key: cord-337105-jlmh79qv
authors: Jacob, Fadi; Pather, Sarshan R.; Huang, Wei-Kai; Zhang, Feng; Hao Wong, Samuel Zheng; Zhou, Haowen; Cubitt, Beatrice; Fan, Wenqiang; Chen, Catherine Z.; Xu, Miao; Pradhan, Manisha; Zhang, Daniel Y.; Zheng, Wei; Bang, Anne G.; Song, Hongjun; Carlos de la Torre, Juan; Ming, Guo-li
title: Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium
date: 2020-09-21
journal: Cell Stem Cell
DOI: 10.1016/j.stem.2020.09.016
sha: 
doc_id: 337105
cord_uid: jlmh79qv

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for the COVID-19 pandemic, has infected over 29 million people and has contributed to over 936,000 deaths world-wide this year so far (WHO, 2020) . While the disease primarily affects the respiratory system, damages and dysfunction have also been found in other organs, including the kidney, heart, liver, and brain (Yang et al., 2020b) . Neurological complications, such as cerebrovascular injury, altered mental status, encephalitis, encephalopathy, dizziness, headache, hypogeusia, and hyposmia, as well as neuropsychiatric ailments, including new onset psychosis, neurocognitive syndrome, and affective disorders, have been reported in a significant number of patients (Mao et al., 2020; Varatharaj et al., 2020) . Viral RNA has been detected in the brain and cerebrospinal fluid (CSF) of some patients with COVID-19 and concomitant neurological symptoms (Helms et al., 2020; Moriguchi et al., 2020; Puelles et al., 2020; Solomon et al., 2020) . Despite numerous reports of neurological findings in patients with COVID-19, it remains unclear whether these symptoms are a consequence of direct neural infection, para-infectious or post-infectious immune-mediated disease, or sequalae of systemic disease (Ellul et al., 2020; Iadecola et al., 2020) . Variability in patient presentation and variable timing of testing for viral RNA in the CSF or brain complicate the interpretation of results in human patients. Limited availability of autopsy brain tissue from patients with COVID-19 and neurological symptoms and the inability to study ongoing disease pathogenesis in the human brain further underscore the need for accessible and tractable experimental models to investigate SARS-CoV-2 neurotropism, its functional impact, and to test therapeutics.

Classic animal models, such as rodents, are limited in their ability to recapitulate human COVID-19 symptoms and usually require viral or transgenic mediated overexpression of human SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) to exhibit symptoms Sun et al., 2020) . Although cell lines, including many human cancer cell lines, have been used to study SARS-CoV-2 infection and test drug efficacy (Hoffmann et al., 2020b; Ou et al., 2020; Shang et al., 2020; Wang et al., 2020) , they do not accurately recapitulate normal human cell J o u r n a l P r e -p r o o f 4 behavior and often harbor tumor-associated mutations, such as TP53, which may affect SARS-CoV-2 replication or the cellular response to SARS-CoV-2 infection (Ma-Lauer et al., 2016) .

Furthermore, human tissue and organ systems contain multiple cell types with variable levels of ACE2 expression and viral susceptibility, which are not adequately represented in these human cell lines. These limitations support the development of human cellular models for SARS-CoV-2 infection that better recapitulates the cellular heterogeneity and function of individual tissues.

Human pluripotent stem cell (hiPSC)-based models provide an opportunity to investigate the susceptibility of various brain cell types to viral infection and their consequences. hiPSCs have been used to generate a variety of monolayer and three dimensional (3D) organoid cultures to study human diseases and potential treatments. For example, hiPSC-derived neural progenitors in monolayer and brain organoids were instrumental in studying the impact of Zika virus infection on human brain development and solidifying the link between Zika virus infection of neural progenitor cells and microcephaly seen in newborns (Ming et al., 2016; Qian et al., 2016; Tang et al., 2016) .

Additionally, these cultures were useful in screening for drugs to treat ZIKV infection (Xu et al., 2016) . Recently, hiPSC-derived organoids have been used to model SARS-CoV-2 infection in many organs, including the intestine, lung, kidney, liver, pancreas, vasculature and brain (Lamers et al., 2020; Monteil et al., 2020; Ramani et al., 2020; Yang et al., 2020a; Zhou et al., 2020) . These studies have shown that SARS-CoV-2 can infect and replicate within cells of multiple organs, leading to transcriptional changes indicative of inflammatory responses and altered cellular functions. Here, we used hiPSC-derived neurons, astrocytes, and microglia in monolayer cultures and region-specific brain organoids of the cerebral cortex, hippocampus, hypothalamus, and midbrain to investigate the susceptibility of brain cells to SARS-CoV-2 infection. We observed sparse infection of neurons and astrocytes, with the exception of regions of organoids with choroid plexus epithelial cells that exhibited high levels of infectivity. Using an optimized protocol to generate choroid plexus organoids (CPOs) from hiPSCs, we showed evidence of productive infection by SARS-CoV-2 and functional consequences at cellular and molecular levels.

To investigate the susceptibility of human brain cells to SARS-CoV-2 infection, we tested various hiPSC-derived neural cells in monolayer cultures and region-specific brain organoids generated using several established (Qian et al., 2018; Qian et al., 2016) and modified protocols (Sakaguchi et al., 2015) ( Figure 1A ). Monolayer hiPSC-derived cortical neurons, astrocytes, and microglia were exposed to either SARS-CoV-2 virus isolate or vehicle control for 12 hours and analyzed at 24, 48, and 120 hours post-infection (hpi) for immunolabelling using convalescent serum from a patient with COVID-19 or SARS-CoV-2 nucleoprotein antibodies (Figures S1A-C). We confirmed the identity of neurons, microglia, and astrocytes by immunostaining for markers MAP2, PU.1, and GFAP, respectively (Figures S1A-C). hiPSC-derived cortical neurons were co-cultured on hiPSCderived astrocytes to improve survival, and analysis at 120 hpi showed rare infection of MAP2 + cells ( Figure S1A ). At 48 and 120 hpi, hiPSC-derived microglia showed no infection of PU.1 + cells ( Figure S1B ), whereas hiPSC-derived astrocytes showed sparse infection of GFAP + cells ( Figure   S1C ). As a validation, we tested infection of primary human astrocytes and observed sparse infection at 24, 48, and 120 hpi ( Figure S1D ). Quantification of hiPSC-derived and primary human astrocytes showed infection of 0.02% and 0.18% of all cells at 120 hpi, respectively ( Figure S1E ).

These results revealed the ability of SARS-CoV-2 to infect monolayer human cortical neurons and astrocytes, but not microglia, although infection of neurons and astrocytes was rare.

To examine the neurotropism of SARS-CoV-2 in a model system that more closely resembles the human brain, we exposed cortical, hippocampal, hypothalamic, and midbrain organoids to SARS-CoV-2 or vehicle control for 8 hours and analyzed samples at 24 and 72 hpi.

