Spatiotemporal Reconstruction of the Human Blastocyst by Single-Cell Gene-Expression Analysis Informs Induction of Naive Pluripotency

Abstract

Human preimplantation embryo development involves complex cellular and molecular events that lead to the establishment of three cell lineages in the blastocyst: trophectoderm, primitive endoderm, and epiblast. Owing to limited resources of biological specimens, our understanding of how the earliest lineage commitments are regulated remains narrow. Here, we examined gene expression in 241 individual cells from early and late human blastocysts to delineate dynamic gene-expression changes. We distinguished all three lineages and further developed a 3D model of the inner cell mass and trophectoderm in which individual cells were mapped into distinct expression domains. We identified in silico precursors of the epiblast and primitive endoderm lineages and revealed a role for MCRS1, TET1, and THAP11 in epiblast formation and their ability to induce naive pluripotency in vitro. Our results highlight the potential of single-cell gene-expression analysis in human preimplantation development to instruct human stem cell biology.

Cell Lineage Specification at Single Cell Resolution

“…Several reports investigated transcriptome of human pre-implantation embryos by single cell sequencing, from which not only a number of genes with conserved expression as in mouse was observed, but also significant difference between human and mouse was identified [11,12]. Moreover, X reactivation and inactivation in female human blastocyst is different as well [13].…”

Development of 3D Tissue Reconstruction Method from Single-cell RNA-seq Data

“…Durruthy-Durruthy et al analyzed 382 individual mouse otocyst and neuroblast cells and reconstructed a mouse otocyst structure by 3D principal component analysis (PCA) [4]. This method was also applied to early and late human blastocysts in [5]. However, each cell was mapped to only the surface of a sphere and it was still insufficient to map cells on the original tissue structure.…”

“…The main contributions of this study are as follows. (i) We obtain at least 18 cells from a blastocyst (E3.5d) in contrast to average 8 cells from a blastocyst (E5.0d -E6.0d) in [5] towards an in silico 3D tissue reconstruction from one individual tissue transcriptome data. (ii) We propose an in silico reconstruction method which can map cells not only to the surface but also to the inner side of a sphere structure by referring expression levels of known marker genes.…”

Single cell analysis of lincRNA expression during human blastocyst differentiation identifiesTERT(+)multi-lineage precursor cells

“…To address these limitations, we implemented an experimental approach ensuring the high likelihood of studying truly viable human embryonic cells and employing the strict morphological criteria for segregation of viable blastocyst cells into the early and late blastocyst developmental stages defined by classic embryological criteria [18]. Using this approach, single-cell gene expression profiling was successfully carried-out in 241 individual cells recovered from early and late human blastocysts to delineate the spatiotemporal dynamics of gene expression changes during human blastocyst differentiation [18].…”

“…To address these limitations, we implemented an experimental approach ensuring the high likelihood of studying truly viable human embryonic cells and employing the strict morphological criteria for segregation of viable blastocyst cells into the early and late blastocyst developmental stages defined by classic embryological criteria [18]. Using this approach, single-cell gene expression profiling was successfully carried-out in 241 individual cells recovered from early and late human blastocysts to delineate the spatiotemporal dynamics of gene expression changes during human blastocyst differentiation [18]. This experimental system faithfully distinguished all three lineages that form the human blastocyst and facilitated development of a threedimensional in silico model of the inner cell mass and trophectoderm, in which individual cells were mapped into distinct expression domains corresponding to the lineage differentiation in human blastocysts [18].…”