key: cord-0796500-1s0l783x
authors: Ramani, Anand; Müller, Lisa; Ostermann, Philipp Niklas; Gabriel, Elke; Abida-Islam, Pranty; Müller-Schiffmann, Andreas; Mariappan, Aruljothi; Goureau, Olivier; Gruell, Henning; Walker, Andreas; Andrée, Marcel; Hauka, Sandra; Houwaart, Torsten; Dilthey, Alexander; Wohlgemuth, Kai; Omran, Heymut; Klein, Florian; Wieczorek, Dagmar; Adams, Ortwin; Timm, Jörg; Korth, Carsten; Schaal, Heiner; Gopalakrishnan, Jay
title: SARS-CoV-2 targets cortical neurons of 3D human brain organoids and shows neurodegeneration-like effects
date: 2020-05-20
journal: bioRxiv
DOI: 10.1101/2020.05.20.106575
sha: b01b752eb569195877576fa12d80202d7b713d90
doc_id: 796500
cord_uid: 1s0l783x

COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within two days of exposure. Using COVID-19 convalescent serum, we identified that SARS-CoV-2 preferably targets soma of cortical neurons but not neural stem cells, the target cell type of ZIKA virus. Imaging cortical neurons of organoids reveal that SARS-CoV-2 exposure is associated with missorted Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Surprisingly, SARS-CoV-2 co-localizes specifically with Tau phosphorylated at Threonine-231 in the soma, indicative of early neurodegeneration-like effects. Our studies, therefore, provide initial insights into the impact of SARS-CoV-2 as a neurotropic virus and emphasize that brain organoids could model CNS pathologies of COVID-19. One sentence summary COVID-19 modeling in human brain organoids

The novel coronavirus disease 2019 caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is spreading worldwide, and the outbreak continues to rise, posing a severe emergency (1) . Understanding the biology of the current COVID-19 pandemic is a high priority for combatting it efficiently. Thus, it is essential to gain initial insights into the infection mechanisms of SARS-CoV-2, including its target cell types and tropism, to contain its short-and long-term effects on human health. Furthermore, it is vital to establish an experimental system that could allow designing measures on how to stop viral replication and protect human health rapidly.

However, practical problems associated with the isolation and handling of highly infective viral strains and lack of reliable in vitro human model systems that can efficiently model COVID-19 hamper these efforts.

Clinical symptoms of COVID-19 patients include upper respiratory tract infection with fever, dry cough, and dyspnea, indicating that the respiratory tract is the first target (2). However, emerging case reports showed that patients infected with SARS-CoV-2 suffered a sudden and complete loss of the olfactory function, stroke, and other severe neurological symptoms (3-7). All of these indicate that SARS-CoV-2 is neurotropic and could infect the central nervous system (CNS) (8-10).

Importantly, it remains unclear whether SARS-CoV-2 is vertically transmitted to fetuses, where it could impair neurodevelopment (11) . Since coronaviruses share a similar structure, it is likely that SARS-CoV-2 exhibits the same infection mechanism and possibly invades into the brain (12) .

Indeed, a clinical report detected the presence of viral RNA in autopsy brain samples (13) . Thus, at this point, it is essential to test whether SARS-CoV-2 infects human neurons and productively replicates in the CNS.

Examining a potential neurotropism of SARS-CoV-2, it is essential to employ a suitable in vitro human model system that can recapitulate the physiological effect of SARS-CoV-2 infection. In this regard, the recently emerged human brain organoids that closely parallel the complex neural epithelium exhibiting a wide diversity of cell types could serve as a suitable model system to test the neurotoxic effects of SARS-CoV-2. Induced Pluripotent Stem Cells (iPSCs)-derived human brain organoids have revealed useful insights into human brain development and helped to model a variety of neurological disorders (14) (15) (16) (17) (18) (19) . Notably, others and our work using brain organoids have revealed unprecedented insights into infection mechanisms, target cell types, and the toxicity effects of the Zika virus (ZIKV) during the recent ZIKV epidemic. These studies validate organoids as a tool for studying not only genetic but also environmental hazards to human brain (20) (21) (22) .