We confirmed the regional identity of cortical, hippocampal, hypothalamic, and midbrain organoids by immunostaining with the markers CTIP2, PROX1, OTP, and TH, respectively ( Figure S1F ). SARS-CoV-2 nucleoprotein was detected sparsely in these organoids derived from two different hiPSC lines in a range that averaged between 0.6% and 1.2% of all cells at 24 and 72 hpi (Figures J o u r n a l P r e -p r o o f 6 1B, 1C, and S1G). Co-immunolabeling with DCX and SARS-CoV-2 nucleoprotein identified most of infected cells as neurons in all organoids ( Figure 1D ) and infection of some GFAP + astrocytes was found in hypothalamic organoids ( Figure S1H ). Because these brain organoids contained mostly neurons, the relative susceptibility of neurons compared to astrocytes could not be assessed with certainty. The number of infected cells did not significantly increase from 24 to 72 hpi ( Figures 1C), indicating that infection may not spread among neurons in brain organoids within this time window.

During development, the choroid plexus develops adjacent to the hippocampus (Lun et al., 2015) and some of our hippocampal organoids contained regions with choroid plexus epithelial cells, which were identified by transthyretin (TTR) expression ( Figure 1E ). Notably, we observed a greater density of infected cells in these regions ( Figure 1E ). Additionally, we observed colocalization of patient convalescent serum and double-stranded RNA (dsRNA) immunolabeling in regions with choroid plexus cells, further supporting SARS-CoV-2 infection ( Figure 1F ).

Together, these results demonstrate that SARS-CoV-2 exhibits limited tropism for neurons and astrocytes of multiple brain regions, but higher infectivity of choroid plexus epithelial cells.

To validate the higher susceptibility and further investigate consequences of SARS-CoV-2 infection of choroid plexus cells, we sought to generate more pure choroid plexus organoids from hiPSCs.

The most dorsal structures of the telencephalon, including the choroid plexus, are patterned by high WNT and BMP signaling from the roof plate (Lun et al., 2015) . An early in vitro study demonstrated the sufficiency of BMP4 exposure to induce choroid plexus fate from neuroepithelial cells (Watanabe et al., 2012) . Furthermore, exposure of human embryonic stem cell-derived embryoid bodies to the GSK3 antagonist CHIR-99021 and BMP4 was shown to generate 3D choroid plexus tissue (Sakaguchi et al., 2015) . Building upon these previous studies, we optimized a simple protocol to generate choroid plexus organoids (CPOs) from hiPSCs ( Figure 2A ). Undifferentiated hiPSCs grown in a feeder-free condition were aggregated into embryoid bodies consisting of J o u r n a l P r e -p r o o f 7 approximately 5,000 cells each using an Aggrewell plate ( Figure 2A ). Embryoid bodies were patterned to the anterior neuroectodermal fate using dual-SMAD inhibition combined with WNT inhibition ( To further characterize these CPOs, we performed transcriptome analysis of CPOs at 50 DIV by bulk RNA sequencing (RNA-seq). Gene expression analysis confirmed the expression of choroid plexus markers, including TTR, AQP1, Chloride Intracellular Channel 6 (CLIC6), Keratin 18 (KRT18), MSX1, and LMX1A, in CPOs at high levels comparable to published transcriptomes of adult human choroid plexus tissue (Rodriguez-Lorenzo et al., 2020) and at much higher levels than those in hippocampal organoids at 45 DIV ( Figure 2D ). In addition, CPOs expressed genes related to adherens junction, signaling, ion channels and solute transporters at high levels comparable to those in adult human choroid plexus tissue ( Figure 2D ), indicating choroid plexus identity and function. At the whole transcriptome level, CPOs, but not hippocampal organoids, showed high correlation to adult human choroid plexus tissue ( Figure 2E ). We validated high levels of expression of a group of choroid plexus signature genes in CPOs at 50 DIV and 100 DIV in comparison to hippocampal organoids at 20 DIV by QPCR ( Figure S2D and Table S1 ).

Given our initial finding of a high rate of infection of choroid plexus cells by SARS-CoV-2, we examined the expression of known SARS-CoV-2 receptors. RNA-seq analysis showed expression J o u r n a l P r e -p r o o f 8 of ACE2 and TMPRSS2 in CPOs at levels similar to the adult human choroid plexus tissue, which were much higher than in hippocampal organoids ( Figure 2F ). Expression levels of NRP1, a newly identified receptor for SARS-CoV-2 (Cantuti-Castelvetri et al., 2020) , were similar in all three samples. Immunohistology confirmed expression of ACE2 at a much higher level in CPOs than in hippocampal organoids ( Figure 2G ). QPCR analysis also showed higher levels of ACE2 and TMPRSS2 expression in CPOs at 50 DIV and 100 DIV than in hippocampal organoids ( Figure S2D ) Together, these results show that our CPOs exhibit a similar transcriptome as adult human choroid plexus tissue and express markers for choroid plexus epithelial cells and SARS-CoV-2 receptors, representing a suitable experimental model to study SARS-CoV-2 infection.

Next, we exposed CPOs at 47 DIV and 67 DIV to either SARS-CoV-2 or vehicle control for 8 hours and analyzed samples at 24 and 72 hpi. Upon SARS-CoV-2, but not vehicle treatment, many TTR + choroid plexus cells showed infection as identified by co-localization of patient convalescent serum and SARS-CoV-2 nucleoprotein immunolabeling in CPOs derived from two independent hiPSC lines ( Figures 3A and S3A ). Additionally, we observed co-localization of patient convalescent serum and abundant dsRNA immunolabeling in TTR + choroid plexus cells ( Figure S3B ). Infected cells identified by SARS-CoV-2 nucleoprotein immunostaining also showed high levels of ACE2 expression, consistent with ACE2 being a critical cell entry receptor for SARS-CoV-2 ( Figure S3C ).

Quantification revealed an average of 9.0% and 12.6 % of TTR + cells infected at 24 hpi and 11.9% and 18.6% of TTR + cells infected at 72 hpi for CPOs infected at 47 DIV and 67 DIV, respectively, across two hiPSC lines ( Figures 3B and S3D ). An increase in the number of infected cells from 24 to 72 hpi suggested that infection could be productive and spread among cells ( Figures 3B and   S3D ). To confirm SARS-CoV-2 productive infection, we examined viral titers using culture supernatants and CPO lysates at 0, 24, and 72 hpi. There was a significant increase in titers of J o u r n a l P r e -p r o o f 9 infectious virus over time after the initial infection ( Figure 3C ). The increase of infectious virus present in CPO lysates superseded the increase in culture supernatants, suggesting a slow release of infectious viruses into the culture medium. The increase in viral titers appeared to be bigger than the increase in the number of infected cells, suggesting that the relationship is not linear. Consistent with this notion, exposure of CPOs at 67 DIV to different amounts of SARS-CoV-2 showed a nonlinear increase in infection with higher viral titers ( Figures S3E and 3F ). Importantly, we observed infection using 10 3 FFU (focus forming units) with an estimated MOI (multiplicity of infection) of 0.001, indicating very high susceptibility of CPOs to SARS-CoV-2 infection ( Figures S3E and 3F ).

