ABSTRACT
A human embryo's legal definition and its entitlement to protection vary greatly worldwide. Recently, human pluripotent stem cells have been used to form in vitro models of early embryos that have challenged legal definitions and raised questions regarding their usage. In this light, we propose a refined legal definition of an embryo, suggest "tipping points" for when human embryo models could eventually be afforded similar protection to that of embryos, and then revisit basic ethical principles that might help to draft a roadmap for the gradual, justified usage of embryo models in a manner that aims to maximize benefits to society.
Subject(s)
Embryo Research , Embryo, Mammalian , Humans , Pluripotent Stem Cells , Embryo Research/ethicsABSTRACT
The segmented body plan of vertebrates is established during somitogenesis, a well-studied process in model organisms; however, the details of this process in humans remain largely unknown owing to ethical and technical limitations. Despite recent advances with pluripotent stem cell-based approaches1-5, models that robustly recapitulate human somitogenesis in both space and time remain scarce. Here we introduce a pluripotent stem cell-derived mesoderm-based 3D model of human segmentation and somitogenesis-which we termed 'axioloid'-that captures accurately the oscillatory dynamics of the segmentation clock and the morphological and molecular characteristics of sequential somite formation in vitro. Axioloids show proper rostrocaudal patterning of forming segments and robust anterior-posterior FGF-WNT signalling gradients and retinoic acid signalling components. We identify an unexpected critical role of retinoic acid signalling in the stabilization of forming segments, indicating distinct, but also synergistic effects of retinoic acid and extracellular matrix on the formation and epithelialization of somites. Comparative analysis demonstrates marked similarities of axioloids to the human embryo, further validated by the presence of a Hox code in axioloids. Finally, we demonstrate the utility of axioloids for studying the pathogenesis of human congenital spine diseases using induced pluripotent stem cells with mutations in HES7 and MESP2. Our results indicate that axioloids represent a promising platform for the study of axial development and disease in humans.
Subject(s)
Body Patterning , Cell Culture Techniques, Three Dimensional , Somites , Humans , Body Patterning/drug effects , Extracellular Matrix/metabolism , Fibroblast Growth Factors/metabolism , In Vitro Techniques , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Mutation , Somites/cytology , Somites/drug effects , Somites/embryology , Somites/metabolism , Spinal Diseases/pathology , Tretinoin/metabolism , Tretinoin/pharmacology , Wnt Signaling Pathway/drug effectsABSTRACT
The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.
Subject(s)
Body Patterning , Gastrula/cytology , Human Embryonic Stem Cells/cytology , Organoids/cytology , Organoids/embryology , Somites/cytology , Somites/embryology , Body Patterning/genetics , Gastrula/embryology , Gastrula/metabolism , Gene Expression Regulation, Developmental , Humans , In Vitro Techniques , Organoids/metabolism , Signal Transduction , Somites/metabolism , TranscriptomeABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner.
Subject(s)
Gastrula , Mouse Embryonic Stem Cells/cytology , Organoids/cytology , Organoids/embryology , Single-Cell Analysis , Somites/cytology , Somites/embryology , Transcriptome , Animals , Collagen , Drug Combinations , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Development , Female , Gastrula/cytology , Gastrula/embryology , Gastrula/metabolism , Gene Expression Regulation, Developmental , Laminin , Male , Mice , Mouse Embryonic Stem Cells/metabolism , Organoids/metabolism , Proteoglycans , RNA-Seq , Somites/metabolism , Time FactorsABSTRACT
Gastrulation is a key milestone in the development of an organism. It is a period of cell proliferation and coordinated cellular rearrangement, that creates an outline of the body plan. Our current understanding of mammalian gastrulation has been improved by embryo culture, but there are still many open questions that are difficult to address because of the intrauterine development of the embryos and the low number of specimens. In the case of humans, there are additional difficulties associated with technical and ethical challenges. Over the last few years, pluripotent stem cell models are being developed that have the potential to become useful tools to understand the mammalian gastrulation. Here we review these models with a special emphasis on gastruloids and provide a survey of the methods to produce them robustly, their uses, relationship to embryos, and their prospects as well as their limitations.
ABSTRACT
Technical and ethical limitations create a challenge to study early human development, especially following the first 3 weeks of development after fertilization, when the fundamental aspects of the body plan are established through the process called gastrulation. As a consequence, our current understanding of human development is mostly based on the anatomical and histological studies on Carnegie Collection of human embryos, which were carried out more than half a century ago. Due to the 14-day rule on human embryo research, there have been no experimental studies beyond the fourteenth day of human development. Mutagenesis studies on animal models, mostly in mouse, are often extrapolated to human embryos to understand the transcriptional regulation of human development. However, due to the existence of significant differences in their morphological and molecular features as well as the time scale of their development, it is obvious that complete knowledge of human development can be achieved only by studying the human embryo. These studies require a cellular framework. Here we summarize the cellular, molecular, and temporal aspects associated with human gastrulation and discuss how they relate to existing human PSCs based models of early development.
Subject(s)
Embryo, Mammalian/metabolism , Gastrulation , Animals , Body Patterning , Embryo, Mammalian/cytology , Humans , Models, AnimalABSTRACT
The EMBO-EMBL Symposium 'Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture' was held in Heidelberg, Germany, in March 2019, with 150 participants seeking to reverse-engineer embryogenesis, emphasizing quantitative simulation and the use of synthetic systems to test models. This highly dynamic, interdisciplinary mix of quantitative developmental genetics, bioengineering, synthetic biology and artificial life aimed to reveal how evolution exploits physical forces and genetics to implement the cell- and tissue-level decision-making required for complex morphogenesis.
Subject(s)
Bioengineering/organization & administration , Congresses as Topic , Developmental Biology/methods , Genetic Engineering , Animals , Bioengineering/methods , Bioengineering/trends , Congresses as Topic/organization & administration , Congresses as Topic/standards , Developmental Biology/organization & administration , Developmental Biology/trends , Embryonic Development/physiology , Gene Regulatory Networks/physiology , Genetic Engineering/methods , Genetic Engineering/trends , Germany , Humans , Models, Biological , Morphogenesis/physiology , Synthetic Biology/methods , Synthetic Biology/organization & administration , Synthetic Biology/trendsABSTRACT
The caudal lateral epiblast of mammalian embryos harbours bipotent progenitors that contribute to the spinal cord and the paraxial mesoderm in concert with the body axis elongation. These progenitors, called neural mesodermal progenitors (NMPs), are identified as cells that co-express Sox2 and T/brachyury, a criterion used to derive NMP-like cells from embryonic stem cells in vitro However, unlike embryonic NMPs, these progenitors do not self-renew. Here, we find that the protocols that yield NMP-like cells in vitro initially produce a multipotent population that, in addition to NMPs, generates progenitors for the lateral plate and intermediate mesoderm. We show that epiblast stem cells (EpiSCs) are an effective source of these multipotent progenitors, which are further differentiated by a balance between BMP and Nodal signalling. Importantly, we show that NMP-like cells derived from EpiSCs exhibit limited self-renewal in vitro and a gene expression signature like their embryonic counterparts.
Subject(s)
Body Patterning/physiology , Germ Layers/cytology , Neural Stem Cells/metabolism , Animals , Body Patterning/genetics , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Lineage , Germ Layers/metabolism , Mesoderm/cytology , Mesoderm/metabolism , Neural Stem Cells/cytology , SOXB1 Transcription Factors/metabolismABSTRACT
The mammalian embryo's caudal lateral epiblast (CLE) harbours bipotent progenitors, called neural mesodermal progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here, we performed a single cell analysis of different in vitro NMP populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them with E8.25 CLE mouse embryos. In our analysis of this region, our findings challenge the notion that NMPs can be defined by the exclusive co-expression of Sox2 and T at mRNA level. We analyse the in vitro NMP-like populations using a purpose-built support vector machine (SVM) based on the embryo CLE and use it as a classification model to compare the in vivo and in vitro populations. Our results show that NMP differentiation from ESCs leads to heterogeneous progenitor populations with few NMP-like cells, as defined by the SVM algorithm, whereas starting with EpiSCs yields a high proportion of cells with the embryo NMP signature. We find that the population from which the Epi-NMPs are derived in culture contains a node-like population, which suggests that this population probably maintains the expression of T in vitro and thereby a source of NMPs. In conclusion, differentiation of EpiSCs into NMPs reproduces events in vivo and suggests a sequence of events for the emergence of the NMP population.
Subject(s)
Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Germ Layers/cytology , Mesoderm/embryology , Pluripotent Stem Cells/cytology , Stem Cells/cytology , Animals , Body Patterning/genetics , Cell Differentiation , Cell Lineage , Female , Gene Expression Profiling , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/metabolism , In Vitro Techniques , Male , Mice , Neural Stem Cells/cytology , Spinal Cord/embryology , Support Vector Machine , TranscriptomeABSTRACT
Organizers, which comprise groups of cells with the ability to instruct adjacent cells into specific states, represent a key principle in developmental biology. The concept was first introduced by Spemann and Mangold, who showed that there is a cellular population in the newt embryo that elicits the development of a secondary axis from adjacent cells. Similar experiments in chicken and rabbit embryos subsequently revealed groups of cells with similar instructive potential. In birds and mammals, organizer activity is often associated with a structure known as the node, which has thus been considered a functional homologue of Spemann's organizer. Here, we take an in-depth look at the structure and function of organizers across species and note that, whereas the amphibian organizer is a contingent collection of elements, each performing a specific function, the elements of organizers in other species are dispersed in time and space. This observation urges us to reconsider the universality and meaning of the organizer concept.
Subject(s)
Organizers, Embryonic/cytology , Organizers, Embryonic/physiology , Amphibians/embryology , Animals , Birds/embryology , Body Patterning/physiology , Chick Embryo , Embryo, Mammalian , Embryo, Nonmammalian , Embryonic Induction/physiology , Gastrula/cytology , Humans , Mammals/embryology , RabbitsABSTRACT
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.
Subject(s)
Organoids/physiology , Translational Research, Biomedical , Growth and Development , Humans , Tissue EngineeringSubject(s)
Embryo Implantation , Embryo Research/ethics , Embryo Research/legislation & jurisprudence , Embryonic Development , Models, Biological , Stem Cell Research/ethics , Stem Cell Research/legislation & jurisprudence , Animals , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Female , Fertilization in Vitro , Humans , Mice , Placenta/cytology , Placentation , Pregnancy , Reproductive Medicine/ethics , Reproductive Medicine/legislation & jurisprudence , Reproductive Medicine/trends , Synthetic Biology/ethics , Synthetic Biology/legislation & jurisprudence , Synthetic Biology/trends , Yolk Sac/cytology , Yolk Sac/growth & developmentABSTRACT
The formation of the spinal cord during early embryonic development in vertebrate embryos is a continuous process that begins at gastrulation and continues through to the completion of somitogenesis. Despite the conserved usage of patterning mechanisms and gene regulatory networks that act to generate specific spinal cord progenitors, there now exists two seemingly disparate models to account for their action. In the first, a posteriorly localized signalling source transforms previously anterior-specified neural plate into the spinal cord. In the second, a population of bipotent stem cells undergo continuous self-renewal and differentiation to progressively lay down the spinal cord and axial mesoderm by posterior growth. Whether this represents fundamental differences between the experimental model organisms utilised in the generation of these models remains to be addressed. Here we review lineage studies across four key vertebrate models: mouse, chicken, Xenopus and zebrafish and relate them to the underlying gene regulatory networks that are known to be required for spinal cord formation. We propose that by applying a dynamical systems approach to understanding how distinct neural and mesodermal fates arise from a bipotent progenitor pool, it is possible to begin to understand how differences in the dynamical cell behaviours such as proliferation rates and cell movements can map onto conserved regulatory networks to generate diversity in the timing of tissue generation and patterning during development.
Subject(s)
Spinal Cord/cytology , Spinal Cord/embryology , Animals , Cell Differentiation , Chickens , Developmental Biology/methods , Mesoderm/embryology , Mice , Models, Animal , Morphogenesis , Neural Plate/cytology , Neural Plate/embryology , Stem Cells/cytology , Xenopus , Zebrafish/embryologyABSTRACT
Intracellular transcriptional regulators and extracellular signaling pathways together regulate the allocation of cell fates during development, but how their molecular activities are integrated to establish the correct proportions of cells with particular fates is not known. Here we study this question in the context of the decision between the epiblast (Epi) and the primitive endoderm (PrE) fate that occurs in the mammalian preimplantation embryo. Using an embryonic stem cell (ESC) model, we discover two successive functions of FGF/MAPK signaling in this decision. First, the pathway needs to be inhibited to make the PrE-like gene expression program accessible for activation by GATA transcription factors in ESCs. In a second step, MAPK signaling levels determine the threshold concentration of GATA factors required for PrE-like differentiation, and thereby control the proportion of cells differentiating along this lineage. Our findings can be explained by a simple mutual repression circuit modulated by FGF/MAPK signaling. This might be a general network architecture to integrate the activity of signal transduction pathways and transcriptional regulators, and serve to balance proportions of cell fates in several contexts.
Subject(s)
Cell Differentiation , Cell Lineage , Fibroblast Growth Factors/metabolism , MAP Kinase Signaling System , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/metabolism , Animals , Biomarkers/metabolism , Cells, Cultured , Endoderm/cytology , Endoderm/metabolism , GATA4 Transcription Factor/metabolism , Gene Expression Regulation , Germ Layers/cytology , Germ Layers/metabolism , Luminescent Proteins/metabolism , Mice , Models, Biological , Red Fluorescent ProteinABSTRACT
Understanding the mechanisms of early embryonic patterning and the timely allocation of specific cells to embryonic regions and fates as well as their development into tissues and organs, is a fundamental problem in Developmental Biology. The classical explanation for this process had been built around the notion of positional information. Accordingly the programmed appearance of sources of Morphogens at localized positions within a field of cells directs their differentiation. Recently, the development of organs and tissues from unpatterned and initially identical stem cells (adult and embryonic) has challenged the need for positional information and even the integrity of the embryo, for pattern formation. Here we review the emerging area of organoid biology from the perspective of Developmental Biology. We argue that the events underlying the development of these systems are not purely linked to self-organization, as often suggested, but rather to a process of genetically encoded self-assembly where genetic programs encode and control the emergence of biological structures.
Subject(s)
Embryonic Stem Cells/physiology , Morphogenesis/genetics , Morphogenesis/physiology , Organoids/physiology , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Developmental Biology/methods , HumansABSTRACT
The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/ß-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation.
Subject(s)
Cell Lineage/physiology , Central Nervous System/embryology , Embryonic Induction/physiology , Fibroblast Growth Factors/physiology , Mesoderm/embryology , Morphogenesis/physiology , Wnt Signaling Pathway/physiology , Animals , Cell Culture Techniques , Flow Cytometry , Fluorescence , Gene Expression Regulation, Developmental/physiology , Mice , Real-Time Polymerase Chain Reaction , Single-Cell Analysis , Time-Lapse ImagingABSTRACT
Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'.
Subject(s)
Body Patterning/physiology , Embryonic Stem Cells/physiology , Germ Layers/embryology , Nervous System/embryology , Animals , Cell Aggregation/physiology , Cell Line , Cell Polarity/physiology , Flow Cytometry , Mice , Microscopy, FluorescenceABSTRACT
The Drosophila adult posterior midgut has been identified as a powerful system in which to study mechanisms that control intestinal maintenance, in normal conditions as well as during injury or infection. Early work on this system has established a model of tissue turnover based on the asymmetric division of intestinal stem cells. From the quantitative analysis of clonal fate data, we show that tissue turnover involves the neutral competition of symmetrically dividing stem cells. This competition leads to stem-cell loss and replacement, resulting in neutral drift dynamics of the clonal population. As well as providing new insight into the mechanisms regulating tissue self-renewal, these findings establish intriguing parallels with the mammalian system, and confirm Drosophila as a useful model for studying adult intestinal maintenance.
Subject(s)
Cell Division , Drosophila melanogaster/physiology , Homeostasis/physiology , Intestines/physiology , Stem Cells/physiology , Animals , Cell Differentiation/physiology , Cell Lineage/physiology , Cell Proliferation , Drosophila melanogaster/cytology , Female , Intestines/cytology , Stem Cells/cytologyABSTRACT
Early mammalian embryos exhibit remarkable plasticity, as highlighted by the ability of separated early blastomeres to produce a whole organism. Recent work in the mouse implicates a network of transcription factors in governing the establishment of the primary embryonic lineages. A combination of genetics and embryology has uncovered the organisation and function of the components of this network, revealing a gradual resolution from ubiquitous to lineage-specific expression through a combination of defined regulatory relationships, spatially organised signalling, and biases from mechanical inputs. Here, we summarise this information, link it to classical embryology and propose a molecular framework for the establishment and regulation of developmental plasticity.