Apical-out airway organoids as a platform for studying viral infections and screening for antiviral drugs.

Airway organoids are polarized 3D epithelial structures that recapitulate the organization and many of the key functions of the in vivo tissue. They present an attractive model that can overcome some of the limitations of traditional 2D and Air-Liquid Interface (ALI) models, yet the limited accessibility of the organoids' apical side has hindered their applications in studies focusing on host-pathogen interactions. Here, we describe a scalable, fast and efficient way to generate airway organoids with the apical side externally exposed. These apical-out airway organoids are generated in an Extracellular Matrix (ECM)-free environment from 2D-expanded bronchial epithelial cells and differentiated in suspension to develop uniformly-sized organoid cultures with robust ciliogenesis. Differentiated apical-out airway organoids are susceptible to infection with common respiratory viruses and show varying responses upon treatment with antivirals. In addition to the ease of apical accessibility, these apical-out airway organoids offer an alternative in vitro model to study host-pathogen interactions in higher throughput than the traditional air-liquid interface model.

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Intestinal region-specific Wnt signalling profiles reveal interrelation between cell identity and oncogenic pathway activity in cancer development.

BACKGROUND: Cancer results from the accumulation of mutations leading to the acquisition of cancer promoting characteristics such as increased proliferation and resistance to cell death. In colorectal cancer, an early mutation leading to such features usually occurs in the APC or CTNNB1 genes, thereby activating Wnt signalling. However, substantial phenotypic differences between cancers originating within the same organ, such as molecular subtypes, are not fully reflected by differences in mutations. Indeed, the phenotype seems to result from a complex interplay between the cell-intrinsic features and the acquired mutations, which is difficult to disentangle when established tumours are studied. METHODS: We use a 3D in vitro organoid model to study the early phase of colorectal cancer development. From three different murine intestinal locations we grow organoids. These are transformed to resemble adenomas after Wnt activation through lentiviral transduction with a stable form of ß-Catenin. The gene expression before and after Wnt activation is compared within each intestinal origin and across the three locations using RNA sequencing. To validate and generalize our findings, we use gene expression data from patients. RESULTS: In reaction to Wnt activation we observe downregulation of location specific genes and differentiation markers. A similar effect is seen in patient data, where genes with significant differential expression between the normal left and right colon are downregulated in the cancer samples. Furthermore, the signature of Wnt target genes differs between the three intestinal locations in the organoids. The location specific Wnt signatures are dominated by genes which have been lowly expressed in the tissue of origin, and are the targets of transcription factors that are activated following enhanced Wnt signalling. CONCLUSION: We observed that the region-specific cell identity has a substantial effect on the reaction to Wnt activation in a simple intestinal adenoma model. These findings provide a way forward in resolving the distinct biology between left- and right-sided human colon cancers with potential clinical relevance.

A Perspective on Organoids for Virology Research.

Animal models and cell lines are invaluable for virology research and host-pathogen interaction studies. However, it is increasingly evident that these models are not sufficient to fully understand human viral diseases. With the advent of three-dimensional organotypic cultures, it is now possible to study viral infections in the human context. This perspective explores the potential of these organotypic cultures, also known as organoids, for virology research, antiviral testing, and shaping the virology landscape.

Cdx and T Brachyury Co-activate Growth Signaling in the Embryonic Axial Progenitor Niche.

In vertebrate embryos, anterior tissues are generated early, followed by the other axial structures that emerge sequentially from a posterior growth zone. The genetic network driving posterior axial elongation in mice, and its disturbance in mutants with posterior truncation, is not yet fully understood. Here, we show that the combined expression of Cdx2 and T Brachyury is essential to establish the core signature of posterior axial progenitors. Cdx2 and T Brachyury are required for extension of a similar trunk portion of the axis. Simultaneous loss of function of these two genes disrupts axial elongation to a much greater extent than each single mutation alone. We identify and validate common targets for Cdx2 and T Brachyury in vivo, including Wnt and Fgf pathway components active in the axial progenitor niche. Our data demonstrate that integration of the Cdx/Hox and T Brachyury transcriptional networks controls differential axial growth during vertebrate trunk elongation.

