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 (ESC-EC), 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-α activation. Functional responses of ESC-EC 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 exhibit increased angiogenic sprouting and are more resistant to inflammatory signals. We further characterized the subphenotype of our ESC-EC and observed both venous and arterial markers on individual cells with a larger percentage of the cells exhibiting a venous phenotype. These data support the hypothesis that the developmental default pathway is toward a venous EC, and that refinement of methods for differentiation towards arterial EC is required to maintain a homogeneous population.

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Endothelial cells (EC) generated in vitro from stem cells are desirable for their potential in a variety of in vitro models and cell-based therapeutic approaches; however, EC can take on a number of functionally and phenotypically distinct specializations. Here, we show the generation of functionally distinct EC subpopulations, including (1) the pro-angiogenic migrating tip-like and proliferative stalk-like EC, and (2) the less migratory cobblestone-shaped phalanx-like EC. Both embryonic stem cell (ESC)-derived EC subpopulations are generated from outgrowths of Flk-1+ vascular progenitor cells with high levels of vascular endothelial growth factor treatment, while the phalanx-like ESC-derived EC (ESC-EC) are subsequently isolated by selecting for cobblestone shape. Compared with the ESC-derived angiogenic endothelial cells (named ESC-AEC) that contain only 14% Flt-1+ and 25% Tie-1+ cells, the selected phalanx-like ESC-EC express higher numbers of cells expressing the phalanx markers Flt-1+ and Tie-1+, 89% and 90%, respectively. The ESC-AEC also contain 35% CXCR4+ tip cells, higher expression levels of stalk marker Notch-1, and lower expression levels of Tie-2 compared with the phalanx-type ESC-EC that do not contain discernible numbers of CXCR4+ tip cells. Perhaps most notably, the ESC-AEC display increased cell migration, proliferation, and 3 times more vessel-like structures after 48 h on Matrigel compared with the phalanx-like ESC-EC. This work analyzes, for the first time, the presence of distinct EC subtypes (tip/stalk, and phalanx) generated in vitro from ESC, and shows that phalanx-like EC can be purified and maintained in culture separate from the tip/stalk-like containing EC.

Vascular progenitor cells derived from stem cells could potentially lead to a variety of clinically relevant applications, including cell‐based therapies and tissue engineering. Here, we describe methods for isolating purified proliferating populations of vascular endothelial cells from mouse embryonic stem cells (mESC) using Flk‐1 positive sorted cells, VEGF supplementation, and a rigorous manual selection technique required for endothelial cell purification and expansion. Using this in vitro derivation procedure, it is possible to obtain millions of cells at various stages of differentiation, with the potential for up to 25 population doublings. Curr. Protoc. Stem Cell Biol. 6:1F.5.1‐1F.5.19. © 2008 by John Wiley & Sons, Inc.

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Heart valvular endothelial cells (VECs) are distinct from vascular endothelial cells (ECs), but have an uncertain context within the spectrum of known endothelial phenotypes, including lymphatic ECs (LECs). Profiling the phenotypes of the heart valve surface VECs would facilitate identification of a proper seeding population for tissue-engineered valves, as well as elucidate mechanisms of valvular disease. Porcine VECs and porcine aortic ECs (AECs) were isolated from pig hearts and characterized to assess known EC and LEC markers. A transwell migration assay determined their propensity to migrate toward vascular endothelial growth factor, an angiogenic stimulus, over 24 h. Compared to AECs, Flt-1 was expressed on almost double the percentage of VECs, measured as 74 versus 38%. The expression of angiogenic EC markers CXCR4 and DLL4 was >90% on AECs, whereas VECs showed only 35% CXCR4+ and 47% DLL4+. AECs demonstrated greater migration (71.5 ± 11.0 cells per image field) than the VECs with 30.0 ± 15.3 cells per image field (p = 0.032). In total, 30% of VECs were positive for LYVE1+/Prox1+, while these markers were absent in AECs. In conclusion, the population of cells on the surface of heart valves is heterogeneous, consisting largely of nonangiogenic VECs and a subset of LECs. Previous studies have indicated the presence of LECs within the interior of the valves; however, this is the first study to demonstrate their presence on the surface. Identification of this unique endothelial mixture is a step forward in the development of engineered valve replacements as a uniform EC seeding population may not be the best option to maximize transplant success.

Most cell culture systems grow and spread as contact-inhibited monolayers on flat culture dishes, but the embryonic stem cell (ESC) is one of the cell phenotypes that prefer to self-organize as tightly packed three-dimensional (3D) colonies. ESC also readily form 3D cell aggregates, called embryoid bodies (EB) that partially mimic the spatial and temporal processes of the developing embryo. Here, the rationale for ESC aggregatation, rather than "spreading" on gelatin-coated or mouse embryonic fibroblast (MEF)-coated dishes, is examined through the quantification of the expression levels of adhesion molecules on ESC and the calculation of the adhesive forces on ESC. Modeling each ESC as a dodecahedron, the adhesive force for each ESC-ESC binding was found to be 9.1 x 10(5) pN, whereas, the adhesive force for ESC-MEF binding was found to be an order of magnitude smaller at 7.9 x 10(4) pN. We also show that E-cadherin is the dominating molecule in the ESC-ESC adhesion and blocking E-cadherin leads to a significant reduction in colony formation. Here, we mathematically describe the preference for ESC to self-assemble into ESC-ESC aggregates and 3D colonies, rather than to bind and spread on gelatin or MEF-coated dishes, and have shown that these interactions are predominantly due to E-cadherin expression on ESC.

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