-induced PAH rats. [Figure 9A] This shows nine clusters obtained by unsupervised clustering of HUVEC, iPSC(GMP1), and GMP1-HE. [Figure 9B] This represents the percentage of HUVEC, iPSC(GMP1), and GMP1-HE ("VPC-feeder active") in each of the nine clusters mentioned above. [Figure 9C]This shows clear clustering of HUVEC, iPSC(GMP1), and GMP1-HE ("VPC feeder activity"). [Figure 10] This represents three clusters identified by VECAD / CDH5 expression. [Figure 11] Figure 11A shows the right ventricular systolic pressure (RVSP) in MCT rats treated with vehicle (control medium), sildenafil (positive control), J1-HE (2.5 × 10⁶ cells), and GMP1-HE (2.5 × 10⁶ cells), as well as in the non-MCT treated control (Cont(Nx)). Figure 11B shows the Fulton index (RV / LV+S) in MCT rats treated with vehicle (control medium), sildenafil (positive control), J1-HE (2.5 × 10⁶ cells), and GMP1-HE (2.5 × 10⁶ cells), as well as in the non-MCT treated control (Cont(Nx)). Figure 11C shows the pulmonary vascular resistance index (PVR index) in MCT rats treated with vehicle (control medium), sildenafil (positive control), J1-HE (2.5 × 10⁶ cells), and GMP1-HE (2.5 × 10⁶ cells), as well as in the non-MCT treated control (Cont(Nx)). Figure 11D shows the number of thickened small vessels in MCT rats treated with vehicle (control medium), sildenafil (positive control), J1-HE (2.5 × 10⁶ cells), and GMP1-HE (2.5 × 10⁶ cells), as well as in the non-MCT treated control (Cont(Nx)). [Figure 12]Figure 12A shows the mean pulmonary artery pressure (mPAP) in Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). Figure 12B shows the right ventricular systolic pressure (RVSP) in Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). Figure 12C shows the Fulton index (RV / LV+S) in Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). Figure 12D shows cardiac output in Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). [Figure 13]Figure 13A shows the mean pulmonary artery pressure (mPAP) in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), GMP1-HE (5 million cells), and sildenafil (positive control), as well as in the non-Sugen-treated control (Nx). Figure 13B shows the right ventricular systolic pressure (RVSP) in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), GMP1-HE (5 million cells), and sildenafil (positive control), as well as in the non-Sugen-treated control (Nx). Figure 13C shows the Fulton index (RV / LV+S) in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), GMP1-HE (5 million cells), and sildenafil (positive control), as well as in the non-Sugen-treated control (Nx). Figure 13D shows the cardiac output in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), GMP1-HE (5 million cells), and sildenafil (positive control), as well as in the non-Sugen-treated control (Nx). [Figure 14]Figure 14A shows the histological features of lung tissue in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), and GMP1-HE (5 million cells), as well as in a non-Sugen-treated control (Nx). Figure 14B shows the pulmonary vascular wall thickness in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), GMP1-HE (5 million cells), and sildenafil (positive control), as well as in a non-Sugen-treated control (Nx). Figure 14C shows the percentages of muscular, semi-muscular, and non-muscular pulmonary vessels in Sugen-treated rats treated with vehicle (negative control), GMP1-HE (1 million cells), GMP1-HE (2.5 million cells), GMP1-HE (5 million cells), and sildenafil (positive control), as well as in non-Sugen-treated controls (Nx). [Figure 15] Figure 15A shows the histological features of Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). Figure 15B shows the pulmonary vessel wall thickness of Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). Figure 15C shows the percentages of muscular, semi-muscular, and non-muscular pulmonary vessels of Sugen-treated rats treated with vehicle (negative control), J1-HE (2.5 million cells), and GMP1-HE (2.5 million cells), as well as in the non-Sugen-treated control (Nx). [Figure 16]Figure 16A shows a micro-CT scan of a normal lung in a non-Sugen-treated rat (Nx control). Figure 16B shows a micro-CT scan of the lung in a vehicle-treated (negative control) SuHx rat. Figure 16C shows a micro-CT scan of the lung in a SuHx rat treated with 1 million GMP-1 HE cells. Figure 16D shows a micro-CT scan of the lung in a SuHx rat treated with 5 million GMP-1 HE cells. Figure 16E shows a micro-CT scan of the lung in a sildenafil-treated SuHx rat. [Figure 17] This shows the expression of CD31 and VECAD in unsorted HE cells ("unsorted") and in VECAD-negative cells (-fraction) and VECAD-positive cells (+fraction) after sorting for VECAD expression. [Figure 18-1] Figure 18A shows mean pulmonary artery pressure (mPAP) in vehicle (negative control), unselected GMP1-HE and selected VECAD+GMP1-HE treated Sugen rats, and in non-Sugen treated control (Nx). Figure 18B shows right ventricular systolic pressure (RVSP) in vehicle (negative control), unselected GMP1-HE and selected VECAD+GMP1-HE treated Sugen rats, and in non-Sugen treated control (Nx). Figure 18C shows Fulton index (RV / LV+S) in vehicle (negative control), unselected GMP1-HE and selected VECAD+GMP1-HE treated Sugen rats, and in non-Sugen treated control (Nx). Figure 18D shows cardiac output in vehicle (negative control), unselected GMP1-HE and selected VECAD+GMP1-HE treated Sugen-treated rats, and in non-Sugen-treated control (Nx). [Figure 18-2]Figure 18E shows the histological features of lung tissue in vehicle (negative control), Sugen-treated rats treated with unselected GMP1-HE and selected VECAD+GMP1-HE, and in non-Sugen-treated controls (Nx). Figure 18F shows the pulmonary vessel wall thickness in vehicle (negative control), Sugen-treated rats treated with unselected GMP1-HE and selected VECAD+GMP1-HE, and in non-Sugen-treated controls (Nx). Figure 18G shows the percentages of muscular, semi-muscular, and non-muscular pulmonary vessels in vehicle (negative control), Sugen-treated rats treated with unselected GMP1-HE and selected VECAD+GMP1-HE, and in non-Sugen-treated controls (Nx). [Figure 19] This represents FLK1 / KDR expression in J1-HE, GMP1-HE, and CD31+ / VECAD+ populations in HUVEC cells. [Modes for carrying out the invention] 【0025】 Detailed description of the invention 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. 【0026】 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. 【0027】 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. 【0028】 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. 【0029】 As used herein, “pluripotent stem cells” broadly refers to cells that, under appropriate conditions, remain undifferentiated, exhibit a normal karyotype (e.g., chromosomes), and possess 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. Pluripotent stem cells are functionally defined as stem cells that are typically (a) capable of inducing teratomas when transplanted into immunodeficient (SCID) mice, (b) capable of differentiating into all three germ layer cell types (e.g., ectoderm, mesoderm, and endoderm), and (c) expressing one or more embryonic stem cell markers (e.g., Oct4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.). In certain embodiments, pluripotent stem cells express one or more markers selected from the group consisting of OCT-4, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated, for example, using methods known in the art. 【0030】 Pluripotent stem cells include, but are not limited to, embryonic stem cells, induced pluripotent stem (iPS) cells, embryo-derived cells (EDCs), adult stem cells, hematopoietic cells, fetal stem cells, mesenchymal stem cells, postpartum stem cells, or embryonic germ cells. In one embodiment, pluripotent stem cells are mammalian pluripotent stem cells. In another embodiment, pluripotent stem cells are human pluripotent stem cells, such as, but not limited to, human embryonic stem (hES) cells, human induced pluripotent stem (iPS) cells, human adult stem cells, human hematopoietic stem cells, human fetal stem cells, human postpartum stem cells, human pluripotent stem cells, or human embryonic germ cells. In one embodiment, pluripotent stem cells are human embryonic stem cells. In another embodiment, pluripotent stem cells are human induced pluripotent stem cells. In another embodiment, pluripotent stem cells may be pluripotent stem cells listed in the Human Pluripotent Stem Cell Registry (hPSCreg). Pluripotent stem cells can be genetically modified or otherwise altered to increase their lifespan, potency, and homing, prevent or reduce allogeneic immune responses, or deliver desired factors to cells differentiating from such pluripotent cells. 【0031】 Pluripotent stem cells can originate from any species. For example, embryonic stem cells have been successfully obtained from mice, several species of non-human primates, and humans, and embryonic stem-like cells have also been generated from numerous other species. Therefore, those skilled in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild felines, e.g., lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (dolphins, whales, etc.). 【0032】 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 or contained within an embryo. This includes blastomeres and aggregated blastomeres obtained as early as the two-cell stage. 【0033】 As used herein, “embryonic stem cells” (ES cells) broadly refer to cells derived from the inner cell mass of a blastocyst or morula and continuously passaged as a cell line. ES cells may be induced by sperm or DNA fertilization of an egg cell, nuclear transfer, parthenogenesis, or by means of generating ES cells homozygous for an HLA region. ES cells may also refer to cells derived from a zygote, blastomeres, or blastocyst-stage mammalian embryo, optionally without destroying the rest of the embryo, produced by sperm-egg fusion, nuclear transfer, parthenogenesis, or chromatin reprogramming and subsequent incorporation of the reprogrammed chromatin into the plasma membrane. Embryonic stem cells can be identified based on one or more of the following characteristics, regardless of their source or the specific method used to produce them: (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. 【0034】 As used herein, “embryonic cells” (EDCs) broadly refer to blastocyst-derived cells, including morula-derived cells, inner cell masses, hyposhields, or epiblastoblast cells, or other pluripotent stem cells of early embryos, including primitive endoderm, ectoderm, and mesoderm, as well as their derivatives. “EDCs” also include blastomeres, aggregated single blastomeres, or cell masses from embryos at various developmental stages, but do not include human embryonic stem cells that have been passaged as cell lines. 【0035】 As used herein, “induced pluripotent stem cells” or “iPS cells” broadly refers to pluripotent stem cells obtained by reprogramming somatic cells. iPS cells can be generated by expressing or inducing the expression of a combination of factors ("reprogramming factors") in somatic cells, such as Oct4 (sometimes called Oct3 / 4), Sox2, Myc (e.g., c-Myc or any Myc variant), Nanog, Lin28, and Klf4. In one embodiment, the reprogramming factors include Oct4, Sox2, c-Myc, and Klf4. In another embodiment, the reprogramming factors include Oct4, Sox2, Nanog, and Lin28. In certain embodiments, at least two reprogramming factors are expressed in somatic cells so that the somatic cells are successfully reprogrammed. In another embodiment, at least three reprogramming factors are expressed in somatic cells so that the somatic cells are successfully reprogrammed. In yet another embodiment, at least four reprogramming factors are expressed in somatic cells so that the somatic cells are successfully reprogrammed. In another embodiment, at least five reprogramming factors are expressed in somatic cells to ensure they are properly reprogrammed. In yet another embodiment, at least six reprogramming factors are expressed in somatic cells, such as Oct4, Sox2, c-Myc, Nanog, Lin28, and Klf4. 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. 【0036】 iPS cells can be generated using somatic cells from a fetus, postnatal, neonatal, young adult, or adult. Somatic cells may include, but are not limited to, fibroblasts, keratinocytes, adipocytes, muscle cells, organ and tissue cells, and various blood cells, such as hematopoietic cells (e.g., hematopoietic stem cells), but are not limited to these. In some embodiments, the somatic cells are fibroblasts, such as dermal fibroblasts, synovial fibroblasts, or lung fibroblasts, or non-fibroblastic somatic cells. 【0037】 iPS cells can be obtained from cell banks. Alternatively, iPS cells can be newly generated by methods known in the art. iPS cells can be specially generated using material from a specific patient or compatible donor for the purpose of generating histocompatible cells. In some embodiments, iPS cells may be generic donor cells that are substantially non-immunogenic. 【0038】 Induced pluripotent stem cells can be produced by expressing or inducing the expression of one or more reprogramming factors in somatic cells. Reprogramming factors can be expressed in somatic cells by infection with viral vectors such as retroviral or lentiviral vectors. CRISPR / Talen / zinc finger nucleases (XFNs) can also be used. Alternatively, reprogramming factors can be expressed in somatic cells using non-integrated vectors such as episomal plasmids or RNA. When expressing reprogramming factors using non-integrated vectors, the factors can be expressed in cells by electroporation, transfection, or transformation of somatic cells using that vector. For example, in mouse cells, reprogramming somatic cells requires expressing four factors (Oct3 / 4, Sox2, c-myc, and Klf4) using integrated viral vectors. In the case of human cells, reprogramming somatic cells only requires expressing four factors (Oct3 / 4, Sox2, NANOG, and Lin28) using an embedded viral vector. 