DOI: 10.5281/zenodo.11487010
title: APP Swedish mutation affects human iPSC-derived pericyte-like cells.

description: Abstract

The APP Swedish (APPswe) mutation causes a familial form of Alzheimer’s disease (AD). While vascular dysfunction is commonly observed in AD and potentially linked to disease progression, the specific contributions of vascular cell types, particularly pericytes, are unclear. Pericytes are mural cells that wrap around endothelial cells in capillaries and play a critical role in modulating vascular functions. We have observed altered functionality in pericyte-like cells with the APPswe mutation, including compromised barrier maintenance, reduced angiogenesis support, increased secretion of pro-inflammatory cytokines upon stimulation, and a hypercontractility phenotype.To further investigate these changes, we aim to analyze gene expression at the transcriptomic level and explore pathway alterations. We harvested cultures from iPSC-derived pericyte-like cells from seven healthy control lines and three APPswe lines and conducted bulk RNA sequencing. We then performed analyses including identifying differentially expressed genes (DEGs) and pathway analysis. Our results revealed significant activation of the cytoskeleton/actin pathway and hypercontractile proteins in APPswe pericyte-like cells, which likely contribute to the observed hypercontractility phenotype. Additionally, we identified DEGs and pathways associated with angiogenesis, metabolism, and inflammation, potentially explaining the changes observed in APPswe cells.Overall, the RNA sequencing data from our study highlight potential pathways through which pericytes may contribute to vascular dysfunction and disease progression in AD.
description: Technical info

RNA sequencing of human induced pluripotent stem cell (hiPSC)-derived pericyte-like cells.

raw data files (.fastq.gz), processed data files (.txt).

Methods

Line information



Genotype: Amyloid precursor protein (APP) KM670/671NL (Swedish) mutation

Reference: Oksanen et al., 2018, Konttinen et al., 2019 and Frederiksen et al., 2019.

Cell culturing: hiPSCs were cultured in matrigel (Corning) coated dishes in E8 medium. Pericyte-like cells were differentiated according to previously published protocol (Blanchard et al., 2020).


RNA sequencing: iPSC-derived pericyte-like cell samples for RNA sequencing (RNA-seq) were collected on day 21 post-differentiation. Cell density was measured from identical wells on 6-well plates, ranging from 0.8-1.6x10^6 cells per well, to ensure comparable confluency. Total RNA was extracted using the RNeasy Mini Kit as per the manufacturer’s instructions, incorporating DNase (Thermo Fisher Scientific) during the isolation to remove DNA. RNase inhibitor RiboLock was added post-elution to preserve RNA quality. RNA quantity and quality were assessed using an Agilent 2100 Bioanalyzer™ (Agilent).

RNA-seq libraries were prepared using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit to deplete ribosomal RNA, and sequenced on a NextSeq500 (Illumina). Library preparation and sequencing services were provided by the Biomedicum Functional Genomics Unit (FuGu) at the Helsinki Institute of Life Science and Biocenter Finland, University of Helsinki.

For gene expression analysis, raw sequence reads were aligned to the human genome GRCh.38 and annotated to gene exons using STAR aligner v2.7.8a56 and HTSeq v0.13.557 with GTF v103, to identify differentially expressed genes (DEGs).