Dynamic Reorganization of Chromatin Accessibility Signatures during Dedifferentiation of Secretory Precursors into Lgr5+ Intestinal Stem Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE83394 | GEO | 2017/06/01
SECONDARY ACCESSION(S): PRJNA325805
REPOSITORIES: GEO
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Project description:Replicating Lgr5+ stem cells and quiescent Bmi1+ cells behave as intestinal stem cells (ISCs) in vivo. Disrupting Lgr5+ ISCs triggers epithelial renewal from Bmi1+ cells, from secretory or absorptive progenitors, and from Paneth cell precursors, revealing a high degree of plasticity within intestinal crypts. Here, we show that GFP+ cells from Bmi1GFP mice are preterminal enteroendocrine cells and we identify CD69+CD274+ cells as related goblet cell precursors. Upon loss of native Lgr5+ ISCs, both populations revert toward an Lgr5+ cell identity. While active histone marks are distributed similarly between Lgr5+ ISCs and progenitors of both major lineages, thousands of cis elements that control expression of lineage-restricted genes are selectively open in secretory cells. This accessibility signature dynamically converts to that of Lgr5+ ISCs during crypt regeneration. Beyond establishing the nature of Bmi1GFP+ cells, these findings reveal how chromatin status underlies intestinal cell diversity and dedifferentiation to restore ISC function and intestinal homeostasis.
| S-EPMC5505276 | biostudies-literature
Project description:The epithelial lining of the small intestine is continuously renewed from a small number of stem cells including Lgr5 expressing crypt base cells that have been shown to be a rapidly cycling stem cell population in homeostasis. Alternative markers of intestinal stem cells have variously identified populations as rapidly cycling or quiescent with proposed roles for the latter as a parallel or reserve stem cell population. However, the exact nature of quiescent crypt cells remains unknown. Here by applying novel mouse models that permit their isolation and characterisation as label-retaining cells (LRCs), and for the first time performing lineage tracing from them, we show quiescent cells to be committed secretory precursors that are capable of recall to the stem cell state. We reveal LRCs to be a small subset of the rapidly cycling Lgr5 expressing population committed along the Paneth-enteroendocrine lineage. Significantly, after injury and subsequent regeneration they are clonogenic, as shown by both lineage tracing and organoid growth demonstrating that they can be recalled to the stem cell pool. These findings in establishing quiescent cells as an effective clonogenic reserve during injury provides a motivation for investigating their role in the maintenance of cancer growth following adjuvant treatment. Six age and sex (female) matched Cyp1a1-H2B-YFP mice ten days post βNF induction were used comparing three cell populations from each. Following epithelial isolation, single cell preparation and staining with anti-CD24 anitbody and UEA-1 lectin, 30,000 cells were flow sorted for each population: Paneth cells (Paneth) were defined as CD24+/UEA+, YFP-LRCs (YFPpos) as CD24+/UEA-/YFP+ and LCCs (YFPneg) as CD24+/UEA-/YFP-. Microarray expression comparison was then performed to identify unique markers of each population. RNA amplification and hybridisation was performed at the Paterson Institute, Manchester, UK, Microarray Facility using Nugen Ovation Pico WTA System for amplification and then hybridisation to a Mouse Exon 1.0 ST Array. Arrays were scanned using an Affymetrix GeneChip scanner 3000 running GCOS software.
2013-02-27 | E-GEOD-43772 | biostudies-arrayexpress
Project description:The epithelial lining of the small intestine is continuously renewed from a small number of stem cells including Lgr5 expressing crypt base cells that have been shown to be a rapidly cycling stem cell population in homeostasis. Alternative markers of intestinal stem cells have variously identified populations as rapidly cycling or quiescent with proposed roles for the latter as a parallel or reserve stem cell population. However, the exact nature of quiescent crypt cells remains unknown. Here by applying novel mouse models that permit their isolation and characterisation as label-retaining cells (LRCs), and for the first time performing lineage tracing from them, we show quiescent cells to be committed secretory precursors that are capable of recall to the stem cell state. We reveal LRCs to be a small subset of the rapidly cycling Lgr5 expressing population committed along the Paneth-enteroendocrine lineage. Significantly, after injury and subsequent regeneration they are clonogenic, as shown by both lineage tracing and organoid growth demonstrating that they can be recalled to the stem cell pool. These findings in establishing quiescent cells as an effective clonogenic reserve during injury provides a motivation for investigating their role in the maintenance of cancer growth following adjuvant treatment.
