key: cord-343317-97n1j0jj
authors: Duan, Xiaohua; Han, Yuling; Yang, Liuliu; Nilsson-Payant, Benjamin E.; Wang, Pengfei; Zhang, Tuo; Xiang, Jenny; Xu, Dong; Wang, Xing; Uhl, Skyler; Huang, Yaoxing; Chen, Huanhuan Joyce; Wang, Hui; tenOever, Benjamin; Schwartz, Robert E.; Ho, David. D.; Evans, Todd; Pan, Fong Cheng; Chen, Shuibing
title: Identification of Drugs Blocking SARS-CoV-2 Infection using Human Pluripotent Stem Cell-derived Colonic Organoids
date: 2020-05-02
journal: bioRxiv
DOI: 10.1101/2020.05.02.073320
sha: 
doc_id: 343317
cord_uid: 97n1j0jj

The current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to screen 1280 FDA-approved drugs against viral infection. Mycophenolic acid and quinacrine dihydrochloride were found to block the infection of SARS-CoV-2 pseudo-entry virus in COs both in vitro and in vivo, and confirmed to block infection of SARS-CoV-2 virus. This study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified drugs that blocks SARS-CoV-2 infection, suitable for rapid clinical testing.

The current COVID-19 pandemic is caused by SARS-coronavirus 2 (SARS-CoV-2).

There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients typically present with respiratory symptoms including cough, dyspnea, and respiratory distress, but nearly 25% of patients have gastrointestinal indications including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with worse COVID-19 outcomes 1 . Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple hESC-derived colonic cell types, but highly enriched in enterocytes. Multiple cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which was further validated in vivo using a humanized mouse model. We used hPSC-derived COs in a high throughput platform to Previously, we reported a chemically-defined protocol to derive COs from hPSCs 2 , which we modified slightly based on published studies 3 . In brief, HUES8 hESCs were induced with CHIR99021 (CHIR) and Activin A to generate definitive endoderm (DE) (Extended Data Fig. 1a) . After 4 days of culture with CHIR +FGF4 to induce hindgut endoderm (HE), cells were treated with BMP2, epidermal growth factor (EGF), and CHIR for 3 days to promote specification of colon progenitors (CPs). Starting on day 11, CPs were treated with a colonic medium containing CHIR, LDN193189 (LDN), and EGF.

After embedding these organoids in Matrigel, spheroids became pseudostratified and progressively cavitated into fully convoluted organoids (Fig. 1a) . The organoids expressed CDX2, Villin and SATB2, confirming colonic identity (Fig. 1b) .

Immunocytochemistry confirmed that COs contain cell types found in normal colon, including keratin 20 (KRT20) + epithelial cells, mucin 2 (MUC2) + goblet cells, EPH receptor B2 (EPHB2) + transit-amplifying (TA) cells, and chromogranin A (CHGA) + neuroendocrine (NE) cells (Fig. 1c) .

Single cell RNA-seq was used to examine global transcript profiles at single cell resolution (Extended Data Fig. 1b) . Consistent with the immunostaining results, most cells express CDX2 and VIL1 (Extended Data Fig. 1c) . Five cell clusters were identified including KRT20 + epithelial cells, MUC2 + goblet cells, EPHB2 + TA cells, CHGA + NE cells, and LGR5 + or BMI1 + stem cells (Fig. 1d-e, Extended Data Fig. 1d) .

We examined the expression of two factors associated with SARS-CoV-2 cell entry, the putative receptor ACE2 and the protease TMPRSS2 4 . Both are expressed in all five cell clusters, but highly enriched in KRT20 + enterocytes ( Fig. 1f-g) . Two-dimensional correlation confirmed the co-expression relationship for ACE2 and KRT20, as well as ACE2 and TMPRSS2 (Fig. 1h) . Immunohistochemistry further validated the coexpression of KRT20 and ACE2 in hPSC-COs (Fig. 1i) .

