Transcriptional profiles of stomach organoids from human embryonic stem cells.
ABSTRACT: Gastric ulcer, which affect many of patients and is deeply related with gastric cancer, is caused by chronic gastric acid stimulation. Stomach fundus, the main body of stomach, is a major source of gastric acid and peptidase for food digestion. Recapturing the main body of stomach requires mainly 3 functionally differentiated cells; parietal (oxyntic) cells, chief (zymogenic) cells, and surface mucous foveolar (pit) cells. We have previously shown the induction of stomach tissue with functional secreting activities by directed differentiation of mouse embryonic stem cells (ES cells) to stomach primordium with both gut epithelium and splanchnic mesoderm. However, generating human stomach with fundus and such functions has been elucidated and a long-desired goal. Here, we describe the method for establishing human embryonic stem cell-derived stomach organoids with fundus gland structure. Along with mouse stomach development and de novo stomach generation from mouse ES cells in vitro, we observed gut-like structure formation from human embryonic stem cells by induction of both endoderm and mesoderm. These human embryonic gut could differentiate into stomach primordium by growth factor stimulation as well as stomach development, and form stomach tissue in three-dimensional organoid culture. Furthermore, these stomach organoids contain fundus-like gland with parietal cells and chief cells, some of secreting activities, and is transcriptionally close to human stomach. Human functional stomach derived from embryonic stem cells represent powerful tools for analying human stomach development, and gastric ulcer related disease including gastric tumorgenesis. Overall design: We generated arrays from stomach organoids derived from human embryonic stem cells.
INSTRUMENT(S): Agilent-028004 SurePrint G3 Human GE 8x60K Microarray (Probe Name Version)
Project description:Background & Aims: Spasmolytic polypeptide/TFF2-expressing metaplasia (SPEM) is known to emerge following parietal cell loss and during Helicobacter pylori infection, however its role in gastric ulcer repair is unknown. Therefore, we sought to investigate if SPEM plays a role in epithelial regeneration. Methods: Acetic acid ulcers were induced in young (2-3 months) C57BL/6 mice to determine the quality of ulcer repair. Gastric tissue was collected and analyzed to determine the expression of SPEM within the regenerating epithelium. As a comparison to native tissue the expression of SPEM was also identified within cultured gastric mouse-derived organoids. Results: Wound healing in the mice coincided with the emergence of SPEM expressing CD44v within the ulcerated region. The emergence of SPEM was also observed in cultured gastric organoids. Conclusions: These data demonstrate the SPEM may play a role in epithelial regeneration. Conclusions: These data demonstrate the SPEM may play a role in epithelial regeneration. 4 samples were used for ulcerated and uninjured tissue. 1 sample was used for intact tissue and organoid-derived RNA. The 'Ulcerated' samples represent C57BL/6 mice with ulcers and the 'Uninjured' samples represent the healthy controls (for "ulcerated" samples). The "Intact stomach tissue" and "Gastric organoids" samples are other types of samples that compared separately. "Gastric organoids" in this comparison are derived from "Intact stomach tissue".
Project description:Successful differentiation methods have been developed in several endodermal organs, including liver, pancreas, and intestine from embryonic stem cell and induced pluripotent stem cells. Compare to these organs, however, little has been known in gastric differentiation from embryonic stem cells. Here, we established a method of gastrointestinal tissue differentiation methods, including stomach regions from embryonic stem cells. These gastrointestinal tissue built gut primordium-like spheroids, which is also able to mature into adult tissue containing all differentiated cells in three-dimensional culture. These embryonic stem cells derived gastrointestinal tissue possessed the similar gene expression profile that of adult stomach. These data indicated that functional gastrointestinal tissue can be derived from embryonic stem cells by mimicking in vivo development and could be useful for investigating gastrointestinal study such as disease modeling. Overall design: We generated arrays from embryonic stem cells-derived stomach tissue cells cultured in FGF10, WNT3A, NOGGIN and R-SPONDIN1 for 42 days, 46 days, and 5 months, or SHH and BMP4 for 4 days after cultured in FGF10, WNT3A, NOGGIN, and R-SPONDIN1 for 42 days. In addition, we also generated arrays from adult mouse stomach, esophagus, lung, intestine, and embryonic stomach as controls. All the datas were normalized to 75% tile shift.
