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Considerable phenotypic variation under identical culture conditions limits the potential of stem-cell-based embryo models (SEMs) in basic and applied research. The biological processes causing this seemingly stochastic variation remain unclear. Here, we investigated the roots of phenotypic variation by parallel recording of transcriptomic states and morphological history in individual structures modeling embryonic trunk formation. Machine learning and integration of time-resolved single-cell RNA sequencing with imaging-based phenotypic profiling identified early features predictive of phenotypic end states. Leveraging this predictive power revealed that early imbalance of oxidative phosphorylation and glycolysis results in aberrant morphology and a neural lineage bias, which we confirmed by metabolic measurements. Accordingly, metabolic interventions improved phenotypic end states. Collectively, our work establishes divergent metabolic states as drivers of phenotypic variation and offers a broadly applicable framework to chart and predict phenotypic variation in organoids and SEMs. The strategy can be used to identify and control underlying biological processes, ultimately increasing reproducibility.

We determined the roles of two coevolved and coexpressed human-specific genes, NBPF14 and NOTCH2NLB, on the abundance of the cortical progenitors that underlie the evolutionary expansion of the neocortex, the seat of higher cognitive abilities in humans. Using automated microinjection into apical progenitors (APs) of embryonic mouse neocortex and electroporation of APs in chimpanzee cerebral organoids, we show that NBPF14 promotes the delamination of AP progeny, by promoting oblique cleavage plane orientation during AP division, leading to increased abundance of the key basal progenitor type, basal radial glia. In contrast, NOTCH2NLB promotes AP proliferation, leading to expansion of the AP pool. When expressed together, NBPF14 and NOTCH2NLB exert coordinated effects, resulting in expansion of basal progenitors while maintaining self-renewal of APs. Hence, these two human-specific genes orchestrate the behavior of APs, and the lineages of their progeny, in a manner essential for the evolutionary expansion of the human neocortex.

Liver regeneration is supported by hepatocytes and, in certain conditions, biliary epithelial cells (BECs). BECs are facultative liver stem cells that form organoids in culture and engraft in damaged livers. However, BEC heterogeneity in the homeostatic liver remains to be fully elucidated. Here, we exploit systemic lentiviral vector (LV) administration to achieve efficient and lifelong gene transfer to BECs in mice. We find that LV-marked BECs retain organoid formation potential and predominantly respond to liver damage; however, they are less clonogenic and display a hepatocyte-primed transcriptome compared to untransduced BECs. We thus identify a BEC subset committed to hepatocyte lineage in the absence of liver damage, characterized by a transcriptional network orchestrated by hepatocyte nuclear factor 4α. We also report in vivo targeting of such BECs in non-human primates. This work highlights intrinsic BEC heterogeneity and that in vivo LV gene transfer to the liver may persist following BEC-mediated repair of hepatic damage.

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

Uncontrolled regeneration leads to neoplastic transformation1-3. The intestinal epithelium requires precise regulation during continuous homeostatic and damage-induced tissue renewal to prevent neoplastic transformation, suggesting that pathways unlinking tumour growth from regenerative processes must exist. Here, by mining RNA-sequencing datasets from two intestinal damage models4,5 and using pharmacological, transcriptomics and genetic tools, we identified liver X receptor (LXR) pathway activation as a tissue adaptation to damage that reciprocally regulates intestinal regeneration and tumorigenesis. Using single-cell RNA sequencing, intestinal organoids, and gain- and loss-of-function experiments, we demonstrate that LXR activation in intestinal epithelial cells induces amphiregulin (Areg), enhancing regenerative responses. This response is coordinated by the LXR-ligand-producing enzyme CYP27A1, which was upregulated in damaged intestinal crypt niches. Deletion of Cyp27a1 impaired intestinal regeneration, which was rescued by exogenous LXR agonists. Notably, in tumour models, Cyp27a1 deficiency led to increased tumour growth, whereas LXR activation elicited anti-tumour responses dependent on adaptive immunity. Consistently, human colorectal cancer specimens exhibited reduced levels of CYP27A1, LXR target genes, and B and CD8 T cell gene signatures. We therefore identify an epithelial adaptation mechanism to damage, whereby LXR functions as a rheostat, promoting tissue repair while limiting tumorigenesis.

Many aspects of neurodegenerative disease pathology remain unresolved. Why do certain neuronal subpopulations acquire vulnerability to stress or mutations in ubiquitously expressed genes, while others remain resilient? Do these neurons harbor intrinsic marks that make them prone to degeneration? Do these diseases have a neurodevelopmental component? Lacking this fundamental knowledge hampers the discovery of efficacious treatments. While it is well established that human organoids enable the modeling of brain-related diseases, we still lack an organoid model that recapitulates the regionalization complexity and physiology of the spinal cord. Here, we describe an advanced experimental protocol to generate neuromuscular organoids composed of a wide rostro-caudal (RC) diversity of spinal motor neurons (spMNs) and mesodermal progenitor-derived muscle cells. This model therefore allows for the robust and reproducible study of neuromuscular unit development and disease.

Organoids and stem-cell-based embryo models (SEMs) are imperfect organ or embryo representations that explore a much larger space of possible forms, or morphospace, compared to their in vivo counterparts. Here, we discuss SEM biology in light of seminal work by Pere Alberch, a leading figure in early evo-devo, interpreting SEMs as developmental 'monstrosities' in the Alberchian sense. Alberch suggested that ordered patterns in aberrant development-i.e. 'the logic of monsters'-reveal developmental constraints on possible morphologies. In the same vein, we detail how SEMs have begun to shed light on structural features of normal development, such as developmental variability, the relative importance of internal versus external constraints, boundary conditions and design principles governing robustness and canalization. We argue that SEMs represent a powerful experimental tool to explore and expand developmental morphospace and propose that the 'monstrosity' of SEMs can be leveraged to uncover the 'hidden' rules and developmental constraints that robustly shape and pattern the embryo.

