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.
Stem cells from development to the clinic
Recent advances in our understanding of stem cell differentiation and fate determination have enabled us to use stem cells in vitro and in vivo in a variety of applications, such as disease modelling, drug screening and for transplantations. This Collection of primary research articles, reviews and protocols from across the Nature journals involves both basic and translational research.
Research articles presented here highlight important work on stem cell differentiation, pluripotency states, lineage specification, development, genetic & epigenetic changes and metabolic regulation. They further explore how this knowledge can be applied to develop stem cell technologies that allow more detailed study of the onset of neural, intestinal, liver, lung, cardiac, muscle and blood diseases, as well as cancer. We showcase papers on the use of stem cells in the clinic to treat a genetic skin disorder and macular degeneration. In addition, the collection includes a number of peer-reviewed protocols from Nature Protocols that focus on iPSC reprogramming and the generation of organoids. Opinion and Review articles discuss the advances in this field, ethical implications of the work, as well as challenges faced by the scientific community as researchers attempt to apply the knowledge gained from basic studies in the clinic.
Disease modelling & Regeneration
Phase 1 clinical study of an embryonic stem cell–derived retinal pigment epithelium patch in age-related macular degeneration
An engineered patch of retinal pigment epithelium generated from human embryonic stem cells is transplanted into the eyes of two patients.
Patients with junctional epidermolysis bullosa (JEB) carry mutations in genes that encode components of the basement membrane, which ensures the integrity between the epidermis and the dermis, such as laminin-332. These mutations cause blistering of the skin and chronic wounds. Following initial treatment of an adult patient with a limited affected region, Michele De Luca and colleagues reconstruct the full epidermis of a 7-year-old patient with autologous transgenic cells transduced with a virus vector carrying the non-mutated form of laminin-322. The integration sites of the virus used for gene delivery provide a tracing tool ex vivo and in vivo and demonstrate that the human epidermis is sustained by a limited number of long-lived stem cells.
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.
Restoring neuronal function by implanting dopaminergic (DA) neurons derived from pluripotent stem cells in patients suffering from Parkinson's disease is a long-term goal in regenerative medicine. In a preclinical study using primate models, Jun Takahashi and colleagues show that such DA progenitors derived from human induced pluripotent stem cells exhibit long-term survival, extended neurites in the host brain and function as midbrain DA neurons when implanted in a monkey model for Parkinson's disease. The implanted cells restored a range of movements and no tumours were observed after two years.
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.
The fusion of patterned cerebral organoids into more complex structures enables modeling of inter-regional processes such as neuronal migration.
Developmental alterations in Huntington's disease neural cells and pharmacological rescue in cells and mice
The Huntington's disease (HD) induced pluripotent stem cell (iPSC) consortium describe the combined use of differentiated patient-derived iPSCs and systems biology to discover underlying mechanisms in HD. They identify neurodevelopmental deficits in HD cells that can be corrected in cells and in vivo with a small molecule.
Striatal neurons directly converted from Huntington’s disease patient fibroblasts recapitulate age-associated disease phenotypes
Direct neuronal conversion of skin fibroblasts from individuals with Huntington’s disease (HD) generates a population of medium spiny neurons that recapitulate hallmarks of HD, including aggregation of mutant huntingtin protein, DNA damage and spontaneous cell death.
Wound healing is essential to repair the skin after injury and distinct stem cells in the epidermis are known to contribute to the process. Here the authors perform molecular, functional and clonal analysis and reveal the individual contribution of stem cells coming from different epidermal compartments to the wound-healing process in mice.
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.
Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells
This protocol describes how to grow a functional and transplantable corneal epithelium and how to generate ocular-like cell lineages resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm derived from human iPS cells.
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.
2017 has witnessed major advances in gut stem cell and cancer stem cell research, delivering key insights into their regulation, more defined culture methods and novel stem cell markers that collectively drive us ever closer to breakthroughs for regenerative medicine and cancer treatment in the clinic.
Endocrine (such as diabetes) and exocrine (such as pancreatitis) disorders of the pancreas have a substantial burden worldwide. This Review explores the potential of regenerative medicine and cell-based approaches to restore both endocrine and exocrine pancreatic function, describing insights into cell replacement, implantation and reprogramming.
The stomach responds to injury via two main patterns, the superficial response and the glandular response. In this Review, Sáenz and Mills discuss cellular plasticity and reprogramming in the stomach in the context of disease (such as gastric cancer) and during repair and homeostasis.
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.
Sinoatrial node cardiomyocytes derived from human pluripotent cells function as a biological pacemaker
Cardiomyocytes derived from human stem cells act as a biological pacemaker in the rat heart.
The conflicting results of cell therapy clinical trials for heart regeneration have led to some confusion over the efficacy of this approach. This Review summarizes the main outcomes of these studies and gives perspectives for future cell-based regenerative trials largely based on the primary therapeutic target: regeneration of lost myocardium by exogenous cells or promotion of intrinsic repair though paracrine signalling.
Human induced pluripotent stem cells (hiPSCs) can be differentiated into many cardiovascular cell types, including cardiomyocytes and endothelial cells. hiPSC-derived cardiovascular cells can recapitulate patient-specific and disease-specific phenotypes. In this Review, Chen et al. discuss how hiPSCs can be used as a platform for cardiovascular drug development and disease modelling, and can facilitate individualized therapy in the era of precision medicine.
Directed differentiation and long-term maintenance of epicardial cells derived from human pluripotent stem cells under fully defined conditions
This protocol differentiates hPSCs into self-renewing epicardial cells by appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and then Gsk3 inhibitor again in a completely defined, xeno-free system.
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.
Mouse tumor organoids are characterized as a model to study tumor biology and drug resistance.
This Review discusses the roles of deregulated RNA processing, including RNA methylation, RNA editing, RNA splicing and RNA binding protein activity, in cancer stem cells and highlights the potential of these events as biomarkers and therapeutic targets.
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.
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.
Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip
An efficient and chemically defined protocol for the differentiation of human induced pluripotent stem cells into podocytes enables the recapitulation of the differential clearance of the human kidney glomerulus in an organ-on-a-chip.
Organoid cystogenesis reveals a critical role of microenvironment in human polycystic kidney disease
Tissue mimics are of great interest in understanding diseases. Here, organoids were developed that resemble polycystic kidney disease cysts and it was demonstrated how material environment and adhesion can affect cystogenesis and disease progression.
Bioengineered constructs combined with exercise enhance stem cell-mediated treatment of volumetric muscle loss
Volumetric muscle loss leads to functional muscle impairment, and current stem cell-based treatments show limited efficacy. Here, the authors generate a stem cell scaffold, implant it in mice, and show that an exercise regimen enhances innervation and restoration of muscle function in mice.
Adult muscles contain quiescent stem cells, known as satellite cells, which are activated upon injury, enabling muscle repair and replenishment of the stem cell pool. Recent studies have shed light on the molecular circuitry regulating satellite cell fate decision and the impairment of this circuitry during degenerative muscle diseases and ageing.
Advances in the derivation of pluripotent stem cells (PSCs) and their differentiation to specific cell types could have diverse clinical applications. Trounson and DeWitt provide an overview of the progress in using embryonic stem cell and induced PSC derivatives for disease treatment and discuss the potential and limitations of such approaches.
The use of cultured human pluripotent stem cells (PSCs) to model human diseases has revolutionized the ways in which we study monogenic, multigenic and epigenetic disorders, by overcoming some of the limitations of animal models. PSC-based disease models are generated using various strategies and can be used for the discovery of new drugs and therapies.
Replacement of pathogenic mitochondrial DNA in human embryos is poised for clinical application.
This protocol describes the generation of mice entirely derived from genome-edited embryonic stem cells, enabling the production of transgenic mice in a single generation.
Injection of embryonic stem cell derived macrophages ameliorates fibrosis in a murine model of liver injury
Macrophages, a type of white blood cell, when derived from embryonic stem cells in the laboratory reduce fibrosis in chronic liver disease. Lesley Forrester and colleagues from the University of Edinburgh found murine embryonic stem-cell-derived macrophages (ESDM) were morphologically similar to bone marrow-derived macrophages (BMDM), previously found to reduce fibrosis and improve liver function in mice with induced liver injury. Using a novel technique, the team found ESDM engulfed fewer particles at a slower rate than BMDM, indicating ESDM were less inflammatory. A higher dose of ESDM was required to have the same effect of BMDM to help liver fibrosis regression. However, they were more efficient in repopulating mouse livers depleted of liver-specific macrophages and also significantly improved liver function, indicating ESDM were similar to resident macrophages in the liver and had therapeutic potential.
