key: cord-0982988-ju6t5nc8
authors: Sano, Emi; Sakamoto, Ayaka; Mimura, Natsumi; Hirabayashi, Ai; Muramoto, Yukiko; Noda, Takeshi; Yamamoto, Takuya; Takayama, Kazuo
title: Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells
date: 2021-02-22
journal: bioRxiv
DOI: 10.1101/2021.02.22.432218
sha: a214b84544ca1c6ba095339b4a7eda806440722c
doc_id: 982988
cord_uid: ju6t5nc8

Genetic differences are a primary reason for differences in the susceptibility and severity of coronavirus disease 2019 (COVID-19). Because induced pluripotent stem (iPS) cells maintain the genetic information of the donor, they can be used to model individual differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro. Notably, undifferentiated human iPS cells themselves cannot be infected bySARS-CoV-2. Using adenovirus vectors, here we found that human iPS cells expressing the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) (ACE2-iPS cells) can be infected with SARS-CoV-2. In infected ACE2-iPS cells, the expression of SARS-CoV-2 nucleocapsid protein, the budding of viral particles, the production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. We also evaluated COVID-19 therapeutic drugs in ACE2-iPS cells and confirmed the strong antiviral effects of Remdesivir, EIDD-2801, and interferon-beta. In addition, we performed SARS-CoV-2 infection experiments on ACE2-iPS/ES cells from 8 individuals. Male iPS/ES cells were more capable of producing the virus as compared with female iPS/ES cells. These findings suggest that ACE2-iPS cells can not only reproduce individual differences in SARS-CoV-2 infection in vitro, but they are also a useful resource to clarify the causes of individual differences in COVID-19 due to genetic differences. Graphical Abstract

The number of coronavirus disease 2019 patients and deaths continues to rise. Interestingly, the symptoms of COVID-19 are known to vary widely among individuals and include asymptomatic cases. Genetic differences are one cause for the differences in susceptibility and severity of COVID-19 (Anastassopoulou et al., 2020; Group, 2020; Pairo-Castineira et al., 2020; Zeberg and Pääbo, 2020) .

Approximately twenty percent of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will present severe symptoms (Weiss and Murdoch, 2020) . To develop drugs for patients with severe COVID-19, it is necessary to identify the causes of the worsening symptoms. Although several models, including cells such as Vero, Calu-3, and Caco-2 cells, organoids, and animals such as angiotensin-converting enzyme 2 (ACE2)-transgenic mice, hamsters, and ferrets have been used to study SARS-CoV-2 infection (Takayama, 2020) , they do not reproduce individual differences well.

individual (Takahashi et al., 2007) . Furthermore, they are widely used as genetic disease models because they inherit the genetic information of their donors (Park et al., 2008) .

Our institute (Center for iPS Cell Research and Application, CiRA) has established iPS cells from more than 500 individuals. Because this iPS cell panel was established from a human population of diverse genetic backgrounds, it can be a resource for reproducing individual differences in response to pathogens and drugs. Accordingly, in this study, we attempted to reproduce the SARS-CoV-2 infection and its individual differences using this panel.

