Unknown,Transcriptomics,Genomics,Proteomics

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Establishment of Porcine and Human Expanded Potential Stem Cells (Single-Cell RNA-seq)

ABSTRACT: Despite intensive efforts, establishing porcine embryonic stem cells have been challenging. We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the activity of critical molecular pathways that predisposes lineage differentiation in the mouse preimplantation embryo. EPSCs had enriched molecular signatures of blastomeres and possessed the developmental potency to all embryonic and extraembryonic cell lineages. In this study, we report the derivation of porcine EPSC (pEPSC) lines either directly from preimplantation embryos or by reprogramming fetal fibroblasts. Under similar culture conditions, human ESCs and iPSCs can be converted, or somatic cells are directly reprogrammed, to EPSCs (hEPSCs) that display the molecular and functional attributes reminiscent of pEPSCs. Here, we performed Single-Cell RNA-seq experiments to characterise the transcriptional heterogeneity of the EPSCs.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Sus scrofa

SUBMITTER: Xi Chen

PROVIDER: E-MTAB-7254 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The transition between morula and blastocyst stage during preimplantation development represents the first differentiation event of embryogenesis. Morula cells undergo the first cellular specialization and produce two well-defined populations of cells, the trophoblast and the inner cell mass (ICM). Embryonic stem cells (ESCs) with unlimited self-renewal capacity are believed to represent the in vitro counterpart of the ICM. Both mouse and rat ESCs can be derived from the ICM cells, but their in vitro stability differs. In this study we performed a microarray analysis in which we compared the transcriptome of mouse and rat morula, blastocyst, and ICM. This cross-species comparison represents a good model for understanding the differences in derivation and cultivation of ESCs observed in the two species. In order to identify alternative regulation of important molecular mechanisms the investigation of differential gene expression between the two species was extended at the level of signaling pathways, gene families, and single selected genes of interest. Some of the genes differentially expressed between the two species are already known to be important factors in the maintenance of pluripotency in ESCs, like for example Sox2 or Stat3, or play a role in reprogramming somatic cells to pluripotency like c-Myc, Klf4 and p53 and therefore represent interesting candidates to further analyze in vitro in the rat ESCs. This is the first study investigating the gene expression changes during the transition from morula to blastocyst in the rat preimplantation development. Our data show that in the pluripotent pool of cells of the rat and mouse preimplantation embryo substantial differential regulation of genes is present, which might explain the difficulties observed for the derivation and culture of rat ESCs using mouse conditions Casanova EA, Okoniewski MJ, Cinelli P. Cross-Species Genome Wide Expression Analysis during Pluripotent Cell Determination in Mouse and Rat Preimplantation Embryos.PLoS One. 2012;7(10):e47107. doi: 10.1371/journal.pone.0047107. Epub 2012 Oct 15. We collected morula and blastocysts stage embryos from mouse and rat and, by immunosurgery, we isolated the ICM cells from the blastocysts. All the embryos and ICMs were pooled into two groups for every developmental stage. Pooling of embryos for RNA extraction in this study was chosen mainly because of the low amounts of RNA that can be isolated from preimplantation embryos, and in addition because of the heterogeneity of the cell populations present in the embryos. For the analysis we pooled a large number (n >100) of the independent isolated embryos to achieve a sufficient accuracy of biological pooling . Due to the difficulties to isolate a larger number of embryos from mice and rats, we performed the microarray study by using two replicate samples per developmental stage.

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Establishment of Porcine and Human Expanded Potential Stem Cells (Single-Cell RNA-seq)

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