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.

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High-Throughput Screening Platform for the Discovery of New Immunomodulator Molecules from Natural Product Extract Libraries

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116635517 ◽

2016 ◽

Vol 21 (6) ◽

pp. 567-578 ◽

Cited By ~ 6

Author(s):

José Pérez del Palacio ◽

Caridad Díaz ◽

Mercedes de la Cruz ◽

Frederick Annang ◽

Jesús Martín ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Natural Product ◽

High Throughput ◽

High Throughput Screening ◽

Viral Infections ◽

Inflammatory Diseases ◽

Immunomodulatory Activity ◽

Inflammatory Drugs ◽

Anti Inflammatory

It is widely accepted that central nervous system inflammation and systemic inflammation play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury, and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that sustained use of anti-inflammatory drugs may prevent or slow down the progression of neurodegenerative diseases. However, the anti-inflammatory drugs and biologics used clinically have the disadvantage of causing side effects and a high cost of treatment. Alternatively, natural products offer great potential for the identification and development of bioactive lead compounds into drugs for treating inflammatory diseases with an improved safety profile. In this work, we present a validated high-throughput screening approach in 96-well plate format for the discovery of new molecules with anti-inflammatory/immunomodulatory activity. The in vitro models are based on the quantitation of nitrite levels in RAW264.7 murine macrophages and interleukin-8 in Caco-2 cells. We have used this platform in a pilot project to screen a subset of 5976 noncytotoxic crude microbial extracts from the MEDINA microbial natural product collection. To our knowledge, this is the first report on an high-throughput screening of microbial natural product extracts for the discovery of immunomodulators.

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Modulating the Barrier Function of Human Alveolar Epithelial (hAELVi) Cell Monolayers as a Model of Inflammation

Alternatives to Laboratory Animals ◽

10.1177/0261192920983015 ◽

2020 ◽

Vol 48 (5-6) ◽

pp. 252-267 ◽

Cited By ~ 1

Author(s):

Julia Katharina Metz ◽

Birgit Wiegand ◽

Sabrina Schnur ◽

Katharina Knoth ◽

Nicole Schneider-Daum ◽

...

Keyword(s):

Barrier Function ◽

Barrier Properties ◽

In Vitro Test ◽

Alveolar Epithelial ◽

Tnf Α ◽

Inflammatory Drugs ◽

Ifn Γ ◽

Anti Inflammatory ◽

The Impact

The incidence of inflammatory lung diseases such as acute respiratory distress syndrome (ARDS) remains an important problem, particularly in the present time with the Covid-19 pandemic. However, an adequate in vitro test system to monitor the barrier function of the alveolar epithelium during inflammation and for assessing anti-inflammatory drugs is urgently needed. Therefore, we treated human Alveolar Epithelial Lentivirus-immortalised cells (hAELVi cells) with the pro-inflammatory cytokines TNF-α (25 ng/ml) and IFN-γ (30 ng/ml), in the presence or absence of hydrocortisone (HC). While TNF-α and IFN-γ are known to reduce epithelial barrier properties, HC could be expected to protect the barrier function and result in an anti-inflammatory effect. We investigated the impact of anti-inflammatory/inflammatory treatment on transepithelial electrical resistance (TEER) and the apparent permeability coefficient (P app) of the low permeability marker sodium fluorescein (NaFlu). After incubating hAELVi cells for 48 hours with a combination of TNF-α and IFN-γ, there was a significant decrease in TEER and a significant increase in the P app. The presence of HC maintained the TEER values and barrier properties, so that no significant P app change was observed. By using hAELVi cells to study anti-inflammatory drugs in vitro, the need for animal experiments could be reduced and pulmonary drug development accelerated.

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