Our protocol details the application of fluorescent cholera toxin subunit B (CTX) derivatives to label intestinal cell membranes whose composition varies with differentiation. In cultured mouse adult stem cell-derived small intestinal organoids, we observe that CTX binding to plasma membrane domains displays a dependence on the differentiation state. Green (Alexa Fluor 488) and red (Alexa Fluor 555) fluorescently labeled CTX derivatives demonstrate variations in fluorescence lifetime, as revealed by fluorescence lifetime imaging microscopy (FLIM), making them suitable for use with other fluorescent dyes and cellular tracers. After fixation, CTX staining is specifically localized within defined regions of the organoids, making it applicable to both live-cell and fixed-tissue immunofluorescence microscopy approaches.

Cells within organotypic cultures experience growth in a setting that mirrors the tissue organization observed in living organisms. HDAC inhibitor 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.

Key signaling pathways, including Wnt, bone morphogenetic protein (BMP), epidermal growth factor (EGF), and Notch, are essential for the intestinal epithelium's maintenance of self-renewal and differentiation capabilities. Given this comprehension, a cocktail of stem cell niche factors, including EGF, Noggin, and the Wnt agonist R-spondin, fostered the proliferation of murine intestinal stem cells and the genesis of organoids exhibiting endless self-renewal and complete differentiation potential. While two small-molecule inhibitors, a p38 inhibitor and a TGF-beta inhibitor, enabled the propagation of cultured human intestinal epithelium, the differentiation ability was compromised. To resolve these problems, advancements have been made in cultivation conditions. Multilineage differentiation was achieved by substituting the EGF and p38 inhibitor with the more effective insulin-like growth factor-1 (IGF-1) and fibroblast growth factor-2 (FGF-2). Apical epithelium monolayer cultures, subjected to mechanical flow, spurred the creation of villus-like structures, featuring a mature enterocyte genetic profile. This paper showcases our recent advancements in human intestinal organoid culture, emphasizing the importance of this development in understanding intestinal homeostasis and related diseases.

During the embryonic stage, the gut tube undergoes substantial morphogenesis, evolving from a simple pseudostratified epithelial tube to the mature intestinal tract, a structure marked by columnar epithelium and its highly specialized crypt-villus architecture. Mice fetal gut precursor cells undergo maturation into adult intestinal cells around embryonic day 165, a process including the formation of adult intestinal stem cells and their derivative progenies. Adult intestinal cells, in contrast to fetal intestinal cells, produce organoids with both crypt-like and villus-like components; the latter develop into simple spheroid-shaped organoids, demonstrating a uniform proliferation pattern. Fetal intestinal spheroids possess the capacity for spontaneous development into complex adult organoid structures, which incorporate intestinal stem cells and differentiated cell types, including enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, thus recapitulating intestinal maturation in a laboratory environment. We detail the procedures for creating fetal intestinal organoids and their maturation into adult intestinal cell types. media supplementation These methodologies allow for the in vitro recreation of intestinal development, providing valuable insights into the mechanisms governing the transition from fetal to adult intestinal cell types.

The creation of organoid cultures enables the study of intestinal stem cell (ISC) function, particularly in the contexts of self-renewal and differentiation. Differentiating, ISCs and early progenitors first decide between a secretory fate (Paneth, goblet, enteroendocrine, or tuft cells) or an absorptive one (enterocytes or M cells). In the adult intestine, the past decade of in vivo studies, which combined genetic and pharmacological approaches, has provided evidence that Notch signaling functions as a binary switch dictating the fate of cells toward secretory or absorptive lineages. Real-time, smaller-scale, and higher-throughput in vitro experiments, made possible by recent organoid-based assay breakthroughs, are starting to shed light on the mechanistic principles underlying intestinal differentiation. This chapter focuses on in vivo and in vitro approaches to modify Notch signaling, scrutinizing their impact on the commitment of intestinal cells. We provide exemplary protocols for utilizing intestinal organoids to evaluate Notch signaling's role in determining intestinal cell lineage identities.

