Abstract
The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.
Highlights
The blood-brain barrier (BBB) is a dynamic and complex system responsible for maintaining homeostasis in the brain by regulating the chemical environment, immune cell transport, and the entry of toxins and pathogens [1, 2]
The purpose of this study is to demonstrate that human brain microvascular endothelial cells (hBMECs) can be derived from the BC1 human induced pluripotent stem cell line, using the approach developed by Lippmann et al [14, 17]
The hBMECs derived from the BC1 cell line show a very similar phenotype to that reported by Lippmann et al, showing that the protocol is robust and generally applicable for BBB research
Summary
The blood-brain barrier (BBB) is a dynamic and complex system responsible for maintaining homeostasis in the brain by regulating the chemical environment, immune cell transport, and the entry of toxins and pathogens [1, 2]. Key characteristics of brain microvascular endothelial cells include: high transendothelial electrical resistance (TEER > 1000 O cm2), low permeability, and expression of tight junction proteins (e.g. claudin-5), transporters (e.g. LAT-1), and efflux pumps (e.g. P-gp) [6, 7]. Cells commonly used in BBB research include primary brain microvascular endothelial cells (BMECs) from vertebrate animals, type II Madin-Darby canine kidney cells (MDCK), immortalized human BMECs, and primary human brain microvascular endothelial cells (hBMECs) [8,9,10]. A fundamental problem in BBB research is that animal-derived cell lines and immortalized human BMECs do not fully recapitulate the characteristics of the human brain [6, 11, 12]. The disadvantages of primary hBMECs are that they are not readily available and lose some of their characteristics when cultured in vitro [13]
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