概要: SARS-CoV-2, the virus responsible for COVID-19, causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. We present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. Monolayers derived from adult lung organoids (ALOs), primary airway cells, or hiPSC-derived alveolar type-II (AT2) pneumocytes were infected with SARS-CoV-2 to create in vitro lung models of COVID-19. Infected ALO-monolayers best recapitulated the transcriptomic signatures in diverse cohorts of COVID-19 patient-derived respiratory samples. The airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (AT2→AT1) was critical for mounting the overzealous host immune response in fatal disease; ALO monolayers with well-mixed proximodistal airway components recapitulated both. Findings validate a human lung model of COVID-19 , which can be immediately utilized to investigate COVID-19 pathogenesis and vet new therapies and vaccines
项目整体设计: Human lung organoids were grown in standard culture, ALI and monolayer conditions. Human small airway epithelium and iPSC-derived alveolar epithelial type 2 cells were infected with SARS-CoV2
Stem cells were isolated from human lung tissue and cultured as 3D organoids. The organoids were broken into single cells and seeded as a monolayer in a 384-well plate. ALI (Air-liquid interphase) model was generated from the single cells collected from the organoid. Alveolar Type 2 cells and alveolospheres were generated from iPSCs.
处理方案:
The lung cells were infected by SARS-CoV2 for 48h and 72h timepoints and uninfected wells were kept as a control
提取方案:
All cells were lysed using TRI-Reagent and RNA was isolated using Zymo Research: Direct-zol RNA MiniPrep Kit
建库方案:
RNA sequencing libraries were generated using the Illumina TruSeq Stranded Total RNA Library Prep Gold with TruSeq Unique Dual Indexes (Illumina, San Diego, CA). Samples were processed following manufacturer’s instructions, except modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 100 basepair (bp) Paired End (PE100) to a depth of approximately 25-40 million reads per sample on an Illumina NovaSeq 6000. Samples were demuxltiplexed using bcl2fastq v2.20 Conversion Software (Illumina, San Diego, CA).