【0039】 The expression of reprogramming factors can be induced by contacting somatic cells with at least one active substance that induces the expression of reprogramming factors, such as an organic small molecule active substance. 【0040】 Furthermore, somatic cells can 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). 【0041】 Once reprogramming factors are expressed or induced in cells, those cells can be cultured. Over time, cells with ES characteristics will appear in the culture dish. Cells can be selected and subcultured, for example, based on the morphology of ES cells or based on the expression of selectable or detectable markers. Cells can be cultured to produce a culture of cells similar to ES cells. 【0042】 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. 【0043】 iPS cells can originate from any species. It has been successful to generate these iPS cells using mouse and human cells. Furthermore, it has also been successful to generate iPS cells using embryonic tissue, fetal tissue, neonatal tissue, and adult tissue. Therefore, it is possible to easily generate iPS cells using donor cells from any species. Thus, iPS cells can be generated from any species, including, but are not limited to, humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild felines, e.g., lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (dolphins, whales, etc.). 【0044】 When a cell is characterized as “positive” or “+” for a given marker, it may be a low (lo), medium (int), and / or high (hi) expressing cell of that marker, depending on the extent to which the marker is present on the cell surface or within a population of cells, in which case these terms relate to the intensity of fluorescence or other color used in the color sorting process of the cell. The differences between lo, int, and hi will be understood in relation to the marker used on the specific cell population being sorted. When a cell is characterized as “negative” or “-” for a given marker, it means that a cell or population of cells may not express the marker, or that the marker may be expressed at a relatively very low level by that cell or population of cells, and that it will produce a very low signal if it is detected and labeled. 【0045】 In one embodiment of the present invention, cells or a population of cells are characterized as expressing a high (hi) level of a marker if the expression level of a certain marker is greater than 60%, 70%, 80%, or 90% compared to a control. In another embodiment, cells or a population of cells are characterized as expressing an intermediate (int) level of a marker if the expression level of a certain marker is about 20%, 30%, 40%, 50% to about 60% compared to a control. In yet another embodiment, cells or a population of cells are characterized as expressing a low (lo) level of a marker if the expression level of a certain marker is about 2%, 5%, 10%, or 15% to about 20% compared to a control. In yet another embodiment, cells or a population of cells are characterized as negative for a certain marker if the expression level of a certain marker is less than about 2%, 1.5%, 1%, or 0.5% compared to a control. In another embodiment, a cell or population of cells is characterized as negative for a certain marker if its expression level is lo, or less than approximately 2%, 1.5%, 1%, or 0.5% compared to a control. The “control” may be any control or standard well known to those skilled in the art and useful for comparative purposes, and may include negative or positive controls. 【0046】 As used herein, “treatment” or “to treat” means treating, curing, reducing, alleviating, altering, correcting, relieving, improving, influencing, preventing, or delaying the onset of a disease or disorder or the symptoms of a disease or disorder. In relation to vascular repair, the term “treatment” or “to treat” includes repairing, replacing, strengthening, improving, rescuing, rearranging, or regenerating defective tissue. 【0047】 As used herein, “angioblasts” or “HB” refers to cells obtained by in vitro differentiation of pluripotent stem cells that have the ability to differentiate into at least hematopoietic cells and endothelial cells. In one embodiment, angioblasts can be generated in vitro from pluripotent stem cells according to the methods described in, for example, U.S. Patent No. 9,938,500, U.S. Patent No. 9,410,123 and WO 2013 / 082543 (all of these documents are incorporated herein by reference in their entirety). Furthermore, angioblasts can be generated in vitro from pluripotent stem cells according to the method described in Example 2 below. In one specific embodiment, embryoid bodies are first obtained from pluripotent stem cells under low-adhesion or non-adhesion conditions, and then angioblasts are generated in vitro from pluripotent stem cells by culturing these embryoid bodies in a culture system containing methylcellulose to create a three-dimensional environment for blast formation. In some embodiments, angioblasts can be generated from pluripotent stem cells under normal oxygen concentration conditions (e.g., 5% CO2 and 20% O2). Angioblasts can also be characterized based on other structural and functional properties, such as, but not limited to, the expression or absence of certain DNA, RNA, microRNA, or protein. 【0048】 In one embodiment, angioblasts are positive for at least one, at least two, at least three, at least four, or at least five cell surface markers selected from the group consisting of CD31 / PECAM1, CD144 / VE-cadh, CD34, CD43, and CD45. In one embodiment, HB is positive for CD31, CD43, and CD45. In another embodiment, HB is positive for CD43 and CD45. In yet another embodiment, HB expresses at low levels or is negative for 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 cell surface markers selected from the group consisting of CD309 / KDR, CXCR4, CXCR7, CD146, Tie2, CD140b, CD90, and CD271. In yet another embodiment, HB expresses at low levels or is negative for CD146. In another embodiment, HB expresses or is negative for Tie2, CD140b, CD90, and CD271 at low levels. In yet another embodiment, HB expresses or is negative for CD146, Tie2, CD140b, CD90, and CD271 at low levels. In one embodiment, HB is positive for CD43 and CD45 and expresses or is negative for CD146, Tie2, CD140b, CD90, and CD271 at low levels. 【0049】 In another embodiment, angioblasts are positive for at least one, at least two, at least three, or at least four miRNAs selected from the group consisting of miRNA-126, miRNA-24, miRNA-223, and miRNA-142-3p. In one embodiment, angioblasts are positive for miRNA-126, miRNA-24, miRNA-223, and miRNA-142-3p. In yet another embodiment, angioblasts are negative for 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 miRNAs selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-196-b, miRNA-214, miRNA-199a-3p, and miRNA-335. In one embodiment, angioblasts are negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-196-b, miRNA-214, miRNA-199a-3p, and miRNA-335. In a further embodiment, angioblasts are positive for miRNA-126, miRNA-24, miRNA-223, and miRNA-142-3p, and negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-196-b, miRNA-214, miRNA-199a-3p, and miRNA-335. 【0050】 As used herein, “hematopoietic endothelial cells” or “HE” refers to cells obtained by in vitro differentiation of pluripotent stem cells that have the ability to differentiate into endothelial cells, smooth muscle cells, pericytes, hematopoietic cells, and mesenchymal cell lineages. HE may be useful in the treatment of vascular diseases as defined herein. In one embodiment, HE may be generated in vitro from pluripotent stem cells according to the methods described in WO 2014 / 100779 and U.S. Patent No. 9,993,503 (both of which are incorporated herein by reference in their entirety). In another embodiment, HE may be generated in vitro from pluripotent stem cells according to the method described in Example 1 below and shown in Figure 1. 【0051】 In one specific embodiment, HE can be generated in vitro from pluripotent stem cells without embryoid body formation or the use of a culture system containing methylcellulose. In one embodiment, the pluripotent stem cells are iPS cells or ES cells. The pluripotent stem cells can be cultured on a feeder cell layer, preferably on a human feeder cell layer, or feeder-free on an extracellular matrix such as Matrigel®. The pluripotent stem cells can be cultured under normal oxygen concentration conditions (e.g., 5% CO2 and 20% O2). For differentiation into HE, the pluripotent stem cells can be cultured in differentiation medium, under hypoxic concentration conditions (e.g., 5% CO2 and 5% O2), and under adhesion conditions. Adhesion conditions may include culturing the cells on an extracellular matrix such as Matrigel®, fibronectin, gelatin, and collagen IV. The differentiation medium may include a basic medium, such as Stemline® II Hematopoietic Stem Cell Expansion Medium (Sigma), Iskov Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium (DMEM), or any other known basic medium. The differentiation medium may further include factors for inducing the differentiation of pluripotent stem cells into HE, such as bone morphogenesis protein 4 (BMP4), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF). Pluripotent stem cells may be cultured in the differentiation medium for about 1 to 12 days, or about 2 to 10 days, or about 3 to 8 days, or about 4, 5, 6, 7, or 8 days, or until the pluripotent stem cells differentiate into HE. In one specific embodiment, pluripotent stem cells are cultured in the differentiation medium for about 6 days or more. 【0052】 In one embodiment, HEs can also be characterized based on certain structural and functional properties, such as, but not limited to, the expression or absence of certain DNA, RNA, microRNA, or protein. In one embodiment, any HE disclosed herein 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, or at least eleven cell surface markers selected from the group consisting of CD31 / PECAM1, CD309 / KDR, CD144, CD34, CXCR4, CD146, Tie2, CD140b, CD90, CD271, and CD105. In one embodiment, the HE of the present invention expresses CD146, CXCR4, CD309 / KDR, CD90, and CD271. In another embodiment, the HE of the present invention expresses CD146. In another embodiment, HE expresses CD31 / PECAM1, CD309 / KDR, CD144, CD34, and CD105. 【0053】 In one embodiment, HE shows limited detection or no detection of at least one, at least two, at least three, or at least four cell surface markers selected from the group consisting of CD34, CXCR7, CD43, and CD45. In another embodiment, HE shows limited detection or no detection of CXCR7, CD43, and CD45. In yet another embodiment, HE shows limited detection or no detection of CD43 and CD45. 【0054】 In one embodiment, the HE of the present invention is CD43(-), CD45(-), and / or CD146(+). In another embodiment, the HE expresses CD31, calponin (CNN1), and NG2, and therefore has the potential to further differentiate into endothelial cells (CD31+), smooth muscle (calponin+), and / or pericytes (NG2+). 【0055】 In one embodiment, CD144(VECAD)-expressing HE cells are isolated from HE cells of the present invention. In one embodiment, the isolated CD144(VECAD)-expressing HE cells further express CD31 and / or CD309 / KDR(FLK-1). In another embodiment, the isolated CD144(VECAD)-expressing HE cells further express at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve cell markers selected from the cell markers listed in Table 22 or Table 23. In one embodiment of the present invention, the isolated CD144(VECAD)-expressing HE cells express at least one, at least two, at least three, at least four, or at least five cell markers selected from the group consisting of PLVAP, GJA4, ESAM, EGFL7, KDR / VEGFR2, and ESAM. In one embodiment, isolated CD144(VECAD)-expressing HE cells further express at least one, at least two, or at least three cellular markers selected from the group consisting of SOX9, PDGFRA, and EGFRA. In another embodiment, isolated CD144(VECAD)-expressing HE cells further express at least one, at least two, at least three, or at least four cellular markers selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. In one embodiment, a composition comprising CD144(VECAD)-expressing HE isolated from the HE of the present invention substantially lacks CD144(VECAD)-negative HE. In one embodiment, a composition containing CD144(VECAD)-expressing HE comprises at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% CD144(VECAD)-expressing HE.In one embodiment, a composition containing CD144(VECAD)-expressing HE contains 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or less than 80% CD144(VECAD)-negative HE. 【0056】 In another embodiment, the HE of the present invention is positive 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, at least nine, or at least ten microRNAs (miRNAs) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p, miRNA-335 (miRNA-335-5p and / or miRNA-335-3p), hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. In one embodiment, the HE is positive for miRNA-214, miRNA-199a-3p, and miRNA-335 (miRNA-335-5p and / or miRNA-335-3p). In another embodiment, HE is positive for miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p, and miRNA-335 (miRNA-335-5p and / or miRNA-335-3p). In one embodiment, HE is positive for miRNA-214. In another embodiment, HE is positive for hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p were identified as uniquely expressed in the HE population compared to meso 3D VPC2 cells described in J1 and U.S. Provisional Application No. 62 / 892,724 and its PCT application (both of which are incorporated herein by reference). 【0057】 In any of these embodiments, the HE disclosed herein may be negative for at least one, at least two, at least three, at least four, at least five, or at least six miRNAs selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. In one embodiment, the HE is negative for miRNA-223 and miRNA-142-3p. In another embodiment, the HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p. 