2013-02-27 | GSE43772 | GEO
Project description:Lgr5+ stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF and Notch signals to neighboring Lgr5+ stem cells. While the colon lacks Paneth cells, Deep Crypt Secretory (DCS) cells are intermingled with Lgr5+ stem cells at crypt bottoms. Here, we report Reg4 as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon mini-gut formation. In agreement, sorted Reg4+ DCS cells promote organoid formation of single Lgr5+ colon stem cells. Stem cells are forced to generate DCS cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4+ DCS cells serve as Paneth cell equivalents in the colon crypt niche.
2016-10-05 | GSE63256 | GEO
Project description:Intestinal stem cell (ISC) differentiation is regulated precisely by a niche in the crypt, where lymphocytes may interact with stem and transient amplifying (TA) cells. However, whether and how lymphocyte-stem/TA cell contact affects ISC differentiation is largely unknown. Here, we uncover a novel role of T cell-stem/TA cell contact in ISC fate decisions. We show that intestinal lymphocyte depletion results in skewed ISC differentiation in mice, which can be rescued by T cell transfer. Mechanistically, integrin αEβ7 expressed on T cells binds to E-cadherin on ISCs and TA cells, triggering E-cadherin endocytosis and the consequent Wnt and Notch signaling alterations. Blocking αEβ7—E-cadherin adhesion suppresses Wnt signaling and promotes Notch signaling in ISCs and TA cells, leading to defective ISC differentiation. Thus, αEβ7+ T cells regulate ISC differentiation at single-cell level through cell-cell contact-mediated αEβ7—E-cadherin adhesion signaling, highlighting a critical role of the T cell-stem/TA cell contact in maintaining intestinal homeostasis.
2021-09-14 | GSE133691 | GEO
Project description:The endodermal lining of the adult gastro-intestinal tract harbors stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation program and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. Conversely, forced expression of Cdx2 in gastric organoids results in their intestinalization. The intestinal genetic program is thus critically dependent on the single transcription factor encoding gene Cdx2.
2014-12-04 | GSE51751 | GEO
Project description:The endodermal lining of the adult gastro-intestinal tract harbors stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation program and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. Conversely, forced expression of Cdx2 in gastric organoids results in their intestinalization. The intestinal genetic program is thus critically dependent on the single transcription factor encoding gene Cdx2. Small intestinal crypts and stomach glands were isolated from Cdx2-/fl / Lgr5-EGFP-CreERT2 mice and cultured for a week in order to generate small intestinal (SI) and stomach (Sto) in vitro organoids. The Lgr5-CreERT2 enzyme activity has been induced by overnight 4-hydroxytamoxifen induction. Tamoxifen treated and untreated Lgr5-EGFPhi SI and Sto stem cells were FACS sorted and seeded back into ENRWfg (Sto med) culture conditions in order to generate Cdx2-/fl small intestinal (Control SI), Cdx2null small intestinal (Cdx2null SI) and Cdx2-/fl stomach (Control Sto) clonal organoids. Cdx2-/fl SI organoids and Cdx2-/fl Sto organoids have been also cultured in ENR (SI med) to induce differentiation. After some passages of clonal organoid expansion, RNA was isolated from Control SI, Cdx2null SI and Control Sto Lgr5-EGFPhi FACS sorted stem cell populations and from smal intestinal and stomach organoids cultured in different conditions and hybridized on Affymetrix Mouse Gene ST 1.1 arrays.
2014-12-04 | E-GEOD-51751 | biostudies-arrayexpress
Project description:Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into 3D epithelial organoids containing all cell types at nearnormal ratios. Culture conditions to generate the main cell types have been established previously, but signals inducing the various types of enteroendocrine cells (EECs) have remained elusive. Here we generate quiescent Lgr5+ stem cells in vitro by inhibition of the EGF-receptor (EGFR) and mitogen-associated protein kinase (MAPK) signaling pathways in organoids, a state that can be readily reversed. Quiescent Lgr5+ stem cells gain a distinct molecular signature, biased towards EEC differentiation. Indeed, combined inhibition of Wnt, Notch and MAPK pathways efficiently generates a diversity of EEC subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and cell fate choice, and provide an in vitro approach for the study of the elusive EECs.
2017-01-03 | GSE80636 | GEO
Project description:Although Lgr5+ intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible so far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5+ intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5+ cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types. Total RNA was isolated from two independently established wild-type mouse small intestinal organoid lines after culture for 6 days in control condition or in the presence of CHIR99021, Valproic Acid (VPA) or both compounds. cRNA was labeled with Cy5 and hybridized against Cy3 labeled cRNA from control organoids (as an internal reference) on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F), resulting in eight individual arrays.
2013-11-22 | E-GEOD-51539 | biostudies-arrayexpress
Project description:Dietary effect on mouse intestinal Lgr5 stem cells
2018-08-09 | GSE102629 | GEO