Human iPSC-derived Brain Microvascular Endothelial Cells

Effect of SARS-CoV-2 Infection on BMEC Integrity

SARS-CoV-2, the virus causing COVID-19, is associated with various neurological symptoms, suggesting its impact on the central nervous system (CNS) through the blood–brain barrier (BBB). The BBB is formed by tight junctions between brain microvascular endothelial cells (BMECs). There have been reports of BMEC infection with SARS-CoV-2, but its mechanism is yet to be determined.

Yamada et al. used the original SARS-CoV-2 strain to infect human iPSC-derived brain microvascular endothelial cells (iPSC-BMELCs) and assessed infection through intracellular RNA detection and viral particle localization. To evaluate the impact of SARS-CoV-2 infection on the BBB, iPSC-BMELCs were seeded into a 24-well Transwell plate to allow the cells to form a monolayer. SARS-CoV-2 infection decreased the TEER by 31% (Fig. 1A) and gradually reduced TEER values over 12 h (Fig. 1B). Immunocytochemical staining revealed that some of the infected cells were caspase-3-positive, indicating apoptosis (Fig. 1C), but they could not observe any distinct changes in TJ structure. RNA-seq analysis of SARS-CoV-2-infected iPSC-BMELCs revealed upregulation of genes related to interferon (IFN) and type I IFN signaling components. RT-qPCR showed that SARS-CoV-2 infection decreased the expression of several TJ markers, such as CLDN3 and CLDN11 (Fig. 2B), and that their protein levels were also slightly reduced (Fig. 2C). These data suggest that SARS-CoV-2 impairs BMEC barrier integrity by downregulating the expression of TJ markers.

Fig. 1. BMEC monolayer barrier examined after SARS-CoV-2 infection (Yamada S, Hashita T, et al., 2024).

Fig. 2. Analysis of human iPSC-BMELC barrier disruption based on RNA-seq (Yamada S, Hashita T, et al., 2024).

BMEC Maturation by Conditioned Media Results in a Barrier Tightness Similar to Co-Cultures

Current in vitro models often fail to mimic the physiological barrier properties of the BBB, especially in scalable formats. Fengler et al. aim to address this challenge by establishing a standardized and scalable multi-well plate format using human iPSC-derived brain endothelial microvessels to screen anti-inflammatory drugs for BBB penetration.

In order to generate functional BBB endothelial cells from human stem cells, they first differentiated IMR90-4 iPSCs into brain microvascular endothelial cells (BMECs) in a four-step protocol according to pervious study (Fig. 3A). After 3 days of iPSC expansion, a mixed cell culture consisting of endothelial and neuronal cell progenitors was generated by exposure to unconditioned medium for 6 days (Fig. 3B). The endothelial cells were then selectively expanded with endothelial cell medium supplemented with all-trans retinoic acid (RA) and bFGF (EC+/+). On day 8 the cultures were subcultured on collagen/fibronectin. In order to characterize the BMEC phenotype, the cells were stained for BBB marker proteins and demonstrated positive staining for typical endothelial markers (PECAM-1, VE-Cadherin), tight-junction proteins (Occludin, ZO-1, Claudin-5), nutrient transporter (Glut-1) and efflux transporters (P-gp, BCRP) (Fig. 4A). TEER measurements between day 9 and 12 revealed peak values of ~2500 Ωxcm² at day 10 (Fig. 4B). Treatment with Staurosporine (STS) abolished tight-junctions, as reflected by the reduced TEER (Fig. 3C). Overall, these results demonstrate that iPSC-derived BMECs express appropriate BBB markers and physiological barrier properties as of day 10.

Fig. 3. (A) BMEC differentiation protocol. (B) Representative brightfield images of differentiating cells from D-3 to D5. (C) Normalized TEER measured on Transwell filters and corresponding immunocytochemistry of STS treated BMEC cultures (Fengler S, Kurkowsky B, et al., 2022).

Fig. 4. Characterization of BMEC cultures and maturation with primary cell conditioned media (Fengler S, Kurkowsky B, et al., 2022).