Project description:Human embryonic stem cells (WA01) were differentiated in a step-wise manner into three-dimensional human gastric organoids (hGOs). At day 34 of differentiation, the hGOs were collected and analyzed by RNA-sequencing.
Project description:Physiological temperature in the human nasal passages peaks at ~34°C, which is lower than internal human body temperature. This experiment explored the effect on the transcriptome of Human Nasal Organoids (HNOs) based on differentiating at 34°C vs. 37°C for 21 days vs. 37°C for 21days followed by a shift to 34°C for 2 additional days. RNAseq was also performed on undifferentiated HNOs.
Project description:To study Aqp5 functions in human gastric cancer organoids, we generated Aqp5 FLIP-Puro organoids allowing for inducible loss of Aqp5 expression upon Cre gesicle administration. We analysed transcriptomes of these organoids at various timepoints following loss of Aqp5 to elucidate potential functions of Aqp5 in this system.
Project description:We previously established long-term 3D organoid culture systems for several murine tissues (intestine, stomach, pancreas and liver) as well as human intestine and pancreas. Here, we describe culture conditions to generate long-term 3D culture from human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. Human gastric cultures can expand indefinitely in 3D Matrigel. Cultures can be generated from normal tissue, from single sorted stem cells, or from tumor tissue. Organoids maintain many characteristics of the respective tissue in terms of histology, marker expression and euploidy. Organoids from normal tissue express markers of four lineages of the stomach and self-organize in gland and pit-domains. They can be directed to specifically express either lineages of the gastric gland, or the gastric pit by addition of Nicotinamide and withdrawal of Wnt. While gastric pit lineages react marginally to bacterial infection, gastric gland lineages mount a strong inflammatory response. The gastric culture system provides a unique tool to study gastric pathologies. We generated 2 sets of experiments. The first set contains organoids in 4 conditions: (1) organoids in expansion condition ENRWFGNiTi ("gland-type organoids") from 3 donors, (2) organoids as in 1 but differentiated for 4 days in differentiation condition ENR_FGNiTi ("pit'type organoids"), (3) organoids as in 1 but infected with Helicobacter pylori strain P12 MOI 50 for 2 h, (4) organoids as in 2 but infected as in 3. All 4 conditions were tested on the same organoid line in parallel. This experiment was conducted independently with cultures from 3 different donors. The second set of experiments compares freshly isolated glands with organoids. Samples from 2 patients were analyzed. Each patient received a total gastrectomy. From each patient, glands from corpus region or pyloric antrum were isolated. From each isolation, one aliquod was stored for microarray analysis and one aliquod used to generate organoids. Organoids and glands were subsequently lysed and analyzed in parallel.
Project description:The directed differentiation of adult gastric stem cells into specific epithelial cell types is crucial for gastric homeostasis. Although it is well appreciated that the niche plays a critical role in gastric epithelium cell differentiation, the relevant molecular factors and the underlying regulatory mechanisms remain poorly understood. In this study, by combining the knowledge of the niche cells obtained from single-cell RNA sequencing and manipulation of signaling pathways, we achieved efficient differentiation of various gastric epithelial cell types in mouse and human gastric organoids. These in vitro differentiated cells showed a similar gene expression profile to those in gastric tissues. Specifically, we showed that BMP4 signaling stimulates parietal cell differentiation, EGF and BMP4 signaling work together to enhance pit cell differentiation, while TGF-β inhibition facilitates chief cell differentiation. In addition, the small molecule Isoxazole 9, which has been shown to stimulate differentiation of neuronal cells, pancreatic β-cells, and intestinal enteroendocrine cells, promotes parietal and endocrine cell differentiation in gastric organoids. Our data also revealed the different requirements of parietal and chief cell differentiation between mouse and human. Together, our findings provide a mechanistic insight into gastric epithelial cell differentiation and uncover its similarities and differences between mouse and human.
Project description:The directed differentiation of adult gastric stem cells into specific epithelial cell types is crucial for gastric homeostasis. Although it is well appreciated that the niche plays a critical role in gastric epithelium cell differentiation, the relevant molecular factors and the underlying regulatory mechanisms remain poorly understood. In this study, by combining the knowledge of the niche cells obtained from single-cell RNA sequencing and manipulation of signaling pathways, we achieved efficient differentiation of various gastric epithelial cell types in mouse and human gastric organoids. These in vitro differentiated cells showed a similar gene expression profile to those in gastric tissues. Specifically, we showed that BMP4 signaling stimulates parietal cell differentiation, EGF and BMP4 signaling work together to enhance pit cell differentiation, while TGF-β inhibition facilitates chief cell differentiation. In addition, the small molecule Isoxazole 9, which has been shown to stimulate differentiation of neuronal cells, pancreatic β-cells, and intestinal enteroendocrine cells, promotes parietal and endocrine cell differentiation in gastric organoids. Our data also revealed the different requirements of parietal and chief cell differentiation between mouse and human. Together, our findings provide a mechanistic insight into gastric epithelial cell differentiation and uncover its similarities and differences between mouse and human.
Project description:The directed differentiation of adult gastric stem cells into specific epithelial cell types is crucial for gastric homeostasis. Although it is well appreciated that the niche plays a critical role in gastric epithelium cell differentiation, the relevant molecular factors and the underlying regulatory mechanisms remain poorly understood. In this study, by combining the knowledge of the niche cells obtained from single-cell RNA sequencing and manipulation of signaling pathways, we achieved efficient differentiation of various gastric epithelial cell types in mouse and human gastric organoids. These in vitro differentiated cells showed a similar gene expression profile to those in gastric tissues. Specifically, we showed that BMP4 signaling stimulates parietal cell differentiation, EGF and BMP4 signaling work together to enhance pit cell differentiation, while TGF-β inhibition facilitates chief cell differentiation. In addition, the small molecule Isoxazole 9, which has been shown to stimulate differentiation of neuronal cells, pancreatic β-cells, and intestinal enteroendocrine cells, promotes parietal and endocrine cell differentiation in gastric organoids. Our data also revealed the different requirements of parietal and chief cell differentiation between mouse and human. Together, our findings provide a mechanistic insight into gastric epithelial cell differentiation and uncover its similarities and differences between mouse and human.
Project description:To understand the molecular basis of the acquisition of 5-FU resistance in gastric cancer stem cells, we established 5-FU-resistant gastric cancer organoids. We used microarrays to detail the global program of gene expression underlying 5-FU resistance and maintenance of stem cell properties in gastric cancer.