Hans Clevers discusses the value of organoids as an experimental platform for understanding the biology of novel viruses.
Stem cells from development to the clinic
Advances in technologies that help to characterize stem cells, including genetic and epigenetic properties and lineage trajectories, have increased our knowledge about their physiological roles and contributions to development, ageing, regeneration and disease. Stem cells, and cells differentiated from them, are now used in vitro and in vivo in a variety of applications, such as disease modelling, drug screening and for transplantations.
Clinical Innovation & Regeneration
Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients
Genetic engineering prevents immune rejection of allogeneic cell transplants derived from iPSCs.
Expression of ETV2 in human cortical organoids induces the formation of vascular-like networks, which reduces cell death within organoids and increases their functional maturation.
Skin organoids generated in vitro from human pluripotent stem cells form complex, multilayered skin tissue with hair follicles, sebaceous glands and neural circuitry, and integrate with endogenous skin when grafted onto immunocompromised mice.
A set of five small molecules can induce the transformation of fibroblasts into rod photoreceptor-like cells, which can partially restore pupil reflex and visual function when transplanted into a rod degeneration mouse model.
‘Tubuloids’ grown from human kidney tissue and urine aid the study of BK virus infection, Wilms tumors and cystic fibrosis.
Pig embryos with a human endothelium are generated through blastocyst complementation using human induced pluripotent stem cells.
Human cerebral organoids undergo vascularization and maturation in the mouse brain.
Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells
Nair et al. report the generation of human ESC-derived mature and functional β cells in vitro with a culture system including a step to induce clustering of immature β-like cells.
Single-cell reconstruction of the adult human heart during heart failure and recovery reveals the cellular landscape underlying cardiac function
Wang, Yu, Zhou, Song et al. profile cardiomyocytes and neighbouring cells from healthy adults and patients with heart failure and in recovery, and delineate their cellular compositions and interaction networks.
Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening
Boretto et al. demonstrate that organoids derived from patients with various types of endometrial pathologies can model disease traits and diversity, and can be used as a drug-screening tool.
Cardiac organoids incorporating an oxygen-diffusion gradient and stimulated with the neurotransmitter noradrenaline can model the structure and function of the human heart after myocardial infarction.
iPSC-derived motor neurons and skeletal muscle cells are co-cultured to establish a model of the human neuromuscular junction (NMJ) within a microfluidic device, which facilitates assessment of axonal outgrowth, NMJ formation and muscle maturation.
Vascular progenitors generated from tankyrase inhibitor-regulated naïve diabetic human iPSC potentiate efficient revascularization of ischemic retina
hPSCs in culture acquire a more naïve pluripotent state upon tankyrase inhibition. Here, the authors show that tankyrase inhibitor-regulated naïve hiPSCs from diabetic donors generate more vascular progenitors and more efficient engraftment into mouse retina than conventional PSCs.
Single cell transcriptomics identifies stem cell-derived graft composition in a model of Parkinson’s disease
What happens to cells on engrafting into the brain in animal models to treat Parkinson’s disease is unclear. Here, the authors use scRNA-seq to examine ventral midbrain (VM)-patterned human embryonic stem cells after functional maturation in a pre-clinical rat model for Parkinson’s disease and identify perivascular-like cells.
A Wnt-mediated transformation of the bone marrow stromal cell identity orchestrates skeletal regeneration
Bone marrow stromal cells (BMSCs) lining sinusoidal blood vessels are mesenchymal cells whose function is critical for the skeleton. Here the authors show that quiescent CXCL12-expressing BMSCs can convert into a skeletal stem cell-like state, and differentiate into cortical bone osteoblasts only in response to injury.
Dual stem cell therapy synergistically improves cardiac function and vascular regeneration following myocardial infarction
Myocardial infarction causes damage to the myocardium and vasculature. Here the authors show in a rat model of myocardial infarction that cardiomyocytes derived from human induced pluripotent stem cells combined with a human mesenchymal stem cell-loaded patch lead to improved cardiac function and promote vessel formation.
Integrin-specific hydrogels modulate transplanted human bone marrow-derived mesenchymal stem cell survival, engraftment, and reparative activities
Mesenchymal stromal cells enhance bone and cartilage repair, but are limited by poor survival and retention after transplantation. Here, the authors show that synthetic hydrogels presenting integrin-specific peptides enhance the survival and persistence of human mesenchymal stromal cells after transplant, as well as bone repair.
Primary stem cells have long been used therapeutically for applications such as bone marrow transplantation. This Review discusses how cell-engineering approaches are enabling the development of next-generation stem cell therapies with improved function, specificity and responsiveness, thereby expanding their applications into areas such as delivering drugs and oncolytic viruses to tumours and promoting tissue repair in various diseases.
