Differentiation and molecular characterization of endothelial progenitor and vascular smooth muscle cells from induced pluripotent stem cells.

INTRODUCTION

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.

METHODS

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 VEGF 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 and genes were simultaneously studied.

RESULTS

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 ; and VSMC-specific markers, including and . Differentiated cells were validated based on their temporal gene expressions, protein synthesis, and localization at certain time points. Significant decreases in and gene expression levels were observed during the EPCs and VSMC differentiation period.

CONCLUSION

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.