The three-dimensional structures, known as intestinal organoids, are formed from adult stem cells found within the tissue. These organoids, embodying critical elements of epithelial biology, allow for the investigation of homeostatic turnover in the corresponding tissue. Enrichment of organoids for mature lineages permits studies of the diverse cellular functions and individual differentiation processes. We explore the processes that dictate intestinal cell fate specification and describe how these can be applied to the generation of mature lineages within mouse and human small intestinal organoids.

In numerous locations throughout the body, there are regions called transition zones (TZs). Epithelial transitions, or transition zones, are strategically positioned at the interface of the esophagus and stomach, the cervix, the eye, and the anal canal and rectum. Due to the heterogeneous composition of TZ's population, a detailed characterization demands single-cell analysis. This chapter presents a protocol for performing primary single-cell RNA sequencing analysis on the epithelium of the anal canal, TZ, and rectum.

To ensure intestinal homeostasis, the process of stem cell self-renewal and subsequent differentiation, alongside the precise lineage specification of progenitor cells, is considered essential. The hierarchical model describes intestinal differentiation as a process of progressively achieving lineage-specific mature cell characteristics, guided by Notch signaling and lateral inhibition, which control cellular fate. Newly published research indicates a broadly permissive condition within intestinal chromatin, which supports the lineage plasticity and adaptation to diet via the Notch transcriptional program's action. The established understanding of Notch signaling in intestinal differentiation is explored in this work, and the potential impact of new epigenetic and transcriptional data on refining or revising this perspective is discussed. We detail sample preparation and data analysis procedures, elucidating the combined application of ChIP-seq, scRNA-seq, and lineage tracing assays to pinpoint the Notch program's dynamics and intestinal differentiation during dietary and metabolic regulation of cellular fate decisions.

Primary tissue serves as the source for organoids, 3D cell clusters cultivated outside the body, and accurately demonstrate the equilibrium of tissues. Organoids offer benefits over 2D cell lines and mouse models, exhibiting particular strengths in both drug screening studies and translational research initiatives. New organoid manipulation methods are continually arising, highlighting the burgeoning importance of organoids in scientific investigation. Despite the advancements in recent times, RNA-sequencing-based drug screening platforms for organoids have yet to achieve widespread adoption. 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. The principle underlying our assay is a confluence of affordability and the sensitive detection of diverse cellular identities, signaling pathways, and crucial cellular phenotype determinants. This method is broadly applicable to various systems, delivering unique insights otherwise inaccessible.

A complex environment, including mesenchymal cells and the gut microbiota, encompasses the epithelial cells that form the intestinal structure. Food's transit through the intestine leads to cell loss, but the intestine's stem cell regeneration system compensates for this by consistently replenishing these lost cells, thus countering apoptosis and abrasion. Stem cell homeostasis has been the subject of intensive investigation over the past ten years, leading to the discovery of signaling pathways, such as the retinoid pathway. Japanese medaka Cell differentiation, a process impacted by retinoids, occurs in both healthy and cancerous cells. This research details multiple in vitro and in vivo strategies to more thoroughly investigate the effect of retinoids on stem, progenitor, and differentiated intestinal cells.

A network of interconnected epithelial cells, manifesting in diverse forms, lines the entire body and its internal organs, establishing a continuous surface. Two differing epithelial types converge at a specialized region termed the transition zone (TZ). TZ regions, small in scale, are strategically positioned in several body parts, such as the juncture between the esophagus and stomach, the cervical region, the eye, and the connection between the anal canal and rectum. While these zones are linked to various pathologies, including cancers, the cellular and molecular mechanisms driving tumor progression remain largely unexplored. In a recent study leveraging an in vivo lineage tracing strategy, we determined the role of anorectal TZ cells in maintaining a healthy state and following injury. A mouse model for lineage tracking of TZ cells, previously developed in our lab, employed cytokeratin 17 (Krt17) as a promoter and GFP as a reporting marker.