To model infection of hPSC-COs with SARS-CoV-2, we used a vesicular stomatitis virus (VSV) based SARS-CoV-2 pseudo-entry virus, with the backbone provided by a VSV-G pseudo-typed Δ G-luciferase virus and the SARS-CoV-2 spike protein incorporated into the surface of the viral particle (See Methods for details) 5, 6 . COs were fragmentized and innoculated with the SARS-CoV-2 pseudo-entry virus. 24 or 48 hr post-infection (hpi), the cells were lysed and monitored for luciferase activity (Extended Data Fig. 2a) . The organoids infected with SARS-CoV-2 pseudo-entry virus at MOI=0.01 showed a strong signal at 24 hpi (Fig. 2a) . Single cell RNA-seq was performed to examine the hPSCderived COs at 24 hpi. The same five cell populations were identified in the COs postinfection ( Fig. 2b and Extended Data Fig. 2b-d) . Compared to uninfected samples, the KRT20 + enterocyte population decreased significantly (Fig. 2c) . Immunostaining confirmed increased cellular apoptosis, suggesting toxicity for these cells (Extended Data Fig. 2e ). In addition, the ACE2 + population was significantly depleted (Fig. 2e) .

The mRNAs of SARS-CoV-2 pseudo-entry virus, including VSV-NS, VSV-N, and VSV-M, were detected in all five cell populations (Fig. 2f) , but not in the uninfected COs (Extended Data Fig. 2f) . Immunostaining further validated the expression of luciferase in ACE2 + , VIL1 + , CDX2 + , KRT20 + , and MUC2 + cells (Fig. 2g) .

Humanized mice carrying hPSC-COs in vivo provide a unique platform for modeling COVID-19. In brief, hPSC-COs were transplanted under the kidney capsule of NODscid IL2Rg null mice. Two weeks after transplantation, the organoid xenograft was removed and examined for cellular identities (Fig. 2h) . Consistent with in vitro culture, ACE2 can be detected in hPSC-derived KRT20 + enterocytes (Fig. 2i) . SARS-CoV-2 pseudo-entry virus was inoculated locally. At 24 hpi, the xenografts were removed and analyzed by immuno-histochemistry. Luciferase was detected in the xenografts inoculated with virus, but not in MOCK-infected controls (Fig. 2j) . Immunohistochemistry detected luciferase in ACE2 + and Villin + cells, suggesting these are permissive to SARS-CoV-2 pseudo-entry virus infection in vivo (Fig. 2k) .

Next, we adapted hPSC-COs to a high throughput screening platform and probed the Prestwick FDA-approved drug library to identify drug candidates capable of blocking SARS-CoV-2 pseudo-virus infection. In brief, hPSC-COs were cultured in 384-well plates. After overnight incubation, organoids were treated with drugs from the library at 10 μ M. One hour post-exposure with drugs, the organoids were innoculated with the SARS-CoV-2 pseudo-entry virus. 24 hpi, the organoids were analyzed for luciferase activity (Fig. 3a) . Drugs that decreased the luciferase activity by at least 75% were chosen as primary hit drugs (Fig. 3b) . Eight drugs (Extended Data Table 1 ) were identified as lead hits and further tested for their capacities to decrease the luciferase signal in a dose-dependent manner (Extended Data Fig. 3) . These drugs could potentially function through blocking virus entry, by decreased cell survival, or even by directly inhibiting luciferase activity. To distinguish these possibilities, the lead hit drugs were tested in comparison to hPSC-COs infected with a control VSVG-luciferase reporter virus. Four of the lead hit drugs showed specificity to SARS-CoV-2 pseudoentry virus, including mycophenolic acid (MPA, Fig. 3c Fig. 3h) . CO-explanted humanized mice were treated with 50 mg/kg MPA by IP injection, followed by local inoculation of SARS-CoV-2 pseudo-entry virus. At 24 hpi, the mice were euthanized, and xenografts were analyzed by immunostaining. Luc + cells in the xenografts of MPA-treated mice were significantly lower than those of vehicletreated mice (Fig. 3i-k) .

