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.
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.
Project description:Genome wide expression profiling to determine the overlap of Affymetrix-signals with SOLID sequencing RNA was extracted using the Qiagen RNeasy kit following the manufacturers guidelines, arrays were prepared and hybridized following the Affymetrix protocol. Mus musculus samples from small intestine and colon, to be compared to transcript data aquired with other techniques
Project description:The small intestinal crypts harbour secretory Paneth cells (PCs), which express bactericidal peptides that are crucial for maintaining intestinal homeostasis. Considering the diverse environmental conditions throughout the course of the small intestine, multiple subtypes of PCs are expected to exist. We applied single cells RNA-sequencing of PCs combined with deep bulk RNA-sequencing on PC populations of different locations (duodenum, jejunum, ileum) and discovered several expression-based clusters. Some of these are discrete and resemble tuft cell-like PCs, goblet cell-like PCs, PCs expressing stem cell markers, and senescent PCs. Other clusters are less discrete, but based on bulk RNA-seq appear to be derived from different locations along the small intestinal tract. Furthermore, a comprehensive spatial analysis was conducted using the Resolve bioscience technique, identifying different PCs clusters along the small intestine, but not along the crypts themselves.
Project description:Although small intestine epithelium has been studied widely in murine, a comprehensive expression landscape in human is still lacking. To explore the cellular diversity of human small intestine epithelium, we sampled the ileal crypts from two patients suffered from right-sided colon cancer while their ilea were relatively normal. scRNA-seq libraries were generated using the 10X v3 Kit, which could guarantee both throughput and quality
Project description:In this experiment, we determined the differences in the transcriptomes of freshly isolated murine small intestine and colon derived crypts. The data shows that the mRNA profiles of the two tissues significantly differ in their ground state. Interestingly, we found that the expression levels of ERK pathway components as well as their positive and negative regulators significantly differ between small intestinal and colonic crypts. This observation indicates that the ERK pathway displays higher basal activity in the small intestine compared to the colon.
Project description:We used single cell RNA sequencing (scRNA-seq) to analyze the diversity of cells within the murine small intestine in the absence of functional Apc.