Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank

ABSTRACT: This study has two components: (1) Human colon adenoma organoids (n=4 patients) were dissociated into single cells. Cells were incubated with a magnetic bead bound to an LGR5 antibody and run through a magnetic column. Magnet bound cells and flow through negative (FTN) cells were obtained. Magnet bound and FTN cells were incubated with an APC-check reagent (which binds to the magnetic bead on the LGR5 antibody) and DAPI, before being sorted by flow cytometry. 3 populations of live (DAPI-) cells were collected: FTN: Flow through negative. LGR5 negative by magnet and by flow cytometry SortedNeg: Magnet bound cells that were negative for LGR5 by flow cytometry SortedPos: Magnet bound cells that were positive for LGR5 by flow cytometry (2) Human colon organoids, as well as the tissue the organoid was derived from and adjcacent normal tissue (from n=19) were also profiled for known colorectal cancer associated mutations using the Qiagen Qiaseq Colorectal Cancer Panel, which provides targeted sequencing information for 71 genes.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Julie Laliberte

PROVIDER: E-MTAB-4698 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

Dame Michael K MK Attili Durga D McClintock Shannon D SD Dedhia Priya H PH Ouillette Peter P Hardt Olaf O Chin Alana M AM Xue Xiang X Laliberte Julie J Katz Erica L EL Newsome Gina M GM Hill David R DR Miller Alyssa J AJ Tsai Yu-Hwai YH Agorku David D Altheim Christopher H CH Bosio Andreas A Simon Becky B Samuelson Linda C LC Stoerker Jay A JA Appelman Henry D HD Varani James J Wicha Max S MS Brenner Dean E DE Shah Yatrik M YM Spence Jason R JR Colacino Justin A JA

Development (Cambridge, England) 20180314 6

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-ass ...[more]

PMID: 29467240

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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.

Dataset Information

RNA-seq of LGR5(+) and LGR5(-) cells isolated from human colon adenoma organoids and targeted DNA-seq of a colorectal biobank

Publications

Identification, isolation and characterization of human LGR5-positive colon adenoma cells.

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