Generating arterial endothelial cell populations

Abstract

Methods for generating human arterial endothelial cells under defined conditions in the absence of insulin are described.

We claim: 
     
         1 - 12 . (canceled) 
     
     
         13 . A substantially pure, isolated population of arterial endothelial cells obtained by culturing mesodermal cells in a serum-free, albumin-free, chemically defined culture medium that is substantially free of insulin and comprises a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), a Notch agonist, a TGF-beta inhibitor, and an inhibitor of inositol monophosphatase, whereby a cell population comprising arterial endothelial cells is obtained;
 wherein the arterial endothelial cells of the population express one or more markers selected from the group consisting of Ephrin B2 (EFNB2), neuropilin1 (NRP-1), Delta-like 4 (DLL4), CD44, CXCR4/CD184, Gap Junction Protein Alpha-4 (GJA4), Hey1, Jagged-1 (JAG1), Notch1, and Notch4.   
     
     
         14 . The isolated population of  claim 13  comprising at least 90% arterial endothelial cells. 
     
     
         15 . The isolated population of  claim 13  comprising at least 99% arterial endothelial cells. 
     
     
         16 . A substantially pure, isolated population of pluripotent stem cell-derived arterial endothelial cells obtained by culturing human pluripotent stem cells for a period of about two days in a serum-free, albumin-free, chemically defined cell culture medium comprising a Bone Morphogenetic Protein (BMP), Activin A, and an activator of Wnt/β-catenin signaling to obtain a cell population comprising mesodermal cells. 
     
     
         17 . The isolated population of  claim 16  comprising at least 90% arterial endothelial cells. 
     
     
         18 . The isolated population of  claim 16  comprising at least 99% arterial endothelial cells. 
     
     
         19 - 23 . (canceled) 
     
     
         24 . A method of in vitro screening of an agent, comprising
 (a) contacting a test agent to the population of arterial endothelial cells of  claim 13 ; and   (b) detecting an effect of the agent on adhesion of leukocytes to the contacted arterial endothelial cells.   
     
     
         25 . The method of  claim 24 , wherein detecting comprises performing a method selected from the group consisting of a leukocyte adhesion assay, RNA sequencing, gene expression profiling, transcriptome analysis, metabolome analysis, detecting reporter or sensor, protein expression profiling, Forster resonance energy transfer (FRET), metabolic profiling, and microdialysis. 
     
     
         26 - 27 . (canceled) 
     
     
         28 . A kit for obtaining arterial endothelial cells, the kit comprising: (i) a serum-free, albumin-free, chemically defined culture medium suitable for differentiation of mesodermal cells into arterial endothelial cells, wherein the culture medium is substantially free of insulin and comprises a fibroblast growth factor (FGF), a vascular endothelial growth factor (VEGF), and at least one of a Notch agonist, a TGF-beta inhibitor, and an inhibitor of inositol monophosphatase; and (ii) instructions describing a method for differentiating mesodermal cells into arterial endothelial cells, the method employing the culture medium. 
     
     
         29 . The kit of  claim 28 , further comprising:
 (a) a serum-free, albumin-free, chemically defined culture medium suitable for differentiation of human pluripotent stem cells into mesodermal cells, wherein the culture medium comprises a BMP, Activin A, and an activator of Wnt/β-catenin signaling; and   (b) instructions describing a method for differentiating human pluripotent stem cells into arterial endothelial cells, the method employing the culture medium of (a).   
     
     
         30 . The isolated population of  claim 13 , wherein the mesodermal cells express one or more mesodermal markers selected from the group consisting of Brachyury (T), EMOS, FOXA2, MIXL1, MSX1, and MSX2. 
     
     
         31 . The arterial endothelial cells of  claim 13 , wherein the cells are cultured for about 6 days until a cell population comprising at least 80% human Ephrin B2 (EFNB2)-positive arterial endothelial and comprising fewer than 20% EphB4 +  cells is obtained. 
     
     
         32 . The isolated population of  claim 16 , wherein the mesodermal cells express one or more mesodermal markers selected from the group consisting of Brachyury (T), EMOS, FOXA2, MIXL1, MSX1, and MSX2. 
     
     
         33 . The method of  claim 16 , wherein the pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells. 
     
     
         34 . The method of  claim 16 , wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor. 
     
     
         35 . The method of  claim 34 , wherein the Gsk3 inhibitor is selected from the group consisting of CHIR 99021, CHIR 98014, BIO-acetoxime, BIO, LiCl, SB 216763, SB 415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide. 
     
     
         36 . The method of  claim 16 , wherein the Notch agonist is selected from the group consisting of Resveratrol (3,4′,5-trihydroxystilbene), valproic acid, and suberoyl bishydroxamic acid. 
     
     
         37 . The method of  claim 16 , wherein the TGF-beta inhibitor is SB431542. 
     
     
         38 . The method of  claim 16 , wherein the inhibitor of inositol monophosphatase is L-690,330.