Next, we examined the cellular consequence of SARS-CoV-2 infection of CPOs. We observed the presence of syncytia in SARS-CoV-2 infected cells, which significantly increased from an average of 4.6% at 24 hpi to 10.5% by 72 hpi (Figures 3D and 3E ). Development of syncytia by cell-cell fusion mediated by interaction between SARS-CoV-2 spike protein and ACE2 expressed on adjacent cells has been reported with SARS-CoV-2 infection of several cell types and is a major mechanism for viral spread (Hoffmann et al., 2020a; Matsuyama et al., 2020; Ou et al., 2020) . In particular, the SARS-CoV-2 spike protein appears to facilitate membrane fusion at a much higher rate than SARS-CoV-1 (Xia et al., 2020) . By examining individual confocal Z-planes we could identify as many as 12 nuclei within a single infected cell at 72 hpi ( Figure 3D ). Interestingly, in addition to syncytia, we observed a significant increase in cell death in both infected and uninfected TTR + choroid plexus cells in CPOs exposed to SARS-CoV-2 as measured by TUNEL immunolabeling ( Figures 3F, 3G and S3G). Cell death increased from an average of 1.4% to 5.9% in infected and from 1.9% to 5.4% in uninfected TTR + cells from 24 to 72 hpi in CPOs infected at 67 DIV across two hiPSC lines ( Figure 3G ). TUNEL + uninfected cells tended to appear adjacent to infected cells, suggesting that infected cells may induce adjacent cells to die through an extrinsic mechanism ( Figure 3F ). Similar results were found for CPOs infected at 47 DIV ( Figure S3G ).

To confirm the susceptibility of choroid plexus epithelial cells to SARS-CoV-2 infection, we examined primary human choroid plexus epithelial cells (pHCPECs). pHCPECs exposed to SARS-J o u r n a l P r e -p r o o f CoV-2, but not vehicle control, for 12 hours showed many infected cells by immunolabeling with SARS-CoV-2 nucleoprotein at 72 and 120 hpi ( Figure S3H ). The number of infected cells increased with higher MOI, with an average of 1.7% and 0.9% of cells infected at 72 and 120 hpi, respectively, using a MOI of 5 ( Figure S3I ).

Together, using CPOs as a 3D human cellular model, our findings reveal SARS-CoV-2 tropism for choroid plexus epithelial cells that results in productive infection, increased cell syncytia that may promote viral spread through cell-cell fusion, and increased cell death among both infected and uninfected adjacent cells.

To gain additional insight into functional consequences of SARS-CoV-2 infection in CPOs at the molecular level, we performed RNA-seq of SARS-CoV-2 and vehicle-treated 47 DIV CPOs at 24 and 72 hpi. Principal component analysis showed clustering of biological replicates within different treatment groups ( Figure S4A ). After aligning reads to the SARS-CoV-2 genome, we detected high numbers of viral transcripts at 24 and 72 hpi, confirming that the virus was replicating within CPOs ( Figure 4A ). We also confirmed the expression of SARS-CoV-2 receptors ACE2, TMPRSS2, and NRP1 in CPOs at all time points ( Figure 4B ). Comparison of SARS-CoV-2 and vehicle-treated CPOs revealed 1,721 upregulated genes and 1,487 downregulated genes at 72 hpi, indicating that SARS-CoV-2 infection leads to large-scale transcriptional dysregulation ( Figure S4B and Table S2 ).

More detailed gene ontology (GO) analysis of upregulated genes at 72 hpi showed enrichment for genes related to viral responses, RNA processing, response to cytokine, cytoskeletal rearrangement, and cell death ( Figure 4C and Table S3 ). Closer examination of upregulated genes revealed an increase in inflammatory cytokines CCL7, IL32, CCL2 (MCP1), IL18, and IL8 ( Figure 4D ). IL32 plays important roles in both innate and adaptive immune responses by inducing TNF-alpha, which activates the signaling pathway of NF-kappa-B and p38

MAPK. This signaling pathway has been shown to be activated following SARS-CoV-2 infection in J o u r n a l P r e -p r o o f 11 other cells (Bouhaddou et al., 2020) . We validated the upregulation of p38 signaling by immunostaining of the phosphorylated form of p38 in infected cells ( Figures S4C and S4D ).

Interestingly, quantification showed elevated levels of phospho-p38 in nearby uninfected cells, although to a lesser degree than in infected cells, suggesting a substantial non-cell autonomous effects ( Figure S4D ). Upregulation of many vascular remodeling genes ( Figure 4D ), such as NPPB and VCAN, suggests that infected choroid plexus cells could signal the vasculature to promote leukocyte invasion (Shechter et al., 2013) .

GO analysis of downregulated genes at 72 hpi, on the other hand, showed enrichment for genes related to ion transport, transmembrane transport, cilium, and cell junction ( Figure 4C and Table S3 ). Closer examination of downregulated genes revealed a decrease in the expression of many transporters and ion channels, such as AQP1, AQP4 and SLC22A8, which are important for normal CSF secretory function ( Figure 4D ) (Brown et al., 2004; Hladky and Barrand, 2016) , suggesting functional deficits of choroid plexus cells. Downregulation of many cell junction genes suggests a remodeling or break-down of normal CSF-blood barrier function, which also occurs during brain inflammation (Engelhardt and Tietz, 2015) . Additionally, the dramatic decrease in TTR production may result in decreased thyroxine transport to the CSF, which has been shown to contribute to neuropsychiatric symptoms and "brain fog" or difficulty in concentrating in patients (Samuels, 2014) . We validated downregulation of TTR levels in infected cells by immunostaining ( Figures S4C and S4D ). We also observed downregulation of TTR levels in uninfected cells at 72 hpi, although to a lesser degree than in infected cells, again suggesting a non-cell autonomous effect ( Figure S4D ).

Transcriptional dysregulation induced by SARS-CoV-2 in CPOs at 24 hpi showed similar gene expression changes as at 72 hpi, although less severe in most cases ( Figures 4D and S4E ).

Comparison of the list of dysregulated genes at 24 and 72 hpi revealed a substantial overlap of 42% and 32% of upregulated and downregulated genes, respectively ( Figure S4F ). We validated the dysregulation of a subset of these genes by QPCR ( Figure S4G ). J o u r n a l P r e -p r o o f 12 To investigate whether organoids from different tissues exhibit similar transcriptional responses upon SARS-CoV-2 infection, we compared SARS-CoV-2-induced transcriptional changes among CPOs and published datasets from hepatocyte organoids (Yang et al., 2020a) and

intestinal organoids . Surprisingly, we found little overlap among upregulated or downregulated genes in the three different organoid types, suggesting a predominantly cell typespecific response to SARS-CoV-2 infection ( Figure 4E ). This finding supports the use of a diverse array of human model systems to investigate effects of SARS-CoV-2 infection. Additionally, we found many more dysregulated genes in CPOs compared to the other two organoid types, suggesting that SARS-CoV-2 may exhibit a greater impact on choroid plexus cells ( Figure 4E ).

Together, these transcriptome analyses suggest that SARS-CoV-2 infection of choroid plexus cells leads to viral RNA production and inflammatory cellular responses with a concomitant compromise of normal barrier and secretory functions as well as increased cell death. Furthermore, responses to SARS-CoV-2 infection are largely tissue organoid specific, at least at the transcriptional level.