Single-cell transcriptomic technologies are transforming our understanding of cellular diversity and function in health and disease. This Review discusses how these technologies have been applied in hepatology, advancing our understanding of cellular heterogeneity and providing novel insights into liver biology such as metabolic zonation and the mechanisms underpinning liver regeneration.
Organoid technology has emerged as a powerful method for studying gastrointestinal cancers. This Review describes organoid models of gastrointestinal cancers, such as colorectal and liver cancer, and discusses how they can be used in basic and translational research in fields such as drug discovery and personalized medicine.
The capacity to regenerate tissue varies across different species and tissue types. The poor regenerative capacity of organs such as the heart and nervous system contributes to the aetiology of a number of serious diseases, including heart failure and Alzheimer disease. In this Review, Goldman and Poss discuss how genetic programmes of regeneration are regulated and how the control mechanisms might be adapted to treat human disease.
Kidney organoids have the potential to advance the field of nephrology. Here, the author discusses progress in the development of kidney organoids and describes remaining challenges to the use of these cultures for the study of kidney physiology and disease.
Chien et al. reflect on lessons learned following the recent announcement that 31 papers from the Anversa laboratory on cardiac cell therapy are being retracted.
Methods & Computational approaches
Fast and efficient generation of knock-in human organoids using homology-independent CRISPR–Cas9 precision genome editing
Artegiani, Hendriks et al. describe a CRISPR–Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.
An electronic interface with 4,096 electrodes can intracellularly record postsynaptic potentials and action potentials from thousands of connected mammalian neurons in vitro.
Unravelling cellular relationships during development and regeneration using genetic lineage tracing
Novel methods for tracking the progeny of single cells involve prospective lineage tracing, in which DNA barcodes are introduced into single cells and tracked over time, or retrospective lineage tracing, in which somatic mutations are used as DNA barcodes. These methods improve our understanding of cell fates in development, cell differentiation and tissue regeneration.
Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis
Transcriptional and epigenomic profiling of osteoblast and adipocyte differentiation shows that adipogenesis is driven by de novo activation of enhancers, whereas osteogenesis involves preestablished enhancers and depends on the activation of pro-osteogenic and antiadipogenic transcription factors.
Single-cell RNA sequencing is used to generate a dataset covering all major human organs in both adult and fetal stages, enabling comparison with similar datasets for mouse tissues.
Single-cell RNA sequencing clarifies the development and specification of neurons in the human cortex and shows that cell stress impairs this process in cortical organoids.
Juxtaposition of region-specific gut spheroids derived from human pluripotent stem cells in the absence of extrinsic factors results in development of segregated hepato-biliary-pancreatic anlages that recapitulate early morphogenetic events.
CRISPR screens in a 3D spheroid cancer model system more accurately recapitulate cancer phenotypes than existing 2D models and were used to identify carboxypeptidase D, acting via the IGF1R, as a 3D-specific driver of cancer growth.
A new computational framework, novoSpaRc, leverages single-cell data to reconstruct spatial context for cells and spatial expression across tissues and organisms, on the basis of an organization principle for gene expression.
Supplying forebrain organoids with a developmental signaling gradient induces topographical patterning along dorso-ventral and antero-posterior axes.
An advanced CellNet pipeline models the dynamics of stem cell differentiation.
Long-term, in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution
Yue, Zong, Li, Li, Zhang, Wu et al. introduce an in toto live-imaging system to track cardiac ventricle chamber formation at single-cell resolution for up to 1.5 days and digitally reconstruct cell dynamics.
The authors generate 3D brain organoids containing oligodendrocytes, astrocytes, and neurons derived from human pluripotent stem cells. These human oligodendrocytes are transcriptionally similar to primary cells and mature to myelinate axons.
Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards
Human pluripotent stem cell derived therapies can have serious safety risks. Here the authors design two drug inducible genetic safeguards to deplete undifferentiated hPSCs and hPSC-derived cell types.
A single-progenitor model as the unifying paradigm of epidermal and esophageal epithelial maintenance in mice
Understanding how cells maintain tissues is challenging. Here, the authors present a single consistent quantitative approach to analyse cell proliferation and lineage tracing data, which shows a single proliferating cell population that maintains epidermal and esophageal epithelial homeostasis.
The signals regulating the establishment and maintenance of the pluripotent epiblast in human embryos are unclear. Here, the authors use a bioinformatics approach to identify the role of IGF1 in human embryo development, and from this, propose a culture medium with IGF1 together with Activin to sustain hESCs in the absence of FGF.
Understanding developmental trajectories has recently been enabled by progress in modern lineage-tracing methods that combine genetic lineage analysis with omics-based characterization of cell states (particularly transcriptomes). In this Review, Wagner and Klein discuss the conceptual underpinnings, experimental strategies and analytical considerations of these approaches, as well as the biological insights gained.
