The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. Neurological symptoms, which range in severity, accompany as many as one-third of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized stem-cell-derived cortical organoids as well as primary human cortical tissue, both from developmental and adult stages. We find significant and predominant infection in cortical astrocytes in both primary tissue and organoid cultures, with minimal infection of other cortical populations. Infected and bystander astrocytes have a corresponding increase in inflammatory gene expression, reactivity characteristics, increased cytokine and growth factor signaling, and cellular stress. Although human cortical cells, particularly astrocytes, have no observable ACE2 expression, we find high levels of coronavirus coreceptors in infected astrocytes, including CD147 and DPP4. Decreasing coreceptor abundance and activity reduces overall infection rate, and increasing expression is sufficient to promote infection. Thus, we find tropism of SARS-CoV-2 for human astrocytes resulting in inflammatory gliosis-type injury that is dependent on coronavirus coreceptors.

Dagmar iber 3,4 , christian Beisel 5 , erik van nimwegen 2 & Verdon taylor 1* Neural stem cells (NSCs) generate neurons of the cerebral cortex with distinct morphologies and functions. How specific neuron production, differentiation and migration are orchestrated is unclear. Hippo signaling regulates gene expression through Tead transcription factors (TFs). We show that Hippo transcriptional coactivators Yap1/Taz and the Teads have distinct functions during cortical development. Yap1/Taz promote NSC maintenance and Satb2 + neuron production at the expense of Tbr1 + neuron generation. However, Teads have moderate effects on NSC maintenance and do not affect Satb2 + neuron differentiation. Conversely, whereas Tead2 blocks Tbr1 + neuron formation, Tead1 and Tead3 promote this early fate. In addition, we found that Hippo effectors regulate neuronal migration to the cortical plate (CP) in a reciprocal fashion, that ApoE, Dab2 and Cyr61 are Tead targets, and these contribute to neuronal fate determination and migration. Our results indicate that multifaceted Hippo signaling is pivotal in different aspects of cortical development. NSCs of the developing cerebral cortex form the ventricular zone (VZ) lining the lumen of the neural tube 1-5. NSCs in the dorsal anterior forebrain are the major source of the projection neurons of the cerebral cortex 4,5. The mechanisms controlling the patterning and cell fate specification of these stem cells during early brain development are not clearly understood. Although various signaling pathways including Notch, Wnt, Shh, FGFs, TGF-β, Retinoic acid, Reelin and Hippo are known to regulate NSC proliferation and to control fate decisions, neurogenesis, and gliogenesis; the crosstalk between the different signaling pathways and the integration of these signals on target genes governing complex cell fate choices is unclear 1-3. Hippo signaling is evolutionarily conserved and a regulator of organ size control and tissue homeostasis 6-9. The pathway is regulated by numerous stimuli including G-protein coupled receptor signaling, mechanical stress, cellular energy status, cell-cell contact and cell-extra-cellular matrix interactions 6-8. Hippo signaling employs a cascade of phosphorylation steps mediated by the kinases Mst1/2 and Lats1/2 8-10. Lats1/2 phosphorylate the transcriptional coregulators Yap1 and Taz to promote cytoplasmic retention and subsequent degradation 6-8. When Hippo signaling is inactive, Yap1/Taz translocate to the nucleus and form multiple complexes with different DNA binding partners including TEADs, SMADs, and Runx TFs (Fig. S1a) 8-10. The Teads are major regulators of Hippo target genes in many systems including cancer 8,11,12. Fat4 and Dchs are receptor and ligand, respectively, of the Hippo pathway in embryonic NSCs. Knockdown of Fat4 results in increased proliferation in the developing nervous system and reduction of neuronal differentiation 13,14. Mutations in FAT4 and DCHS cause Van Maldergem syndrome in humans, an autosomal-recessive disorder characterized by in...

Dynamical control of cellular microenvironments is highly desirable to study complex processes such as stem cell differentiation and immune signaling. We present an ultra-multiplexed microfluidic system for high-throughput single-cell analysis in precisely defined dynamic signaling environments. Our system delivers combinatorial and time-varying signals to 1500 independently programmable culture chambers in week-long live-cell experiments by performing nearly 106 pipetting steps, where single cells, two-dimensional (2D) populations, or 3D neurospheres are chemically stimulated and tracked. Using our system and statistical analysis, we investigated the signaling landscape of neural stem cell differentiation and discovered “cellular logic rules” that revealed the critical role of signal timing and sequence in cell fate decisions. We find synergistic and antagonistic signal interactions and show that differentiation pathways are highly redundant. Our system allows dissection of hidden aspects of cellular dynamics and enables accelerated biological discovery.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) readily infects a variety of cell types impacting the function of vital organ systems, with particularly severe impact on respiratory function. It proves fatal for one percent of those infected. Neurological symptoms, which range in severity, accompany a significant proportion of COVID-19 cases, indicating a potential vulnerability of neural cell types. To assess whether human cortical cells can be directly infected by SARS-CoV-2, we utilized primary human cortical tissue and stem cell-derived cortical organoids. We find significant and predominant infection in cortical astrocytes in both primary and organoid cultures, with minimal infection of other cortical populations. Infected astrocytes had a corresponding increase in reactivity characteristics, growth factor signaling, and cellular stress. Although human cortical cells, including astrocytes, have minimal ACE2 expression, we find high levels of alternative coronavirus receptors in infected astrocytes, including DPP4 and CD147. Inhibition of DPP4 reduced infection and decreased expression of the cell stress marker, ARCN1. We find tropism of SARS-CoV-2 for human astrocytes mediated by DPP4, resulting in reactive gliosis-type injury.