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Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

ABSTRACT: We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.

SUBMITTER: Wong WT

PROVIDER: S-EPMC5391207 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

Wong Wing Tak WT Matrone Gianfranco G Tian XiaoYu X Tomoiaga Simion Alin SA Au Kin Fai KF Meng Shu S Yamazoe Sayumi S Sieveking Daniel D Chen Kaifu K Burns David M DM Chen James K JK Blau Helen M HM Cooke John P JP

eLife 20170321

We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcription ...[more]

PMID: 28323620

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Project description:BackgroundUnderstanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out.ResultsA whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.ConclusionThe mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns. These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate. These data will form the basis for understanding not only cell fate determination and cellular homeostasis in the normal mammary epithelium but also the contribution of different mammary epithelial cell types to the etiology and molecular pathology of breast disease.

Dataset Information

Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

Publications

Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

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