Transcriptome analyses of inner cell mass and trophectoderm cells isolated by magnetic‐activated cell sorting from bovine blastocysts using single cell RNA‐seq

Abstract

Research on bovine embryonic stem cells (bESCs) has been hampered because bESCs are cultured in conditions that are based on information obtained from culturing mouse and human inner cell mass (ICM) cells. The aim of this study was to compare gene expression in ICM and trophectoderm (TE) cell lineages of bovine embryos and to discuss the findings relative to information available for mice and humans. We separated a high-purity (>90%) ICM and TE from bovine blastocysts by magnetic-activated cell sorting and analysed their transcriptomes by single cell RNA-seq. Differentially expressed genes (DEGs) were assessed using Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. Finally, qRT-PCR was performed to validate the RNA-seq results. From 207 DEGs identified (adjusted p ≤ .05; fold change ≥2), 159 and 48 had greater expression in the ICM and TE cells respectively. We validated 27 genes using qRT-PCR and found their expression patterns were mostly similar to those of RNA-seq, including 12 novel ICM-dominant (HNF4A, CCL24, FGFR4, IFITM3, PTCHD2, GJB5, FN1, KLK7, PRDM14, GRP, FGF19 and GCM1) and two novel TE-dominant (SLC10A1 and WNT4) genes. Bioinformatics analysis showed that these DEGs are involved in many important pathways, such as MAPK and cancer cell pathways, and these pathways have been shown to play essential roles in mouse and human ESCs in the self-renewal and pluripotent maintenance. As a conclusion, there were sufficient differences to allow us to conclude that the control of pluripotency in bovine ICM cells is species-specific.

The expression level of SOX2 at the blastocyst stage regulates the developmental capacity of bovine embryos up to day-13 of in vitro culture

“…SOX2 expression is not affected by lack of OCT4 (Daigneault et al, 2018), but OCT4 is required for NANOG expression (Simmet et al, 2018). Both markers SOX2 and NANOG are highly expressed in cells of the ICM during the blastocyst stage in mice (Avilion, et al, 2003), humans (Adjaye, 2005) and bovine embryos (Ozawa et al, 2012;Zhao et al, 2016). The absence of SOX2 promotes the differentiation of stem cells to cells that are similar to trophoblasts (Masui et al, 2007).…”

Single-cell gene expression of the bovine blastocyst

“…2015; Hosseini et al . 2015; Zhao et al . 2016), and genes involved in important processes for the developing embryo including the Hippo signaling pathway, epigenetic regulation, tight junction formation, cell polarity and chemokine signaling.…”

Establishment of bovine expanded potential stem cells

“…We and others recently reported establishment of mouse, human, and porcine expanded potential stem cells (EPSCs) (7)(8)(9)(10). We posited that deriving stem cells from earlier preimplantation embryos, for example, mouse fourcell or eight-cell cleavage embryos, might help overcome the substantial species differences reported in the blastocyst-stage embryos (11)(12)(13)(14)(15)(16). Following this line of reasoning, EPSCs were established by inhibiting molecules and pathways operating in preblastocyst embryos (7,9,10).…”