Differentiation of disease-specific induced pluripotent stem cells into a blood-brain barrier system for the analysis of Alzheimer's disease

Introduction:

The risk for Alzheimer's disease (AD), the most common type of dementia, increases with age resulting in a major challenge for the ageing society. Dysfunction of the blood-brain barrier (BBB) contributes to AD progression and the characteristic accumulation of amyloid-β (Aβ) peptides. Recently, genome-wide association studies (GWAS) with participation of our research team revealed a significant association between the ATP-binding cassette transporter A7 (ABCA7) and the late-onset form of AD (LOAD). It was shown that ABCA7 is involved in Aβ homeostasis, lipid metabolism, and phagocytosis, but the detailed role in LOAD still remains unclear. The generation of patient-derived, disease-specific induced pluripotent stem (iPS) cells followed by the differentiation into cells of the BBB is a valuable approach to study disease mechanisms related to ABCA7 during the pathogenesis of AD.

Methods:

First, we determined single nucleotide polymorphisms (SNPs), previous identified by GWAS within the ABCA7 gene, in samples from AD patients. Of particular interest were risk variants leading to an amino acid exchange in functional protein domains. Secondly, episomal vectors were used for the generation of iPS cells from B-lymphoblastoid cell lines of patients carrying AD-associated SNPs and matched controls without the risk variants. After a successful verification of pluripotency the established iPS cells were differentiated into cells of the BBB system, notably endothelial cells and astrocytes. The expression of cell specific markers was analyzed by flow cytometry, transcript, and immunofluorescence analysis.

Results:

Following the generation of AD-specific iPS cells, pluripotency was proved by alkaline phosphatase staining and the occurrence of pluripotency-related transcription factors like OCT4 and NANOG. The obtained iPS cells displayed the ability of neural induction and the spontaneous differentiation into derivatives of the three germ layers. The efficient differentiation of iPS cells into endothelial cells and astrocytes was characterized by transcript analysis of cell-specific markers, including KDR and GFAP. The endothelial cells also expressed BBB-relevant markers, like OCLN and TJP1. Barrier functionality analysis by measuring the transendothelial electrical resistance demonstrated values > 1000 Ω*cm2.

Conclusion:

In summary, the generated iPS cells and subsequently differentiated endothelial cells and astrocytes were suitable to establish a BBB model with the great advantage that they carry a patient- and disease-specific background. Therefore, they are a promising opportunity to study AD-associated genetic risk variants and the resulting pathogenic phenotype in vitro, which provides a powerful tool to investigate underlying AD disease mechanisms.