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Organoids are microscopic self-organizing, three-dimensional structures that are grown from stem cells in vitro. They recapitulate many structural and functional aspects of their in vivo counterpart organs. This versatile technology has led to the development of many novel human cancer models. It is now possible to create indefinitely expanding organoids starting from tumor tissue of individuals suffering from a range of carcinomas. Alternatively, CRISPR-based gene modification allows the engineering of organoid models of cancer through the introduction of any combination of cancer gene alterations to normal organoids.When combined with immune cells and fibroblasts, tumor organoids become models for the cancer microenvironment enabling immune-oncology applications. Emerging evidence indicates that organoids can be used to accurately predict drug responses in a personalized treatment setting. Here, we review the current state and future prospects of the rapidly evolving tumor organoid field.

The coronavirus disease-19 (COVID-19) pandemic underscores the threat posed by newly emerging viruses. Understanding the biology of novel viruses rests in large part on in vitro models that allow viral replication. Human and animal organoids are now proving their value as an experimental virology platform.Hans Clevers discusses the value of organoids as an experimental platform for understanding the biology of novel viruses.

Intestinal stem cells at the bottom of crypts fuel the rapid renewal of the different cell types that constitute a multitasking tissue. The intestinal epithelium facilitates selective uptake of nutrients while acting as a barrier for hostile luminal contents. Recent discoveries have revealed that the lineage plasticity of committed cells — combined with redundant sources of niche signals — enables the epithelium to efficiently repair tissue damage. New approaches such as single-cell transcriptomics and the use of organoid models have led to the identification of the signals that guide fate specification of stem cell progeny into the six intestinal cell lineages. These cell types display context-dependent functionality and can adapt to different requirements over their lifetime, as dictated by their microenvironment. These new insights into stem cell regulation and fate specification could aid the development of therapies that exploit the regenerative capacity and functionality of the gut.The intestinal epithelium undergoes rapid turnover and is constantly exposed to hostile luminal contents. Recent insights from single-cell transcriptomics and organoid models have revealed that tissue repair is dependent on cell lineage plasticity and signals originating from different niche components.

Inflammation underlies many chronic and degenerative diseases, but it also mitigates infections, clears damaged cells and initiates tissue repair. Many of the mechanisms that link inflammation to damage repair and regeneration in mammals are conserved in lower organisms, indicating that it is an evolutionarily important process. Recent insights have shed light on the cellular and molecular processes through which conventional inflammatory cytokines and Wnt factors control mammalian tissue repair and regeneration. This is particularly important for regeneration in the gastrointestinal system, especially for intestine and liver tissues in which aberrant and deregulated repair results in severe pathologies.

Recent examples have highlighted how stem cells have the capability to initiate morphogenesis in vitro; that is, to generate complex structures in culture that closely parallel their in vivo counterparts. Lgr5, the receptor for the Wnt-agonistic R-spondins, marks stem cells in multiple adult organs of mice and humans. In R-spondin-based three-dimensional cultures, these Lgr5 stem cells can grow into ever-expanding epithelial organoids that retain their original organ identity. Single Lgr5 stem cells derived from the intestine can be cultured to build epithelial structures that retain hallmarks of the in vivo epithelium. Here, we review the mechanisms that support this notable example of self-organization and discuss applications of this technology for stem cell research, disease modeling (e.g., for colorectal cancer and cystic fibrosis), and regenerative medicine.

Adult stem cell–based organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting organoid cultures. Patient-derived cancer organoids therefore hold great promise for personalized medicine. Here, we provide an overview of the protocols used by different groups to establish organoids from various epithelial tissues and cancers, plus the different protocols subsequently used to test the in vitro therapy sensitivity of these patient-derived organoids. We also provide an in-depth protocol for the generation of head and neck squamous cell carcinoma organoids and their subsequent use in semi-automated therapy screens. Establishment of organoids and subsequent screening can be performed within 3 months, although this timeline is highly dependent on a.o. starting material and the number of therapies tested. The protocol provided may serve as a reference to successfully establish organoids from other cancer types and perform drug screenings thereof. This protocol summarizes the various approaches available to derive organoids from cancer patients and use these for screening of possible treatments. An optimized protocol for using head and neck cancer organoids is also described.

Much of our knowledge regarding the interactions between epithelial tissues and the immune system has been gathered from animal models and co-cultures with cell lines. However, unique features of human cells cannot be modelled in mice, and cell lines are often transformed or genetically immortalized. Organoid technology has emerged as a powerful tool to maintain epithelial cells in a near-native state. In this Review, we discuss how organoids are being used in immunological research to understand the role of epithelial cell–immune cell interactions in tissue development and homeostasis, as well as in diseases such as cancer.Organoid technology has emerged as a powerful tool to maintain epithelial cells in a near-native state that can be used to better understand the interactions between epithelial cells and the immune system in tissue development, homeostasis, infection and cancer.

Organogenesis, tissue homeostasis and organ function involve complex spatial cellular organization and tissue dynamics. The underlying mechanisms of these processes, and how they are disrupted in disease, are challenging to address in vivo and ethically impossible to study in human. Organoids, three-dimensional (3D) stem cell cultures that self-organize into ex vivo 'mini-organs', now open a new window onto cellular processes within tissue. Light microscopy is a powerful approach to probe the cellular complexity that can be modeled with organoids. This combination of tools is already leading to exciting synergies in stem cell and cancer research.

The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR–Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR–Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1–2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2–3 months. Culture conditions are described for long-term expansion of human fetal hepatocytes as 3D organoids. Gene knockin and knockout approaches are also described for organoids derived from human fetal hepatocytes and human adult liver ductal cells.

Adult organs such as the intestines and skin continually renew themselves every few days or weeks. In several mammalian tissues, this renewal relies on Wnt signaling. Clevers et al. review this crucial role in stem cell self renewal. Wnt plays a pivotal role in tissue regeneration even in the earliest animals. Wnt proteins function mainly as short-range signals between adjacent cells. The short-range, spatially-constrained nature of Wnt signals underpins mammalian stem cell niche architecture and tissue self-organization. Science , this issue 10.1126/science.1248012 Stem cells fuel tissue development, renewal, and regeneration, and these activities are controlled by the local stem cell microenvironment, the “niche.” Wnt signals emanating from the niche can act as self-renewal factors for stem cells in multiple mammalian tissues. Wnt proteins are lipid-modified, which constrains them to act as short-range cellular signals. The locality of Wnt signaling dictates that stem cells exiting the Wnt signaling domain differentiate, spatially delimiting the niche in certain tissues. In some instances, stem cells may act as or generate their own niche, enabling the self-organization of patterned tissues. In this Review, we discuss the various ways by which Wnt operates in stem cell control and, in doing so, identify an integral program for tissue renewal and regeneration.