Gene Expression
Nebulas
Gene Expression NebulasSummary: Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells
Overall Design: 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.
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| Growth Protocol: | The human iPS cells were maintained on 0.5 μg/cm2 recombinant human laminin 511 E8 fragments (iMatrix-511, Nippi) with StemFit AK02N medium (Ajinomoto) containing 10 μM Y-27632 (from day 0 to day 1, FUJIFILM Wako Pure Chemical). To passage human iPS cells, near-confluent human iPS cell colonies were treated with TrypLE Select Enzyme (Thermo Fisher Scientific) for 5 min at 37°C. After the centrifugation, human iPS cells were seeded at an appropriate cell density (1.3×104 cells/9 cm2) onto iMatrix-511. Human ES/iPS cells on iMatrix-511 were subcultured every 6 days. |
| Treatment Protocol: | Undifferentiated human iPS cells (1383D6) were transduced with 600 VP/cells of LacZ-, ACE2-, or TMPRSS2-expressing Ad vectors (Ad-LacZ, Ad-ACE2, or Ad-TMPRSS2, respectively) for 2 hr, and then cultured with AK02 medium for 2 days. ACE2-expressing human iPS (ACE2-iPS) cells were infected with SARS-CoV-2 (5×104 TCID50/well) for 2 hr, and then cultured with AK02 medium. |
| Extract Protocol: | Total RNA was isolated from uninfected or infected ACE2-iPS cells using ISOGENE (NIPPON GENE). |
| Library Construction Protocol: | Library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific |