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Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

ABSTRACT: Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

PROVIDER: PRJNA715256 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13995141.fastq.gz	Fastqsanger.gz
	SRR13995142.fastq.gz	Fastqsanger.gz
	SRR13995143.fastq.gz	Fastqsanger.gz
	SRR13995144.fastq.gz	Fastqsanger.gz
	SRR13995145.fastq.gz	Fastqsanger.gz

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Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids

Project description:We applied BMP4 to circular micropattern cultures to differentiate human embryonic stem cells (H1 hESCs) into microcolonies termed ‘gastruloids’, comprising germ layer and extraembryonic cells. Time-course single cell RNA-sequencing was performed at 0h (immediately before BMP4 treatment), 12h, and 24h after BMP4 treatment. Combining these 0h, 12h, and 24h datasets with previously generated 44h datasets, we report transcriptomic dynamics over the course of BMP4-driven differentiation in micropatterned cultures.

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Gene expression dynamics underlying cell fate emergence in 2D micropatterned human embryonic stem cell gastruloids.

Project description:Human embryonic stem cells cultured in 2D micropatterns with BMP4 differentiate into a radial arrangement of germ layers and extraembryonic cells. Single-cell transcriptomes demonstrate generation of cell types transcriptionally similar to their in vivo counterparts in Carnegie stage 7 human gastrula. Time-course analyses indicate sequential differentiation, where the epiblast arises by 12 h between the prospective ectoderm in the center and the cells initiating differentiation toward extraembryonic fates at the edge. Extraembryonic and mesendoderm precursors arise from the epiblast by 24 h, while nascent mesoderm, endoderm, and primordial germ cell-like cells form by 44 h. Dynamic changes in transcripts encoding signaling components support a BMP, WNT, and Nodal hierarchy underlying germ-layer specification conserved across mammals, and FGF and HIPPO pathways being active throughout differentiation. This work also provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation.

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YAP1 Drives Lineage Specification and Patterning in hESC-derived Gastruloids

Project description:The gastrulation process is controlled by the interplay between morphogenetic signals from BMP, WNT and NODAL pathways. Increasing evidences support an emerging role of the Hippo-YAP signaling in the cell-fate decisions that guide lineage specification in mouse and human Embryonic Stem Cells (hESCs). However, the contribution of YAP to the process of gastrulation in hESCs remains unknown. Here, we show that YAP1 regulates the specification, size and patterning of the three-germ layers. Using hESC-derived 2D-micropatterned gastruloids and directed differentiation approaches we show that YAP maintains a semi-active NODAL signaling during gastrulation essential to regulate the exit of pluripotency. In absence of YAP1, a hyperactive NODAL signaling retains SMAD2.3 in the nuclei, impeding the exit of pluripotency and the acquisition of the ectodermal gene program. Accordingly, the inhibition of NODAL signaling is sufficient to rescue the gastrulation-defective phenotype of the YAP1 KO hESCs. Our work revealed that Hippo-YAP1 signaling is an important component of the developmental network that coordinate hESC pluripotency and gastrulation.

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Project description:Gastruloids are a powerful in vitro model of early human development. However, although elongated and composed of all three germ layers, human gastruloids do not morphologically resemble post-implantation human embryos. Here we show that an early pulse of retinoic acid (RA), together with later Matrigel, robustly induces human gastruloids with posterior embryo-like morphological structures, including a neural tube flanked by segmented somites, and diverse cell types including neural crest, neural progenitors, renal progenitors, and myocytes. Through in silico staging based on single-cell RNA-seq (scRNA-seq), we find that human RA-gastruloids progress further than other human or mouse embryo models, aligning to E9.5 mouse and CS11 cynomolgus monkey embryos. We leverage chemical and genetic perturbations of RA-gastruloids to confirm that WNT and BMP signalling regulate somite formation and neural tube length in the human context, while transcription factors TBX6 and PAX3 underpin presomitic mesoderm and neural crest, respectively. Looking forward, RA-gastruloids are a robust, scalable model for decoding early human embryogenesis.

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Symmetry breaking and germ layer emergence through optogenetic Wnt activation in gastruloids

Project description:Human gastrulation is a dynamic, complex, and poorly understood process. Starting from a seemingly homogenous population, cell and tissue organization during this process establishes the 3D organismal body plan. However, due to technical and ethical limitations, mechanisms of human gastrulation are largely unexplored. For example, it is generally unclear whether a spatially ordered, external cue is necessary for patterning, or whether the epiblast harbors the innate ability to differentiate and self-organize in the absence of asymmetric cues. Human pluripotent stem cells (hPSCs) fortunately offer the potential to model gastrulation, most notably through generation of 3D “gastruloids”. Here we use optogenetic control of Wnt signaling to establish a 3D hPSC gastruloid model and demonstrate the spatially-ordered emergence of the three germ layer lineages in the absence of exogenous asymmetrical cues.

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