Whether neurodevelopmental defects underlie postnatal neuronal death in neurodegeneration is an intriguing hypothesis only recently explored. Here, we focus on spinal muscular atrophy (SMA), a neuromuscular disorder caused by reduced survival of motor neuron (SMN) protein levels leading to spinal motor neuron (MN) loss and muscle wasting. Using the first isogenic patient-derived induced pluripotent stem cell (iPSC) model and a spinal cord organoid (SCO) system, we show that SMA SCOs exhibit abnormal morphological development, reduced expression of early neural progenitor markers, and accelerated expression of MN progenitor and MN markers. Longitudinal single-cell RNA sequencing reveals marked defects in neural stem cell specification and fewer MNs, favoring mesodermal progenitors and muscle cells, a bias also seen in early SMA mouse embryos. Surprisingly, SMN2-to-SMN1 conversion does not fully reverse these developmental abnormalities. These suggest that early neurodevelopmental defects may underlie later MN degeneration, indicating that postnatal SMN-increasing interventions might not completely amend SMA pathology in all patients.

Collective cell dynamics play a crucial role in many developmental and physiological contexts. While two-dimensional (2D) cell migration has been widely studied, how three-dimensional (3D) geometry and topology interplay with collective cell behavior to determine dynamics and functions remains an open question. In this work, we elucidate the biophysical mechanism underlying rotation in spherical tissues, a phenomenon widely reported both in vivo and in vitro. Using murine pancreas-derived organoids as a model system, we find that epithelial spheres exhibit persistent rotation, rotational axis drift, and rotation arrest. Using a 3D vertex model, we demonstrate how the combined action of traction force and polarity alignment can account for these distinct rotational dynamics near a solid to flow transition. Furthermore, our analysis shows that the spherical tissue rotates as an active solid occasionally switching to a flowing state and exhibits spontaneous chiral symmetry breaking. Using a continuum model, we demonstrate how the topological defects in the polarity field underlie this symmetry breaking process, which is revealed by asymmetries in the cell elongation pattern. For cell elongation to reveal the chiral asymmetry, shear flow is required in addition to the solid body rotation. Altogether, our work reveals a robust chiral symmetry breaking mechanism with potential implications for left-right symmetry breaking processes in morphogenetic events.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Neocortex expansion during evolution is linked to higher numbers of neurons, which are thought to result from increased proliferative capacity and neurogenic potential of basal progenitor cells during development. Here, we show that EREG, encoding the growth factor EPIREGULIN, is expressed in the human developing neocortex and in gorilla cerebral organoids, but not in the mouse neocortex. Addition of EPIREGULIN to the mouse neocortex increases proliferation of basal progenitor cells, whereas EREG ablation in human cortical organoids reduces proliferation in the subventricular zone. Treatment of cortical organoids with EPIREGULIN promotes a further increase in proliferation of gorilla but not of human basal progenitor cells. EPIREGULIN competes with the epidermal growth factor (EGF) to promote proliferation, and inhibition of the EGF receptor abrogates the EPIREGULIN-mediated increase in basal progenitor cells. Finally, we identify putative cis-regulatory elements that may contribute to the observed inter-species differences in EREG expression. Our findings suggest that species-specific regulation of EPIREGULIN expression may contribute to the increased neocortex size of primates by providing a tunable pro-proliferative signal to basal progenitor cells in the subventricular zone.

Human tissue three-dimensional (3D) organoid cultures have the potential to reproduce in vitro the physiological properties and cellular architecture of the organs from which they are derived. The ability of organoid cultures derived from human stomach, liver, kidney, and colon to metabolically activate three dietary carcinogens, aflatoxin B1 (AFB1), aristolochic acid I (AAI), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was investigated. In each case, the response of a target tissue (liver for AFB1; kidney for AAI; colon for PhIP) was compared with that of a nontarget tissue (gastric). After treatment cell viabilities were measured, DNA damage response (DDR) was determined by Western blotting for p-p53, p21, p-CHK2, and γ-H2AX, and DNA adduct formation was quantified by mass spectrometry. Induction of the key xenobiotic-metabolizing enzymes (XMEs) CYP1A1, CYP1A2, CYP3A4, and NQO1 was assessed by qRT-PCR. We found that organoids from different tissues can activate AAI, AFB1, and PhIP. In some cases, this metabolic potential varied between tissues and between different cultures of the same tissue. Similarly, variations in the levels of expression of XMEs were observed. At comparable levels of cytotoxicity, organoids derived from tissues that are considered targets for these carcinogens had higher levels of adduct formation than a nontarget tissue.

Three-dimensional cell and organoid cultures rely on the mechanical support of viscoelastic matrices. However, commonly used matrix materials lack control over key cell-instructive properties. Here we report on fully synthetic hydrogels based on DNA libraries that self-assemble with ultrahigh-molecular-weight polymers, forming a dynamic DNA-crosslinked matrix (DyNAtrix). DyNAtrix enables computationally predictable and systematic control over its viscoelasticity, thermodynamic and kinetic parameters by changing DNA sequence information. Adjustable heat activation allows homogeneous embedding of mammalian cells. Intriguingly, stress-relaxation times can be tuned over four orders of magnitude, recapitulating mechanical characteristics of living tissues. DyNAtrix is self-healing, printable, exhibits high stability, cyto- and haemocompatibility, and controllable degradation. DyNAtrix-based cultures of human mesenchymal stromal cells, pluripotent stem cells, canine kidney cysts and human trophoblast organoids show high viability, proliferation and morphogenesis. DyNAtrix thus represents a programmable and versatile precision matrix for advanced approaches to biomechanics, biophysics and tissue engineering.

Mechanics is known to play a fundamental role in many cellular and developmental processes. Beyond active forces and material properties, osmotic pressure is believed to control essential cell and tissue characteristics. However, it remains very challenging to perform in situ and in vivo measurements of osmotic pressure. Here we introduce double emulsion droplet sensors that enable local measurements of osmotic pressure intra- and extra-cellularly within 3D multicellular systems, including living tissues. After generating and calibrating the sensors, we measure the osmotic pressure in blastomeres of early zebrafish embryos as well as in the interstitial fluid between the cells of the blastula by monitoring the size of droplets previously inserted in the embryo. Our results show a balance between intracellular and interstitial osmotic pressures, with values of approximately 0.7 MPa, but a large pressure imbalance between the inside and outside of the embryo. The ability to measure osmotic pressure in 3D multicellular systems, including developing embryos and organoids, will help improve our understanding of its role in fundamental biological processes.