Here, we report that SARS-CoV-2 readily targets cortical neurons but not neural stem cells of 3D human brain organoids. Neurons invaded with SARS-CoV-2 at the cortical area display Tau missorting, Tau hyperphosphorylation and apparent neuronal death. Moreover, we show that although SARS-CoV-2 can readily target brain organoids, SARS-CoV-2 does not productively replicate, suggesting that the CNS may not support the replication of SARS-CoV-2.

We isolated SARS-CoV-2 from an infected patient admitted to our university hospital, University of Düsseldorf (see method section for culturing and propagation). To investigate whether SARS-CoV-2 replicates in inoculated African green monkey kidney cells (Vero CCL-81), we performed realtime quantitative polymerase chain reaction (qPCR) analysis with cell culture supernatant. As expected, the amount of SARS-CoV-2 RNA drastically increased from 0-dpi until 3-dpi ( Figure   S1A ). Next, we analyzed the infectivity of generated SARS-CoV-2 particles by propagating viruscontaining supernatant to new Vero cells. We confirmed the infection of new Vero cells by the emergence of virus-induced cytopathic effects (CPEs) and an increase in SARS-CoV-2 RNA over 4-dpi. Taken together, we demonstrated the successful generations of a novel SARS-CoV-2 isolate, SARS-CoV-2 NRW-42, derived from a nasopharyngeal and oropharyngeal swab specimen. The sequence access (number PRJNA627229) showed only eight nucleotide exchanges compared to SARS-CoV-2 Wuhan-Hu-1 isolate. To test for potential adaption to the used Vero CCL-81, we whole-genome sequenced virus samples collected from the clinical specimen used for inoculation and virus samples from the supernatant after four days of propagation. Sequence analysis revealed no nucleotide substitution after isolation and propagation in Vero cells. In summary, we isolated an infectious SARS-CoV-2 isolate (NRW-42) with high sequence similarity to Wuhan-Hu-1.

First, we set out to identify antibodies that can specifically determine SARS-CoV-2 infection. We isolated COVID-19 convalescent serum from blood samples of four independent individuals who recently recovered from COVID-19 (AB1, AB2, AB3, and AB4). Testing them in an enzyme-linked immunosorbent assay (ELISA) that used the S1 domain of the spike protein of SARS-CoV-2 as an antigen revealed that except for AB2, the rest of the convalescent serum contained SARS-CoV-2-specific IgG ( Figure S1B ).

We chose to work with AB4 as it displayed the best immunoreactivity to SARS-CoV-2exposed brain organoid tissues than the other serum. AB4 specifically recognized SARS-CoV-2-infected Vero cells ( Figure 1A ).

To further validate the specificity of the AB4, we performed co-immunostaining with a mouse monoclonal Anti-SARS-CoV-2 S and a polyclonal Anti-SARS-CoV-2 NP. These commercial antibodies were raised against the AA 1029-1192 of the S2 domain and AA-1-100 of the nucleoprotein of SARS-CoV-2, respectively. All of these antibodies recognized only SARS-CoV-2-infected Vero cells ( Figure 1A ). In Western blots that used SARS-CoV-2-infected Vero cell extracts, AB4 detected a protein band with an apparent molecular weight of 75 kD, a size similar of the cleaved-spike protein. Both rabbit polyclonal and mouse monoclonal antibodies recognized protein bands around 50 and 180 kDs, sizes similar to the nucleoprotein and uncleaved spike proteins ( Figure 1B) . Together, these experiments validate that AB4 detects SARS-CoV-2 infection.

Although the apparent target of SARS-CoV-2 is the respiratory tract, recently reported neurological phenotypes indicate that SARS-CoV-2 has detrimental effects on the CNS and could infect the CNS directly. To test this hypothesis, we generated 3D human brain organoids from two different iPSC lines and exposed them to our SARS-CoV-2 NRW-42 isolate. As described before, organoids exhibit their specific neuronal cell types, which are spatially restricted. For example, apical neural progenitors cells displaying elongated nuclei (NPCs) are aligned to form a lumen that is similar to the ventricular zone (VZ) of the mammalian brain. Cortical neurons are positioned basally to the VZ forming cortical plate (Figure 2A ) (23) (24, 25) . We exposed at least two different age groups of organoids (Day-15 and Day-60) to SARS-CoV-2 (TCID 50 /mL of 50) and analyzed after 2-and 4-dpi.

