Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cellextrusion. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. However, accessing the apical enteroid surface ischallenging because it is enclosed within the spheroid. Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation.
This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules.
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