A therapy for suppressing canonical and noncanonical SARS-CoV-2 viral entry and an intrinsic intrapulmonary inflammatory response

Significance New severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants continue to emerge with differing effects on the lung, yet there is a lack of readily available, biologically relevant in vitro models for studying their impact on lung epithelial cells. We have harnessed our patient-specific, induced pluripotent stem cell–derived, multicell lung organoid model to investigate critical aspects of COVID-19, including viral tropism and routes of entry, lung-specific immune response, and a role for surfactant protein B in innate immunity, viral surveillance, cell survival, and inflammation. These findings can potentially revolutionize our understanding of the lung’s intrinsic response to viral infections, leading to innovative therapies.

Table S5: Recipes of Medias used in lung differentiations.

FIGURE S4
FIGURE S5: Compounds blocking various routes of viral entry and processing show different efficacies in reducing viral infections.Western blot of PLOs from different racial groups, pretreated with the endocytic blocking agent (apilimod ["API"]), the cathepsin B blocking agent (ONO5334 ["ONO"]), or an agent that blocks viral replication (Remdesivir ["REM"]) 24 hpi with SARS-CoV-2 ("V").The data are representative of three independent experiments.PLO GM23450 was derived from hiPSCs generated from an African-American female, HYRO103 from a Hispanic male, GM2370 from a Caucasian female, and ALDA31616 from a Caucasian male (Supplementary Table 1).Actin served as a loading control.Interestingly, even at this cursory level of analysis, one can see the suggestion of possible disparity in the greater degree of infection (the DMSO control lane) between the Hispanic male and the others, as well as a disparate response to anti-viral agents: Apilimod may have worked less effectively in the Hispanic male and Remdesivir may have worked most effectively in the Caucasian male.

FIGURE S5
FIGURE S6: Single cell transcriptional characteristics of the hPSC-LOs.(A) Uniform manifold approximation and projection (UMAP) of hPSC-PLOs 16 hpi and (B) characteristic genes per cluster.were derived.The SP-B mutant lines were previously published (7).These cell lines have been validated by immunofluorescence for markers of pluripotency, karyotype, and genomic analysis if applicable.The hiPSCs and the human embryonic stem cell line H9 (WiCell), were cultured on matrigel coated (Matrigel GFR; Corning, #354230) plates in mTeSR medium (StemCell Technologies #85850).Medium was changed daily, and cells were passed using ReLeSR (StemCell Technologies #05872) every 5-7 days.Cultures were maintained in an undifferentiated state in a 5% CO2/ambient air environment.

FIGURE S6
Authentication of Cell Lines is based on guidelines from the International Cell Line Authentication Committee (iclac.org):Authentication testing was performed on established cell lines regardless of the application.The pluripotency of the ESC and iPSC cell lines were confirmed through immunofluorescence for pluripotent markers (Oct4, Nanog, TRA1-81, SOX2, TRA 1-60) and normal karyotype was also confirmed prior to use.Early passages at the time of verification of pluripotency and karyotype are cryopreserved and those stocks utilized for further experimentation.
Different cell lines and derivatives were never manipulated together to avoid the possibility of cross-contamination.
Furthermore, all cell lines were used at early passage.Upon reaching higher passage number, cells were discarded, and cultures restarted from early passage cryovials.Monthly screening for mycoplasma using the MyoSEQ ThermoFisher Mycoplasma Detection kit was performed, and new cells/cell lines were maintained in quarantine until confirmed to be mycoplasma negative.
Transwells were pre-coated with 50µg/mL Collagen type I from Rat tail (BD Biosciences #354236) at 7.5 µg/cm 2 .Collagencoated transwells were rehydrated with 100 µL PneumaCult TM Ex-Plus media at 37°C, 5% CO2 for 30 min prior to seeding NHBECs for ALI culture.NHBECs were then seeded in the apical chamber at 5x10 4 cells in 100uL of media, for a total of 5x10 4 cells/ 100 µL of media per collagen-coated transwell with 500µL of media in the basolateral chamber.The cells were then incubated at 37°C, 5% CO2 overnight.On Day 1, the media in the apical and basolateral chambers were exchanged with 100 µL and 500 µL, respectively, with fresh PneumaCult TM Ex-Plus media.NHBECs were 80-100% confluent the day after seeding.The next PneumaCult TM Ex-Plus media change was on Day 3. On Day 4-7, or until culture reached confluency, the apical and basolateral chambers were fed PneumaCult TM ALI media (StemCell #05021) daily supplemented with 10µM ROCK inhibitor (Tocris Y-27632).On Day 8, the apical media was removed, and the basal media replaced with PneumaCult TM ALI media without Y-27632.Subsequent media changes were every other day thereafter.On Day 14 postairlift, the apical surfaces were washed with DPBS, once per week.Cells were grown in 37°C, 5% CO2 incubator until four weeks post airlift.