Cryo-sectioning followed by immunofluorescence analysis using convalescent serum indicated that except AB2, all of them could specifically recognize somas of SARS-CoV-2-positive cells in SARS-CoV-2 exposed organoids ( Figure S2A ). In our opinion, among others, AB4 displayed the best immunoreactivity. Interestingly, AB2, which did not contain S1-reactive IgG, did not label cells in SARS-CoV-2 exposed organoids ( Figure S2Aiv) . A sub-population of cells recognized by AB4 was also labeled by the monoclonal anti-SARS-CoV-2 S antibody, indicating that SARS-CoV-2 could target organoids within 2-dpi ( Figure S2B ).

First, we began analyzing Day-15 organoids, a developmental stage that we used to study ZIKV infections (20) . At this developmental stage, organoids mostly constitute actively proliferating NPCs at the VZs and a primitive cortical plate containing early neurons (Figure 2A) . Testing the target cell types of SARS-CoV-2 in these organoids revealed that SARS-CoV-2 could mostly target the cortical plate specified by pan-neuronal marker TUJ-1 ( Figure 2B-C) . Importantly, the perinuclear localization of SARS-CoV-2 in somas of cortical neurons is similar to the localization pattern of the virus in Vero cells, indicating that SARS-CoV-2 can enter into neuronal cells of brain organoids ( Figure 2D ).

Of note, although these organoids are abundant with NPCs, we did not detect SARS-CoV-2positive NPCs at the VZ, suggesting that SARS-CoV-2 preferably targets the cortical region of the brain organoid ( Figure. 2Biii) . In this respect, the infection route of SARS-CoV-2 is strikingly differing from the recent ZIKV isolate that has a robust neurotropism preferably infecting neural NPCs at the VZ (20) (26) . Analyzing Day-60 organoids revealed that the number of SARS-CoV-2positive cells was significantly higher than in Day-15 organoids. This suggests that SARS-CoV-2 prefers relatively mature neuronal cell types present in older organoids differentiated from both iPSCs donors ( Figure. 2C and E). Since we did not find differences between donors, the rest of the experiments utilized Day-60 organoids generated from donor-2, which displayed signs of cortical maturation as judged by the presence of more MAP2-positive neurons ( Figure S2C ).

Turning our analysis to the later time point of infection (dpi-4) revealed that there was no apparent increase in SARS-CoV-2-positive cells ( Figure 2E ). Corroborating to this, we could not detect an increase in viral RNA in the supernatants between 2-and 4-dpi, indicating that SARS-CoV-2 does not productively replicate in brain organoids until 4-dpi but suggestive of an abortive replication ( Figure 2F ). This is in contrast to recent reports showing SARS-CoV-2 productively infects vascular, kidney, and gut organoids (27) (28) (29) . Notably, angiotensin-converting enzyme 2 (ACE-2), an entry receptor of SARS-CoV-2 is highly expressed in these organoid types. Testing the ACE-2 expression at the level of mRNA via a qRT-PCR revealed that both iPSCs-derived brain organoids and neurons exhibited ~12.5 and 50 folds lesser than human respiratory epithelial cells (hREC), which served as a positive control ( Figure 2G ).

