Abstract
BackgroundIn vitro blood–brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. For the differentiation of iBMELCs, Matrigel, which is a gelatinous protein mixture, is often used as a coating substrate. However, the components of Matrigel can vary among lots, as it is obtained from mouse sarcoma cells with the use of special technics and also contains various basement membranes. Therefore, fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots.MethodsiBMELCs were differentiated from human iPS cells on several matrices. The barrier integrity of iBMELCs was evaluated based on transendothelial electrical resistance (TEER) values and permeability of fluorescein isothiocyanate-dextran 4 kDa (FD4) and Lucifer yellow (LY). Characterization of iBMELCs was conducted by RT-qPCR and immunofluorescence analysis. Functions of efflux transporters were defined by intracellular accumulation of the substrates in the wells of multiwell plates.ResultsiBMELCs differentiated on laminin 221 fragment (LN221F-iBMELCs) had higher TEER values and lower permeability of LY and FD4 as compared with iBMELCs differentiated on Matrigel (Matrigel-iBMELCs). Besides, the gene and protein expression levels of brain microvascular endothelial cells (BMEC)-related markers were similar between LN221F-iBMELCs and Matrigel-iBMELCs. Moreover, both Matrigel- and LN221F-iBMELCs had functions of P-glycoprotein and breast cancer resistance protein, which are essential efflux transporters for barrier functions of the BBB.ConclusionThe fully defined substrate LN221F presents as an optimal coating matrix for differentiation of iBMELCs. The LN221F-iBMELCs had more robust barrier function for a longer period than Matrigel-iBMELCs with characteristics of BMECs. This finding will contribute the establishment of an iBMELC supply system for pharmacokinetic and pathological models of the BBB.
Highlights
In vitro blood–brain barrier (BBB) models using human induced pluripotent stem cell-derived brain microvascular endothelial-like cells have been developed to predict the BBB permeability of drug candidates
laminin 221 fragment (LN221F) coating enhanced the barrier integrity of iBMELCs We attempted to identify basement membranes that were more suitable to promote the differentiation of iBMELCs than Matrigel using previously reported protocols (Fig. 1a)
To examine the effect of LN221F on human mature brain microvascular endothelial cells (BMEC), we measured the transendothelial electrical resistance (TEER) values of primary BMECs derived from human and monkey, and hCMEC/D3 cells on several a Relative TEER value induced pluripotent stem (iPS) cells b
Summary
In vitro blood–brain barrier (BBB) models using human induced pluripotent stem (iPS) cell-derived brain microvascular endothelial-like cells (iBMELCs) have been developed to predict the BBB permeability of drug candidates. Fully defined substrates as substitutes for Matrigel are needed for a stable supply of iBMELCs with less variation among lots. Brain microvascular endothelial cells (BMECs), which are one of the constituents of the blood–brain barrier (BBB), regulate the transition of substances between the vasculature and brain parenchyma via strong intercellular adhesions and expression of multidrug efflux transporters [1]. The robustness of the BBB often abrogates drug development for the treatment of central nervous system disorders because many candidates cannot reach the brain parenchyma. It is difficult to predict the permeability of all drug candidates using in vivo models. It is necessary to establish models that can and precisely predict drug permeability of the BBB for pre-screening, especially for drug discovery
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