Aims Endothelial cell (EC) dysfunction plays a key role in the initiation and progression of cardiovascular disease. However, studying these disorders in ECs from patients is challenging, hence the use of human induced pluripotent stem cells (hiPSCs) and their in vitro differentiation into ECs represents a very promising approach. Still, the generation of hiPSC-derived ECs (hECs) remains demanding as a cocktail of growth factors and an intermediate purification step are required for hEC enrichment. Therefore, we probed the utility of a forward programming approach using transgenic hiPSC lines. Methods and Results We have used the transgenic hiPSC line PGP1 ETV2 iso2 to explore the in vitro differentiation of hECs via doxycycline-dependent induction of the transcription factor ETV2 and compared these with a standard differentiation protocol for hECs using non-transgenic control hiPSCs. The transgenic hECs were highly enriched without an intermediate purification step and expressed – as non-transgenic hECs and HUVECs – characteristic EC markers. The viability and yield of transgenic hECs were strongly improved by applying EC growth medium during differentiation. This protocol was successfully applied in two more transgenic hiPSC lines yielding reproducible results with low line-to-line variability. Transgenic hECs displayed typical functional properties, such as tube formation and LDL uptake, and a more mature phenotype than non-transgenic hECs. Transgenic hiPSCs preferentially differentiated into the arterial lineage, this was further enhanced by adding a high VEGF concentration to the medium. We also demonstrate that complexing lentivirus with magnetic nanoparticles and application of a magnetic field enables efficient transduction of transgenic hECs. Conclusions We have established a highly efficient, cost-effective, and reproducible differentiation protocol for the generation of functional hECs via forward programming. The transgenic hECs can be genetically modified and are a powerful tool for disease modelling, tissue engineering, and translational purposes.

中文翻译：

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通过转录因子 ETV2 对 hiPSC 进行正向编程：快速、可重复且经济高效的生成高度富集的功能性内皮细胞


内皮细胞 （EC） 功能障碍在心血管疾病的发生和发展中起关键作用。然而，研究患者 EC 中的这些疾病具有挑战性，因此使用人诱导多能干细胞 （hiPSC） 及其体外分化为 ECs 代表了一种非常有前途的方法。尽管如此，hiPSC 衍生的 EC （hEC） 的生成仍然要求很高，因为 hEC 富集需要生长因子混合物和中间纯化步骤。因此，我们探讨了使用转基因 hiPSC 系的正向编程方法的效用。方法和结果我们使用转基因 hiPSC 系 PGP1 ETV2 iso2 来探索通过多西环素依赖性诱导转录因子 ETV2 的体外分化 hECs，并将其与使用非转基因对照 hiPSCs 的 hECs 标准分化方案进行了比较。转基因 hECs 在没有中间纯化步骤的情况下高度富集，并表达为非转基因 hEC 和 HUVEC 特征性 EC 标志物。在分化过程中施用 EC 生长培养基大大提高了转基因 hECs 的活力和产量。该方案已成功应用于另外两个转基因 hiPSC 系，产生具有低线间变异性的可重复结果。转基因 hECs 表现出典型的功能特性，例如管形成和 LDL 摄取，并且比非转基因 hECs 更成熟。转基因 hiPSC 优先分化为动脉谱系，通过向培养基中添加高浓度的 VEGF 进一步增强了这一点。我们还证明，将慢病毒与磁性纳米颗粒复合并应用磁场可以有效转导转基因 hEC。 结论 我们已经建立了一种高效、经济且可重复的分化方案，用于通过正向编程生成功能性 hEC。转基因 hEC 可以进行基因改造，是疾病建模、组织工程和转化目的的强大工具。