Next, we identified that the SARS-CoV-2-positive region of the cortical plate is further substantiated by Tau, a microtubule-associated protein that stabilizes neuronal microtubules and promotes axonal outgrowth (Figure 3 ) (30) . Tau dysfunction is implicated in Alzheimer's disease and other Tauopathies. Aberrant Tau proteostasis is also a sensitive marker for unspecific lesions of the CNS, such as traumatic brain disorder (31) . To investigate the consequences of SARS-CoV-2's effect on cortical neurons, we imaged the Tau localization on SARS-CoV-2-exposed organoids using a pan Tau antibody. Under physiological conditions, Tau is mainly an axonal protein that labels the axons of mature neurons ( Figure. 3Ai-iv and S4A). Applying high-resolution imaging followed by de-convolution, we could visualize the localization of Tau exclusively in axons the cortical neurons (Figure 3Aiv ). The term Tau missorting is used when Tau protein is mislocalized into a somatodendritic compartment and is observed at the early stages of Tau pathology (32). We then imaged cortical neurons for additional phosphorylated Tau using AT8 antibodies (specific for S202 and T205 of Tau) and p396 (specific for S396 of Tau). Although they label the axons of SARS-CoV-2-exposed organoids with slightly increased intensity, unlike AT180, none of these phospho-specific antibodies recognized Tau in the soma of SARS-CoV-2-positive neurons ( Figure   S4 ). In summary, these results demonstrate the presence of very specific phosphorylation of Tau at position T231 and the missorting of Tau to the soma of SARS-CoV-2-positive neurons implying neuronal stress reactions upon virus entry.

Phosphorylation of Tau at T231 allows for isomerization of the following proline residue into distinct cis-and trans-conformations by the propyl-isomerase PIN1 (36). Cis-pT231Tau is acutely produced by neurons after traumatic brain injury, leading to disruption of the axonal microtubule network and apoptosis (37) (38). Accordingly, analyzing the nuclei of SARS-CoV-2-positive cells ( Figure 4A ), we realized that they are highly condensed or fragmented exhibiting a strong reaction to 4′,6-diamidino-2-phenylindole (DAPI) that labels nuclei, a feature quite frequently observed in dead cells. To test neuronal cell death as a consequence of SARS-CoV-2 infection, we stained the SARS-CoV-2-exposed samples with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) that detects fragmented DNA in dead cells. We identified a significant number of cell death, suggesting that SARS-CoV-2-positive cells have undergone cell death within 2-dpi ( Figure 4B ).

We used 3D human brain organoids as an experimental system to study the COVID-19 and identified unexpected neurodegeneration-like effects of SARS-CoV-2 on human neurons. So far, the possible direct effect of SARS-CoV-2 on the CNS has been debated but not experimentally demonstrated. Thus, it was essential to examine whether SARS-CoV-2 can directly target human neurons and whether this leads to productive infection.

The finding that SARS-CoV-2 preferentially targets cortical neurons but not actively proliferating NPCs may suggest that developing embryonic brains are potentially less susceptible or free from the neurotoxic effects of SARS-CoV-2. This is indeed in striking contrast to ZIKV, a neurotropic virus that directly infects NPCs and triggers them to prematurely differentiate into neurons leading to congenital microcephaly (20) (21) (22) .

In contrast to vascular, kidney, and intestinal organoids (27) (28) (29) , SARS-CoV-2 cannot actively replicate in brain organoids at least until 4-dpi. There are several reasons why the human brain organoids might not support the active replication of SARS-CoV-2. Firstly, the developmental stages of brain organoids used in this work may not contain the full complement of SARS-CoV-2's host cell replication factors. As an example, efficient replication of SARS-CoV requires ACE-2 (39) whose expression appears to be relatively low in brain organoids ( Figure 2G) . Next, the cell tropism of SARS-CoV-2 in brain organoids is post-mitotic neurons, which may not support the generation of viral progeny (Figure 2B-C) . Future experiments using aged organoids and bioengineered organoids with SARS-CoV-2 replication factors are required to conclude if brain organoids can support productive infection of SARS-CoV-2.

ACE-2 is an entry receptor for SARS-CoV, and efficient replication of SARS-CoV (SARS outbreak in the year 2003, also depends on the expression level of ACE-2 (39, 40). Curiously, SARS-CoV could only infect the brain of transgenic mice expressing an elevated level of human ACE-2 but not non-transgenic mice. This key finding suggests that the neurotropism of SARS-CoV, to some extent, depend on the expression level of human ACE-2 in the brain (12) . From this, although unexpectedly, the brain is susceptible to SARS-CoV-2 infections, our experiments using human brain organoids reveal that human neurons are indeed vulnerable to SARS-CoV-2 infections even though they express a low level of ACE2. This finding offers a couple of possibilities. First, even a basal level of ACE2 expression is sufficient for viral entry into the neurons. Second, the presence of yet unknown neuron-specific viral entry factors has to be elucidated. Even a low level of ACE-2 is sufficient for the viral entry is an intriguing phenomenon as it could explain why SARS-CoV-2 has a broad spectrum of target organs and cell types (13) .