Using a platform of hiPSC-derived monolayer neurons, microglia, astrocytes, and region-specific brain organoids, we showed limited tropism of SARS-CoV-2 for neurons and astrocytes with a particularly high rate of infection of choroid plexus epithelial cells. Using an optimized protocol for generating CPOs from hiPSCs that resemble the morphology, marker expression, and transcriptome of adult human choroid plexus, we revealed productive infection of SARS-CoV-2 in choroid plexus epithelial cells, which we confirmed using primary human choroid plexus epithelial cells. Analysis of the consequence of SARS-CoV-2 infection of CPOs showed an increase in both cell autonomous and non-cell autonomous cell death and transcriptional dysregulation related to increased inflammation and altered barrier and secretory function. Our study provides an organoid platform to investigate the cellular susceptibility, pathogenesis, and treatment strategies of SARS-J o u r n a l P r e -p r o o f 13 CoV-2 infection of brain cells and further implicates the choroid plexus as a potentially important site for pathophysiology. Notably, the choroid plexus is known to be a site of infection for several viruses and agents that affect the brain (Schwerk et al., 2015) .

Although COVID-19 primarily manifests as respiratory distress resulting from inflammatory damage to the lung epithelium, there is mounting clinical evidence implicating other organs, such as the kidney, intestine, and brain, in disease pathogenesis (Renu et al., 2020) . The currently widely used cellular model systems, such as Vero E6 cells or human cancer cell lines, do not adequately recapitulate the cellular diversity and breadth of the disease. Although mice engineered to express human ACE2 have provided an in vivo model to study SARS-CoV-2, the system relies on human ACE2 overexpression and does not fully recapitulate symptoms of COVID-19 in humans. Recently, organoids have emerged as a model to study human cells and organogenesis in vitro (Clevers, 2016) . Organoids for various tissues have been used to better understand SARS-CoV-2 viral tropism and pathology Monteil et al., 2020; Ramani et al., 2020; Yang et al., 2020a; Zhou et al., 2020) . These studies support cell-type specific susceptibility to SARS-CoV-2 infection. Our finding that dysregulated gene expression varies widely among hepatocyte, intestinal, and choroid plexus organoids infected with SARS-CoV-2 suggests unique responses in different cell types and highlights the need for diverse human cellular model systems when studying the disease. Brain organoid models for SARS-CoV-2 infection are particularly useful since it is not feasible to take brain biopsies from patients with COVID-19. The load of SARS-CoV-2 that may enter the brain is likely to be variable throughout the course of illness and may be difficult to accurately assess in brain samples. For example, viral particles or RNA has been detected in the CSF or brain of some but not the majority of patients with neurological symptoms (Helms et al., 2020; Moriguchi et al., 2020; Puelles et al., 2020; Solomon et al., 2020; Virani et al., 2020) , although the amount of viral particles in the CSF may be insufficient for detection .

J o u r n a l P r e -p r o o f 14 Brain organoids allow analyses of the acute and long-term impact of SARS-CoV-2 infection in real time, with the ability to introduce perturbations, such as therapeutic agents, to assess cellular responses. Use of region-specific brain organoids, including CPOs, expands the organoid toolset for investigating SARS-CoV-2 infection, and provides a platform for testing new therapeutics.

Building upon previous findings (Sakaguchi et al., 2015; Watanabe et al., 2012) , we optimized a CPO model that is simple, robust and reproducible with the initial goal to study SARS-CoV-2 infection. These CPOs express high levels of SARS-CoV-2 receptors and exhibit productive infection of SARS-CoV-2 and pathogenesis at cellular and molecular levels. CPOs may provide a better model system than primary human choroid plexus epithelial cells, which are not widely available and often lose some characteristics upon multiple rounds of expansion in culture and showed a lower infection rate than our CPOs. Unlike a very recently developed protocol that utilizes cultures with Matrigel extracellular matrix (Pellegrini et al., 2020) , our CPOs do not obviously polarize or develop fluid-filled, cystic structures, but do offer the benefit of not relying on Matrigel, which has batch-to-batch variation and can lead to increased inter-organoid variability. Our protocol uses feeder-free hiPSCs to further improve organoid reproducibility, and CPOs are cultured on an orbital shaker to enhance oxygen and nutrient diffusion throughout the organoids. With these improvements, CPOs reproducibly generate projections of cuboidal cells and exhibit uniform expression of choroid plexus markers OTX2, AQP1, and TTR in most of the cells within the organoids. They also exhibit a transcriptome similar to adult human choroid plexus tissue.

Importantly, these CPOs express many transporters that are essential for CSF secretion. These CPOs provide a platform for future investigations of cell type-specific pathogenesis, mechanisms and treatment of SARS-CoV-2 infection. Our CPO platform can also be used to model human choroid plexus development and associated brain disorders in the future (Lun et al., 2015) .

Spread in the CNS J o u r n a l P r e -p r o o f 15 ACE2 has been identified as a key cell entry receptor for SARS-CoV-2 (Hoffmann et al., 2020b).

Although moderate ACE2 expression has been detected in various brain regions, particularly high levels have been reported in the choroid plexus in humans and mice . The high levels of ACE2 expression in CPOs may explain their higher susceptibility to SARS-CoV-2 infection compared to other brain organoids. Moreover, the productive infection and high incidence of syncytia in infected choroid plexus cells may contribute to the viral spread. On the other hand, despite relatively low ACE2 expression, we did detect sparse infection of neurons by SARS-CoV-2.

Recent studies point to the potential for other proteins that are expressed more highly in neural cells, such as NRP1, to serve as receptors for SARS-CoV-2. We did not observe an obvious increase in the number of infected neurons over time, suggesting that the virus may not spread efficiently from neuron-to-neuron.

Central nervous system disorders, including seizures and encephalopathy, have been reported with SARS-CoV-1 with detection of infectious virus and RNA in the CSF (Hung et al., 2003; Lau et al., 2004; Xu et al., 2005) . A substantial portion of patients with COVID-19 exhibit various neurological symptoms, which are highly variable, probably due to many factors (Helms et al., 2020; Moriguchi et al., 2020; Puelles et al., 2020; Solomon et al., 2020; Virani et al., 2020) . How SARS-CoV-2 may enter the brain is currently unknown. The main hypotheses are either neuron-toneuron spread via bipolar cells located in the olfactory epithelium that extend axons and dendrites to the olfactory bulb, or a hematogenous route across the blood-CSF-barrier (Desforges et al., 2014) . Neuron-to-neuron propagation has been described for other coronaviruses (Dube et al., 2018; Netland et al., 2008) , but it was not obvious in our study. Our finding that the choroid plexus is particularly susceptible to productive infection with SARS-CoV-2, raises the possibility that choroid plexus epithelial cells could be infected from virus circulating in closely associated capillaries. SARS-CoV-2 may replicate within choroid plexus cells and shed viral copies into the CSF. Indeed, we detected increased viral load in culture supernatants overtime. Alternatively, virus may enter the CSF via the cribiform plate in nasal passages and then replicate in choroid plexus J o u r n a l P r e -p r o o f 16 cells, increasing viral availability in the central nervous system. Viral propagation through the CSF may also explain cases of spinal cord pathology in some patients with COVID-19 (Giorgianni et al., 2020; Valiuddin et al., 2020) . More detailed analyses of autopsy brain samples from patients with COVID-19 and better animal models of SARS-CoV-2 infection are necessary to understand potential routes of viral entry into the brain.

