Division of Thrombosis and Hemostasis, Department of Internal Medicine, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
PLoS One. 2024 Feb 23;19(2):e0297465. doi: 10.1371/journal.pone.0297465. eCollection 2024.
Endothelial cells generated from induced pluripotent stem cells (hiPSC-ECs) show the majority of endothelial cell characteristics and markers, such as cobblestone morphology and the expression of VEGF and VE-cadherin. However, these cells are failing to show a mature endothelial cell phenotype, which is represented by the low expression and production of von Willebrand Factor (VWF) leading to the round morphology of the Weibel Palade Bodies (WPBs). The aim of this study was to improve the maturation process of hiPSC-ECs and to increase the levels of VWF.
hiPSC-ECs were differentiated by a standard differentiation protocol from hiPSCs generated from healthy control donors. To induce maturation, the main focus was to increase the expression and/or production of VWF by the adjustment of potential parameters influencing differentiation and maturation. We also compared alternative differentiation protocols. Cells were analyzed for the expression of endothelial cell markers, WPB structure, and the production and secretion of VWF by flow cytometry, confocal microscopy and ELISA.
The generated hiPSC-ECs have typical endothelial cell surface expression profiles, with low expression levels of non-endothelial markers as expected. Co-culture with pericytes, varying concentrations and timing of differentiation factors, applying some level of flow, and the addition of HDAC inhibitors did not substantially improve maturation of hiPSC-ECs. Transfection with the transcription factor ETV2 to induce a faster hiPSC-EC differentiation process resulted in a limited increase in VWF production, secretion, and elongation of WPB structure. Alternative differentiation protocols had limited effect.
hiPSCs-ECs have the potential to show a more mature endothelial phenotype with elongated WPBs after >30 days in culture. However, this comes with limitations as there are very few cells detected, and cells are deteriorating after being in culture for extended periods of time.
诱导多能干细胞（hiPSC）分化而来的内皮细胞表现出大多数内皮细胞的特征和标志物，如鹅卵石形态以及 VEGF 和 VE-钙黏蛋白的表达。然而，这些细胞未能表现出成熟的内皮细胞表型，这表现为血管性血友病因子（VWF）的低表达和产生，导致 Weibel Palade 小体（WPB）呈圆形。本研究旨在改善 hiPSC-EC 的成熟过程并增加 VWF 的水平。
通过从健康对照供体中生成的 hiPSC 的标准分化方案将 hiPSC-EC 进行分化。为了诱导成熟，主要关注通过调整影响分化和成熟的潜在参数来增加 VWF 的表达和/或产生。我们还比较了替代分化方案。通过流式细胞术、共聚焦显微镜和 ELISA 分析细胞内皮细胞标志物的表达、WPB 结构以及 VWF 的产生和分泌。
生成的 hiPSC-EC 具有典型的内皮细胞表面表达谱，与预期的一样，非内皮标志物的表达水平较低。与周细胞共培养、改变分化因子的浓度和时间、施加一定程度的流动以及添加 HDAC 抑制剂并没有显著改善 hiPSC-EC 的成熟。转录因子 ETV2 的转染可诱导更快的 hiPSC-EC 分化过程，但仅导致 VWF 产生、分泌和 WPB 结构伸长的有限增加。替代分化方案的效果有限。
hiPSC-EC 具有在培养超过 30 天后表现出更成熟的内皮表型和拉长的 WPB 的潜力。然而，这存在局限性，因为检测到的细胞非常少，并且细胞在培养较长时间后会恶化。
