Extended culture of 2D gastruloids to model human mesoderm development
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ABSTRACT:
PROVIDER: PRJNA1090137 | ENA |
REPOSITORIES: ENA
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Project description:Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4, known as a 2D gastruloid, are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h.
2025-01-15 | GSE262081 | GEO
Project description:Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 (2D gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h. We discovered a phase of highly reproducible morphogenesis during which directed migration from the primitive streak-like region gives rise to a mesodermal layer beneath an epiblast-like layer. Multiple types of mesoderm arise with striking spatial organization including lateral mesoderm-like cells on the colony border and paraxial mesoderm-like further inside the colony. Single cell transcriptomics showed strong similarity of these cells to mesoderm in human and non-human primate embryos. However, our data suggest that the annotation of the reference human embryo may need to be revised. This illustrates that extended culture of 2D gastruloids provides a powerful model for human mesoderm differentiation and morphogenesis.
| S-EPMC10996563 | biostudies-literature
Project description:Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Indeed, there is a lack of an in vitro model replicating the differentiation of both heart and head muscles to study these mechanisms. Such models are required to allow live-imaging and high throughput genetic and drug screening. Here, we show that the formation of self-organizing or pseudo-embryos from mouse embryonic stem cells (mESCs), also called gastruloids, reproduces cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm establishment and differentiation in gastruloids and comparing it to mouse embryos, we present the first evidence for skeletal myogenesis in gastruloids. By inferring lineage trajectories from the gastruloids single-cell transcriptomic data, we further suggest that heart and head muscles formed in gastruloids derive from cardiopharyngeal mesoderm progenitors. We identify different subpopulations of cardiomyocytes and skeletal muscles, which most likely correspond to different states of myogenesis with “head-like” and “trunk-like” skeletal myoblasts. These findings unveil the potential of mESC-derived gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases.
2024-11-01 | GSE232773 | GEO
Project description:Patterning and growth are fundamental features of embryonic development that must be tightly coordinated during morphogenesis. While metabolism is known to control cell growth, how it impacts patterning and links to morphogenesis is poorly understood. To understand how metabolism impacts early mesoderm specification during gastrulation, we used in vitro mouse embryonic stem (ES) cell-derived gastruloids, due to ease of metabolic manipulations and high-throughput nature. Gastruloids showed mosaic expression of glucose transporters co-expressing with the mesodermal marker T/Bra. To understand the significance of cellular glucose uptake in development, we used the glucose metabolism inhibitor 2-deoxy-D-glucose (2-DG). 2-DG blocked the expression of T/Bra and abolishes axial elongation in gastruloids. Surprisingly, removing glucose completely from the medium did not phenocopy 2-DG treatment despite a significant decline in glycolytic intermediates occurring under both conditions. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification. We corroborated these results in vivomouse embryos where supplementing mannose rescued the 2-DG mediated phenotype of mesoderm specification and proximo-distal elongation of the primitive streak. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. At molecular level, proteomics analysis revealed that mannose reversed glycosylation of the Wnt pathway regulator, Secreted Frizzled Receptor, Frzb, expressed in the primitive streak of the mouse embryo. Our study showed how mannose linked metabolism to glycosylation of a developmental pathway component, crucial in patterning of mesoderm and morphogenesis of gastruloids.
2024-01-12 | MSV000093849 | MassIVE
Project description:Patterning and growth are fundamental features of embryonic development that must be tightly coordinated during morphogenesis. As metabolism can control cell growth while also providing mechanistic links to developmental signalling pathways, it is ideally placed to enable this coordination. To understand how metabolism impacts early mesoderm specification, we used mouse embryonic stem (ES) cell-derived gastruloids, as these enable temporal control over metabolic manipulations and can be generated in large quantities. Gastruloids show mosaic expression of two glucose transporters,Slc2a1andSlc2a3both of which co-express with the expression of both the mesodermal markerT/Braand the neural markerSox2. To understand the significance of cellular glucose uptake in development, we used the glucose metabolism inhibitor 2-deoxy-D-glucose (2-DG). 2-DG specifically blocks the expression ofT/Brawithout affecting the expression ofSox2and abolishes axial elongation in gastruloids. Surprisingly, removing glucose completely from the medium did not phenocopy 2-DG treatment despite a significant decline in glycolytic intermediates occurring under both conditions. As 2-DG can also act as a competitive inhibitor of mannose, we added mannose together with 2-DG and found that it could rescue the mesoderm specification. Together, our results show that while mannose is crucial for mesoderm specification, the glycolytic pathway is dispensable at early stages ofT/Braexpression in gastruloids.
2024-03-11 | GSE236887 | GEO
Project description:TBXT dose sensitivity and the decoupling of mesoderm specification from EMT progression in 2D human gastruloids
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Project description:Mannose is crucial for mesoderm specification and symmetry breaking in gastruloids
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Project description:Cardiopharyngeal Mesoderm specification into cardiac and skeletal muscle lineages in gastruloids
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Project description:In the nascent mesoderm, levels of Brachyury (TBXT) expression must be precisely regulated to ensure cells exit the primitive streak and pattern the anterior-posterior axis, but how this varying dosage informs morphogenesis is not well understood. In this study, we define the transcriptional consequences of TBXT dose during early gastrulation using human induced pluripotent stem cell (hiPSC)-based models of early gastrulation and mesoderm differentiation. Multiomic single-nucleus RNA and single-nucleus ATAC sequencing reveal that TBXT is required for the temporal progression of epithelial-to-mesenchymal transition (EMT) in a dose-dependent manner and that this transition occurs independently of the acquisition of mesodermal identity. These results demonstrate that EMT can be decoupled from mesoderm development in the early gastrula and shed light on the mechanisms underlying human embryogenesis.
2024-02-14 | GSE245998 | GEO