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

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.

Figure 1

Illumina ® (Saffron Walden, UK) whole genome expression microarray. (A) Study design with different timepoints under directed human embryonic stem cell (hESC)–endothelial cell (EC) differentiation. Arrows represent pairwise comparisons performed. (B) Principle component analysis (PCA) showing separation of primary mature human saphenous vein endothelial cells (HSVECs) and hESC-derived cells. (C) Summary of differentially expressed probe sets. Numbers in brackets represent total number of differentially expressed probe sets for the associated pairwise comparison.

Figure 2

Validation of human embryonic stem cell–endothelial cell day 2 transcription factor differential expression. (A) Differential mRNA expression of 12 of the 22 transcription factors was confirmed by TaqMan® quantitative RT-PCR. (B) Differential expression of FOXA2 mRNA was confirmed in H1s and RC10s by TaqMan® quantitative RT-PCR. *P <0.05, **P <0.01 and ***P <0.001 versus pluripotent control. RQ, relative quantitation. (C) Immunocytofluorescent staining showing upregulation of FOXA2 protein expression at human embryonic stem cell–endothelial cell (hESC–SC) day 2. DAPI, 4′,6-diamidino-2-phenylindole; HSVEC, primary human saphenous vein endothelial cell. Scale bars = 20 μm.

Figure 3

In vitro validation of predicted FOXA2 bivalency. Immunoprecipitation of H3K4me3, H3K27me3 and total H3 from chromatin of pluripotent SA461s and H9s revealed enrichment of the histone modifications at the FOXA2 transcriptional start site, as identified by quantitative PCR using primers for three regions, covering around 2.5 kb in total. *P <0.05 versus IgG control.