Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.

Physiological‐relevant in vitro tissue models with their promise of better predictability have the potential to improve drug screening outcomes in preclinical studies. Despite the advances of spheroid models in pharmaceutical screening applications, variations in spheroid size and consequential altered cell responses often lead to nonreproducible and unpredictable results. Here, a microfluidic multisize spheroid array is established and characterized using liver, lung, colon, and skin cells as well as a triple‐culture model of the blood‐brain barrier (BBB) to assess the effects of spheroid size on (a) anticancer drug toxicity and (b) compound penetration across an advanced BBB model. The reproducible on‐chip generation of 360 spheroids of five dimensions on a well‐plate format using an integrated microlens technology is demonstrated. While spheroid size‐related IC50 values vary up to 160% using the anticancer drugs cisplatin (CIS) or doxorubicin (DOX), reduced CIS:DOX drug dose combinations eliminate all lung microtumors independent of their sizes. A further application includes optimizing cell seeding ratios and size‐dependent compound uptake studies in a perfused BBB model. Generally, smaller BBB‐spheroids reveal an 80% higher compound penetration than larger spheroids while verifying the BBB opening effect of mannitol and a spheroid size‐related modulation on paracellular transport properties.

The postnatal subventricular zone (SVZ) harbors neural stem cells (NSCs) that exhibit robust neurogenesis. However, the epigenetic mechanisms that maintain NSCs and regulate neurogenesis remain unclear. We report that label-retaining SVZ NSCs express Eed, the core component of Polycomb repressive complex 2. In vivo and in vitro conditional knockout and knockdown show Eed is necessary for maintaining NSC proliferation, neurogenesis and neurosphere formation. We discovered that Eed functions to maintain p21 protein levels in NSCs by repressing Gata6 transcription. Both Gata6 overexpression and p21 knockdown reduced neurogenesis, while Gata6 knockdown or p21 overexpression partially rescued neurogenesis after Eed loss. Furthermore, genetic deletion of Eed impaired injury induced SVZ proliferation and emigration. These data reveal a novel epigenetic regulated pathway and suggest an essential role for Eed in SVZ homeostasis and injury.

Background: The blood-brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases' progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. Methods: Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. Results: In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. Conclusions: The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.

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Significant advancements in the field of preclinical in vitro blood-brain barrier (BBB) models have been achieved in recent years, by developing monolayer-based culture systems towards complex multi-cellular assays. The coupling of those models with other relevant organoid systems to integrate the investigation of blood-brain barrier permeation in the larger picture of drug distribution and metabolization is still missing. Here, we report for the first time the combination of a human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier model with a cortical brain and a liver spheroid model from the same donor in a closed microfluidic system (MPS). The two model compounds atenolol and propranolol were used to measure permeation at the blood–brain barrier and to assess metabolization. Both substances showed an in vivo-like permeation behavior and were metabolized in vitro. Therefore, the novel multi-organ system enabled not only the measurement of parent compound concentrations but also of metabolite distribution at the blood-brain barrier.

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