Accelerated reprogramming of hiPSCs into functional brain endothelial-like cells using multiplexed CRISPR activation.

Human induced pluripotent stem cells (hiPSCs) offer a renewable and scalable source for generating brain endothelial cells (BECs), enabling the development of in vitro blood-brain barrier (BBB) models that closely reflect human physiology. In this study, we demonstrate a streamlined differentiation strategy for producing hiPSC-derived BEC-like cells (iBECs) using multiplex CRISPR/dCas9 activation (CRISPRa) to transcriptionally reprogram iPSCs by selectively and simultaneously upregulating BEC transcription factors (ERG, ETV2, FLI1) and BEC genes (CLDN5, CDH5, PECAM1, KDR). We observe that this approach significantly accelerates differentiation from 13 to five days while maintaining BBB characteristics. CRISPRa iBECs exhibit high transendothelial electrical resistance (TEER ~ 400 Ω·cm2), low transcellular permeability (< 0.027 × 10⁻3 cm/min), and expression of key BBB markers including Claudin-5, ZO-1, CDH5, PECAM1, Occludin, and GLUT-1, as well as receptor-mediated transporters TfR1, IGF1R, and TMEM30A. Our study demonstrates a novel CRISPRa-directed differentiation strategy that not only accelerates BEC differentiation but also demonstrates the utility of CRISPR gene regulation as an option in modifying the phenotype of iPSC-derived cells.