Abstract 18031: Identification of Novel Determinants Towards Endothelial Differentiation Using a Heterokaryon Approach

Abstract

For directed differentiation of embryonic stem cells (ESCs) to endothelial cells (ECs), we need more information regarding the genes that are activated or suppressed, and their temporal order of activation, during EC specification. Accordingly, we sought to use heterokaryons (the stable fusion product of two different cell types) to identify critical determinants of EC differentiation. The premise is that cytoplasmic factors in the EC that maintain its phenotype will act on the ESC nucleus in the heterokaryon to reprogram it toward EC lineage. Using murine specific probes, or RNAseq, we will identify key genes that are modulated during early EC determination. Accordingly, we employed murine ESC labelled by transduction with retroviruses encoding GFP, and human ECs labelled with CellTracker Red, and induced heterokaryon formation using sendai virus-enveloped protein. Multi-nucleated heterokaryons were generated, which contained punctate murine nuclei and diffusely staining human nuclei. The dual-stained heterokaryons were sorted from the unfused cells and homokaryons by FACS. The extent and timing of differentiation of the murine ESCs within the heterokaryons were examined by probing for EC specific genes (KDR, CD144, vWF and CD31) and ES pluripotency markers (Nanog, Sox2 and Oct4) using murine specific primers. Intriguingly, heterokaryons appear to recapitulate ontogeny, in that early mesodermal genes (eg. KDR) are upregulated in murine ESC at 6h, whereas more EC-specific genes are not upregulated until later (eg. vWF at 24h). Data from RNAseq studies further confirmed the major transcriptional factors and regulators and identified some novel factors for endothelial differentiation. These novel genes will be confirmed using gain- and loss- of function studies of ESCs undergoing differentiation. These studies shall provide new knowledge regarding the hierarchy of genes regulating differentiation to the EC lineage and thus provide insight into vascular development, and ultimately new therapeutic avenues for vascular regeneration.