Abstract 5438: Directed Differentiation of Endothelial Cells From Coronary Artery Disease (CAD) Patients-derived iPS Cells
Background. We recently generated Induced pluripotent stem (iPS) cells generated from peripheral blood mononuclear cells of coronary artery disease (CAD) patients. In this study, we sought to differentiate these iPS cells into endothelial cells (ECs), and to confirm their vasculogenic potential in vivo using multiple animal models.
Methods and Results. In these experiments, iPS cells were spontaneously differentiated through embryoid body (EB) formation and were sorted for KDR using FACS. Next, we further cultured these KDR+ cells for 21 days and sorted for KDR and CD31. These KDR+CD31+ cells showed a higher rate (~70%) of double positivity for UEA1 lectin and ac-LDL uptake. The KDR+CD31+ cells expressed endothelial specific proteins such as VE-cadherin and von Willebrand factor (vWF) under immunohistochemical examination, formed tubes when cultured in Matrigel, and expressed EC-specific genes (KDR, CD31, CD34, eNOS, VE-cadherin, vWF) as shown by qRT-PCR analysis. In this culture condition, the EC phenotype was maintained > 2 months. Next, to verify their EC characteristics in vivo and provide bona fide evidence of EC commitment of iPS cells, Matrigel plug assay and a hindlimb ischemia model were employed. First, we injected a mixture of Matrigel and the KDR+CD31+ cells labeled with a red fluorescent dye, DiI, into the backs of athymic mice subcutaneously. Two months later, the mice were perfused with an endothelial cell marker, isolectin B4 (ILB4) to stain functional endothelium. The injected cells gave rise to vascular structures and were stained positive for ILB4. Second, we surgically induced hindlimb ischemia in nude mice and injected the KDR+CD31+ cells labeled with DiI into the hindlimb muscles immediately after the surgery. After sacrificing the mice nine days later, muscles were harvested and the tissues were subjected to immunohistochemistry with ILB4. Injected KDR+CD31+ cells expressed ILB4 and were incorporated into vascular structures, suggesting vasculogenesis.
In conclusion. iPS cells derived from CAD patients can generate functionally competent ECs in vivo, Together the present study suggest that endothelially differentiated cells from iPS cells can be used therapeutically in an autologous manner to treat tissue ischemia.