key: cord-0732436-c2j1ri3t
authors: Krüger, Jana; Groß, Rüdiger; Conzelmann, Carina; Müller, Janis A.; Koepke, Lennart; Sparrer, Konstantin M.J.; Weil, Tatjana; Schütz, Desirée; Seufferlein, Thomas; Barth, Thomas F.E.; Stenger, Steffen; Heller, Sandra; Münch, Jan; Kleger, Alexander
title: Drug inhibition of SARS-CoV-2 replication in human pluripotent stem cell-derived intestinal organoids
date: 2020-11-10
journal: Cell Mol Gastroenterol Hepatol
DOI: 10.1016/j.jcmgh.2020.11.003
sha: 102ced10910bbeaaeb376df2a5e09cc923becd04
doc_id: 732436
cord_uid: c2j1ri3t

Background and aims The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in COVID-19 patients are of urgent need. Methods Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. Results Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. Conclusions Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects the respiratory tract with mostly mild symptoms, but up to 20% of patients develop severe pneumonia eventually followed by multi-organ failure and death 1 . Intriguingly, up to 50% of patients present with gastrointestinal symptoms, associated with prolonged disease duration and increased severity [2] [3] [4] [5] . Viral RNA is detected in rectal swabs and feces long after nasopharyngeal swabs tested negative 6, 7 . Infection of host cells with SARS-CoV-2 requires angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). Both proteins mediate multiorgan tropism, as they are detected in esophagus, ileum, and colon 5, [8] [9] [10] [11] . The advent of organotypic cell culture systems provides a roadmap to successfully set up personalized medicine approaches. Organotypic cultures, also referred to as organoids, can be defined as three-dimensional (3D) structures derived either from pluripotent or organ-restricted stem cells harboring the ability to mimic in vivo architecture and multilineage differentiation of terminally differentiated tissues [12] [13] [14] [15] . Both model systems are derived from a cell source with unlimited self-renewal potentially allowing unlimited replenishments of the desired cell type or tissue such as the gut 16 . While single layered human intestinal organoids (HIOs) derived from human adult gut stem cells contain only epithelial cell types 17 , pluripotent stem cells derived intestinal organoids (PSC-HIO) comprise endodermal and mesodermal progeny 18 resembling epithelium as well as fibroblasts or blood vessels of the gut, respectively 19 . Thus, HIOs are less complex in architecture and lack in vivo transplantability as well as analytical access to developmental intermediate stages as compared to PSC-HIOs, while the latter are not fully mature [17] [18] [19] . In turn, both models are comparable and complement each other with model-specific advantages and limitations. HIOs express ACE2 and are susceptible to SARS-CoV-2 [20] [21] [22] [23] . Albeit the exploration of new drugs is rapidly evolving, knowledge on their efficiency to inhibit intestinal infection of SARS-CoV-2 is at first unknown.

However, drug testing might require a more complex organotypic culture system to reflect the true value of a given drug 24 . Remdesivir is up to now the sole agent showing benefit on pulmonary phenotypes in COVID-19 patients 25 . The histamine-2-blocker famotidine has been suggested to reduce severe COVID-19 course 26, 27 . Meanwhile, recent in silico predictions suggest that famotidine could interact with the catalytic site of three proteases (chymotrypsin-like, papain-like and TMPRSS2 protease) involved in SARS-CoV-2 replication. Notably, famotidine binding affinity was low to these proteases suggesting that additional interventions such as direct antivirals might be necessary to reach full efficiency 28 . In contrast, a recent case series provided promising results by high-dose oral famotidine administration being well tolerated and associated with improved patient-reported outcomes in non-hospitalized patients with COVID-19 29 . Here, we employ PSC-HIOs to study SARS-CoV-2 tropism with respect to distinct intestinal cell types and test such drug candidates in a human organotypic culture system resembling a natural 3D environment. 

First, we studied expression of ACE2 and TMPRSS2 in organs of the gastrointestinal tract.

