Using human pluripotent stem cell differentiated endothelial cells to study the differential regulation in PAH

Abstract

Aims Endothelial cells (ECs) have been applied in clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, lack of enough transplantable cells before the deterioration of patient condition is current limitation to apply cell therapy in cardiovascular diseases. So, we thought it necessary to continue to differentiate embryo stem cells (ESCs)/induced-pluripotent stem cells (iPSCs) into endothelial cells (ECs) and identify their characteristics. Methods and results Comparing previous reported methods of human pluripotent cell differentiation toward vascular cells/ Hemogenic Endothelial cells/ endothelial colony-forming cells, we established a highly efficient differentiation protocol to get cells that match phenotype of isolated ECs from health donors. This protocol, including two stages, early mesoderm endothelial progenitor stage and EC marker expression stage. In the first stage, Rock inhibitor Y27632 and DMSO plays an important role in inducing-APLNR + mesoderm and promotes EC differentiation potential; later on SB431542 and BMP4 drives cells toward EC lineage. Meanwhile, an improved protocol with chemically-defined medium (CDM) has similar differentiation efficiency,again demonstrating that a large number of clinically needed cells could be obtained with simple factors. ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have the characteristics of early EPCs marked by CD133 and mesenchymal stem cells. Microarray analysis further revealed IPAH-derived EPCs features of rapid proliferation and weak immune regulation. At last, a model Zebrafish xenograft was utilized to assess the functionality of differentiated ESC/iPSC-ECs. ConclusionsWe established a highly efficient differentiation protocol to get ESC/iPSC-ECs with characteristics of phenotype and molecular matched with early-EPCs from health donors, and revealed the molecular pathogenesis in PAH.