【0058】 In one embodiment, HE is positive for miRNA-214, miRNA-199a-3p, and miRNA-335 (miRNA-335-5p and / or miRNA-335-3p), and negative for miRNA-223 and miRNA-142-3p. 【0059】 In one embodiment, HE cells are genetically modified. HE cells may be genetically modified to express gene products that are thought to promote or intended to promote the therapeutic response that these cells produce. For example, HE cells may be genetically modified to express heterologous proteins, such as vascular endothelial growth factor (VEGF) and its isoforms, fibroblast growth factor (FGF, acidic and basic), angiopoietin-1 and other angiopoietins, erythropoietin, heme oxygenase, transforming growth factor α (TGF-α), transforming growth factor β (TGF-β) or other members of the TGF-β superfamily, such as BMP1, 2, 4, 7 and their receptors MBPR2 or MBPR1, hepatic growth factor (cell dispersal factor), hypoxia-inducible factor (HIF), endothelial nitric oxide synthase, prostaglandin I synthase, Krupple-like factors (KLF-2, 4, etc.), and any other heterologous proteins useful in promoting the therapeutic response to vascular disease. 【0060】 As used herein, “vascular disease” means any abnormal condition or injury of the heart, lungs and / or blood vessels (arteries, veins and capillaries). Vascular disease includes, but is not limited to, diseases, disorders and / or injuries of the pericardium (i.e., the pericardium), heart valves (e.g., insufficient valves, stenotic valves, rheumatic heart disease, mitral valve prolapse, aortic valve regurgitation), myocardium (coronary artery disease, myocardial infarction, heart failure, ischemic heart disease, angina), blood vessels (e.g., arteriosclerosis, aneurysms), or veins (e.g., varicose veins, hemorrhoids). Vascular diseases as used herein include, but are not limited to, coronary artery disease (e.g., arteriosclerosis, atherosclerosis, and other diseases or injuries of arteries, arterioles and capillaries, or related conditions), myocardial infarction (e.g., acute myocardial infarction), organized myocardial infarction, ischemic heart disease, arrhythmias, left ventricular dilation, embolism, heart failure, congestive heart failure, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditis, chronic coronary ischemia, dilated cardiomyopathy, restenosis, arrhythmias, angina, hypertension (e.g., pulmonary hypertension, glomerular hypertension, portal hypertension), myocardial hypertrophy, peripheral artery disease including severe limb ischemia, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cor pulmonale, dyscardial infarction, inflammatory heart disease, congenital heart disease, rheumatic heart disease, diabetic vascular disease, and pulmonary endothelial injury (e.g., acute lung injury (ALI), acute respiratory distress syndrome (ARDS)). Vascular diseases can result from congenital defects, genetic defects, environmental influences (e.g., dietary influences, lifestyle, stress, etc.), and other defects or influences. 【0061】 In one embodiment, vascular disease is pulmonary hypertension (PH). Pulmonary hypertension includes pulmonary artery hypertension (PAH), pulmonary hypertension with left heart disease, pulmonary hypertension with lung disease and / or chronic hypoxemia, chronic arterial occlusion, and pulmonary hypertension with unknown or multifactorial mechanisms, such as sarcoidosis, histiocytosis X, lymphangiomatosis, and compression of pulmonary vessels. Galie et al. 4 See 2016;37(1):67-119. In one specific example, vascular disease is PAH. 【0062】 Exemplary therapeutic uses The HE of the present invention is useful for the treatment of vascular diseases. Accordingly, the present invention provides a method for treating vascular diseases in a subject by administering a composition comprising the HE of the present invention to the subject. In one embodiment, vascular diseases include, but are not limited to, diseases, disorders, or injuries of the pericardium (i.e., pericardium), heart valves (i.e., insufficient valves, stenotic valves, rheumatic heart disease, mitral valve prolapse, aortic valve regurgitation), myocardium (coronary artery disease, myocardial infarction, heart failure, ischemic heart disease, angina), blood vessels (i.e., arteriosclerosis, aneurysms), or veins (i.e., varicose veins, hemorrhoids). In another embodiment, vascular diseases include, but are not limited to, coronary artery disease (i.e., arteriosclerosis, atherosclerosis, and other diseases or related conditions of arteries, arterioles and capillaries), myocardial infarction (e.g., acute myocardial infarction), organized myocardial infarction, ischemic heart disease, arrhythmias, left ventricular dilation, embolism, heart failure, congestive heart failure, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditis, chronic coronary ischemia, dilated cardiomyopathy, restenosis, arrhythmias, angina, hypertension, myocardial hypertrophy, peripheral artery disease including severe ischemic limb, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cor pulmonale, dyscardial infarction, inflammatory heart disease, congenital heart disease, rheumatic heart disease, diabetic vascular disease, and pulmonary endothelial injury diseases (e.g., acute lung injury (ALI), acute respiratory distress syndrome (ARDS)). 【0063】 In one embodiment, vascular disease is pulmonary hypertension (PH). In a specific embodiment, vascular disease is PAH. 【0064】 HE of the present invention may also be useful in treating symptoms of vascular disease. For example, HE may be used to treat symptoms of myocardial infarction, chronic coronary ischemia, arteriosclerosis, congestive heart failure, dilated cardiomyopathy, restenosis, coronary artery disease, heart failure, arrhythmia, angina, atherosclerosis, hypertension, severe limb ischemia, peripheral vascular disease, pulmonary hypertension, or myocardial hypertrophy. Treatment of one or more symptoms of vascular disease may provide clinical benefits such as the reduction of one or more of the following symptoms: shortness of breath, fluid retention, headache, vertigo, chest pain, left shoulder or arm pain, and ventricular dysfunction. 【0065】 The HE of the present invention may exhibit certain properties that contribute to reducing and / or minimizing damage and promoting the repair and regeneration of blood vessels after injury. These include, among other things, the ability to synthesize and secrete growth factors that stimulate the formation of new blood vessels, the ability to synthesize and secrete growth factors that stimulate cell survival and proliferation, the ability to proliferate and differentiate into cells that are directly involved in the formation of new blood vessels, the ability to engraft damaged myocardium and inhibit scar formation (collagen deposition and cross-linking), and the ability to proliferate and differentiate into cells of the vascular lineage. In one embodiment, the HE of the present invention has vascular repair ability. In one embodiment, the HE contributes to post-injury progenitor cell replenishment under normal conditions. In another embodiment, the HE of the present invention has the ability to hom to the site of vascular injury, facilitate reendothelialization, and prevent neointima formation. Therefore, the HE of the present invention can be used to treat vascular tissue damaged by injury, inflammation, or disease. 【0066】 The effects of HE treatment according to the present invention may be demonstrated by, but are not limited to, one of the following clinical measures: increased ejection fraction, decreased heart failure rate, decreased infarct size, decreased associated morbidity (pulmonary edema, renal failure, arrhythmia), improved exercise tolerance or other measures of quality of life, and decreased mortality. The effects of cell therapy may be evident over several days to several weeks after the procedure. However, beneficial effects may be observed within just a few hours after the procedure and may persist for several years. 【0067】 Subjects treated with the HE of the present invention according to the methods described herein would typically have, be suspected of having, or be diagnosed as being at risk of vascular disease. Vascular disease is typically diagnosed and / or monitored by a physician using a standard set of methods. The terms “subject” and “patient” are used interchangeably herein and refer to any vertebrate, including mammals, rodents and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc. In one specific embodiment, the subject is a primate. In another embodiment, the subject is a human. 【0068】In one embodiment, the methods of the present invention may be performed in conjunction with existing vascular therapies to effectively treat vascular diseases. The methods and compositions of the present invention include concurrent or sequential treatment with non-biological and / or biological agents.Non-biological and / or biological agents are not limited to analgesics, e.g., nonsteroidal anti-inflammatory drugs, opioid agonists and salicylates; anti-infective agents, e.g., antiparasitic agents, anti-anaerobic agents; antibiotics, aminoglycoside antibiotics, antifungal antibiotics, cephalosporin antibiotics, macrolide antibiotics, various β-lactam antibiotics, penicillin antibiotics, quinolone antibiotics, sulfonamide antibiotics, tetracycline antibiotics, and anti-myelin antibiotics. Cobacterial drugs, anti-tuberculous anti-mycobacterial drugs, antiparasitic drugs, antimalarial antiparasitic drugs, antiviral drugs, antiretroviral drugs, scabies insecticides, anti-inflammatory drugs, corticosteroid anti-inflammatory drugs, antipruritic drugs / local anesthetics, topical anti-infective drugs, antifungal topical anti-infective drugs, antiviral topical anti-infective drugs; electrolytic nephroactive substances, e.g., acidifying agents, alkalizing agents, diuretics, carbonic anhydrase inhibitory diuretics, loop diuretics, osmotic diuretics, potassium-sparing diuretics, thiazide diuretics, electrolyte replacement drugs (electrolyte (replacement) and uric acid excretion agents; enzymes, e.g., pancreatic enzymes and thrombolytic enzymes; gastrointestinal drugs, e.g., antidiarrheals, gastrointestinal anti-inflammatory agents, antacids and anti-ulcer agents, gastric acid pump inhibitor anti-ulcer agents, gastric mucosal anti-ulcer agents, H2 blocker anti-ulcer agents, gallstone dissolving agents, digestive drugs, emetics, laxatives and stool softeners, and intestinal motility promoters; general anesthetics, e.g., inhalation anesthetics, halogenated inhalation anesthetics, intravenous anesthetics, barbiturate intravenous anesthetics, benzodiazepine intravenous anesthetics and opioid agonist intravenous anesthetics; hormones and hormone regulators, e.g., abortifacients, adrenal glands Examples include active agents, corticosteroid adrenal agents, androgens, antiandrogens, immunobiological agents such as immunoglobulins, immunosuppressants, toxoids, and vaccines; local anesthetics such as amide and ester local anesthetics; musculoskeletal agents such as antigout anti-inflammatory agents, corticosteroid anti-inflammatory agents, gold compound anti-inflammatory agents, immunosuppressant anti-inflammatory agents, nonsteroidal anti-inflammatory drugs (NSAIDs), salicylate anti-inflammatory agents, minerals; and vitamins such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K. 【0069】 Administration As disclosed herein, HE of the present invention may be administered by a variety of routes, including systemic administration by intravenous or arterial infusion (including regurgitation), or by direct injection into cardiac or peripheral tissue. Systemic administration, particularly via peripheral venous access, has the advantage of being minimally invasive and utilizing the natural perfusion of the heart and the ability of vascular endothelial precursors to target the site of injury. Cells may be injected in a single bolus, by slow infusion, or in a series of applications spaced several hours apart, or the prepared cells may be appropriately stored for several days or weeks. Cells may also be applied using catheterization so as to enhance the initial pass of cells through the heart by managing myocardial blood flow with a balloon. As with peripheral venous access, cells may be injected in a single bolus through a catheter or in multiple small injections. Cells may also be applied directly to the myocardium by epicardial injection. This would be used under direct visualization in connection with open-heart surgery (e.g., coronary artery bypass grafting) or the placement of ventricular assist devices. A catheter fitted with a needle may be used to deliver cells directly to the myocardium endocardially, which would be a less invasive direct application method. 【0070】 In one embodiment, the delivery route includes intravenous delivery via a standard peripheral intravenous catheter, central venous catheter, or pulmonary artery catheter. In another embodiment, cells may be delivered by an intracoronary route accessed in a currently acceptable manner. Cell flow is controlled by the sequential inflation / deflation of distal and proximal balloons placed within the patient's vascular system, thereby creating a temporary no-flow zone that promotes cell engraftment or the action of cell therapy. In yet another embodiment, cells may also be delivered by an endocardial (inner surface of the cardiac chambers) approach, which may require the use of a suitable catheter and the ability to image or detect the intended target tissue. Alternatively, cells may be delivered by an epicardial (outer surface of the heart) approach. This delivery may be achieved under direct visualization during open-heart surgery or by a thoracoscopic approach requiring dedicated cell delivery equipment. Furthermore, cells can also be delivered using the following routes alone or in combination with one or more of the approaches described above: subcutaneous, intramuscular, intratracheal, sublingual, retrograde coronary perfusion, coronary bypass devices, extracorporeal membrane oxygenation (ECMO) devices, and pericardial fenestration. 