Directed differentiation of hESC and hiPSCs to lung organoids (LOs)
For human iPSC-LO generation, we used 6 iPSC cell lines [Supplementary Table 1] which were cultured in feeder free conditions upon Matrigel (Corning #354230) coated plates in mTeSR Plus medium (StemCellTech #100-0276).Media was changed daily, and stem cells were passaged weekly using enzyme free dissociation reagent ReLeSR™ (Stem Cell Tech #05872).Cultures were maintained in an undifferentiated state, in a 5% CO2 incubator at 37°C.For lung progenitor spheroid generation, human PSCs were dissociated into single cells with exposure to 20 min of accutase, and approximately 2.0 x 10 5 cells were seeded on Matrigel-coated plates in mTeSR supplemented with 10μM Y-27632 and incubated for 24 hours.Each iPSC cell line required seeding density optimization with the goal of 50-70% confluency the day after seeding.
The matrigel containing cells were polymerized for 30-60 min in a 37°C incubator and then LPC media (SFBM supplemented with 10 ng/mL of human recombinant BMP4, 0.1 µM of retinoic acid (RA), 3 µM of GSK3β inhibitor/Wnt activator CHIR99021 and 10 µM of Rock Inhibitor Y-27632) was added to submerge the matrigel droplets.LPC media was changed every other day for 9-11 days without Y-27632.Analysis of the surface antigen CPM, or the intracellular marker NKX2-1 should be performed at the end of this differentiation period to determine the efficacy of differentiation.If the LPC spheroids express > 50% CPM/NKX2-1, LO differentiation may proceed.
To generate LOs (proximal, distal, and whole lung), LPC spheroids were incubated in 2 U/ml dispase for 30 min at 37°C with manual pipetting every 15 min to break apart the matrigel.Cold PBS was added, transferred to a 15 mL conical tube, and then centrifuged at 400 x g for 5 min.The supernatant was carefully removed then 3 mL of cold PBS was added to the pellet, mixed, and then centrifuged again at 400 x g for 5 min.The supernatant was carefully removed, and the pellet resuspended in 2-3 mL of TrypLE Express (Gibco # 12605010) for 12 min at 37°C to keep the LPC spheroids as aggregates.
The reaction was quenched with 2% FBS in DMEM/F12 then centrifuged at 400 x g for 5 min.The supernatant was removed, and the cell pellet resuspended in 1ml of quenching media supplemented with 10 µM Rock inhibitor (Y-27632) and a cell count was performed.Cells were embedded into cold matrigel as aggregates at 5 × 10 4 cells per 200 µL of Matrigel and transferred into the apical portion of 0.4 µm translucent 12-well inserts (VWR 10769-208).
For infections in intact 3D LOs, the matrigel droplets were washed with PBS, then 1 ml of Cell Recovery Solution (Corning #354253) was added per 200 µL Matrigel drops.Matrigel was mechanically dissociated using wide bore P1000 tips, titurating gently to maintain the integrity of the organoids.The mixture was placed at 4°C for 1 h, with manual resuspension every 15 min.After 1 hour, the sample was transferred to a 15 mL conical tube, using a wide bore pipette.
The organoids were centrifuged at 200 x g for 1 min.Chilled PBS was added to the pellet and gently mixed then centrifuged at 200 x g for 1 min.The pellet was resuspended in 500 µL of LO media supplemented with 10 µM Y-27632 in 24 well ultralow attachment (ULA) plate (Corning #3473) at approximately 20-30 organoids per well.On the day of infection, the 3D organoids were placed in LO medium without DCI.
For infections in 2D monolayers, 3D LOs were dissociated into single cells and seeded at 2x10 4 cells per well of a matrigel coated 96-well plate approximately 3 days before infection.Matrigel containing LOs was incubated in 2 U/ml dispase for 30 min at 37 °C with manual pipetting every 15 min to break apart the matrigel.Cold PBS was added to the well, transferred to a 15 mL conical tube and then centrifuged at 400 x g for 5 min.The supernatant was carefully removed then 3 mL of cold PBS was added to the pellet, resuspended, and then centrifuged again at 400 x g for 5 min.The supernatant was carefully removed, and the pellet resuspended in 2-3 mL of TrypLE Express (Gibco # 12605010) for 15-20 min at 37°C to dissociate the organoids into single cells.The reaction was quenched with 2% FBS in DMEM/F12 then centrifuged at 400 x g for 5 min.The supernatant was removed, and the cell pellet resuspended in 1ml of quenching media supplemented with 10 µM Rock inhibitor (Y-27632) and a cell count was performed.Cold PBS was added to the mixture then centrifuged at 400 x g for 5 min.Supernatant was carefully removed and resuspended in 2-3 mL of TrypLE Express (Gibco # 12605010) for 20 min at 37 °C.Reaction was quenched with 2% FBS in DMEM/F12 then centrifuged at 400 x g for 5 min.The supernatant was aspirated, and the cell pellet resuspended in 1 mL of quenching media supplemented with 10 µM Rock inhibitor (Y-27632).Cell count was performed, and 2 x 10 4 cells were seeded per well of a matrigel coated 96-well plate with 100 µL of respective LO media.The wells were 90-100% confluent prior to infections.On the day of infection, the media was replaced with LO medium without DCI.
All materials used can be found in Supplementary Table 4.All media recipes can be found in Supplementary Table 5.
The 3D organoids were prepped for flow cytometry/FACS or single cell RNA sequencing per the protocol below.The monolayer infected lung cells were washed with PBS three times then fixed in 4% PFA for immunohistochemistry (see below).

