Biology subjects

Zhu, J.

Publications and source records attributed to Zhu, J..

At least 505 records · Page 28Linked to original sources

bioRxiv2020

Parietal cell-Specific SLC26A9 Deletion induces spontaneous Gastric Carcinogenesis in Mice

Previous study showed that Slc26a9 loss impairs parietal cell function and survival. We investigated whether Slc26a9 loss causes spontaneous gastric carcinogenesis in mice and plays a role in the development and progression in human gastric cancer (GC). Gastric histopathology and potential molecular mechanism were explored in Slc26a9 knockout mice and wild-type littermates as well as Slc26a9fl/fl/Atp4b-Cre and Slc26a9fl/fl mice from 8 days to 18 months by histological and immunohistochemical analyses, quantitative PCR, in situ hybridization, and RNA microarray analysis, respectively. We demonstrated that loss of parietal cell expression of Slc26a9 is the key event to induce spontaneous gastric carcinogenesis in mice, and clarified the sequence of events leading to malignant transformation, including Slc26a9 deficiency in parietal cells resulted in dysregulated differentiation of stem cells in an inflammatory environment, activated Wnt signaling pathway to induce gastric epithelia cell hyperproliferation and apoptosis inhibition, as well as spontaneous epithelial to mesenchymal transition-induced cancer stem cell phenotypes. Downregulation of SLC26A9 correlated with GC patients short survival. Graphical AbstractLoss of parietal cell expression of Slc26a9 is the key event to induce spontaneous gastric carcinogenesis in transgenic mice. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/316398v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@33a2dcorg.highwire.dtl.DTLVardef@94ce8org.highwire.dtl.DTLVardef@9851eforg.highwire.dtl.DTLVardef@2b5fc3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Transcriptome analysis of potato leaves in response to infection with the necrotrophic pathogen Alternaria solani

The potato early blight was caused by Alternaria solani (Aso). At present, potato early blight resistant varieties are lacking. The experiment of Aso inoculation on a.k.a. Favorita showed that the content of chlorophyll decreased continuously within 2 days after inoculation. In addition, the contents of JA-Ile, SA, ABA and IAA were determined by LC-MS.The results of two defense-related hormones showed that jasmonic acid content decreased significantly, while jasmonic acid content did not change significantly. In order to understand the difference in gene expression of potatoes infected by Aso, the 24 hpi, 36 hpi and 48 hpi transcriptome were determined. The results showed that the gene annotation and cluster analysis of DEGs revealed a variety of defense responses to Aso infection, especially the plant hormone signal transduction pathway. The differential expression of 10 DEGs was confirmed by qPCR analysis. Finally, through weighted gene co-expression network analysis (WGCNA), "samonl" was determined as a key hormone-related network in the process of Aso infection in potatoes. This study provides an important clue for understanding the interaction between potato and Aso and for disease resistance breeding.

plant biology↗

bioRxiv2020

Myeloid cell-associated resistance to PD-1/PD-L1 blockade in urothelial cancer revealed through bulk and single-cell RNA sequencing

Adaptive immunity and tumor-promoting inflammation exist in delicate balance in individual tumor microenvironments; however, the role of this balance in defining sensitivity and resistance to PD-1/PD-L1 blockade therapy in urothelial cancer and other malignancies is poorly understood. We pursued an unbiased systems biology approach using bulk RNA sequencing data to examine pre-treatment molecular features associated with sensitivity to PD-1/PD-L1 blockade in patients with metastatic urothelial cancer and identified an adaptive_immune_response module associated with response and an inflammatory_response module and stromal module associated with resistance. We mapped these gene modules onto single-cell RNA sequencing data demonstrating the adaptive_immune_response module emanated predominantly from T, NK, and B cells, the inflammatory_response module from monocytes/macrophages, and the stromal module from fibroblasts. The adaptive_immune_response:inflammatory_response module expression ratio in individual tumors, reflecting the balance between antitumor immunity and tumor-associated inflammation and coined the 2IR score, best correlated with clinical outcomes and was validated in an independent cohort. Individual monocytes/macrophages with low 2IR scores demonstrated upregulation of proinflammatory genes including IL1B and downregulation of antigen presentation genes, were unrelated to classical M1 versus M2 polarization, and were enriched in pre-treatment peripheral blood from patients with PD-L1 blockade-resistant metastatic urothelial cancer. Single sentence summaryProinflammatory monocytes/macrophages, present in tumor and blood, are associated with resistance to immune checkpoint blockade in urothelial cancer.