Organoids are 3D structures derived from stem cells that recapitulate some key characteristics of real organs. The authors review recent progress in organoid derivation and applications and outline how advances in other disciplines might lead to more physiologically relevant organoids.
This Review discusses how stem cell bioengineering can advance regenerative medicine by giving insight into the design principles that underlie different levels of stem cell systems — from the inner circuitry in single cells and the stem cell niche to systemic interactions between organs and tissues.
In this Perspective, Lea and Niakan describe advances in CRISPR/Cas9 genome editing techniques and discuss ethical questions and potential clinical implications of this technology.
Harnessing DNA repair pathways in genome editing In this Review, Yeh, Richardson and Corn discuss the DNA repair pathways that underlie genome editing and recent improvements and strategies to yield desired genomic alterations in cells and organisms.
Embryogenesis & Development
Matrigel is widely used for cell culture. However, its ill-defined composition, batch-to-batch variability and animal-derived nature lead to experimental uncertainty and a lack of reproducibility. In this Review, we discuss the limitations of Matrigel and highlight synthetic alternatives for stem-cell culture, regenerative medicine and organoid assembly.
This protocol describes how to differentiate human induced pluripotent stem cells into oogonia in vitro. It is suitable for investigating the mechanisms of human primordial germ cell specification and epigenetic reprogramming.
Organoids are 3D cell culture systems that mimic the structural and functional characteristics of organs. In this Review, the authors discuss the biochemical and mechanical material properties relevant for organoid formation and highlight materials designed with the aim to establish organoid cultures as powerful research platforms.
Single-cell RNA sequencing and spatial transcriptomics reveal that the somitogenesis clock is active in mouse gastruloids, which can be induced to generate somites with the correct rostral–caudal patterning.
Human gastruloids—three-dimensional aggregates derived from human embryonic stem cells—show features of human embryos at around 19–21 days, and provide a model for the study of early human development.
Self-organizing neuruloids model developmental aspects of Huntington’s disease in the ectodermal compartment
Micropatterning generates the four ectodermal cell types of human neurulation.
Modeling neural tube development by differentiation of human embryonic stem cells in a microfluidic WNT gradient
Patterning of the human neural tube is modeled in a microfluidic device.
Genome-scale screens identify JNK–JUN signaling as a barrier for pluripotency exit and endoderm differentiation
CRISPR screens identify JNK–JUN family genes as repressors of definitive endoderm differentiation in human pluripotent stem cells. JUN co-occupies stem cell enhancers with OCT4, NANOG, SMAD2 and SMAD3 and inhibits the exit from pluripotency.
Chromatin interaction analysis identifies PRC2-bound silencers in mESCs, which, when deleted in mice, can lead to developmental phenotypes. Silencers in pluripotent cells can transition into active tissue-specific enhancers during development.
Maternal SETD2 deficiency leads to loss of H3K36me3, aberrant DNA methylation and ectopic H3K4me3 and H3K27me3 in mouse oocytes. Maternal depletion of SETD2 causes oocyte defects and subsequent zygotic arrest.
Simunovic et al. use human embryonic stem cells to generate a three-dimensional model of a human pre-gastrulation epiblast and show that anterior–posterior symmetry breaking can be induced by BMP4 and WNT signalling.
Eomes and Brachyury control pluripotency exit and germ-layer segregation by changing the chromatin state
The T-box factors Eomes and Brachyury activate mesoderm and endoderm programs by establishing accessible chromatin at mesoderm and endoderm enhancers, and bind and repress enhancers of pluripotency and neuroectoderm genes.
A modified brain-organoid culture generates extensive axon outgrowth with specific tract-like patterns. Organoid tracts connect neurons across distant sites and can innervate and stimulate co-cultured mouse spinal cord tissue to elicit muscle contractions.
Morphogenesis of extra-embryonic tissues directs the remodelling of the mouse embryo at implantation
How the shape of the pre-implantation murine embryo changes dramatically upon implantation is unclear. Here, the authors use live imaging with a cdx2-GFP reporter line in combination with loss of function experiments to demonstrate that FGF signalling mediated trophectoderm morphogenesis orchestrates this process.
The identity of the earliest murine in vivo lung epithelial progenitors (marked by NKX2-1 expression) is unclear. Here, the authors use single-cell RNA sequencing to define the genetic program of these lung primordial progenitors, which will improve in vitro lung specification of pluripotent stem cells.
Silencing of maternal hepatic glucocorticoid receptor is essential for normal fetal development in mice
Matthew Quinn et al showed diminished hepatic stress responses in pregnant mice due to epigenetic-mediated decreases in glucocorticoid receptor expression. This decrease is necessary for fetal development and highlights the importance of understanding the tissue-specific effects of glucocorticoid activation in models of maternal stress.