It has been reported that SARS-CoV-2 infection is dependent on the expression of ACE2 and transmembrane protease, serine 2 (TMPRSS2) in host cells (Hoffmann et al., 2020) . Type II alveolar epithelial cells, bronchial ciliated cells, pharyngeal epithelial cells, and intestinal epithelial cells all express high levels of ACE2 (Ziegler et al., 2020) and can be easily infected by SARS-CoV-2. It has also been reported that embryonic stem (ES) cell-derived type II alveolar epithelial cells and intestinal epithelial cells can be infected with SARS-CoV-2 (Han et al., 2021) . However, the investigation of infection differences at the individual level and large scale using iPS cell-derived somatic cells is hindered by the long time (generally more than 3 weeks) to differentiate the iPS cells and the variable differentiation efficiency among iPS cell lines (Kajiwara et al., 2012) . SARS-CoV-2 infection experiments in undifferentiated iPS cells would reduce the time. However, due to the low expression of ACE2 and TMPRSS2, SARS-CoV-2 does not infect iPS cells. Therefore, in this study, we identified SARS-CoV-2-related genes that enable SARS-CoV-2 to infect undifferentiated human iPS cells. To facilitate gene overexpression experiments with multiple iPS cell lines, we used adenovirus (Ad) vectors. Because the gene transfer efficiency of Ad vectors to undifferentiated iPS cells is almost 100%, there was no need to obtain clones expressing SARS-CoV-2-related genes. Next, we investigated whether the life cycle of SARS-CoV-2 can be reproduced in iPS cells expressing these SARS-CoV-2-related genes. We also show the value of ES/iPS cells to test drugs and gender effects. Our findings indicate that iPS cells expressing SARS-CoV-2-related genes can be a useful to study SARS-CoV-2 infection and its individual patient differences.

First, we examined whether undifferentiated iPS cells could be infected by SARS-CoV-2 (Fig. S1A) . Before conducting this experiment, we examined the expression levels of viral receptors and proteases in undifferentiated iPS cells (Fig.   S1B) . The gene expression level of ACE2 was low, but that of CD147 was high. CD147 is reported as a coronavirus receptor (Wang et al., 2020) . TMPRSS2, a protease, was also expressed in undifferentiated iPS cells. We thus tried to infect undifferentiated iPS cells with SARS-CoV-2, but the morphology of the iPS cell colonies did not change ( Fig. S1C ). In addition, virus genome in the cell culture supernatant (Fig. S1D ) and the production of infectious virus (Fig. S1E) were not detected. The gene expression levels of undifferentiated markers (Fig. S2A ) and innate immune response-related markers ( Fig. S2B) were also unchanged. Furthermore, the expression of SARS-CoV-2 nucleocapsid (N) protein was not detected (Fig. S2C) . Together, these results indicated that SARS-CoV-2 does not infect undifferentiated iPS cells.

Because human ACE2 and TMPRSS2 are known to be important for SARS-CoV-2 to infect cells, we overexpressed human ACE2 and TMPRSS2 in undifferentiated iPS cells by using Ad vectors (Fig. 1A) . The overexpression of ACE2 in iPS cells (ACE2-iPS cells) caused a large amount of SARS-CoV-2 infection (Fig.   1B) . Additionally, the amount of virus genome in the cell culture supernatant increased (Fig. 1C) . This was not the case if only overexpressing TMPRSS2. Furthermore, two days after the ACE2-iPS cells were infected with SARS-CoV-2, cell fusion was observed (Fig. 1D) , and four days after many of the cells died. Therefore, these results indicate that ACE2 expression is required for SARS-CoV-2 to infect undifferentiated iPS cells.

Transmission electron microscope (TEM) images of ACE2-iPS cells infected with SARS-CoV-2 were obtained ( Figs. 2 and S3 ). Zippered endoplasmic reticulum ( Fig. 2B) , double-membrane spherules (DMS) (Fig. 2B ) (Maier et al., 2013) , and viral particles near the cell membrane (black arrow) (Fig. 2D) black arrows) were also observed in infected ACE2-iPS cells (Fig. S3) . DMVs are the central hubs for viral RNA synthesis (Klein et al., 2020) . These structures were not observed in the uninfected ACE2-iPS cells. These TEM images show that the life cycle of SARS-CoV-2 can be observed in ACE2-iPS cells.

