This protocol describes, using fluorescent cholera toxin subunit B (CTX) derivatives, the method for labeling intestinal cell membrane compositions which change depending on differentiation. Employing mouse adult stem cell-derived small intestinal organoid cultures, we observe that CTX's binding to specific plasma membrane domains is correlated with the progression of differentiation. Fluorescence lifetime imaging microscopy (FLIM) measurements highlight differences in fluorescence lifetimes between green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescent CTX derivatives, which can also be used with other fluorescent dyes and cell trackers. Crucially, CTX staining is spatially limited to particular regions within the organoids following fixation, allowing its application in live-cell and fixed-tissue immunofluorescence microscopy.
Cells within organotypic cultures experience growth in a setting that mirrors the tissue organization observed in living organisms. AZD4547 research buy Employing the intestine as a model, we outline the procedure for establishing three-dimensional organotypic cultures, followed by techniques for examining cell morphology and tissue architecture using histology, and molecular expression analysis through immunohistochemistry. Additionally, molecular analyses like PCR, RNA sequencing, or FISH are applicable to this system.
Via the interplay of key signaling pathways such as Wnt, bone morphogenetic protein (BMP), epidermal growth factor (EGF), and Notch, the intestinal epithelium sustains its self-renewal and differentiation capacities. From this perspective, the interplay of stem cell niche factors, in conjunction with EGF, Noggin, and the Wnt agonist R-spondin, demonstrated the ability to cultivate mouse intestinal stem cells and to form organoids with persistent self-renewal and complete differentiation. Two small-molecule inhibitors, a p38 inhibitor and a TGF-beta inhibitor, were employed to propagate cultured human intestinal epithelium, yet this resulted in a diminished capacity for differentiation. Cultural conditions have been enhanced to address these problems. The switch from EGF and a p38 inhibitor to insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2) unlocked the potential for multilineage differentiation. By applying mechanical flow to the apical region of monolayer cultures, the development of villus-like structures was promoted, manifesting in mature enterocyte gene expression. Our recent technological innovations in human intestinal organoid cultures are highlighted here, promising a deeper insight into intestinal homeostasis and diseases.
From a simple pseudostratified epithelial tube, the gut tube dramatically alters during embryonic development, morphing into a sophisticated intestinal tract characterized by columnar epithelium and intricate crypt-villus structures. Fetal gut precursor cells in mice mature into adult intestinal cells around embryonic day 165, a time when adult intestinal stem cells and their derived progeny are formed. Adult intestinal cells create organoids possessing both crypt and villus-like regions; unlike this, fetal intestinal cells are able to culture simple, spheroid-shaped organoids showing a uniform proliferation. The in-vitro maturation of intestinal cells is mirrored by the spontaneous transition of fetal intestinal spheroids into adult organoid structures, which contain intestinal stem cells and differentiated cells like enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. Comprehensive procedures for the derivation of fetal intestinal organoids and their subsequent transformation into adult intestinal cell lineages are elaborated upon. tibio-talar offset In vitro models of intestinal development, facilitated by these methods, offer opportunities to understand the regulatory mechanisms driving the transition between fetal and adult intestinal cell states.
Intestinal stem cell (ISC) self-renewal and differentiation are replicated in organoid cultures, which have been designed for that specific purpose. Differentiation prompts the initial lineage commitment of ISCs and early progenitor cells, requiring a selection between secretory fates (Paneth, goblet, enteroendocrine, or tuft cells) and absorptive fates (enterocytes or M cells). Past decade in vivo studies, utilizing genetic and pharmacological methodologies, have demonstrated Notch signaling's function as a binary switch regulating secretory versus absorptive lineage commitment in the adult intestine. Recent breakthroughs in organoid-based assays enable in vitro, real-time observation of smaller-scale, high-throughput experiments, which are now contributing to a deeper comprehension of the underlying mechanistic principles of intestinal differentiation. Using in vivo and in vitro models, this chapter outlines methods for modulating Notch signaling and analyzes the impact on intestinal cell fate decisions. We demonstrate, via example protocols, how to use intestinal organoids to investigate Notch pathway activity in shaping intestinal cell lineage.
