Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh.
Centre for Discovery Brain Sciences, University of Edinburgh.
J Vis Exp. 2023 Jul 28(197). doi: 10.3791/65492.
The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.
心脏是在发育过程中第一个具有功能的器官，因此在妊娠早期就开始进行血液循环。除了运输氧气和营养物质以确保胎儿生长外，胎儿循环还通过机械线索控制内皮细胞层中发生的许多关键发育事件。生物力学信号诱导血管结构变化，建立动静脉规范，并控制造血干细胞的发育。发育组织的不可及性限制了对循环在人类早期发育中作用的理解；因此，体外模型是研究血管力学生物学的重要工具。本文描述了一种从人诱导多能干细胞中分化内皮细胞及其随后接种到流体设备中以研究它们对机械线索反应的方案。这种方法允许在机械刺激下对内皮细胞进行长期培养，然后回收内皮细胞进行表型和功能特征分析。这里建立的体外模型对于阐明转导机械线索介导的信号的细胞内分子机制将是至关重要的，这些机制最终协调了人类胎儿生命期间血管的发育。
