RNA-seq of sorted intestinal stem and Paneth cells
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PROVIDER: PRJNA660763 | ENA |
REPOSITORIES: ENA
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Project description:The impact of Mll1 removal on the intestinal stem cells and its direct effect on neighbouring Paneth cells was evaluated in sorted intestinal stem and Paneth cells from Mll1FC/+; Lgr5-eGFP-CreERT2/+ (control) and Mll1FC/FC; Lgr5-eGFP-CreERT2/+ (knockout) mice, 4 and 10 days after tamoxifen-induced mutagenesis. Using 75-base-pair reads, 30 million reads per sample with comparable unique mapped reads for stem (70-77%) and Paneth (60-76%) cells were obtained. To analyze differentially expressed genes, we applied DESeq2 analysis to the RNA-seq dataset. Analysis by DAVID and GSEA at a false discovery rate (FDR) of 5% was conducted.The stem cell transcriptome revealed that Mll1 knockout stem cells exhibited a decreased expression of several transcription factors and stem cell genes. Additionally, Mll1 ablation in stem cells had an impact on Paneth cells. Downregulation of Paneth cell specific markers indicated a loss of Paneth cell identity.
2021-11-10 | GSE157285 | GEO
Project description:Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, small cycling cells located at crypt bottoms1, 2. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells, that are known to produce bactericidal products such as lysozyme and cryptdins/defensins3. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells4. Here, we note a close physical association of Lgr5 stem cells with Paneth cells in vivo and in vitro. CD24+ Paneth cells express EGF, TGF?, Wnt3 and the Notch-ligand Dll4, all essential signals for stem cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell. We used intestinal cell fractions from Lgr5-EGFP-ires-CreERT2 mice, expressing GFP under the control of the Lgr5 promoter. RNA was isolated from two FACS sorted cell populations: stem cells were sorted based on high level of GFP expression (GFPhi) and Paneth cells were sorted based on high level of CD24 expression (CD24hi) and high side-scatter (SSChi). Differentially labelled cRNA from GFPhi and CD24hi/SSChi cells from two different sorts (each combining ten individual mice) were hybridized on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays.
2010-11-29 | E-GEOD-25109 | biostudies-arrayexpress
Project description:The experiment shows the aging related expression changes in Paneth and Lgr5Hi cells between young and old mice. These expression changes are related to the role of stem cell niche in aging and regeneration of intestinal epithelium.
2019-07-08 | E-MTAB-7916 | biostudies-arrayexpress
Project description:Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, small cycling cells located at crypt bottoms1, 2. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells, that are known to produce bactericidal products such as lysozyme and cryptdins/defensins3. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells4. Here, we note a close physical association of Lgr5 stem cells with Paneth cells in vivo and in vitro. CD24+ Paneth cells express EGF, TGFα, Wnt3 and the Notch-ligand Dll4, all essential signals for stem cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells dramatically improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24+ cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.
2010-11-29 | GSE25109 | GEO
Project description:Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their co-existence with stem cells making it difficult to culture Panth cells alone in vitro. Using a simplied toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region and surfactant assisted one pot protein digestion, we identified more than 1,300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues, a comparable proteomic coverage can be achieved with 3,600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplied workflow enables profiling of Paneth cell associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.
2022-10-12 | PXD034143 | Pride
Project description:Purpose: Characterize functional alterations in stem cells and paneth cells obtained from young and aged mice, focusing on age-based impairment of intestinal regeneration due to a decline in canonical Wnt signaling. Methods: mRNA profiles of young and aged stem and paneth cells were generated in triplicate (with one additional young paneth sample) using the Illumina HiSeq 2500. Reads that passed quality filters were aligned to the mm10 mouse genome with annotations provided by UCSC. Results: Approximately 10 millions reads were aligned per sample, corresponding to 36186 transcripts -- of these, 19574 exhibited reasonable expression. The effect of age was tested wtihin paneth and stem cells, using unpaired t-tests with a p-value cutoff of 0.05 and fold change cutoff of 1.5. Within paneth cells, 1025 genes were significant; within stem cells, 750 genes exhibited differential regulation. Among the downregulated genes in paneth and stem cells, we observed significant enrichment of canonical Wnt signaling genes. Conclusion: Age-related downregulation of canonical Wnt signaling is involved in the impairment of intestinal regulation upon aging.
2017-03-14 | GSE84061 | GEO
Project description:The impact of Mll1 removal on intestinal stem cells expressing an oncogenic form of beta-catenin (beta-cateninGOF) was analysed in 4 pairs of sorted intestinal stem cells of Lgr5-CreERT2; beta-cateninGOF;Mll1+/- (control) and Lgr5-CreERT2; beta-cateninGOF;Mll1-/- (knockout) at 10 days after tamoxifen-induced mutagenesis. Using 75-base-pair reads, around 30 million reads per sample with comparable unique mapped reads (73-78%) were obtained. To analyze differentially expressed genes, we applied DESeq2 analysis to the RNA-seq dataset. Differentially expressed genes in beta-cateninGOF; Mll1-/- versus beta-cateninGOF; Mll1+/- stem cells showed both up- and downregulation of genes at a false discovery rate (FDR) of 10%. This included a global increase in the expression of goblet cell-specific genes. Downregulated genes included specific markers of Paneth cells, indicating that ablation of Mll1 shifted the Paneth-like identity of beta-cateninGOF stem cells towards a goblet cell fate. The stem cell transcriptome further revealed that beta-cateninGOF; Mll1-/- stem cells exhibited a decreased expression of several transcription factors and stem cell genes.
2020-10-09 | GSE148394 | GEO
Project description:The impact of removal of Mll1 in intestinal Paneth cells expressing an constitutively active form of beta-catenin (beta-cateninGOF) was analysed in 4 pairs of sorted Paneth cells of Lgr5-CreERT2; beta-cateninGOF;Mll1+/- (control) and Lgr5-CreERT2; beta-cateninGOF;Mll1-/- (knockout) mice at 10 days after tamoxifen-induced mutagenesis. Using 75-base-pair reads, around 30 million reads per sample with comparable unique mapped reads (52-93%) were obtained. To analyze differentially expressed genes, we applied DESeq2 analysis to the RNA-seq dataset. Differentially expressed genes in beta-cateninGOF; Mll1-/- versus beta-cateninGOF; Mll1+/- Paneth cells showed both up- and downregulation of genes at a false discovery rate (FDR) of 10%. This included a global increase in the expression of goblet cell-specific genes. Downregulated genes included specific markers of Paneth cells, indicating that ablation of Mll1 in Paneth cells shifted their identity towards a mixed Paneth-goblet cell fate.
2022-01-08 | GSE177047 | GEO
Project description:RNA-seq of mouse Paneth and Lgr5 expressing intestinal stem cells (ISCs)
| PRJEB32330 | ENA
Project description:The goal of this project is to generate transcriptome profiling of Lgr5-GFP high intestinal stem cells (High)/Lgr5-GFP low progenitors (Low) and CD24+C-kit+ Paneth (PA) cells for a systemic analysis of cellular pathways involved in responses to fasting and aging.
2019-07-19 | GSE122230 | GEO