Human liver organoids for disease modeling of fibrolamellar carcinoma.

Fibrolamellar carcinoma (FLC) is a rare, often lethal, liver cancer affecting adolescents and young adults, for which there are no approved therapeutics. The development of therapeutics is hampered by a lack of in vitro models. Organoids have shown utility as a model system for studying many diseases. In this study, tumor tissue and the adjacent non-tumor liver were obtained at the time of surgery. The tissue was dissociated and grown as organoids. We developed 21 patient-derived organoid lines: 12 from metastases, three from the liver tumor and six from adjacent non-tumor liver. These patient-derived FLC organoids recapitulate the histologic morphology, immunohistochemistry, and transcriptome of the patient tumor. Patient-derived FLC organoids were used in a preliminary high-throughput drug screen to show proof of concept for the identification of therapeutics. This model system has the potential to improve our understanding of this rare cancer and holds significant promise for drug testing and development.

Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.

The molecular events that drive hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV-infected organoids produced covalently closed circular DNA (cccDNA) and HBV early antigen (HBeAg), expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV-infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity and drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor sodium taurocholate co-transporting polypeptide (NTCP) after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the hepatocellular carcinoma (HCC) cohort on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV-infected patients.

The Intestinal Epithelium - Fluid Fate and Rigid Structure From Crypt Bottom to Villus Tip.

The single-layered, simple epithelium of the gastro-intestinal tract controls nutrient uptake, coordinates our metabolism and shields us from pathogens. Despite its seemingly simple architecture, the intestinal lining consists of highly distinct cell populations that are continuously renewed by the same stem cell population. The need to maintain balanced diversity of cell types in an unceasingly regenerating tissue demands intricate mechanisms of spatial or temporal cell fate control. Recent advances in single-cell sequencing, spatio-temporal profiling and organoid technology have shed new light on the intricate micro-structure of the intestinal epithelium and on the mechanisms that maintain it. This led to the discovery of unexpected plasticity, zonation along the crypt-villus axis and new mechanism of self-organization. However, not only the epithelium, but also the underlying mesenchyme is distinctly structured. Several new studies have explored the intestinal stroma with single cell resolution and unveiled important interactions with the epithelium that are crucial for intestinal function and regeneration. In this review, we will discuss these recent findings and highlight the technologies that lead to their discovery. We will examine strengths and limitations of each approach and consider the wider impact of these results on our understanding of the intestine in health and disease.

Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.

Hepatic, pancreatic, and biliary (HPB) organoids are powerful tools for studying development, disease, and regeneration. As organoid research expands, the need for clear definitions and nomenclature describing these systems also grows. To facilitate scientific communication and consistent interpretation, we revisit the concept of an organoid and introduce an intuitive classification system and nomenclature for describing these 3D structures through the consensus of experts in the field. To promote the standardization and validation of HPB organoids, we propose guidelines for establishing, characterizing, and benchmarking future systems. Finally, we address some of the major challenges to the clinical application of organoids.

Exploring the human lacrimal gland using organoids and single-cell sequencing.

The lacrimal gland is essential for lubrication and protection of the eye. Disruption of lacrimal fluid production, composition, or release results in dry eye, causing discomfort and damage to the ocular surface. Here, we describe the establishment of long-term 3D organoid culture conditions for mouse and human lacrimal gland. Organoids can be expanded over multiple months and recapitulate morphological and transcriptional features of lacrimal ducts. CRISPR-Cas9-mediated genome editing reveals the master regulator for eye development Pax6 to be required for differentiation of adult lacrimal gland cells. We address cellular heterogeneity of the lacrimal gland by providing a single-cell atlas of human lacrimal gland tissue and organoids. Finally, human lacrimal gland organoids phenocopy the process of tear secretion in response to neurotransmitters and can engraft and produce mature tear products upon orthotopic transplantation in mouse. Together, this study provides an experimental platform to study the (patho-)physiology of the lacrimal gland.

Introductions to the Community: Early-Career Researchers in the Time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease.

The development, homeostasis, and repair of intrahepatic and extrahepatic bile ducts are thought to involve distinct mechanisms including proliferation and maturation of cholangiocyte and progenitor cells. This study aimed to characterize human extrahepatic cholangiocyte organoids (ECO) using canonical Wnt-stimulated culture medium previously developed for intrahepatic cholangiocyte organoids (ICO). Paired ECO and ICO were derived from common bile duct and liver tissue, respectively. Characterization showed both organoid types were highly similar, though some differences in size and gene expression were observed. Both ECO and ICO have cholangiocyte fate differentiation capacity. However, unlike ICO, ECO lack the potential for differentiation towards a hepatocyte-like fate. Importantly, ECO derived from a cystic fibrosis patient showed no CFTR channel activity but normal chloride channel and MDR1 transporter activity. In conclusion, this study shows that ECO and ICO have distinct lineage fate and that ECO provide a competent model to study extrahepatic bile duct diseases like cystic fibrosis.