Finally, hPSC-COs were infected with SARS-CoV-2 virus at MOI=0.1 or 0.01. At 24 hpi, immunostaining detected the expression of SARS-CoV membrane protein in the infected hPSC-COs, which partially co-localized with CDX2 and KRT20 (Fig. 4a) . Bulk RNA sequencing confirmed viral transcripts in the SARS-CoV-2 infected hPSC-COs (MOI=0 .1, Fig. 4b) . The MOCK and infected hPSC-COs separated clearly into two distinct clusters in a PCA plot (Fig. 4c) . Differential gene expression analysis showed striking induction of chemokine gene expression, including for IL1A, CXCL8, CXCL6, CXCL11, and IL1B, yet with no detectable levels of IFN-I or IFN-III, which is consistent with recent reports [8] [9] [10] (Fig. 4d) . Ingenuity Pathway Analysis of the differential gene expression list highlighted the production of nitric oxide and reactive oxygen species, oxidative phosphorylation, as well as IL-15 production (Fig. 4e) . The hPSC-COs were pre-treated with MPA or QNHC and infected with a relatively high titer of SARS-CoV-2 virus (MOI=0.1). Immunostaining confirmed the decrease of SARS-CoV-2 + cells in MPA or QNHC-treated hPSC-COs (Fig. 4f) . In summary, we report that hPSC-derived COs express ACE2 and TMPRS2S2 and are permissive to SARS-CoV-2 infection. There is currently a lack of physiologically relevant models for COVID-19 disease that enable drug screens. Previous studies were based on clinical data or transgenic animals, for example mice that express human ACE2.

However, such transgenic animals fail to fully recapitulate the cellular phenotype and host response of human cells 11, 12 . We adapted a hPSC-derived CO platform for high throughput drug screening. Using disease-relevant normal colonic human cells, we screened 1280 FDA-approved compounds and identified MPA and QNHC, two drugs that can block the entry of SARS-CoV-2 into human cells. Strikingly, in this assay, the efficacies of MPA and QNHC for blocking viral entry are more than 5 times higher than chloroquine, a drug recently authorized by the FDA for emergency use to treat COVID-19 patients. Moreover, the MPA concentrations effective in blocking viral entry and replication are below that which is routinely used in clinical therapy 13 . MPA is a reversible, non-competitive inhibitor of inosine-5′-monophosphate dehydrogenase and is used widely and safely as an immunosuppressive drug (mycophenolate mofetil; CellCept) to prevent organ rejection after transplantation and for the treatment of autoimmune diseases 14 . MPA has been reported to block replication of human immunodeficiency virus 15 , dengue 16 , as well as Middle East respiratory syndrome coronavirus (MERS-CoV) 17 . Several studies on MERS-CoV suggest that MPA may noncompetitively inhibit the viral papain-like protease while also altering host interferon response 17, 18 . A recent study also predicts MPA would modulate the interaction between host protein inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) and SARS-CoV-2 protein nonstructural protein 14 (nsp14) 19 . Furthermore, a clinical study of MERS-CoV suggested that the patients treated with mycophenolate mofetil has 0% mortality rate, which is significantly lower than the overall mortality rate as 37% 20 . QNHC (Acriquine ® , Atabrine ® , Atebrin ® , Mepacrine ® ) is an FDA-approved antimalarial drug used more recently as an anthelmintic, antiprotozoal, antineoplastic agent, and antirheumatic 21 .

Recent studies have shown that quinacrine protects mice against Ebola virus infection in vivo 22 . Both MPA and QNHC can be considered candidates for clinical trials of COVID-19 therapy. 

Recombinant Indiana VSV (rVSV) expressing SARS-CoV-2 spikes were generated as previously described 23 . HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV2-spike (kindly provided by Dr. Peihui Wang, Shandong University, China) using FuGENE 6 (Promega). Cells were cultured overnight at 37°C with 5% CO2. The next day, medium was removed and VSV-G pseudotyped Δ G-luciferase (G*ΔG-luciferase, Kerafast) was used to infect the cells in DMEM at an MOI of 3 for 1 hr before washing the cells with 1X DPBS three times. DMEM supplemented with 2% fetal bovine serum and 100 I.U./mL penicillin and 100 μ g/mL streptomycin was added to the infected cells and they were cultured overnight as described above. The next day, the supernatant was harvested and clarified by centrifugation at 300g for 10 min and aliquots stored at −80°C.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), isolate USA- 