Next, we analyzed gene and protein expressions in uninfected and infected iPS cells 3 days after the viral infection. The intracellular viral genome and ACE2 expression levels in ACE2-iPS cells infected with SARS-CoV-2 were high ( Fig. 3A) . At the same time, ACE2 overexpression and SARS-CoV-2 infection did not alter the gene expression levels of undifferentiated markers (Fig. 3B ) or innate immune responserelated markers (Fig. 3C) . The gene expression levels of endoderm markers except for CER1 ( Fig. S4A ) and SARS-CoV-2-related genes (CD147, NRP1, and TMPRSS2) (Figs. S4B, 3A) were also unchanged. Immunostaining data showed that SARS-CoV-2 N protein was strongly expressed in ACE2-iPS cells 2 days after the infection (Figs. 3D,

We also performed RNA-seq analysis in uninfected and infected ACE2-iPS cells. The colored dots in the volcano plot in figure 4A indicate genes whose expression levels changed significantly more than 4-fold. In total, this change occurred in 6.7% of all genes (Fig. S6A) . A GO term analysis was performed for these genes (Figs. S6B, S6C). None of the genes included undifferentiated markers (Fig. 4B ) or innate immuneresponse markers (Fig. 4C) . The gene expression levels of ectoderm, mesoderm, and endoderm markers were also unchanged after infection with SARS-CoV-2 (Fig. S7) .

Overall, these results suggest that human iPS cells maintain an undifferentiated state even when SARS-CoV-2 replicates in large numbers.

Evaluation of COVID-19 candidate drugs using ACE2-iPS cells Next, we examined whether ACE2-iPS cells could be used for drug screening.

We tried eight drugs used in COVID-19 clinical trials. Vero cells were used as the control. After exposing the cells to various concentrations of a drug, the number of viral RNA copies in the culture supernatant was quantified (Fig. 5A) . Data fitting resulted in sigmoid curves, and half-maximal effective concentrations (EC50) were calculated (Fig.   5B ). Among the eight drugs, the antiviral effect of Remdesivir was strongest. On the other hand, Chloroquine and Favipiravir did not inhibit viral replication, and Ivermectin was highly cytotoxic (Fig. S8A) . The EC50 and half-maximal cytotoxic concentration (CC50) values of Ivermectin were almost the same between control and infected ACE2-iPS cells (Fig. S8B) . With the exception of interferon-beta, drug effects were stronger in ACE2-iPS cells than in Vero cells. Lastly, we confirmed the anti-viral effects of RNAdependent RNA Polymerase (RdRp) inhibitors (Remdesivir and EIDD-2801) and

TMPRSS2 inhibitors (Camostat and Nafamostat) in ACE2-iPS cells, indicating that ACE2-iPS cells can be used to evaluate COVID-19 drug candidates.

Finally, we performed SARS-CoV-2 infection experiments using human ACE2-iPS/ES cells established from eight donors. The replication efficiency of the virus was different among the ACE2-iPS/ES cell lines (Fig. 6A) . Note that there was no significant difference in ACE2 expression levels in the ACE2-iPS/ES cell lines (Fig.   S9 ). Interestingly, the viral replication capacity of male ACE2-iPS/ES cells was higher than that of female ACE2-iPS/ES cells (Fig. 6B) , suggesting that sex differences in the susceptibility to SARS-CoV-2 can be reproduced using ACE2-iPS/ES cells. Recently, it has been speculated that the expression levels of androgen receptor and its target gene, TMPRSS2, are involved in the sex differences in the SARS-CoV-2 infection (Wambier et al., 2020) . The TMPRSS2 expression levels appeared to be higher in male iPS/ES cells than in female iPS/ES cells (Fig. 6C) , but there was no significant difference ( Fig.   6D ).

In this study, we showed that the life cycle of SARS-CoV-2 can be reproduced in human iPS cells overexpressing ACE2. In addition, we were able to confirm the effects of two TMPRSS2 inhibitors (Camostat and Nafamostat) and two RdRp inhibitors (Remdesivir and EIDD-2801) using these ACE2-iPS cells. Finally, we showed a difference in the efficiency of infection of SARS-CoV-2 among ACE2-iPS/ES cells from 8 donors. These results suggest that by using our iPS cell panel, it will be possible to investigate the effects of race and blood type as well as gender on SARS-CoV-2 infection. By conducting SARS-CoV-2 infection experiments using a panel of iPS cells for which genomic information has been obtained, it will also be possible to find genomic mutations that appear with high frequency in susceptible cells.

