Project description:The purpose of this study is to compare the cellular populations present within granuloma (Sca1+) and non-granuloma (Sca-1–) crypt epithelum arising from parasitic H polygyrus infections.
Project description:c-Kit+ cells mark deep crypt secretory cells (DCSs) and supply Wnt3, EGF, and Notch signals to support their neighboring crypt bottom-intermingled Lgr5+ cells. We used single cells sequencing (scRNA-seq) to analyse the diversity and stemness of c-Kit+ cells in the colonic epithelium.
Project description:The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of approximately six cycling Lgr5+ stem cells at the bottoms of small intestinal crypts1. We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. Keywords: expression profiling
Project description:The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of approximately six cycling Lgr5+ stem cells at the bottoms of small intestinal crypts1. We have now established long-term culture conditions under which single crypts undergo multiple crypt fission events, whilst simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt-villus organoids. Tracing experiments indicate that the Lgr5+ stem cell hierarchy is maintained in organoids. We conclude that intestinal crypt-villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche. Keywords: expression profiling Freshly isolated small intestinal crypts from two mice were divided into two parts. RNA was directly isolated from one part (RNeasy Mini Kit, Qiagen), the other part was cultured for one week according to the conditions described in the associated paper, followed by RNA isolation. We prepared labeled cRNA following the manufacturer’s instruction (Agilent Technologies). Differentially labelled cRNA from small intestinal crypts and organoids were hybridised separately for the two mice on a 4X44k Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts and villus from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (cKO) (Villin-Cre+; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% deletion efficiency. RNA was directly isolated from intestinal crypt and villus, and this was used for RNAseq. Gene expression analysis of cKO derived crypt and villus provides a spatially restricted outlook on the maturation status of the intestinal epithelium in the villi and the absence of Paneth cells in the crypt.
Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we isolated small intestinal crypts and villus from wild type (WT) (Villin-Cre -; Lsd1f/f) and intestinal-epithelial-specific knock-out (cKO) (Villin-Cre+; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% recombination efficiency. RNA was directly isolated from the crypt and villus, and this was used for RNAseq. Gene expression analysis of cKO derived crypt and villus provides a spatially restricted outlook on the maturation status of the intestinal epithelium in the villi and the absence of Paneth cells in the crypt. Additionally, these mice were treated with antibiotics to study epithelium intrinsic changes related to LSD1 deletion but independent of the bacterial microbiome.
Project description:Mycobacterial granulomas are categorical manifestations of tuberculosis pathogenesis. They result from an ensemble of immune responses to Mycobacterium tuberculosis infection, but the identities, arrangement, cellular interactions and regulation of the cells that comprise them has thus far been incompletely understood. We conducted spatial and single-cell RNA sequencing of clinical granuloma biopsy specimens from tuberculosis patients. We found that granulomas consist of concentric transcriptional laminae surrounding areas of central necrosis. Gene expression programs associated with regional architecture were conserved among samples. We identified distinct populations of granuloma-associated stromal cells, fibroblasts, lymphocytes, mast cells, dendritic cells, neutrophils and macrophages. Populations further differentiate into multiple granuloma-specific states that correlate with location. We used inferential analysis to predict dominant granuloma cell-cell interactions, the activity of major signaling pathways, and differential transcription factor activities. Using spatial deconvolution, we mapped a conserved pattern of cellular organization dominated by macrophages rich in osteopontin/SPP1 expression. Trajectory analysis of macrophage subtypes mapped their differentiation and supported the importance of SPP1 to granuloma macrophage polarization. Cumulatively, we have identified a dominant macrophage granuloma population as well as its central regulatory gene in human samples and confirmed its importance to granuloma biology in vivo.
Project description:Mycobacterial granulomas are categorical manifestations of tuberculosis pathogenesis. They result from an ensemble of immune responses to Mycobacterium tuberculosis infection, but the identities, arrangement, cellular interactions and regulation of the cells that comprise them has thus far been incompletely understood. We conducted spatial and single-cell RNA sequencing of clinical granuloma biopsy specimens from tuberculosis patients. We found that granulomas consist of concentric transcriptional laminae surrounding areas of central necrosis. Gene expression programs associated with regional architecture were conserved among samples. We identified distinct populations of granuloma-associated stromal cells, fibroblasts, lymphocytes, mast cells, dendritic cells, neutrophils and macrophages. Populations further differentiate into multiple granuloma-specific states that correlate with location. We used inferential analysis to predict dominant granuloma cell-cell interactions, the activity of major signaling pathways, and differential transcription factor activities. Using spatial deconvolution, we mapped a conserved pattern of cellular organization dominated by macrophages rich in osteopontin/SPP1 expression. Trajectory analysis of macrophage subtypes mapped their differentiation and supported the importance of SPP1 to granuloma macrophage polarization. Cumulatively, we have identified a dominant macrophage granuloma population as well as its central regulatory gene in human samples and confirmed its importance to granuloma biology in vivo.