GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION

Abstract

Damage to vascular endothelial cells (EC) is the core of various vascular diseases such as stroke and cardiovascular complications. Understanding molecular mechanisms that govern endothelial gene expression and function is crucial for developing novel therapeutic approaches for vascular endothelial dysfunction-related complications. Induced pluripotent stem cells (iPS) provide a valuable in vitro system to study the molecular processes, including EC-specific gene regulation, that contribute to establishment of EC phenotype. We aimed to study endothelial gene regulation by generating iPS from human umbilical vein EC (HUVECs) and differentiating the resulting iPS back into EC. This model provides a system with a homogenous genetic Background to explore how EC phenotype is revoked (HUVEC to iPS), and reestablished (iPS to EC). Colonies (iPS) were generated using originally reported transcription factors. Quantitative RT-PCR and immunofluorescence analysis were used to characterize iPS and cell lineages derived from iPS. Pluripotency of the iPS were demonstrated by generation of embryoid bodies (EB) and detection of the three germ layer markers. Additionally a novel method was used to directly differentiate iPS into neuronal cell lineages with high efficiency without the use of EB. Next EB generated from iPS were induced to differentiate into EC, thus establishing the EC to iPS back to EC system for analyses. In this system we explored the pattern of activation and repression of a highly EC-specific gene, von Willebrand factor (VWF), as well as transacting factors and cofactors that regulate the VWF promoter activity. Our analyses demonstrated that expression of transacting factors that function as activators of the VWF promoter mirrored that of VWF gene, while those functioning as repressors where either reversed or not significantly altered. Temporal expression of activators were observed during various stages of differentiation. Specifically during the progression from EB to EC we observed upregulation of GATA6 followed by Ets transactivators. Furthermore, the ability of iPS to incorporate into microvasculature was demonstrated in a mouse model of kidney vascular injury. Generation of iPS from EC and differentiation of iPS back to EC provides a unique opportunity to explore the molecular mechanisms that are involved in establishment of EC specific gene regulation and consequently the establishment of EC phenotype. This system will also provide an opportunity for exploring the potential of these EC derived iPS and their differentiated EC towards developing cell- therapy approaches for vascular diseases.