A Human Organoid Model of Aggressive Hepatoblastoma for Disease Modeling and Drug Testing.

Hepatoblastoma is the most common childhood liver cancer. Although survival has improved significantly over the past few decades, there remains a group of children with aggressive disease who do not respond to current treatment regimens. There is a critical need for novel models to study aggressive hepatoblastoma as research to find new treatments is hampered by the small number of laboratory models of the disease. Organoids have emerged as robust models for many diseases, including cancer. We have generated and characterized a novel organoid model of aggressive hepatoblastoma directly from freshly resected patient tumors as a proof of concept for this approach. Hepatoblastoma tumor organoids recapitulate the key elements of patient tumors, including tumor architecture, mutational profile, gene expression patterns, and features of Wnt/ß-catenin signaling that are hallmarks of hepatoblastoma pathophysiology. Tumor organoids were successfully used alongside non-tumor liver organoids from the same patient to perform a drug screen using twelve candidate compounds. One drug, JQ1, demonstrated increased destruction of liver organoids from hepatoblastoma tumor tissue relative to organoids from the adjacent non-tumor liver. Our findings suggest that hepatoblastoma organoids could be used for a variety of applications and have the potential to improve treatment options for the subset of hepatoblastoma patients who do not respond to existing treatments.

Enteroendocrine Dynamics - New Tools Reveal Hormonal Plasticity in the Gut.

The recent intersection of enteroendocrine cell biology with single-cell technologies and novel in vitro model systems has generated a tremendous amount of new data. Here we highlight these recent developments and explore how these findings contribute to the understanding of endocrine lineages in the gut. In particular, the concept of hormonal plasticity, the ability of endocrine cells to produce different hormones over the course of their lifetime, challenges the classic notion of cell types. Enteroendocrine cells travel in the course of their life through different signaling environments that directly influence their hormonal repertoire. In this context, we examine how enteroendocrine cell fate is determined and modulated by signaling molecules such as bone morphogenetic proteins (BMPs) or location along the gastrointestinal tract. We analyze advantages and disadvantages of novel in vitro tools, adult stem cell or iPS-derived intestinal organoids, that have been crucial for recent findings on enteroendocrine development and plasticity. Finally, we illuminate the future perspectives of the field and discuss how understanding enteroendocrine plasticity can lead to new therapeutic approaches.

Long-Term Survival of Transplanted Autologous Canine Liver Organoids in a COMMD1-Deficient Dog Model of Metabolic Liver Disease.

The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson's disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor.

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/ß-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.

Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping.

Homeostatic regulation of the intestinal enteroendocrine lineage hierarchy is a poorly understood process. We resolved transcriptional changes during enteroendocrine differentiation in real time at single-cell level using a novel knockin allele of Neurog3, the master regulator gene briefly expressed at the onset of enteroendocrine specification. A bi-fluorescent reporter, Neurog3Chrono, measures time from the onset of enteroendocrine differentiation and enables precise positioning of single-cell transcriptomes along an absolute time axis. This approach yielded a definitive description of the enteroendocrine hierarchy and its sub-lineages, uncovered differential kinetics between sub-lineages, and revealed time-dependent hormonal plasticity in enterochromaffin and L cells. The time-resolved map of transcriptional changes predicted multiple novel molecular regulators. Nine of these were validated by conditional knockout in mice or CRISPR modification in intestinal organoids. Six novel candidate regulators (Sox4, Rfx6, Tox3, Myt1, Runx1t1, and Zcchc12) yielded specific enteroendocrine phenotypes. Our time-resolved single-cell transcriptional map presents a rich resource to unravel enteroendocrine differentiation.

Tales from the crypt: new insights into intestinal stem cells.

