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.DOI: http://dx.doi.org/10.7554/eLife.23588.001

TBX20 Regulates Angiogenesis Through the Prokineticin 2–Prokineticin Receptor 1 Pathway

“…To examine the Tbx20 gene expression in endothelial differentiation, two model systems were used, one is the mESC differentiation to ECs and the other one is heterokaryon induced differentiation of pluripotent stem cells into endothelial lineage. During the mESC differentiation into endothelial lineage 18 , Tbx20 gene expression was upregulated by 16-fold at day 8 of differentiation compared with mESCs (Fig. S1a).…”

“…S1a). In a heterokaryon model 18 , fusing mESC with HUVECs induced differentiation of pluripotent stem cells toward endothelial lineage. We found that Tbx20 gene expression was upregulated and peaked at 4-fold at 6 hours after heterokaryon formation compared with co-culture control (CT, Fig.…”

“…In this model, murine embryonic stem cells (ESCs) are fused with adult human ECs, and RNA-seq is performed at intervals to assess changes in global gene expression of the resulting hybrid cells. We found that factors maintaining endothelial phenotype act on the ESCs to induce the expression of genes associated with endothelial lineage 18 . Using this model, we discovered novel genes implicated in endothelial development.…”

“…The chimeric heterokaryon model was first used to identify novel genes in nuclear reprogramming to pluripotency 17 . We have adopted the model to identify novel genes in endothelial development 18 . In this model, murine embryonic stem cells (ESCs) are fused with adult human ECs, and RNA-seq is performed at intervals to assess changes in global gene expression of the resulting hybrid cells.…”

A Missing LNC in Vascular Diseases

“…This is an excellent approach to obtaining a global view of the transcriptional activity of a cell. An analysis of RNA-seq data can reveal novel transcripts that are determinants of phenotype and function (2). However, the amount of data obtained from RNA-seq can be overwhelming.…”