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Mus musculus

ABSTRACT: Single-cell RNA-seq of murine small intestine crypts

PROVIDER: PRJNA401770 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6007360_1.fastq.gz	Fastqsanger.gz
	SRR6007360_2.fastq.gz	Fastqsanger.gz
	SRR6007361_1.fastq.gz	Fastqsanger.gz
	SRR6007361_2.fastq.gz	Fastqsanger.gz
	SRR6007362_1.fastq.gz	Fastqsanger.gz

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Long-term in vitro expansion of epithelial organoids from human colon, adenoma, adenocarcinoma and Barrett's Esophagus

Project description:Background & Aims: We have recently established long-term culture conditions under which single crypts or stem cells derived from murine small intestine expand over long periods of time. Growing crypts undergo multiple crypt fission events, whilst simultaneously generating villus-like epithelial domains in which all differentiated cell types are present. We have now adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Methods: Based on the murine small intestinal culture system, we optimized the murine and human colon culture system. Results: Addition of Wnt3A to the growth factor cocktail allowed mouse colon crypts to expand indefinitely. Further addition of nicotinamide, a small molecule Alk inhibitor and a p38 inhibitor was essential for long-term human small intestine and colon culture. The culture system also allowed growth of murine Apcmin adenomas, human colorectal cancer and human esophageal metaplastic Barrett’s epithelium. Conclusion: The culture technology should be widely applicable as a research tool for infectious, inflammatory and neoplastic pathologies of the human gastrointestinal tract. Moreover, regenerative applications may become feasible with ex vivo expanded intestinal epithelia. Human organoids were grown embedded in Matrigel in HISC (Human intestinal stem cell culture) medium. Additionally, human small intestinal crypts and villi were isolated independently from a freshly operated sample. RNA was isolated using the RNeasy Micro kit (Qiagen). Samples were labled according to Agilent guidelines with Cy3, whereas human reference RNA (Stratagene) was labeled in Cy5. Feature Extraction Software was used to extract and normalize data.

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