GENERATING BRAIN MICROVASCULAR ENDOTHELIAL CELLS FROM PLURIPOTENT STEM CELLS TO MODEL THE HUMAN BLOOD-BRAIN BARRIER AND THE NEUROVASCULAR UNIT

摘要

The invention is directed to a method for producing brain microvascular endothelial cells comprising: (A) providing pluripotent stem cells (PSCs); (B) inducing the formation of an early mesoderm cell culture comprising incubation of the PSCs in a chemically defined medium (CDM) comprising i) mesodermal inducing agent, ii) fibroblast growth factor, iii) phosphoinositide 3 -kinases pathway inhibitor, and iv) TGF-beta pathway inhibitor; (C) further patterning mesoderm formation comprising incubating the early mesoderm cell culture in a chemically defined medium (CDM) comprising i) fibroblast growth factor, ii) phosphoinositide The invention is directed to a method for producing brain microvascular endothelial cells comprising: (A) providing pluripotent stem cells (PSCs); (B) inducing the formation of an early mesoderm cell culture comprising incubation of the PSCs in a chemically defined medium (CDM) comprising i) mesodermal inducing agent, ii) fibroblast growth factor, iii) phosphoinositide 3 -kinases pathway inhibitor, and iv) TGF-beta pathway inhibitor; (C) further patterning mesoderm formation comprising incubating the early mesoderm cell culture in a chemically defined medium (CDM) comprising i) fibroblast growth factor, ii) phosphoinositide 3 -kinases pathway inhibitor, and iii) TGF-beta pathway inhibitor; (D) induction of endothelial cell formation comprising incubating the mesodermal cells in a chemically defined medium (CDM) comprising: i) an agent capable of inducing adherens junction marker and vascular endothelial cell makers, and ii) a cAMP signalling activator; (E) induction of brain microvascular endothelial cell (BMEC) formation comprising incubating the endothelial cells in a chemically defined medium (CDM) comprising: an agent capable of inducing adherens junction markers and vascular endothelial cell makers; a Wnt signalling pathway activator; and an agent capable of activating retinoic acid signalling. 3 -kinases pathway inhibitor, and iii) TGF-beta pathway inhibitor; (D) induction of endothelial cell formation comprising incubating the mesodermal cells in a chemically defined medium (CDM) comprising: i) an agent capable of inducing adherens junction marker and vascular endothelial cell makers, and ii) a cAMP signalling activator; (E) induction of brain microvascular endothelial cell (BMEC) formation comprising incubating the endothelial cells in a chemically defined medium (CDM) comprising: an agent capable of inducing adherens junction markers and vascular endothelial cell makers; a Wnt signalling pathway activator; and an agent capable of activating retinoic acid signalling.