Specialized Tip/Stalk-Like and Phalanx-Like Endothelial Cells from Embryonic Stem Cells

Abstract

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.

Endothelial cells expressing low levels of CD143 (ACE) exhibit enhanced sprouting and potency in relieving tissue ischemia

“…Moreover, the profile of cytokines secreted by the sprouting cells was very similar to that observed for non-sorted endothelial cells, which is again consistent with previous studies[26]. Several genes and pathways have been previously described to exhibit increased expression in sprouting cells, including Notch signaling[40], VEGF receptors[41], various adhesion molecules [42] and cell surface receptors [43,7]. However, in our study, VEGFR1 and VEGFR2 were in the group of 54 genes that displayed similar levels of expression in the sprouting cells and non-sorted endothelial cells.…”

“…Endothelial tip cells are the leading cells of a sprout, and are highly polarized and migratory, minimally proliferative, and display numerous extended filopodia[6]. Endothelial stalk cells follow the tip cells, and are characterized by fewer filopodia, higher proliferative capacity and lumen formation and coordination[7]. Although there exists plasticity and reversibility between these phenotypes during sprouting[8], very little is known about whether cells that participate in formation of new sprouts, as compared to those that do not, were previously committed to a more angiogenic phenotype, or if this is a stochastic process.…”

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

“…Blancas et al reported a chemically defined protocol using growth factors, including VEGF-A and BMP-4, along with ECMs such as fibronectin. 6 Wu et al employed not only soluble inducing factors of CHIR-99021and VEGF-A, but also the anti-absorptive agent polyvinyl alcohol to preferentially detach non-endothelial lineages, resulting in high yields of iPSC-ECs. 54 Whereas high-efficiency differentiation protocols such as that from Wu et al 54 might mask the potential benefit of 3D scaffolds, in this study we employed a differentiation protocol 45 that, in our hands, yielded ~20% efficiency of CD31 + iPSC-ECs in conventional tissue culture dishes, and thereby allow for the possibility of observing an enhancement in differentiation in 3D scaffolds.…”

Induced Pluripotent Stem Cell–Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome

“…Our analysis proposed Hox genes as a key driver determinant of the differentiation variability. Other works have shown that extracellular matrix coating, serum and seeding density can also affect differentiation of pluripotent cells to the endothelial lineage 41, 42 .…”