RNA sequencing of hiPSC-derived endothelial cells via doxycycline-induced ETV2 transcription factor overexpression. RNA sequencing of hiPSC-derived endothelial cells via doxycycline-induced ETV2 transcription factor overexpression

Endothelial cells are known to express tissue-specific phenotypes to support the organ functionality due to the residing tissue microenvironment niche. The objective of this study is to compare the transcriptomic profiling of the generated endothelial cells (called iETV2-ECs) in neuro maintenance medium (NMM) with endothelial cells derived from primary human brain and other organs, as well as human induced pluripotent stem cells. The iETV2-ECs were generated from human induced pluripotent stem cells (hiPSC) that were genome engineered with a doxycycline-inducible overexpression cassette for the master regulator of endothelial differentiation (i.e. ETV2). The cells were treated with doxycycline for 8 days in the NMM supplemented with bFGF, FBS and endothelial cell growth supplements and subsequently maintained in NMM up to 21 days. Cells were harvested on day 8, 14 and 21 days for total RNA extraction, and their transcriptomic profiles were analyzed by bulk RNA sequencing technique. P ublicly-available RNA sequencing datasets of different endothelial cell types were integrated into the iETV2-EC data set for differentially expressed genes analysis. The ultimate goal of this project is to develop vascularized 3D neural model to study human brain vascularization and disease modelling. Overall design: The goal of this study is to analyze the transcriptomic profiling of the generated endothelial cells (iETV2-Ecs) in neuro maintenance medium. RNA profiles of endothelial cells at day 8, 14 and 21 days. Three independent samples for each time points (n=3)

内皮细胞（endothelial cells）会因所处组织微环境龛的调控，表达组织特异性表型，从而维持器官的正常功能，这一特性已被学界广泛证实。本研究的核心目标为：对比在神经维持培养基（neuro maintenance medium, NMM）中培养的诱导型内皮细胞（命名为iETV2-ECs）的转录组谱，与原代人源脑组织、其他器官来源的内皮细胞，以及人诱导多能干细胞（human induced pluripotent stem cells, hiPSC）来源内皮细胞的转录组差异。iETV2-ECs由经基因组工程改造的人诱导多能干细胞诱导产生：该干细胞搭载了多西环素诱导型过表达元件，用于调控内皮分化的核心转录因子ETV2的表达。细胞在添加了碱性成纤维细胞生长因子（bFGF）、胎牛血清（FBS）及内皮细胞生长添加剂的神经维持培养基中，经多西环素诱导培养8天，随后继续维持于该培养基中至第21天。分别于第8、14、21天收集细胞，提取总RNA，通过批量RNA测序（bulk RNA sequencing）技术分析其转录组谱。将公开获取的不同内皮细胞类型的RNA测序数据集整合至iETV2-EC数据集，用于差异表达基因分析。本项目的最终目标为构建血管化三维神经模型，以研究人脑血管生成机制及疾病建模。实验设计概述：本研究旨在分析神经维持培养基中培养的诱导型内皮细胞（iETV2-ECs）的转录组谱。检测节点为第8、14、21天的内皮细胞转录组，每个时间点设置3次独立生物学重复（n=3）