Organoid technologies offer unique insights into the biological processes of the tissues they mimic and are being developed at a rapid pace. Here, we introduce a Collection of content from across the Nature Journals, outlining recent progress and challenges in the organoid field.
Organoids are cell-derived in vitro 3D organ models and allow the study of biological processes, such as cell behaviour, tissue repair and response to drugs or mutations, in an environment that mimics endogenous cell organisation and organ structures. Starting as a major technological breakthrough they are now firmly established as an essential tool in biological research and also have important implications for clinical use. A major advantage is that organoids can be grown from a limited supply of starting material, e.g. biopsies, and used for drug screening to develop individual therapies. They have further shown potential in the modelling of diseases, gene editing and transplantations and not least helped to answer many important biological questions. Efforts are underway to setup cryopreserved biobanks of human organoids as a resource for researchers and clinicians.
Editorial and comment
Current advances in biotechnology open up unprecedented possibilities to transform human tissues into complex, valuable tissue products, such as organoids. Here, we propose consent for governance as a leading paradigm for the derivation, storage and use of complex human tissue products to ensure adjustment to changing ethical requirements.
Advances in stem cell research offer unprecedented insights into human biology and opportunities for clinical translation. They also raise many questions with social and ethical implications.
Nature journals formalize ethics standards for human-embryo and stem-cell papers.
Three-dimensional brain organoid models have come into the spotlight as in vitro tools to recapitulate complex features of the brain. Four recent papers now leverage current technologies to generate new flavours of brain organoids and address aspects of brain biology which, to date, have been challenging to explore.
Organoids are a powerful tool to study both physiological and disease processes. A completely synthetic matrix assembled from exchangeable modular parts has been developed and not only supports proliferation of human intestinal organoids derived from pluripotent embryonic stem cells, but also augments subsequent ad vivo implantation into injured murine colon.
Difficult questions will be raised as models of the human brain get closer to replicating its functions, explain Nita A. Farahany, Henry T. Greely and 15 colleagues.
Single-cell analyses in cancer are limited by the small biomass of individual cells. In vitro production of 3D organoid structures from single tumour-derived cells generates sufficient biomass for in-depth analyses.
Transplantation of cerebral organoids into the mouse brain overcomes some limitations of in vitro systems.
Inner ear organoids will facilitate disease studies and drug screening.
In an article published recently in Nature Medicine, the authors generate organoid models of liver neoplasia. In doing so, they highlight both the diversity of current organoid methodologies and their application to cancer modeling and therapeutics discovery.
Cruz-Acuña et al. develop synthetic hydrogels that support the generation and expansion of viable human intestinal organoids from pluripotent stem cells and can be used as injectable vehicles for organoid engraftment and wound healing.
Leushacke et al. provide insights into the role of Lgr5 cells in the oxyntic stomach, demonstrating that they label a subpopulation of chief cells that function as reserve stem cells during regeneration and cells-of-origin of gastric cancer.
Chen et al. generate lung bud organoids from human pluripotent stem cells that recapitulate early lung development, such as branching airway formation and early alveolar structures, which could potentially be used to model lung disease.
Turco et al. derive long-term genetically stable organoids from normal endometrium and the decidua that recapitulate characteristics of in vivo uterine glands, respond to hormones and differentiate into secretory and ciliated endometrial cells.
Direct reprogramming of fibroblasts into renal tubular epithelial cells by defined transcription factors
Kaminski et al. demonstrate that combined expression of the transcription factors Emx2, Hnf1b, Hnf4a and Pax8 converts mouse and human fibroblasts into induced renal tubular epithelial cells.
Organoids derived from individual cells from colorectal cancers and adjacent normal tissue are used to investigate intra-tumour diversification at the genomic, epigenetic and functional levels.
Some types of colon tumour are considered immunologically cold owing to their limited response to immunotherapy. Here, the authors model metastatic colorectal tumours using compound genetic mouse models and organoid transplantation and find that their immunogenicity is at least partly regulated by TGFβ signalling in the tumour microenvironment. Stromal-derived TGFβ seems to regulate T-cell differentiation and exclude immune infiltration from tumours. Inhibition of TGFβ can effectively reduce the growth of metastatic colorectal cancer, and synergizes with anti-PD1 blockade, suggesting potential combination strategies for more potent immunotherapy for colorectal cancer.
