Abstract
We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.
Highlights
In contrast to the parental Amniotic fluid (AF) cells, the OCT4 and NANOG promoters were hypomethylated in the amniotic fluid derived induced pluripotent stem cells (AF-induced pluripotent stem cells (iPSCs)) and PCR analysis demonstrated the absence of the vector and transgene sequences in the established AF-iPSCs confirming the endogenous expression of reprogramming genes (Supplementary Fig. 1B,C)
In an improvement over existing human stem cell/iPSC brain endothelial cells (BECs) differentiation protocols[22,23,24,26], iPSC-derived BECs (i-BEC) were derived from AF-iPSCs using an efficient and simplified monolayer differentiation protocol without the requirement for cell surface marker enrichment or co-differentiation and purification steps to yield a pure population of BECs
transendothelial electrical resistance (TEER) values, averaging 500 Ωcm[2], were achieved in i-BEC monolayer maintenance cultures in the absence of conditioned medium or hydrocortisone/retinoic acid (RA) treatments used to increase TEER in other human blood-brain barrier (BBB) models[42,53]. This high baseline TEER values translate into a low sucrose permeability (2.8 × 10−5 cm/min) similar to that observed in other human iPSC-derived BBB models (3.4 × 10−5 cm/min)[26] and much lower than that observed in hCMEC/D3 (1.65 × 10−3 cm/min)[18]
Summary
We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In a study evaluating more than 7 000 drug compounds, only 5% could cross the BBB and produce a pharmacological response in the CNS9,10
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