Accelerated Reprogramming of hiPSCs into Functional Brain Endothelial Cells with Multiplexed CRISPR Activation

Despite physiological shortcomings, in vitro BBB models derived from human induced pluripotent stem cells (hiPSC) are favored due to their species specificity and superior barrier qualities. Here, we demonstrate the generation of hiPSCs-derived brain endothelial cells (iBEC) using transcriptional reprogramming with a multiplex CRISPR/dCas9 activation (CRISPRa) strategy that selectively activates essential BEC genes.

Transcriptional reprogramming simplified the differentiation of iPSCs into mature iBECs and accelerated differentiation from 13 to only five days. Furthermore, CRISPRa reprogrammed iBECs exhibit robust barrier formation with TEER values > 230 Ω x cm2 and correspondingly low transcellular permeability values (< 0.015 x10-3 cm/min). The iBECs expressed a panel of key BBB markers (Claudin-5, ZO-1, CDH5, PECAM1, Occludin, GLUT-1) and transporters involved in receptor-mediated transcytosis (TFR1, IGF1R, TMEM30A).