Methods for differentiation of human pluripotent stem cells to brain microvascular endothelial cells
Methods for generating functional brain microvascular endothelial cells (BMECs) under chemically defined, serum-free conditions are provided. In particular, efficient and cost-effective methods for generating functional BMECs under chemically defined culture conditions are provided. BMECs obtained according to the methods provided herein are suitable for in vitro blood brain barrier (BBB) formation.
1. A method for generating a population of human brain microvascular endothelial cells (BMECs) from human pluripotent stem cells, wherein the method comprises, in order,
(a) culturing human pluripotent stem cells for about 24 hours in a chemically defined, serum-free culture medium that comprises an activator of Wnt/β-catenin signaling, whereby cells that express mesodermal markers are obtained;
(b) culturing the cells expressing mesodermal markers for about 5 days in the presence of a chemically defined, serum-free culture medium comprising a neuronal cell culture supplement, whereby cells that express endothelial progenitor marker Flk-1 are obtained; and
(c) culturing the Flk-1+ cells of (b) for about two days in the presence of a chemically defined, serum-free endothelial medium comprising a neuronal cell culture supplement, bFGF/FGF2, and retinoic acid (RA), whereby a cell population comprising human BMECs is obtained.
2. The method of claim 1 , wherein the human pluripotent stem cells of (a) are cultured in a chemically defined, serum-free culture medium at a cell density of about 35×10 3 cells/cm′ for about 3 days prior to exposure to the chemically defined, serum-free culture medium comprising an activator of Wnt/β-catenin signaling.
3. The method of claim 1 , wherein at least 95% of cells of the cell population comprising human BMECs are BMECs positive for expression of one or more of CD31, p-glycoprotein (Pgp), occludin, and claudin-5.
4. The method of claim 1 , further comprising growing the human BMECs of step (c) as a monolayer to confluence.
5. The method of claim 4 , comprising the step of taking an initial transendothelial electrical resistance (TEER) measurement of the confluent monolayer, wherein the TEER measurement is greater than 2000 Ohm (Ω)×cm 2 .
6. The method of claim 1 , wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.
7. The method of claim 6 , wherein the Gsk3 inhibitor is a small molecule selected from the group consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide.
8. The method of claim 6 , wherein the Gsk3 inhibitor is CHIR99021 and is present in a concentration of about 3 μM to about 12 μM.
9. The method of claim 1 , wherein no selecting, separating, or enriching steps are applied to the cells of step (a), (b), and (c) to generate the cell population of human BMECs.