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Cell Stem Cell Jun 2023The emergence of the three germ layers and the lineage-specific precursor cells orchestrating organogenesis represent fundamental milestones during early embryonic...
The emergence of the three germ layers and the lineage-specific precursor cells orchestrating organogenesis represent fundamental milestones during early embryonic development. We analyzed the transcriptional profiles of over 400,000 cells from 14 human samples collected from post-conceptional weeks (PCW) 3 to 12 to delineate the dynamic molecular and cellular landscape of early gastrulation and nervous system development. We described the diversification of cell types, the spatial patterning of neural tube cells, and the signaling pathways likely involved in transforming epiblast cells into neuroepithelial cells and then into radial glia. We resolved 24 clusters of radial glial cells along the neural tube and outlined differentiation trajectories for the main classes of neurons. Lastly, we identified conserved and distinctive features across species by comparing early embryonic single-cell transcriptomic profiles between humans and mice. This comprehensive atlas sheds light on the molecular mechanisms underlying gastrulation and early human brain development.
Topics: Humans; Mice; Animals; Gastrulation; Germ Layers; Cell Differentiation; Organogenesis; Brain
PubMed: 37192616
DOI: 10.1016/j.stem.2023.04.016 -
Advances in Experimental Medicine and... 2019The field of regenerative medicine is looking for a pluripotent/multipotent stem cell able to differentiate across germ layers and be safely employed in therapy.... (Review)
Review
The field of regenerative medicine is looking for a pluripotent/multipotent stem cell able to differentiate across germ layers and be safely employed in therapy. Unfortunately, with the exception of hematopoietic stem/progenitor cells (HSPCs) for hematological applications, the current clinical results with stem cells are somewhat disappointing. The potential clinical applications of the more primitive embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have so far been discouraging, as both have exhibited several problems, including genomic instability, a risk of teratoma formation, and the possibility of rejection. Therefore, the only safe stem cells that have so far been employed in regenerative medicine are monopotent stem cells, such as the abovementioned HSPCs or mesenchymal stem cells (MSCs) isolated from postnatal tissues. However, their monopotency, and therefore limited differentiation potential, is a barrier to their broader application in the clinic. Interestingly, results have accumulated indicating that adult tissues contain rare, early-development stem cells known as very small embryonic-like stem cells (VSELs), which can differentiate into cells from more than one germ layer. This chapter addresses different sources of stem cells for potential clinical application and their advantages and problems to be solved.
Topics: Cell Differentiation; Embryonic Stem Cells; Germ Layers; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells; Regenerative Medicine
PubMed: 31898779
DOI: 10.1007/978-3-030-31206-0_1 -
Nature Jan 2020Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available...
Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms that do not recapitulate the in vivo conditions. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior-posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.
Topics: Amnion; Blastocyst; Cell Culture Techniques; Cell Differentiation; Cell Lineage; Cell Polarity; Collagen; Drug Combinations; Embryo, Mammalian; Embryonic Development; Epithelium; Gastrulation; Germ Layers; Humans; Laminin; Primitive Streak; Proteoglycans; RNA-Seq; Single-Cell Analysis; Transcription Factors; Transcriptome; Trophoblasts; Yolk Sac
PubMed: 31830756
DOI: 10.1038/s41586-019-1875-y -
Cell Stem Cell Apr 2023Human stem cell-derived blastoids display similar morphology and cell lineages to normal blastocysts. However, the ability to investigate their developmental potential...
Human stem cell-derived blastoids display similar morphology and cell lineages to normal blastocysts. However, the ability to investigate their developmental potential is limited. Here, we construct cynomolgus monkey blastoids resembling blastocysts in morphology and transcriptomics using naive ESCs. These blastoids develop to embryonic disk with the structures of yolk sac, chorionic cavity, amnion cavity, primitive streak, and connecting stalk along the rostral-caudal axis through prolonged in vitro culture (IVC). Primordial germ cells, gastrulating cells, visceral endoderm/yolk sac endoderm, three germ layers, and hemato-endothelial progenitors in IVC cynomolgus monkey blastoids were observed by single-cell transcriptomics or immunostaining. Moreover, transferring cynomolgus monkey blastoids to surrogates achieves pregnancies, as indicated by progesterone levels and presence of early gestation sacs. Our results reveal the capacity of in vitro gastrulation and in vivo early pregnancy of cynomolgus monkey blastoids, providing a useful system to dissect primate embryonic development without the same ethical concerns and access challenges in human embryo study.
Topics: Pregnancy; Animals; Female; Humans; Gastrulation; Macaca fascicularis; Embryo, Mammalian; Germ Layers; Embryonic Development; Endoderm; Cell Differentiation
PubMed: 37028403
DOI: 10.1016/j.stem.2023.03.009 -
Nature Communications Oct 2018Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells...