Periventricular neuronal heterotopia (PH) is one of the most common forms of cortical malformation in the human cortex. We show that human neuronal progenitor cells (hNPCs) derived from PH patients with a DCHS1 or FAT4 mutation as well as isogenic lines had altered migratory dynamics when grafted in the mouse brain. The affected migration was linked to altered autophagy as observed in vivo with an electron microscopic analysis of grafted hNPCs, a Western blot analysis of cortical organoids, and time-lapse imaging of hNPCs in the presence of bafilomycin A1. We further show that deficits in autophagy resulted in the accumulation of paxillin, a focal adhesion protein involved in cell migration. Strikingly, a single-cell RNA-seq analysis of hNPCs revealed similar expression levels of autophagy-related genes. Bolstering AMPK-dependent autophagy by metformin, an FDA-approved drug, promoted migration of PH patients-derived hNPCs. Our data indicate that transcription-independent homeostatic modifications in autophagy contributed to the defective migratory behavior of hNPCs in vivo and suggest that modulating autophagy in hNPCs might rescue neuronal migration deficits in some forms of PH.

Hepatocytes form bile canaliculi that dynamically respond to the signalling activity of bile acids and bile flow. Little is known about their responses to intraluminal pressure. During embryonic development, hepatocytes assemble apical bulkheads that increase the canalicular resistance to intraluminal pressure. Here, we investigate whether they also protect bile canaliculi against elevated pressure upon impaired bile flow in adult liver. Apical bulkheads accumulate upon bile flow obstruction in mouse models and patients with primary sclerosing cholangitis (PSC). Their loss under these conditions leads to abnormally dilated canaliculi, resembling liver cell rosettes described in other hepatic diseases. 3D reconstruction reveals that these structures are sections of cysts and tubes formed by hepatocytes. Mathematical modelling establishes that they positively correlate with canalicular pressure and occur in early PSC stages. Using primary hepatocytes and 3D organoids, we demonstrate that excessive canalicular pressure causes the loss of apical bulkheads and formation of rosettes. Our results suggest that apical bulkheads are a protective mechanism of hepatocytes against impaired bile flow, highlighting the role of canalicular pressure in liver diseases.

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.

Within the peri-portal region of the adult liver, portal fibroblasts exist in close proximity to epithelial ductal/cholangiocyte cells. However, the cellular interactions between them are poorly understood. Here, we provide two co-culture techniques to incorporate liver portal mesenchyme into ductal cell organoids, which recapitulate aspects of their cellular interactions in vitro. We integrate several techniques from mesenchyme isolation and expansion to co-culture by microfluidic cell co-encapsulation or 2D-Matrigel layer. The protocol is easily adaptable to other cells from other organs. For complete information on the generation and use of this protocol, please refer to Cordero-Espinoza et al.1.

The functional relevance of an organoid is dependent on the differentiation, morphology, cell arrangement and biophysical properties, which collectively define the state of an organoid. For an organoid culture, an individual organoid or the cells that compose it, these state variables can be characterised, most easily by transcriptomics and by high-content image analysis. Their states can be compared to their in vivo counterparts. Current evidence suggests that organoids explore a wider state space than organs in vivo due to the lack of niche signalling and the variability of boundary conditions in vitro. Using data-driven state inference and in silico modelling, phase diagrams can be constructed to systematically sort organoids along biochemical or biophysical axes. These phase diagrams allow us to identify control strategies to modulate organoid state. To do so, the biochemical and biophysical environment, as well as the cells that seed organoids, can be manipulated.

Motor neuron diseases (MNDs) are a heterogeneous group of disorders that affect the cranial and/or spinal motor neurons (spMNs), spinal sensory neurons and the muscular system. Although they have been investigated for decades, we still lack a comprehensive understanding of the underlying molecular mechanisms; and therefore, efficacious therapies are scarce. Model organisms and relatively simple two-dimensional cell culture systems have been instrumental in our current knowledge of neuromuscular disease pathology; however, in the recent years, human 3D in vitro models have transformed the disease-modeling landscape. While cerebral organoids have been pursued the most, interest in spinal cord organoids (SCOs) is now also increasing. Pluripotent stem cell (PSC)-based protocols to generate SpC-like structures, sometimes including the adjacent mesoderm and derived skeletal muscle, are constantly being refined and applied to study early human neuromuscular development and disease. In this review, we outline the evolution of human PSC-derived models for generating spMN and recapitulating SpC development. We also discuss how these models have been applied to exploring the basis of human neurodevelopmental and neurodegenerative diseases. Finally, we provide an overview of the main challenges to overcome in order to generate more physiologically relevant human SpC models and propose some exciting new perspectives.

Neurodegenerative diseases remain incompletely understood and therapies are needed. Stem cell-derived organoid models facilitate fundamental and translational medicine research. However, to which extent differential neuronal and glial pathologic processes can be reproduced in current systems is still unclear. Here, we tested 16 different chemical, physical, and cell functional manipulations in mouse retina organoids to further explore this. Some of the treatments induce differential phenotypes, indicating that organoids are competent to reproduce distinct pathologic processes. Notably, mouse retina organoids even reproduce a complex pathology phenotype with combined photoreceptor neurodegeneration and glial pathologies upon combined (not single) application of HBEGF and TNF, two factors previously associated with neurodegenerative diseases. Pharmacological inhibitors for MAPK signaling completely prevent photoreceptor and glial pathologies, while inhibitors for Rho/ROCK, NFkB, and CDK4 differentially affect them. In conclusion, mouse retina organoids facilitate reproduction of distinct and complex pathologies, mechanistic access, insights for further organoid optimization, and modeling of differential phenotypes for future applications in fundamental and translational medicine research.