We observed a difference in the infection efficiency of ACE2-iPS/ES cells between donors, but we do not know if this difference reflects the sensitivity of the original donors to COVID-19, since none were COVID-19 patients. Therefore, we are currently establishing iPS cells from severe and mild COVID-19 patients. We plan to Because single nucleotide mutations can be easily introduced into iPS cells (Kim et al., 2018) , the function of these mutations can be studied using genome-edited iPS cells.

To infect iPS cells with SARS-CoV-2, we overexpressed ACE2. However, if ACE2 and its related genes are responsible for the individual differences in SARS-CoV-2 infection, our system will not be effective. To analyze the mutation and expression of ACE2 and its related genes, it is essential to use somatic cells expressing ACE2. Also, our system is not effective for non-genetic causes of individual differences in COVID-19 severity. For example, it has been speculated that age-related differences in COVID-19 symptoms are due to impaired cytotoxic CD8+T cell responses (Westmeier et al., 2020) . For such studies, it is more suitable to use blood samples.

Accordingly, there may be multiple causes of the individual differences in COVID-19 symptoms. The ACE2-iPS cells that we have developed in this study will be one tool to elucidate the cause of these individual differences, which will help identify vulnerable populations and develop new drugs.

The SARS-CoV-2 strain used in this study (SARS-CoV-2/Hu/DP/Kng/19-027) was kindly provided by Dr. 

The authors declare no competing financial interests. and near the cell membrane (black arrows) (D) were observed.

LacZ-or ACE2-expressing human iPS cells (LacZ-iPS cells and ACE2-iPS cells, respectively) were infected with SARS-CoV-2 (5×10 4 TCID50/well) for 2 hr and then cultured with AK02 medium for 2 or 3 days. Control human iPS cells were not The gene expression levels of pluripotent markers (NANOG, OCT3/4, and SOX2) (A) and innate immunity-related markers (IFNα, MxA, and ISG15) (B) in uninfected and infected human iPS cells were examined by qPCR. (C) Immunofluorescence analysis of SARS-CoV-2 NP (green), Tra1-81 (red), and OCT3/4 (red) in uninfected and infected human iPS cells. Nuclei were counterstained with DAPI (blue). Data are shown as means ± SD (n=3).

Undifferentiated human iPS cells (1383D6) were transduced with 600 VP/cell of LacZor ACE2-expressing Ad vectors (Ad-LacZ and Ad-ACE2, respectively) for 2 hr and then cultured with AK02 medium for 2 days. Control human iPS cells were not transduced with Ad vectors. The LacZ-and ACE2-expressing human iPS cells were then infected with SARS-CoV-2 (5×10 4 TCID50/well) for 2 hr and cultured with AK02 medium for 3 days. (A, B) The gene expression levels of endoderm markers (FOXA2, SOX17, and CER1) (A) and viral receptors (CD147 and NRP1) (B) were examined by qPCR. Data are shown as means ± SD (n=3). One-way ANOVA followed by Tukey's post hoc test (*p<0.05, compared with Ad-LacZ). 

All unique/stable reagents generated in this study are available from the corresponding authors with a completed Materials Transfer Agreement.

The human ES/iPS cell lines 1383D6 (Nakagawa et al., 2014) (provided by Dr.

Masato Nakawaga, Kyoto University), 201B7 (Takahashi et al., 2007) , 

The SARS-CoV-2 strains used in this study (SARS-CoV-2/Hu/DP/Kng/19-027) were provided from the Kanagawa Prefectural Institute of Public Health. SARS-CoV-2 was isolated from a COVID-19 patient (GenBank: LC528233.1). The isolation and analysis of the virus will be described elsewhere (manuscript in preparation). The virus was plaque-purified and propagated in Vero cells and stored at -80°C. All experiments including virus infections were done in a biosafety level 3 facility at Kyoto University strictly following regulations.