Intestinal organoids, which are three-dimensional structures, are generated from adult stem cells found within the tissue. The homeostatic turnover of the corresponding tissue is a focus of study, which these organoids—representing key elements of epithelial biology—can enable. Investigations into the differentiation processes and diverse cellular functions are facilitated by the enrichment of organoids for mature lineages. We present an analysis of intestinal fate specification mechanisms, and strategies for manipulating these to cause mouse and human small intestinal organoids to differentiate into each of their respective mature, functional types.
Transition zones (TZs), designated as specialized regions, are present in multiple areas of the body. Transition zones, markers of where two distinct epithelial forms meet, are situated at the boundary between the esophagus and the stomach, within the cervix, the eye, and at the rectoanal junction. TZ's population is diverse, and a comprehensive understanding necessitates single-cell analysis. Within this chapter, we outline a procedure for conducting a primary single-cell RNA sequencing analysis of anal canal, TZ, and rectal epithelium samples.
For the preservation of intestinal homeostasis, the equilibrium of stem cell self-renewal and differentiation, coupled with appropriate progenitor cell lineage specification, is deemed crucial. In the hierarchical model, the development of intestinal differentiation relies on a progressive acquisition of mature, lineage-specific cell features, precisely managed by Notch signaling and lateral inhibition to determine cell fates. Intestinal chromatin, operating in a broadly permissive manner, is revealed by recent research to be a key element in the lineage plasticity and dietary adaptation driven by the Notch transcriptional program. This review scrutinizes the established understanding of Notch signaling in intestinal development, emphasizing how new epigenetic and transcriptional findings might potentially reshape or amend current interpretations. Our comprehensive guide encompasses sample preparation, data analysis, and the application of ChIP-seq, scRNA-seq, and lineage tracing to chart the Notch program's evolution and intestinal differentiation in response to dietary and metabolic factors influencing cell fate.
Primary tissue serves as the source for organoids, 3D cell clusters cultivated outside the body, and accurately demonstrate the equilibrium of tissues. Organoids' advantages over 2D cell lines and mouse models are particularly evident in drug-screening and translational research applications. New organoid manipulation methods are continually arising, highlighting the burgeoning importance of organoids in scientific investigation. Despite recent progress in the field, RNA-sequencing drug screening methods using organoids are not yet routinely employed. This detailed protocol describes the execution of TORNADO-seq, a drug screening technique based on targeted RNA sequencing within organoid models. Intricate phenotypic analyses with meticulously chosen readouts allow for the direct grouping and classification of drugs, regardless of structural similarities or pre-existing knowledge of shared modes of action. Our assay is designed with both cost-effectiveness and sensitive detection in mind, pinpointing multiple cellular identities, signaling pathways, and key drivers of cellular phenotypes. This high-content screening approach can be utilized across multiple systems to extract data otherwise unattainable.
A complex environment, including mesenchymal cells and the gut microbiota, encompasses the epithelial cells that form the intestinal structure. The intestine's remarkable stem cell regeneration system continually replaces cells lost due to apoptosis or the abrasive action of food passage. Signaling pathways, such as the retinoid pathway, have been identified through research on stem cell homeostasis conducted over the last decade. PacBio Seque II sequencing The mechanisms of cell differentiation are affected by retinoids in both healthy and cancerous tissues. The impact of retinoids on intestinal stem cells, progenitors, and differentiated cells is explored through several in vitro and in vivo approaches in this study.
Epithelial cells, forming various types, unite to create a seamless layer encompassing all body surfaces and internal organs. A special region, the transition zone (TZ), is defined by the convergence of two various types of epithelia. Scattered throughout the body are small TZ regions, including those situated between the esophagus and stomach, the cervix, the eye, and the space between the anal canal and rectum. These zones are found to be associated with multiple pathologies, such as cancers, yet the cellular and molecular mechanisms driving tumor progression are poorly investigated. Our recent in vivo lineage tracing study investigated the role of anorectal TZ cells in maintaining homeostasis and in the aftermath of injury. In our prior work, a mouse model for the tracing of TZ cell lineages was established. This model employed cytokeratin 17 (Krt17) as a promoter and GFP as the reporter molecule.