Differentiation of disease-specific induced pluripotent stem cells into a blood-brain barrier system analyzing the role of APOE4 in Alzheimerʼs disease

Introduction Accumulation of amyloid-β (Aβ) peptides is one pathological hallmark of late-onset Alzheimerʼs disease (LOAD), the most common type of dementia. Dysregulation and later on breakdown of the blood-brain barrier (BBB) contributes and worsens the course of the disease. The ε4 allele of apolipoprotein E (APOE) was identified by GWAS as the most associated genetic risk factor for developing LOAD. APOE participates in several metabolic pathways including lipid transport, Aβ aggregation, and Aβ clearance. However, the molecular and cellular signaling pathways regulated by APOE4 are currently poorly understood. Therefore, we use patient-derived induced pluripotent stem cells (iPSCs) for the differentiation into BBB cells studying APOE, and in particular its role in AD disease mechanisms.

Methods First, we determined the APOE status of our AD patients and healthy matched controls. Secondly, B-lymphoblastoid cell lines of patients carrying the APOE4 allele and chosen controls with homozygous APOE3 alleles were used for reprogramming of iPSCs with episomal vectors. Then, subsequent to a successful verification of pluripotency and detailed characterization, the generated iPSCs lines were differentiated into cells of the BBB system. Expression levels of cell specific markers and barrier functionality were analyzed.

Results Following the generation of AD-specific and matched control iPSCs pluripotency was verified, inter alia, by alkaline phosphatase staining and a successful passed PluriTest microarray analysis. Then, characterized iPSCs were differentiated into brain microvascular endothelial-like cells and astrocytes. Existence of cell- and BBB-specific markers including TJP1, GLUT1 or GFAP, and GLAST confirmed efficient differentiation. Low volume quantitative RT‐PCR analysis revealed significant differences in the expression level of BBB specific markers in AD-specific cells compared to healthy controls. Barrier functionality was demonstrated by transendothelial electrical resistance values > 1000 Ω*cm² and hindered permeability of the small molecule sodium fluorescein for both AD patients and matching controls. Co-culture with astrocytes strengthens barrier function for matched controls but not for LOAD-specific cells.

Conclusion Overall, we established a patient-specific BBB model suitable to research genetic risk variants (e.g. APOE4) and investigate underlying AD disease mechanisms. Co-culture with astrocytes seems to induce the observed pathogenic phenotype of the LOAD-specific BBB model compared to the control BBB model.