Our findings reveal that SARS-CoV-2 does not merely infect cortical neurons but has a consequence. SARS-CoV-2 infection induces pathological effects similar to early Tauopathies and neuronal cell death. Detection of early Tau phosphorylation at T231 in SARS-CoV-2-positive neurons is remarkable as it can trigger a cascade of downstream effects that finally could initiate neurodegenerative-like diseases. Early Tau phosphorylation could be reversible (31) . However, phosphorylation events observed in conjunction with apparent neuronal cell death suggest that SARS-CoV-2 has detrimental effects on cortical neurons (Figure 3 and 4) . Another interesting aspect to consider is that the current pandemic is the acute phase of SARS-CoV-2 infection. Thus, it is unclear what chronic or long-term effects it may cause in the CNS.

In conclusion, COVID-19 research has taken center stage in biomedical research. It is noteworthy that three coronavirus epidemics have occurred within the last two decades, and thus the future zoonotic coronavirus outbreak cannot be unexpected. With the advent of emerging human organoid research, which did not exist twenty years ago, we should be able to model the current SARS-CoV-2 infections and sufficiently prepare us for the future. Recent works utilizing kidney and gut organoids have already revealed insights into infection mechanisms (27) (28) (29) . Adding to them is the current work that establishes brain organoids as a test system for SARS-CoV-2 infection and provides indications for potential neurotoxic effects of SARS-CoV-2. Since organoids are experimentally tractable human in vitro system and convenient to culture as well as to infect, organoid systems may well serve as a test-bed to screen for anti-SARS-CoV-2 agents. The presented work only provides initial insights. Future experiments utilizing mature state of brain organoids, bioengineered organoids, and orthogonal experiments with complementary in vivo experimental models are assured to dissect the neuropathology of SARS-CoV-2. 

We want to thank Dr. Boris Görg for offering generous support with their microscope facility.

We want to thank Ms Gladiola Goranci and Nazlican Altnisk for their excellent technical assistance. This work was financially supported by a grant from Fritz-Thyssen Foundation.

"We would like to thank the diagnostics department of the Institute of Virology, University

Hospital Düsseldorf. Authors declare that they have no competing financial interests. SARS-CoV-2-exposed organoids. Four organoids from two (n=2) independent batches were examined. Unpaired t test, *P<0.05. Error bars represent mean + SD.

For the isolation of infectious SARS-CoV-2 particles, nasopharyngeal, and oropharyngeal swab specimens from one individual with positive qRT-PCR results for SARS-CoV-2 infection was used. The Swab specimen was transported in a viral cultivation medium and stored at 4°C overnight. Freezing at -20°C was found to interfere with the infectivity of viral particles. Before the inoculation of susceptible cells, 500 µL maintenance medium (Dulbecco's Modified Eagle Medium (Thermo Fisher), 2 % Fetal Calf Serum (PAN Biotech), 100 U/mL Penicillin and 100 µg/mL Streptomycin (Gibco) was added to the swab specimen. To get rid of major impurities, samples were briefly centrifuged (3000 x g; 60 sec) and the supernatant was transferred to new vials.

To obtain respiratory epithelia, a MedScand Cytobrush Plus GT (Cooper Surgical, Trumbull, USA) with a gentle-touch tip was rinsed with isotonic saline before use. Afterward the brush was inserted into the inferior nasal meatus followed by rotatory and linear motions against the medial and superior side. Isolated cells were transferred into a 15 mL centrifuge tube (Corning Incorporated, New York, USA) with 5 mL prewarmed RPMI 1640 medium containing 2 % Antibiotic-Antimycotic 100 x (Gibco® Life Technology, Grand Island, USA). The brushes were vigorously shaken several times within the tube and cells were pelletized by centrifugation at 900 rpm for five minutes at room temperature. hRECs were re-suspended in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, Gibco® Life Technology, New York, USA) supplemented with 2 % UltroserTM G Serum Substitute (Pall Corporation, Port Washington, USA) and 2 % Antibiotic-Antimycotic 100 x and seeded on T-25 or T-75 rat-tail collagen-coated tissue flasks (Greiner Bio-One, Kremsmünster, Austria), according to the pellet size respectively, and incubated at 37°C, 5 % CO2.

