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Transcriptome analyses of inner cell mass and trophectoderm cells isolated by magnetic‐activated cell sorting from bovine blastocysts using single cell RNA ‐seq
Author(s) -
Zhao XM,
Cui LS,
Hao HS,
Wang HY,
Zhao SJ,
Du WH,
Wang D,
Liu Y,
Zhu HB
Publication year - 2016
Publication title -
reproduction in domestic animals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.546
H-Index - 66
eISSN - 1439-0531
pISSN - 0936-6768
DOI - 10.1111/rda.12737
Subject(s) - kegg , biology , inner cell mass , transcriptome , embryonic stem cell , cell sorting , gene , cell , microbiology and biotechnology , gene expression , rna , rna seq , induced pluripotent stem cell , genetics , blastocyst , embryogenesis
Contents Research on bovine embryonic stem cells ( bESC s) has been hampered because bESC s 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 ( DEG s) 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 DEG s 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 ( HNF 4A , CCL 24 , FGFR 4 , IFITM 3 , PTCHD 2 , GJB 5 , FN 1 , KLK 7 , PRDM 14 , GRP , FGF 19 and GCM 1 ) and two novel TE ‐dominant ( SLC 10A1 and WNT 4 ) genes. Bioinformatics analysis showed that these DEG s 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 ESC s 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.