The transcriptional dysregulation in CPOs after SARS-CoV-2 infection indicates an inflammatory response and perturbed choroid plexus cellular function. Our study further suggested both cell autonomous and non-cell autonomous impact of SARS-CoV-2 infection. Many pro-inflammatory cytokines were upregulated, including CCL7, IL32, CCL2, IL18, and IL8, which are important for recruiting immune cells to sites of infection. Future studies are needed to validate the release of these cytokines by ELISA analyses. Notably, one case report examining inflammatory cytokines present in the serum and CSF of a patient with COVID-19 presenting with severe seizures identified CCL2 as the only cytokine enriched in the patient's CSF compared to serum (Farhadian et al., 2020) . CCL2 was one of the most highly upregulated genes after SARS-CoV-2 infection, suggesting that the choroid plexus may be the source of this cytokine in the CSF. Upregulation of vascular and extracellular matrix remodeling genes also suggests that during infection, choroid plexus cells can signal the adjacent vasculature to recruit inflammatory cells. Downregulation of many genes important for CSF secretion, including AQP1, and many ion transporters, such as ATP1A2, may lead to altered CSF production and composition. Mutations in many of these dysregulated transporter genes have been implicated in neurological complications in humans, such as migraine and encephalopathy (Murphy et al., 2018) . Increased expression of cell death genes supports our finding of increased numbers of TUNEL + cells after SARS-CoV-2 infection. Downregulation of many genes related to the cilium suggests an impaired cytoskeleton and dysfunctional sensing of the extracellular environment. There was also a significant downregulation of TTR, which is necessary for carrying thyroid hormone from the blood into the CSF, and lowered TTR production by the choroid plexus has been associated with neuropsychiatric disorders, such as schizophrenia and depression (Huang et al., 2006; Sullivan et al., 1999) . Furthermore, low CSF thyroid hormone has been associated with many neurological symptoms, including slowing of thought and speech, decreased attentiveness, and reduced cognition, which have all been reported by patients with COVID-19 (Ellul et al., 2020) . Our study implicates the choroid plexus epithelium as a potential target of SARS-CoV-2 infection and a contributor to COVID-19 disease pathogenesis that warrants further investigation.

Our study demonstrates clear susceptibility of hiPSC-derived neurons, astrocytes, and choroid plexus cells, as well as primary astrocytes and choroid plexus epithelial cells, to SARS-CoV-2 infection in vitro, however, a thorough analysis of brain samples from patients with COVID-19 is necessary to confirm neural susceptibility in vivo. Our methodology has a number of constraints that limit result interpretation. First, since brain organoids more closely resemble the developing fetal brain than the mature adult brain, there may exist important differences in SARS-CoV-2 susceptibility between immature and mature cells. There has been recent evidence of vertical transmission of SARS-CoV-2 from mother to fetus (Dong et al., 2020; Vivanti et al., 2020; Zeng et al., 2020) , but the impact on the fetal brain remains unclear. Brain organoids may serve as a useful model system to explore the potential effects of fetal SARS-CoV-2 exposure on brain development.

Second, direct exposure of brain organoid cultures to SARS-CoV-2 in the culture medium may not accurately recapitulate the physiological amount and duration of viral exposure in humans. Different viral titers may reveal different viral tropism in vitro. To avoid the use of high titers that may cause J o u r n a l P r e -p r o o f 18 non-physiological viral infection, we exposed different brain region-specific organoids of the same estimated MOI of 0.1 and found a differential infection rate. Importantly, SARS-CoV-2 at an estimated MOI as low as 0.001 can lead to detectable infection of CPOs, indicating very high susceptibility of choroid plexus cells, at least in vitro. Third, brain organoid models lack an intact blood-brain-barrier or blood-CSF-barrier which may modulate SARS-CoV-2 access to the brain. Our brain organoids also lack additional cell types, such as oligodendrocytes, microglia, stromal cells, immune cells, and endothelial cells, which may modulate susceptibility to infection and contribute to disease pathogenesis in vivo. Immune cells, such as T cells and monocytes, have been shown to mediate host responses to SARS-CoV-2 infection, including tissue-specific inflammation (Tay et al., 2020) . Endothelial cells are major targets of SARS-CoV-2 and may be the primary cause of SARS-CoV-2-related effects in the brain with neurological symptoms being secondary to vascular changes and hypoxia (Ellul et al., 2020) . Future studies of SARS-CoV-2 susceptibility can extend to a more diverse selection of brain cells, as well as peripheral neurons. Also see Figure S2 and Table S1. Table S2 for the complete list of differentially expressed genes and Table S3 for the complete list of GO terms for differentially expressed genes. tmem.: transmembrane. (E) Venn diagrams comparing the overlap of upregulated and downregulated genes following S-CoV-2 infection in CPOs, human hepatocyte organoids (Yang et al., 2020a) , and human intestinal organoids . Differentially expressed genes at both 24 and 72 hpi were combined for CPOs and differentially expressed genes for both expansion and differentiation intestinal organoid types were combined for intestinal organoids. Also see Figure S4 and Tables S1, S2 and S3.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Guo-li Ming (gming@pennmedicine.upenn.edu) .

This study did not generate new unique reagents.

The access number for the RNA-seq data reported in this study is deposited in GEO (GSE157852). 

Human iPSC lines used in the current study were derived from healthy donors and were either obtained from commercial sources or previously generated and fully characterized (Chiang et al., 2011; Wen et al., 2014; Yoon et al., 2014) . C1-2 hiPSCs were generated from fibroblasts obtained from ATCC (CRL-2097). C3-1 hiPSCs were generated from fibroblasts obtained from an individual in a previously characterized American family (Sachs et al., 2005) . WTC11 hiPSC were obtained from Coriell (GM25256). HT268A hiPSCs were generated from dermal fibroblasts obtained from Coriell (GM05659) (Vu et al., 2018) , and PCS201012 hiPSCs were generated from dermal fibroblasts obtained from ATCC (PCS-201-012) (Beers et al., 2015) . Generation of hiPSC lines (Bardy et al., 2015) . Primary human astrocytes (ScienCell Research Laboratories) were cultured and expanded for two passages, according to the supplier's instructions. After the second expansion, astrocytes were cryopreserved at 250,000 cells per vial. Banked astrocytes were thawed directly onto 96 well imaging plates (Greiner Bio-one) coated with Poly-L-ornithine (Sigma-Aldrich) at 2 µg/cm 2 and laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane (Sigma-Aldrich) at 5 µg/mL at a density of 16,000 cells per well for shorter infection times (48 hours or less) and 8000 cell per well for 120 hour infections. Primary human choroid plexus epithelial cells (ScienCell Research Laboratories) were cultured according to the supplier's instructions. Cells were either passaged once or plated directly out of thaw onto 96-well imaging plates coated with Poly-L-ornithine at 2 µg/cm 2 at a density of 4000 cells per well.

