Human induced pluripotent stem cells (hiPSCs) possess the potential to differentiate toward vascular cells including endothelial cells (ECs), pericytes, and smooth muscle cells. Epigenetic mechanisms including DNA methylation and histone modification play a crucial role in regulating lineage differentiation and specification. Herein, we utilized a three-stage protocol to induce differentiation of mesoderm, vascular progenitors, and ECs from hiPSCs and investigated the regulatory effects of histone acetylation on the differentiation processes. We found that the expression of several histone deacetylases (HDACs), including HDAC1, HDAC5, and HDAC7, were greatly upregulated at the second stage and downregulated at the third stage. Interestingly, although HDAC1 remained in the nucleus during the EC differentiation, HDAC5 and HDAC7 displayed cytosol/nuclear translocation during the differentiation process. Inhibition of HDACs with sodium butyrate (NaBt) or BML210 could hinder the differentiation of vascular progenitors at the second stage and facilitate EC induction at the third stage. Further investigation revealed that HDAC may modulate the stepwise EC differentiation via regulating the expression of endothelial transcription factors ERG, ETS1, and MEF2C. Opposite to the expression of EC markers, the smooth muscle/pericyte marker ACTA2 was upregulated at the second stage and downregulated at the third stage by NaBt. The stage-specific regulation of ACTA2 by HDAC inhibition was likely through regulating the expression of TGFβ2 and PDGFB. This study suggests that HDACs play different roles at different stages of EC induction by promoting the commitment of vascular progenitors and impeding the later stage differentiation of ECs.

Human induced pluripotent stem cells (hiPSCs) not only provide an abundant source of vascular cells for potential therapeutic applications in vascular disease but also constitute an excellent model for understanding the mechanisms that regulate the differentiation and the functionality of vascular cells. Here, we reported that myocyte enhancer factor 2C (MEF2C) transcription factor, but not any other members of the MEF2 family, was robustly upregulated during the differentiation of vascular progenitors and endothelial cells (ECs) from hiPSCs. Vascular endothelial growth factors (VEGF) strongly induced MEF2C expression in endothelial lineage cells. The specific upregulation of MEF2C during the commitment of endothelial lineage was dependent on the extracellular signal regulated kinase (ERK). Moreover, knockdown of MEF2C with shRNA in hiPSCs did not affect the differentiation of ECs from these hiPSCs, but greatly reduced the migration and tube formation capacity of the hiPSC-derived ECs. Through a chromatin immunoprecipitation-sequencing, genome-wide RNA-sequencing, quantitative RT-PCR, and immunostaining analyses of the hiPSC-derived endothelial lineage cells with MEF2C inhibition or knockdown compared to control hiPSC-derived ECs, we identified TNF-related apoptosis inducing ligand (TRAIL) and transmembrane protein 100 (TMEM100) as novel targets of MEF2C. This study demonstrates an important role for MEF2C in regulating human EC functions and highlights MEF2C and its downstream effectors as potential targets to treat vascular malfunction-associated diseases.