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Open Access Research

Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment

Lynsey Howard1, Ruth M Mackenzie1, Nikolay A Pchelintsev2, Tony McBryan2, John D McClure1, Martin W McBride1, Nicole M Kane1, Peter D Adams2, Graeme Milligan3 and Andrew H Baker1*

Author Affiliations

1 British Heart Foundation Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, 126 University Place, Glasgow, UK G12 8TA

2 Institute of Cancer Sciences, Garscube Estate, Beatson Institute for Cancer Research, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK, G61 1BD

3 Institute of Neuroscience and Psychology, 58 Hillhead Street, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK G12 8QB

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Stem Cell Research & Therapy 2013, 4:36  doi:10.1186/scrt192

Published: 24 April 2013

Abstract

Introduction

Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage.

Methods

We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes.

Results

We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation.

Conclusion

This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease.

Keywords:
Gene expression; Epigenetics; Endothelial differentiation; Embryonic stem cells; Transcription factors