Project description:In vitro human pluripotent stem cell derived intestinal organoids (HIOs) are immature and lack for diverse differentiated secretory cell types. We would like to test the hypothesis whether addition of a mesenchyme secreting ligand which is depleted in canonical organoid culture media could increase the maturity and secretory cell type diversity in HIOs in vitro. To do this, we adapted the directed differentiation protocol of HIOs by growing HIOs in media with EGF, NOGGIN, R-spondin-1 (ENR) for 30 days, isolated epithelial cells with dispase, recovered them with adult intestinal enteroid media with Wnt-3A (WENR). Then we introduced the mesenchyme secreting ligand NRG1 to the established enteroid culture (WENR+NRG1) and compared them to the enteroids grown in control condition (WENR).
Project description:When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as host-pathogen intreaction shows great promise for modelling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modelling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a dramatic reduction in parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.
Project description:Recently, three-dimensional small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small-molecule drug treatment can skew organoid epithelial cell differentiation towards particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the use and characterisation of enteroid models has not yet been fully explored, such that the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. In this study, we have performed label-free quantitative proteomics to determine whether skewing murine enteroid cultures towards the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Our data confirm that skewed enteroids recapitulate important features of the in vivo gut environment, confirming that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, by comparison of our mass spectrometry data with histology data contained within the Human Protein Atlas, we identify putative novel markers for goblet and Paneth cells.
Project description:To further the development of an in vitro model that can more faithfully recapitulate drug disposition of orally administered drugs, we investigated the utility of human enteroid monolayers to simultaneously assess intestinal drug absorption and first-pass metabolism processes. We cultured human enteroid monolayers from three donors, derived via biopsies containing duodenal stem cells that were propagated and then differentiated atop permeable Transwell® inserts, and confirmed transformation into a largely enterocyte population via RNA-seq analysis and immunocytochemical (ICC) assays. Proper cell morphology was assessed and confirmed via bright field microscopy and ICC imaging of tight junction proteins and other apically and basolaterally localized proteins. Enteroid monolayer barrier integrity was demonstrated by elevated transepithelial electrical resistance (TEER) that stabilized after 10 days in culture and persisted for 42 days. These results were corroborated by low paracellular transport probe permeability at 7 and 21 days in culture. The activity of a prominent drug metabolizing enzyme, CYP3A, was confirmed at 7, 21, and 42 days culture under basal, 1?,25(OH)2 vitamin D3-induced, and 6',7'-dihydroxybergamottin-inhibited conditions. The duration of these experiments is particularly noteworthy, as this is the first study assessing DMET function for enteroids cultured for greater than 12 days. The sum of these results suggests enteroid monolayers are a promising in vitro model to investigate and quantitatively predict an orally administered drug's intestinal absorption and/or metabolism and associated drug-drug/natural product-drug interactions.
Project description:Breastfeeding has been associated with long lasting health benefits. Nutrients and bioactive components of human breast milk promote cell growth, immune development, and shield the infant gut from insults and microbial threats. The molecular and cellular events involved in these processes are ill defined. We have established human pediatric enteroids and interrogated maternal milk’s impact on epithelial cell maturation and function in comparison with commercial infant formula. Colostrum applied apically to pediatric enteroid monolayers reduced ion permeability, stimulated epithelial cell differentiation, and enhanced tight junction function by upregulating occludin amount. Breast milk heightened the production of antimicrobial peptide -defensin 5 by goblet and Paneth cells, and modulated cytokine production, which abolished apical release of pro-inflammatory GM-CSF. These attributes were not found in commercial infant formula. Epithelial cells exposed to breast milk elevated apical and intracellular pIgR amount and enabled maternal IgA translocation. Proteomic data revealed a breast milk-induced molecular pattern associated with tissue remodeling and homeostasis. Using a novel ex vivo pediatric enteroid model, we have identified cellular and molecular pathways involved in human milk-mediated improvement of human intestinal physiology and immunity.
Project description:Unbiased and FACS was performed on human intestinal organoid cells and coupled to single cell sorting by the SortSeq protocol (Muraro et al., 2016).
Project description:We focused on a rare cell population of human intestine, the M cells. Using human intestinal organoid as a model, the M cells could be differentiated, FACS-enriched and analyzed by RNA-seq.
Project description:We focused on a rare cell population of human intestine, the M cells. Using human intestinal organoid as a model, the M cells could be differentiated, FACS-sorted from organoids and analyzed by scRNA-seq.
Project description:We focused on a rare cell population of human intestine, the BEST4+ cells. Using human intestinal organoid as a model, the BEST4+ cells could be differentiated, FACS-enriched and analyzed by scRNA-seq, together with the BEST4- cell lineages such as the enterocytes, goble cells and EECs.