The mechanisms by which interactions between different cell types influence lineage identity and cell maturation during human development are unknown. Barbara Treutlein and colleagues use single-cell RNA-sequencing to analyse the emergence of hepatocytes lineages in a three-dimensional organoid system that is based on the reconstitution of hepatic, stromal and endothelial interactions. They compare their findings in vitro with data they obtain from fetal and adult human livers, and show that hepatocytes from the organoids closely resemble fetal liver cells. Through a chemical screen, they show that the three-dimensional system can be used to explore how signalling pathways influence endothelial network and hepatoblast formation.
Three-dimensional cellular models of the human brain, or organoids, enable the in vitro study of cerebral development and disease, but exactly which cells are generated and how much of the brain's complexity they recreate is undefined. To investigate in depth the nature of cells in human cerebral organoids differentiated from pluripotent stem cells, Paola Arlotta and colleagues carried out droplet-based single-cell expression analysis on cells isolated from over 30 organoids at developmental stages ranging from 3 to 9 months and beyond. They identify a wide diversity of neurons and progenitors and show that the more mature organoids formed dendritic spines as well as electrically active networks, which responded to light stimulation. The authors suggest that organoids may facilitate the study of circuit function using physiological sensory mechanisms. Elsewhere in this issue, Sergiu Paşca and colleagues show that re-assembling ventral and dorsal forebrain spheroids obtained separately in vitro allows the migration of human interneurons and the formation of functional synapses.
GABAergic neurons play important roles in brain function and are implicated in numerous psychiatric disorders. They migrate long distances from the ventral to the dorsal forebrain before integrating to cortical circuits. In vitro modelling of GABAergic neuronal differentiation during this interaction would allow us to investigate the cause of human brain disorders associated with defects in neuronal migration, but this has so far been difficult. Sergiu Paşca and colleagues have developed an approach for generating neural three-dimensional spheroids resembling either the ventral or dorsal forebrain. They show that assembling the two types of spheroids separately in vitro allows the saltatory migration of human interneurons into the cortex, as seen in human development, and the formation of functional synapses with the dorsally derived cortical glutamatergic neurons. In this context, they find that interneurons from Timothy syndrome patients exhibit perturbation in migration patterns. Elsewhere in this issue, Paola Arlotta and colleagues carried out single cell expression analysis on cells from human brain organoids to investigate the nature of cells generated by these three-dimensional models.
Epithelial organoids are being used in the laboratory to model organ development and function. So far these systems have relied on animal-derived matrices, which can be highly variable and are poorly defined, a problem that also makes them unsuitable for clinical application. Matthias Lutolf and colleagues have now designed modular synthetic hydrogen networks to support the formation of intestinal organoids from mouse and human intestinal stem cells. The authors produced dynamic matrices, initially optimal for intestinal stem cell expansion, which depends on high stiffness, and subsequently become permissive to intestinal differentiation and organoid formation through softening of their mechanical properties.
The only current treatment for cataracts, the leading cause of blindness, is to extract the damaged lens surgically and implant an artificial intraocular lens. The technique has its limitations, so there is great interest in the possibility of a regenerative medicine approach. Two papers published in this issue of Nature report advances that could bring that prospect a little closer. Kang Zhang and colleagues isolate mammalian lens epithelial stem/progenitor cells and show that Pax6 and Bmi1 are required for their renewal. They have also developed a removal procedure for cataract-affected tissue that preserves these cells, and achieved lens regeneration in rabbits, macaques and in human infants with cataracts. In the second paper, Kohji Nishida and colleagues describe a protocol for in vitro generation of a self-formed ectodermal autonomous multi-zone (SEAM) from human induced pluripotent stem cells. The SEAM includes distinct cell lineages from the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. Previous experiments had focused mainly on obtaining one cell type. These authors show that cells from the SEAM can be expanded to form a functional corneal epithelium when transplanted to an animal model of blindness.