Transformation of intestinal stem cells into gastric stem cells on loss of transcription factor Cdx2.

The endodermal lining of the adult gastro-intestinal tract harbours stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation programme and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2(null) small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. We show that the intestinal genetic programme is critically dependent on the single transcription factor encoding gene Cdx2.

Region-specific regulation of posterior axial elongation during vertebrate embryogenesis.

BACKGROUND: The vertebrate body axis extends sequentially from the posterior tip of the embryo, fueled by the gastrulation process at the primitive streak and its continuation within the tailbud. Anterior structures are generated early, and subsequent nascent tissues emerge from the posterior growth zone and continue to elongate the axis until its completion. The underlying processes have been shown to be disrupted in mouse mutants, some of which were described more than half a century ago. RESULTS: Important progress in elucidating the cellular and genetic events involved in body axis elongation has recently been made on several fronts. Evidence for the residence of self-renewing progenitors, some of which are bipotential for neurectoderm and mesoderm, has been obtained by embryo-grafting techniques and by clonal analyses in the mouse embryo. Transcription factors of several families including homeodomain proteins have proven instrumental for regulating the axial progenitor niche in the growth zone. A complex genetic network linking these transcription factors and signaling molecules is being unraveled that underlies the phenomenon of tissue lengthening from the axial stem cells. The concomitant events of cell fate decision among descendants of these progenitors begin to be better understood at the levels of molecular genetics and cell behavior. CONCLUSIONS: The emerging picture indicates that the ontogenesis of the successive body regions is regulated according to different rules. In addition, parameters controlling vertebrate axial length during evolution have emerged from comparative experimental studies. It is on these issues that this review will focus, mainly addressing the study of axial extension in the mouse embryo with some comparison with studies in chick and zebrafish, aiming at unveiling the recent progress, and pointing at still unanswered questions for a thorough understanding of the process of embryonic axis elongation.

Evolutionarily conserved requirement of Cdx for post-occipital tissue emergence.

Mouse Cdx genes are involved in axial patterning and partial Cdx mutants exhibit posterior embryonic defects. We found that mouse embryos in which all three Cdx genes are inactivated fail to generate any axial tissue beyond the cephalic and occipital primordia. Anterior axial tissues are laid down and well patterned in Cdx null embryos, and a 3' Hox gene is initially transcribed and expressed in the hindbrain normally. Axial elongation stops abruptly at the post-occipital level in the absence of Cdx, as the posterior growth zone loses its progenitor activity. Exogenous Fgf8 rescues the posterior truncation of Cdx mutants, and the spectrum of defects of Cdx null embryos matches that resulting from loss of posterior Fgfr1 signaling. Our data argue for a main function of Cdx in enforcing trunk emergence beyond the Cdx-independent cephalo-occipital region, and for a downstream role of Fgfr1 signaling in this function. Cdx requirement for the post-head section of the axis is ancestral as it takes place in arthropods as well.

The miR-144/451 locus is required for erythroid homeostasis.

The process of erythropoiesis must be efficient and robust to supply the organism with red bloods cells both under condition of homeostasis and stress. The microRNA (miRNA) pathway was recently shown to regulate erythroid development. Here, we show that expression of the locus encoding miR-144 and miR-451 is strictly dependent on Argonaute 2 and is required for erythroid homeostasis. Mice deficient for the miR-144/451 cluster display a cell autonomous impairment of late erythroblast maturation, resulting in erythroid hyperplasia, splenomegaly, and a mild anemia. Analysis of gene expression profiles from wild-type and miR-144/451-deficient erythroblasts revealed that the miR-144/451 cluster acts as a "tuner" of gene expression, influencing the expression of many genes. MiR-451 imparts a greater impact on target gene expression than miR-144. Accordingly, mice deficient in miR-451 alone exhibited a phenotype indistinguishable from miR-144/451-deficient mice. Thus, the miR-144/451 cluster tunes gene expression to impart a robustness to erythropoiesis that is critical under conditions of stress.