Explore the vast range of titles available.

Podocytes, a key component of the glomerular filtration barrier, adhere tightly to the glomerular basement membrane (GBM) through the actions of extracellular ligands within the GBM, transmembrane podocyte adhesion receptors, and intracellular linker proteins. This Review summarizes recent advances in our understanding of the cell biology and genetics of podocyte adhesion with a focus on its functional relevance in physiology and disease. Cell–matrix adhesion is crucial for maintaining the mechanical integrity of epithelial tissues. Podocytes—a key component of the glomerular filtration barrier—are exposed to permanent transcapillary filtration pressure and must therefore adhere tightly to the underlying glomerular basement membrane (GBM). The major cell–matrix adhesion receptor in podocytes is the integrin α3β1, which connects laminin 521 in the GBM through various adaptor proteins to the intracellular actin cytoskeleton. Other cell–matrix adhesion receptors expressed by podocytes include the integrins α2β1 and αvβ3, α-dystroglycan, syndecan-4 and type XVII collagen. Mutations in genes encoding any of the components critical for podocyte adhesion cause glomerular disease. This Review highlights recent advances in our understanding of the cell biology and genetics of podocyte adhesion with special emphasis on glomerular disease. Key Points Podocytes are constantly exposed to mechanical stress; therefore, strong adhesion of podocytes to the glomerular basement membrane (GBM) is required for blood ultrafiltration Podocyte adhesions to the GBM are mediated by transmembrane receptors, such as integrin α3β1, which link extracellular GBM proteins to the intracellular cytoskeleton Mutations in components that mediate podocyte–matrix adhesions may cause glomerular disease characterized by GBM abnormalities, podocyte foot process effacement, and proteinuria Glomerular disease caused by defective podocyte adhesion might be ameliorated by drugs that decrease transcapillary filtration pressure

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk. Organoid technology has enabled the generation of several breast cancer organoids. Here, the authors combine propagation of normal human mammary tissues with mass cytometry to evaluate the ability of organoid culture technologies to preserve stem cells and differentiated cell types.

Novel reassortant avian influenza H7N9 virus and pandemic 2009 H1N1 (H1N1pdm) virus cause human infections, while avian H7N2 and swine H1N1 virus mainly infect birds and pigs, respectively. There is no robust in vitro model for assessing the infectivity of emerging viruses in humans. Based on a recently established method, we generated long-term expanding 3D human airway organoids which accommodate four types of airway epithelial cells: ciliated, goblet, club, and basal cells. We report differentiation conditions which increase ciliated cell numbers to a nearly physiological level with synchronously beating cilia readily discernible in every organoid. In addition, the differentiation conditions induce elevated levels of serine proteases, which are essential for productive infection of human influenza viruses and low-pathogenic avian influenza viruses. We also established improved 2D monolayer culture conditions for the differentiated airway organoids. To demonstrate the ability of differentiated airway organoids to identify human-infective virus, 3D and 2D differentiated airway organoids are applied to evaluate two pairs of viruses with known distinct infectivity in humans, H7N9/Ah versus H7N2 and H1N1pdm versus an H1N1 strain isolated from swine (H1N1sw). The human-infective H7N9/Ah virus replicated more robustly than the poorly human-infective H7N2 virus; the highly human-infective H1N1pdm virus replicated to a higher titer than the counterpart H1N1sw. Collectively, we developed differentiated human airway organoids which can morphologically and functionally simulate human airway epithelium. These differentiated airway organoids can be applied for rapid assessment of the infectivity of emerging respiratory viruses to human.

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7–21 d; generation of genetically manipulated clonal organoid cultures takes 14–21 d; and organoid expansion for xenotransplantation takes >4 weeks. Dekkers et al. provide a toolbox for the long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5⁺) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5⁺ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5⁺ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4⁺ DCS cells promote organoid formation of single Lgr5⁺ colon stem cells. DCS cells can be massively produced from Lgr5⁺ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4⁺ DCS cells serve as Paneth cell equivalents in the colon crypt niche.

Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3β1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3β1. In the absence of α3β1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis.

Integrins are transmembrane αβ glycoproteins that connect the extracellular matrix to the cytoskeleton. The laminin-binding integrin α3β1 is expressed at high levels in lung epithelium and in kidney podocytes. In podocytes, α3β1 associates with the tetraspanin CD151 to maintain a functional filtration barrier. Here, we report on a patient homozygous for a novel missense mutation in the human ITGA3 gene, causing fatal interstitial lung disease and congenital nephrotic syndrome. The mutation caused an alanine-to-serine substitution in the integrin α3 subunit, thereby introducing an N-glycosylation motif at amino acid position 349. We expressed this mutant form of ITGA3 in murine podocytes and found that hyperglycosylation of the α3 precursor prevented its heterodimerization with β1, whereas CD151 association with the α3 subunit occurred normally. Consequently, the β1 precursor accumulated in the ER, and the mutant α3 precursor was degraded by the ubiquitin-proteasome system. Thus, these findings uncover a gain-of-glycosylation mutation in ITGA3 that prevents the biosynthesis of functional α3β1, causing a fatal multiorgan disorder.

Human enteroviruses frequently cause severe diseases in children. Human enteroviruses are transmitted via the fecal-oral route and respiratory droplets, and primary replication occurs in the gastro-intestinal and respiratory tracts; however, how enteroviruses infect these sites is largely unknown. Human intestinal organoids have recently proven to be valuable tools for studying enterovirus-host interactions in the intestinal tract. In this study, we demonstrated the susceptibility of a newly developed human airway organoid model for enterovirus 71 (EV71) infection. We showed for the first time in a human physiological model that EV71 replication kinetics are strain-dependent. A glutamine at position 145 of the VP1 capsid protein was identified as a key determinant of infectivity, and residues VP1-98K and VP1-104D were identified as potential infectivity markers. The results from this study provide new insights into EV71 infectivity in the human airway epithelia and demonstrate the value of organoid technology for virus research.

The authors have designed modular synthetic hydrogel networks for mouse and human intestinal stem cell cultures that support intestinal organoid formation. Synthetic matrices for organoid culture Epithelial organoids are being used in the laboratory to model organ development and function. So far these systems have relied on animal-derived matrices, which can be highly variable and are poorly defined, a problem that also makes them unsuitable for clinical application. Matthias Lutolf and colleagues have now designed modular synthetic hydrogen networks to support the formation of intestinal organoids from mouse and human intestinal stem cells. The authors produced dynamic matrices, initially optimal for intestinal stem cell expansion, which depends on high stiffness, and subsequently become permissive to intestinal differentiation and organoid formation through softening of their mechanical properties. Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease 1 , 2 , 3 . However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown 4 . Here we used modular synthetic hydrogel networks 5 , 6 to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes ( APC , P53 (also known as TP53 ), KRAS and SMAD4 ) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression. Using the CRISPR/Cas9 system, up to four frequently occurring colorectal cancer mutations were introduced alone or in combination into stem cell organoids derived from human small intestinal or colon tissue, allowing an in-depth investigation of the contribution of these mutations to cancer progression. An organoid-based model of evolving tumours Using the precise genome-editing capabilities of the CRISPR/Cas9 system, Hans Clevers and colleagues have introduced four of the most common colorectal cancer mutations into human organoid cultures formed by small intestinal or colon cells. In the resulting in vitro colorectal cancer progression model, oncogenic mutation of APC , P53 , KRAS and SMAD4 removes dependency on stem-cell-niche factors and converts normal organoids into tumoroids that grow as adenocarcinomas upon xenotransplantation into mice. This system will be invaluable in the future to study the biology of human cancers and develop new therapeutic approaches.