SARS-CoV-2 viruses
All work with SARS-CoV-2 was conducted in Biosafety Level-3 conditions at the University of California San Diego Antibiotic-Antimycotic was also added to the media in basal chambers.After 24 h, the material was removed, and apical surfaces were washed.Apical washes (passage 1) were taken daily and stored at -80.Fresh NHBECs at ALI were inoculated with passage 1 virus as above to produce passage 2 and with passage 2 to produce passage 3. Titers were expanded by infecting TMPRSS2-Vero cells with passage 3 virus.Gamma (hCoV-19/Japan/TY7-503/2021, P.1, Gamma), and Delta (hCoV-19/USA/PHC658/2021, B.1.617.2,Delta) were acquired from BEI and expanded on TMPRSS2-VeroE6 cells.
Omicron (hCoV-19/USA/CA-SEARCH-59467/2021, BA.1, Omicron) was isolated from a patient sample under UC San Diego IRB #160524.Original patient isolate sequence can be found at GISAID (EPI_ISL_8186377).Serial dilutions in DMEM + 3% FBS were made from a positive nasopharyngeal swab stored in viral transport media.Dilutions were incubated on TMPRSS2-VeroE6 cells and monitored for CPE.When CPE was observed, the contents were transferred to fresh cells for a total of 3 passages on TMPRSS2-VeroE6 cells.
For single cell RNAseq of the mixed organoids, SARS-CoV-2 isolate WA1 (USA-WA1/2020, A) was acquired from BEI and propagated on VeroE6 cells resulting in mutation of the furin cleavage site (142, 143).
All viral stocks were clarified and stored at -80ºC.Titers were determined by plaque assay or fluorescent focus assay on TMPRSS2-VeroE6 cells.All stocks are verified by full genome sequencing.Original sample sequencing links can be found in Supplementary Table 6.

Infection of NHBECs at ALI
After two 30 min incubations with PBS at 37°C, 5% CO2, virus diluted in PBS was added to the apical chamber in 100 µL.Virus was removed after 24 h and apical washes (150 µL PBS with 10 min incubation at 37°C, 5% CO2,) were taken daily and stored at -80ºC.Titer was determined by fluorescent focus assay on TMPRSS2-Vero cells.Infected transwells were fixed in 4% PFA for immunofluorescence staining.