To assay pseudo-typed virus infection on colon organoids, COs were seeded in 24 well plates. Pseudo-typed virus was added at MOI=0.01 plus polybrene at a final concentration of 8 μ g/mL, and the plate centrifuged for 1 hr at 1200g. At 3 hpi, the infection medium was replaced with fresh medium. At 24 hpi, colon organoids were harvested for luciferase assays or immunostaining analysis. For chemical screening analysis, colon organoids were digested by TrypLE and seeded in 384 well plates at 1x10 4 cells per well. After chemical treatment, pseudo-typed virus was added at MOI=0.01 and the plate centrifuged for 1 hr at 1200g. At 24 hpi, hPSC-COs were harvested for luciferase assays according to the Luciferase Assay System protocol (Promega). Cells were lysed in RIPA buffer for protein analysis or fixed in 5% formaldehyde for 24 h for immunofluorescent staining, prior to safe removal from the BSL-3 facility.

The colon organoids were released from Matrigel using Cell Recovery Solution (Corning) on ice for 1 hr, followed by fixation in 4% paraformaldehyde for 4 hr at 4°C, washed twice with 1X PBS and allowed to sediment in 30% sucrose overnight. The organoids were then embedded in OCT (TissueTek) and cryo-sectioned at 10 μ m thickness. For indirect immunofluorescence staining, sections were rehydrated in 1X PBS for 5 min, permeabilized with 0.2% Triton in 1X PBS for 10 min, and blocked with blocking buffer containing 5% normal donkey serum in 1X PBS for 1 hr. The sections were then incubated with the corresponding primary antibodies diluted in blocking buffer at 4°C overnight. The following day, sections were washed three times with 1X PBS before incubating with fluorophore-conjugated secondary antibody for one hr at RT. The sections were washed three times with 1X PBS and mounted with Prolong Gold Antifade mounting media with DAPI (Life technologies). Images were acquired using an LSM880 Laser Scanning Confocal Microscope (Zeiss) and processed with Zen or Imaris (Bitplane) software.

Organoids and tissues were fixed in 4% PFA for 20 min at RT, blocked in Mg 2+ /Ca 2+ free PBS plus 5% horse serum and 0.3% Triton-X for 1 hr at RT, and then incubated with primary antibody at 4°C overnight. The information for primary antibodies is provided in Extended Data Table 2 . Secondary antibodies included donkey anti-mouse, goat, rabbit or chicken antibodies conjugated with Alexa-Fluor-488, Alexa-Fluor-594 or Alexa-Fluor-647 fluorophores (1:500, Life Technologies). Nuclei were counterstained by DAPI. antibodies. Antibody-mediated fluorescence was detected on a LI-COR Odyssey CLx imaging system and analyzed using Image Studio software (LI-COR).

The colon organoids cultured in Matrigel domes were dissociated into single cells using 0.25% Trypsin (Gibco) at 37°C for 10 min, and the trypsin was then neutralized with DMEM F12 supplemented with 10% FBS. The dissociated organoids were pelleted and resuspended with L15 Medium (Gibco) supplemented with 10 mM HEPES, and 10 ng/ml DNaseI (Sigma). The resuspended organoids were then placed through a 40 µm filter to obtain a single cell suspension, and stained with DAPI followed by sorting of live 

