Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

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.

Snake Venom Gland Organoids.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids.

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.

Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium.

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."

Cryo-electron tomography on focused ion beam lamellae transforms structural cell biology.

Cryogenic electron microscopy and data processing enable the determination of structures of isolated macromolecules to near-atomic resolution. However, these data do not provide structural information in the cellular environment where macromolecules perform their native functions, and vital molecular interactions can be lost during the isolation process. Cryogenic focused ion beam (FIB) fabrication generates thin lamellae of cellular samples and tissues, enabling structural studies on the near-native cellular interior and its surroundings by cryogenic electron tomography (cryo-ET). Cellular cryo-ET benefits from the technological developments in electron microscopes, detectors and data processing, and more in situ structures are being obtained and at increasingly higher resolution. In this Review, we discuss recent studies employing cryo-ET on FIB-generated lamellae and the technological developments in ultrarapid sample freezing, FIB fabrication of lamellae, tomography, data processing and correlative light and electron microscopy that have enabled these studies. Finally, we explore the future of cryo-ET in terms of both methods development and biological application.

An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.

Druggable redox pathways against Mycobacterium abscessus in cystic fibrosis patient-derived airway organoids.

Mycobacterium abscessus (Mabs) drives life-shortening mortality in cystic fibrosis (CF) patients, primarily because of its resistance to chemotherapeutic agents. To date, our knowledge on the host and bacterial determinants driving Mabs pathology in CF patient lung remains rudimentary. Here, we used human airway organoids (AOs) microinjected with smooth (S) or rough (R-)Mabs to evaluate bacteria fitness, host responses to infection, and new treatment efficacy. We show that S Mabs formed biofilm, and R Mabs formed cord serpentines and displayed a higher virulence. While Mabs infection triggers enhanced oxidative stress, pharmacological activation of antioxidant pathways resulted in better control of Mabs growth and reduced virulence. Genetic and pharmacological inhibition of the CFTR is associated with better growth and higher virulence of S and R Mabs. Finally, pharmacological activation of antioxidant pathways inhibited Mabs growth, at least in part through the quinone oxidoreductase NQO1, and improved efficacy in combination with cefoxitin, a first line antibiotic. In conclusion, we have established AOs as a suitable human system to decipher mechanisms of CF-driven respiratory infection by Mabs and propose boosting of the NRF2-NQO1 axis as a potential host-directed strategy to improve Mabs infection control.

The crystal structure of the EspB-EspK virulence factor-chaperone complex suggests an additional type VII secretion mechanism in Mycobacterium tuberculosis.

Pathogenic species from the Mycobacterium genus are responsible for a number of adverse health conditions in humans and animals that threaten health security and the economy worldwide. Mycobacteria have up to five specialized secretion systems (ESX-1 to ESX-5) that transport virulence factors across their complex cell envelope to facilitate manipulation of their environment. In pathogenic species, these virulence factors influence the immune system's response and are responsible for membrane disruption and contributing to cell death. While structural details of these secretion systems have been recently described, gaps still remain in the structural understanding of the secretion mechanisms of most substrates. Here, we describe the crystal structure of Mycobacterium tuberculosis ESX-1 secretion-associated substrate EspB bound to its chaperone EspK. We found that EspB interacts with the C-terminal domain of EspK through its helical tip. Furthermore, cryogenic electron microscopy, size exclusion chromatography analysis, and small-angle X-ray scattering experiments show that EspK keeps EspB in its secretion-competent monomeric form and prevents its oligomerization. The structure presented in this study suggests an additional secretion mechanism in ESX-1, analogous to the chaperoning of proline-glutamate (PE)-proline-proline-glutamate (PPE) proteins by EspG, where EspK facilitates the secretion of EspB in Mycobacterium species.

Understanding the invisible hands of sample preparation for cryo-EM.

Cryo-electron microscopy (cryo-EM) is rapidly becoming an attractive method in the field of structural biology. With the exploding popularity of cryo-EM, sample preparation must evolve to prevent congestion in the workflow. The dire need for improved microscopy samples has led to a diversification of methods. This Review aims to categorize and explain the principles behind various techniques in the preparation of vitrified samples for the electron microscope. Various aspects and challenges in the workflow are discussed, from sample optimization and carriers to deposition and vitrification. Reliable and versatile specimen preparation remains a challenge, and we hope to give guidelines and posit future directions for improvement.

Transcription factor achaete scute-like 2 controls intestinal stem cell fate.

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism.

The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves' disease patient sera, demonstrating that organoids can model human autoimmune disease.

Mycobacteria-host interactions in human bronchiolar airway organoids.

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.

Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

Modelling of primary ciliary dyskinesia using patient-derived airway organoids.

Patient-derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long-term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient-specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient-specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.

In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

Visualization of a short-range Wnt gradient in the intestinal stem-cell niche.

Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.

Enteroendocrine and tuft cells support Lgr5 stem cells on Paneth cell depletion.

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.