Functional Characterization of Embryonic Stem Cell-Derived Endothelial Cells

Endothelial cells (EC) derived from embryonic stem cells (ESC) require additional functional characterization before they are used as a cell therapy in order to enhance their potential for engraftment and proliferation. We explore several physiologically relevant functions of ESC-derived EC (ESCEC), such as its capacity to produce nitric oxide (NO), regulate permeability, activate and express surface molecules for the recruitment of leukocytes in response to inflammatory stimuli, migrate and grow new blood vessels, lay down extracellular matrix, and take up low-density lipoproteins. We also examined the ESC-EC ability to upregulate NO in response to shear stress and downregulate NO in response to pro-inflammatory TNF activation. Functional responses of ESCEC were compared with those of cultured mouse aortic ECs. The ESC-EC exhibit most aspects of functional endothelium, but interesting differences remain. The ESC-EC produced less NO on a per cell basis, but the same amount of NO if quantified based on the area of endothelial tissue. They also Received: May 18, 2010 Accepted after revision: January 12, 2011 Published online: May 31, 2011 Dr. Kara E. McCloskey School of Engineering University of California, Merced P.O. Box 2039, Merced, CA 95344 (USA) Tel. +1 209 228 7885, E-Mail kmccloskey @ ucmerced.edu © 2011 S. Karger AG, Basel Accessible online at: www.karger.com/jvr Glaser /Gower /Lauer /Tam /Blancas /Shih / Simon /McCloskey J Vasc Res 2011;48:415–428 416 characterized these cells for a variety of endothelial specific markers using immunofluorescent labeling [2–5] . Lineage-specific markers that correlate with histological and phenotypic characteristics of somatic cells is routinely applied for the classification of many types of differentiated stem cells. Common EC markers include endothelial nitric oxide synthase (eNOS), receptors for vascular endothelial growth factors (VEGF) Flk-1 and Flt-1, vascular endothelial cadherin (VE-cadherin), CD34 and platelet EC adhesion molecule (PECAM-1). As previously published, our ESC-derived EC (ESC-EC) express these markers indicating their lineage-specific commitment, and do not contain cells expressing nondifferentiated ESC or smooth muscle cells [3, 4] . In addition to surface marker characterization, functional assays are also essential determinants of appropriate cellular maturation. Since EC or endothelial progenitor cells (EPC) differentiated in culture from stem cells are exposed to a more limited repertoire of the differentiation cues that are experienced in vivo , it is especially important to assess functional capability in addition to marker analysis. For the EC, physiologically relevant functional assays include the ability to synthesize nitric oxide (NO), regulate permeability (that is, the flux of molecules across an intact endothelium), respond to inflammatory activation by tumor necrosis factor(TNF), migrate and grow new blood vessels, and produce appropriate extracellular matrix (ECM) for developing a basal lamina. Characterization of in vitro-derived ESCEC and their response to environmental cues and inflammation is a critical step in assessing the quality of cultured cells for clinical application in cell-based therapies. EC Synthesize and Release NO EC regulate blood pressure and blood flow by releasing the vasodilators including: NO and prostacyclin, as well as vasoconstrictors including: endothelin and platelet-activating factor. Production of these molecules is critical for maintaining vascular homeostatic function [6] . Release of NO by the EC relaxes the smooth muscle cells in the walls of the arterioles, and is the principal factor that regulates dilation of the vessel wall and, in turn, blood flow. NO also inhibits the aggregation of platelets and thus keeps inappropriate clotting from interfering with blood flow. NO is synthesized within EC (among other cells) by eNOS. eNOS and NO secretion is constitutively active in EC, but is also upregulated by certain stimuli, such as laminar shear stress [7–10] . The mechanisms underlying the shear stress-induced eNOS expression include enhancing eNOS gene transcription and stabilizing eNOS mRNA. In both of the cases, the tyrosine kinase c-Src played a central role, and a complex kinase cascade including Raf, Ras, MEK1/2 and ERK1/2 seems to be involved in the signal transduction leading to eNOS transcription by shear stress [11] . In addition, TNF stimulation has been shown to downregulate eNOS mRNA, protein and activity in EC via destabilization of eNOS mRNA [12] . EC Regulate Vascular Permeability to Macromolecules and Protein In addition to regulating blood flow, endothelium plays a vital role in regulating the transport of fluids, molecules and cells between the bloodstream and surrounding tissue. An EC monolayer is relatively impermeable, ! 1% flux, to large molecules (1–100 kDa). The presence of membrane-bound receptors helps facilitate this gatekeeping role for numerous molecules including proteins, lipid-transporting particles, metabolites and hormones [6] . A number of investigators have successfully grown EC on porous supports to create in vitro models of permeability [13–15] for studying the permeability of the EC monolayer under various conditions. Transient increases in endothelial permeability do occur normally after tissue injury; however, chronic increases in permeability is abnormal and has been implicated in atherosclerosis, diabetic retinopathy and tumor growth [16] . EC Are Building Blocks for Generating New Blood Vessels It is widely accepted that the new blood vessels arise through two mechanisms during development, called vasculogenesis and angiogenesis [17] . Vasculogenesis is a process in which hemangioblasts, EC precursors from mesodermal origins, grow and organize to form vascular networks. Angiogenesis is the formation of new blood vessels by sprouting from existing vessels. Recently, an alternative view has demonstrated the presence of circulating EC in the adult [18] and their incorporation into ischemic tissue [19] , suggesting a role for vasculogenesis in the adult as well as in the embryo. It is now thought that both angiogenesis and vasculogenesis occur during postnatal life and that they are not mutually exclusive events. Both angiogenesis and vasculogenesis require EC proliferation, migration and three-dimensional organization [20] . The ability of EC to form lines and tube-like formations in vitro has been demonstrated to be a good measure of vasculogenic and angiogenic potential in tissue. A variety of culture conditions for generating these vascular structures have been employed, including culturing cells on the surface of collagen or fibrin gels, on a