Zika virus infection has been linked to an increase in the number of infants born with microcephaly in Brazil, but direct experimental proof that Zika virus causes birth defects was lacking. Here Alysson Muotri and colleagues show that the Brazilian Zika virus strain can cross the placenta and cause intrauterine growth restriction, including signs of microcephaly, in the SJL strain of mice. They also show that the virus can infect human brain organoids, inducing cell death by apoptosis and disrupting cortical layers.
Human cerebral organoids undergo vascularization and maturation in the mouse brain.
Engineering human brain organoids with floating scaffolds enhances the maturity and reproducibility of cortical tissue structure.
Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells
Human pluripotent stem cells are differentiated into inner ear organoids containing cells similar to hair cells and sensory neurons.
Transplantation of engineered organoids enables rapid generation of metastatic mouse models of colorectal cancer
Genetically engineered colon organoids form tumors that undergo a stepwise progression toward metastatic disease after orthotopic transplantation.
Metastatic progression of colorectal cancer is modeled in mice using in vivo genome editing and transplantation of engineered organoids.
Tumor organoids derived from the most common subtypes of primary liver cancer recapitulate the histologic and molecular features of the tissues of origin, even after long-term culture. These in vitro models, as well as those for colorectal cancer reported in Crespo et al. in a previous issue, are amenable for drug screening and allow the identification of therapeutic approaches with potential for cancer treatment.
Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids
Repair of defects in the common bile duct is hampered by a lack of healthy donor tissue. Developing human extrahepatic cholangiocyte organoids and testing them in mouse models may provide a way to overcome this limitation.
Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing
A protocol based on chemical modulation of WNT activity is used to efficiently generate colonic organoids that recapitulate the molecular features of human colon tissue. Colonic organoids generated from induced pluripotent stem cells from patients with familial adenomatous polyposis provide an in vitro platform for disease modeling and preclinical drug testing.
Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system
Organoids formed by combining pluripotent-stem-cell-derived human neural crest cells with pluripotent-stem-cell-derived intestinal tissue show functional interstitial cells of Cajal and undergo waves of contraction; these tissues reveal insights into the molecular defects characterizing Hirschsprung's disease.
Mouse tumor organoids are characterized as a model to study tumor biology and drug resistance.
This paper reports the efficient generation of human alveolar cells from induced pluripotent stem cells and their expansion in 3D culture.
The fusion of patterned cerebral organoids into more complex structures enables modeling of inter-regional processes such as neuronal migration.
Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids
This paper describes an in vitro method to generate human T cells from hematopoietic stem and progenitor cells (HSPCs). It should be useful for both basic and applied studies using T cells.
EZH2 enables germinal centre formation through epigenetic silencing of CDKN1A and an Rb-E2F1 feedback loop
The histone methyltransferase EZH2 silences genes by generating H3K27me3 marks. Here the authors use a 3D GC organoid and show EZH2 mediates germinal centre (GC) formation through epigenetic silencing of CDKN1A and release of cell cycle checkpoints.
The dynamics of progenitor cells in human neocortex development has not been studied directly. Here, the authors timelapse image human neuroepithelial (NE) and radial glial (RG) cells in embryonic brain slices and find properties of NE cells and RG that are mimicked in cerebral organoids.
Three-dimensional culture systems and organoids for mammary glands are important to understand mammary gland development. Here, the authors identify conditions (including Neuregulin 1 and R-spondin 1) that allow the culture of organoids that are responsive to hormonal stimulation for up to 2.5 months.
Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells
Sensory hair cells from the mammalian inner ear do not regenerate. Here, the authors induce direct hair cell formation from mouse embryonic stem cells using a three-dimensional culture system and observe differentiation of Type I and Type II vestibular hair cells and establishment of synapses with neurons.
Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6
There has been limited success in generating tissues from human induced pluripotent stem cells (hiPSCs). Here, the authors genetically engineer expression of the transcription factor Gata6 in a single isogenic hiPSC population resulting in complex tissue structures that exhibit liver bud-like properties.
