A Stable and Reproducible Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells

Abstract

The human blood brain barrier (BBB) is a selective barrier formed by human brain endothelial cells (hBECs), which is important to ensure adequate neuronal function and protect the central nervous system (CNS) from disease. The development of human in vitro BBB models is thus of utmost importance for drug discovery programs related to CNS diseases. Here, we describe a method to generate a human BBB model using cord blood-derived hematopoietic stem cells. The cells were initially differentiated into ECs followed by the induction of BBB properties by co-culture with pericytes. The brain-like endothelial cells (BLECs) express tight junctions and transporters typically observed in brain endothelium and maintain expression of most in vivo BBB properties for at least 20 days. The model is very reproducible since it can be generated from stem cells isolated from different donors and in different laboratories, and could be used to predict CNS distribution of compounds in human. Finally, we provide evidence that Wnt/β-catenin signaling pathway mediates in part the BBB inductive properties of pericytes.

Adapting coculture in vitro models of the blood–brain barrier for use in cancer research: maintaining an appropriate endothelial monolayer for the assessment of transendothelial migration

“…11 In compliance with French legislation, written, informed consent was obtained for the collection of human umbilical cord blood. The study's objectives and protocol were approved by the French Ministry of Higher Education and Research (reference; CODECOH DC2011-1321).…”

“…11,13 At this stage, the cells have a cobblestone-like morphology and express high levels of EC markers, including CD31, vascular endothelial cadherin, and von Willebrand factor. 11 The ECs differentiated into BBB ECs after 5 to 6 days. Pericytes were seeded in the morning in 12-well plates (50 000 cells per well) and ECs were seeded on inserts in the afternoon of the same day (80 000 cells per insert).…”

“…Next, the inserts were transferred onto pericytes (50 000 cells per well, seeded in 12-well plates 2 days before the transfer). The resulting coculture was maintained for 5 days-the minimum length of time required for the induction of barrier properties 11 under standard conditions (a humidified 5% CO 2 atmosphere, with renewal of the ECM-5 medium every 2 days).…”

“…In Vitro Models of Non-Barrier-Forming Peripheral-Like Endothelium: CD34 + -Derived ECs and HUVECs Monocultures As models of peripheral endothelium with no BBB function, we used CD34 + -derived ECs 11,13 and HUVECs (below passage 5). They were cultured in ECM-5 and ECM-2 medium (composed of ECM basal medium supplemented with 2% (v/v) heat-inactivated fetal calf serum, 1% (v/v) EC growth supplement (Sciencell), and 0.5% (v/v) gentamycin), respectively.…”

“…9,10 In our recently developed in vitro model of the human BBB, ECs derived from human cord blood hematopoietic stem cells are cultured on the surface of 0.4-μm-pore inserts, together with bovine brain pericytes in the lower chamber. 11 After 6 days of coculture, the BBB phenotype (ie, expression of BBB markers and functional tightness, assessed by permeability studies using hydrophilic molecules such as Lucifer yellow (LY; carbohydrazide dilithium salt), sodium fluorescein, or sucrose) is induced in the ECs. 11 However, studies of transendothelial migration 1 (where cancer cells cross an endothelial monolayer, mimicking extravasation, or intravasation) require inserts with a larger pore size (at least 3 μm).…”