High efficient serum free differentiation of endothelial cells from human iPS cells without sorting

Introduction: Endothelial cells (ECs) form the inner lining of all blood vessels of the body, play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in-vitro investigations of ECs’ physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. Method: hiPSC are cultured under serum free conditions, and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 hours. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro), and melatonin (Mel) for 48 hours. Result: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two dimensional (2D) mono layer into three dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von-Willebrand-factor, and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). Conclusion: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at an efficiency eliminating the need for further enrichment strategies, hence providing a scalable source of hiPSC-ECs. Human induced pluripotent stem cels (hiPSC) were differentiated ad under serum free conditions into mesodermal progenitor cells via stimulation of Wnt signaling for 24 hours. Mesodermal progenitor cells were further differentiated into endothelial-like cells (ECs) by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro), and melatonin (Mel) for 48 hours.