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Georgios Kontos, Panagiota Antonopoulou, Dawn Sim, Jenny Mckenzie, Marcus Fruttiger; De novo formation of vascular structures in vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2216.
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© ARVO (1962-2015); The Authors (2016-present)
Embryonic stem cells are pluripotent; they have the ability to differentiate to tissues of all three germ layers. We describe the development of an adaptive model of early vascular development using embryoid bodies (EBs).
Undifferentiated human embryonic stem cell (hES) lines were thawed and expanded on a feeder cell layer consisting of inactivated mouse embryonic fibroblasts (MEFs). Once a potent number of healthy undifferentiated hES colonies were achieved; the cultures were disrupted. Pluripotent hES were aggregated into EBs.Uniform EBs were achieved by culturing hES suspensions in microwell plates for 24 h. Through a controlled differentiation protocol containing human Vascular Endothelial Growth Factor-165 (VEGF-165) and foetal calf serum (FCS), EBs were cultured in suspension and induced towards vascular linage. Polymerase chain reaction (PCR) primers for CD34, VE-Cadherin and VEGFR2 were used. Endothelial cells co-expressing CD34 and VE-Cadherin were immunostained; light microscopy visualized the recurring structures. Confocal microscopy allowed the 3D reconstruction of these structures. Some EBs were dissociated and CD34+ cells were selected by magnetic sorting. CD34+ cells were expanded and cultured.
CD34, VE-Cad expressing structures in the form of a vascular plexus were evident in less than two weeks of differentiation. A primary vascular plexus was formed within the EBs. Sequential immunohistochemistry suggested that the vascular structures were in a continuous state of remodeling characterized by sprouting angiogenesis. PCR confirmed the expression of CD34, VE-Cadherin and VEGFR2 genes in EBs. Confocal microscopy and software processing depicted the presence of lumen possessing 3D structures within the EBs.
Uniformly sized EBs is a potential model for induction of vasculogenesis i.e. de novo formation of vascular structures. This process mimics the formation of the primary vascular plexus in the developing embryo. Future applications of this method include the treatment of vasodegenerative diseases, either as transplanted cells or thevascularization of lab-engineered tissues.
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