HDACs regulate the differentiation of endothelial cells from human iPSCs

Abstract

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.

Keywords: HDAC inhibitor; differentiation; endothelial cells; histone deacetylase; induced pluripotent stem cells.

Figure 1.. Differentiation of hiPSCs toward ECs.

(A) Schematic protocol for the induction of ECs from hiPSCs. (B) Typical microscope images of hiPSCs and differentiating cells at different stages. (C) Fluorescent images of hiPSC-ECs immunostained for CD31 and ACTA2. (D) The expression of CD31 and CD144 in hiPSCs and the differentiating cells was assessed by qRT-PCR. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 versus hiPSCs. (E) Tubes formed by hiPSC-ECs on matrigel in vitro.

Figure 2.. The expression of HDAC genes during EC differentiation.

(A) The expression of Class I HDACs (HDAC1, 2, 3, and 8) in hiPSCs and differentiating cells was assessed by qRT-PCR. (B) The expression of Class II HDACs (HDAC4, 5, 6, and 7) in hiPSCs and differentiating cells was assessed by qRT-PCR.

Figure 3.. Expression regulation of HDAC1, 5, and 7 at the second differentiation stage.

(A) qRT-PCR analysis of the expression of HDAC1, 5, and 7 in differentiating cells cultured with the indicated growth factors from day 2 to day 4. (B) Differentiating cells were cultured with either ERK1/2 or PI3K inhibitor, U0126 (10 μM) and LY294002 (10 μM) from day 2 to day 4. The expression of HDAC1, 5, and 7 was assessed by qRT-PCR.

Figure 4.. The localization of HDAC1, 5, and 7 during EC differentiation from iPSCs.

Immunofluorescent staining for HDAC1, 5, and 7 in iPSCs (Day 0), the vascular progenitors (day 4) and ECs (day 8). Scale bar=100μm.

Figure 5.. Effects of HDAC inhibition on EC differentiation at the second stage.

(A) Schematic representation of the protocol for HDAC inhibition. (B) After treatment with NaBt (1 or 2 mM) from day 2 to day 4, the cells were assessed for their expression of ACTA2, CNN1, CD31, and CD144 by qRT-PCR. (C) After treatment with BML210 (10 μM) from day 2 to day 4, , the cells were assessed for their expression of ACTA2, CNN1, CD31, and CD144 by qRT-PCR. (D) After treatment of NaBt (1 or 2 mM) from day 2 to day 4, the cells were allowed to further culture and differentiation. The cells at day 8 were immunostained with antibodies against CD31 (green) and ACTA2 (red). The nuclei were stained with DAPI (blue).

Figure 6.. Effects of HDAC inhibition on EC differentiation at the third stage.

(A) Schematic representation of the protocol for HDAC inhibition. (B) After treatment with NaBt (1 or 2 mM) from day 5 to day 8, the cells were assessed for their expression of ACTA2, CNN1, CD31, and CD144 by qRT-PCR. (C) After treatment with BML210 (10 μM) from day 5 to day 8, the cells were assessed for their expression of ACTA2, CNN1, CD31, and CD144 by qRT-PCR.

Figure 7.. Effects of NaBt on the expression of endothelial transcription factors.

(A) The expression of endothelial transcription factors such as ERG, ETS1, GATA2, and MEF2C, was assessed by qRT-PCR. (B) After treatment with NaBt from day 2 to day 4, the cells were assessed for their expression of endothelial transcription factors by qRT-PCR. (C) After treatment with NaBt from day 5 to day 8, the cells were assessed for their expression of endothelial transcription factors by qRT-PCR.

Figure 8.. Effects of NaBt on the expression of the TGFβ and the PDGFB genes.

(A) After treatment with NaBt from day 2 to day 4, the cells were assessed for their expression of TGFβ1, TGFβ2, and PDGFB by qRT-PCR. (B) After treatment with NaBt from day 5 to day 8, the cells were assessed for their expression of TGFβ1, TGFβ2, and PDGFB by qRT-PCR. (C) After treatment with PDGF-BB (100ug/ml) from day 2 to day 4, the cells were assessed for their expression of ACTA2 and CNN1 by qRT-PCR. (D) After treatment with PDGF-BB (100ug/ml) from day 5 to day 8, the cells were assessed for their expression of ACTA2 and CNN1 by qRT-PCR.