Our understanding of diabetes mellitus has benefited from a combination of clinical investigations and work in model organisms and cell lines. Organoid models for a wide range of tissues are emerging as an additional tool enabling the study of diabetes mellitus. The applications for organoid models include studying human pancreatic cell development, pancreatic physiology, the response of target organs to pancreatic hormones and how glucose toxicity can affect tissues such as the blood vessels, retina, kidney and nerves. Organoids can be derived from human tissue cells or pluripotent stem cells and enable the production of human cell assemblies mimicking human organs. Many organ mimics relevant to diabetes mellitus are already available, but only a few relevant studies have been performed. We discuss the models that have been developed for the pancreas, liver, kidney, nerves and vasculature, how they complement other models, and their limitations. In addition, as diabetes mellitus is a multi-organ disease, we highlight how a merger between the organoid and bioengineering fields will provide integrative models.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders. As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Animal organs exhibit complex topologies involving cavities and tubular networks, which underlie their form and function1-3. However, how topology emerges during the development of organ shape, or morphogenesis, remains elusive. Here we combine tissue reconstitution and quantitative microscopy to show that tissue topology and shape is governed by two distinct modes of topological transitions4,5. One mode involves the fusion of two separate epithelia and the other involves the fusion of two ends of the same epithelium. The morphological space is captured by a single control parameter that can be traced back to the relative rates of the two epithelial fusion modes. Finally, we identify a pharmacologically accessible pathway that regulates the frequency of two modes of epithelial fusion, and demonstrate the control of organoid topology and shape. The physical principles uncovered here provide fundamental insights into the self-organization of complex tissues6.

Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[a]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, CYP1A1 and NQO1, were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP-t-7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3-6, 2022, experts in organoid development and biology convened at the Keystone Symposium "Organoids as Tools for Fundamental Discovery and Translation" to discuss recent advances and insights from this relatively new model system into human development and disease.

The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia-the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution.

How cells build embryos is still a major mystery. Many unresolved questions require the study of the processes that pattern and shape the embryo in live specimens, in toto, across spatial and temporal scales. In mammalian embryogenesis, this remains a major challenge as the embryo develops in utero, precluding easy accessibility. For human embryos, technical, ethical and legal limitations further hamper the in-depth investigation of embryogenesis, especially beyond gastrulation stages. This has resulted in an over-reliance on model organisms, particularly mice, to understand mammalian development. However, recent efforts show critical differences between rodent and primate embryos, including timing, architecture and transcriptional regulation. Thus, a human-centric understanding of embryogenesis is much needed. To empower this, novel in vitro approaches, which coax human pluripotent stem cells to form embryonic organoids that model embryo development, are pivotal. Here, we summarize these emergent technologies that recapitulate aspects of human development "in a dish". We show how these technologies can provide insights into the molecular, cellular and morphogenetic processes that fuel the formation of a fully formed fetus, and discuss the potential of these platforms to revolutionize our understanding of human development in health and disease. Despite their clear promise, we caution against over-interpreting the extent to which these in vitro platforms model the natural embryo. In particular, we discuss how fate, form and function - a tightly coupled trinity in vivo, can be disconnected in vitro. Finally, we propose how careful benchmarking of existing models, in combination with rational protocol design based on an increased understanding of in vivo developmental dynamics and insights from mouse in vitro models of embryo development, will help guide the establishment of better models of human embryo development.

Organoids have become one of the fastest progressing and applied models in biological and medical research, and various organoids have now been developed for most of the organs of the body. Here, we review the methods developed to generate pancreas organoids in vitro from embryonic, fetal and adult cells, as well as pluripotent stem cells. We discuss how these systems have been used to learn new aspects of pancreas development, regeneration and disease, as well as their limitations and potential for future discoveries.

Organoids are miniaturized and simplified versions of an organ produced in vitro from stem or progenitor cells. They are used as a model system consisting of multiple cell types forming an architecture relevant to the organ and carrying out the function of the organ. They are a useful tool to study development, homeostasis, regeneration, and disease. The imaging of organoids has become a pivotal method to visualize and understand their self-organization, symmetry breaking, growth, differentiation, and function. In this review, we discuss imaging methods, how to analyze these images, and challenges in organoid research.

Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Comparing the biology of humans to that of other primates, and notably other hominids, is a useful path to learn more about what makes us human. Some of the most interesting differences among hominids are closely related to brain development and function, for example behaviour and cognition. This makes it particularly interesting to compare the hominid neural cells of the neocortex, a part of the brain that plays central roles in those processes. However, well-preserved tissue from great apes is usually extremely difficult to obtain. A variety of new alternative tools, e.g. brain organoids, are now beginning to make it possible to search for such differences and analyse their potential biological and biomedical meaning. Here we present an overview of recent findings from comparisons of the neural stem and progenitor cells (NSPCs) and neurons of hominids. In addition to differences in proliferation and differentiation of NSPCs, and maturation of neurons, we highlight that the regulation of the timing of these processes is emerging as a general foundational difference in the development of the neocortex of hominids.

Reliable establishment of tumor organoids is paramount to advance applications of organoid technology for personalized medicine. Here, we share our multi-center experience on initiation and tumorigenic confirmation of hepatobiliary cancer organoids. We discuss current concerns, propose potential solutions, and provide future perspectives for improvements in hepatobiliary cancer organoid establishment.

Epithelial organoids can recapitulate many processes reminiscent of morphogenesis in vivo including lumen and multilayer formation, folding, branching, delamination and elongation. While being noisier in vitro than in vivo, these processes can be monitored live and subjected to interferences, a field that is emerging. We elaborate on the signalling molecules controlling morphogenesis, from the medium and their emergence as signalling centers in the organoids. Further, we discuss how organoid shape is controlled by mechanical cues within the organoid and their interplay with the material properties of the environment.

Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC.

RNF43/ZNRF3 negatively regulate WNT signalling. Both genes are mutated in several types of cancers, however, their contribution to liver disease is unknown. Here we describe that hepatocyte-specific loss of Rnf43/Znrf3 results in steatohepatitis and in increase in unsaturated lipids, in the absence of dietary fat supplementation. Upon injury, Rnf43/Znrf3 deletion results in defective hepatocyte regeneration and liver cancer, caused by an imbalance between differentiation/proliferation. Using hepatocyte-, hepatoblast- and ductal cell-derived organoids we demonstrate that the differentiation defects and lipid alterations are, in part, cell-autonomous. Interestingly, ZNRF3 mutant liver cancer patients present poorer prognosis, altered hepatic lipid metabolism and steatohepatitis/NASH signatures. Our results imply that RNF43/ZNRF3 predispose to liver cancer by controlling the proliferative/differentiation and lipid metabolic state of hepatocytes. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 mutated individuals at risk of developing fatty liver and/or liver cancer.

