Robust Generation of Hemangioblastic Progenitors from Human Embryonic Stem Cells

Abstract

Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However, successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models, the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. In addition to eliminating several expensive factors that are unnecessary, we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury, KDR, CD31 and Lmo2 gene expression, while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development, consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.