To reduce the risk of contamination, the medium was replaced after 24 hours, and the flasks were then integrated into the regular feeding procedure (exchange of medium every 48 -72 hours). After one week, the concentration of Antibiotic-Antimycotic was reduced to 1 %. Reaching confluency of 90 %, the collagen layer was digested by incubating with 200 U/mL collagenase type IV (Worthington Biochemical Company, New Jersey, USA) for 30 -60 min, followed by several washing steps with DMEM/F-12 supplemented with 1 % Antibiotic-Antimycotic. To reduce the number of fibroblasts, the pellet was re-suspended in 7 mL DMEM/F12 supplemented with 2 % UltroserTM G, seeded on tissue culture treated T-25 flasks (Corning Incorporated, New York, USA) and incubated for 1 hour at 37°C, 5 % CO2. The cells were then separated by incubating with Trypsin-EDTA 0,05 % for five minutes before the reaction was stopped with FBS followed by centrifugation at 900 rpm for five minutes at room temperature.

After re-suspending in PneumaCultTM-Ex Medium (STEMCELLTM Technologies, Vancouver, Canada), 4 x 105 cells/mL were seeded on collagen-coated 6,5 mm Transwell®, 0.4 µm pore Polyester membrane inserts (Corning Incorporated, New York, USA) with 250 µL medium on the apical side and 500 µL on the basolateral side, respectively. Before airlift, after three to five days, depending on cell confluency, PneumaCultTM-Ex Medium was replaced every day at the apical and basolateral side. To perform airlift, the medium on the apical side was carefully removed, whereas the basolateral medium was exchanged with PneumaCultTM-ALI Medium. The airlifted inserts were then integrated into the regular feeding procedure and incubated at 37°C, 5% CO2. Fully differentiated, the pseudostratified epithelium is expected 15-30 days after airlift and resembles human airway epithelium (in vivo) with respect to function and morphology.

In compliance with the decision of the German committee on biological agents (ABAS) of the Federal Institute for Occupational Safety and Health, all experimental studies involving infectious SARS-CoV-2 were performed within the biosafety level 3 (P3) facility at the University Hospital

Düsseldorf. For isolation of SARS-CoV-2 from a clinical specimen, 2.5x105 Vero cells (ATCC-CCL-81, obtained from LGC Standards) were seed into T25 cell culture flasks in maintenance medium and cultured at 37°C in a humidified cell culture incubator. The following day, SARS-CoV-2 inoculum was prepared by diluting 200 µL of a clinical specimen with 800 µL maintenance medium. The medium was removed from Vero cells, and 1 mL inoculum (1 mL of maintenance medium for control Vero cells) was added onto the Vero cell monolayer. Vero cells were incubated for one h on a laboratory shaker at 37°C in a humidified incubator. Afterward, 4 mL of maintenance medium were added. To monitor viral replication, 100 µL of supernatant were directly harvested as the first sample (0 h post-inoculation) and every 24 h for four days post-inoculation. Additionally, cells were imaged by light microscopy. For gene expression analysis of ACE2, quantitative RT-PCR analysis was performed by using qPCR MasterMix (PrimerDesign Ltd) and fluorescence emission was monitored by LightCycler 1.5 (Roche). For normalization, primers #5163 (5′ CCA CTC CTC CAC CTT TGA 3′) and #5164 (5′ ACC CTG TTG CTG TAG CCA 3′) were used monitoring cellular GAPDH expression. Expression was then calculated as 2 (-∆ct) .

For propagation of infectious SARS-CoV-2 particles from Vero cell culture supernatant, 2.5x105 Vero cells were seeded into T25 cell culture flasks in maintenance medium and incubated at 37°C in a humidified cell culture incubator. The next day, the supernatant of inoculated Vero cells at day four post-inoculation (see above) was diluted with maintenance medium (1:2, 1:10, 1:100, 1:1000) in a total volume of 5 mL and added to the cells, which were incubated for four days at 37°C.

