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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 -
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 -
Cell Stem Cell Jun 2023Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic...
Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic analysis, we provide a resource mapping cell states and types during gastruloid development and compare them with the in vivo embryo. We developed a high-throughput handling and imaging pipeline to spatially monitor symmetry breaking during gastruloid development and report an early spatial variability in pluripotency determining a binary response to Wnt activation. Although cells in the gastruloid-core revert to pluripotency, peripheral cells become primitive streak-like. These two populations subsequently break radial symmetry and initiate axial elongation. By performing a compound screen, perturbing thousands of gastruloids, we derive a phenotypic landscape and infer networks of genetic interactions. Finally, using a dual Wnt modulation, we improve the formation of anterior structures in the existing gastruloid model. This work provides a resource to understand how gastruloids develop and generate complex patterns in vitro.
Topics: Mice; Animals; Embryo, Mammalian; Primitive Streak; Pluripotent Stem Cells; Embryonic Development
PubMed: 37209681
DOI: 10.1016/j.stem.2023.04.018 -
Nature Oct 2023Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of...
Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.
Topics: Female; Humans; Pregnancy; Cell Differentiation; Cell Lineage; Embryo Implantation; Embryonic Development; Germ Layers; Human Embryonic Stem Cells; Placenta; Pluripotent Stem Cells; Primitive Streak; Yolk Sac
PubMed: 37369348
DOI: 10.1038/s41586-023-06354-4 -
Cell Stem Cell Sep 2023Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the...
Naive human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures ("blastoids") that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate the defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick three-dimensional (3D) extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that the modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.
Topics: Humans; Gastrulation; Embryo Implantation; Embryo, Mammalian; Blastocyst; Epithelial Cells
PubMed: 37683602
DOI: 10.1016/j.stem.2023.08.005 -
Nature Dec 2022Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans,...
Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, nonhuman primates are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we collected six Carnegie stage 8-11 cynomolgus monkey (Macaca fascicularis) embryos and performed in-depth transcriptomic analyses of 56,636 single cells. Our analyses show transcriptomic features of major perigastrulation cell types, which help shed light on morphogenetic events including primitive streak development, somitogenesis, gut tube formation, neural tube patterning and neural crest differentiation in primates. In addition, comparative analyses with mouse embryos and human embryoids uncovered conserved and divergent features of perigastrulation development across species-for example, species-specific dependency on Hippo signalling during presomitic mesoderm differentiation-and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the nonhuman primate research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.
Topics: Animals; Humans; Mice; Gastrulation; Macaca fascicularis; Organogenesis; Single-Cell Analysis; Embryoid Bodies; Gene Expression Profiling; Primitive Streak; Neural Tube; Neural Crest; Hippo Signaling Pathway; Mesoderm; Stem Cells
PubMed: 36517595
DOI: 10.1038/s41586-022-05526-y -
Nature Sep 2022Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling...
Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development and to advance stem-cell-based regenerative approaches. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.
Topics: Animals; Callithrix; Cell Differentiation; Embryo, Mammalian; Endoderm; Female; Gastrulation; Gene Expression Profiling; Germ Layers; Humans; Pluripotent Stem Cells; Uterus
PubMed: 35709828
DOI: 10.1038/s41586-022-04953-1 -
Developmental Cell Feb 2021The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN)...
The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN) stem cells to generate embryo-like structures called ETX embryos. Although ETX embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ESCs transiently expressing transcription factor Gata4, which drives the extra-embryonic endoderm fate, and combined them with ESCs and TS cells to generate induced ETX embryos (iETX embryos). We show that iETX embryos establish a robust anterior signaling center that migrates unilaterally to break embryo symmetry. Furthermore, iETX embryos gastrulate generating embryonic and extra-embryonic mesoderm and definitive endoderm. Our findings reveal that replacement of XEN cells with ESCs transiently expressing Gata4 endows iETX embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.
Topics: Animals; Biomarkers; Cell Line; Cell Lineage; Embryo, Mammalian; Embryonic Stem Cells; Endoderm; Epithelial-Mesenchymal Transition; GATA4 Transcription Factor; Gastrulation; Induced Pluripotent Stem Cells; Mice; Morphogenesis; Primitive Streak; Signal Transduction
PubMed: 33378662
DOI: 10.1016/j.devcel.2020.12.004 -
Development (Cambridge, England) Apr 2023During gastrulation, early embryos specify and reorganise the topology of their germ layers. Surprisingly, this fundamental and early process does not appear to be... (Review)
Review
During gastrulation, early embryos specify and reorganise the topology of their germ layers. Surprisingly, this fundamental and early process does not appear to be rigidly constrained by evolutionary pressures; instead, the morphology of gastrulation is highly variable throughout the animal kingdom. Recent experimental results demonstrate that it is possible to generate different alternative gastrulation modes in single organisms, such as in early cnidarian, arthropod and vertebrate embryos. Here, we review the mechanisms that underlie the plasticity of vertebrate gastrulation both when experimentally manipulated and during evolution. Using the insights obtained from these experiments we discuss the effects of the increase in yolk volume on the morphology of gastrulation and provide new insights into two crucial innovations during amniote gastrulation: the transition from a ring-shaped mesoderm domain in anamniotes to a crescent-shaped domain in amniotes, and the evolution of the reptilian blastoporal plate/canal into the avian primitive streak.
Topics: Animals; Gastrulation; Gastrula; Mesoderm; Germ Layers; Primitive Streak
PubMed: 37067451
DOI: 10.1242/dev.200885 -
Seminars in Cell & Developmental Biology May 2023Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures,... (Review)
Review
Recent advances in pluripotent stem cell culture allow researchers to generate not only most embryonic cell types, but also morphologies of many embryonic structures, entirely in vitro. This recreation of embryonic form from naïve cells, known as synthetic morphogenesis, has important implications for both developmental biology and regenerative medicine. However, the capacity of stem cell-based models to recapitulate the morphogenetic cell behaviors that shape natural embryos remains unclear. In this review, we explore several examples of synthetic morphogenesis, with a focus on models of gastrulation and surrounding stages. By varying cell types, source species, and culture conditions, researchers have recreated aspects of primitive streak formation, emergence and elongation of the primary embryonic axis, neural tube closure, and more. Here, we describe cell behaviors within in vitro/ex vivo systems that mimic in vivo morphogenesis and highlight opportunities for more complete models of early development.
Topics: Gastrulation; Morphogenesis
PubMed: 35817656
DOI: 10.1016/j.semcdb.2022.07.002