A bipotential organoid culture of respiratory epithelium for modeling SARS-CoV-2 infection

The airways and the alveoli of the human respiratory tract are lined by two distinct types of epithelium. We previously established long-term expanding human lung epithelial organoids from lung tissues and developed a 'proximal' differentiation protocol to generate mucociliary airway organoids, yet the derivation of alveolar organoids from adult lung has remained a challenge. Here we defined a 'distal' differentiation approach to generate alveolar organoids from the same source that allows the establishment of airway organoids. Alveolar organoids are enriched for AT1 and AT2 cells and functionally simulate the alveolar epithelium. AT2 cells in lung organoids act as the progenitor cells from which alveolar organoids emerge. Moreover, we demonstrate productive SARS-CoV-2 infection of alveolar organoids. We further optimize 2-dimensional (2D) airway organoids. When differentiated under a slightly acidic pH, these 2D airway organoids sustain enhanced viral replication and better recapitulate the high infectivity of SARS-CoV-2. Moreover, the optimized 2D airway organoids can model IgG transcytosis across the airway epithelium. Collectively, we establish a bipotential organoid culture system that can reproducibly expand the entire human respiratory epithelium in vitro for modeling respiratory diseases, including COVID-19. Overall design: Human lung organoids were induced to generate 3D airway organoids, 2D airway organoids and 3D alveolar organoids in triplicate as described above or previously (Zhou et al., 2018). Three biological replicates of lung organoids and derived organoids were harvested for RNA extraction using RNeasy Mini Kit (Qiagen, Cas.74106). After a quality check with Bioanalyzer, RNA specimens were applied to library preparation by KAPA mRNA HyperPrep Kit. In brief, Poly-A containing mRNA was collected by using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented to 200 ~ 300 bp by incubating at 94°C for 6 min in the presence of magnesium ions. The fragmented mRNA was then applied as a template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent 3' adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 15 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration. Illumina NovaSeq 6000 was used for Pair-End 151bp sequencing. Sequencing reads were assigned into individual samples, with each sample having an average throughput of 9.6 GB and a total throughput of 111.5 Gb. In terms of sequence quality, an average of 93% of the bases achieved a quality score of Q30 where Q30 denotes the accuracy of a base call to be 99.9%. Sequencing reads were mapped to the human reference genome GRCh38 with RNA-Seq aligner STAR. Clean reads were quantified based on the number of reads spanning the back-splicing junction, and their fragments per kb for a million reads (FPKM) were calculated using HTSeq 0.13.5. Aligned BAM files were sorted by SAMtools. GenomicAlignments and DESeq2 R packages (version 1.18.1) were used for read counting and differential expression analysis. Genes with an absolute value of log2 fold change greater than 1 and adjusted P-value (Benjamini-Hochberg) less than 0.05 were considered as differentially expressed.