Plaque assay
Ten-fold serial dilutions were made in DMEM and added to 12-well plates of confluent VeroE6 cells for 1 h at 37˚C with occasional rocking.Input was removed and monolayers were overlaid with 1mL of a 1:1 mixture of 1.2% agarose and 2 x MEM complete (2 x MEM supplemented with 8% FBS, 2 x L-glutamine, 2 x non-essential amino acids, and 2 x sodium bicarbonate).Plates were incubated 48 h at 37˚C and then fixed with 2 mL 10% formaldehyde in PBS for 24 h at RT.
Overlays were removed, and plaques were visualized by staining with 0.025% crystal violet in 2% EtOH.Plaque assays were performed and counted by a blinded experimenter.Plaque assays were counted and recorded by a blinded observer.

Fluorescent focus assay
Ten-fold serial dilutions were made in DMEM and added to 96-well plates of confluent TMPRSS2-VeroE6 cells.
After 1 h at 37˚C, input was removed, and wells were overlaid with 100 µL 1% methylcellulose in DMEM + 1% FBS and 1 x Pen/Strep.Plates were fixed after 24 h incubation at 37˚C by addition of 150 µL 8% formaldehyde for at least 30 min at RT.
Cells were stained with anti-nucleocapsid primary antibody (GeneTex, gtx135357) and anti-rabbit AlexaFluor secondary (Thermo Fisher Scientific) and plates were imaged on the Incucyte S3 imager.Foci were counted using the Incucyte onboard software tools.

Virus quantification, FFU immunofluorescence imaging and analysis
3D hPSC-LOs were removed from matrigel intact as described above.They were plated in 24 well ultra-low attachment plates in LO base media, without DCI, at a concentration of 20 organoids per well.hPSC-LOs that were dissociated and plated as a monolayer were also changed to LO base media without DCI, prior to infection.3D LOs were infected with SARS-CoV-2 at an MOI of 1-3 and incubated for 24 -72 h at 37 °C.Dissociated LOs were infected with SARS- CoV-2 at an MOI of 0.1.
For the compound experiments, 3D LOs in suspension were pretreated with DMSO (control), cathepsin inhibitors ONO5334 and Apilimod and remdesivir (all at a concentration of 5 µM).Two hours after the addition of compounds, SARS-CoV-2 was added for 24 h.Mock infections were performed using viral growth media (DMEM + 3% FBS).Infected and mock controls were washed three times in PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore 20-188) at 2 x with protease inhibitor for western blot analysis.
For infections with poractant alfa and recombinant SP-B, hiPSC LOs were dissociated & plated as a monolayer ( 4x 10 4 cells/well) (along with VeroE6 cells as a positive control), pre-incubated with exogenous surfactant (poractant alfa [Curosurf] 1 mg/ml) or with recombinant SP-B [rSP-B] (5 µM) for 30 min prior to infection with SARS-CoV-2 at an MOI of 0.1.After 1 hour of infection, cells were overlayed in media supplemented with 1.2% carboxymethylcellulose as discussed below.
For infections of VeroE6 cells using carboxymethylcellulose, media was prepared using 500 mL LO base media or DMEM-glutamax supplemented with 3% FCS, 2% gentamycin, 1% penicillin/streptomycin plus 6 g autoclaved carboxy methyl cellulose.iPS-derived human LOs were dissociated and plated as a monolayer then infected with SARS-CoV-2 at the indicated MOI.After 1 h of infection, cells were overlayed in carboxymethylcellulose media.After incubation for up to 72 h, overlay was removed and cells were fixed in 4% formaldehyde for 30 min at RT.Fixed cells were washed in BD CytoPerm, and then stained overnight with a primary nucleocapsid antibody (GeneTex GTX135357), counterstained with an anti-rabbit AF647 secondary antibody (ThermoFisher), and nuclei counterstained with 1 µM Hoechst or 1 µM Sytox Green.
Whole well scans were captured with an Incucyte S3 or 10 x 0.45 NA objective using a Nikon Ti2-E microscope with a Qi-2 camera and Nikon Elements 5.02.02 acquisition software utilizing the Jobs module.Wells were imaged for red, green and/or far-red fluorescence using a SpectraX light engine (Lumencor) with individual LFOV filter cubes (Semrock) (excitation/emission maxima at 554/609, 470/525 and 618/698 nm, respectively).FFU analysis was carried out using a custom macro written in the Fiji distribution of ImageJ.The macro leverages machine learning using Ilastik (144), StarDist (arXiv:1806.03535v2)and GPU acceleration using CLIJ to process data files (145).Nuclei were segmented using StarDist, and positive cell stain masks generated using a trained model in Ilastik.Masks were used to count the number of nuclei in clusters, and to define the staining intensity of the positive clusters, and morphological characteristics and size of FFU, and the distribution of the clusters within the well and from each other.The cluster shape was also assessed by generating a ratio of the perimeter and convex hull of each cluster, with values approaching 1 being circular and values approaching zero becoming more irregular.