Adult stem cells
Transplantation-based assays of haematopoietic stem cells (HSCs) and progenitors isolated on the basis of the expression of their surface markers have inferred that the haematopoietic lineage follows a tree-like structure that starts from a long-term multipotent HSC at its base and splits into a few major branches. However, recent data question the existence of this structure, instead supporting the idea that the blood lineage is sustained by several fate-restricted progenitors. Hans-Reimer Rodewald and colleagues have developed a DNA recombination locus based on the Cre–loxP system that can tag single cells using several hundred thousand barcodes. They introduce the labelling in mouse embryos and track HSCs during their life. Surprisingly, the adult HSC compartment is a mosaic of HSC clones derived from embryos and contributes with different proportion to blood lineage, some multilineage and others of restricted fates, according to a pattern that is consistent within clones. However, they define an early split of fate between myeloid erythroid and lymphocyte development which agrees with the tree-like structure.
It is generally believed that a very small number of haematopoietic stem cells (HSCs) maintain multilineage haematopoiesis by stably producing a hierarchy of short-lived progenitor cells. This theory is historically based on transplantation experiments in lethally irradiated hosts. Using a new transposon-based labelling technique that enables unique tagging of individual cells and their progeny in vivo, Fernando Camargo and colleagues now show that this might not be the case during native non-transplant haematopoiesis. The authors found that the main drivers of steady state haematopoiesis during most of adulthood are a large number of long-lived progenitors, rather than classically defined haematopoietic stem cells.
Many blood disorders can be treated with haematopoietic (blood-generating) stem cell (HSC) transplants, but such treatment does not always lead to efficient replenishment of all blood lineages. Through single-cell transplantation of HSCs in mice, Sten Eirik Jacobsen and colleagues define lineage-restricted fates of long-term self-renewing cells. They identify a class of HSC that effectively replenishes the megakaryocyte and platelet lineages over other lineages, and other HSCs that are more able to participate in megakaryocyte, erythroid and myeloid lineages despite being able to sustain lymphoid potential. Genetic lineage tracing also shows that platelet-biased HSCs are able to support unperturbed adult haematopoiesis.
Allon Klein, Merav Socolovsky and colleagues examine the emergence of distinct blood cell lineages from mouse haematopoietic progenitors. Their approach combines single-cell transcriptomics, cell fate potential assays and population balance analysis—a computational method for predicting cell fate probabilities from population snapshots. They use a new flow-cytometry strategy to sort cells with newly defined markers of erythroid differentiation and validate the findings at the single-cell level. The results show that differentiation is a continuous, albeit hierarchical, process. They also reveal that erythroid and mast cell fates are coupled, and that remodelling the expression of cell cycle regulators is very important as erythroid cells proceed to terminal differentiation.
The effect of changes in metabolite levels on stem-cell fate in vivo has been unclear. Sean Morrison and colleagues survey the metabolites of haematopoietic stem cells (HSCs) and progenitors. They show that each type of blood cell has a specific signature and that human and mouse HSCs have high levels of ascorbate, which drop during differentiation. Depletion of ascorbate in mice increases the number and function of HSCs and cooperates with a mutation associated with leukaemia to accelerate tumorigenesis. Analysis of the phenotypes indicates that ascorbate can act in a non-cell-autonomous fashion, partly by modulating the function of the tumour suppressor Tet2.
This paper describes a fully defined, nonxenogeneic in vitro niche for the differentiation of haematopoietic stem and progenitor cells to progenitor T cells in mouse and human.
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.
Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage
Gutierrez-Martinez et al. show that an impaired DNA damage response and reduced apoptotic priming in old haematopoietic stem cells (HSCs) contribute to the survival and expansion of damaged HSCs in the bone marrow of aged mice.
Velten et al. use single-cell transcriptomics and functional data to map the early lineage commitment of human haematopoietic stem cells as a continuous process of cells passing through transitory states rather than demarcating discrete progenitors.
Lineage-tracing experiments in the mouse show that Lgr6, but not Lgr5, functions as a cancer stem marker in skin squamous cell carcinomas (SCCs). The authors also show that Lgr6-knockout mice are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5.
Functional screen of MSI2 interactors identifies an essential role for SYNCRIP in myeloid leukemia stem cells
Michael Kharas and colleagues characterize the MSI2 protein interactome in leukemia cells and subsequently perform a functional screen identifying 24 genes required for leukemia in vivo. They focus on the RNA-binding protein SYNCRIP, showing that it regulates Hoxa9 and other transcripts involved in a myeloid leukemia stem cell program.
Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia
Applying a new, more sensitive single-cell transcriptomics method to diagnosis, remission and progression samples from patients with chronic myeloid leukemia reveals insight into the heterogeneity of cells that resist treatment with targeted therapy, as well as into the dynamics of disease progression and its effects on nontransformed hematopoietic stem cells.
Microenvironmental pressures in glioblastoma select for glioma stem cells (GSCs) subpopulations that are maintained through preferential activation of BMI1 and EZH2 in different niches. Given the high degree of intratumor heterogeneity, combined pharmacological inhibition of Polycomb repressive complexes targets proneural and mesenchynmal GSCs and expands lifespan in mice, warranting the therapeutic evaluation of this approach in patients with glioblastoma.
Adult stem cells are essential for the maintenance of tissue homeostasis and wound repair, but cancer can hijack their tissue regenerative functions to promote malignancy. Ge and Fuchs review recent insights into the determinants and general principles underlying stem cell plasticity under homeostasis, stress and cancer.
Stem cells are long-lived and possess unique mechanisms related to quiescence, DNA damage response and apoptosis that protect them throughout their lifespan and during tissue repair. These mechanisms may also have a role in cancer stem cells and tumorigenesis.
Notch signalling is a fundamental negative regulator of epidermal stemness. Here, the authors show that cell mechanics through YAP/TAZ activity prevent primary human keratinocytes from differentiating by inhibiting cell-autonomous Notch signals.
A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells
Individual human epidermal cells differ in their self-renewal ability. Here the authors perform genome-wide pooled RNAi screens to uncover the molecular basis for this heterogeneity, and identify genes conferring a clonal growth advantage on normal and neoplastic human epidermal cells.
This paper reports the efficient generation of human alveolar cells from induced pluripotent stem cells and their expansion in 3D culture.
This paper describes methods for the 3D culture of mouse lung progenitor cells that can differentiate in vitro and in vivo along all epithelial lineages.
Decline in stem cell function causes loss of tissue homeostasis and increased incidence of age-related diseases. During ageing, adult stem cells accumulate damage and the niche in which they reside malfunctions. These defects are associated with changes in the epigenome that contribute to organ dysfunction and disease.
The role of epigenetic regulation in adult stem cell function depends on the specific tissue and factor, but it commonly affects stem cell maintenance, self-renewal and differentiation without disrupting germ-layer fate.
Some terminally differentiated cells have the capacity to de-differentiate or transdifferentiate under physiological conditions as part of a normal response to injury. Recent insights have been gained into the role of this cell plasticity in maintaining tissue and organ homeostasis, and this has important implications for cell-based therapies.
The mechanism by which cell geometry regulates cell signalling is reported to be modulated by lipid rafts within the plasma membrane, which are now shown to be responsible for geometry-dependent mesenchymal stem cell differentiation.
A select group of bone marrow cells (BMCs) with the capacity to regenerate the heart are not all the same. Working with mouse cells, a team led by Annarosa Leri used single cell-based analytical techniques to test whether all BMCs that express a cell surface marker called c-kit possess the ability to form new heart tissue. They found that these BMCs, despite their shared expression of c-kit, were not a uniform population. Only a subset could give rise to various cell lineages in the heart. Others remained in an undifferentiated state and retained their bone marrow identity, even within the damaged heart. The findings could help explain why researchers have reported such disparate results in the past when assessing the heart repairing potential of c-kit-positive BMCs.
Co-culture of meniscal cartilage-forming cells with fat-derived stem cells can lead to enhanced cartilage matrix production when cultured under simulated microgravity. Adetola Adesida from the University of Alberta in Edmonton, Canada, and colleagues cultured two types of cells found together in the knee—cartilage-forming chondrocyte cells (taken from the meniscus) and mesenchymal stem cells (isolated from the infrapatellar fat pad)—in a rotary cell culture system designed to model weightlessness on Earth. Simulated microgravity enhanced the synergistic interaction between the two types of cells in culture, resulting in more matrix production, but it also prompted the cartilage-forming cells to differentiate towards bone-forming cells, as evidenced by gene expression analysis. These findings suggest that microgravity and simulated microgravity-based culture technologies could help bioengineers grow knee replacements for people with meniscus tears, but increased bone-directed differentiation could pose a possible problem for astronauts on prolonged missions.