All studies involving hiPSCs were performed under approved protocols of the University of Pennsylvania and NIH. Refer to the Key Resources Table for the source of each hiPSCs. All hiPSC lines were confirmed to have a normal karyotype and were confirmed free of Mycoplasma, Acholeplasma, and Spiroplasma with a detection limit of 10 CFU/mL by targeted PCR (Biological Industries). For cortical, hippocampal, hypothalamic, and midbrain organoid generation, hiPSCs were cultured on mouse embryonic fibroblast feeder (MEF) cells in stem cell medium consisting of DMEM:F12 supplemented with 20% KnockOut Serum Replacement, 1X MEM-NEAAs, 1X

GlutaMAX, 1X Penicillin-Streptomycin, 1X 2-mercaptoethanol, and 10 ng/mL bFGF in a 5% CO 2 , 37°C, 90% relative humidity incubator as previously described (Qian et al., 2018) . Culture medium was replaced every day. hiPSCs were passaged every week onto a new tissue-treated culture plate coated with 0.1% gelatin for 2 hours and pre-seeded with γ-irradiated CF1 MEF cells one day in advance. Colonies were detached by washing with DPBS and treating with 1 mg/mL Collagenase Type IV for 30-60 minutes. Detached colonies were washed 3 times with 5 mL DMEM:F12 and dissociated into small clusters by trituration with a P1000 pipette. For CPO generation, hiPSCs J o u r n a l P r e -p r o o f 33 were maintained in feeder-free conditions on plates pre-coated with hESC-qualified Matrigel (Corning) using mTeSR Plus medium (Stemcell Technologies). Culture medium was replaced every other day and hiPSCs were passaged every week by washing with DPBS and incubating with ReLeSR reagent (Stemcell Technologies) for 5 minutes. Detached hiPSCs were broken into smaller clusters by gentle trituration using a 5 mL pipette and seeded onto fresh coated plates.

Generation of cortical organoids from C3-1 hiPSCs was performed as previously described (Qian et al., 2018; Qian et al., 2016) . Briefly, hiPSC colonies were detached with Collagenase Type IV, For generation of hypothalamic organoids from C1-2 and C3-1 hiPSC lines, hiPSC colonies were processed similarly to those for cortical organoids followed by exposure to Shh signaling and Wntinhibition in induction medium with 50 ng/mL recombinant Sonic Hedgehog, 1 µM Purmorphamine, 10 µM IWR1-endo and 1 µM SAG, and then transferred to differentiation medium for further culture.

Organoids were maintained with media changes every other day.

At 70% confluence, undifferentiated hiPSCs (C1-2 and WTC11 lines) grown in feeder-free conditions were detached using ReLeSR reagent and gently triturated into small clusters using a 5 mL pipette and pipette aid. hiPSC clusters were centrifuged at 200 g for 3 minutes and the supernatant was aspirated and replaced with mTeSR Plus medium supplemented with 10 µM Y-27632. Approximately 5,000 hiPSCs were aggregated into each EB by following instructions using the Aggrewell 800 plate and cultured overnight in mTeSR Plus medium supplemented with 10 µM Y-27632. The next day (Day 1), embryoid bodies were gently removed from the Aggrewell plate using a P1000 pipettor with a wide-bore tip, washed three times with DMEM:F12, and transferred to neural induction medium containing DMEM:F12 supplemented with 20% KnockOut Serum On day 30, medium was replaced with differentiation medium containing a 1:1 mixture of DMEM/F12 and Neurobasal supplemented with 1X N2 supplement, 1X B27 supplement w/o vitamin A, 1X Penicillin/Streptomycin, 1X Non-essential Amino Acids, 1X GlutaMax, 10 ng/mL BDNF, and 10 ng/mL GDNF. Differentiation medium was replaced every three days and organoids could be maintained for over 100 days.

SARS-CoV-2 USA-WA1/2020 (Gen Bank: MN985325.1) (Harcourt et al., 2020) viral isolate was obtained from BEI Resources. SARS-CoV-2 virus was expanded and titered using Vero E6 cells.

Culture cells were infected in a 96-well plate with 100 µL of medium per well using multiplicity (MOI) of 0.2, 1, or 5, and vehicle-treated cells as controls. After virus exposure for 12 hours, cells were washed 3 times with fresh medium and returned to culture. Brain organoids were infected in a 24well ultra-low attachment plate (Corning) with 0.5 mL of medium per well containing vehicle, 10 3 , 10 4 , or 10 5 focus forming units (FFU) of SARS-CoV-2 for 8 hours on an orbital shaker rotating at approximately 100 rpm. After virus exposure, organoids were washed 3 times with fresh medium and returned to culture. For infection of organoids, we controlled the virus titer rather than the MOI since it is not feasible to obtain an accurate cell count on each batch of organoids before infection.

According to our estimate, each organoid contains approximately 250-500,000 cells, corresponding J o u r n a l P r e -p r o o f 36 to an estimated MOI of 0.1-0.05 when using 10 5 FFU SARS-CoV-2 virus and infecting 4 organoids together.

At experimental endpoints, brain organoids were placed directly in 4% methanol-free formaldehyde (Polysciences) diluted in DPBS (Thermo Fisher Scientific) overnight at 4 o C on a rocking shaker.

After fixation, brain organoids were washed in DPBS and cryoprotected by overnight incubation in 30% sucrose (Sigma-Aldrich) in DPBS at 4°C. Brain organoids were placed in a plastic cryomold (Electron Microscopy Sciences) and snap frozen in tissue freezing medium (General Data) on dry ice. Frozen tissue was stored at −80°C until processing. Monolayer cells grown on glass coverslips were fixed in 4% methanol-free formaldehyde (Polysciences), diluted in DPBS (ThermoFisher Scientific) for 5 hours at room temperature, washed with DPBS, and stored at 4°C until ready for immunohistology. Serial tissue sections (25 µm for brain organoids) were sliced using a cryostat (Leica, CM3050S), and melted onto charged slides (Thermo Fisher Scientific). Slides were dried at room temperature and stored at −20°C until ready for immunohistology. For immunofluorescence staining, the tissue sections were outlined with a hydrophobic pen (Vector Laboratories) and washed with TBS containing 0.1% . Tissue sections were permeabilized and nonspecific binding was blocked using a solution containing 10% donkey serum (v/v), 0.5% Triton X-100 (v/v), 1% BSA (w/v), 0.1% gelatin (w/v), and 22.52 mg/ml glycine in TBST for 1 hour at room temperature. The tissue sections were incubated with primary antibodies (see the Key Resources Table) diluted in TBST with 5% donkey serum (v/v) and 0.1% Triton X-100 (v/v) overnight at 4°C.

After washing in TBST, tissue sections were incubated with secondary antibodies (see the Key

Resources Table) diluted in TBST with 5% donkey serum (v/v) and 0.1% Triton X-100 (v/v) for 1.5 hours at room temperature. After washing with TBST, sections were washed with TBS to remove detergent and incubated with TrueBlack reagent (Biotium) diluted 1:20 in 70% ethanol for 1 minute J o u r n a l P r e -p r o o f 37 to block autofluorescence. After washing with TBS, slides were mounted in mounting solution (Vector Laboratories), cover-slipped, and sealed with nail polish.

Monolayer cultures were imaged with a Perkin-Elmer Opera Phenix high-content automatic imaging system with a 20x air objective in confocal mode. At least 9 fields were acquired per well. Images were analyzed using Columbus Image Data Storage and Analysis System. Brain organoid sections were imaged as z stacks using a Zeiss LSM 810 confocal microscope or a Zeiss LSM 710 confocal microscope (Zeiss) using a 10X, 20X, 40X, or 63X objective with Zen 2 software (Zeiss). Images were analyzed using either Imaris 7.6 or ImageJ software. Images were cropped and edited using Adobe Photoshop (Adobe) and Adobe Illustrator (Adobe).

