Abstract 16809: Robust Vessel Forming Capability Of Endothelial Cells Which Are Differentiated From Human Pluripotent Stem Cells via a Novel Two-Dimensional Serum Free Differentiation System
Background: Human pluripotent stem cells (hPSCs) which include human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have emerged as an important source of endothelial cells (ECs) that can used for cardiovascular regenerative therapy. However, current EC differentiation systems have limitation in their efficiency and their clinical applicability due to the use of animal serum or feeders. To overcome this pitfall, we developed a novel two-dimensional differentiation system in which hPSCs can be efficiently differentiated into ECs without using embryoid body (EB) formation and animal derivatives.
Methods and Results: We used three lines of hPSCs: two hESCs (H1 and H9) and one hiPSC line. First, these cells were induced to differentiate into the mesodermal lineage by being cultured onto collagen-coated plates with GSK3-β inhibitor for 3 days. These cells were then cultured under DMEM-F12 supplemented with KSR and defined angiogenic factors (VEGF, EGF, bFGF, heparin) followed by mechanical shear stress to the cultured cells. These cells expressed endothelial specific proteins such as CHD5 (VE-cadherin)(22±4%) and von Willebrand factor (vWF) (18±5%) by FACS, formed tubes when cultured in Matrigel, and expressed EC-specific genes (KDR, TIE2, CD34, eNOS, CDH5, vWF) measured by qRT-PCR analysis. We next sorted CDH5+ cells using MACS. To verify in vivo EC characteristics and vasculogenic potential of sorted CDH5+ cells in vivo, a Matrigel plug assay and a hindlimb ischemia model were employed. The sorted CDH5+ cells, pre-labeled with a DiI were injected into the ischemic limbs in the HLI model or injected into the backs of athymic mice after being mixed with Matrigel. Tissues were harvested 7 to 14 days after perfusion with an EC marker, isolectin B4. Confocal examination of the tissues showed robust incorporation of CDH5+ cells into vascular structures, suggesting functional vasculogenesis.
Conclusions: We developed a novel stepwise, animal component-free, two-dimensional system to differentiate hPSCs into functional ECs. This system will serve as a useful platform for investigating endothelial development and the resultant ECs can be useful for treating ischemic cardiovascular diseases.
- © 2011 by American Heart Association, Inc.