Pericytes reside outside of capillary blood vessels. In the brain, pericytes, brain microvascular endothelial cells, and astrocytes form the neurovascular unit (NVU). A normally functioning NVU regulates cerebral blood flow and the permeability of the blood-brain-barrier (BBB). Breakdown of the NVU and dysfunction of the BBB are increasingly recognised early biomarkers of dementia, including Alzheimer’s disease. An isoform of Apolipoprotein E (APOE), namely APOE4, which is the strongest genetic risk factor for Alzheimer’s disease1, has a particular importance in BBB dysfunction compared to the E3 variant. Pericyte dysfunction has received increasing recognition in Alzheimer’s disease where pericytes contract capillaries and thus reduce cerebral blood flow, in a downstream response to amyloid-β2. However, pericytes in health and disease remain poorly understood, partially due to a lack of adequate models that can recapitulate the complexity of the multi-cellular NVU. The aim of this study is to investigate the effect of APOE4, compared to APOE3, on the contraction of human pericytes in vitro.
iPSCs from a donor diagnosed with Alzheimer’s disease carrying the APOE e4/e4 allele (UKBi011-A, EBiSC) as well as the isogenic control with the APOE e3/e3 isotype (UKBi011-A-3, EBiSC) were differentiated into pericytes3. Success of differentiation was confirmed by immuno-histochemistry of pericyte markers. The labelled calcium indicator Fluo4 was used to measure increase in fluorescence, thus increase in calcium release, within the iPSC-derived pericytes.
APOE3- and APOE4-pericytes stained positively for NG2, PDGFR-β, calponin, and SM22. Genomic sequencing confirmed the isoforms of APOE3 and APOE4. Fluo4 staining was used to observe calcium release, which was triggered by two known vasoconstrictors Endothelin-1 and U46619, the latter one being a thromboxane A2 receptor agonist. Calcium firing was observed for a period of at least 10 minutes. Change in cell area and calcium increase were quantified using an automated image analysis pipeline in CellProfiler.
These results suggest that functional pericyte-like cells were obtained and that their calcium release could be triggered. We will build on these findings to develop a robust model to study the effect of APOE4 on pericyte function and dysfunction in health and Alzheimer’s disease.
1. Blanchard J.W., et al., Nat Med. 26(6):952-963 (2020).
2. Nortley R., et al., Science 365(6450):eaav9518 (2019).
3. Faal T., et al., Stem Cell Reports. 2019;12(3):451-460 (2019)."