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Establishment of a Novel Patient hiPSC-derived in vitro Blood-Brain Barrier Model of Collagen IV Small Vessel Disease


Type

Thesis

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Authors

Goodwin-Trotman, Mary 

Abstract

Cerebral small vessel disease (SVD) is a prevalent cause of stroke (25-30%) and dementia (45%). Despite its frequency, little is known about the cause and disease progression of SVD. Matrisome alteration leading to blood-brain barrier (BBB) dysfunction is thought to play a role. Sporadic cases are exacerbated by age and vascular risk factors, predominantly affecting the elderly. However, mutations in Collagen IV α1/α2 (COL4A1/2), a highly abundant matrisome protein, cause monogenic SVD, which shares many clinical features with the sporadic form. In order to better understand the pathomolecular mechanisms leading to SVD, human induced pluripotent stem cells (hiPSC) generated from patients with mutations in COL4A1/2 genes (COL4A1G755R and COL4A2G702D), isogenic controls and multiple wild-type (WT) control lines were used to establish a novel in vitro BBB model of Collagen IV SVD. Encompassing hiPSC-derived brain microvascular endothelial cells (BMEC), mural cells (MC) and astrocytes, the model was used to probe the phenotype of COL4A1/2 SVD. Differentiation of hiPSC into BMEC was extensively optimised to overcome high variability between different hiPSC lines. Once established, hiPSC-BMEC were tested with functional assays including transendothelial electrical resistance (TEER), permeability assays (4kDa FITC-Dextran and Sodium Fluorescein), LDL-uptake and tube formation. hiPSC-BMEC were then combined with hiPSC-MC and hiPSC-astrocytes into a Transwell® co-culture model.
WT hiPSC-derived BMEC exhibit high TEER of ~3000-4000Ωxcm2, which is increased and maintained over two weeks in co-culture with astrocytes. Limited, short-term increase in TEER is seen in co-culture with MC. Aspects of the COL4A1/2 SVD disease phenotype were reproduced in vitro. COL4A2G702D BMEC exhibit lower tight junction protein expression; while COL4A1G755R BMEC demonstrate discontinuous tight junctions. COL4A2G702D BMEC also show defective P-glycoprotein (P-gp)-mediated Rhodamine123 efflux compared to the isogenic control, which is suggestive of a transport deficiency across the barrier. COL4A1G755R and COL4A2G702D BMEC and MC have increased levels of MMPs. Notably, these findings replicate what is seen in patients, strengthening the hypothesis that matrisome alterations are important in SVD.

Description

Date

2020-09-01

Advisors

Granata, Alessandra

Keywords

Blood-brain barrier, Small Vessel Disease, Collagen IV, hiPSC, Matrisome

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
Rosetrees Trust, British Heart Foundation, Alzheimer’s Association and Stroke Association.