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Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2.
摘要：
背景：B细胞CLL/淋巴瘤6成员B（BCL6B）在细胞核内作为序列特异性转录抑制因子，在各种生物功能中发挥关键作用，包括肿瘤抑制，免疫反应，干细胞自我更新,血管生成。然而,BCL6B是否参与内皮细胞(EC)的发育在很大程度上还不清楚.已知ETS变体转录因子2(ETV2)促进EC分化。本研究旨在确定BCL6B在EC分化中的重要作用及其潜在机制。
方法：建立具有BCL6B过表达或BCL6B敲低的多西环素诱导的人诱导多能干细胞（hiPSC）系，并分化成EC和血管类器官（VOs）。进行RNA测序分析以鉴定在EC从hiPSC分化期间由BCL6B调节的潜在信号通路。实时定量PCR(qRT-PCR)用于检测多能性和血管特异性标记基因的表达。通过流式细胞术分析确定EC分化效率。通过体外管形成测定评价EC的性能。使用免疫荧光分析或蛋白质印迹评估蛋白质表达和血管样结构。荧光素酶报告基因测定和染色质免疫沉淀（ChIP）-PCR分析用于确定BCL6B和ETV2之间的调节关系。
结果:从hiPSC成功地产生了功能性ECs和V0s。值得注意的是,BCL6B的过表达被抑制,而BCL6B的敲低改善了从hiPSC的EC分化。此外,BCL6B的过表达减弱了衍生的hiPSC-EC形成管状结构的能力。此外，与对照VOs相比，BCL6B过表达抑制了VOs的生长，而BCL6B敲除对VOs大小影响不大。RNA测序分析证实，我们的分化方案诱导细胞/组织/系统发育过程的景观变化，特别是血管发育和管形态发生，BCL6B显著调节。随后的实验证实，BCL6B与ETV2启动子区的结合促进了BCL6B的抑制作用，从而抑制了ETV2的转录活性。重要的是,BCL6B过表达对hiPSCs向EC分化的抑制作用可以通过ETV2过表达来挽救。
结论：BCL6B通过抑制ETV2的转录活性抑制EC分化并阻碍VO发育。
方法：建立具有BCL6B过表达或BCL6B敲低的多西环素诱导的人诱导多能干细胞（hiPSC）系，并分化成EC和血管类器官（VOs）。进行RNA测序分析以鉴定在EC从hiPSC分化期间由BCL6B调节的潜在信号通路。实时定量PCR(qRT-PCR)用于检测多能性和血管特异性标记基因的表达。通过流式细胞术分析确定EC分化效率。通过体外管形成测定评价EC的性能。使用免疫荧光分析或蛋白质印迹评估蛋白质表达和血管样结构。荧光素酶报告基因测定和染色质免疫沉淀（ChIP）-PCR分析用于确定BCL6B和ETV2之间的调节关系。
结果:从hiPSC成功地产生了功能性ECs和V0s。值得注意的是,BCL6B的过表达被抑制,而BCL6B的敲低改善了从hiPSC的EC分化。此外,BCL6B的过表达减弱了衍生的hiPSC-EC形成管状结构的能力。此外，与对照VOs相比，BCL6B过表达抑制了VOs的生长，而BCL6B敲除对VOs大小影响不大。RNA测序分析证实，我们的分化方案诱导细胞/组织/系统发育过程的景观变化，特别是血管发育和管形态发生，BCL6B显著调节。随后的实验证实，BCL6B与ETV2启动子区的结合促进了BCL6B的抑制作用，从而抑制了ETV2的转录活性。重要的是,BCL6B过表达对hiPSCs向EC分化的抑制作用可以通过ETV2过表达来挽救。
结论：BCL6B通过抑制ETV2的转录活性抑制EC分化并阻碍VO发育。
