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.

A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.

Blastocyst-like structures generated solely from stem cells.

The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells 1 . From mouse blastocysts, it is possible to derive both trophoblast 2 and embryonic stem-cell lines 3 , which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal-KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.

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.

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.

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.

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.

Mouse and human urothelial cancer organoids: A tool for bladder cancer research.

Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.

LGR6 marks nephron progenitor cells.

BACKGROUND: Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists. RESULTS: Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre-tubular aggregates, renal vesicles, and in segments of S-shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single-cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long-term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons. CONCLUSIONS: Given the profound early mesenchymal expression and MET signature of LGR6+ cells, together with the lineage tracing of mesenchymal LGR6+ cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.

Sequential cancer mutations in cultured human intestinal stem cells.

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.

Correction: The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/ß-Catenin Coactivators Essential for Intestinal Homeostasis.

[This corrects the article DOI: 10.1371/journal.pbio.1000539.].

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67-RFP allele.

Cycling Lgr5+ stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5+ cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67RFP knock-in allele that identifies dividing cells. Using Lgr5-GFP;Ki67RFP mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5high stem cells are continuously in cell cycle, while a fraction of Lgr5low progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5low Ki67- cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.

Derivation of functional thymic epithelial organoid lines from adult murine thymus.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire-expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development.

Human conjunctiva organoids to study ocular surface homeostasis and disease.

The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.

The leukemia-associated Mllt10/Af10-Dot1l are Tcf4/ß-catenin coactivators essential for intestinal homeostasis.

Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/ß-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/ß-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and ß-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/ß-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a ß-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published ß-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.

CCBE1 is essential for mammalian lymphatic vascular development and enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo.

RATIONALE: Collagen- and calcium-binding EGF domains 1 (CCBE1) has been associated with Hennekam syndrome, in which patients have lymphedema, lymphangiectasias, and other cardiovascular anomalies. Insight into the molecular role of CCBE1 is completely lacking, and mouse models for the disease do not exist. OBJECTIVE: CCBE1 deficient mice were generated to understand the function of CCBE1 in cardiovascular development, and CCBE1 recombinant protein was used in both in vivo and in vitro settings to gain insight into the molecular function of CCBE1. METHODS AND RESULTS: Phenotypic analysis of murine Ccbe1 mutant embryos showed a complete lack of definitive lymphatic structures, even though Prox1(+) lymphatic endothelial cells get specified within the cardinal vein. Mutant mice die prenatally. Proximity ligation assays indicate that vascular endothelial growth factor receptor 3 activation appears unaltered in mutants. Human CCBE1 protein binds to components of the extracellular matrix in vitro, and CCBE1 protein strongly enhances vascular endothelial growth factor-C-mediated lymphangiogenesis in a corneal micropocket assay. CONCLUSIONS: Our data identify CCBE1 as a factor critically required for budding and migration of Prox-1(+) lymphatic endothelial cells from the cardinal vein. CCBE1 probably exerts these effects through binding to components of the extracellular matrix. CCBE1 has little lymphangiogenic effect on its own but dramatically enhances the lymphangiogenic effect of vascular endothelial growth factor-C in vivo. Thus, our data suggest CCBE1 to be essential but not sufficient for lymphangiogenesis.

Identification of stem cells in small intestine and colon by marker gene Lgr5.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. It is currently believed that four to six crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49) was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Here, using two knock-in alleles, we reveal exclusive expression of Lgr5 in cycling columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cell generated all epithelial lineages over a 60-day period, suggesting that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.