【0071】 In one embodiment, the cells are administered to the patient as an intravascular bolus or timed infusion. 【0072】 composition The present invention provides a composition containing HE. In certain embodiments, the composition is at least 1 × 10 3 It contains 1 HE. In another embodiment, the composition contains at least 1 × 10 4 It contains 1 HE. In another embodiment, the composition contains at least 1 × 10 5 , at least 1 × 10 6 , at least 1 × 10 7 or at least 1 × 10 8 The composition contains HE. The composition may further include additives known in the art to enhance, control, or otherwise direct the intended therapeutic effect. 【0073】 In one embodiment, the composition of the present invention further comprises a biocompatible matrix such as a solid support matrix, a bioadhesive or biobandage, or a bioscaffold, or a bioink used in 3D bioprinting. The biocompatible matrix can facilitate in vivo operations by supporting and / or directing the fate of implanted cells. Non-limiting examples of biocompatible matrices include absorbable and / or non-absorbable solid matrices, e.g., the submucosa (SIS) of the small intestine, e.g., porcine (and other SIS sources); cross-linked or non-cross-linked arginates, hydrophilic colloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, polyglutinin (PGL) meshes, fleece, foam dressings, bioadhesives (e.g., fibrin glue and fibrin gel), dead de-epidermized skin equivalents, hydrogels, albumin, polysaccharides, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, polyacrylates, polymethacrylates, ethylene vinyl acetate, polyvinyl alcohol, and the like. 【0074】 The HE of the present invention can be formulated into a pharmaceutical composition comprising HE and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include saline solution, aqueous buffer solution, solvent, dispersion medium, or any combination thereof. Non-limiting examples of pharmaceutically acceptable carriers include sugars, e.g., lactose, glucose, and sucrose; starches, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients, e.g., cocoa butter and suppository waxes; oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, e.g., propylene glycol; polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; esters, e.g., ethyl oleate and ethyl laurate; agar; buffers, e.g., magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations. In some embodiments, pharmaceutically acceptable carriers are stable under manufacturing and storage conditions. In one embodiment, the HE of the present invention is formulated in GS2 medium as described in WO 2017 / 031312, which is incorporated herein by reference. 【0075】 The present invention further provides a cryopreserved composition containing HE. The cryopreserved composition may further comprise a cryopreservant. Cryopreservants are known in the art and include, but are not limited to, dimethyl sulfoxide (DMSO) and glycerol. The cryopreserved composition may also comprise an isotonic solution such as a cell culture medium. 【0076】 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] 【0077】 Example 1: Generation of hematopoietic endothelial cells (HE) Hematopoietic endothelial cells were generated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (iPSCs) as shown in Figure 1. hESCs (e.g., J1 hESCs) or iPSCs (e.g., GMP-1 iPSCs) were cultured in 6-well plates for 4 days on human dermal fibroblast feeder cells in mTeSR1 (Stemcell Technology) + 1% penicillin / streptomycin, with daily medium changes. To plate the hESCs or iPSCs for differentiation (day 1), the mTeSR1 medium was removed from each well of the 6-well plate. Each well was washed with 2 mL of DMEM / F12 (Gibco) or D-PBS, the DMEM / F12 or D-PBS was aspirated, and 1 mL of enzyme-free Gibco® Cell Dissociation Buffer (CDB) was added to each well. The plate was incubated in a normal oxygen concentration CO2 incubator (5% CO2 / 20% O2) for approximately 5–8 minutes until the cells showed a detached form. Next, the CDB was carefully removed by pipetting, taking care not to disturb the loosely attached cells. Cells were collected by adding 2 mL of mTeSR1 to each well and collected in a collection tube. The remaining cells in the wells were gently washed with another 2 mL of mTeSR1 and transferred to the collection tube. The tube was centrifuged at 120 × g for 3 minutes to remove the culture medium. The cells were resuspended in mTeSR1 medium containing Y-27632 (Stemgent) at a final concentration of 10 μM at a final density of 400,000 cells / 10 mL. 10 mL of this cell suspension was transferred to a collagen IV coated 10 cm plate. The plate was placed in a normal oxygen pressure incubator overnight. 【0078】 On the following day (day 0), the mTeSR1 / 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. 【0079】 On the second day, the culture medium was aspirated, and 10–12 mL of fresh BVF-M was added to each 10 cm plate. 【0080】 On the fourth day, the culture medium was aspirated again, and 10-15 mL of fresh BVF-M was added to each 10 cm plate. 【0081】 On day 6, 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 aspirating the D-PBS. 5 mL of StemPro Accutase (Gibco) was added to each 10 cm plate, and the plates were incubated in a normal oxygen-pressure CO2 incubator (5% CO2 / 20% O2) for 3–5 minutes. The 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 EGM2 medium (Lonza) or Stemline® II hematopoietic stem cell expansion medium (Sigma), and the cells were collected in collection tubes by passing them through a 30 μM cell strainer. The tubes were centrifuged at 120–250 g for 5 minutes. Next, the cells were resuspended in EGM2 medium or Stemline® II hematopoietic stem cell expansion medium (Sigma) and counted. After counting, the cells were centrifuged (250 × g, 5 minutes) and frozen in 3 × 10⁶ cells of frozen medium (10% DMSO + heat-inactivated FBS). 6The 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). 【0082】 Example 2: Generation of angioblasts (HB) Angioblasts were generated from human embryonic stem cells (e.g., J1 hESCs) or human induced pluripotent stem cells (e.g., GMP-1 iPSCs) as shown in Figure 2. In a 6-well plate, hESCs or iPSCs cultured on human dermal fibroblast feeder cells in mTeSR1 (Stemcell Technology) + 1% penicillin / streptomycin were detached from the wells by incubating each well in an incubator with DMEM / F12 (Gibco) containing 4 mg / mL collagenase IV (Gibco) at 37°C (5% CO2 / 20% O2) for approximately 10 minutes until the cells detached from the plate. The DMEM / F12 containing collagenase IV was removed from each well, washed with DMEM / F12, and 2 mL of mTeSR1 was added to each well. The cells were then detached using a cell scraper if necessary. The cell suspension was transferred to a conical tube, and each well was washed again with 2 mL of mTeSR1 and transferred back into the conical tube. The tubes were centrifuged at 300 × g for 2 minutes, and the supernatant was removed. The cell pellet was 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 an ultra-low adhesion surface 6-well plate (Corning) at a density of approximately 750,000 to 1,200,000 cells per well. Embryoid bodies were formed by incubating the plate 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 of 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 cells were placed in a CO2 incubator at normal oxygen pressure for a further 48 hours (days 2-4) to allow embryoid body formation to continue. 【0083】 On the 4th day, embryoid bodies were collected into 15 mL tubes, centrifuged at 120 - 300×g for 2 minutes, washed with D-PBS, and dissociated into a single cell suspension using StemPro Accutase (Gibco). Accutase was inactivated using FBS (Hyclone), and the single cells were passed through a cell strainer, centrifuged, and resuspended in Stemline II medium (Sigma) to a concentration of approximately 1×10 6 cells / mL. Approximately 3×10 6 cells were mixed in 30 mL of Methocult BGM medium [MethoCult™ SF H4536 (without EPO) (StemCell Technologies), penicillin / streptomycin (Gibco), ExCyte cell growth supplement (1:100) (Millipore), 50 ng / mL Flt3 ligand (PeproTech), 50 ng / mL VEGF (Humanzyme), 50 ng / mL TPO (PeproTech), 30 ng / mL bFGF (Humanzyme)], replated in a 10 cm dish with a low attachment surface (Corning), and incubated in a CO2 incubator under normal oxygen pressure for 7 days (days 4 - 11) to form angioblasts. 【0084】 On the 11th day, angioblasts were collected 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 Stemline II, and the cells were counted and frozen as described above. 【0085】 Example 3: Cell Marker Analysis Hemangioblasts (HE) collected on day 6 according to Example 1 and hemangioblasts (HB) collected on day 11 according to Example 2 were analyzed by FACS for endothelial cell markers, blood / hematopoietic markers, and pericyte markers. Briefly, the collected cells were resuspended in 50 μL of FACS buffer (2% FBS / PBS) at a density of 100 k / tube. Flow cytometry antibodies were added according to Table 1 and incubated at 4°C for 20 minutes. Next, 1 mL of FACS buffer was added to each tube and centrifuged at 250 × g for 5 minutes. The cells were resuspended in 200 μL of propidium iodide (PI)-free FACS buffer per tube. Samples were analyzed using MACS Quant Analyzer 10 (Miltenyi Biotec: 130-096-343). HUVEC was used as a positive control, and HDF or undifferentiated hESCs were used as negative controls. In addition, HUVEC was used as a single-stain (SS) control for compensation. 【0086】 (Table 1) Antibody staining table for MACS Quant Analyzer 10 TIFF0007876440000001.tif96156 【0087】 Alternatively, FACs analysis was performed using a Sony SA3800 spectral analyzer. Briefly, the harvested cells were resuspended in 100 μL of FACS buffer (2% FBS / PBS) at 100-200 k / tube. Flow cytometry antibodies were added according to Table 2, and incubated at 4°C for 20 minutes. Next, 1 mL of FACS buffer was added to each tube, and the tubes were centrifuged at 300 × g for 5 minutes. The cells were resuspended in 100 μL per tube of FACS buffer with or without PI (diluted 1:1000 in FACS buffer). The samples were analyzed using a Sony SA3800 spectral analyzer. HUVEC cells were used as a positive control, and undifferentiated hESCs were used as a negative control. 【0088】 (Table 2) Antibody staining table for Sony SA3800 spectral analyzer TIFF0007876440000002.tif48164 【0089】 result As shown in Tables 3-4, HB was positive for the blood markers CD43 and CD45, as well as the endothelial cell markers CD31, CD144, and CD34, but the expression levels of Tie2, CD140b, CD90, and CD271 were low or undetectable. 【0090】 In contrast, as shown in Tables 3-4, HE was positive for CD146, CXCR4, and Flk1 (CD309 / KDR), as well as pericyte / mesenchymal markers CD90 and CD271, but negative for blood / hematopoietic markers CD43 and CD45. 【0091】 (Table 3) Summary of cell surface markers on HB and HE from J1 and GMP1 strains, analyzed with MACS Quant Analyzer 10 and / or Sony SA3800 spectral analyzer. TIFF0007876440000003.tif104170 【0092】 (Table 4) Summary of surface markers on J1-derived HB and HE cells analyzed with MACS Quant Analyzer 10 TIFF0007876440000004.tif35156 【0093】 Time course of cell marker expression in various cells during the differentiation process showed that cells upregulate mesodermal lineage markers, with surface expression of PDGFRA and APLNR reaching its peak on day 2. Subsequently, the expression of these markers decreased, which correlated with an increase in the vascular cell marker CD31 on day 6 (Figure 3). Examination with a light microscope suggested that this differentiation method generates a mixture of cells, which then exhibit either endothelial or mesenchymal morphology. 【0094】 Further characterization of HE cells produced on day 6 revealed that the majority of cells were CD146+ and expressed VECAD+ (CD144+) or CD140B+ (PDGFRB+), but did not express the hematopoietic markers CD43 and CD45, demonstrating that this protocol produces distinct vascular and perivascular cells. Further characterization of HE cells produced on day 6 was performed for CD31, CD43, CD34, KDR (FLK1), CXCR4, CD144, CD146, CD105, CD140b (PDGFRb), and NG2. These are shown in Figures 4A and 4B. 【0095】 The putative vascular endothelial fractions identified by CD31 expression were positive for FLK1 / CD309 (also known as VEGFR2), VECAD, CD34, and CD105 (Figure 5). When HE cells on day 6 were transferred to a medium that supports vascular endothelial proliferation and kept under normal oxygen conditions for an additional 5–7 days, CD31, CD34, and FLK1 / CD309 (VEGFR2) expression were maintained or increased. 【0096】 Example 4: HE expresses endothelial, smooth muscle, and pericyte cell markers. Further analysis using immunocytochemistry (ICC) was performed as described below, with HUVEC used as a control. HE was plated for at least 24 hours, then washed twice with D-PBS (Gibco) containing Ca2+ and Mg2+. The cells were then fixed at room temperature with 4% PFA (Electron Microscopy Science) for 10 minutes. After fixation, the cells were washed three times for 5 minutes each with D-PBS containing Ca2+ and Mg2+. Next, the cells were treated for 1 hour with 1× Perm / Wash buffer (BD) containing 5% normal goat serum (Cell Signaling Technology). After aspirating Perm / Wash / Blocking buffer, the cells were treated overnight with primary antibody-containing Perm / Wash / Blocking buffer (human CD31, 1:50, Invitrogen; human NG2, 1:50, PD Pharmagen; human calponin, 1:100, Millipore). The following day, the cells were washed three times for 5 minutes each with Perm / Wash buffer. Next, the cells were treated at room temperature with secondary and DAPI-containing Perm / Wash / Blocking buffer (DAPI, 1:1000, Invitrogen; goat anti-Ms-Cy3, goat anti-Rb-Alexaflour488) for 1 hour. The cells were washed three times with Perm / Wash buffer for 5 minutes each and imaged with Keyence BZ-X710 (Keyence). As shown in Figure 6, HE cells expressed endothelial markers (CD31), smooth muscle markers (calponin), and pericyte markers (NG2), indicating their ability to differentiate into endothelial cells, smooth muscle cells, and pericytes. Furthermore, when HE cells on day 6 were transferred to a medium that supports pericyte proliferation, CD140B expression slightly decreased, while NG2, CD90, CD73, CD44, and CD274 expression were maintained or increased (data omitted). 