genomics↗

bioRxiv2020

An expanded gene catalog of mouse gut metagenome

High-quality and comprehensive reference gene catalogs are essential for metagenomic research. The rather low diversity of samples used to construct existing catalogs of mouse gut metagenomes limits the size and numbers of identified genes in existing catalogs. We therefore established an expanded gene catalog of genes in the mouse gut metagenomes (EMGC) containing >5.8 million genes by integrating 88 newly sequenced samples, 86 mouse-gut-related bacterial genomes and 3 existing gene catalogs. EMGC increases the number on non-redundant genes by more than one million genes compared to the so far most extensive catalog. More than 50% of the genes in EMGC were taxonomically assigned and 30% were functionally annotated. 902 Metagenomic species (MGS) assigned to 122 taxa are identified based on the EMGC. The EMGC-based analysis of samples from groups of mice originating from different animal providers, housing laboratories and genetic strains substantiated that diet is a major contributor to differences in composition and functional potential of the gut microbiota irrespective of differences in environment and genetic background. We envisage that EMGC will serve as an efficient and resource-saving reference dataset for future metagenomic studies in mice.

bioinformatics↗

bioRxiv2020

Self-assembling nanoparticles presenting receptor binding domain and stabilized spike as next-generation COVID-19 vaccines

Vaccination against SARS-CoV-2 provides an effective tool to combat the COIVD-19 pandemic. Here, we combined antigen optimization and nanoparticle display to develop vaccine candidates for SARS-CoV-2. We first displayed the receptor-binding domain (RBD) on three self-assembling protein nanoparticle (SApNP) platforms using the SpyTag/SpyCatcher system. We then identified heptad repeat 2 (HR2) in S2 as the cause of spike metastability, designed an HR2-deleted glycine-capped spike (S2G{Delta}HR2), and displayed S2G{Delta}HR2 on SApNPs. An antibody column specific for the RBD enabled tag-free vaccine purification. In mice, the 24-meric RBD-ferritin SApNP elicited a more potent neutralizing antibody (NAb) response than the RBD alone and the spike with two stabilizing proline mutations in S2 (S2P). S2G{Delta}HR2 elicited two-fold-higher NAb titers than S2P, while S2G{Delta}HR2 SApNPs derived from multilayered E2p and I3-01v9 60-mers elicited up to 10-fold higher NAb titers. The S2G{Delta}HR2-presenting I3-01v9 SApNP also induced critically needed T-cell immunity, thereby providing a promising vaccine candidate. ONE-SENTENCE SUMMARYThe SARS-CoV-2 receptor binding domain and S2G{Delta}HR2 spike elicited potent immune responses when displayed on protein nanoparticles as vaccine candidates.

microbiology↗

bioRxiv2020

Synthetic mammalian signaling circuits for robust cell population control

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed Paradaux, in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales, of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, the same cells limited population growth, but were more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system, and demonstrate that paradoxical circuit architectures can provide robust population control.

synthetic biology↗

bioRxiv2020

The global landscape of SARS-CoV-2 genomes, variants, and haplotypes in 2019nCoVR

