key: cord-0949079-4say8tj1
authors: Sano, Emi; Deguchi, Sayaka; 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: 2021-04-16
journal: iScience
DOI: 10.1016/j.isci.2021.102428
sha: ed9c68d932ff0e370a16f90d8c8640d8434f1c0f
doc_id: 949079
cord_uid: 4say8tj1

Genetic differences are a primary reason for differences in the susceptibility and severity of COVID-19. Because iPS cells maintain the genetic information of the donor, they can be used to model individual differences in SARS-CoV-2 infection in vitro. 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, budding of viral particles, production of progeny virus, double membrane spherules, and double-membrane vesicles were confirmed. 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 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.

does not infect iPS cells. Therefore, in this study, we identified SARS-CoV-2-related 92 genes that enable SARS-CoV-2 to infect undifferentiated human iPS cells. To facilitate 104 First, we examined whether undifferentiated iPS cells could be infected by 105 SARS-CoV-2 (Fig. S1A) . Before conducting this experiment, we examined the 106 expression levels of viral receptors and proteases in undifferentiated iPS cells (Fig.   107   S1B) . The gene expression level of ACE2 was low, but that of CD147 was high. CD147 108 is reported as a coronavirus receptor (Wang et al., 2020). TMPRSS2, a protease, was 109 also expressed in undifferentiated iPS cells. We thus tried to infect undifferentiated iPS 110 cells with SARS-CoV-2, but the morphology of the iPS cell colonies did not change 111 (Fig. S1C ). In addition, virus genome in the cell culture supernatant (Fig. S1D ) and the 112 production of infectious virus (Fig. S1E) were not detected. The gene expression levels 113 of undifferentiated markers (Fig. S2A ) and innate immune response-related markers 114 ( Fig. S2B) were also unchanged. Furthermore, the expression of SARS-CoV-2 115 nucleocapsid (N) protein was not detected (Fig. S2C) . Together, these results indicated 116 that SARS-CoV-2 does not infect undifferentiated iPS cells.

117 118 ACE2 expression is required for SARS-CoV-2 to infect human iPS cells 119 Because human ACE2 and TMPRSS2 are known to be important for 120 SARS-CoV-2 to infect cells, we overexpressed human ACE2 and TMPRSS2 in 121 undifferentiated iPS cells by using Ad vectors (Fig. 1A) . The overexpression of ACE2 (Fig. 1C) . This was not the case if only overexpressing TMPRSS2. Furthermore, two 125 days after the ACE2-iPS cells were infected with SARS-CoV-2, cell fusion was 126 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 128 iPS cells. 151 We also performed an RNA-seq analysis of uninfected and infected ACE2-iPS 152 cells. The colored dots in the volcano plot in figure 4A indicate genes whose expression 153 levels changed significantly more than 4-fold. In total, this change occurred in 6.7% of 154 all genes (Fig. S6A) . A GO term analysis was performed on these genes (Figs. S6B, 155 S6C). None of the genes included undifferentiated markers (Fig. 4B) or innate 156 immune-response markers (Fig. 4C) . The gene expression levels of ectoderm, 157 mesoderm, and endoderm markers were also unchanged after infection with 158 SARS-CoV-2 (Fig. S7) . Overall, these results suggest that human iPS cells maintain an 159 undifferentiated state even when SARS-CoV-2 replicates in large numbers. 160 We tried eight drugs used in COVID-19 clinical trials. Vero cells were used as the 164 control. After exposing the cells to various concentrations of a drug, the number of viral 165 RNA copies in the culture supernatant was quantified (Fig. 5A) . Data fitting resulted in 166 sigmoid curves, and half-maximal effective concentrations (EC50) were calculated (Fig.   167   5B ). Among the eight drugs, the antiviral effect of Remdesivir was strongest. On the 168 other hand, Chloroquine and Favipiravir did not inhibit viral replication, and Ivermectin 169 was highly cytotoxic (Fig. 5C) . The EC50 and half-maximal cytotoxic concentration In this study, we confirmed sex differences in the susceptibility to To infect iPS cells with SARS-CoV-2, we overexpressed ACE2. However, if 238 ACE2 and its related genes are responsible for the individual differences in 239 SARS-CoV-2 infection, our system will not be effective. To analyze the mutation and 240 expression of ACE2 and its related genes, it is essential to use somatic cells expressing 241 ACE2. Also, our system is not effective for studying the non-genetic causes of 242 individual differences in COVID-19 severity. For example, it has been speculated that The authors declare no competing financial interests. The amount of infectious virus in the supernatant was measured by the TCID50 assay. Table S1 . 

ACE2 expression is required for SARS-CoV-2 to infect human iPS cells 2. SARS-CoV-2 life cycle can be reproduced in the ACE2-iPS cells