【0097】 Example 5: Single-cell miRNA profile To evaluate the expression levels of 96 microRNAs associated with pluripotency or vascular cell identity, further analysis using single-cell qRT-PCR was performed on J1-derived HB and HE cells as described below. TaqMan gene expression assays (Applied Biosystems) were ordered for the 96 human miRNAs. For a final stock volume of 50 μL, a 10× assay was prepared 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 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 pre-loaded 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 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 PCR was performed using the protocol "GE96x96 miRNA Standard 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" procedure was followed. 【0098】 (Table 5) miRNA marker profiles TIFF0007876440000005.tif109164 【0099】 result As shown in Figure 7, J1-HE cells had a unique miRNA expression profile compared to undifferentiated embryonic stem cells (J1), human vascular cells (HUVECs), and J1-derived HB cells. Specific examples of miRNA markers are shown in Table 5. 【0100】 Example 6: In vitro differentiation of endothelial cells and angiogenesis HE and HB cells derived from J1 and GMP-1 were further tested in vitro for their respective ability to differentiate into endothelial cells. Approximately 300k HE cells and 500-600k HB cells were resuspended in 18 mL of EGM2 or Vasculife VEGF medium kit (Lifeline Cell Tech), and 3 mL of the suspension was dispensed into each well of a fibronectin-coated 6-well plate (Corning). After 2 days of culture, the medium was changed and fresh EGM2 or Vasculife VEGF medium was added. Photographs were taken when the cells reached approximately 60-70% confluence. HB (day 5) and HE (day 3) differentiated toward the endothelial lineage in the fibronectin-coated plate, both exhibiting characteristic endothelial cobblestone morphology (data omitted). 【0101】 To test for angiogenesis, cells were harvested after photography. Briefly, each well was washed with D-PBS, 1 mL of StemPro Accutase (Gibco) was added to each well, and incubated at 37°C for 3–5 minutes. Single-cell suspensions were prepared by pipetting the cultures several times. The plates were washed with EGM2 medium or Vasculife VEGF medium, transferred to conical tubes, and centrifuged at 250 g for 5 minutes. Cell counts were performed using a Nexcelom Cellometer K2. 【0102】 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. The collected HB and HE were added to 250 μL of EGM2 medium or Vasculife VEGF medium per well, at a concentration of approximately 5.0 × 10⁶. 4 Cells were seeded at a specific density. After 2-3 hours of plating, the medium was replaced with 250 μL of fresh medium containing AcLDL (Molecular Probes) (5 μL AcLDL + 245 μL of medium). The plates were then 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 EGM2 medium or Vasculife VEGF medium per well was added. Finally, micrographs of each well were taken at 4x magnification using a Keyence microscope. HB and HE formed vascular-like networks on Matrigel (data omitted). 【0103】 Example 7: In vivo study in a pulmonary hypertension model The objective of this study was to evaluate the effect of hematopoietic endothelial cells on sugen-hypoxia (SuHx)-induced pulmonary hypertension (PAH) in rats. This study also evaluated the potential efficacy of hematopoietic endothelial cells in treating SuHx-induced pulmonary hypertension (PAH) in nude rats. SuHx-induced pulmonary hypertension in rats is a well-documented model and is useful for investigating the effects of antihypertensive agents on pulmonary artery pressure and right ventricular remodeling in rats with pulmonary hypertension. 【0104】 seed Male nude (RNU) rats (Charles River Laboratories) weighing 200-250g at the time of enrollment in the study. 【0105】 Test item VPC1 = J1-HB prepared in Example 2 above. VPC2 = J1-HE prepared in Example 1 above. 【0106】 Vehicle (negative control) Distilled sterile water 【0107】 Preparation of Sugen Solution For administration on day 0, a solution of Sugen was prepared in DMSO at a concentration of 10 mg / mL. 【0108】 Experimental Procedure The animals were randomized based on their individual body weight to ensure an even distribution among the treatment groups. 【0109】 Animals from groups 2 through 8 (see Table 6) were subjected to a sugen / hypoxic / normal oxygen protocol for 21 days. Animals from group 1 were given DMSO (the vehicle for sugen) and subjected to hypoxic / normal oxygen using the same protocol. The animals were observed daily for any changes in their behavior and overall health. 【0110】 Treatment with the test material or vehicle was administered as scheduled on day 1 or day 9, as described in Table 6. Food and water were provided freely. The animals' behavior and overall health were observed daily. Weekly weight was recorded. 【0111】 On the day of surgery, rats were anesthetized with a mixture of 2-2.5% isoflurane USP (Abbot Laboratories, Montreal, Canada) in oxygen. Hemodynamic and functional parameters (systemic arterial blood pressure, right ventricular blood pressure, pulmonary artery blood pressure, oxygen saturation, and heart rate) were continuously recorded until 5 minutes had elapsed or the pulmonary artery pressure signal was lost, whichever came first. 【0112】 Next, the rats were induced to lose blood and flushed with 0.9% NaCl into the pulmonary circulation. The lungs and heart were removed together from the thoracic cavity. The lung (left lobe) was inflated with 10% NBF. The left lobe was prepared on a slide for histopathological analysis. The heart was removed to measure the wet weight of the left ventricle, including the right ventricle and septum, as part of the Fulton index. 【0113】 (Table 6) Assignment of treatment group and treatment information TIFF0007876440000006.tif123163 【0114】 Data Analysis Heart rate. Heart rate was measured using an N-595 pulse oximeter (Nonin, Plymouth, Minnesota) attached to the left foreleg of the animals. The heart rate values ​​obtained from the pulse oximeter were measured as beats per minute (bpm) using the cursor reading in Clampfit 10.2.0.14 (Axon Instrument Inc., Foster City, California, USA [now Molecular Devices Inc.]). 【0115】 Saturation (SO2). Blood oxygen saturation (SO2) was read from the signal of a pulse oximeter (Nonin, Plymouth, Minnesota) attached to the left foreleg of the animal. The saturation value was measured as a percentage (%) using the cursor reading in Clampfit 10.2.0.14. 【0116】 Arterial blood pressure. Arterial blood pressure was continuously recorded throughout the experiment using an intraarterial fluid-filled catheter (AD Instruments, Colorado Springs, Colorado, USA). Diastolic and systolic pressure values ​​were measured in mmHg using cursor readings in Clampfit 10.2.0.14. The mean arterial blood pressure was calculated using the following formula. Mean arterial pressure = diastolic pressure + (Systolic pressure - Diastolic pressure) / 3 【0117】 Pulse pressure was calculated as the difference between the systolic and diastolic readings. 【0118】 Ventricular pressure and pulmonary blood pressure. Right ventricular pressure and pulmonary blood pressure were recorded using an intraventricular fluid-filled catheter (AD Instruments, Colorado Springs, Colorado, USA). Diastolic and systolic pressure values ​​were measured in mmHg using cursor readings in Clampfit 10.2.0.14. Mean ventricular pressure and mean pulmonary blood pressure values ​​were calculated using the following formulas. Mean ventricular pressure or mean pulmonary blood pressure = diastolic pressure + (pulse pressure / 3) 【0119】 Fulton Index. At the end of the physiological recording, the lungs and hearts of each animal were removed. The hearts were dissected so that the right ventricle was separated from the left ventricle with a septum, and their weights were measured separately. The Fulton index was then calculated using the following formula. TIFF0007876440000007.tif9128 【0120】 Statistical analysis. Values ​​are expressed as mean ± SEM (standard error of the mean). One-way ANOVA and repeated independent Student t-tests were performed for all experimental conditions using Microsoft Excel 2007, comparing the treatment group to the control, healthy animals, or Sugen-hypoxic animals (vehicles). A difference was considered statistically significant if p ≤ 0.05. 【0121】 In the results, * consistently indicates that the value is significantly different from the normal oxygen control group (Figure 1), and ** indicates that it is significantly different from the SuHx control group (Group 2). In other words, * indicates that the animal is significantly different from a healthy animal, and ** indicates that the animal is significantly different from a fully diseased animal that has not benefited from any therapeutic treatment. 【0122】 result The Sugen + hypoxia (SuHx)-induced PAH rat model is a widely used model for studying pulmonary arterial hypertension. Sugen is a VEGF receptor antagonist known to cause pulmonary endothelial lesions by first damaging approximately 50% of endothelial cells in the pulmonary vascular system at the exposure levels used in this study (a single dose of 20 mg / kg). Remodeling of damaged endothelial and vascular cells, as well as vasoconstriction, occur, occluding the pulmonary arterioles, thereby restricting blood flow through the pulmonary arteries and increasing pulmonary arterial pressure. The decrease in blood flow through the pulmonary arteries and the increase in pulmonary arterial pressure increase right ventricular afterload, which leads to the development of marked right ventricular hypertrophy, which is specific to SuHx-treated rats and is also observed in clinical patients with PAH. 【0123】 In this study, all animals with only SuHx+ vehicles developed severe PAH, as expected. Affected animals exhibited all the features of the PAH model, and SuHx animals had statistically higher pulmonary blood pressure (systolic, diastolic, and mean) compared to healthy animals (Tables 7, 8, and 9). The mean pulmonary blood pressure was 41.2 mmHg (Table 9), which was three times higher in SuHx+ vehicles than in healthy animals, corresponding to broad-spectrum moderate / severe luminary arterial hypertension. 【0124】 (Table 7) Effects of VPC1 and VPC2 on systolic pulmonary blood pressure in sugen-hypoxia-induced PAH rats TIFF0007876440000008.tif56161 【0125】 (Table 8) Effects of VPC1 and VPC2 on diastolic pulmonary blood pressure in sugen-hypoxia-induced PAH rats TIFF0007876440000009.tif59161 【0126】 (Table 9) Effects of VPC1 and VPC2 on mean pulmonary blood pressure in sugen-hypoxic-induced PAH rats TIFF0007876440000010.tif56161 【0127】 Increased pulmonary blood pressure led to increased right ventricular afterload, which directly resulted in right ventricular (RV) hypertrophy, as indicated by the Fulton index (right ventricle-to-left ventricle ratio), being 2.7 times higher in the SuHx vehicle group than in the normal oxygen-rich group (Group 1) (Table 10). PAH is characterized by short-term right ventricular hypertrophy with a significant increase in myocardial thickness, followed by prolonged right ventricular distension and fibrosis. Within the 21-day study period, this rat model generally did not exhibit significant right ventricular distension for long enough to be observed. In this study, the increase in the Fulton index clearly indicates significant right ventricular hypertrophy. These data also show that cell injection had no effect on the development of right ventricular hypertrophy. 【0128】 (Table 10) Effects of VPC1 and VPC2 on Fulton Index in sugen-hypoxia-induced PAH rats TIFF0007876440000011.tif56163 【0129】 Pulse pressure is considered normal if it is higher than 25% of systolic pressure. In the normal group, pulse pressure is 26% of systolic pressure (Table 11). In SuHx+ vehicle animals, pulse pressure decreased to 22% of systolic pressure. Although Sugen hypoxia-induced PAH is not thought to affect myocardial inotropy, insufficient gas exchange due to PAH causes a biphasic hypoxic effect on the left ventricle, ultimately leading to chronic hypoxia and loss of contractility. 【0130】 (Table 11) Effects of VPC1 and VPC2 on pulse pressure in sugen-hypoxia-induced PAH rats TIFF0007876440000012.tif56161 【0131】 Oxygen saturation (SO2) of 95-100% is considered normal. In the control group, SO2 was 98.6%, but in the vehicle group, it decreased to 88.4% (Table 12), confirming that hypertension originating in the lungs affects systemic oxygenation. 【0132】 (Table 12) Effects of VPC1 and VPC2 on SO2 in sugen-hypoxia-induced PAH rats TIFF0007876440000013.tif56163 【0133】 During the 21-day study, the body weight of normal healthy rats increased by 68 g, while the body weight of SuHx-vehicle animals increased by an average of 21 g (Table 13). While slower body weight gain should imply relatively smaller increases in organ weight, lung weight measurement is a basic but rapid means of estimating inflammation / edema and remodeling, as remodeling and inflammation / edema contribute to organ weight gain. The lungs of vehicle-treated rats were 1.8 times heavier than those of normal rats (Table 14). A significant increase in lung weight suggests significant pulmonary edema, embolism, or fibrosis, all of which are characteristic of PAH. SuHx-induced PAH is characterized by early vasoconstriction of the pulmonary vascular system, which may be considered partly responsible for the increase in lung weight (vascular smooth muscle hypertrophy). 