On 22 January 2020, the National Genomics Data Center (NGDC), part of the China National Center for Bioinformation (CNCB), created the 2019 Novel Coronavirus Resource (2019nCoVR), an open-access SARS-CoV-2 information resource. 2019nCoVR features a comprehensive integration of sequence and clinical information for all publicly available SARS-CoV-2 isolates, which are manually curated with value-added annotations and quality evaluated by our in-house automated pipeline. Of particular note, 2019nCoVR performs systematic analyses to generate a dynamic landscape of SARS-CoV-2 genomic variations at a global scale. It provides all identified variants and detailed statistics for each virus isolate, and congregates the quality score, functional annotation, and population frequency for each variant. It also generates visualization of the spatiotemporal change for each variant and yields historical viral haplotype network maps for the course of the outbreak from all complete and high-quality genomes. Moreover, 2019nCoVR provides a full collection of SARS-CoV-2 relevant literature on COVID-19 (Coronavirus Disease 2019), including published papers from PubMed as well as preprints from services such as bioRxiv and medRxiv through Europe PMC. Furthermore, by linking with relevant databases in CNCB-NGDC, 2019nCoVR offers data submission services for raw sequence reads and assembled genomes, and data sharing with National Center for Biotechnology Information. Collectively, all SARS-CoV-2 genome sequences, variants, haplotypes and literature are updated daily to provide timely information, making 2019nCoVR a valuable resource for the global research community. 2019nCoVR is accessible at https://bigd.big.ac.cn/ncov/.

genomics↗

bioRxiv2020

The immunodominant and neutralization linear epitopes for SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes a tremendous threat to global health. Although vaccines against the virus are under development, the antigen epitopes on the virus and their immunogenicity are poorly understood. Here, we simulated the three-dimensional structures of SARS-CoV-2 proteins with high performance computer, predicted the B cell epitopes on spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins of SARS-CoV-2 using structure-based approaches, and then validated the epitope immunogenicity by immunizing mice. Almost all 33 predicted epitopes effectively induced antibody production, six of which were immunodominant epitopes in patients identified via the binding of epitopes with the sera from domestic and imported COVID-19 patients, and 23 were conserved within SARS-CoV-2, SARS-CoV and bat coronavirus RaTG13. We also found that the immunodominant epitopes of domestic SARS-CoV-2 were different from that of the imported, which may be caused by the mutations on S (G614D) and N proteins. Importantly, we validated that eight epitopes on S protein elicited neutralizing antibodies that blocked the cell entry of both D614 and G614 pseudo-virus of SARS-CoV-2, three and nine epitopes induced D614 or G614 neutralizing antibodies, respectively. Our present study shed light on the immunodominance, neutralization, and conserved epitopes on SARS-CoV-2 which are potently used for the diagnosis, virus classification and the vaccine design tackling inefficiency, virus mutation and different species of coronaviruses.

immunology↗

bioRxiv2020

A CAR RNA FISH assay reveals functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue

Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness. Brief summaryWe developed an imaging platform and analysis pipeline to study large populations of engineered cells at a single cell level in situ. One Sentence SummaryWe developed a CAR RNA FISH assay to study chimeric antigen receptor T cell trafficking and function in mouse tissue.

cancer biology↗

bioRxiv2020

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GP{Delta}muc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GP{Delta}muc trimers in their unliganded state. We then display a modified GP{Delta}muc trimer on reengineered nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

microbiology↗

bioRxiv2020

Lanthipeptide Synthetases Participate the Biosynthesis of 2-Aminovinyl-Cysteine Motifs in Thioamitides

Thioamitides are a group of ribosomally synthesized and post-translational modified peptides with potent antiproliferative and pro-apoptotic activities. Their biosynthesis remains largely unknown, especially for the characteristic C-terminal 2-aminovinyl-Cysteine (AviCys) motifs. Herein, we report the discovery that homologs of class III lanthipeptide synthetases (LanKCts)encoded outside putative thioamitide biosynthetic gene clusters (BGCs) fully dehydrate the precursor peptides. Remarkably, LanKCt enzymes bind tightly to cysteine decarboxylases encoded inside thioamitide BGCs, and the resulting complex complete the macrocyclization of AviCys rings. Furthermore, LanKCt enzymes are present in the genomes of many thioamitide-producing strains and are functional when in complex with cysteine decarboxylases to produce AviCys macrocycles. Thus, our study reveals the participation of lanthipeptide synthetases as a general strategy for dehydration and AviCys formation during thioamitides biosynthesis and thus paves the way for the bioengineering of this class of bioactive natural products.