In the liver, ductal cells rarely proliferate during homeostasis but do so transiently after tissue injury. These cells can be expanded as organoids that recapitulate several of the cell-autonomous mechanisms of regeneration but lack the stromal interactions of the native tissue. Here, using organoid co-cultures that recapitulate the ductal-to-mesenchymal cell architecture of the portal tract, we demonstrate that a subpopulation of mouse periportal mesenchymal cells exerts dual control on proliferation of the epithelium. Ductal cell proliferation is either induced and sustained or, conversely, completely abolished, depending on the number of direct mesenchymal cell contacts, through a mechanism mediated, at least in part, by Notch signaling. Our findings expand the concept of the cellular niche in epithelial tissues, whereby not only soluble factors but also cell-cell contacts are the key regulatory cues involved in the control of cellular behaviors, suggesting a critical role for cell-cell contacts during regeneration.

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 cccDNA, 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 as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor 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 HCC cohort on the TCGA LIHC 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 evolutionary expansion of the neocortex, the seat of higher cognitive functions in humans, is primarily due to an increased and prolonged proliferation of neural progenitor cells during development. Basal progenitors, and in particular basal radial glial cells, are thought to have a key role in the increased generation of neurons that constitutes a foundation of neocortex expansion. Recent studies have identified primate-specific and human-specific genes and changes in gene expression that promote increased proliferative capacity of cortical progenitors. In many cases, the cell biological basis underlying this increase has been uncovered. Model systems such as mouse, ferret, nonhuman primates, and cerebral organoids have been used to establish the relevance of these genes for neocortex expansion.

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.

Organoid cultures hold the promise of transforming basic and clinical research. Here, we provide a storyline of the highlights and breakthroughs that have an impact in present clinical research. We also discuss the bottlenecks that delay their full exploitation for the next generation of biomedical research.

Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days.

How epithelial cells coordinate their polarity to form functional tissues is an open question in cell biology. Here, we characterize a unique type of polarity found in liver tissue, nematic cell polarity, which is different from vectorial cell polarity in simple, sheet-like epithelia. We propose a conceptual and algorithmic framework to characterize complex patterns of polarity proteins on the surface of a cell in terms of a multipole expansion. To rigorously quantify previously observed tissue-level patterns of nematic cell polarity (Morales-Navarette et al., eLife 2019), we introduce the concept of co-orientational order parameters, which generalize the known biaxial order parameters of the theory of liquid crystals. Applying these concepts to three-dimensional reconstructions of single cells from high-resolution imaging data of mouse liver tissue, we show that the axes of nematic cell polarity of hepatocytes exhibit local coordination and are aligned with the biaxially anisotropic sinusoidal network for blood transport. Our study characterizes liver tissue as a biological example of a biaxial liquid crystal. The general methodology developed here could be applied to other tissues and in-vitro organoids.

Organoids constitute biological systems which are used to model organ development, homeostasis, regeneration, and disease in vitro and hold promise for use in therapy. Reflecting in vivo development, organoids form from tissue cells or pluripotent stem cells. Cues provided from the media and individual cells promote self-organization of these uniform starting cells into a structure, with emergent differentiated cells, morphology, and often functionality that resemble the tissue of origin. Therefore, organoids provide a complement to two-dimensional in vitro culture and in vivo animal models of development, providing the experimental control and flexibility of in vitro methods with the three-dimensional context of in vivo models, with fewer ethical restraints than human or animal work. However, using organoids, we are only just beginning to understand on the cellular level how the external conditions and signaling between individual cells promote the emergence of cells and structures. In this review, we focus specifically on organoids derived from endodermal tissues: the starting conditions of the cells, signaling mechanisms, and external media that allow the emergence of higher order self-organization.

The biophysical properties of polymer based gels, for instance the commonly used Matrigel, crucially depend on polymer concentration. Only certain polymer concentrations will produce a gel optimal for a specific purpose, for instance for organoid development. Hence, in order to design a polymer scaffold for a specific purpose, it is important to know which properties are optimal and to control the biophysical properties of the scaffold. Using optical tweezers, we perform a biophysical characterization of the biologically relevant Matrigel while systematically varying the polymer concentration. Using the focused laser beam we trace and spectrally analyze the thermal fluctuations of an inert tracer particle. From this, the visco-elastic properties of the Matrigel is quantified in a wide frequency range through scaling analysis of the frequency power spectrum as well as by calculating the complex shear modulus. The viscoelastic properties of the Matrigel are monitored over a timespan of 7 h. At all concentrations, the Matrigel is found to be more fluid-like just after formation and to become more solid-like during time, settling to a constant state after 1-3 h. Also, the Matrigel is found to display increasingly more solid-like properties with increasing polymer concentration. To demonstrate the biological relevance of these results, we expand pancreatic organoids in Matrigel solutions with the same polymer concentration range and demonstrate how the polymer concentration influences organoid development. In addition to providing quantitative information about how polymer gels change visco-elastic properties as a function of polymer concentration and time, these results also serve to guide the search of novel matrices relevant for organoid development or 3D cell culturing, and to ensure reproducibility of bio-relevant Matrigels.

High-throughput drug screening of patient-derived organoids offers an attractive platform to determine cancer treatment efficacy. Here, selective plane illumination microscopy (SPIM) was used to determine treatment response in organoids with endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD. Rapid 3-D autofluorescence imaging of colorectal cancer organoids was achieved. A quantitative image analysis approach was developed to segment each organoid and quantify changes in endogenous fluorescence caused by treatment. Quantitative analysis of SPIM volumes confirmed the sensitivity of patient-derived organoids to standard therapies. This proof-of-principle study demonstrates that SPIM is a powerful tool for high-throughput screening of organoid treatment response.

Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus.

Alpha-1 antitrypsin (AAT) is a product of SERPINA1 gene mainly expressed by hepatocytes. Clinically relevant mutations in the SERPINA1 gene, such as Z (Glu342Lys), results in an expression of misfolded AAT protein having high propensity to polymerize, accumulate in hepatocytes and thus to enhance a risk for hepatocyte damage and subsequent liver disease. So far, the relationship between the Z-AAT accumulation and liver cell damage remains not completely understood. We present three-dimensional organoid culture systems, as a novel tool for modeling Z-AAT-related liver diseases.