Expression of both SARS-CoV-2 entry factors was most prominent in the epithelial lining of duodenum, gallbladder, and colon as compared to Caco-2 cells (Figure 1A-C) . Duodenal cells showed apical ACE2 expression in the glycocalyx and a strong cytoplasmic TMPRSS2 signal ( Figure   1A 

To determine susceptibility to in vitro infection, PSC-HIOs were exposed to SARS-CoV-2, and expression of viral spike (S) and nucleocapsid (N) protein together with the epithelial marker E cadherin (Ecad) or respective labels for more specialized cell types (CHGA, LYZ, MUC2) were analyzed. Upon SARS-CoV-2 infection of PSC-HIOs, S protein was detected in 10% of the cells after 24 h, which increased to 57% at 48 h, suggesting viral replication and spreading infection (Figure   2A ,B). Co-staining for N protein confirmed this finding ( Figure 2C) The over-the-counter histamine-2 receptor antagonist famotidine is a putative therapeutic agent for COVID-19 28, 29 . Thus, we tested famotidine concentrations of up to 2.5 mM but did not observe These tissue-specific differences should be subject to further investigation. deterioration. This would be in line with low binding affinity of famotidine to SARS-CoV-2 proteases 28 , however, its effect on host-specific immune response as well as tissue specific efficiency might be of relevance to ultimately judge its clinical value particularly to treat life-limiting lung infection. In line, histamine-2 receptor antagonists are able to modulate histamine effector pathways by receptor binding, thus directing immunomodulatory effects 36 . Specifically, they can boost the innate immune function and revert negative effects of histamine on production and release of cytokines such as TNF-α and interferon-γ possibly supporting immune response during viral infection 37, 38 . Accordingly, a recent case series suggested efficiency to treat mild/moderate COVID-19 with pulmonary parameters as a readout 29 . Thus, the current clinical study landscape remains in support of famotidine 26 albeit our in vitro data appear contrary and warrants further investigation in randomized controlled trials, best with taking gastrointestinal facets of COVID-19 into account.

Overall, our data demonstrate that SARS-CoV-2 can infect all investigated cell types of the gut, with the exception of goblet cells, and can perturb intestinal integrity, which might lead to diarrhea amongst many other gastrointestinal symptoms frequently reported by COVID-19 patients. Most importantly, we tested and validated our culture platform as an indefinite resource to organ specific 

ORF1b-nsp14 (ORF1b) 42 deparaffinized, rehydrated and subjected to heat mediated antigen retrieval in tris Buffer (pH 9) or citrate buffer (pH 6). Tissue was permeabilized with 0.5% Triton-X for 30 min at room temperature, stained over night with primary antibodies in antibody diluent (Zytomed) in a wet chamber at 4°C.

After washing with PBS-T, slides were incubated with secondary antibodies (Alexa Fluor IgG H+L, Invitrogen, 1:500) and 500 ng/ml DAPI in Antibody Diluent for 90 min in a wet chamber at room temperature. After washing with PBS-T and water, slides were mounted with Fluoromount-G (Southern Biotech). Negative controls were performed using IgG controls or irrelevant polyclonal serum (anti-mycobacterium tuberculosis) for polyclonal antibodies, respectively. Absence of background staining confirmed specificity of the primary antibodies. Images were acquired with Keyence BZ 9000 microscope, quantified with ImageJ. Laser Scanning confocal images were acquired using the Zeiss LSM710. Z-stacks were deconvoluted and edited in Huygens (Scientific Volume Imaging) and Fiji (ImageJ).

Statistics were performed using GraphPad Prism. Inhibitory concentration 50 (IC50) was determined by nonlinear regression (Inhibitor) vs. normalized response. For Figure 3G , 4D and 5D one-way ANOVA analysis and Dunnett's multiple comparison test were performed. Data of Figure   2E was analyzed with a paired T-test. P-values < 0.05 were classified as significant (*p < 0,05, ** p < 0,01, *** p < 0,001, **** p < 0,0001). Time course (d) Viral genome copies/ml (log 10 ) CoV-N CoV-ORF1b 

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