It is difficult to generate functional human anterior pituitary tissues in vitro. Here, Ozone et al. generate human anterior pituitary from embryonic stem cells by recapitulating in vivodevelopment, and demonstrate this tissue secretes hormones and rescues hypopituitarism when grafted into mice.
Reviews and Perspectives
Barker and colleagues review the history and recent developments of organoid cultures derived from pluripotent stem cells and adult epithelia, and discuss how the technology can be used for basic research as well as translational applications.
Psychiatric disorders are difficult to model owing to their inherent complexity and heterogeneity. This Perspective focuses on the use of 3D brain organoids in modeling these disorders, considering both their advantages and their limitations.
In this Review, Drost and Clevers discuss the recent advances in organoid models of cancer and how they can be exploited to drive the translation of basic cancer research into novel patient-specific treatment regimens in the clinic.
The development of indefinitely propagating human 'mini-guts' has led to a rapid advance in gastrointestinal research. This Review highlights the uses of enteroids, colonoids and organoids in functional transport physiology studies and host–pathogen studies.
By capturing and manipulating the self-organizing capacity of pluripotent stem cells, researchers have established protocols for the production ofin vitrobrain-like 'organoids'. Di Lullo and Kriegstein evaluate approaches to organoid generation and consider their potential as models of brain development and disease.
3D organoids are valuable tools for increasing understanding of disease biology. In this Review, the authors describe how successful application of organoids into urological cancer research can further our understanding of these diseases and provide preclinical cancer models to aid precision medicine.
Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells
This protocol describes the generation of early-developing cardiac organoids from human pluripotent stem cells. Geometric confinement of the hiPSCs drives spatial organization of the cells from a 2D layer into 3D cardiac microchambers.
This protocol describes procedures for building the SpinΩ bioreactor for 3D tissue culture and differentiating human iPSCs into different brain region–specific organoids resembling developing human dorsal forebrain, midbrain and hypothalamus.
This protocol uses a three-layer system to organize rat primary testicular cells into organoids that can both establish and maintain germ cells in an environment containing a functional blood–testis barrier.
A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression
In this protocol, mouse or human colorectal cancer organoids are transplanted into the cecal epithelium. The transplanted organoids grow into a single primary tumor mass within the intestinal tract and tumor cells are capable of metastasis.
Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation
Colonoscopy-guided mucosal injection is used to induce site-directed tumors and to transplant tumors into the distal colons of mice. Tumors for engraftment are obtained from cancer organoids derived from mouse or human tissue and can be genetically modified before use.
Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture
This protocol describes the synthesis and application of hydrogel matrices comprising a poly(ethylene glycol) backbone, functionalized with cell adhesion cues and laminin-111. Uses include expanding stem cells and differentiating them into organoids.
Directed differentiation of human induced pluripotent stem cells into functional cholangiocyte-like cells
This protocol describes how to recapitulate biliary development by differentiation of hPSCs into endoderm, foregut progenitor cells, hepatoblasts, cholangiocyte progenitors and mature 3D cholangiocyte-like cell organoids.
Immuno-engineered organoids for regulating the kinetics of B-cell development and antibody production
This protocol describes how to generate mouse 3D immune organoids consisting of germinal-center-like B cells in a gelatin matrix.
This protocol describes how to differentiate human pluripotent stem cells into nephron progenitor cells with subsequent generation of 2D and 3D kidney organoids.
Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation
This protocol describes the long-term culture of liver and pancreas 3D organoids from human and mouse, and differentiation of liver organoids in vitro and in vivo. Methodology for genetic manipulation of these self-renewing organoids is also detailed.
This protocol describes stepwise differentiation of human pluripotent stem cells into 3D kidney organoids that contain segmented nephrons connected to collecting ducts, which are surrounded by renal interstitial cells and an endothelial network.
This protocol describes a strategy for generating 3D prostate organoid cultures from healthy mouse and human prostate cells (either bulk or FAC-sorted single luminal and basal cells), metastatic prostate cancer lesions and circulating tumor cells.