多能干细胞已被各个研究者用于分化和表征内皮细胞（EC）和血管平滑肌细胞（VSMC），以用于血管损伤的临床治疗。研究持续发展以分化和表征具有更高血管化潜能和低恶性转化风险的细胞，以适应受体的需求。与以往研究不同，本研究旨在使用逐步分化技术将诱导多能干细胞（iPS细胞）分化为内皮祖细胞（EPC）和血管平滑肌细胞（VSMC）。通过阐明分化过程中特定阶段基因和蛋白的时空表达，实现了这一目标。还在分化期间研究了iPS细胞中高表达的致癌基因的存在情况。 诱导iPS细胞分化为侧板中胚层细胞（Flk1+）。在中胚层分化期间的第5.5天，分离了Flk1+细胞群体。利用VEGF165和血小板源性生长因子-BB（PDGF-BB）分别将Flk1+细胞分化为EPC和VSMC，并使用基因表达水平、免疫细胞化学（ICC）和免疫印迹（WB）方法对其进行表征。在分化过程中，同时研究了标记基因和原癌基因Myc和Klf4基因的表达水平。 Flk1+细胞的最佳分离时间为第5.5天。通过EPC特异性标记物，如Kdr、Pecam1、CD133、Cdh5、Efnb2、Vcam1；以及VSMC特异性标记物，如Acta2、Cnn1、Des和Myh11，对EPC和VSMC进行了分化和表征。根据它们的时间基因表达、蛋白合成和定位进行了验证。在EPC和VSMC分化期间，Myc和Klf4基因表达水平显著下降。 根据基因表达水平、ICC和WB的特征，我们成功分化了EPC和VSMC，并观察到这两种分化细胞中致癌基因表达水平的显著下降。在安全性方面，所描述的方法提供了更好的安全保障。利用这种方法获得的EPC和VSMC可能是移植和血管再生的良好候选物。 © 2023作者

Pluripotent stem cells have been used by various researchers to differentiate and characterize endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) for the clinical treatment of vascular injuries. Studies continue to differentiate and characterize the cells with higher vascularization potential and low risk of malignant transformation to the recipient. Unlike previous studies, this research aimed to differentiate induced pluripotent stem (iPS) cells into endothelial progenitor cells (EPCs) and VSMCs using a step-wise technique. This was achieved by elucidating the spatio-temporal expressions of the stage-specific genes and proteins during the differentiation process. The presence of highly expressed oncogenes in iPS cells was also investigated during the differentiation period.Induced PS cells were differentiated into lateral mesoderm cells (Flk1+). The Flk1+ populations were isolated on day 5.5 of the mesodermal differentiation period. Flk1+ cells were further differentiated into EPCs and VSMCs using VEGF165 and platelet-derived growth factor-BB (PDGF-BB), respectively, and then characterized using gene expression levels, immunocytochemistry (ICC), and western blot (WB) methods. During the differentiation steps, the expression levels of the marker genes and proto-oncogenic Myc and Klf4 genes were simultaneously studied.The optimal time for the isolation of Flk1+ cells was on day 5.5. EPCs and VSMCs were differentiated from Flk1+ cells and characterized with EPC-specific markers, including Kdr, Pecam1, CD133, Cdh5, Efnb2, Vcam1; and VSMC-specific markers, including Acta2, Cnn1, Des, and Myh11. Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in Myc and Klf4 gene expression levels were observed during the EPCs and VSMC differentiation period.EPCs and VSMCs were successfully differentiated from iPS cells and characterized by gene expression levels, ICC, and WB. We observed significant decreases in oncogene expression levels in the differentiated EPCs and VSMCs. In terms of safety, the described methodology provided a better safety margin. EPCs and VSMC obtained using this method may be good candidates for transplantation and vascular regeneration.© 2023 The Author(s).