Denner, P. | Search bioRxiv

bioRxiv2021

Human iPSC-derived brain endothelial microvessels in a multi-well format enable permeability screens of anti-inflammatory drugs

Optimizing drug candidates for blood-brain barrier (BBB) penetration in humans remains one of the key challenges and many devastating brain diseases including neurodegenerative diseases still do not have adequate treatments. So far, it has been difficult to establish state-of-the-art human stem cell derived in vitro models that mimic physiological barrier properties including a 3D microvasculature in a format that is scalable enough to screen drugs for BBB penetration in early drug development phases. To address this challenge, we established human induced pluripotent stem cell (iPSC)-derived brain endothelial microvessels in a standardized and scalable multi-well plate format. iPSC-derived brain microvascular endothelial cells (BMECs) were supplemented with primary cell conditioned media and grew to intact microvessels in 10 days of culturing. Produced microvessels show a typical BBB phenotype including endothelial protein expression, tight-junctions and polarized localization of efflux transporter. Microvessels exhibited physiological relevant trans-endothelial electrical resistance (TEER), were leak-tight for 10 kDa dextran-Alexa 647 and strongly limited the permeability of sodium fluorescein (NaF). Permeability tests with reference compounds confirmed the suitability of our model as platform to identify potential BBB penetrating anti-inflammatory drugs. In summary, the here presented brain microvessel platform recapitulates physiological properties and allows rapid screening of BBB permeable anti-inflammatory compounds that has been suggested as promising substances to cure so far untreatable neurodegenerative diseases.

cell biology↗

bioRxiv2020

Highly efficient intercellular spreading of protein misfolding mediated by viral ligand - receptor interactions

Pathological protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble proteins of the same kind. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contributes to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell-to-cell contact as well as via EV depends on receptor-ligand interactions and subsequent release of cargo into the cytosol. We hypothesized that enabling these interactions through viral ligands enhances the aggregate-inducing capacity of EV-associated proteopathic seeds. Using different cellular models propagating model prion-like protein aggregates, mouse-adapted prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase protein aggregate induction by direct cell-to-cell contact or via viral glycoprotein-decorated EV. Thus, receptor-ligand interactions are major determinants of intercellular aggregate dissemination. Further, our data raise the intriguing possibility that acute or latent viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer. HIGHLIGHTSO_LIDifferent types of proteopathic seeds are secreted in association with extracellular vesicles C_LIO_LIReceptor-ligand interactions are important drivers of direct cell-to-cell and extracellular vesicle-mediated spreading of protein misfolding C_LIO_LIViral glycoproteins mediating attachment and membrane fusion strongly enhance aggregate inducing capacity in recipient cells C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/173070v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1735b86org.highwire.dtl.DTLVardef@7ddaf5org.highwire.dtl.DTLVardef@b222aborg.highwire.dtl.DTLVardef@1022dd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