Prolonged culturing of iPSC-derived brain endothelial-like cells is associated with quiescence, downregulation of glycolysis, and resistance to disruption by an Alzheimer\u2019s brain milieu

Lindsey M Williams,Jessica E Young,William A Banks,Aric F Logsdon,Takashi Fujimoto,Kira M Evitts,Michelle A Erickson,Riley R Weaver

doi:10.1186/s12987-022-00307-1

Abstract

BackgroundHuman induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (iBECs) are a robust, scalable, and translatable model of the human blood–brain barrier (BBB). Prior works have shown that high transendothelial electrical resistance (TEER) persists in iBECs for at least 2 weeks, emphasizing the utility of the model for longer term studies. However, most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. In this study, we characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points.MethodshiPSCs were differentiated into iBECs using fully defined, serum-free medium. The proportion of proliferating cells was determined by BrdU assays. We evaluated TEER, expression of glycolysis enzymes and tight and adherens junction proteins (TJP and AJP), and glucose transporter-1 (GLUT1) function by immunoblotting, immunofluorescence, and quantifying radiolabeled tracer permeabilities. We also compared barrier disruption in response to TNF-α and conditioned medium (CM) from hiPSC-derived neurons harboring the Alzheimer’s disease (AD)-causing Swedish mutation (APPSwe/+).ResultsA significant decline in iBEC proliferation over time in culture was accompanied by adoption of a more quiescent endothelial metabolic state, indicated by downregulation of glycolysis-related proteins and upregulation GLUT1. Interestingly, upregulation of GLUT1 was associated with reduced glucose transport rates in more quiescent iBECs. We also found significant decreases in claudin-5 (CLDN5) and vascular endothelial-cadherin (VE-Cad) and a trend toward a decrease in platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas zona occludens-1 (ZO-1) increased and occludin (OCLN) remained unchanged. Despite differences in TJP and AJP expression, there was no difference in mean TEER on day 2 vs. day 9. TNF-α induced disruption irrespective of iBEC proliferative state. Conversely, APPSwe/+ CM disrupted only proliferating iBEC monolayers.ConclusioniBECs can be used to study responses to disease-relevant stimuli in proliferating vs. more quiescent endothelial cell states, which may provide insight into BBB vulnerabilities in contexts of development, brain injury, and neurodegenerative disease.

Highlights

The vascular blood–brain barrier (BBB) is a vital interface that limits the unregulated transfer of circulating substances into the brain and facilitates the regulated transport of substances such as ions, nutrients, and signaling molecules that are essential for CNS homeostasis [1]
We focused on: (1) metabolic markers of endothelial quiescence, because an altered relation between glucose metabolism and transendothelial glucose transport may be important to consider when using iBECs to study dysfunctional glucose transporter-1 (GLUT1)-mediated glucose transport at the BBB in Alzheimer’s disease (AD) [16,17,18], (2) markers of BBB integrity, such as transendothelial electrical resistance (TEER) and expression of Tight junction protein (TJP) and Tight and adherens junction proteins (AJP), because Brain endothelial cell (BEC) proliferation has been associated with BBB leakiness [19, 20], and (3) responses to inflammatory and AD-associated insults, which are insults that can happen concurrently with increased BEC proliferation [21, 22]
In vitro studies using primary human endothelial cells (ECs) have shown that ECs adopt a quiescent state upon contact inhibition, which is associated with altered regulation of glucose metabolism and transport [29]

Summary

Introduction

The vascular blood–brain barrier (BBB) is a vital interface that limits the unregulated transfer of circulating substances into the brain and facilitates the regulated transport of substances such as ions, nutrients, and signaling molecules that are essential for CNS homeostasis [1]. The interface functions of the BBB include transporter-mediated passage of nutritive and regulatory substances, secretory functions, and the ability of BECs to modulate their activities in response to signals from the blood or brain compartments. Each of these functions can facilitate communication between the brain and the periphery, regulating CNS activities [5]. Most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. We characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points

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