biochemistry↗

bioRxiv2020

Integrative Spatial Single-cell Analysis with Graph-based Feature Learning

We propose GLISS, a strategy to discover spatially-varying genes by integrating two data sources: (1) spatial gene expression data such as image-based fluorescence in situ hybridization techniques, and (2) dissociated whole-transcriptome single-cell RNA-sequencing (scRNA-seq) data. GLISS utilizes a graph-based association measure to select and link genes that are spatially-dependent in both data sources. GLISS can discover new spatial genes and recover cell locations in scRNA-seq data from landmark genes determined from SGE data. GLISS also offers a new dimension reduction technique to cluster the genes, while accounting for the inferred spatial structure of the cells. We demonstrate the utility of GLISS on simulated and real datasets, including datasets on the mouse olfactory bulb and breast cancer biopsies, and two spatial studies of the mammalian liver and intestine.

bioinformatics↗

bioRxiv2020

Anatomical modeling of brain vasculature in two-photon microscopy by generalizable deep learning

Objective and Impact StatementSegmentation of blood vessels from two-photon microscopy (2PM) angiograms of brains has important applications in hemodynamic analysis and disease diagnosis. Here we develop a generalizable deep learning technique for accurate 2PM vascular segmentation of sizable regions in mouse brains acquired from multiple 2PM setups. The technique is computationally efficient, thus ideal for large-scale neurovascular analysis. IntroductionVascular segmentation from 2PM angiograms is an important first step in hemodynamic modeling of brain vasculature. Existing segmentation methods based on deep learning either lack the ability to generalize to data from different imaging systems, or are computationally infeasible for large-scale angiograms. In this work, we overcome both these limitations by a method that is generalizable to various imaging systems, and is able to segment large-scale angiograms. MethodsWe employ a computationally efficient deep learning framework with a loss function that incorporates a balanced binary-cross-entropy loss and a total variation regularization on the networks output. Its effectiveness is demonstrated on experimentally acquired in-vivo angiograms from mouse brains of dimensions up to 808x808x702 m. ResultsTo demonstrate the superior generalizability of our framework, we train on data from only one 2PM microscope, and demonstrate high-quality segmentation on data from a different microscope without any network tuning. Overall, our method demonstrates 10x faster computation in terms of voxels-segmented-per-second and 3x larger depth compared to the state-of-the-art. ConclusionOur work provides a generalizable and computationally efficient anatomical modeling framework for brain vasculature, which consists of deep learning based vascular segmentation followed by graphing. It paves the way for future modeling and analysis of hemodynamic response at much greater scales that were inaccessible before.

neuroscience↗

bioRxiv2020

A Uropathogenic E. coli UTI89 model of prostatic inflammation and collagen accumulation for use in studying aberrant collagen production in the prostate

Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation utilizing transurethral instillations of E. coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 {micro}L) distributes exclusively to bladder, 100 and 200 {micro}L volumes distributes to bladder and prostate, and a 500 {micro}L volume distributes to bladder, prostate and ureter. A threshold optical density (OD) of 0.4 E. coli UTI89 in the instillation fluid is necessary for significant (p < 0.05) prostate colonization. E. coli UTI89 infection results in a low frequency, high volume spontaneous voiding pattern. This phenotype is due to exposure to E. coli UTI89, not catheterization alone, and is minimally altered by a 50 {micro}L increase in instillation volume and doubling of E. coli concentration. Prostate inflammation is isolated to the dorsal prostate and is accompanied by increased collagen density. This is partnered with increased density of PTPRC+, ProCOL1A1+ co-positive cells and decreased density of ACTA2+, ProCOL1A1+ co-positive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.

physiology↗

bioRxiv2020

Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including unambiguous O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30 O-glycosites were identified in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

biochemistry↗

bioRxiv2020

Tet1 isoforms differentially regulate gene expression, synaptic transmission and memory in the mammalian brain.

