概要: The current COVID-19 pandemic is caused by the novel coronavirus SARS-coronavirus 2 (SARS-CoV-2). There are currently no therapeutic options for mitigating this disease due to lack of a vaccine and limited knowledge of SARS-CoV-2 virus biology. As a result, there is an urgent need to create new disease models to study SARS-CoV-2 biology and to screen for therapeutics using human disease-relevant tissues. COVID-19 patients often present with respiratory symptoms including cough, dyspnea, and respiratory distress but upwards of 25% of respiratory dysfunction, many COVID-19 patients have digestive system indications, including anorexia, diarrhea, vomiting, and abdominal pain. Moreover, these symptoms are associated with more severe COVID-19 outcomes1. Here, we report using human pluripotent stem cell-derived colonic organoids (hPSC-COs) to explore the permissiveness of different colonic cell types to SARS-CoV-2 infection. Single cell RNA-seq and immunostaining showed that the putative viral entry receptor ACE2 is expressed in multiple types of hESC-derived colon cells, but are highly enriched in hPSC-derivedKRT20+ enterocytes. Distinct cell types in the COs can be infected by a SARS-CoV-2 pseudo-entry virus, which is further validated in vivo using a humanized mouse model. Finally, we adapted hPSC-derived COs to a high throughput platform to screen 1280 FDA-approved drugs. Mycophenolic acid was found to block the entry of SARS-Cov-2 pseudo-entry virus in COs, and confirmed to block infection of SARS-CoV-2 virus. In summary, this study established both in vitro and in vivo organoid models to investigate infection of SARS-CoV-2 disease-relevant human colonic cell types and identified a drug suitable for rapid clinical testing that blocks SARS-CoV-2 infection.
项目整体设计: scRNA-seq analysis of human pluripotent stem cell-derived colonic organoids (hPSC-COs) infected with SARS-CoV-2 pseudo-entry virus.
The colon progenitor organoids were collected from the initial 2D cultures and embedded in a 100% Matrigel dome in a 24-well plate. Differentiation to mature colonic cell types was achieved by culturing these colon progenitor organoids in differentiation medium containing 600 nM LDN193189 (Axon), 3 CHIR99021 and 100 ng/ml hEGF in Advance DMEM F12 medium supplemented with 1X B27 supplement, 1X GlutaMax, 10 mM HEPES and 1X Pen-Strep. The differentiation medium were refreshed every 3 days for at least 40 days to achieve full colonic differentiation. The colon organoids were passaged and expanded every 10 14 days at 1:6 density. To passage the organoids, the Matrigel domes containing the organoids were scrapped off the plate and resuspended in cold splitting media (Advance DMEM F12 medium supplemented with 1X GlutaMax, 10 mM HEPES and 1X Pen-Strep). The organoids were mechanically dislodged from the Matrigel dome and fragmented by pipetting in cold splitting media. The old Matrigel and splitting media were removed after spinning down cells and the organoids were resuspended in 100% Matrigel. 50 Matrigel containing fragmentized colon organoids were plated in one well of a pre-warmed 24-well plate.
处理方案:
To assay pseudotyped virus infection on colon organoids, COs were seeded in 24 well plates. Pseudotyped virus was added at MOI=0.01 plus polybrene at a final concentration of 8 /mL, and the plate centrifuged for 1 hr at 1200g. At 3i, the infection medium was replaced with fresh medium.
提取方案:
hPSC-COs were harvested by cell scraper, mixed with 20 l Matrigel (Corning) and transplanted under the kidney capsule of 7-9 weeks old male NSG mice. At two weeks post-transplantation, SARS-Cov-2 pseudo-entry virus was inoculated locally at 1x10e3 FFU.
建库方案:
Library was constructed according to the user manual of Chromium Single Cell 3Library & Gel Bead Kit v2
测序信息
分子类型:
Poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina NovaSeq 6000
链特异性:
Specific
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文章
Identification of SARS-CoV-2 inhibitors using lung and colonic organoids.