Project description:Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bi-directionally towards pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of chief cells. By lineage tracing using a Troy-eGFP-ires-CreERT2 allele, single marked cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy+ chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific, 'plastic' subset of differentiated chief cells capable of replenishing entire gastric units, essentially serving as a quiescent ‘reserve’ stem cell. An EGFP-ires-CreERT2 cassette was introduced into the transcriptional start side of Tnfrsf19 (Troy), resulting in the expression of EGFP and CreERT2 under the control of endogenous Troy-regulatory sequences. Troy is expressed in basally located chief and parietal cells. Using FACS, Troy positive chief and parietal cells can be isolated separately. Expression profiling was performed on these two cell types: three arrays of sorted chief cells, two arrays of sorted parietal cells. As a reference, two arrays of separately isolated whole gastric corpus glands were added. In addition, single sorted Troy positive chief cells can be placed in culture, forming 3D cultures called organoids. Three arrays were generated from different organoid cultures, one time in normal medium containing the factors EGF, Gastrin, FGF10, Noggin, Wnt3a and Rspondin (EGFNWR medium) and one time in medium containing only EGF and Rspondin (ER medium).
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:In adult stomach, Lgr5 is a marker of epithelial glandular stem cells that grow in the three dimensional ex vivo culture system as organoids. However, little is known about the markers that characterize fetal progenitors before cytodifferentiation and their potential involvement in regenerative processes in adults. Using the ex vivo culture system, we isolated epithelial progenitors from the fetal stomach. These cells generated stable undifferentiated immortal spheroids showing lower growth factor requirements as compared to the adult-type gastric Lgr5-expressing organoids. Although very similar in their gene expression profiles, cultured fetal gastric and intestinal spheroids differentially expressed Sox2/Cdx2 genes involved in regional pre-patterning. Accordingly, gastric but not intestinal spheroids, spontaneously converted into the cell lineages of the adult glandular stomach ex vivo. In vivo, the Trop2 marker enriched in the fetal gastric spheroids was transiently expressed in the gastric epithelium before cytodifferentiation while it remained barely detectable under glandular homeostasis in adults. However, upon specific ablation of the Lgr5 stem cell pool, highly proliferative Trop2-expressing cells rapidly emerged in the adult epithelium, qualifying Trop2 as a marker of Lgr5-independent gastric stem cells. Together, these data indicate that the Trop2 receptor identifies gastric fetal progenitors and adult stem/progenitor cells involved in regeneration of glandular stomach. Overall design: 1. Mouse E15.5 stomach and small intestine epithelial progenitors were isolated from same pool of embryos and cultured ex vivo, growing as undifferentiated immortal spheroids. At passage 2 day 6 or passage 3 day 2 or day3 , spheroid-generating cells were harvested; 2. Mouse small intestine organoids were derived from E15.5 embryos and adult (3 month-old) mice cultures. Cells were isolated at passage 4 day5 (embryos) and passage 1 day5 (adult); 3. Trop2-expressing cells from mouse E14.5 proximal or distal stomach and intestine were isolated by flow cytometry; 4. Trop2-expressing or Lgr5-expressing cells from adult (2 month-old) Lgr5-DTR heterozygous mice pre-treated with or without diphtheria toxin were isolated by flow cytometry; and the the mRNA profiles of these cells were determined
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. Overall design: 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.
Project description:Induced pluripotent stem (iPS) cells are a novel stem cell population induced from mouse and human adult somatic cells through reprogramming by transduction of defined transcription factors. Recently, researchers success in generation various cells from iPSCs. Lung progenitor cells are able to differentiate to all of the cells exist in adult lung, attract attention for regenerative medicine. It is known to present between embryonic day 9.5 from 13.5 in mice. We can realized at regenerative medicine in lung using lung progenitor cell surface markers defined by compering each endoderm primordia of organ formation. We investigated the differences in gene expression of each endoderm organ. Mouse gut organs morphogenesis begin at E9.5-E11.5. The primordium lung, esophagus, stomach, and intestine at E11.5 were dissected and analysed transcription profile.
Project description:Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers. Overall design: We analyzed gene expression of 36 patient-derived gastric tumor, 9 normal-like, 1 IM, 8 normal stomach, 1 normal colon, 5 engineered normal stmomach and 1 engineered normal colon organoids. mRNA were extracted from organoids and hybridized to GeneChip PrimeView Human Gene Expression Array, and scanned using GeneChip Scanner 3000 7G.
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.