The dynamic regulation of DNA methylation in post-mitotic neurons is necessary for memory formation and other adaptive behaviors. Ten-eleven translocation 1 (TET1) plays a part in these processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating active DNA demethylation. However, attempts to pinpoint its exact role in the nervous system have been hindered by contradictory findings, perhaps due in part, to a recent discovery that two isoforms of the Tet1 gene are differentially expressed from early development into adulthood. Here, we demonstrate that both the shorter transcript (Tet1S) encoding an N-terminally truncated TET1 protein and a full-length Tet1 (Tet1FL) transcript encoding canonical TET1 are co-expressed in the adult brain. We show that Tet1S is the predominantly expressed isoform, and is highly enriched in neurons, whereas Tet1FL is generally expressed at lower levels and more abundant in glia, suggesting their roles are at least partially cell-type specific. Using viral-mediated, isoform- and neuron-specific molecular tools, we find that Tet1S repression enhances, while Tet1FL impairs, hippocampal-dependent memory. In addition, the individual disruption of the two isoforms leads to contrasting changes in basal synaptic transmission and the dysregulation of unique gene ensembles in hippocampal neurons. Together, our findings demonstrate that each Tet1 isoform serves a distinct role in the mammalian brain.

neuroscience↗

bioRxiv2020

Panoramic Insights into the Microevolution and Macroevolution of Prevotella copri-containing Lineage in Primate Guts

Prevotella copri and related taxa are widely detected in mammalian gut microbiomes and have been linked with one human enterotype. However, their microevolution and macroevolution among hosts are poorly characterized. In this study, extensively collected marker genes and genomes were analyzed to trace their evolutionary history, host specificity, and biogeographic distribution. Investigations based on 16S rRNA gene, gyrB, and genomes suggested that a multi-specific P. copri-containing lineage (PCL) harbors diverse species in higher primates. Firstly, P. copri is the dominant species of PCL in the human gut and consists of multiple groups exhibiting high genomic divergence and conspicuous but non-strict biogeographic pattern. Most African strains with high genomic divergence from other strains were phylogenetically placed near the species root, indicating the co-evolutionary history of P. copri and Homo sapiens. Secondly, although long-term co-evolution between PCL and higher primates was revealed, sporadic signals of co-speciation and extensive host jumping of PCL members were observed among higher primates. Metagenomic and phylogenetic analyses indicated that P. copri and other PCL species found in captive mammals have been recently transmitted from humans. Thirdly, strong evidence was found on the extensively horizontal transfer of genes (e.g., carbohydrate-active enzyme encoding genes) among sympatric P. copri groups and PCL species in the same primate host. Our study provides panoramic insights into the complex effects of vertical and horizontal transmission, and potential niche adaption on speciation, host, and biogeographical distribution spanning microevolutionary and macroevolutionary history for a certain gut bacterial lineage. ImportancePrevotella copri and its related taxa, which we designated as Prevotella copri-containing lineage (PCL) in the present study, are widely detected in guts of human, non-human primates and many captive mammals, showing positive or negative correlation to some human diseases. However, a comprehensive understanding on its microevolutionary (within P. copri) and macroevolutionary (among PCL members) history across host species and host biogeography is still lacking. According to our analysis based on 16S rRNA gene, gyrB and genomes, we provided the panoramic insights into the putative effects of vertical transfer, horizontal transmission and potential niche selection on host and biogeographical distribution of this gut bacterial lineage and P. copri. To our knowledge, it is the first time that a gut bacterial lineage was studied at both micro- and macroevolutionary levels, which can aid our systematic understanding on the host-microbe co-evolutionary interactions.

microbiology↗

bioRxiv2020

Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 "apical-out" organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

cell biology↗