FFUTrackR data visualization
Image J datalogs from analyses of whole well scans were visualized using the R Shiny app FFUTrackR (https://croker.shinyapps.io/ffutrackr).The FFUTrackR pipeline was developed using Shiny in R 3.6.3., the R packages Tidyverse and plotrix for basic data loading and filtering utilities.shinydashboardPlus and shinyWidgets are used for module management and custom input controls.FFUTrackR requires R packages dplyr, ggpubr, and ggbeeswarm for basic data handling and plotting utilities.The code for FFUTrackR is available at: https://github.com/WeiqiPeng0/FFU.TrackR.

Virus-induced cell death
SARS-CoV-2-triggered cell death of Vero E6 cells was monitored using an Incucyte S3.Virus was added to Vero E6 cells at the indicated MOI for 1 h in DMEM/10% FBS at 37ºC/5%CO2, then BH3 mimetics added for up to 48 h.Cell viability was monitored using 1 µg/mL propidium iodide.Five fields of view at 10x magnification representing 33.6% well coverage was monitored for changes in cell viability every 6 h.PI-positive cells were identified using Incucyte software.

Bulk RNA sequencing preparation and analysis:
RNA was purified from TRIzol lysates using Direct-zol RNA Microprep kits (Zymo Research R2061) according to manufacturer recommendations that included DNase treatment.RNAseq assay was performed using an Illumina NEXTSeq 500 platform in the SBP Genomics Core.Briefly, PolyA RNA was isolated using the NEBNext® Poly(A) mRNA Magnetic Isolation Module and barcoded libraries were made using the NEBNext® Ultra II™ Directional RNA Library Prep Kit for Illumina® (NEB, Ipswich MA).Libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2 kit (Illumina Inc., San Diego CA).
For viral RNA analysis, sequencing reads were aligned to the SARS-CoV-2/human/USA/WA-CDC-WA1/2020 genome (GenBank: MN985325.1)using Bowtie2 and were visualized using IGV software.After further filtering and quality control, the R package edgeR29 was used to calculate reads per kilobase of transcript per million mapped reads (RPKM) and log2 [counts per million] matrices as well as to perform differential expression analysis.PCA was performed using log2 [counts per million] values and gene set analysis was run with WebGestalt30.Heatmaps and bar plots were generated using Graphpad Prism software, v.7.0d.In the volcano plots, differentially expressed genes (P-adjusted value < 0.05) with a log2 [fold change] > 1 are indicated.Read data was processed in BaseSpace (basespace.illumina.com).Reads were aligned to Homo sapiens genome (hg19) using STAR aligner (https://code.google.com/p/rna-star/)with default settings.
We and fold change ≥ 2.0 or ≤ -2.0 were selected as differentially expressed genes.Differentially expressed genes were then analyzed using Ingenuity Pathway Analysis (Qiagen, Redwood City, USA) and Gene Set Enrichment Analysis (GSEA) (146).