Platelet-derived growth factor signaling modulates adult hair follicle dermal stem cell maintenance and self-renewal
Signals from a protein that regulates cell division are essential to maintain the stem cells that regenerate hair follicles. Jeff Biernaskie and colleagues at Canada’s University of Calgary found signals from platelet-derived growth factor (PDGF) promote self-renewal of ‘hair follicle dermal stem cells’ (hfDSCs)—cells present at the bottom of hair follicles important for their regeneration. They ‘turned off’ the gene responsible for PDGF production in hfDSCs in mice. This led to a significant reduction in the stem cells with successive hair cycles. They also tested the effects of PDGF signaling molecules on isolated hfDSCs and found they improved their ability to proliferate and to induce follicle regeneration. The results suggest disruption to PDGF signaling may contribute to hair loss. PDGF could be an important additive to rapidly expand hfDSCs ex vivo for cell-based therapies.
Reprogramming & Pluripotency
Copy number variants at particular genomic locations have been shown to arise in human pluripotent stem cells (hPSCs) under certain culture conditions, but the extent of acquired mutations in such culture remains to be determined. Kevin Eggan and colleagues surveyed the exomes of 140 human embryonic stem cell (hESC) lines, some of which are in the pipeline for clinical use.They identified mosaic mutations in the TP53 gene in a subset of cells for five unrelated hESC lines and show that the cells carrying the mutations outcompeted the non-mutant cells and could readily differentiate. Similar mutations were also identified by mining published datasets for an additional 14 hESC lines and more than 100 human induced PSC lines. The study highlights the need for in-depth characterization of cells derived from hPSCs before their use in the clinic.
The Human Induced Pluripotent Stem Cells Initiative (HipSci) has resulted in the generation, genotyping and phenotyping of more than 700 human induced pluripotent stem (iPS) cell lines derived from 300 healthy individuals. Although analysis of these data indicates that most of the variations in phenotypes between cells arise from variations between individuals, the authors also assess the consequences of the rare genetic defects that are recurrently seen in iPS cells after reprogramming and provide a map of the common regulatory variants that can change the transcriptome of human pluripotent cells. This resource will be useful for genetic studies of complex traits and cancer.
A screen in which combinatorial pairs of transcription factors are exogenously expressed in fibroblasts identifies different combinations that reprogram these cells into induced neuronal cells with diverse functional properties.
Mouse fibroblasts are reprogrammed to induced pluripotent stem cells using single-chain antibodies in place of transcription-factor genes.
Expression of the minimally polymorphic HLA-E molecule prevents NK-cell-mediated rejection of cells lacking expression of HLA-A, B and C.
Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector
Human iPSC-derived neurons are generated from individuals with or without Alzheimer's disease carrying different APOE alleles and reveal a toxic, neuron-intrinsic gain of function of the ApoE4 variant that is a strong genetic risk factor for AD.
Human ALS/FTD patient iPSC-derived neurons are used to uncover mechanisms by which C9ORF72 mutations cause neurodegeneration.
Transcription factors orchestrate dynamic interplay between genome topology and gene regulation during cell reprogramming
The authors analyze time-resolved changes in genome topology, gene expression, transcription-factor binding, and chromatin state during iPSC generation. They conclude that 3D genome reorganization generally precedes gene expression changes and that removal of locus-specific topological barriers explains why pluripotency genes are activated sequentially during reprogramming.
TET1, TET2 and TET3 triple-knockout (TKO) human embryonic stem cells (hESCs) exhibit bivalent promoter hypermethylation without a corresponding decrease in gene expression in the undifferentiated state. However, PAX6 promoter hypermethylation in TKO hESCs impairs neural differentiation.
Ernesto Guccione and colleagues report that the transcription factor PRDM15 regulates naive pluripotency in mouse embryos and embryonic stem cells and in derivation of mouse and human iPSCs. They further show that PRDM15 promotes WNT signaling and inhibits MAPK–ERK signaling by directly regulating the expression of R-spondin1 and Sprouty1, respectively.
This study identifies regulatory variants in sensory neurons derived from induced pluripotent stem cells. Despite differentiation-induced variability, an allele-specific method allowed detection of loci influencing gene expression, chromatin accessibility and RNA splicing.
Transient transcription factor expression rapidly induces a homogenous population of mature GABAergic neurons from human pluripotent stem cells, aiding the study of inhibitory neuron function and disease.
Combining each sgRNA with a unique molecular identifier in a genome-wide screen increases sensitivity and robustness in both positive and negative selection.
Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming
This study compares naive human pluripotent stem cells, either reprogrammed directly from somatic cells or converted from primed cells, under a variety of culture conditions.
Pastor et al. demonstrate a role for TFAP2C in the promotion and maintenance of human naive pluripotency by facilitating the opening of enhancers close to pluripotency factors.
Yilmaz et al. generate a genome-wide loss-of-function library using human haploid embryonic stem cells and define genes that are essential for cell survival, growth and pluripotency maintenance, as well as growth-restricting genes.
Pandya et al. describe a protocol to differentiate human and mouse iPSCs into cells with the phenotype, transcriptional profile and functional properties of microglia. The treatment of murine intracranial malignant gliomas with these cells demonstrates their potential clinical use. These microglia-like cells will enable further studies into the role of microglia in health and disease.
Chemically defined and growth-factor-free culture system for the expansion and derivation of human pluripotent stem cells
A new chemically defined culture medium for the long-term culture of human pluripotent stem cells uses only three chemical compounds and a lower number of recombinant proteins than used in commercially available media.
Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods
Epigenetic and transcriptomic differences in human induced pluripotent stem cells generated from the same fibroblast population reveals that the reprogramming method affects the cells’ gene-expression levels but not their differentiation potential.
Cell state transitions during embryonic development are associated with epigenetic changes that alter chromatin structure and gene expression. Interplay between epigenetic regulatory layers can be studied using genomic technologies and embryonic stem cell cultures that reflectin vivocell states.
In this article, the authors review the mechanisms by which the pluripotency gene regulatory network governs the acquisition, maintenance and dissolution of the pluripotent state, including the interaction of these networks with chromatin-mediated and RNA-mediated regulatory mechanisms. They discuss recent evidence for alternative pluripotency states and the factors that affect transitions between these states.
The ectopic expression of a defined set of transcription factors can experimentally reprogramme somatic cells into other cell types, including pluripotent cells. This method enables exploration of the molecular characteristics of pluripotency, cell specification, differentiation and cell fate stability, as well as their transcriptional and epigenetic regulation.
Recent advances in our understating of the molecular underpinnings of alternative primed- and naive-like pluripotent states in rodents and humans highlight potential functional benefits of naive pluripotency and identify key unanswered questions in this rapidly evolving field.
This year marks the tenth anniversary of the generation of induced pluripotent stem cells (iPSCs) by transcription factor-mediated somatic cell reprogramming. Takahashi and Yamanaka portray the path towards this ground-breaking discovery and discuss how, since then, research has focused on understanding the mechanisms underlying iPSC generation and on translating such advances to the clinic.
Regulation of pluripotency: Li and Belmonte review the pluripotency gene regulatory network, the molecular principles of pluripotency gene function, regulation by RNA-binding proteins and alternative splicing, heterogeneity and alternative pluripotency states.
Stem cells in development
The molecular mechanisms that direct early cell fate decisions in human embryos are currently unclear. Kathy Niakan and colleagues have used CRISPR–Cas9-mediated genome editing to analyse the role of the pluripotency transcription factor OCT4 during human embryogenesis, and uncover some unexpected functions. They first defined the most efficient OCT4-targeting single-cell RNA and delivery method using a combination of analysis in human embryonic stem cells and mouse embryos, before moving to donated diploid human zygotes. They find that OCT4 is required early in development to regulate the expression of genes in extra-embryonic trophectoderm, which makes up the placenta, and of pluripotent genes such as NANOG, which define the pluripotent epiblast.
Trophoblast and embryonic stem cells interact in vitro to form structures that resemble early blastocysts, and the embryo provides signals that drive early trophectoderm development and implantation.
During mammalian development, embryonic pluripotent stem cells form a cavitated epithelium at the time of implantation. Magdalena Zernicka-Goetz and colleagues show that, in spheroids made from mouse embryonic stem cells, the cells must leave their unrestricted naive pluripotent state for the events leading to cavity formation. The transcription factor Oct4 activates this exit and the expression of genes that code for proteins involved in lumenogenesis. The authors also show that these events are conserved in spheroids from human embryonic stem cells.
Kian Peng Koh and colleagues report that TET1 regulates lineage-specific genes in the mouse postimplantation embryo, many of them independently of DNA methylation changes, through regulation of JMJD8 expression. They show that Tet1 deletion causes embryonic defects, which are partially penetrant in an inbred strain but fully lethal in non-inbred mice.
