Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells

Abstract

Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type-specific gene expression, is not yet understood. We studied the regulation of a highly endothelial-specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re-established (HiPS back to endothelial cells [EC-Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC-Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC-Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC-Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. STEM CELLS 2019;37:542-554 SIGNIFICANCE STATEMENTIn these studies, a novel approach involving generation and differentiation of induced pluripotent stem cells is used to revoke and subsequently re-establish a specific cell phenotype, namely endothelial cells, to explore transcriptional regulatory networks that are associated with establishing cell type specific gene regulation. It provides a novel approach, compared with classic analyses of differential gene regulation among cells of distinct phenotypes, toward determining how cell type specific gene expression is regulated. The process allows for determining simultaneously what the critical components that contribute to silencing are, as well as activating target cell-type specific gene regulation.

BMP9 exhibits dual and coupled roles in inducing osteogenic and angiogenic differentiation of mesenchymal stem cells

“…It is reported that 3D scaffolds hold the potential to promote blood vessel formation and bone formation [60][61][62], although BMP9-mediated blood vessel formation was identified by both in vitro and in vivo tests, it is still necessary to investigate BMP9-mediated bone formation with the use of 3D scaffolds to further construct BMP9-mediated bone tissue engineering.…”

In Silico Analysis to Explore Lineage-Independent and -Dependent Transcriptional Programs Associated with the Process of Endothelial and Neural Differentiation of Human Induced Pluripotent Stem Cells

“…The initial dataset contained 49,400 observations for 19,929 unique genes [ 17 , 18 ]. Each observation had multiple gene expression values corresponding to induced pluripotent cells, differentiated endothelial cells and differentiated neuronal cells.…”

“…A potential approach towards addressing this gap in knowledge is to compare the molecular signature of two distinct cell lineages that are derived from one parental stem cell, thus possessing a similar genetic background with each other as well as with the parental stem cell. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells (HUVEC) the and subsequent differentiation of the resulting iPS (referred to as HiPS) back into a highly pure population of endothelial cells (Ec-Diff), as well as a population of neural cells that contained both neurons and astrocytes (Nn-Diff) [ 17 , 18 ]. Although the Nn-Diff were a mix of both neurons and astrocytes, their common neural phenotype is clearly distinct from endothelial cells, and thus they provide an appropriate comparator to the highly pure endothelial population of Ec-Diff.…”

“…A comparison of HUVEC, HiPS and EC-Diff transcriptome by gene array provided information regarding transcription regulatory factors, including chromatin and DNA modifiers, as well as cofactors as potential contributors to the establishment of endothelial cell phenotype [ 18 ]. Furthermore, we had performed gene array analyses of Nn-Diff that were derived from HiPS to confirm their neural lineage identity [ 17 ].…”

“…Furthermore, we had performed gene array analyses of Nn-Diff that were derived from HiPS to confirm their neural lineage identity [ 17 ]. Here, using the two datasets generated from these two analyses [ 17 , 18 ], we have identified genes that are specifically associated with the establishment of endothelial compared to neural phenotypes. We aimed to gain insight into transcriptional machinery that is activated commonly to allow a general differentiation process compared to those that are associated with differentiation specifically towards endothelial or non-endothelial neural lineage.…”