The intestinal epithelium withstands continuous mechanical, chemical and biological insults despite its single-layered, simple epithelial structure. The crypt-villus tissue architecture in combination with rapid cell turnover enables the intestine to act both as a barrier and as the primary site of nutrient uptake. Constant tissue replenishment is fuelled by continuously dividing stem cells that reside at the bottom of crypts. These cells are nurtured and protected by specialized epithelial and mesenchymal cells, and together constitute the intestinal stem cell niche. Intestinal stem cells and early progenitor cells compete for limited niche space and, therefore, the ability to retain or regain stemness. Those cells unable to do so differentiate to one of six different mature cell types and move upwards towards the villus, where they are shed into the intestinal lumen after 3-5 days. In this Review, we discuss the signals, cell types and mechanisms that control homeostasis and regeneration in the intestinal epithelium. We investigate how the niche protects and instructs intestinal stem cells, which processes drive differentiation of mature cells and how imbalance in key signalling pathways can cause human disease.

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.

Generation and characterization of rat liver stem cell lines and their engraftment in a rat model of liver failure.

The rat is an important model for liver regeneration. However, there is no in vitro culture system that can capture the massive proliferation that can be observed after partial hepatectomy in rats. We here describe the generation of rat liver stem cell lines. Rat liver stem cells, which grow as cystic organoids, were characterized by high expression of the stem cell marker Lgr5, by the expression of liver progenitor and duct markers, and by low expression of hepatocyte markers, oval cell markers, and stellate cell markers. Prolonged cultures of rat liver organoids depended on high levels of WNT-signalling and the inhibition of BMP-signaling. Upon transplantation of clonal lines to a Fah(-/-) Il2rg(-/-) rat model of liver failure, the rat liver stem cells engrafted into the host liver where they differentiated into areas with FAH and Albumin positive hepatocytes. Rat liver stem cell lines hold potential as consistent reliable cell sources for pharmacological, toxicological or metabolic studies. In addition, rat liver stem cell lines may contribute to the development of regenerative medicine in liver disease. To our knowledge, the here described liver stem cell lines represent the first organoid culture system in the rat.

Repairing organs: lessons from intestine and liver.

The concept of organ regeneration has fascinated humanity from ancient mythology to modern science fiction. Recent advances offer the potential to soon bring such technology within the grasp of clinical medicine. Rapidly expanding insights into the intrinsic repair processes of the intestine and liver have uncovered significant plasticity in epithelial tissues. Harnessing this knowledge, researchers have recently created culture systems that enable the expansion of stem cells into transplantable tissue in vitro. Here we discuss how the growing tool set of stem cell biology can bring organ repair from fictitious narrative to medical practice.

Long-term culture of genome-stable bipotent stem cells from adult human liver.

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Saving the neck from scission.

Secretory granule biogenesis is a pivotal process for regulated release of hormones and neurotransmitters. A prominent example is the pancreatic ß cell that secretes insulin, a major anabolic hormone controlling cellular metabolism upon nutrient availability. We recently described a checkpoint mechanism that halts scission of nascent secretory granules at the trans-Golgi network (TGN) until complete loading of insulin is achieved. We demonstrated that the Bin/Amphiphysin/Rvs (BAR) domain-containing protein Arfaptin-1 prevents granule scission until it is phosphorylated by Protein Kinase D (PKD). Arfaptin-1 phosphorylation releases its binding to ADP-rybosylation factor (ARF) allowing scission to occur. Lack of this control mechanism in ß cells resulted in premature scission, generation of dysfunctional insulin granules and impaired regulated insulin secretion without affecting constitutive release of other transport carriers. Here we discuss two important questions related to this work: How might completion of granule loading be sensed by PKD, and how does Arfaptin-1 specifically regulate insulin granule formation in beta cells?

Regulation of PTEN activity by p38δ-PKD1 signaling in neutrophils confers inflammatory responses in the lung.

Despite their role in resolving inflammatory insults, neutrophils trigger inflammation-induced acute lung injury (ALI), culminating in acute respiratory distress syndrome (ARDS), a frequent complication with high mortality in humans. Molecular mechanisms underlying recruitment of neutrophils to sites of inflammation remain poorly understood. Here, we show that p38 MAP kinase p38δ is required for recruitment of neutrophils into inflammatory sites. Global and myeloid-restricted deletion of p38δ in mice results in decreased alveolar neutrophil accumulation and attenuation of ALI. p38δ counteracts the activity of its downstream target protein kinase D1 (PKD1) in neutrophils and myeloid-restricted inactivation of PKD1 leads to exacerbated lung inflammation. Importantly, p38δ and PKD1 conversely regulate PTEN activity in neutrophils, thereby controlling their extravasation and chemotaxis. PKD1 phosphorylates p85α to enhance its interaction with PTEN, leading to polarized PTEN activity, thereby regulating neutrophil migration. Thus, aberrant p38δ-PKD1 signaling in neutrophils may underlie development of ALI and life-threatening ARDS in humans.