Cerebral organoids are 3D stem cell-derived models that can be utilized to study the human brain. The current consensus is that cerebral organoids consist of cells derived from the neuroectodermal lineage. This limits their value and applicability, as mesodermal-derived microglia are important players in neural development and disease. Remarkably, here we show that microglia can innately develop within a cerebral organoid model and display their characteristic ramified morphology. The transcriptome and response to inflammatory stimulation of these organoid-grown microglia closely mimic the transcriptome and response of adult microglia acutely isolated from post mortem human brain tissue. In addition, organoid-grown microglia mediate phagocytosis and synaptic material is detected inside them. In all, our study characterizes a microglia-containing organoid model that represents a valuable tool for studying the interplay between microglia, macroglia, and neurons in human brain development and disease.
Topics: Adult; Aged; Aged, 80 and over; Cerebrum; Female; Germ Layers; Humans; Immunity; Male; Mesoderm; Microglia; Middle Aged; Neurons; Organoids; Stem Cells; Transcriptome; Young Adult
PubMed: 30301888
DOI: 10.1038/s41467-018-06684-2 -
Cell Stem Cell Sep 2022Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids)...
Despite its clinical and fundamental importance, our understanding of early human development remains limited. Stem cell-derived, embryo-like structures (or embryoids) allowing studies of early development without using natural embryos can potentially help fill the knowledge gap of human development. Herein, transcriptome at the single-cell level of a human embryoid model was profiled at different time points. Molecular maps of lineage diversifications from the pluripotent human epiblast toward the amniotic ectoderm, primitive streak/mesoderm, and primordial germ cells were constructed and compared with in vivo primate data. The comparative transcriptome analyses reveal a critical role of NODAL signaling in human mesoderm and primordial germ cell specification, which is further functionally validated. Through comparative transcriptome analyses and validations with human blastocysts and in vitro cultured cynomolgus embryos, we further proposed stringent criteria for distinguishing between human blastocyst trophectoderm and early amniotic ectoderm cells.
Topics: Animals; Blastocyst; Cell Lineage; Ectoderm; Embryo, Mammalian; Germ Layers; Humans; Single-Cell Analysis
PubMed: 36055194
DOI: 10.1016/j.stem.2022.08.009 -
Nature Oct 2023The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after...
The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs). Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions). Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.
Topics: Humans; Embryo Implantation; Embryo, Mammalian; Embryonic Development; Fertilization; Gastrulation; Germ Layers; Human Embryonic Stem Cells; Trophoblasts; Yolk Sac; Giant Cells
PubMed: 37673118
DOI: 10.1038/s41586-023-06604-5 -
Developmental Biology Jun 2021During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important... (Review)
Review
During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important prerequisite for the emergence of patterns within the developing organism is to establish asymmetries at various scales, ranging from individual cells to the entire embryo, eventually giving rise to the different body structures. This becomes especially apparent during gastrulation, when the earliest major lineage restriction events lead to the formation of the different germ layers. Traditionally, the unfolding of the developmental program from symmetry breaking to germ layer formation has been studied by dissecting the contributions of different signaling pathways and cellular rearrangements in the in vivo context of intact embryos. Recent efforts, using the intrinsic capacity of embryonic stem cells to self-assemble and generate embryo-like structures de novo, have opened new avenues for understanding the many ways by which an embryo can be built and the influence of extrinsic factors therein. Here, we discuss and compare divergent and conserved strategies leading to germ layer formation in embryos as compared to in vitro systems, their upstream molecular cascades and the role of extrinsic factors in this process.
Topics: Animals; Embryonic Stem Cells; Extraembryonic Membranes; Gastrulation; Germ Layers; Humans; Signal Transduction
PubMed: 33352181
DOI: 10.1016/j.ydbio.2020.12.014 -
Cell Stem Cell Sep 2022A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue...
A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.
Topics: Animals; Cell Differentiation; Embryo, Mammalian; Germ Layers; Humans; Mesoderm; Pluripotent Stem Cells; Primates
PubMed: 36055191
DOI: 10.1016/j.stem.2022.08.001 -
Nature Dec 2019Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major...
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Topics: Animals; Cell Differentiation; Cell Lineage; Chromatin; DNA Methylation; Demethylation; Embryoid Bodies; Endoderm; Enhancer Elements, Genetic; Epigenesis, Genetic; Epigenome; Erythropoiesis; Factor Analysis, Statistical; Gastrula; Gastrulation; Gene Expression Regulation, Developmental; Mesoderm; Mice; Pluripotent Stem Cells; RNA; Single-Cell Analysis; Time Factors; Zinc Fingers
PubMed: 31827285
DOI: 10.1038/s41586-019-1825-8