【0134】 (Table 13) Effects of VPC1 and VPC2 on weight gain in sugen-hypoxic-induced PAH TIFF0007876440000014.tif56163 【0135】 (Table 14) Effects of VPC1 and VPC2 on lung weight in sugen-hypoxic-induced PAH TIFF0007876440000015.tif56163 【0136】 The survival rate of the SuHx+ vehicle was measured at 80%, with 2 out of 10 rats dying before the day of surgery (Figure 8). This is consistent with the internal historical mortality rate of RNU rats in this model. 【0137】 VPC1. VPC1 was tested at two different doses, with 2.5 million and 5 million cells. Each dose was injected into one animal group on day 1 (groups 3 and 5, respectively) and into one group on day 9 (groups 6 and 8, respectively). None of the tested doses caused a statistically significant change in pulmonary blood pressure (systolic, diastolic, and mean) compared to the untreated SuHx group (Tables 7, 8, and 9). As a result, none of the VPC1 doses significantly prevented an increase in the Fulton index (Table 10), suggesting that VPC1 would not prevent right ventricular (RV) hypertrophy associated with PAH. 【0138】 Pulse pressure, mean arterial pressure, and heart rate did not change with VPC1 treatment compared to the vehicle group (Tables 11, 9, and 15). 【0139】 (Table 15) Effects of VPC1 and VPC2 on heart rate in sugen-hypoxa-induced PAH rats TIFF0007876440000016.tif56163 【0140】 In the negative control SuHx group, the SO2 level was 88%, which is lower than the normal saturation range (95-100%) (Table 12). In the groups treated with 2.5M and 5M VPC1 cells on day 1, the SO2 levels were 93% and 92%, respectively, which were slightly higher than in the negative control group (Table 12). In the group treated with 5M VPC1 cells on day 9, the SO2 level was 95%, which is within the range considered to be a normal, healthy animal (Table 12). 【0141】 Relative lung weight was no different in the VPC1-treated group compared to the vehicle group (Table 14), suggesting that VPC1 may not prevent pulmonary fibrosis and / or associated edema. 【0142】 During the 21-day study, the body weight of normal, healthy rats increased by 68 g, while the body weight of animals treated with SuHx alone (vehicle group) increased by an average of 21 g (Table 13). No animals treated with VPC1 gained more body weight than the vehicle group (Table 13). 【0143】 The survival rate was 80% in the group treated with the vehicle, while it was 100% in the groups treated with 2.5M cells of VPC1 on day 1 and 5M cells of VPC1 on day 9 (Figure 8). 【0144】 Animal survival rates, along with general health status and physiological parameters, suggest that VPC1 at a dose of 2.5 million cells injected on either day 1 or day 9 had no significant effect on SuHx-induced PAH in rats. A dose of 5 million cells injected on day 9 appeared to provide a small benefit, as indicated by increased hemoglobin oxygen saturation and increased animal survival rates. 【0145】 It is noteworthy that the animals showed no intolerance or adverse effects as a result of VPC1 injection. Cage-side observations revealed no discomfort in the animals, aside from symptoms related to PAH. 【0146】 VPC2. VPC2 was tested at a cell dose of 2.5 million cells. The cells were injected into one animal group (Group 4) on day 1 and into another animal group (Group 7) on day 9. 【0147】 In the group treated with VPC2 on day 1, systolic, diastolic, and mean pulmonary blood pressure were statistically lower (22%, 24%, and 23%, respectively) compared to the vehicle animals (Tables 7, 8, and 9). This suggests that the 2.5 million cellular VPC2 injected on day 1 improved blood flow through the pulmonary arteries by preventing tissue remodeling and / or preventing pulmonary artery vasoconstriction caused by sugen-hypoxia and its endothelial cell damage. 【0148】 However, the Fulton index increased (Table 10), suggesting that the effect of VPC2 on the hemodynamics of the animals was insufficient to prevent right ventricular (RV) hypertrophy associated with PAH. Furthermore, pulse pressure, mean arterial pressure, and heart rate did not differ statistically between the group treated with VPC2 (on day 1 or day 9) and the group treated with vehicle alone (Tables 11, 9, and 15). 【0149】 In the negative control SuHx group, SO2 levels were 88%, lower than the normal saturation range (95-100%). In the group treated with 2.5M VPC2 on day 1, SO2 levels returned to the normal range. This represents a statistically and clinically significant benefit (Table 12). 【0150】 Relative lung weight in the VPC2-treated group was not statistically significant compared to the vehicle-treated group (Table 14). 【0151】 The weight gain in animals given VPC2 was no different from that of animals given vehicle (Table 13). The survival rate was 80% in the group treated with vehicle alone, while it was 100% in the group treated with 2.5 million VPC2 cells on day 1 or day 9 (Figure 8), suggesting that VPC2 provides some degree of protection to animals suffering from PAH. 【0152】 The reduction in pulmonary blood pressure and improvement in saturation, along with improved animal survival rates, suggest that VPC2 offers some benefit in SuHx-induced PAH in rats. 【0153】 Consideration This pulmonary hypertension study used RNU rats, which have been shown to develop a very severe and rapid form of PAH under these experimental conditions. The final experimental condition used in this study was found to induce severe pulmonary hemodynamic impairment while maintaining a mortality rate of less than 20% over 21 days. 【0154】 The rapidity of disease progression and the severity of symptoms just three weeks later were a problem, and since the disease progressed so rapidly, it was a considerable challenge to produce a therapeutic benefit for the animals. It was thought that the onset and early progression of the disease could be prevented by a potent vasodilator, but the mechanism of action of the test article in this study was not favorable for such rapid testing. 【0155】 Nevertheless, injection of 2.5 million VPC2 cells on day 1 resulted in a decrease in systolic, diastolic, and mean pulmonary blood pressure. The latter decreased from 41.2 mmHg to 31.6 mmHg, which was a statistically favorable benefit. More importantly, the mean pulmonary blood pressure of the animals returned to the range (25 - 35 mmHg) that allows normal physical activity. This, together with the increased oxygen saturation, suggests that 2.5 million VPC2 cells administered on day 1 can significantly improve pulmonary hemodynamics and remove the persistent hypoxia that leads to chronic ischemia and pulmonary remodeling in clinical PAH patients. 【0156】 Furthermore, when considering the functional endpoints of this study, a difference between VPC1 and VPC2 became apparent, and in both cases, VPC2 cells injected at a density of 2.5 million on day 1 produced better results than injection of 2.5 million VPC1 cells on the same day. This is surprising because HB has previously been shown to have an effect in the mouse hindlimb ischemia model and the mouse myocardial infarction model. See U.S. Patent No. 9,938,500. Additionally, when considering pulmonary hemodynamics and all other measured functional parameters, injection of 2.5 million VPC2 cells on day 1 produced better results than injection of 2.5 million VPC2 cells on day 9. 【0157】 Overall, this study demonstrated the efficacy of VPC2(HE) cells in the very aggressive and rapid induction of PAH syndrome involving RNU rats. Although several reports have associated higher severity of PAH in immunocompromised patients, a rapid and severe progression such as that induced in this study is not known clinically. With more time and a less extreme pulmonary arterial hypertension, the functional benefits associated with a single IV injection of VPC2 cells (HE) are expected to be more favorable than what this dataset suggests. 【0158】 Example 8: Histopathological Analysis Pulmonary arterial hypertension (PAH) is characterized by a marked and persistent increase in pulmonary arterial pressure. Chronic alveolar hypoxia due to lung disease or other causes of reduced oxygen availability in animal models leads to a persistent increase in pulmonary vascular resistance and pulmonary hypertension. Multiple factors are involved in the pathophysiology of PAH, and here, persistent vasoconstriction and remodeling of the pulmonary vascular wall are thought to be the most important. Vasoconstriction is a reversible response of smooth muscle cells to various stimuli but is required to maintain the remodeling that occurs in all layers of the vascular wall and ultimately leads to a more persistent constriction of the lumen diameter. 【0159】 In this study, in the animals tested in Example 7, by analyzing various parameters, it was determined whether the tested hematopoietic endothelial cells prevent the development of structural lesions that characterize pulmonary vascular changes in the PAH model. 【0160】 material and method The left lobe of the lung recovered from all rats in all experimental groups (shown in Table 6) was fixed by perfusion with 10% formalin and then sent to IRIC (The Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada) to prepare slides for histopathological analysis. 【0161】 A transverse section of the left middle lobe was cut, embedded in paraffin, sliced ​​to a thickness of 5 μm, mounted, and stained with hematoxylin and eosin (H&E). 【0162】 Each slice was visualized at 200x magnification using a Nikon Eclipse T100 microscope. A minimum of 10 non-overlapping fields were randomly selected from each lung. Micrographs were taken using a Nikon DS-Fi1 digital camera with Nikon NIS Elements 4.30. Photographers were blinded regarding the treatments performed on the rats and the features of interest. For each of the 10 fields, one well-focused micrograph of each region was taken and saved. All blood vessels found in each field were analyzed, from the largest to the smallest, without any threshold or limitation on vessel size. 【0163】 We identified intralobular vessels within the gas exchange areas of the lung, associated with the alveoli, alveolar ducts, and respiratory bronchioles. All vessels associated with terminal bronchioles and larger airways were excluded. 【0164】 Based on the lumen diameter, the vessels were divided into three size groups: small (10–50 microns), medium (50–100 microns), or large (>100 microns) by measuring the longest axis of the transversely segmented lumen. The diameter was measured at the widest point of the lumen using "Infinity Analyze 5.0.3." and perpendicular to the long axis of the vessel. The lumen is located between the inner edges of the internal elastic lamina; that is, the internal elastic lamina was considered to be part of the vessel wall rather than forming part of the lumen. 【0165】 Each blood vessel was classified as non-muscular, semi-muscular, or muscular. 【0166】 Completely muscular. Completely surrounded (over >90% of the periphery) by a smooth muscle layer identified by staining and the internal and external elastic lamina. In muscularized vessels, the outer diameter was measured at the same point as the inner diameter in non-muscular vessels, extending from the outer edge of the external elastic lamina to the opposite outer edge. 【0167】 Partially muscular: Incompletely surrounded by crescent-shaped smooth muscle (10-90% of the circumference), with part of the circumference surrounded by two elastic laminas. In partially muscularized vessels, the outer diameter was measured at the same point as the inner diameter in non-muscular vessels, extending from the outer edge of the outer elastic lamina to the opposite outer edge of the outermost elastic lamina at that point (regardless of whether it is an inner or outer elastic lamina). 【0168】 Non-muscular: The entire outer periphery of the blood vessel (<10%) is a single elastic lamina, lacking a distinct smooth muscle layer. 【0169】 analysis The values ​​are expressed as mean ± SEM (standard error of the mean). Repeated independent Student t-tests were performed to compare the following groups for all experimental conditions: 【0170】 To confirm successful disease induction, the SuHx group (negative control) animals were compared to healthy animals (normal oxygen control). The treatment group and the negative control animals (SuHx) were compared. A difference of p ≤ 0.05 was considered statistically significant. 【0171】 In all cases, * indicates that the value is significantly different from the control (no SuHx) group, and ** indicates that the value is significantly different from the negative control (SuHx) group. 【0172】 result Sugen's effects Sugen injections induced a combination of severe arteriovenous lesions, including medial and adventitia thickening of pulmonary arterioles, as well as concentric neointimal lesions and complex plexiform-like lesions. Two patterns of complex lesion formation were observed: one in which lesions formed within the vascular lumen, and the other in which they protruded outside the vessel (aneurysmal). A third structural consequence of Sugen induction of PAH occurred much later in disease progression, and its essence lay in the appearance of fibrosis within the pulmonary parenchyma. While preclinical Sugen-induced PAH is not a fibrosis model in itself, scrutiny of late-stage embedded stained tissue allows for a definitive assessment of fibrosis. The appearance of fibrosis indicates irreversible PAH, similar to that observed in long-term affected patients. Unfortunately, these patients tend not to respond to the current class of vasodilator treatments for PAH. 【0173】 To determine the severity of morphometric changes that can be observed between healthy lungs and PAH lungs, we selected pulmonary arteriole wall thickness, vascular classification, proliferative cells (progenitor cells) surrounding these arteries, and relative diameter of the arterial lumen. Infiltration of mononuclear inflammatory cells (alveolar macrophages) and leukocytes (clusters of lymphocyte-like cells and eosinophils) in the lungs, interstitial / alveolar edema and fibrosis in the lungs, and plexus-like lesions were also used as indicators of the pathophysiological state of the lungs. 