Tissue culture supernatants and lysates from SARS-CoV-2 treated CPOs were collected at 0, 24, and 72 hpi for 2 biological replicates per timepoint containing 4 organoids each. Tissue culture supernatants were stored at -80°C until titration. For lysates, organoids were washed with cold PBS, mechanically dispersed and disrupted by freeze-thaw cycles, and the clarified homogenate was stored at -80°C until titration. Viral titers were determined in Vero E6 cells using an immune focus forming unit assay. Briefly, Vero E6 cells (3 x 10 4 cells/96-well) were inoculated with 100 µl of serially diluted organoid supernatant or lysate in a 96-well plate at 37°C and 5% CO 2 for 20 hours.

Cells were fixed with 4% methanol-free paraformaldehyde overnight at 4°C. Infected foci were labeled using immunofluorescence staining for SARS-CoV-2 nucleoprotein. SARS-CoV-2 nucleoprotein positive cells were counted in each well. Readouts were performed on wells with the highest dilution displaying observable infection.

J o u r n a l P r e -p r o o f 38 To minimize variability due to sampling and processing, each biological replicate consisted of 3 organoids and replicates for all experimental conditions were processed in parallel for RNAextraction, library preparation and sequencing. At the desired experimental endpoints, organoids were homogenized in TRIzol (Thermo Fisher Scientific) using a disposable pestle and handheld mortar and stored at -80 C until processing. RNA clean-up was performed using the RNA Clean & Concentrator kit (Zymo Research) after TRIzol phase separation according to the manufacturer's protocol. RNA concentration and quality were assessed using a Nanodrop 2000 (Thermo Fisher Scientific).

Library preparation was performed as previously described with some minor modifications (Weng et al., 2017) . About 300 ng of RNA in 3.2 µL was combined with 0.25 µL RNase inhibitor (NEB) and 1 µL CDS primer (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3') in an 8-well PCR tube strip, heated to 70 ºC for 2 minutes, and immediately placed on ice. 5.55 µL RT mix, containing 2 µL of 5X SMARTScribe RT buffer (Takara), 0.5 µL of 100 mM DTT (Millipore Sigma), 0.3 µL of 200 mM MgCl2 (Thermo Fisher Scientific), 1 µL of 10 mM dNTPs (Takara), 1 µL of 10 µM TSO primer (5'-AAGCAGTGGTATCAACGCAGAGTACATrGrGrG-3'), 0.25 µL of RNase inhibitor (NEB), and 0.5 µL SMARTScribe reverse transcriptase (Takara) was added to the reaction. RT was performed under the following conditions: 42 ºC for 90 minutes, 10 cycles of 50 ºC for 2 minutes and 42 ºC for 2 minutes, 70 ºC for 15 minutes, and 4 ºC indefinitely. For cDNA amplification, 2 µL of the RT reaction was combined with 2.5 µL of 10X Advantage 2 buffer (Takara), 2.5 µL of 2.5 mM dNTPs (Takara), 0.25 µL of 10 µM IS PCR primer (5'-AAGCAGTGGTATCAACGCAGAGT-3'), 17.25 µL nuclease free water (ThermoFisher), and 0.5 µL Advantage 2 DNA Polymerase (Takara).

Thermocycling conditions were as follows: 94 ºC for 3 minutes, 8 cycles of 94 ºC for 15 s, 65 ºC for 30 s, and 68 ºC for 6 minutes, 72 ºC for 10 minutes, and 4 ºC indefinitely. Amplified cDNA was purified using 0.8X AMPure XP beads (Beckman Coulter), eluted in 15 µL nuclease-free water, and quantified using Qubit dsDNA HS assay kit (Thermo Fisher Scientific). cDNA was fragmented by combining 100 pg cDNA in 1 µL nuclease free water, 2X TD buffer (20 mM Tris, pH 8.0; Thermo J o u r n a l P r e -p r o o f 39 Fisher Scientific), 10 mM MgCl 2 , and 16% PEG 8000 (MilliporeSigma), and 0.5 µL Tn5 (Lucigen).

The mixture was heated to 55 ºC for 12 minutes, and the reaction was terminated upon the addition of 1.25 µL of 0.2% SDS (Fisher) and incubated at room temperature for 10 minutes. Fragments were amplified by adding 16.75 µL nuclease free water (Thermo Fisher Scientific), 1 µL of 10 mM Nextera i7 primer, 1 µL of 10 mM Nextera i5 primer, and 25 µL KAPA HiFi hotstart readymix (EMSCO/FISHER). Thermocycling conditions were as follows: 72 ºC for 5 minutes, 95 ºC for 1 minute, 14 cycles of 95 ºC for 30 s, 55 ºC for 30 s, and 72 ºC for 30 s, 72 ºC for 1 minute, and 4 ºC indefinitely. DNA was purified twice with 0.8X AMPure XP beads (Beckman Coulter) and eluted in 10 µL of 10 mM Tris, pH 8 (Thermo Fisher Scientific). Samples were quantified by qPCR (KAPA) and pooled at equal molar amounts. Final sequencing library fragment sizes were quantified by bioanalyzer (Agilent) with an average size of ~420 bp, and concentrations were determined by qPCR (KAPA). Samples were loaded at concentrations of 2.7 pM and sequenced on a NextSeq 550 (Illumina) using 1x72 bp reads to an average depth of 40 million reads per sample.

Choroid plexus organoid raw sequencing data were demultiplexed with bcl2fastq2 v2.17.1.14 (Illumina) with adapter trimming using Trimmomatic v0.32 software (Bolger et al., 2014) . Alignment was performed using STAR v2.5.2a (Dobin et al., 2013) . A combined reference genome consisting of GENCODE human genome release 28 (GRCh38.p12) and Ensembl SARS-CoV-2 Wuhan-Hu-1 isolate genome (genome assembly: ASM985889v3, GCA_009858895.3; sequence: MN908947.3) was used for alignment. Multimapping and chimeric alignments were discarded, and uniquely mapped reads were quantified at the exon level and summarized to gene counts using STAR -quantMode GeneCounts. All further analyses were performed in R v3.6.0. Transcript lengths were retrieved from GTF annotation files using GenomicFeatures v1.36.4 (Lawrence et al., 2013) and raw counts were converted to units of transcripts per million (TPM) using the calculateTPM function in scater v1.12.2 (McCarthy et al., 2017) . A combined TPM normalization using both human and

