The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The “oval cell” response emanates from the biliary tree when all hepatocytes are ...affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.
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•Human and mouse hepatocytes can be grown long-term as organoids•Hepatocyte organoids consist of progenitors and differentiated hepatocytes•Murine hepatocyte organoids reflect regeneration after partial hepatectomy•Organoids from primary human hepatocytes engraft into damaged mouse liver
Modeling the regenerative ability of the liver in response to acute damage using long-term 3D organoid cultures in mice and human cells yields proliferative hepatocytes that are able to successfully engraft in animal models.
Outer-inner membrane vesicles (O-IMVs) were recently described as a new type of membrane vesicle secreted by the Antarctic bacterium Shewanella vesiculosa M7T. Their formation is characterized by the ...protrusion of both outer and plasma membranes, which pulls cytoplasmic components into the vesicles. To demonstrate that this is not a singular phenomenon in a bacterium occurring in an extreme environment, the identification of O-IMVs in pathogenic bacteria was undertaken. With this aim, a structural study by Transmission Electron Microscopy (TEM) and Cryo-transmission electron microscopy (Cryo-TEM) was carried out, confirming that O-IMVs are also secreted by Gram-negative pathogenic bacteria such as Neisseria gonorrhoeae, Pseudomonas aeruginosa PAO1 and Acinetobacter baumannii AB41, in which they represent between 0.23% and 1.2% of total vesicles produced. DNA and ATP, which are components solely found in the cell cytoplasm, were identified within membrane vesicles of these strains. The presence of DNA inside the O-IMVs produced by N. gonorrhoeae was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. A proteomic analysis of N. gonorrhoeae-derived membrane vesicles identified proteins from the cytoplasm and plasma membrane. This confirmation of O-IMV extends the hitherto uniform definition of membrane vesicles in Gram-negative bacteria and explains the presence of components in membrane vesicles such as DNA, cytoplasmic and inner membrane proteins, as well as ATP, detected for the first time. The production of these O-IMVs by pathogenic Gram-negative bacteria opens up new areas of study related to their involvement in lateral gene transfer, the transfer of cytoplasmic proteins, as well as the functionality and role of ATP detected in these new vesicles.
Respiratory infections remain a major global health concern. Tuberculosis is one of the top 10 causes of death worldwide, while infections with Non‐Tuberculous Mycobacteria are rising globally. ...Recent advances in human tissue modeling offer a unique opportunity to grow different human “organs” in vitro, including the human airway, that faithfully recapitulates lung architecture and function. Here, we have explored the potential of human airway organoids (AOs) as a novel system in which to assess the very early steps of mycobacterial infection. We reveal that Mycobacterium tuberculosis (Mtb) and Mycobacterium abscessus (Mabs) mainly reside as extracellular bacteria and infect epithelial cells with very low efficiency. While the AO microenvironment was able to control, but not eliminate Mtb, Mabs thrives. We demonstrate that AOs responded to infection by modulating cytokine, antimicrobial peptide, and mucin gene expression. Given the importance of myeloid cells in mycobacterial infection, we co‐cultured infected AOs with human monocyte‐derived macrophages and found that these cells interact with the organoid epithelium. We conclude that adult stem cell (ASC)‐derived AOs can be used to decipher very early events of mycobacteria infection in human settings thus offering new avenues for fundamental and therapeutic research.
Flowchart of host–pathogen interactions in microinjected human bronchiolar airway organoids. Organoids were injected using a micromanipulator coupled to a microinjection system (left). The capillary was pushed into the lumen of airway organoids (AOs) to deliver mycobacteria (middle). The readouts used to study host–pathogen interactions within the organoids are summarized (right). Created with Biorender.com.
The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate ...histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1’s tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.
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•BAP1 loss affects cell polarity and epithelial organization in human liver tissue•BAP1 controls chromatin accessibility of junctional and cytoskeletal genes•Rescuing BAP1 catalytic activity in the nucleus restores epithelial organization•In an engineered human cancer model, malignant features are induced upon BAP1 loss
Artegiani et al. show that BAP1 mutation in human liver organoids coincides with loss of multiple epithelial characteristics through impairment of chromatin accessibility and gene expression, and this is critical for the acquisition of malignant features in a human model of cholangiocarcinoma.
Alzheimer's disease (AD) is the most prevalent form of dementia. Key AD symptoms include memory and cognitive decline; however, comorbid symptoms such as depression and sensory‐perceptual dysfunction ...are often reported. Among these, a deterioration of olfactory sensation is observed in approximately 90% of AD patients. However, the precise pathophysiological basis underlying olfactory deficits because of AD remains elusive. The olfactory glomeruli in the olfactory bulb (OB) receive sensory information in the olfactory processing pathway. Maintaining the structural and functional integrity of the olfactory glomerulus is critical to olfactory signalling. Herein, we conducted an in‐depth histopathological assessment to reveal detailed structural alterations in the olfactory glomeruli in AD patients. Fresh frozen post‐mortem OB specimens obtained from six AD patients and seven healthy age‐matched individuals were examined. We used combined immunohistochemistry and stereology to assess the gross morphology and histological alterations, such as those in the expression of Aβ protein, microglia, and neurotransmitters in the OB. Electron microscopy was employed to study the ultrastructural features in the glomeruli. Significant accumulation of Aβ, morphologic damage, altered neurotransmitter levels, and microgliosis in the olfactory glomeruli of AD patients suggests that glomerular damage could affect olfactory function. Moreover, greater neurodegeneration was observed in the ventral olfactory glomeruli of AD patients. The synaptic ultrastructure revealed distorted postsynaptic densities and a decline in presynaptic vesicles in AD specimens. These findings show that the primary olfactory pathway is affected by the pathogenesis of AD, and may provide clues to identifying the mechanism involved in olfactory dysfunction in AD.
Herein we show that the primary olfactory pathway is affected by the pathogenesis of AD. This may provide clues to identifying the mechanism involved in olfactory dysfunction in AD.
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.
Cycling intestinal Lgr5⁺ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as ...surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5⁺ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5⁺ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.
Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established ...decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.
The increasing demand for cryo-electron microscopy (cryo-EM) reveals drawbacks in current sample preparation protocols, such as sample waste and lack of reproducibility. Here, we present several ...technical developments that provide efficient sample preparation for cryo-EM studies. Pin printing substantially reduces sample waste by depositing only a sub-nanoliter volume of sample on the carrier surface. Sample evaporation is mitigated by dewpoint control feedback loops. The deposited sample is vitrified by jets of cryogen followed by submersion into a cryogen bath. Because the cryogen jets cool the sample from the center, premounted autogrids can be used and loaded directly into automated cryo-EMs. We integrated these steps into a single device, named VitroJet. The device's performance was validated by resolving four standard proteins (apoferritin, GroEL, worm hemoglobin, beta-galactosidase) to ~3 Å resolution using a 200-kV electron microscope. The VitroJet offers a promising solution for improved automated sample preparation in cryo-EM studies.
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