【0174】 The severity of histopathological changes, such as thickening of the arterial media, infiltration of "progenitor" cells in the adventitia of arterioles, and infiltration of alveolar macrophages, alveolar edema and fibrosis, as well as plexus-like lesion formation in the lung parenchyma, was scored from 0 to 3. Here, 0 = none, 1 = mild, 2 = moderate, and 3 = severe. 【0175】 Arterial size, lumen diameter, and presence or absence of arteriole muscularization were compiled from the lungs of SuHx-induced PAH rats treated with VPC1 and VPC2, as well as negative control animals, as shown in Table 6. 【0176】 Negative control rats As expected, the lung tissue of control (normoxic) animals was mainly composed of non-muscular arterioles (88.3%) (Tables 16, 17, and 18). In contrast, the lung tissue of negative control (SuHx) animals was mainly composed of muscular arterioles (83.9%) (Tables 16, 17, and 18). This observation is consistent with the hyperplasia observed in the 56-day Sugen-hypoxia model in Sprague-Dawley rats. Eleven days of hypoxia at 17% oxygen following Sugen injection is sufficient to cause continuous pulmonary VSM contraction leading to hypertrophy and hyperplasia of vascular smooth muscle (VSM), and the proliferation of VSM cells changed arterioles that are normally non-muscular into arterioles that are partially or completely muscularized. As a result, in these blood vessels, the wall thickness increased and the lumen space decreased. In addition, 10 days in a normoxic environment still maintained hypoxic conditions in the lungs due to pulmonary smooth muscle remodeling. The hypoxia period of this study was characterized by rapid endothelial proliferation, which resulted in various grades of plexiform lesions. By the end of 21 days, these lesions were often sufficient to completely eliminate small-diameter arterioles. 【0177】 (Table 16) Effects of VPC1 and VPC2 on the percentage of non-muscular blood vessels in SuHx-induced PAH rats TIFF0007876440000017.tif56170 【0178】 (Table 17) Effects of VPC1 and VPC2 on the percentage of muscular blood vessels in SuHx-induced PAH rats TIFF0007876440000018.tif56170 【0179】 (Table 18) Effects of VPC1 and VPC2 on the percentage of semi-muscular blood vessels in SuHx-induced PAH rats TIFF0007876440000019.tif56170 【0180】 In the control (normal oxygen) group, the majority of vessels (≒88%) were characterized as "small" size (less than 50 microns in diameter) and were primarily non-muscular (Tables 16, 17, and 18). Nearly 12% of vessels were described as "medium" size, and the remaining very small number were considered "large." PAH induction by SuHx altered vessel thickness and led to a shift in the distribution of vessels based on size (at the end of the study, ≒60% were characterized as small vessels, 38% as medium vessels, and the remainder as large vessels). The changes induced by SuHx were evident in the thickening of the muscular layer within vessels (as shown in Tables 19, 20, and 21), with the muscle tissue of small and medium-sized pulmonary vessels significantly increasing by 16–42% and 20–33%, respectively, compared to control (normal oxygen) animals. The wall thickness of large vessels did not change significantly. 【0181】 (Table 19) Effects of VPC1 and VPC2 on small vessel wall thickness in SuHx-induced PAH rats TIFF0007876440000020.tif55170 【0182】 (Table 20) Effects of VPC1 and VPC2 on medium vessel wall thickness in SuHx-induced PAH rats TIFF0007876440000021.tif55170 【0183】 (Table 21) Effects of VPC1 and VPC2 on large vessel wall thickness in SuHx-induced PAH rats TIFF0007876440000022.tif55170 【0184】 The increase in wall thickness reduces the diameter of the arterial lumen, increasing pulmonary artery pressure, which the right ventricle must counteract (right ventricular afterload). 【0185】 Plexiform lesions were not observed in healthy, uninducible animals. In contrast, animals induced by Sugen but not benefiting from any treatment presented grade 2 and grade 3 plexiform lesions corresponding to moderate (grade 2) to severe endothelial hyperplasia and complete disappearance of some vascular lumen (grade 3). In addition to the plexiform lesions characteristic of human PAH, the animals also showed signs of fibrosis and interstitial / alveolar edema. 【0186】 VPC1 VPC1 was tested at two different doses, 2.5 million cells and 5 million cells. Each dose was injected into one animal group on day 1 and into another animal group on day 9. 【0187】 Just as PAH induction alters the size-based distribution of blood vessels, treatment with VPC1 also alters the size-based distribution of blood vessels. VPC1 slightly increased the number of "small" blood vessels and decreased the number of "medium" blood vessels compared to SuHx rats (data omitted). 【0188】 The wall thickness of pulmonary small vessels (mostly determined by the thickness of the smooth muscle layer) in rats treated with 2.5M and 5M VPC1 cells on day 1 was statistically significantly lower compared to vehicle-treated rats. The wall thickness of medium and large vessels did not change significantly (Tables 19, 20, and 21). Treatment with VPC1 on day 9 had no effect on vessel wall thickness. 【0189】 The percentage of muscular blood vessels was significantly lower in animals treated with 2.5M and 5M VPC1 cells on day 1, from 83.9% in negative control SuHx-treated animals to 64% and 69%, respectively, in VPC1-treated animals (Tables 16, 17, and 18). 【0190】 The same dose of VPC1 administered on day 9 did not have a statistically significant effect on the percentage of muscular blood vessels in lung tissue. 【0191】 Furthermore, alveolar macrophage infiltration, edema / fibrosis, and pulmonary artery lesions observed in the group treated with VPC1 on day 1 were less frequent than in the vehicle animals. Plexiphenous lesions in the group treated with VPC1 on day 1 were classified as mild / moderate (score 1 or 2). 【0192】 VPC2 VPC2 was tested at a cell dose of 2.5 million cells. The cells were injected into one animal group on day 1 and into another animal group on day 9. 【0193】 Just as PAH induction alters the size-based distribution of blood vessels, treatment with VPC2 on day 1 also alters the size-based distribution of blood vessels. VPC2 injected on day 1 increased the number of "small" size vessels and decreased the number of "medium" size vessels compared to SuHx rats. Treatment with VPC2 on day 1 brought the ratio of "small" size vessels to "medium" and "large" size vessels very close to that observed in perfectly healthy, heterooxygenous rats. 【0194】 The wall thickness of pulmonary small vessels in rats treated with 2.5M cells of VPC2 on day 1 was statistically smaller compared to vehicle-treated rats. VPC2 administered on day 9 had no effect on vessel wall thickness. There was no significant change in the wall thickness of medium and large vessels (Tables 19, 20, and 21). Treatment with VPC2 on day 9 had no effect on vessel wall thickness. 【0195】 The percentage of muscular blood vessels was significantly lower in animals treated with VPC2 on day 1, from 83.9% in vehicle-treated animals to 45% in VPC2-treated animals. As a result, the percentage of non-muscular blood vessels increased from 7% to 46%. VPC2 on day 9 had no significant effect on muscular blood vessels. See Tables 16, 17, and 18. 【0196】 These results support functional findings indicating that the severity of PAH symptoms in SuHx-induced animals is significantly lower in animals treated with VPC2. VPC2 prevented pulmonary vascular remodeling in the SuHx-induced PAH rat model. 【0197】 Furthermore, alveolar macrophage infiltration, edema / fibrosis, and pulmonary artery lesions observed in VPC2-treated animals were far less frequent and classified as none / mild (score 0-1) compared to negative control SuHx animals, suggesting that VPC2 prevents the development of PAH-related lung changes. 【0198】 This study demonstrated the high efficacy of VPC2(HE) against functional and structural findings in highly aggressive and rapidly induced PAH syndrome in RNU rats. 【0199】 Example 9: HE contains a unique vascular endothelial fraction that is VECAD+. The flow cytometry and transcript analysis described above indicated that a significant amount of vascular endothelial components appear to be generated by the HE differentiation protocol. To further clarify the similarities and differences between PSC-derived EC-like cells and mature EC cells, single-cell RNA sequencing was performed comparing HE, HUVEC, and undifferentiated iPSCs (GMP1). 【0200】 Unsupervised clustering revealed nine clusters among the tested cell types (Figure 9A). As expected, undifferentiated iPSCs formed a distinct cluster from HE cells ("VPC feeder activity") and HUVECs (Figures 9B and 9C). HE cells were grouped into multiple clusters, but overall, they formed a population largely distinct from iPSCs and HUVECs. Examination of the expression of specific vascular endothelial cell markers identified three clusters (clusters 2, 4, and 5) based on the presence of VECAD / CDH5 (Figure 10). Clusters 2 and 4 consisted mainly of HUVECs, while cluster 5 consisted of HE cells (Figure 9B). As cluster 5 appeared to consist of VECAD+ cells, differential gene expression analysis was performed comparing VECAD+ HE cells from cluster 5 with cells from the other clusters. Cluster 5 was found to have a strong vascular endothelial signature, as indicated by the function of the most differentially expressed gene (Table 22). Many of the top 50 significantly upregulated genes in Cluster 5 were genes with known vascular expression and activity, including PLVAP, GJA4, ESAM, EGFL7, KDR / VEGFR2, ESAM, and VECAD (CDH5) (Table 22). Gene ontology analysis showed that the most enriched pathways were EC migration, endothelial development, sprouting angiogenesis, and other EC-related processes. Similarly, gene set enrichment analysis revealed pathways important for endothelial development and function, including TGF beta signaling and hypoxia. 【0201】 Clustering analysis also showed that HE cells differ significantly from HUVECs. The contribution of HUVECs to cluster 5 was negligible but not zero, and clusters 2 and 4 were mainly composed of HUVECs with little HE expression (<15%) (Figure 9B). Differential gene expression analysis comparing cluster 5 to clusters mainly composed of HUVECs revealed that VECAD+HE cells in cluster 5 were immature ECs or progenitor ECs (Table 23). Genes with elevated expression levels in VECAD+HE cells included SOX9, PDGFRA, and EGFRA, which are markers for replicative vessel-borne progenitor vascular cells, the precursors of terminally differentiated ECs. A recent study comparing endothelial colony-forming cells (ECFCs) with mature vascular endothelial cells (EC) (Kutikhin, AGet al. Cells 9:876 (2020)) identified KDR / VEGFR2, NOTCH4, and collagen I and IV subunits as ECFC enrichment factors, and their transcripts were similarly upregulated in VECAD+HE cells of cluster 5 compared to HUVECs. However, other ECFC enrichment genes, such as CD34, were not elevated in HE cells. Both HE cells and HUVECs expressed VECAD / CDH5 and PECAM1 / CD31, but HUVEC levels were higher, which is also consistent with HE cells being more immature or progenitor EC-like cells. Gene ontology analysis using differentially expressed sets of genes in VECAD+HE cells and HUVECs showed that the most enriched pathways were sterol biosynthesis, protein kinase A signaling, gastrointestinal tract, and ventricular development. Gene set enrichment analysis revealed that differentially expressed genes, compared to iPSCs, are associated with pathways crucial for endothelial development and homeostasis, including MTORC1, WNT, and TGF beta signaling.In summary, single-cell RNA sequencing revealed a cluster of HEs similar to HUVECs, possessing the characteristics of true ECs but also exhibiting discriminant features suggesting immature or progenitor phenotypes. 【0202】 (Table 22) Top 50 genes significantly upregulated in cluster 5 compared to cells in other clusters TIFF0007876440000023.tif176164TIFF0007876440000024.tif198164 【0203】 (Table 23) Top 100 genes significantly upregulated in cluster 5 compared to HUVEC cells TIFF0007876440000025.tif23164TIFF0007876440000026.tif241164TIFF0007876440000027.tif241164TIFF0007876440000028.tif234164 【0204】 Example 10: HE attenuates hemodynamic parameters and vascular remodeling in a rat model of pulmonary artery hypertension. Treatment of rodents with monoclotaline (MCT) induces vascular resistance and cardiac dysfunction (Rabinovitch, M. Toxicol Pathol 19, 458-469 (1991)), and the Sugen / hypoxia model induces the aforementioned clinical markers as well as plexiform lesions, a prominent clinical feature of progressive disease in humans (Ciuclan, L. et al. Am J Respir Crit Care Med 184, 1171-1182 (2011)). 【0205】 In MCT rats, treatment with HE derived from J1-ESCs and treatment with HE derived from GMP-1 iPSCs both attenuated PAH symptoms. Briefly, rnu / rnu rats were given a single dose of MCT (50 mg / kg, iPSC) on day 0. On day 3, the rats were divided into vehicle, J1-HE, and GMP-1 HE groups and administered intravenously to control medium or cells (2.5 × 10⁶).6 Rats were administered sildenafil (15 mg / kg / day). As a positive control, another group was given a high dose of sildenafil (approximately 15 mg / kg / day) in their drinking water. On day 28, hemodynamic analysis was performed by right and left heart catheterization. As expected, vehicle-treated rats showed increased right ventricular systolic pressure (RVSP), Fulton index, and pulmonary vascular resistance index (PVR index) (Figures 11A-C). Rats treated with J1-HE had lower RVSP and PVR index values ​​(Figures 11A and 11C). Rats treated with GMP-1-HE also had lower RVSP, Fulton index, and PVR index values ​​(Figures 11A-C). Histological analysis revealed that rats in the J1-HE and GMP-1-HE groups had fewer thickened blood vessels compared to vehicle-treated rats, which was quantitatively supported (Figure 11D). 