Ad vectors were constructed using Adeno-X TM Adenoviral System 3 (Takara Bio). The ACE2 and TMPRSS2 genes were amplified by PCR using cDNA generated from Pulmonary Alveolar Epithelial Cell Total RNA (ScienCell Research Laboratories) as a template. The ACE2 and TMPRSS2 genes were inserted into Adeno-X TM Adenoviral System 3, resulting in pAdX-ACE2 and pAdX-TMPRSS2, respectively. The ACE2-and TMPRSS2-expressing Ad vectors (Ad-ACE2 and Ad-TMPRSS2, respectively) were propagated in HEK293 cells (JCRB9068, JCRB Cell Bank). LacZexpressing Ad vectors were purchased from Vector Biolabs. The vector particle (VP) titer was determined by using a spectrophotometric method (Maizel Jr et al., 1968) . Table S1 . Table S2 . 

Uninfected and infected ACE2-iPS cells were fixed in phosphate buffer with 2% glutaraldehyde and subsequently post-fixed in 2% osmium tetra-oxide for 2 hr at 4°C. After fixation, the cells were dehydrated in a graded series of ethanol and embedded in epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead staining solution, and examined using an electron microscope (HITACHI H-7600) at 100 kV.

For the immunofluorescence staining of human iPS cells, the cells were fixed with 4% paraformaldehyde in PBS at 4°C. After blocking the cells with PBS containing 2% bovine serum albumin and 0.2% Triton X-100 at room temperature for 45 min, the cells were incubated with a primary antibody at 4°C overnight and then with a secondary antibody at room temperature for 1 hr. All antibodies used in this report are described in Table S3 . 

Total RNA was prepared using the RNeasy Mini Kit (Qiagen). RNA integrity was assessed with a 2100 Bioanalyzer (Agilent Technologies). The library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina) according to the manufacturer's instructions. Sequencing was performed on an Illumina NextSeq500.

The fastq files were generated using bcl2fastq-2.20. Adapter sequences and low-quality bases were trimmed from the raw reads by Cutadapt ver 1.14 (Martin, 2011) . The trimmed reads were mapped to the human reference genome sequences (hg38) using STAR ver 2.5.3a (Dobin et al., 2013) with the GENCODE (release 36, GRCh38.p13) (Frankish et al., 2019) gtf file. The raw counts for protein-coding genes were calculated using htseq-count ver 0.12.4 (Anders et al., 2015) with the GENCODE gtf file. Gene expression levels were determined as transcripts per million (TPM) with DEseq2 (Love et al., 2014) . Raw data concerning this study were submitted under Gene Expression

Omnibus (GEO) accession number GSE166990.

Statistical significance was evaluated by unpaired two-tailed Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. Statistical analyses were performed using GraphPad Prism8 and 9. Data are representative of three independent experiments. Details are described in the figure legends.

Human genetic factors associated with susceptibility to SARS-CoV-2 infection and COVID-19 disease severity

HTSeq-a Python framework to work with highthroughput sequencing data

STAR: ultrafast universal RNA-seq aligner

GENCODE reference annotation for the human and mouse genomes

Genomewide association study of severe Covid-19 with respiratory failure

Identification of SARS-CoV-2 inhibitors using lung and colonic organoids

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells

Microhomology-assisted scarless genome editing in human iPSCs

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

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

Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes

The polypeptides of adenovirus: I

Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12

Cutadapt removes adapter sequences from high-throughput sequencing reads

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

A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells

Genetic mechanisms of critical illness in Covid-19

Disease-specific induced pluripotent stem cells

Induction of pluripotent stem cells from adult human fibroblasts by defined factors

In vitro and animal models for SARS-CoV-2 research

Androgen sensitivity gateway to COVID-19 disease severity

CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

Clinical course and mortality risk of severe COVID-19

Impaired cytotoxic CD8+ T cell response in elderly COVID-19 patients

The major genetic risk factor for severe COVID-19 is inherited from Neanderthals

SARS-CoV-2 receptor ACE2 is an interferonstimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues