Embryonic Stem Cell Populations Research Articles

Neural crest origin of sympathetic neurons at the dawn of vertebrates.

The neural crest is an embryonic stem cell population unique to vertebrates1 whose expansion and diversification are thought to have promoted vertebrate evolution by enabling emergence of new cell types and structures such as jaws and peripheral ganglia2. Although jawless vertebrates have sensory ganglia, convention has it that trunk sympathetic chain ganglia arose only in jawed vertebrates3-8. Here, by contrast, we report the presence of trunk sympathetic neurons in the sea lamprey, Petromyzon marinus, an extant jawless vertebrate. These neurons arise from sympathoblasts near the dorsal aorta that undergo noradrenergic specification through a transcriptional program homologous to that described in gnathostomes. Lamprey sympathoblasts populate the extracardiac space and extend along the length of the trunk in bilateral streams, expressing the catecholamine biosynthetic pathway enzymes tyrosine hydroxylase and dopamine β-hydroxylase. CM-DiI lineage tracing analysis further confirmed that these cells derive from the trunk neural crest. RNA sequencing of isolated ammocoete trunk sympathoblasts revealed gene profiles characteristic of sympathetic neuron function. Our findings challenge the prevailing dogma that posits that sympathetic ganglia are a gnathostome innovation, instead suggesting that a late-developing rudimentary sympathetic nervous system may have been characteristic of the earliest vertebrates.

Transcription factor co-expression mediates lineage priming for embryonic and extra-embryonic differentiation

In early mammalian development, cleavage stage blastomeres and inner cell mass (ICM) cells co-express embryonic and extra-embryonic transcriptional determinants. Using a protein-based double reporter we identify an embryonic stem cell (ESC) population that co-expresses the extra-embryonic factor GATA6 alongside the embryonic factor SOX2. Based on single cell transcriptomics, we find this population resembles the unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence. To relate transcription factor binding in these cells to future fate, we describe a complete enhancer set in both ESCs and naive extra-embryonic endoderm stem cells and assess SOX2 and GATA6 binding at these elements in the ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers, suggesting that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.

Embryonic stem cell ERK, AKT, plus STAT3 response dynamics combinatorics are heterogeneous but NANOG state independent.

Signaling is central in cell fate regulation, and relevant information is encoded in its activity over time (i.e., dynamics). However, simultaneous dynamics quantification of several pathways in single mammalian stem cells has not yet been accomplished. Here we generate mouse embryonic stem cell (ESC) lines simultaneously expressing fluorescent reporters for ERK, AKT, and STAT3 signaling activity, which all control pluripotency. We quantify their single-cell dynamics combinations in response to different self-renewal stimuli and find striking heterogeneity for all pathways, some dependent on cell cycle but not pluripotency states, even in ESC populations currently assumed to be highly homogeneous. Pathways are mostly independently regulated, but some context-dependent correlations exist. These quantifications reveal surprising single-cell heterogeneity in the important cell fate control layer of signaling dynamics combinations and raise fundamental questions about the role of signaling in (stem) cell fate control.

Emergent Criticality in Coupled Boolean Networks.

Early embryonic development involves forming all specialized cells from a fluid-like mass of identical stem cells. The differentiation process consists of a series of symmetry-breaking events, starting from a high-symmetry state (stem cells) to a low-symmetry state (specialized cells). This scenario closely resembles phase transitions in statistical mechanics. To theoretically study this hypothesis, we model embryonic stem cell (ESC) populations through a coupled Boolean network (BN) model. The interaction is applied using a multilayer Ising model that considers paracrine and autocrine signaling, along with external interventions. It is demonstrated that cell-to-cell variability can be interpreted as a mixture of steady-state probability distributions. Simulations have revealed that such models can undergo a series of first- and second-order phase transitions as a function of the system parameters that describe gene expression noise and interaction strengths. These phase transitions result in spontaneous symmetry-breaking events that generate new types of cells characterized by various steady-state distributions. Coupled BNs have also been shown to self-organize in states that allow spontaneous cell differentiation.

Transcriptomics, regulatory syntax, and enhancer identification in mesoderm-induced ESCs at single-cell resolution.

Embryonic stem cells (ESCs) can adopt lineage-specific gene-expression programs by stepwise exposure to defined factors, resulting in the generation of functional cell types. Bulk and single-cell-based assays were employed to catalog gene expression, histone modifications, chromatin conformation, and accessibility transitions in ESC populations and individual cells acquiring a presomitic mesoderm fate and undergoing further specification toward myogenic and neurogenic lineages. These assays identified cis-regulatory regions and transcription factors presiding over gene-expression programs occurring at defined ESC transitions and revealed the presence of heterogeneous cell populations within discrete ESC developmental stages. The datasets were employed to identify previously unappreciated genomic elements directing the initial activation of Pax7 and myogenic and neurogenic gene-expression programs. This study provides a resource for the discovery of genomic and transcriptional features of pluripotent, mesoderm-induced ESCs and ESC-derived cell lineages.

(Fish) tales of congenital diseases: molecularly distinguishing cranial neural crest cell populations

Congenital diseases that affect the anterior segment are typically due to cranial neural crest defects. This transient embryonic stem cell population ultimately gives rise to the bone, cartilage, and connective tissue of the jaw and mid face, and within the eye contributes to the corneal, iris, ciliary body, aqueous outflow pathways, and sclera. Thus, due to their common origin, congenital eye anomalies are often associated with facial defects.

Time to go: neural crest cell epithelial-to-mesenchymal transition.

ABSTRACTNeural crest cells (NCCs) are a dynamic, multipotent, vertebrate-specific population of embryonic stem cells. These ectodermally-derived cells contribute to diverse tissue types in developing embryos including craniofacial bone and cartilage, the peripheral and enteric nervous systems and pigment cells, among a host of other cell types. Due to their contribution to a significant number of adult tissue types, the mechanisms that drive their formation, migration and differentiation are highly studied. NCCs have a unique ability to transition from tightly adherent epithelial cells to mesenchymal and migratory cells by altering their polarity, expression of cell-cell adhesion molecules and gaining invasive abilities. In this Review, we discuss classical and emerging factors driving NCC epithelial-to-mesenchymal transition and migration, highlighting the role of signaling and transcription factors, as well as novel modifying factors including chromatin remodelers, small RNAs and post-translational regulators, which control the availability and longevity of major NCC players.

Expression atlas of avian neural crest proteins: Neurulation to migration

Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent Stem Cells.

Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.

Ribosomal stress induces 2-cell embryo-like state transition of the mouse ESCs through p53 activation

Embryonic stem cells (ESCs) maintain a pluripotent state and genome integrity in long-term culture. A rare population of ESCs showing 2-cell embryo-specific gene expression is believed to play critical roles in sustainable pluripotency and genome stability. However, the molecular mechanism controlling this transition to a 2-cell embryo-like (2CL) state remains unclear. We carried out screening to search for the factors involved in 2CL state induction and found a ribosomal RNA processing factor, Pum3 to be a candidate. Increased 2CL state population accompanied with an accumulation of pre-ribosomal RNA and activated p53 in the Pum3-KO ESC. Furthermore, the increase of 2CL state cells in the Pum3-KO ESCs was completely abrogated by the deletion of p53. The DNA damage induced by the Ultraviolet light (UV) irradiation and Zeocin promoted the transition to a 2CL state in a p53-dependent manner. Thus, our study provides new insights into a 2CL state transition mechanism through stress-dependent p53 activation of ESCs.

Retinoic acid induces NELFA‐mediated 2C‐like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media

ObjectivesMouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst‐stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2‐cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2‐cell embryo‐like (2C‐like) cells in the chemically defined media remains largely unknown.Materials and MethodsWe established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C‐like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter‐positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA‐seq.ResultsRetinoic acid drives a NELFA (negative elongation factor A)‐mediated 2C‐like state in mouse ESCs, characterized with 2C‐specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs.ConclusionsWe showed that RA has a broader role in 2C‐like cells state, not only is one of the upstream regulators of the 2C‐like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C‐like transition.

Transcription factor OTX2 silences the expression of cleavage embryo genes and transposable elements.

Upon mammalian fertilization, zygotic genome activation (ZGA) and activation of transposable elements (TEs) occur in early embryos to establish totipotency and support embryogenesis. However, the molecular mechanisms controlling the expression of these genes in mammals remain poorly understood. The 2-cell-like population of mouse embryonic stem cells (mESCs) mimics cleavage-stage embryos with transient Dux activation. In this study, we demonstrated that deficiency of the transcription factor OTX2 stimulates the expression of ZGA genes in mESCs. Further analysis revealed that OTX2 is incorporated at the Dux locus with corepressors for transcriptional inhibition. We also found that OTX2 associates with TEs and silences the subtypes of TEs. Therefore, OTX2 protein plays an important role in ZGA and TE expression in mESCs to orchestrate the transcriptional network.

Xenopus, an emerging model for studying pathologies of the neural crest.

Neural crest cells are a multipotent embryonic stem cell population that emerges from the lateral border of the neural plate after an epithelium-to-mesenchyme transition. These cells then migrate extensively in the embryo and generate a large variety of differentiated cell types and tissues. Alterations in almost any of the processes involved in neural crest development can cause severe congenital defects in humans. Moreover, the malignant transformation of one of the many neural crest derivatives, during childhood or in adults, can cause the development of aggressive tumors prone to metastasis such as melanoma and neuroblastoma. Collectively these diseases are called neurocristopathies. Here we review how a variety of approaches implemented using the amphibian Xenopus as an experimental model have shed light on the molecular basis of numerous neurocristopathies, and how this versatile yet underused vertebrate animal model could help accelerate discoveries in the field. Using the current framework of the neural crest gene regulatory network, we review the pathologies linked to defects at each step of neural crest formation and highlight studies that have used the Xenopus model to decipher the cellular and molecular aspects of neurocristopathies.

Distinct molecular profile and restricted stem cell potential defines the prospective human cranial neural crest from embryonic stem cell state.

Neural crest cells are an embryonic multipotent stem cell population. Recent studies in model organisms have suggested that neural crest cells are specified earlier than previously thought, at blastula stages. However, the molecular dynamics of early neural crest specification, and functional changes from pluripotent precursors to early specified NC, remain to be elucidated. In this report, we utilized a robust human model of cranial neural crest formation to address the distinct molecular character of the earliest stages of neural crest specification and assess the functional differences from its embryonic stem cell precursor. Our human neural crest model reveals a rapid change in the epigenetic state of neural crest and pluripotency genes, accompanied by changes in gene expression upon Wnt-based induction from embryonic stem cells. These changes in gene expression are directly regulated by the transcriptional activity of β-catenin. Furthermore, prospective cranial neural crest cells are characterized by restricted stem cell potential compared to embryonic stem cells. Our results suggest that human neural crest induced by Wnt/β-catenin signaling from human embryonic stem cells rapidly acquire a prospective neural crest cell state defined by a unique molecular signature and endowed with limited potential compared to pluripotent stem cells.

On the road again: Establishment and maintenance of stemness in the neural crest from embryo to adulthood.

Unique to vertebrates, the neural crest (NC) is an embryonic stem cell population that contributes to a greatly expanding list of derivatives ranging from neurons and glia of the peripheral nervous system, facial cartilage and bone, pigment cells of the skin to secretory cells of the endocrine system. Here, we focus on what is specifically known about establishment and maintenance of NC stemness and ultimate fate commitment mechanisms, which could help explain its exceptionally high stem cell potential that exceeds the “rules set during gastrulation.” In fact, recent discoveries have shed light on the existence of NC cells that coexpress commonly accepted pluripotency factors like Nanog, Oct4/PouV, and Klf4. The coexpression of pluripotency factors together with the exceptional array of diverse NC derivatives encouraged us to propose a new term “pleistopotent” (Greek for abundant, a substantial amount) to be used to reflect the uniqueness of the NC as compared to other post‐gastrulation stem cell populations in the vertebrate body, and to differentiate them from multipotent lineage restricted stem cells. We also discuss studies related to the maintenance of NC stemness within the challenging context of being a transient and thus a constantly changing population of stem cells without a permanent niche. The discovery of the stem cell potential of Schwann cell precursors as well as multiple adult NC‐derived stem cell reservoirs during the past decade has greatly increased our understanding of how NC cells contribute to tissues formed after its initial migration stage in young embryos.

Hypoxia inducible factor-2α importance for migration, proliferation, and self-renewal of trunk neural crest cells.

BackgroundThe neural crest is a transient embryonic stem cell population. Hypoxia inducible factor (HIF)‐2α is associated with neural crest stem cell appearance and aggressiveness in tumors. However, little is known about its role in normal neural crest development.ResultsHere, we show that HIF‐2α is expressed in trunk neural crest cells of human, murine, and avian embryos. Knockdown as well as overexpression of HIF‐2α in vivo causes developmental delays, induces proliferation, and self‐renewal capacity of neural crest cells while decreasing the proportion of neural crest cells that migrate ventrally to sympathoadrenal sites. Reflecting the in vivo phenotype, transcriptome changes after loss of HIF‐2α reveal enrichment of genes associated with cancer, invasion, epithelial‐to‐mesenchymal transition, and growth arrest.ConclusionsTaken together, these results suggest that expression levels of HIF‐2α must be strictly controlled during normal trunk neural crest development and that dysregulated levels affects several important features connected to stemness, migration, and development.

Using Xenopus Neural Crest Explants to Study Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to posttranslational modifications, to protein turnover. It involves remodeling of polarity and adhesion and enhances migratory capabilities. During physiological events such as embryogenesis or wound healing EMT is used to initiate cell migration, but EMT can also occur in pathological settings. In particular, EMT has been linked tofibrosis and cancer. Neural crest (NC) cells, an embryonic stem cell population whose behavior recapitulates the main steps of carcinoma progression, are a great model to study EMT. In this chapter, we provide a fully detailed protocol to extract NC cells from Xenopus embryos and culture them to study the dynamics of cell-cell adhesion, cell motility, and dispersion.

Cellulose Nanofibers for Encapsulation and Pluripotency Preservation in the Early Development of Embryonic Stem Cells.

Materials for three-dimensional cultures aim to reproduce the function of the extracellular matrix, enabling cell adhesion and growth by remodeling the environment. However, embryonic stem cells (ESCs) must develop in environments that prevent adhesion and preserve their pluripotency. In this study, we used cellulose nanofiber hydrogels to mimic the developing conditions required for ESCs. These plant-based hydrogels are simultaneously biocompatible and exogenous to mammalian cells, preventing remodeling and attachment. The storage modulus of these hydrogels could be fine-tuned by varying the degree of oxidation to enable selective degradation. The ESCs proliferated in the artificial environment, forming increasingly large embryoid bodies for 15 days. Unlike traditional cultures in which ESCs begin differentiating upon the removal of the chemical inhibition, the expression of pluripotency markers in the ESC population remained high for the entire two weeks. Cellulase from Trichoderma reesei was used to retrieve the ESC cultures selectively. The proposed unique system is a prospective model with which to study the early development of embryonic cells, as well as a nonchemical method of preserving undifferentiated populations of ESCs.

From pluripotency to totipotency: an experimentalist's guide to cellular potency.

Embryonic stem cells (ESCs) are derived from the pre-implantation mammalian blastocyst. At this point in time, the newly formed embryo is concerned with the generation and expansion of both the embryonic lineages required to build the embryo and the extra-embryonic lineages that support development. When used in grafting experiments, embryonic cells from early developmental stages can contribute to both embryonic and extra-embryonic lineages, but it is generally accepted that ESCs can give rise to only embryonic lineages. As a result, they are referred to as pluripotent, rather than totipotent. Here, we consider the experimental potential of various ESC populations and a number of recently identified in vitro culture systems producing states beyond pluripotency and reminiscent of those observed during pre-implantation development. We also consider the nature of totipotency and the extent to which cell populations in these culture systems exhibit this property.

Rules of collective migration: from the wildebeest to the neural crest.

Collective migration, the movement of groups in which individuals affect the behaviour of one another, occurs at practically every scale, from bacteria up to whole species' populations. Universal principles of collective movement can be applied at all levels. In this review, we will describe the rules governing collective motility, with a specific focus on the neural crest, an embryonic stem cell population that undergoes extensive collective migration during development. We will discuss how the underlying principles of individual cell behaviour, and those that emerge from a supracellular scale, can explain collective migration.This article is part of the theme issue ‘Multi-scale analysis and modelling of collective migration in biological systems’.