Abstract 14006: VEGF Signaling Enhances Endothelial Differentiation of Induced Pluripotent Stem Cells: Invitro and in vivo Characterization
Background: We have previously reported spontaneous cardiac differentiation of skeletal myoblast derived iPS cells generated by our novel non viral epigenetic approach. Given the pivotal role of vascular endothelial growth factor (VEGF) in angiogenic cascade, we propose that directed differentiation of iPS cells by VEGF to adopt endothelial phenotype will enhance angiogenic/vasculogenic repair of ischemic myocardium.
Methods & Results In these experiments, iPS cells were pretreated with VEGF (50ng/ml) for 5 days. After culturing, these cells were analyzed at various time points for two weeks. After 5 days flow cytometry revealed a high percentage of the endothelial cells (EC) expressing CD31(85%),VE-cadherin(47%),vWF(36%)which was confirmed by immunohistochemistry. RT-PCR analysis showed sequential upregulation of EC-specific genes (CD31, Tie2, eNOS, VE-cadherin, vWF). iPS cell derived ECs exhibit higher proliferation and migration rate (p<0.01vs non differentiated iPS) and tube formation when cultured in matrigel. These cells showed significant reduction in LDH release and Annexin-v positivty (p<0.01) when exposed to oxidant stress (100μ mol/l H2O2 for 60 minutes). VEGF treatment enhanced Notch and jagged activation through PI3 kinase/Akt pathway enriching their specific functional profile and stemness feature. These beneficial effects were abolished when treated with specific anti-VEGF antibody. To provide bona fide evidence of EC commitment of iPS cells in vivo, ECs at different time points labeled with a red fluorescent dye, PKH 26 were injected in the infarcted mouse myocardium after LAD ligation. At 7 days and 4 weeks the extent of neovasularization resulting from engraftment of endothelial progenitor cells of 5-7 days age was significantly superior as compared to other groups(p<0.01). Similarly endothelial cells apoptosis and myocardial fibrosis were significantly attenuated in treated hearts together with hemodynamic recovery.
Conclusion: This study provides understanding of mechanistic insights into angiogenic potential of IPS cells derived endothelial cells in invivo murine myocardial infarction model. These cells can be used readily for different purposes including cell based therapeutics and drug screening
- © 2011 by American Heart Association, Inc.