Primer sequences are listed in Figure S1 . About 0.5-1.5 µg RNA was used to generate the cDNA for QPCR with SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific) according to manufacturer's protocol. For QPCR reaction, 2 µL of cDNA was mixed with 6 µL of nuclease free water, 1 µL of 10 mM forward primer, 1 µL of 10 mM reverse primer and 10 µL of SYBR Green qPCR Master Mix (Thermo Fisher Scientific), and the QPCR reactions were performed on the StepOnePlus Real-Time PCR System (Applied Biosystems). Thermocycling conditions were as follows: 95 ºC for 20 s, 44 cycles of 95 ºC for 3 s and 60 ºC for 30 s. The difference between the Ct values (∆Ct) of the genes of interest and ACTIN gene was calculated for each experimental sample, and 2^(-∆∆Ct) was used to calculate the fold-change in expression of the genes of interest between samples using 20 DIV HOs as the reference for Figure S2D and 72 hpi Vehicle-treated CPOs as the reference for Figure S4G . shown as mean ± SEM or mean + SD as stated in the Figure Legends . In all cases, the p values are represented as follows: * * * p < 0.001, * * p < 0.01, * p < 0.05, and ns, not statistically significant when p > 0.05. In all cases, the stated ''n'' value is either separate wells of monolayer cultures or individual organoids with multiple independent images used to obtain data points for each. No statistical methods were used to pre-determine sample sizes. For all quantifications of immunohistology, the samples being compared were processed in parallel and imaged using the same settings and laser power for confocal microscopy. For monolayer cultures, cells were segmented by first identifying nuclei, and then cytoplasmic area. Mean cytoplasmic intensities for SARS-CoV-2 nucleoprotein immunostaining were then measured in the segmented cytoplasmic regions, and a mean cytoplasmic intensity threshold was determined to identify SARS-CoV-2 infected cells. Cell numbers were then automatically counted for SARS-CoV-2 infected DAPI + cells, and total DAPI + cells. For brain organoids, immuno-positive cells were quantified manually using the cell counter function in ImageJ (NIH) or Imaris (Bitplane). Ming and colleagues tested SARS-CoV-2 neurotropism using monolayer neural cells and brain organoids generated from human pluripotent stem cells and show minimal neuron and astrocyte infection, but efficient choroid plexus infection, leading to cell death and functional deficits. CCL7  IL32  CCL2  IL18  IL8  CASP8  IGFBP3  ANXA3  ANXA2  PAWR  PLAU  TNC  VCAN  THBS1  NPPB  CTGF  TAGLN  ACTG2  CDCP1  ACTA2  CD274  TNFAIP6  ITGB6  YWHAE  DYNLT1  AQP1  AQP4  ATP2B3  SLC7A8  SLC39A12   RGS9BP  ARR3  CDHR1  USH2A  CNGB1   SLC22A8  SLC5A5  ATP1A2  ATP8A1  ATP2B2   ANXA9  PTPRC  KCNA1  SLC17A8  CAMK4  APOE  HPGD  TTR 

Gene ontology: tool for the unification of biology. The Gene Ontology Consortium

The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice

Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro

A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture

Trimmomatic: a flexible trimmer for Illumina sequence data

The Global Phosphorylation Landscape of SARS-CoV-2 Infection

Molecular mechanisms of cerebrospinal fluid production

Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system

VennDiagram: a package for the generation of highlycustomizable Venn and Euler diagrams in R

The spatial and cell-type distribution of SARS-CoV-2 receptor ACE2 in human and mouse brain

Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation

Modeling Development and Disease with Organoids

Human coronaviruses: viral and cellular factors involved in neuroinvasiveness and neuropathogenesis

STAR: ultrafast universal RNA-seq aligner

SARS-CoV-2 productively infects human gut enterocytes

Possible central nervous system infection by SARS coronavirus

Software for computing and annotating genomic ranges

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Development and functions of the choroid plexus-cerebrospinal fluid system

p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease

Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R

PANTHER version 14: more genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools

Advances in Zika Virus Research: Stem Cell Models, Challenges, and Opportunities

Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2

A first case of meningitis/encephalitis associated with SARS-Coronavirus-2

Familial Hemiplegic Migraine With Asymmetric Encephalopathy Secondary to ATP1A2 Mutation: A Case Series

Severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ACE2

Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune crossreactivity with SARS-CoV

Human CNS barrier-forming organoids with cerebrospinal fluid production

Multiorgan and Renal Tropism of SARS-CoV-2

Generation of human brain region-specific organoids using a miniaturized spinning bioreactor

Brain-Region-Specific Organoids Using Mini-bioreactors for Modeling ZIKV Exposure

SARS-CoV-2 targets neurons of 3D human brain organoids

Coronaviruses pathogenesis, comorbidities and multi-organ damage -A review

Altered secretory and neuroprotective function of the choroid plexus in progressive multiple sclerosis

A frameshift mutation in Disrupted in Schizophrenia 1 in an American family with schizophrenia and schizoaffective disorder

Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue

Psychiatric and cognitive manifestations of hypothyroidism. Current opinion in endocrinology, diabetes, and obesity 21

The choroid plexus-a multi-role player during infectious diseases of the CNS

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Orchestrated leukocyte recruitment to immuneprivileged sites: absolute barriers versus educational gates

Neuropathological Features of Covid-19

Low levels of transthyretin in the CSF of depressed patients

A Mouse Model of SARS-CoV-2 Infection and Pathogenesis

Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth

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The Gene Ontology Resource: 20 years and still GOing strong

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Syndrome associated with SARS-CoV-2 infection. IDCases

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Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

BMP4 sufficiency to induce choroid plexus epithelial fate from embryonic stem cell-derived neuroepithelial progenitors

Synaptic dysregulation in a human iPS cell model of mental disorders

An Intrinsic Epigenetic Barrier for Functional Axon Regeneration

Coronavirus disease (COVID-19) situation dashboard

Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis

Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids

Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study

Encephalitis as a clinical manifestation of COVID-19

Modeling a genetic risk for schizophrenia in iPSCs and mice reveals neural stem cell deficits associated with adherens junctions and polarity

Antibodies in Infants Born to Mothers With COVID-19 Pneumonia

Infection of bat and human intestinal organoids by SARS-CoV-2

2020) was downloaded from the NCBI Gene Expression Omnibus (GEO: accession number GSE137619). The data was aligned to GRCh38.p12 and raw counts TPM-normalized to parallel CPO and HO datasets. TPM expression values were log 10 (TPM + 1) transformed for plotting individual gene expression values

2014) and correlation analysis was performed by calculating Spearman correlation coefficients in the space of shared genes across the whole transcriptome of all datasets

Differential gene expression analysis for CPOs between 72 hpi vehicle, 24 hpi SARS-CoV-2 infection, and 72 hpi SARS-CoV-2 infection was performed using DESeq2

SARS-CoV-2 viral transcripts were removed for differential gene expression analysis

Upregulated and downregulated genes for 24 hpi SARS-CoV-2 compared to 72 hpi vehicle, and 72 hpi SARS-CoV-2 compared to 72 hpi vehicle were filtered by adjusted p-value < 0.05. Volcano plots for 24 hpi SARS-CoV-2 and 72 hpi SARS-CoV-2 were plotted using EnhancedVolcano v1.7.8. Shared and unique signatures for upregulated and downregulated genes between 24 hpi SARS-CoV-2 and 72 hpi SARS-CoV-2 were visualized using VennDiagram v1

TPM + 1) normalized and converted to row Z-scores per gene for visualization. For SARS-CoV-2 cross-organoid comparison analysis, a list of upregulated and downregulated differentially expressed genes (DEGs) after SARS-CoV-2 infection were downloaded for adult hepatocyte organoids at 24 hpi (Yang et al., 2020a), intestinal organoids at 60 hpi in expansion (EXP) medium (Lamers et al., 2020), and intestinal organoids at 60 hpi in differentiation (DIF) medium

We thank members of Ming 

F.J. contributed to histological analysis of various brain organoids and development of the CPO model. S.R.P. and F.Z. contributed to RNA-seq data analysis. F.Z. contributed to QPCR validation. 

The authors declare no competing interests.J o u r n a l P r e -p r o o f