The study investigates individual differences in SARS-CoV-2 infection in ACE2-expressing stem cells

The clinical phenotype of coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is noteworthy due to the wide severity of individual patients. Genetic variation is known to mediate some of these differences.

To investigate these differences, researchers in the new study used humans Induced pluripotent stem cells (IPSC) From a variety of genetically diverse individuals. These cells contain the genetic information of the donor and are therefore used to model genetic disorders.

Research published as a preprint bioRxiv* The server uses iPSC panels from over 500 individuals. Researchers preferred undifferentiated iPSCs to reduce the time required for differentiation, especially because infections are not always reliable.

Establishment of SARS-CoV-2 infection in iPS cells

Human iPSCs express low levels of the viral entry receptor angiotensin converting enzyme 2 (ACE2) and therefore do not infect SARS-CoV-2. These cells carry high levels of the coronavirus receptor CD147 and the serine protease TMPRSS2.

To make them susceptible to SARS-CoV-2 infection, iPSCs were engineered with near 100% efficiency using related genes inserted by the adenovirus vector (Ad). These genes encoded human ACE2 and TMPRSS2 at overexpression levels, causing large-scale viral infections. Overexpression of TMPRSS2 alone was not sufficient to produce this effect.

Cell fusion was observed 2 days after infection with ACE2-positive iPS cells, often with cell death 4 days later. Under transmission electron microscopy (TEM), infected cells showed characteristic changes such as zippered endoplasmic reticulum (ER), double membrane globules (DMS), and viral particles near the cell membrane.

Viral particles were found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), along with dual-membrane vesicles (DMV). The latter is central to the synthesis of viral RNA and is not present in uninfected cells.

Therefore, TEM is a useful tool for investigating the life cycle of this virus.

TEM image of infected ACE2-iPS cells (A) TEM image of infected ACE2-iPS cells. Viral particles were observed in the endoplasmic reticulum with a zipper, double membrane globules (DMS) (B), and ERGIC (black arrow) (C) and near the cell membrane (black arrow) (D).

Undifferentiated state persists after infection

Gene and protein expression was assessed 3 days after infection in both infected and non-infected cells. This indicated that the infected cells had high levels of the viral genome and ACE2, but no undifferentiated cell markers or innate immune markers. The viral nucleocapsid (N) protein was also strongly expressed in infected cells 2 days after infection.

RNA-seq analysis of these cells showed that about 7% of all genes had more than 4-fold changes in expression levels and were free of these encoded undifferentiated or innate immune markers. ..

Endoderm marker ( CER1 With genes CD147, NRP1,and TMPRSS2 The gene known as the SARS-CoV-2 related marker has also not changed. The mesoderm and ectoderm markers have also not changed.

These results suggest that human iPS cells remain undifferentiated even when SARS-CoV-2 is replicated in large numbers... “

Evaluation of candidate drugs

Compared to Vero cells, iPS cells have been shown to be more potent against SARS-CoV-2 infection, with the exception of interferon beta. Of the eight drugs tested, remdesivir showed the greatest antiviral activity and ivermectin showed high cytotoxicity.

Chloroquine and favipiravir were unable to suppress viral replication. RNA-dependent RNA polymerase (RdRp) inhibitors (remdesivir and EIDD-2801) and TMPRSS2 inhibitors (camostat and nafamostat) have been shown to have antiviral activity in these cells and value them in drug evaluation. is showing.

Individual differences

The researchers then infected iPSCs from eight different donors and investigated the differences in infection purely by individual factors. The virus replicated with varying efficiencies among the eight donor cell lines, all of which had comparable expression of ACE2.

The virus exhibits greater replication capacity in cells of male origin than in females, indicating possible gender differences in the susceptibility to replication in these cells. Both the androgen receptor and its target TMPRSS2 gene, the latter being expressed at slightly higher levels in male iPS cells, may be involved in this difference.

What is the impact?

If the ACE2 receptor is overexpressed, the viral life cycle can be investigated in these human iPS cells. This study showed the effect of gender differences on the efficiency of infection and viral replication. The inhibitory effect of the drug could also be observed in this cell model.

These results suggest that the iPS cell panel can be used to investigate the effects of race, blood type, and gender on SARS-CoV-2 infection. “

In addition, the identification of gene mutations that occur frequently in cells that maintain infection is made possible by the use of iPSCs from panels with documented genomic information.

Infection efficiency varies from donor to donor, but the fact that none of these donors had COVID-19 limits the inference that they had different susceptibility to the disease. This study is being conducted using iPSCs in patients with mild and severe COVID-19, and the findings of infected cells can be compared with the patient’s clinical manifestations.

Mutations in certain innate immune genes may be associated with the severity of symptoms, but further research is needed to understand how these genes function during COVID-19. This can be conveniently done with iPSCs, making it easy to introduce single-nucleotide mutations.

It is noteworthy that the role of ACE2 overexpression is not ruled out by individual differences in SARS-CoV-2 infection within iPSCs from different donors. In that case, another system using somatic cells expressing ACE2 should be used to first assess the effects of expression and mutations on ACE2 and other related genes.

Causes of non-genetic individual differences, including those due to aging and associated CD8 reduction, are not addressed by this system. T cells Cytotoxicity. Studies on blood samples may help clarify the causes of such fluctuations in susceptibility and viral replication. ACE2-positive iPSCs continue to be a useful platform for investigating and determining the causes of individual differences, identifying high-risk groups, and assessing new drugs.

*Important Notices

bioRxiv publishes unpeer-reviewed preliminary scientific reports and should not be considered definitive, guide clinical / health-related behaviors, or be treated as established information.