Single-cell lung organoid preparation for scRNA-seq
3D hPSC-LOs in ultra-low attachment plates were dissociated into single cells using accutase (Gibco) at 37 °C for 10 min, 24hpi.The dissociated organoids were pelleted and resuspended with DMEM F12 + 10% FBS.The resuspended organoids were then placed through a 40 µm filter to obtain a single-cell suspension.To remove the dead cells, 1 µL Dead Cell Removal cocktail (Annexin V, STEMCELL Technology) was added into the dissociated CO/PCCO single cells suspension together with 1 µL Biotin selection cocktail (Annexin V, STEMCELL Technology), gently mixed and incubated at RT for 3 min.After 3 min incubation, RapidSpheresTM (STEMCELL Technology) was vortexed for 30 s at RT and 2 µL added to the above mixture.At the same time, 850 µL LO base medium was added and gently pipetted 2~3 times.Tubes were then placed into a magnetic holder and incubated for 3 min at RT.After incubation, the supernatant was collected in a new tube for 10X GEM generation.For each of the SC-RNA-seq library, we pooled 20 individual LOs derived from H9 cells and iPSC-lines specified in the manuscript.
For the hPSC LOs outside the BSL3, they were dissociated from matrigel as described above using dispase and trypsin.The resuspended organoids were then placed through a 40 µm filter to obtain a single-cell suspension and stained with DAPI followed by sorting of live cells using the FACS Aria Fusion Flow Cytometer (BD Biosciences).The sorted cells were washed with 1× PBS + 0.04% BSA, counted manually after sorting and the cell viability was assessed with trypan blue.

Single cell library preparation and sequencing
10X sc-RNA-seq-3'-V3.1 kit (10X Genomics) was used to generate the GEM, cDNA, and sequencing library according to the manufacturer's instructions (10X Genomics).Briefly, live cells were partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs) with the 10x Chromium Controller (10X Genomics), 1000 cells were targeted.Upon cell lysis and dissolution of the Single Cell 3′-V3.1 Gel Bead within the droplet, primers containing an Illumina P7 and R2 sequence, a 14 bp 10XBarcode, a 10 bp randomer, and a poly-dT primer sequence were released and mixed with the cell lysates and bead-derived Master Mix.Barcoded, full-length cDNA from poly-adenylated mRNA was then generated in each individual bead, then individual droplets were broken and homogenized before the remaining non-cDNA components were removed with silane magnetic beads (Invitrogen).The libraries were then size-selected, and the R2, P5 and P7 sequences were added to each selected cDNA during end repair and adapter ligation.After Illumina bridge amplification of cDNA, each library was sequenced with the PE100 reads on the Novaseq6000 at the IGM Core in UCSD (mixed LO samples), and The Scripps Oceanography Genomics Core (PLO samples), to a depth of approximately 120M reads per sample.
The FASTQ files were imported to a 10x Cell Ranger data analysis pipeline (v.3.0.2) to align reads, generate feature-barcode matrices and perform clustering and gene expression analysis.In the first step, cellranger mkfastq demultiplexed samples and generated fastq files; and in the second step, cellranger count aligned fastq files to the reference genome and extracted gene expression unique molecular identifier (UMI) count matrix.To measure viral gene expression in LOs, we built a custom reference genome by integrating the four virus genes and GFP into the 10x pre-built human reference (GRCh38 v.3.0.0) using cellranger mkref.The sequences of four pseudo-viral genes (VSV-N, VSV-P, VSV-M and VSV-L) and SARS-CoV-2 genes (E, M, N, S, ORF1ab, ORF3a, ORF6, ORF7a, ORF8, and ORF10) were retrieved from NCBI https://www.ncbi.nlm.nih.gov/nuccore/335873).

scRNA-seq data analysis for lung organoids
We prepared a combined human genome version hg38 and SARS-CoV-2 genome version NC_045512v2 for alignment of 10X scRNA-seq raw data.We used human Gencode gene annotations v32 (Ensembl 98) augmented with SARS-CoV-2 predicted genes from NCBI for the Cell Ranger processing step.scRNA-seq reads from each sample were aligned to the combined genome using Cell Ranger v5.0.1.Data QC, filtering, integration, clustering, and differential expression were performed using Seurat v4.0.5 (147) and Harmony (148).Prior to integration of samples, cells with <40% mitochondrial content and < 98 percentile of genes detected (to remove potential doublets/multiplets) were kept in each sample for downstream analysis.The samples were merged and gene counts for the merged dataset were normalized using NormalizeData().3000 variable features were determined using FindvariableFeatures() and the data scaled using ScaleData().PCA components were computed using RunPCA() and subsequently the samples were integrated using RunHarmony() function.Clusters of cells were computed using RunUMAP(reduction="harmony," dims=1:40), FindNeighbors(reduction="harmony", dims=1:40), and FindClusters(resolution=0.3).Cluster markers were found using FindAllMarkers().Infection and IFNome scores were computed using AddModuleScore().Differential expression analyses were performed using FindMarkers().Feature, violin, and other plots were prepared using Seurat or ggplot2.Pathway analyses of differentially expressed genes were performed using Ingenuity Pathway Analysis (Qiagen, Redwood City, USA) and Gene Set Enrichment Analysis (GSEA) (146).
To obtain a list of top interferon-stimulated genes (ISGs), all human type I interferon datasets were taken from the Interferome v2.0 database (87).For each dataset, significantly up-regulated genes (log2 fold change > 1; unadjusted pvalue < 0.05) were ranked by the magnitude of their log2 fold changes.These ranks were then combined across datasets by geometric mean using the TopKLists (149) R package to produce an overall ranked list of type I ISGs (Interferon Stimulated Genes) identified.