【0206】 Next, PSC-derived HE was tested again in a Sugen / hypoxic model of PAH. In these studies, rnu / rnu rats were subjected to Sugen / hypoxic / normal oxygen conditions for 21 days. On day 0, the rats received a single dose of Sugen, followed on day 1 by intravenous injection of vehicle, J1-HE, or GMP-1-HE in doses of 1 million, 2.5 million, or 5 million cells. As an additional control, another group received sildenafil (50 mg / kg) twice daily via oral gastric tube. Rats treated with 2.5 million J1-HE and GMP-1-HE per injection showed reductions in mPAP, RVSP, and Fulton index, as well as improvements in cardiac function such as stroke volume and cardiac output, compared to vehicle-treated rats (Figures 12A-D). Furthermore, GMP-1-HE dose-dependently improved its efficacy in pulmonary hemodynamics, RV remodeling, and cardiac function (Figures 13A-D). Histological analysis of lung tissue revealed differences between control rats and J1-HE-treated or GMP-1-HE-treated rats in the Sugen / hypoxic model (Figures 14A-C and 15A-C). HE-treated animals showed fewer plexiform lesions compared to vehicle-treated animals (Figures 14A and 15A). Pulmonary vascular wall thickness was also reduced in HE-treated animals compared to vehicle-treated animals (Figures 14B and 15B). In HE-treated animals, the percentage of pulmonary vessels classified as muscular and semi-muscular was lower than in the vehicle-treated group (Figures 14C and 15C). Finally, HE-treated lungs showed less immune cell infiltration compared to vehicle-treated animals (data omitted). 【0207】 Whole-transcriptome analysis of lungs from HE-treated and vehicle-treated rats in a Sugen / hypoxic model supported physiological data suggesting that HE treatment attenuates pathological vascular remodeling. RNA was collected from rat lungs on day 21 and differential gene expression analysis was performed. Pathway analysis of genes downregulated by ≥1.25-fold by cell treatment showed reductions in genes associated with smooth muscle cell development, immune cell infiltration, and inflammation, among others. Conversely, genes upregulated by ≥1.25-fold by cell treatment were associated with favorable metabolic states, i.e., metabolic states that benefit oxidative phosphorylation, whose disruption is associated with the PAH disease state. Overall, these data suggest that HE protects rats in a PAH model by reducing vascular resistance, vascular remodeling, and cardiac hypertrophy at dose ranges of 2.5 million to 5 million units per single injection. 【0208】 Example 11: HE repairs the pulmonary microvascular system. Endothelial progenitor cells have been reported to preserve the microvascular system in MCT-treated lungs (Zhao et al. Cir.Res. 96:442-450 (2005)). Therefore, micro-CT scans were performed on lungs of SuHx models treated with Nx control, vehicle, sildenafil, and 1 million and 5 million GMP1-HE cells. Micro-CT scans revealed uniform filling of the distal arteriole bed and a uniform pattern of capillary perfusion in normal lungs (Figure 16A). In contrast, vehicle-treated SuHx lungs showed stenosis of the distal arteriole bed and capillary occlusion (Figure 16B). Treatment with 5 million HE cells (Figure 16D) preserved the microvascular system as visualized by contrast injection, but treatment with 1 million HE cells (Figure 16C) did not. In 5 million HE cells, a significant improvement in the appearance of the pulmonary microvascular system was observed, along with the preservation of arterial continuity and enhanced capillary perfusion (Figure 16D). Sildenafil treatment showed moderate improvement in capillary perfusion (Figure 16E). 【0209】 Example 12: HE contains a unique vascular endothelial fraction that is therapeutically active. Based on the single-cell profiling of HE described above and the similarities between the VECAD+HE fraction and HUVEC, it was suggested that this subpopulation is likely the active component that confers its therapeutic effect on HE in PAH. To verify this, another study using the Sugen / hypoxia model was conducted using 2.5 million "bulk" or unsorted HE cells, and 2.5 million VECAD+HE cells purified from "bulk" HE cells by magnetic sorting of VECAD+ cells (Figure 17). The sorted fraction of VECAD+ cells showed that the majority also expressed CD31 (Figure 17). VECAD+HE improved clinical measurements: mPAP (Figure 18A), RVSP (Figure 18B), RV remodeling (Figure 18C), and cardiac output (Figure 18D) compared to vehicle-treated animals. The pulmonary vascular system was also maintained compared to vehicle-treated animals, with fewer plexiform lesions (Figure 18E), reduced wall thickness (Figure 18F), and reduced vascular muscularization (Figure 18G). Similar results were obtained by delivery of true mature endothelial cells (HUVECs). 【0210】 Analysis of VECAD+ / CD31+ populations in J1-HE and GMP1-HE regarding FLK1 / KDR expression revealed that these HEs include populations that are CD31+ / VECAD+ / FLK1+ (Figure 19). 【0211】 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

[Claim 1] A composition comprising hematopoietic endothelial cells (HE) for use in the treatment of vascular disease in subjects suffering from or suspected of suffering from vascular disease, HE is CD73(+) and also positive for miRNA-214, miRNA-199a-3p, and miRNA-335. composition. [Claim 2] (a) Vascular diseases are selected from the group consisting of coronary artery disease, myocardial infarction, organizing myocardial infarct, ischemic heart disease, arrhythmia, left ventricular dilation, embolism, heart failure, congestive heart failure, subendocardial fibrosis, left or right ventricular hypertrophy, myocarditis, chronic coronary ischemia, dilated cardiomyopathy, restenosis, arrhythmia, angina, hypertension, myocardial hypertrophy, peripheral artery disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cor pulmonale, dyscardial rhythm disorders, inflammatory heart disease, congenital heart disease, rheumatic heart disease, diabetic vascular disease, and endothelial lung injury disease. (b) The vascular disease is pulmonary hypertension, (c) The vascular disease is pulmonary hypertension, (d) Vascular disease is peripheral artery disease, (e) The vascular disease is severe limb ischemia, (f) Vascular disease is a pulmonary endothelial disease, (g) The vascular disease is acute respiratory distress syndrome (ARDS), (h) The vascular disease is arteriosclerosis, atherosclerosis, disease or injury of the arteries, arterioles and capillaries, or related conditions, acute myocardial infarction, glomerular hypertension, portal hypertension, or acute lung injury, and / or (i) Mean pulmonary blood pressure is reduced in the subjects, A composition for use according to claim 1. [Claim 3] A composition comprising hematopoietic endothelial cells (HE) for use in increasing blood flow in subjects suffering from or suspected of suffering from vascular disease, HE is CD73(+) and also positive for miRNA-214, miRNA-199a-3p, and miRNA-335. composition. [Claim 4] (a) Vascular diseases are selected from the group consisting of pulmonary hypertension, peripheral artery disease, and pulmonary endothelial injury, (b) The vascular disease is pulmonary hypertension, (c) The vascular disease is acute respiratory distress syndrome (ARDS), and / or (d) The vascular disease is severe limb ischemia, The composition for use according to claim 3. [Claim 5] A composition comprising hematopoietic endothelial cells (HE) for use in reducing blood pressure in subjects with or suspected of having vascular disease, HE is CD73(+) and also positive for miRNA-214, miRNA-199a-3p, and miRNA-335. composition. [Claim 6] (a) Vascular diseases are selected from the group consisting of pulmonary hypertension, peripheral artery disease, and pulmonary endothelial injury, (b) The vascular disease is pulmonary hypertension, (c) The vascular disease is acute respiratory distress syndrome (ARDS), (d) The vascular disease is severe limb ischemia, (e) Blood pressure is diastolic pressure, systolic pressure, or mean pulmonary artery pressure, and / or (f) Blood pressure is reduced by at least 20% in the subjects. A composition for use according to claim 5. [Claim 7] (a) HE is further positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p, (b) HE is positive for miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, (c)HE is positive for miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. (d) HE is negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p, (e)HE is negative for miRNA-223 and miRNA-142-3p, (f) HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223 and miRNA-142-3p, (g)HE further expresses at least one cell surface marker selected from the group consisting of CD24, CD31 / PECAM1, CD309 / KDR, CD144, CD34, CXCR4, CD146, Tie2, CD140b, CD90, CD271, and CD105. (h)HE further expresses CD146, CXCR4, CD309 / KDR, CD90 and CD271, (i) HE further expresses CD146, (j)HE further expresses CD144 (VECAD), (k)HE further expresses at least one cell marker selected from the group consisting of CD24, CD31, CD309 / KDR (FLK-1), PLVAP, GJA4, EGFL7, and ESAM. (l)HE further expresses at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA. (m)HE further expresses at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. (n)HE further expresses CD31 / PECAM1, CD309 / KDR, CD144, CD34 and CD105, (o)HE shows limited detection or no detection of at least one cell surface marker selected from the group consisting of CD34, CXCR7, CD43, and CD45. (p)HE indicates limited detection of CXCR7, CD43 and CD45, or no detection at all. (q)HE indicates limited detection of CD43 and CD45, or no detection at all. (r)HE is CD73(+), CD43(-), CD45(-) and CD146(+), (s)HE further expresses (i) CD144 (VECAD) and (ii) CD31 and / or CD309 / KDR (FLK-1), and / or (t)HE further expresses CD24, A composition for use according to any one of claims 1 to 6. [Claim 8] (a) A process to obtain HE by in vitro differentiation of pluripotent stem cells, (b) A step of obtaining HE by culturing pluripotent stem cells under adhesion conditions, in differentiation medium, and / or in the absence of methylcellulose. (c) Process for obtaining HE by in vitro differentiation of pluripotent stem cells without embryoid body formation. A method for producing a composition for use according to any one of claims 1 to 7, including the composition described in any one of claims 1 to 7. [Claim 9] (a) The subject is a human being, (b) Pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells, (c) The pluripotent stem cells are human pluripotent stem cells, and / or (d) HE is human HE, A composition for use according to any one of claims 1 to 7. [Claim 10] A composition comprising hematopoietic endothelial cells (HE), wherein the HE is positive for miRNA-214, miRNA-199a-3p, and miRNA-335, and is CD73(+), CD43(-), CD45(-), and CD146(+). [Claim 11] (a) HE is further positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p, (b) HE is positive for miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, (c)HE is positive for miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. (d) HE is negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p, (e) HE is negative for miRNA-223 and miRNA-142-3p, and / or (f) HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223 and miRNA-142-3p. The composition according to claim 10. [Claim 12] The composition according to claim 10 or 11, wherein HE expresses CD144 (VECAD) and CD73. [Claim 13] (a) HE further expresses at least one cell marker selected from the group consisting of CD24, CD31, CD309 / KDR (FLK-1), PLVAP, GJA4, EGFL7, and ESAM, (b) HE further expresses at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA, (c)HE further expresses at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. (d) The composition substantially lacks CD73-negative HE cells and CD144 (VECAD)-negative HE cells, (e) HE expresses (i) CD144 (VECAD) and (ii) CD73 and (iii) CD31 and / or CD309 / KDR (FLK-1), and / or (f) HE further expresses CD24, The composition according to claim 12. [Claim 14] A pharmaceutical composition comprising hematopoietic endothelial cells (HE) and a pharmaceutically acceptable carrier, wherein the HE is positive for miRNA-214, miRNA-199a-3p, and miRNA-335, and is CD73(+), CD43(-), CD45(-), and CD146(+). [Claim 15] (a) HE is further positive for at least one microRNA (miRNA) selected from the group consisting of miRNA-126, miRNA-24, miRNA-196-b, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690 and hsa-miR-7151-3p, (b) HE is positive for miRNA-126, miRNA-24, miRNA-196-b, miRNA-214, miRNA-199a-3p and miRNA-335, (c)HE is positive for miRNA-214, miRNA-199a-3p, miRNA-335, hsa-miR-11399, hsa-miR-196b-3p, hsa-miR-5690, and hsa-miR-7151-3p. (d) HE is negative for at least one miRNA selected from the group consisting of miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223, and miRNA-142-3p, (e) HE is negative for miRNA-223 and miRNA-142-3p, and / or (f) HE is negative for miRNA-367, miRNA-302a, miRNA-302b, miRNA-302c, miRNA-223 and miRNA-142-3p. The pharmaceutical composition according to claim 14. [Claim 16] The pharmaceutical composition according to claim 14 or 15, wherein HE expresses CD73 and CD144 (VECAD). [Claim 17] (a) HE further expresses at least one cell marker selected from the group consisting of CD24, CD31, CD309 / KDR (FLK-1), PLVAP, GJA4, EGFL7, and ESAM, (b) HE further expresses at least one cell marker selected from the group consisting of SOX9, PDGFRA, and EGFRA, (c)HE further expresses at least one cell marker selected from the group consisting of KDR / VEGFR2, NOTCH4, collagen I, and collagen IV. (d) The composition substantially lacks CD73-negative HE cells and CD144 (VECAD)-negative HE cells, (e) HE expresses (i) CD144 (VECAD) and (ii) CD73 and (iii) CD31 and / or CD309 / KDR (FLK-1), and / or (f) HE further expresses CD24, The pharmaceutical composition according to claim 16.