Neural progenitor cells (NPCs) play a central role during the development and evolution of the mammalian neocortex. Precise temporal and spatial control of NPC proliferation by a concert of cell-intrinsic and cell-extrinsic factors is essential for the correct formation and proper function of the neocortex. In this review, we focus on the regulation of NPC proliferation by neurotransmitters, which act as a group of cell-extrinsic factors during mammalian neocortex development. We first summarize, from both in vivo and in vitro studies, our current knowledge on how γ-aminobutyric acid (GABA), glutamate and serotonin modulate NPC proliferation in the developing neocortex and the potential involvements of different receptors in the underlying mechanisms. Another focus of this review is to discuss future perspectives using conditionally gene-modified mice and human brain organoids as model systems to further our understanding on the contribution of neurotransmitters to the development of a normal neocortex, as well as how dysregulated neurotransmitter signaling leads to developmental and psychiatric disorders.

Organoid cultures have emerged as an alternative in vitro system to recapitulate tissues in a dish. While mouse models and cell lines have furthered our understanding of liver biology and associated diseases, they suffer in replicating key aspects of human liver tissue, in particular its complex architecture and metabolic functions. Liver organoids have now been established for multiple species from induced pluripotent stem cells, embryonic stem cells, hepatoblasts and adult tissue-derived cells. These represent a promising addition to our toolbox to gain a deeper understanding of this complex organ. In this perspective we will review the advances in the liver organoid field, its limitations and potential for biomedical applications.

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.

The human brain has undergone substantial change since humans diverged from chimpanzees and the other great apes1,2. However, the genetic and developmental programs that underlie this divergence are not fully understood. Here we have analysed stem cell-derived cerebral organoids using single-cell transcriptomics and accessible chromatin profiling to investigate gene-regulatory changes that are specific to humans. We first analysed cell composition and reconstructed differentiation trajectories over the entire course of human cerebral organoid development from pluripotency, through neuroectoderm and neuroepithelial stages, followed by divergence into neuronal fates within the dorsal and ventral forebrain, midbrain and hindbrain regions. Brain-region composition varied in organoids from different iPSC lines, but regional gene-expression patterns remained largely reproducible across individuals. We analysed chimpanzee and macaque cerebral organoids and found that human neuronal development occurs at a slower pace relative to the other two primates. Using pseudotemporal alignment of differentiation paths, we found that human-specific gene expression resolved to distinct cell states along progenitor-to-neuron lineages in the cortex. Chromatin accessibility was dynamic during cortex development, and we identified divergence in accessibility between human and chimpanzee that correlated with human-specific gene expression and genetic change. Finally, we mapped human-specific expression in adult prefrontal cortex using single-nucleus RNA sequencing analysis and identified developmental differences that persist into adulthood, as well as cell-state-specific changes that occur exclusively in the adult brain. Our data provide a temporal cell atlas of great ape forebrain development, and illuminate dynamic gene-regulatory features that are unique to humans.

The past decade has seen an explosion in the field of in vitro disease modelling, in particular the development of organoids. These self-organizing tissues derived from stem cells provide a unique system to examine mechanisms ranging from organ development to homeostasis and disease. Because organoids develop according to intrinsic developmental programmes, the resultant tissue morphology recapitulates organ architecture with remarkable fidelity. Furthermore, the fact that these tissues can be derived from human progenitors allows for the study of uniquely human processes and disorders. This article and accompanying poster highlight the currently available methods, particularly those aimed at modelling human biology, and provide an overview of their capabilities and limitations. We also speculate on possible future technological advances that have the potential for great strides in both disease modelling and future regenerative strategies.

Malformations of the human neocortex in development constitute a heterogeneous group of complex disorders, resulting in pathologies such as intellectual disability and abnormal neurological/psychiatric conditions such as epilepsy or autism. Advances in genomic sequencing and genetic techniques have allowed major breakthroughs in the field, revealing the molecular basis of several of these malformations. Here, we focus on those malformations of the human neocortex, notably microcephaly, and macrocephaly, where an underlying basis has been established at the level of the neural stem/progenitor cells (NPCs) from which neurons are directly or indirectly derived. Particular emphasis is placed on NPC cell biology and NPC markers. A second focus of this review is on experimental model systems used to dissect the underlying mechanisms of malformations of the human neocortex in development at the cellular and molecular level. The most commonly used model system have been genetically modified mice. However, although basic features of neocortical development are conserved across the various mammalian species, some important differences between mouse and human exist. These pertain to the abundance of specific NPC types and/or their proliferative capacity, as exemplified in the case of basal radial glia. These differences limit the ability of mouse models to fully recapitulate the phenotypes of malformations of the human neocortex. For this reason, additional experimental model systems, notably the ferret, non-human primates and cerebral organoids, have recently emerged as alternatives and shown to be of increasing relevance. It is therefore important to consider the benefits and limitations of each of these model systems for studying malformations of the human neocortex in development.

During mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocytes and cholangiocytes. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous progenitor population that initiate differentiation from E13.5. Recently, scRNA-seq analysis has identified sub-populations of transcriptionally distinct hepatoblasts at E11.5. Here, we show that hepatoblasts are not only transcriptionally but also functionally heterogeneous, and that a subpopulation of E9.5-E10.0 hepatoblasts exhibit a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a subpopulation constituting 2% of E9.5-E10.0 hepatoblasts that express the adult stem cell marker Lgr5, and generate both hepatocyte and cholangiocyte progeny that persist for the lifespan of the mouse. Combining lineage tracing and scRNA-seq, we show that Lgr5 marks E9.5-E10.0 bipotent liver progenitors residing at the apex of a hepatoblast hierarchy. Furthermore, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to either hepatocyte or cholangiocyte fates. Our study demonstrates functional heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a subpopulation of bipotent liver progenitors.