LINE-1 activation after fertilization regulates global chromatin accessibility in the early mouse embryo
Maria-Elena Torres-Padilla and colleagues use a targeted epigenomic approach to investigate the role of LINE-1 retroelements during early mouse development. Their data suggest that timely activation of LINE-1 regulates global chromatin accessibility and is integral to the mouse developmental program.
Didier Trono and colleagues show that both human DUX4 and mouse Dux are expressed before zygotic genome activation (ZGA) and lead to activation of ZGA-associated genes. Dux knockout in mouse embryonic stem cells prevents cycling through a 2-cell-like state, and zygotic depletion of Dux impairs embryonic development.
Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons
Bradley Cairns, Douglas Carrell, Stephen Tapscott and colleagues transcriptionally profile human oocytes and preimplantation embryos and highlight DUX4-family proteins as activators of cleavage-stage genes and repetitive elements. They show that Dux expression converts mouse embryonic stem cells into two-cell (2C) embryo-like cells, thus suggesting mouse DUX and human DUX4 as drivers of the mammalian cleavage/2C state.
Fang et al. identify a PAX5–OCT4–PRDM1 transcriptional network that acts as a developmental switch in the transition from human pluripotent stem cells to the primordial germ cell lineage.
Is it time to reassess the 14-day rule for human embryo research?
Single-cell technologies are transforming our understanding of pre-implantation and early post-implantation development and of in vitro pluripotency. Specifically, single-cell transcriptomics and imaging and the accompanying bioinformatics methods have enabled precision interrogation of cell fate choices and cell lineage diversification, which occur at the level of the individual cell.
This protocol extension describes how to obtain functionally mature oocytes from embryonic or induced pluripotent stem cells. These oocytes can be used to produce live mouse offspring.
A deep learning approach enables fast and robust prediction of hematopoietic stem cell lineage choice in time-lapse imaging three generations before conventional marker onset.
Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation
Ibarra-Soria et al. study cellular diversity, transcriptional signatures, lineage specification and somitogenesis on a single-cell level in E8.25 mouse embryos, and reveal the regulation of blood progenitor formation by the leukotriene pathway.
Human pluripotent stem cells constitute a unique system to study the earliest stages of human embryonic haematopoiesis and the origins of human blood cell diseases, and they are an invaluable tool for the generation of haematopoietic stem and progenitor cell populations for cell-based regenerative therapies.
Previous lines of evidence have suggested that neural precursors are present in adult humans and continue to generate new neurons in the hippocampus even after full maturation. Here, Arturo Alvarez-Buylla and colleagues re-visit that concept and come to a different conclusion. Using a more comprehensive and larger set of samples of human hippocampus than those analysed in previous studies, the authors find evidence for the production of new neurons early in life, but note that hippocampal neurogenesis rates decline rapidly within the first few years of childhood. The authors were unable to detect the production of any new neurons in adults. The same patterns of neurogenesis were observed in rhesus macaques.
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.
Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing
Using single-cell RNA-seq, the authors show that early developmental neurogenesis in the dentate gyrus of the hippocampus is largely conserved in the adult, but with a perinatal transformation of stem cells to an adult type.
Mechanical cues play critical roles in embryonic development. A micropatterned neuroectoderm developmental model based on human pluripotent stem cells now reveals how morophogenetic signals such as cell shape and contractility regulate neural tissue development.
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.
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.
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.
FateID identifies fate biases in early multipotent progenitor populations from single-cell RNA-seq data.
Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring
Jiang et al. trace two embryonic fibroblast lineages in the mouse, one that does not express engrailed and mediates early dermal development and one that expresses engrailed and mediates scar tissue formation.
Long-lived unipotent Blimp1-positive luminal stem cells drive mammary gland organogenesis throughout adult life
The role of stem/progenitor cell populations in mammary gland morphogenesis is not well understood. Here, the authors show that a transcriptional repressor, Blimp1, is expressed in a rare luminal stem cell population, which contribute to duct formation, and survive multiple rounds of pregnancy and involution.
HoangDinh Huynh and Yihong Wan investigate the role of the mTORC1 pathway during osteoclastogenesis and find that the cytokine RANKL inactivates mTORC1 via calcineurin-mediated dephosphorylation, leading to activation of NFATc1 by reducing its phosphorylation. These findings have implications for bone diseases and mTORC1/NFATc1 signaling.
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.