Naoko Kogata et al. generated murine mammary progenitor cell lines that form spheres and secrete milk upon hormonal stimulation. Deletion of Sox9 increased the ability of these cells to forms spheres but decreased milk production induced by lactogenic stimuli, consistent with the role of this transcription factor on maintaining the stem cell state.
This Review summarizes our current understanding of the molecular, genetic and epigenetic regulation of normal placentation, largely based on insights from the mouse model, and outlines the new opportunities provided by recent successes in deriving human trophoblast stem cells.
The respiratory system comprises multiple cell types, and consequently its development and regeneration involves intricate cellular crosstalk. Better understanding of these complex cellular interactions will improve the treatment of respiratory diseases and tissue repair after injury.
The 3D organization of the genome is crucial for gametogenesis, embryogenesis and cell differentiation through its modulation of transcription, DNA replication and cell division. Recent studies have highlighted the roles of 3D chromatin dynamics, such as the formation of enhancer–promoter interactions in mammalian development.
Stem Cells & Ageing
Mass cytometry in combination with a thiol-reactive barcoding strategy allows analysis and comparison of cell-type-specific signaling networks in organoids.
Tumor-reactive T cells are generated by coculturing tumor organoids and autologous peripheral blood lymphocytes and are evaluated for their capacity to carry out effector functions after recognition of tumor cells and whether they kill tumor organoids.
Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization
Baccin, Al-Sabah, Velten et al. use single-cell and spatially resolved transcriptomics to map the cellular, molecular and spatial organization of the endosteal, sinusoidal and arteriolar bone marrow niches.
Stress induces hair greying in mice through depletion of melanocyte stem cells, which is mediated by the activation of sympathetic nerves rather than through immune attack or adrenal stress hormones.
MLLT3 is identified as a crucial regulator of the self-renewal of human haematopoietic stem cells, and helps to maintain an active chromatin state in haematopoietic stem-cell regulatory genes during culture.
A dual genetic strategy enables the labelling and in vivo imaging of native long-term haematopoietic stem cells in the mouse calvarial bone marrow.
Epicardial cells derived from human embryonic stem cells augment cardiomyocyte-driven heart regeneration
Cell therapy in a rat model of heart attack is enhanced by stem-cell-derived epicardium.
Markers that distinguish pancreatic and hepatic progenitors improve stem-cell differentiation to pancreatic β cells.
NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells
A new method generates astrocytes from human pluripotent stem cells in 5 days.
Generation of pancreatic β cells from stem cells is enhanced by manipulating the cytoskeleton.
A Tppp3+Pdgfra+ tendon stem cell population contributes to regeneration and reveals a shared role for PDGF signalling in regeneration and fibrosis
Using single-cell transcriptomics and in vivo injury models, Harvey et al. identify a Tppp3+Pdgfra+ stem cell population in the tendon sheath and demonstrate the role of PDGFRα signalling in regeneration and fibrosis.
Saçma, Pospiech and co-workers show that sinusoidal niches are uniquely preserved on ageing, that they are the predominant niche for label-retaining (LR)-HSCs in aged mice and display higher reconstitution capacity compared with non-LR HSCs.
Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells
Short- and long-term cultures of human stem-cell-derived neurons reveal that a pattern of restricted selection of clustered protocadherin isoforms, pre-established in pluripotent cells, distinguishes immature from mature neurons.
Human stem cell-derived microglia integrate into mouse brain, displaying transcriptome signatures of microglia directly isolated from human brain and providing a chimeric model to study human-specific aspects of Alzheimer’s disease and other brain diseases.
This protocol enables isolation of viable and pure stem cell populations from muscle, and describes how to transplant the cells and follow repopulation and differentiation potential in vivo.
In utero exposure to acetaminophen and ibuprofen leads to intergenerational accelerated reproductive aging in female mice
Moïra Rossitto et al. find that exposure of pregnant mice to acetaminophen and ibuprofen leads to delayed meiosis in the germline of female offspring and accelerated ovarian aging in F2 females. Their results suggest the use of these analgesics in pregnancy may adversely affect female reproductive health spanning multiple generations.
Wilkinson and colleagues discuss haematopoietic stem cell (HSC) self-renewal in mice and humans. Experimental techniques for assaying HSC self-renewal are addressed, along with biological mechanisms regulating HSC self-renewal in vivo and ex vivo, and the therapeutic implications of this understanding.
Ageing is characterized by the functional decline of tissues and organs and increased risk of ageing-associated disorders, and this decline is associated with epigenetic changes. Recently, ‘rejuvenating’ interventions, such as metabolic manipulation, partial cell reprogramming, heterochronic parabiosis and senescent cell ablation, have been proposed to extend healthspan and lifespan by modulating the epigenome.