BCL6B-dependent suppression of ETV2 hampers endothelial cell differentiation.

in Stem cell research & therapy by Zhonghao Li, Wei Wu, Qiushi Li, Xin Heng, Wei Zhang, Yinghong Zhu, Lin Chen, Ziqi Chen, Mengcheng Shen, Ning Ma, Qingzhong Xiao, Yi Yan

TLDR

  • This study investigates the role of BCL6B in endothelial cell (EC) differentiation and its potential mechanisms. The study uses doxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown to determine the important role of BCL6B in EC differentiation and its potential mechanisms. The study finds that overexpression of BCL6B suppresses while knockdown of BCL6B improves EC differentiation from hiPSCs. The study also finds that the overexpression of BCL6B attenuates the capacity of derived hiPSC-ECs to form a tubular structure and represses the growth of VOs, whereas BCL6B knockdown has little effect on the size of VOs. The study confirms that the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression. The study suggests that BCL6B plays a crucial role in EC differentiation and its potential mechanisms, and could be a potential therapeutic target for EC-related diseases.

Abstract

B-cell CLL/lymphoma 6 member B (BCL6B) operates as a sequence-specific transcriptional repressor within the nucleus, playing crucial roles in various biological functions, including tumor suppression, immune response, stem cell self-renew, and vascular angiogenesis. However, whether BCL6B is involved in endothelial cell (EC) development has remained largely unknown. ETS variant transcription factor 2 (ETV2) is well known to facilitate EC differentiation. This study aims to determine the important role of BCL6B in EC differentiation and its potential mechanisms. Doxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown were established and subjected to differentiate into ECs and vessel organoids (VOs). RNA sequencing analysis was performed to identify potential signal pathways regulated by BCL6B during EC differentiation from hiPSCs. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pluripotency and vascular-specific marker genes expression. EC differentiation efficiency was determined by Flow cytometry analysis. The performance of EC was evaluated by in vitro Tube formation assay. The protein expression and the vessel-like structures were assessed using immunofluorescence analysis or western blot. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP)-PCR analysis were used to determine the regulatory relationship between BCL6B and ETV2. Functional ECs and VOs were successfully generated from hiPSCs. Notably, overexpression of BCL6B suppressed while knockdown of BCL6B improved EC differentiation from hiPSCs. Additionally, the overexpression of BCL6B attenuated the capacity of derived hiPSC-ECs to form a tubular structure. Furthermore, compared to the control VOs, BCL6B overexpression repressed the growth of VOs, whereas BCL6B knockdown had little effect on the size of VOs. RNA sequencing analysis confirmed that our differentiation protocol induced landscape changes for cell/tissue/system developmental process, particularly vascular development and tube morphogenesis, which were significantly modulated by BCL6B. Subsequent experiments confirmed the inhibitory effect of BCL6B is facilitated by the binding of BCL6B to the promoter region of ETV2, led to the suppression of ETV2's transcriptional activity. Importantly, the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression. BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.

Overview

  • The study investigates the role of BCL6B in endothelial cell (EC) differentiation and its potential mechanisms using doxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown. The study aims to determine the important role of BCL6B in EC differentiation and its potential mechanisms. The study uses RNA sequencing analysis, quantitative real-time PCR (qRT-PCR), flow cytometry analysis, in vitro Tube formation assay, immunofluorescence analysis or western blot, Luciferase reporter gene assay, and chromatin immunoprecipitation (ChIP)-PCR analysis to identify potential signal pathways regulated by BCL6B during EC differentiation from hiPSCs. The study finds that overexpression of BCL6B suppresses while knockdown of BCL6B improves EC differentiation from hiPSCs. The study also finds that the overexpression of BCL6B attenuates the capacity of derived hiPSC-ECs to form a tubular structure and represses the growth of VOs, whereas BCL6B knockdown has little effect on the size of VOs. The study confirms that the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression. The study suggests that BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.

Comparative Analysis & Findings

  • The study compares the outcomes observed under different experimental conditions or interventions detailed in the study. The study identifies that overexpression of BCL6B suppresses while knockdown of BCL6B improves EC differentiation from hiPSCs. The study also finds that the overexpression of BCL6B attenuates the capacity of derived hiPSC-ECs to form a tubular structure and represses the growth of VOs, whereas BCL6B knockdown has little effect on the size of VOs. The study confirms that the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression.

Implications and Future Directions

  • The study's findings suggest that BCL6B plays a crucial role in EC differentiation and its potential mechanisms. The study identifies that BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2. The study suggests that BCL6B could be a potential therapeutic target for EC-related diseases. The study also identifies that the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression. The study suggests that further research is needed to explore the regulatory relationship between BCL6B and ETV2 in EC differentiation and its potential therapeutic implications.
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