For determination of viral titer in TCID50/mL, 5*10 3 Vero cells were seeded in the first ten columns of 96-well plate in 100 µL maintenance medium and incubated at 37°C in a humidified cell culture incubator for 24 h. In a new 96-well plate, 180 µL maintenance medium was added to all wells of the first ten columns. For serial dilutions of the virus stock, 20 µL of the stock solution were added to the wells of the first column. Then 20 µL of the first dilution were transferred to the wells of the next column to obtain ten-fold serial dilutions up to 10 -9 . The tenth column of the 96-well plate serves as a control. After exchanging the medium of the previously prepared Vero cell plate with 100 µL fresh maintenance medium, 100 µL of each virus dilution were transferred to the Vero cell plate. After incubation at 37°C for four days, microscopic inspection of the plate was used to monitor cytopathic effects (CPEs) in the form of detached cells. TCID50/mL was determined as:

The gel electrophoretic separation of proteins was performed under denaturing conditions in the presence of SDS in a non-continuous gel system, which consisted of a 5% stacking gel and 10% resolving gel, which was then transferred to nitrocellulose membranes. Once the transfer was finished, the membrane was soaked into 5% milk in TRIS/HCl based buffer (TBST) for a minimum of 30 min at RT. After incubating with primary antibodies overnight at 4°C, the blots were treated with secondary antibodies at RT for 1 h. Super Signal West Pico or Femto Chemiluminescent substrates (Pierce) were used for detection. Anti-body dilutions for Western blots: Human convalescent serum AB4 (1:400), polyclonal rabbit anti SARS COV-2 (1:500), monoclonal mouse anti SARS COV-2 (1:500), and mouse anti GAPDH (1:20,000, 60004-I-Ig, Proteintech). As the secondary antibodies goat anti-Mouse IgG (H+ L) HRP (1:5000, 31430, Thermo Fisher Scientific), goat anti-Rabbit IgG (H+L) HRP (1:5000, 31466, Invitrogen), Anti-human secondary antibodies conjugated to HRP (1:5000, Thermoscientific).

For light microscopy analysis, monolayer cells (Vero and aspics-derived neurons) were fixed for 10 minutes. Brain organoids were fixed for 30 minutes. We used 4% paraformaldehyde/PBS as a fixative (17) . Organoids were incubated in 30% sucrose overnight at 4°C, embedded in Tissue-Tek O.C.T. compound (Sakura, Netherlands). Organoids were cryofrozen at -80°C before sectioning into 10 to 15 µm thin slices using Cryostat Leica CM3050 S. Thin sections and cells were permeabilized with a buffer containing 0.5% Triton X100 for 10 minutes. Specimens were blocked with 0.5% fish gelatin/PBS for 1 hr, both at room temperature. For SOX2 stainings, antigen retrieval was required. For this, sections were treated with repeated heating (microwave) in Sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) was applied before permeabilization and blocking. We used different antibodies as follows: Human convalescent serum (AB1 to AB3 ( For secondary antibodies, Alexa Fluor Dyes conjugated either with goat/donkey anti-mouse, antihuman, or anti-rabbit (1:500 or 1:1000, molecular probes, Invitrogen) was used. For DNA staining, DAPI at a concentration of 1 µg/mL (Sigma Aldrich, USA) was used, and the coverslips were mounted using Mowiol (Carl Roth, Germany). The raw images were collected using a Leica SP8 confocal system (Leica microsystems, Germany) and processed with the help of Adobe Photoshop (Adobe Systems, USA). For deconvolution, the captured image files were processed using ZEN software (2.3, SP1, black, 64bit, release version 14.0.0.0; ZEISS, Oberkochen, Germany) for 3D reconstruction and deconvolution. After deconvolution, files were imported into Fiji and further processed using Image J, Adobe photoshop CC 2018, and Adobe Illustrator CC 2018. For 3D surface and volume rendering, raw image files were processed using Imaris (x64 version 7.7.1).