We filtered cells with less than 300 or more than 8000 genes detected as well as cells with mitochondria gene content greater than 30%, and used the remaining cells (6175 cells for the uninfected sample and 2962 cells for the infected sample) for downstream analysis. We normalized the gene expression UMI counts for each sample separately using a deconvolution strategy 24 implemented by the R scran package (v.1.14.1). In particular, we pre-clustered cells in each sample using the quickCluster function; we computed size factor per cell within each cluster and rescaled the size factors by normalization between clusters using the computeSumFactors function; and we normalized the UMI counts per cell by the size factors and took a logarithm transform using the normalize function. We further normalized the UMI counts across samples using the multiBatchNorm function in the R batchelor package (v1.2.1). We identified highly variable genes using the FindVariableFeatures function in the R Seurat (v3.1.0) 25 , and selected the top 3000 variable genes after excluding mitochondria genes, ribosomal genes and dissociation-related genes. The list of dissociation-related genes was originally built on mouse data 26 , we converted them to human ortholog genes using Ensembl BioMart. We aligned the two samples based on their mutual nearest neighbors (MNNs) using the fastMNN function in the R batchelor package, this was done by performing a principal component analysis (PCA) on the highly variable genes and then correcting the principal components (PCs) according to their MNNs. We selected the corrected top 50 PCs for downstream visualization and clustering analysis. We ran the uniform manifold approximation and projection (UMAP) dimensional reduction using the RunUMAP function in the R Seurat 25 package with training epochs setting to 2000. We clustered cells into eight clusters by constructing a shared nearest neighbor graph and then grouping cells of similar transcriptome profiles using the FindNeighbors function and FindClusters function (resolution set to 0.2) in the R Seurat package. We identified marker genes for each cluster by performing differential expression analysis between cells inside and outside that cluster using the FindMarkers function in the R Seurat package. After reviewing the clusters, we merged four clusters that were likely from stem cell population into a single cluster (LGR5 + or BMI1 + stem cells) and kept the other four clusters (KRT20 + epithelial cells, MUC2 + goblet cells, EPHB2 + TA cells, and CHGA + NE cells) for further analysis. We re-identified marker genes for the merged five clusters and selected the top 10 positive marker genes per cluster for heatmap plot using the DoHeatmap function in the R Seurat package 25 .

hPSC-COs were harvested by cell scraper, mixed with 20 µl Matrigel (Corning) and transplanted under the kidney capsule of 7-9 weeks old male NSG mice. Two weeks post-transplantation, SARS-CoV-2 pseudo-entry virus was inoculated locally at 1x10 3 FFU. At 24 hpi, the mice were euthanized and used for immunohistochemistry analysis.

To determine the MPA's activity in vivo, the mice were treated with 50 mg/kg MPA in (10%DMSO/90% corn oil) by IP injection. Two hours after drug administration, SARS-CoV-2 pseudo-entry virus was inoculated locally at 1x10 3 FFU. At 24 hpi, the mice were euthanized and used for immunohistochemistry analysis.

All animal work was conducted in agreement with NIH guidelines and approved by the WCM Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC).

To perform the high throughput small molecule screen, hPSC-COs were dissociated using TrypLE for 20 min in a 37℃ waterbath and replated into 10% Matrigel-coated 384well plates at 20,000 cells/40 µl medium/well. After 6 hr, cells were treated with compounds from an in-house library of ~1280 FDA-approved drugs (Prestwick) at 10 µM. DMSO treatment was used as a negative control. One hour late, cells will be infected with SARS-CoV-2 pseudo virus (MOI=0.01). After 24 hpi, hPSC-COs were harvested for luciferase assay following the Luciferase Assay System protocol (Promega). 

The authors declare the following competing interests: R.E.S. is on the scientific advisory board of Miromatrix Inc. The other authors have no competing of interest.

scRNA-seq and RNA-seq data is available from the GEO repository database, accession number GSE147975. Expression level Identity a. NPM1  CIQBP  LDHB  MGST1  SNHG8  NCL  PLIN2  HIST1H4C  LCN15  GDF15  PHLDA2  IGFBP3  LRRC75A  S100A16  EMP3  CD24  B4GALT1  CEACAM6  HNRNPH1  ANPEP  ALDOB  RBP2  TM4SF2O  SELENOP  FABP6  TSPAN1  MUC13  APOA1  KCTD12  SCGN  TUBAIA  OR51E1  NPW  CYBA2  TPH1  CHGA  MDK  CDKN1C  CLCA1  FCGBP  MUC2  GUCA2A  SELENOM  FRZB  ST6GALNAC1  HES6  TFF3  REG4 a. 

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