Identifying & Labeling Endosomes (Macropinosomes)
Endosomes (specifically macropinosomes) were labeled and quantitated utilizing previously established procedures (150-152).Macropinosomes in live cells were labelled by their uptake of high molecular weight fluorescent dextran (FITC-Dextran).In brief, live cells are incubated with 1 mg/mL high molecular weight FITC-Dextran for 30 min, washed with cold PBS, fixed with 3.7% paraformaldehyde and nuclei stained with DAPI.Quantitation was as previously described using image analysis facilitated by Cytation 5 automated imaging and the Gen 5 software.Macropinocytosis in lung cells is also dependent on PI3K; uptake in these cells was inhibited by treatment with LY294002 (25 mM), an inhibitor of PI3K.Cells were pre-treated with the respective inhibitors for 0.5-3 hours prior to infection.At least 25,000 cells-per condition were analyzed for n=3 replicates.

Western blot
Cell samples were washed 3 times with PBS to remove traces of media components, pelleted and lysed with ice cold RIPA buffer containing protease and phosphatase inhibitors (Thermoscientific Halt kit).Samples were vigorously vortexed and centrifuged at 16,000 x g for 25 min at 4°C to separate the proteinous supernatant from the cellular debris.
The protein concentration of the supernatant was calculated using a BCA assay kit (ThermoScientific, 23225).10 µg of protein and Nupage sample buffer with diothiothreitol was boiled at 95°C for 3 min then loaded into a bis-tris gel and run with MES running buffer.The gel was transferred to 0.45 µm PVDF membrane for 60 min at 20V, then blocked in 5% w/v nonfat dry milk (NFDM) in tris buffered saline (TBS) for 60 min.Primary antibodies were diluted in 1% NFDM in TBS with 0.1% Tween-20 (TBST) and incubated overnight at 4°C.The membranes were then washed in TBST 3 times for 5 min on an orbital shaker.Secondary antibodies (LICOR goat anti rabbit 800CW and goat anti-mouse 680, 1:10,000) were diluted in 1% NFDM for 60 min then rinsed with TBST before imaging on the odyssey near infrared scanner.

Immunofluorescence
Dissociated lung cells were washed twice in PBS, then fixed in BD Fix/Perm containing 4% formaldehyde for 30 min at RT.Fixed cells were washed twice in BD Cytofix/Cytoperm and stained for SARS-CoV-2 with a primary Nucleocapsid antibody at 1:2000 (GeneTex GTX135357) conjugated to AF594 (ThermoFisher A20185) and nuclei counterstained with Hoechst or Sytox Green at 1:5000.3D Organoids were fixed in 4% paraformaldehyde for 1 h, then washed twice with PBS.
Fixed organoids were embedded in a 30% w/v solution of sucrose to preserve the fine cytoarchitecture from cryo-damage.
Organoids were then imbedded into OTC and sliced at 10 µm.Serial sections were mounted onto glass slides and allowed to air dry before staining.Sections were incubated in primary antibody diluted in 5% v/v donkey serum and 5% w/v bovine serum albumin with TX-100 for 24 h at 4°C then secondary antibodies at room temperature for 1 h.Nuclei were counterstained by DAPI (1:5000).The information for the primary and secondary antibodies is provided in Supplementary Table 7.The figures were processed using ImageJ software.

