The development of human organoid culture models has led to unprecedented opportunities to generate self-organizing, three-dimensional miniature organs that closely mimic in vivo conditions. The ...ability to expand, culture, and bank such organoids now provide researchers with the opportunity to generate next-generation living biobanks, which will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine as well as the development of a personalized treatment approach. However, compared to traditional tissue repositories, the generation of a living organoid biobank requires a much higher level of coordination, additional resources, and scientific expertise. In this short review, we discuss the opportunities and challenges associated with the generation of a living organoid biobank. Focusing on human intestinal organoids, we highlight some of the key aspects that need to be considered and provide an outlook for future development in this exciting field.
Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of ...cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.
The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through ...progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated.
Among the endodermal tissues of adult mammals, the gastrointestinal (GI) epithelium exhibits the highest turnover rate. As the ingested food moves along the GI tract, gastric acid, digestive enzymes, ...and gut resident microbes aid digestion as well as nutrient and mineral absorption. Due to the harsh luminal environment, replenishment of new epithelial cells is essential to maintain organ structure and function during routine turnover and injury repair. Tissue-specific adult stem cells in the GI tract serve as a continuous source for this immense regenerative activity. Tissue homeostasis is achieved by a delicate balance between gain and loss of cells. In homeostasis, temporal tissue damage is rapidly restored by well-balanced tissue regeneration, whereas prolonged imbalance may result in diverse pathologies of homeostasis and injury repair. Starting with a summary of the current knowledge of GI tract homeostasis, we continue with providing models of acute injury and chronic diseases. Finally, we will discuss how primary organoid cultures allow new insights into the mechanisms of homeostasis, injury repair, and disease, and how this novel 3D culture system has the potential to translate into the clinic.
Human organoid systems recapitulate key features of organs offering platforms for modelling developmental biology and disease. Tissue-derived organoids have been widely used to study the impact of ...extrinsic niche factors on stem cells. However, they are rarely used to study endogenous gene function due to the lack of efficient gene manipulation tools. Previously, we established a human foetal lung organoid system (Nikolić et al., 2017). Here, using this organoid system as an example, we have systematically developed and optimised a complete genetic toolbox for use in tissue-derived organoids. This includes 'Organoid Easytag', our efficient workflow for targeting all types of gene loci through CRISPR-mediated homologous recombination followed by flow cytometry for enriching correctly targeted cells. Our toolbox also incorporates conditional gene knockdown or overexpression using tightly inducible CRISPR interference and CRISPR activation which is the first efficient application of these techniques to tissue-derived organoids. These tools will facilitate gene perturbation studies in tissue-derived organoids facilitating human disease modelling and providing a functional counterpart to many ongoing descriptive studies, such as the Human Cell Atlas Project.
The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer
. ...However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.
The intestinal epithelium forms the inner layer of the human intestine and serves a wide range of diverse functions. Its constant exposure to a vast amount of complex microbiota highlights the ...critical interface that this single-cell layer forms between the host and our environment. Importantly, the well-documented contribution of environmental factors towards the functional development of the human intestinal epithelium directly implies epigenetic mechanisms in orchestrating this complex interplay. The development of intestinal epithelial organoid culture systems that can be generated from human tissue provides researchers with unpresented opportunities to study functional aspects of human intestinal epithelial pathophysiology. In this brief review, we summarise existing evidence for the role of epigenetics in regulating intestinal epithelial cell function and highlight the great potential for human gut organoids as translational research tools to investigate these mechanisms in vitro.
The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical ...reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.
Human intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular ...characteristics and behaviour in culture remains limited.
We performed genome-wide DNA methylation and transcriptomic profiling of human IEOs derived from paediatric/adult and fetal small and large bowel as well as matching purified human gut epithelium. Furthermore, organoids were subjected to in vitro differentiation and genome editing using CRISPR/Cas9 technology.
We discovered stable epigenetic signatures which define regional differences in gut epithelial function, including induction of segment-specific genes during cellular differentiation. Established DNA methylation profiles were independent of cellular environment since organoids retained their regional DNA methylation over prolonged culture periods. In contrast to paediatric and adult organoids, fetal gut-derived organoids showed distinct dynamic changes of DNA methylation and gene expression in culture, indicative of an in vitro maturation. By applying CRISPR/Cas9 genome editing to fetal organoids, we demonstrate that this process is partly regulated by TET1, an enzyme involved in the DNA demethylation process. Lastly, generating IEOs from a child diagnosed with gastric heterotopia revealed persistent and distinct disease-associated DNA methylation differences, highlighting the use of organoids as disease-specific research models.
Our study demonstrates striking similarities of epigenetic signatures in mucosa-derived IEOs with matching primary epithelium. Moreover, these results suggest that intestinal stem cell-intrinsic DNA methylation patterns establish and maintain regional gut specification and are involved in early epithelial development and disease.
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of ...human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
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•A human organoid biobank combines hormone labeling and enteroendocrine cell generation•Transcriptomic profiling of human enteroendocrine cells uncovers differences with mice•Functional validation of EEC receptors and transcription factors•Secretome analysis reveals the repertoire of enteroendocrine secreted products
An organoid-based platform for studying human enteroendocrine cells, which sense intestinal content and release hormones to regulate many processes throughout the body, is developed by Beumer et al. and used to describe the landscape of mRNA expression and secreted products.
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