Apoptotic cells were detected by using DeadEnd™ Fluorometric TUNEL System (Promega, G3250, USA) according to the manufacturer´s protocol.

Serum samples AB1 and AB2 were obtained under a protocol approved by the ethical commitee, medical faculty, University Hospital Düsseldorf, Heinrich-Heine-University (study number 5350). Serum samples AB3 and AB4 were obtained under a protocol approved by the Institutional Review Board of the University of Cologne (protocol 16-054). Human respiratory epithelial cells (hREC)

were obtained by nasal brush biopsy from healthy control individuals. The study was endorsed by the local ethical committee at the University of Münster, and each patient gave informed written consent (Study number, 2015-104-f-S, Flimmerepithel) and 2020-274-f-S (COVID-19). Trained physicians from the Department of General Pediatrics, University Hospital of Münster, performed biopsies. In SARS-CoV-2-exposed 2D cultures (ii), SARS-CoV-2 (green) localizes at the soma of neurons 

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Multiorgan and Renal Tropism of SARS-CoV-2. The New England journal of medicine

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Dilution factor of consecutive dilutions

N = Total number of wells showing CPE

V = Volume per well in µL

SARS-CoV-2 infection

Each well contained one organoid in 2 mL differentiation medium and infected with SARS-CoV-2. was incubated as stationary suspension culture and treated. To exclude the effects observed were not induced by SARS-CoV-2, the control organoids (control, uninfected) were treated with supernatants of non-infected Vero cells. Generation of convalescent serum and ELISA validation AB1 and AB2 were obtained 23 and 16 days after the diagnosis of SARS-CoV-2 infection. AB3 and AB4 were obtained 27 and 28 days after the diagnosis of SARS-CoV-2 infection (by PCR). 1. Blood samples were drawn directly into serum collection tubes and spun for 15 minutes at 3.000 rpm. After centrifugation, the clear supernatant was aliquoted and stored at -80o C. ELISA was performed using semi-quantitative SARS-CoV-2-IgA and SARS-CoV-2-IgG ELISAs that detect binding against the recombinant S1 domain of the SARS-CoV-2 spike protein (Euroimmun, Lübeck, Germany). iPSCs into NPCs using STEMdiff Neural Induction Medium (Stem cell technologies, USA). Five days later, the formed neurospheres were collected and cultured on poly-L-ornithine (PLO)-/laminin coated dishes. Seven days later, using a neural rosette selection medium (Stem cell technologies, USA), we re-cultured neural rosettes to generate NPCs. NPCs were differentiated into cortical neurons as described previously (37). Briefly, NPCs were seeded on poly-L-ornithine (PLO)-/laminin coated coverslips. Forty-eight hours later, NPCs were switched to cortical neuronal differentiation medium consisting of BrainPhys basal medium(38) supplemented with 1x B27

Five days old neurospheres were harvested and embedded in matrigel (Corning, USA) drops. Differentiation medium mixture of DMEM/F12 and Neural Basal Medium (in 1:1 ratio), supplemented with 1:200 N2, 1:100 L-glutamine, 1:100 B27 w/o vitamin A, 100 U/mL penicillin, 100 µg/mL streptomycin

Assaying of SARS-CoV-2 replication and convalescent serum

CoV-2 productively replicates in inoculated Vero cells. Real-time quantitative polymerase chain reaction (qPCR) analysis of Vero cell culture supernatant shows that SARS-CoV-2 RNA drastically increases from 0-dpi until 3-dpi

Isolation of COVID-19 convalescent serum and their analysis in an enzyme-linked immunosorbent assay (ELISA)

The convalescent serum AB4 detects SARS-CoV-2-positive cells

In contrast, AB1 does not recognize cells in SARS-CoV-2-exposed organoids. None of these serums identify cells in control un-exposed organoids

Further validation of AB4. Mouse monoclonal Anti-SARS-CoV-2 S detects (magenta) SARS-CoV-2-positive cells labeled by AB4 (green). Figures display scale bars

Day-60 organoids (ii) exhibit signs of cortical maturation as distinguished by the abundance of MAP2-positive neurons in their cortical plate (CP)