Intracellular flow cytometry analysis
Flow cytometry intracellular staining was performed following the instruction manual for the Fixation/Permeabilization Solution Kit (BD Biosciences).In brief, infected cells were washed twice with PBS, then incubated in Zombie UV (BioLegend 423107) per the manufacturer's recommendations for 30 min at room temperature.Cells were washed in FACS buffer (PBS + 2% FBS) once and resuspended in Fixation/Permeabilization solution at 4 °C for 30 min.
The cells were washed twice in 1× Perm/Wash buffer, incubated with primary Nucleocapsid antibody (GeneTex GTX135357) conjugated to AF594 (ThermoFisher A20185) at 4 °C for 30 min in the dark, and washed twice before flow cytometry analysis.Flow cytometry was performed using mock infected LOs as a negative control and gates were set to exclude the dead cells (Zombie UV stained) using the LSR Fortessa X-20 (BD Biosciences).All experiments were done a minimum of three separate times in three technical replicates.Data analysis was performed using FlowJo software and statistical analyses were done in Prism 8 (GraphPad).The information for the primary antibodies and antibodies is provided in Supplementary Table 7.
Poractant Alfa and recombinant SP-B Vero cells were plated in a monolayer in a 96 well plate and 3 titrations (1 mg/mL, 0.1 mg/mL, and 0.01 mg/mL) of Poractant alfa (Curosurf) were added 60 min prior to infection.The SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, WA, and NY) were added at 150 FFU and the plate was placed on a shaker for 1 h.The plate was then overlayed with 150 µL of DMEM/Carboxymethylcellulose/FBS mix and placed in the incubator for 24 h.The hPSC derived LOs were plated as a monolayer in a 96 well plate as previously described.They were exposed to 1 mg/mL of Poractant Alfa or 6.6 µM of recombinant SP-B (Prospec #PRO-2585) for 60 min prior to infection.SARS-CoV-2 was added at an MOI of 0.1 and the plate was placed on a shaker for 1 h.The plate was then overlayed with 150 µL of SFBM/Carboxymethylcellulose/FBS mix and placed in the incubator for 24 h.After 24h, the plates were washed twice with PBS and fixed and stained as per the immunofluorescence protocol above.

Quantification And Statistical Analysis
Statistical analysis was performed with Prism 8 (GraphPad Software).We compared all time course data related to cell death by two-way analysis of variance (ANOVA) followed by a Šidák multiple comparisons test.For the poractant alfa and rSP-B experiments, a one-way ANOVA or one-tailed t-test was used to determine significance.This was followed by a Šidák multiple comparisons test correction.

FIGURE S1 :FIGURE S2 :FIGURE S2 FIGURE S3 :FIGURE S4 :
FIGURE S1: Differentiation of human pluripotent stem cells (hPSC) into multi-cell type 3D lung organoids (LOs).(A) Schematic for directed differentiation of hPSCs into 3D LOs with corresponding phase contrast images of each step, including undifferentiated hPSCs, definitive endoderm, anterior foregut endoderm, and lung progenitor cells (LPC).Image of 3D LPCs shows NKX2-1 via GFP expression.(B) Live cell phase contrast images of the representative morphology of 3 different 3D lung organoid types: proximal (PLO), distal (DLO) and whole lung organoids (WLO).(C) Immunostaining of dissociated 3D WLOs with some representative lung epithelial and mesenchymal markers.Their respective cell types are shown in the schematic in (D).(D) Schematic of the epithelial cells in the PLOs and DLOs with representative immunofluorescent images of the epithelial markers found in the DLOs (alveolar type 2 cells) and PLOs (airway cells).

FIGURE S7: Acute infection with SARS-CoV-2 induces a lung-autonomous intrapulmonary inflammatory cascades. (
A, B) UMAPs of a subset of interferon genes differentially-expressed in (A) mock vs (B) infected LOs.Highest expression levels are in red.(C) Dot plot showing differentiallyexpressed chemokine and cytokine genes by cluster, comparing infected vs mock LOs.See text for significance of each.(D) Canonical pathway analysis of infected vs mock LOs by cluster using IPA.(E) Disease and Biological Functions using IPA.(F) Upstream Regulators using IPA.

FIGURE S7 FIGURE S8: Transcriptional changes and tropism in SP-B deficient hiPSC-LOs 16 hpi with SARS-CoV-2 (
A) UMAP of 3D SP-B deficient PLOs, 24 hpi with the Alpha variant.Ratios of each cluster in the wild type and SP-B mutant PLOs are presented in the right-hand figure.