概要: Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells
项目整体设计: 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.
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.
处理方案:
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.
提取方案:
Total RNA was isolated from uninfected or infected ACE2-iPS cells using ISOGENE (NIPPON GENE).
建库方案:
Library preparation was performed using a TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA) according to the manufacturer’s instructions.
测序信息
分子类型:
polyA(+) RNA
库的片段类型:
SINGLE
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina NextSeq 500
链特异性:
Specific
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文章
Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells.
