Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Single cell RNA sequencing of endometrial organoids in the IVMC protocol

ABSTRACT: Accessing endometrial tissue during the early phases of the menstrual cycle is challenging. With this experiment we are capturing the dynamic epithelial cell states after breakdown using endometrial organoids (EOs) subjected to the in vitro menstrual cycle (IVMC) protocol. EOs are collected for scRNAseq analysis before breakdown (Hormonal Withdrawal 48h), 12h after breakdown (Post-breakdown 12h), 24h after breakdown (Post-breakdown 24h), 48h after breakdown (Post-breakdown 48h) as well as 24h (E2 differentiation 24h) and 48h after estrogen treatment (E2 differentiation 48h).

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Charlotte Soneson

PROVIDER: E-MTAB-15330 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In Rspondin-based three-dimensional cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. Here we report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely resemble the original tumor. The spectrum of genetic changes within the 'living biobank' agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell line- and xenograft-based drug studies and allow personalized therapy design. Self-renewal of the intestinal epithelium is driven by Lgr5 stem cells located in crypts. We have recently developed a long-term culture system that maintains basic crypt physiology. Wnt signals are required for the maintenance of active crypt stem cells. Indeed, the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo. R-spondin-1 is the ligand for Lgr5. Epidermal growth factor (EGF) signaling is associated with intestinal proliferation, while transgenic expression of Noggin induces a dramatic increase in crypt numbers. The combination of R-spondin-1, EGF, and Noggin in Matrigel sustains ever-expanding small intestinal organoids, which display all hallmarks of the original tissue in terms of architecture, cell type composition, and self-renewal dynamics. We adapted the culture condition for long-term expansion of human colonic epithelium and primary colonic adenocarcinoma, by adding nicotinamide, A83-01 (Alk inhibitor), Prostaglandin E2 and the p38 inhibitor SB202190. Of note, a two-dimensional culture method for cells from normal and malignant primary tissue has been described by Schlegel and colleagues. Here, we explore organoid technology to routinely establish and phenotypically annotate ‘paired organoids’ derived from adjacent tumor and healthy epithelium from CRC patients.

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Single cell RNA sequencing of endometrial organoids in the IVMC protocol

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