Malformations of the human cortex represent a major cause of disability1. Mouse models with mutations in known causal genes only partially recapitulate the phenotypes and are therefore not unlimitedly suited for understanding the molecular and cellular mechanisms responsible for these conditions2. Here we study periventricular heterotopia (PH) by analyzing cerebral organoids derived from induced pluripotent stem cells (iPSCs) of patients with mutations in the cadherin receptor-ligand pair DCHS1 and FAT4 or from isogenic knockout (KO) lines1,3. Our results show that human cerebral organoids reproduce the cortical heterotopia associated with PH. Mutations in DCHS1 and FAT4 or knockdown of their expression causes changes in the morphology of neural progenitor cells and result in defective neuronal migration dynamics only in a subset of neurons. Single-cell RNA-sequencing (scRNA-seq) data reveal a subpopulation of mutant neurons with dysregulated genes involved in axon guidance, neuronal migration and patterning. We suggest that defective neural progenitor cell (NPC) morphology and an altered navigation system in a subset of neurons underlie this form of PH.

Brain organoids have become increasingly used systems allowing 3D-modeling of human brain development, evolution, and disease. To be able to make full use of these modeling systems, researchers have developed a growing toolkit of genetic modification techniques. These techniques can be applied to mature brain organoids or to the preceding embryoid bodies (EBs) and founding cells. This review will describe techniques used for transient and stable genetic modification of brain organoids and discuss their current use and respective advantages and disadvantages. Transient approaches include adeno-associated virus (AAV) and electroporation-based techniques, whereas stable genetic modification approaches make use of lentivirus (including viral stamping), transposon and CRISPR/Cas9 systems. Finally, an outlook as to likely future developments and applications regarding genetic modifications of brain organoids will be presented.

Three-dimensional (3D) tissues grown in culture from human stem cells offer the incredible opportunity to analyze and manipulate human development, and to generate patient-specific models of disease. Methods to sequence DNA and RNA in single cells are being used to analyze these so-called 'organoid' systems in high-resolution. Single-cell transcriptomics has been used to quantitate the similarity of organoid cells to primary tissue counterparts in the brain, intestine, liver, and kidney, as well as identify cell-specific responses to environmental variables and disease conditions. The merging of these two technologies, single-cell genomics and organoids, will have profound impact on personalized medicine in the near future.

Since their recent development, organoids that emulate human brain tissue have allowed in vitro neural development studies to go beyond the limits of monolayer culture systems, such as neural rosettes. We present here a review of organoid studies that focuses on cortical wall development, starting with a technical comparison between pre-patterning and self-patterning brain organoid protocols. We then follow neocortex development in space and time and list those aspects where organoids have succeeded in emulating in vivo development, as well as those aspects that continue to be pending tasks. Finally, we present a summary of medical and evolutionary insight made possible by organoid technology.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

In light microscopy, refractive index mismatches between media and sample cause spherical aberrations that often limit penetration depth and resolution. Optical clearing techniques can alleviate these mismatches, but they are so far limited to fixed samples. We present Iodixanol as a non-toxic medium supplement that allows refractive index matching in live specimens and thus substantially improves image quality in live-imaged primary cell cultures, planarians, zebrafish and human cerebral organoids.

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena.

Organoids representing a diversity of tissues have recently been created, bridging the gap between cell culture and experiments performed in vivo Being small and amenable to continuous monitoring, they offer the opportunity to scrutinize the dynamics of organ development, including the exciting prospect of observing aspects of human embryo development live. From a physicist's perspective, their ability to self-organize - to differentiate and organize cells in space - calls for the identification of the simple rules that underlie this capacity. Organoids provide tractable conditions to investigate the effects of the growth environment, including its molecular composition and mechanical properties, along with the initial conditions such as cell number and type(s). From a theoretical standpoint, different types of in silico modeling can complement the measurements performed in organoids to understand the role of chemical diffusion, contact signaling, differential cell adhesion and mechanical controls. Here, we discuss what it means to take a biophysical approach to understanding organogenesis in vitro and how we might expect such approaches to develop in the future.

The recent increase in organoid research has been met with great enthusiasm, as well as expectation, from the scientific community and the public alike. There is no doubt that this technology opens up a world of possibilities for scientific discovery in developmental biology as well as in translational research, but whether organoids can truly live up to this challenge is, for some, still an open question. In this Spotlight article, Meritxell Huch and Juergen Knoblich begin by discussing the exciting promise of organoid technology and give concrete examples of how this promise is starting to be realised. In the second part, Matthias Lutolf and Alfonso Martinez-Arias offer a careful and considered view of the state of the organoid field and its current limitations, and lay out the approach they feel is necessary to maximise the potential of organoid technology.

Self-organization of cells is a fundamental design principle in biology, yet the inherent non-linearity of self-organizing systems often poses significant challenges in deciphering the underlying mechanisms. Here, we discuss recent progress in this respect, focusing on examples from development, regeneration and organoid differentiation. Together, these three paradigms emphasize the active material properties of tissues that result from the functional coupling between individual cells as active units. Further, we discuss the challenge of obtaining reproducible outcomes on the basis of self-organizing systems, which development and regeneration, but not the current organoid culture protocols, achieve.

The evolutionary expansion of the mammalian brain, notably the neocortex, provides a platform for the higher cognitive abilities that characterize humans. Cortical expansion is accompanied by increased folding of the pial surface, which gives rise to a gyrencephalic (folded) rather than lissencephalic (unfolded) neocortex. This expansion reflects the prolonged and increased proliferation of neural stem and progenitor cells (NPCs). Distinct classes of NPCs can be distinguished based on either cell biological criteria (apical progenitors [APs], basal progenitors [BPs]) or lineage (primary progenitors and secondary progenitors). Cortical expansion in development and evolution is linked to an increased abundance and proliferative capacity of BPs, notably basal radial glial cells, a recently characterized type of secondary progenitor derived from apical radial glial cells, the primary progenitors. To gain insight into the molecular basis underlying the prolonged and increased proliferation of NPCs and in particular BPs, comparative genomic and transcriptomic approaches, mostly for human versus mouse, have been employed and applied to specific NPC types and subpopulations. These have revealed two principal sets of molecular changes. One concerns differences in the expression of common genes between species with different degrees of cortical expansion. The other comprises human-specific genes or genomic regulatory sequences. Various systems that allow functional testing of these genomic and gene expression differences between species have emerged, including transient and stable transgenesis, genome editing, cerebral organoids, and organotypic slice cultures. These provide future avenues for uncovering the molecular basis of cortical expansion. For further resources related to this article, please visit the WIREs website.

