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Single-cell RNA-sequencing of iBlastoids generated by reprogramming

ABSTRACT: Here, we report an in vitro model of the human blastocyst by reprogramming fibroblasts, termed iBlastoids. Comprehensive characterization of these iBlastoids demonstrated that they can accurately represent the overall architecture of human blastocysts including an inner cell mass-like structure, marked by classic EPI-associated genes surrounded by an outer layer of cells with key molecular characteristics of the TE and a cavity resembling the blastocoel. Single-cell transcriptomics confirmed the presence of EPI, PE and TE-like cells and unveiled a high correlation between blastocysts and iBlastoids. Finally, iBlastoids are capable of modeling in vitro, several molecular and morphological aspects of blastocyst implantation. In summary, we have developed a scalable and tractable model to study the human blastocyst, and we envision that iBlastoids will facilitate further studies of the molecular and functional processes during early human development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156596 | GEO | 2021/01/19

REPOSITORIES: GEO

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Project description:Differentiation of totipotent embryonic cells into the first cell lineages (trophectoderm (TE) and inner cell mass (ICM), and further into epiblast (EPI) and primitive endoderm (PE)), although well studied, is only beginning to be understood at the molecular level. An in depth understanding of the regulation of these early differentiation events would enable improvements in the quality of in vitro produced embryos, as well as the study and application of bovine embryonic stem cells. The objective of this work was to perform single cell RNASeq on bovine blastocysts derived in vivo (IVV), in vitro in a conventional in vitro production system (IVC), and in vitro in reduced nutrient media (IVR). All in vitro culture was serum free. In vivo embryonic cells could be assigned based on Leiden clustering analysis, although there was some overlap in expression of marker genes. The two TE groups identified clearly separated in a uniform manifold approximation and projection (UMAP) plot from EPI groups. Putative epiblast cells could be identified. In in vitro embryos produced in a conventional system, considerable overlap in expression of marker genes made Leiden clustering difficult, but a selected count could be used for UMAP to tentatively identify one TE, 2 EPI and 2 PE groups. In assigned cell types we observed higher expression of the identifying marker genes, but some marker genes were expressed in all cell types. In IVC embryos, TE markers clustered while PE and EPI marker genes overlapped. In IVR embryos, again there was considerable overlap between marker gene expression. Like IVC embryos, TE was easily identified by clustering in IVR, but EPI and PE were too similar to distinguish. In summary, the methods used here could partially distinguish putative cell types in bovine blastocysts, although marker gene expression was not always discreet. This may not be surprising given differentiation events are in progress in the blastocyst stage embryo. TE cells were more easily distinguished than EPI and PE cells, which would support this hypothesis.

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Single-cell RNA-sequencing of iBlastoids generated by reprogramming

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