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Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Increased extracellular matrix (ECM) stiffness has been implicated in esophageal adenocarcinoma (EAC) progression, metastasis, and resistance to therapy. However, the underlying protumorigenic pathways are yet to be defined. Additional work is needed to develop physiologically relevant in vitro 3D culture models that better recapitulate the human tumor microenvironment and can be used to dissect the contributions of matrix stiffness to EAC pathogenesis. Here, we describe a modular, tumor ECM-mimetic hydrogel platform with tunable mechanical properties, defined presentation of cell-adhesive ligands, and protease-dependent degradation that supports robust in vitro growth and expansion of patient-derived EAC 3D organoids (EAC PDOs). Hydrogel mechanical properties control EAC PDO formation, growth, proliferation, and activation of tumor-associated pathways that elicit stem-like properties in the cancer cells, as highlighted through in vitro and in vivo environments. We also demonstrate that the engineered hydrogel serves as a platform for identifying potential therapeutic targets to disrupt the contribution of protumorigenic matrix mechanics in EAC. Together, these studies show that an engineered PDO culture platform can be used to elucidate underlying matrix-mediated mechanisms of EAC and inform the development of therapeutics that target ECM stiffness in EAC., Introduction Over the past 30 years, the incidence of esophageal adenocarcinoma (EAC) has risen dramatically, by 300% to 600% in the US (1). Previous studies have demonstrated that changes in [...]

Bronchospheres have emerged as a promising in vitro model toward probing questions on organ development and disease. Several organoid models, including from airway (e.g., bronchial, tracheal) cells, require three‐dimensional (3D) Matrigel, a complex mouse tumor‐derived matrix that typically leads to heterogeneous size and structures. Synthetic and naturally derived polymeric hydrogels show increased opportunities as an alternative to Matrigel culture. In addition, recent advances in hydrogel‐based microcavities (i.e., microwells) have shown improved control over organoid size, structure, and composition. Here, we build upon this approach and describe the fabrication and characterization of microwell hydrogels based on other polymers, including diacrylated poly(ethylene glycol), agarose, methacrylated gelatin, and norbornene‐modified hyaluronic acid. Using these microwell hydrogels, human bronchial epithelial cells and lung fibroblasts readily assemble into viable cyst‐like bronchospheres. The study shows that the cellular composition regulates the formation and structure of the bronchosphere which also depends on the type and adhesiveness of the hydrogel. Furthermore, both hydrogel type and cellular composition influence the amount and composition of deposited extracellular matrix within the microwells. This hydrogel fabrication platform provides an accessible in vitro culture platform for the formation and growth of bronchospheres which can be extended to the culture of other organoid systems. [ABSTRACT FROM AUTHOR]

The urgent need for effective treatments for acute and chronic lung diseases underscores the significance of developing innovative preclinical human research tools. The 2023 American Thoracic Society Workshop on Precision-Cut Lung Slices (PCLSs) brought together 35 experts to discuss and address the role of human tissue–derived PCLSs as a unique tool for target and drug discovery and validation in pulmonary medicine. With increasing interest and usage, together with advancements in methods and technology, there is a growing need for consensus on PCLS methodology and readouts. The current document recommends standard reporting criteria and emphasizes the requirement for careful collection and integration of clinical metadata. We further discuss current clinically relevant readouts that can be applied to PCLSs and highlight recent developments and future steps for implementing novel technologies for PCLS modeling and analysis. The collection and correlation of clinical metadata and multiomic analysis will further advance the integration of this preclinical platform into patient endotyping and the development of tailored therapies for patients with lung disease. [ABSTRACT FROM AUTHOR]