The liver and pancreas are critical organs maintaining whole body metabolism. Historically, the expansion of adult-derived cells from these organs in vitro has proven challenging and this in turn has hampered studies of liver and pancreas stem cell biology, as well as being a roadblock to disease modelling and cell replacement therapies for pathologies in these organs. Recently, defined culture conditions have been described which allow the in vitro culture and manipulation of adult-derived liver and pancreatic material. Here we review these systems and assess their physiological relevance, as well as their potential utility in biomedicine.

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution.

Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

A rediscovery of three-dimensional culture has led to the development of organ biogenesis, homeostasis and disease models applicable to human tissues. The so-called organoids that have recently flourished serve as valuable models bridging between cell lines or primary cells grown on the bottom of culture plates and experiments performed in vivo. Though not recapitulating all aspects of organ physiology, the miniature organs generated in a dish are useful models emerging for the pancreas, starting from embryonic progenitors, adult cells, tumour cells and stem cells. This review focusses on the currently available systems and their relevance to the study of the pancreas, of β-cells and of several pancreatic diseases including diabetes. We discuss the expected future developments for studying human pancreas development and function, for developing diabetes models and for producing therapeutic cells.

A mutation in the centrosomal-P4.1-associated protein (CPAP) causes Seckel syndrome with microcephaly, which is suggested to arise from a decline in neural progenitor cells (NPCs) during development. However, mechanisms ofNPCs maintenance remain unclear. Here, we report an unexpected role for the cilium inNPCs maintenance and identifyCPAPas a negative regulator of ciliary length independent of its role in centrosome biogenesis. At the onset of cilium disassembly,CPAPprovides a scaffold for the cilium disassembly complex (CDC), which includes Nde1, Aurora A, andOFD1, recruited to the ciliary base for timely cilium disassembly. In contrast, mutatedCPAPfails to localize at the ciliary base associated with inefficientCDCrecruitment, long cilia, retarded cilium disassembly, and delayed cell cycle re-entry leading to premature differentiation of patientiPS-derivedNPCs. AberrantCDCfunction also promotes premature differentiation ofNPCs in SeckeliPS-derived organoids. Thus, our results suggest a role for cilia in microcephaly and its involvement during neurogenesis and brain size control.

Many organs such as the vasculature, kidney, lungs, pancreas and several other glands form ramified networks of tubes that either maximize exchange surfaces between two compartments or minimize the volume of an organ dedicated to the production and local delivery of a cell-derived product. The structure of these tubular networks can be stereotyped, as in the lungs, or stochastic with large variations between individuals, as in the pancreas. The principles driving stereotyped branching have attracted much attention and several models have been proposed and refined. Here we focus on the pancreas, as a model of non-stereotyped branching. In many ramified tubular organs, an important role of the mesenchyme as a source of branching signals has been proposed, including in the pancreas. However, our previous work has shown that in the absence of mesenchyme, epithelial cells seeded in vitro in Matrigel form heavily branched organoids. Here we experimentally show that pancreatic organoids grow primarily at the tips. Furthermore, in contrast to classical 'depletion of activator' mechanisms, organoids growing in close vicinity seem not to affect each other's growth before they get in contact. We recapitulate these observations in an in silico model of branching assuming a 'local inhibitor' is secreted by the epithelium. Remarkably this simple mechanism is sufficient to generate branched organoids similar to those observed in vitro, including their transition from filled spheres to a tree like structure. Quantifying the similarity between in silico and in vitro development through a normalized surface to volume ratio, our in silico model predicts that inhibition is likely to be cooperative and that the diffusing inhibitor decays within a length scale of 10-20 μm.

Cerebral organoids-3D cultures of human cerebral tissue derived from pluripotent stem cells-have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and previously unidentified interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single-cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

In vitro three-dimensional (3D) cultures are emerging as novel systems with which to study tissue development, organogenesis and stem cell behavior ex vivo. When grown in a 3D environment, embryonic stem cells (ESCs) self-organize into organoids and acquire the right tissue patterning to develop into several endoderm- and ectoderm-derived tissues, mimicking their in vivo counterparts. Tissue-resident adult stem cells (AdSCs) also form organoids when grown in 3D and can be propagated in vitro for long periods of time. In this Review, we discuss recent advances in the generation of pluripotent stem cell- and AdSC-derived organoids, highlighting their potential for enhancing our understanding of human development. We will also explore how this new culture system allows disease modeling and gene repair for a personalized regenerative medicine approach.

The endoderm gives rise to diverse tissues and organs that are essential for the homeostasis and metabolism of the organism: the thymus, thyroid, lungs, liver and pancreas, and the functionally diverse domains of the digestive tract. Classically, the endoderm, the 'innermost germ layer', was in the shadow of the ectoderm and mesoderm. However, at a recent Keystone meeting it took center stage, revealing astonishing progress in dissecting the mechanisms underlying the development and malfunction of the endodermal organs. In vitro cultures of stem and progenitor cells have become widespread, with remarkable success in differentiating three-dimensional organoids, which - in a new turn for the field - can be used as disease models.

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.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells (1). The whole embryonic organ can be cultured at multiple stages of development (2-4). These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.

In the context of a cellular therapy for diabetes, methods for pancreatic progenitor expansion and subsequent differentiation into insulin-producing beta cells would be extremely valuable. Here we establish three-dimensional culture conditions in Matrigel that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the medium composition we generate either hollow spheres, which are mainly composed of pancreatic progenitors, or complex organoids that spontaneously undergo pancreatic morphogenesis and differentiation. The in vitro maintenance and expansion of pancreatic progenitors require active Notch and FGF signaling, thus recapitulating in vivo niche signaling interactions. Our experiments reveal new aspects of pancreas development, such as a community effect by which small groups of cells better maintain progenitor properties and expand more efficiently than isolated cells, as well as the requirement for three-dimensionality. Finally, growth conditions in chemically defined biomaterials pave the way for testing the biophysical and biochemical properties of the niche that sustains pancreatic progenitors.

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.