Organoids, Volume 4, Issue 1 (March 2025) – 6 articles

In cell biology, the stem cell niche is the dynamic microenvironment in which stem cells reside and receive signals that determine their behavior and fate. The stem cell niche has largely been a theoretical construct due to the difficulty in identifying and manipulating individual stem cells and their surroundings. Recent technical advances have made it possible to characterize the niches that maintain and control stem cell activity in several organs, including the small intestine. Although the small intestine has a relatively simple architecture, it has an extraordinary capacity for fast self-renewal. Thus, the organ is a unique model for studying intestinal stem cells (ISCs) and their niche. The intestinal epithelium maintains the intestine, enabling it to perform its absorption, secretion, and barrier functions. ISCs reside at the base of crypts adjacent to Paneth cells. In vivo, ISCs are surrounded by the microenvironment that makes up the niche, which provides a variety of stimuli that determine the fate of the cells. Research on stem cell niches is beginning to deepen our understanding of ISC regulation at the cellular and molecular levels and is expected to provide insights that can be applied to ISC therapy. Intestinal organoids originate from a group of crypt base ISCs. These organoids possess a three-dimensional (3D) cell structure made up of the lumen facing inward. Therefore, 3D intestinal organoids are often digested and seeded in a two-dimensional (2D) manner to form confluent organoid monolayers. Here, we not only review our current understanding of ISC niches with a focus on systems that are well-characterized at the cellular and mechanistic levels, but we also summarize the current applications of intestinal organoids. Full article

In 2023, at the Center for Biological Resources (CRB) at the Institute of Genetics and Biophysics (IGB, Naples, Italy) of the National Research Council (CNR), the Breast Cancer Tissues and Organoids Biobank (BCTO BioBank) was founded. This is a new generation Biobank, dedicated to the collection, characterization, storage, and distribution of tissues and their 3D ‘organoid’ patients-derived. Tumor and healthy tissues from breast cancer patients have been collected from surgeons at Monaldi Hospital (Naples, Italy) and used to generate the corresponding tumor and healthy organoids from the same patient. After their establishment in culture, both organoids were characterized for their receptor status on a microfluidic 2-lane OrganoPlate, by immunofluorescence. The resulting data were compared with the expression profile obtained by immunohistochemistry on respective parental tissues. These data allowed us to phenotypically validate the generated organoids and classify them in a dedicated database, where also the clinical data of the corresponding patients were collected. During the six months of activities, we collected and characterized 27 samples. The continuous BCTO BioBank activity is fundamental to generating a high number of samples, for a broader and efficiently elaborated patient stratification at molecular level, biomarker discovery investigations, and for tailored treatment protocols design. Full article

The healthy gut masks a dynamic balance between pro- and anti-inflammatory activities, largely due to microbial factors in the lumen. IL-10 is vital among the anti-inflammatory mediators, yet confirming constitutive versus stimulated secretion in any cell type is difficult due to the cellular complexity in the gut. Seeking to determine whether intestinal epithelial cells are programmed to constitutively make IL-10, we confirmed that IL-10 mRNA was present in enteroids from C57BL/6 mice and IL-10 protein was co-localized with a Paneth cell marker but not with markers for goblet or tuft cells. Paneth cells positive for IL-10 also possessed apical and basal IL-10RA, while cells negative for IL-10 had only basal IL-10RA, suggesting a possible autocrine role for IL-10. Indeed, Paneth cells in IL-10 gene knockout (IL-10KO) enteroids possessed lower levels of anti-microbial protein mRNAs, which could not be restored by adding IL-10. Enteroids passaged onto Transwell^® filters to form monolayers were treated with IL-10 and STAT3 phosphorylation was measured. Apically applied IL-10 resulted in a stronger STAT3 signal than basally applied cytokine. Our results indicate that a subpopulation of Paneth cells constitutively secrete IL-10 apically, which binds apical IL-10RA, impacting the expression of anti-microbial proteins unique to Paneth cells. Full article

Intestinal organoids are useful for the in vitro investigation of the properties of intestinal epithelial cells and their interaction with the gut microbiome. In this study, we cultured cecal and colonic organoids from common marmosets, which are highlighted as model nonhuman primates but are susceptible to gastrointestinal diseases. The organoids established were capable of passaging and long-term culture. The results of quantitative reverse transcription PCR and immunostaining showed that the organoids differentiated into major cell types (colonocytes, goblet cells, and enteroendocrine cells) in the intestinal epithelium, enabling the in vitro analysis of these cells in marmosets. The organoids could therefore represent a useful model for the investigation of gut physiology in relation to gastrointestinal diseases and host-microbiome interactions, further expanding medical, biological, and veterinary research in the future. Full article

Peritoneal carcinomatosis from gastrointestinal tumours is considered a poor prognostic factor, with a median overall survival of six to nine months in the absence of intervention. The advent of patient-derived organoid cultures (PDOs) has provided a breakthrough in personalised medicine, allowing researchers and clinicians to model the complexity and heterogeneity of individual tumours in vitro. PDOs hold great promise in this field, as variations in the management of peritoneal carcinomatosis due to differences in the method of delivery of chemotherapeutics, drug selection, exposure duration, and tumour pathology make it impractical to use a single, standardised treatment regimen. We aim to summarise the methodologies and limitations of studies encapsulating organoids derived from peritoneal metastases to encourage design considerations that may improve future clinical relevance, standardise protocols, and address translational challenges in personalising treatment strategies. Full article

Summary Statement: A tailored image analysis workflow was applied to quantify cortical organoid health, development, morphology and cellular composition over time. The assessment of cellular composition and viability of stem cell-derived organoid models is a complex but essential approach to understanding the mechanisms of human development and disease. Aim: Our study was motivated by the need for an image-analysis workflow, including high-cell content, high-throughput methods, to measure the architectural features of developing organoids. We assessed stem cell-derived cortical organoids at 4 and 6 months post-induction using immunohistochemistry-labelled sections as the analysis testbed. The workflow leveraged fluorescence imaging tailored to classify cells as viable and dying or non-viable and assign neuronal and astrocytic perinuclear markers to count cells. Results/Outcomes: Image acquisition was accelerated by capturing the organoid slice in 3D using widefield-fluorescence microscopy. This method used computational clearing to resolve nuclear and perinuclear markers and retain their spatial information within the organoid’s heterogeneous structure. The customised workflow analysed over 1.5 million cells using DAPI-stained nuclei, filtering and quantifying viable and non-viable cells and the necrotic-core regions. Temporal analyses of neuronal cell number derived from perinuclear labelling were consistent with organoid maturation from 4 to 6 months of in vitro differentiation. Overall: We have provided a comprehensive and enhanced image analysis workflow for organoid structural evaluation, creating the ability to gather cellular-level statistics in control and disease models. Full article