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.

FIG. 1.

EC derived in chemically defined conditions express appropriate endothelial makers. We examined the expression of endothelial markers Flk-1, VE-cadherin, Flt-1, and Tie-1. The histograms include the ESC-AEC (left) and the purified ESC-EC (right) derived using R1 ESC under chemically defined conditions. EC, endothelial cell; ESC, embryonic stem cell; AEC, angiogenic endothelial cells.

FIG. 2.

Cell morphology and proliferation of ESC-AEC and ESC-EC. Differences in cell morphology were very apparent between the ESC-AEC (A, C) and ESC-EC (B, D). Interestingly, (A) both self-assembled into lines, but cultures (C) the ESC-AEC maintained with stable loops at confluence, forming vacuoles (indicated by arrows) in the monolayer. As expected, (D) the ESC-EC exhibited the cobblestone morphology of the purified cells at confluence. Note that this morphology is not apparent in (B) subconfluent cultures of ESC-EC. (E) This graph reports the average population proliferation rates of the ESC-AEC and ESC-EC. The ESC-AEC also proliferate at faster rates, that is, shorter doubling times, compared with the other EC. *Statistically significant. Error bars=SD. Analysis of the cell cycle stages indicate that the (F) ESC-AEC are largely in proliferative S-phase, while most of the (G) ESC-EC are in quiescent G0 phases.

FIG. 3.

Increased migration and sprouting activity in ESC-AEC compared with ESC-EC. (A, B) After 12 h on Matrigel, both cell populations exhibit angiogenic sprouting activity. However, after 48 h, the (C) ESC-AEC sprouts continue increasing in number and size, while the (D) ESC-EC sprouts show signs of regression. (E) The quantification of vessel coverage area indicates that a significant increase in angiogenic sprouting of the ESC-AEC is evident after only 12 h, and becomes even more pronounced after 48 h when the sprouts from ESC-AEC continue to increase compared with the regressing ESC-EC. Statistically significant differences (*P>0.05) between the 2 cell populations were observed at both 12 and 48 h. (F) Diagram of the transwell assay shows that the chemoattractants are added to the bottom well. The cells migrate from the top transwell though the pores in the transwell insert onto the bottom surface of the insert well. (G) The number of migrating cells were then quantified. Note that ESC-AEC were much more responsive to the proangiogenic chemoattractant compared with serum only. All cell populations were compared for statistical significance. *Statistically significant from ESC-AEC; ^#statistically significant from ESC-EC; ^∼statistically significant from serum-free; ^{^}statistically significant from 10% fetal bovine serum. Error bars=SEM.

FIG. 4.

ESC-EC express VE-cadherin and ESC-AEC express increased levels of actin stress fibers and HSP27 phosphorylation. The presence of extracellular VE-cadherin adhesion molecule is not observed to be expressed in many of the (A) ESC-AEC but is expressed in the (B) ESC-EC, while both cell populations (C, D) express EC marker PECAM-1. Blue=DAPI, green=VE-cadherin, scale bar=100 μm. The cytoskeleton organization and presence of stress fibers of the (E) ESC-AEC is noticeably increased compared with the (F) ESC-EC. Blue=DAPI, green=phalloidin 488, scale bar=50 μm. HSP27 phosphorylation, a factor in endothelial actin organization and migration, was also examined. Phosphorylated HSP27 was (G) very high in ESC-AEC (blue) compared with IgG control (black), but there was (H) almost no expression in the ESC-EC (blue) compared with IgG control (black). DAPI, 4′,6-diamidino-2-phenylindole. Color images available online at www.liebertpub.com/scd

FIG. 5.

ESC-AEC expressed increased numbers of stalk and tip cell markers compared with ESC-EC. The 2 cell populations were stained for both extracellular (stalk cells) and total (intra- and extracellular) Notch1, tip cell marker CXCR4 and Tie-2. Compared with isotype controls (left histogram), (A, C) ESC-AEC and (B, D) ESC-EC populations expressed extracellular and total Notch1, but the ESC-AEC expressed higher numbers, especially extracellular Notch1. CXCR4 was expressed in 35% of the (E) ESC-AEC, but not in the (F) ESC-EC. Lastly, both cell populations contained Tie-2+ cells, but fewer Tie-2 molecules (indicated by smaller right shift) were observed in the (G) ESC-AEC compared with the (H) ESC-EC.

FIG. 6.

ESC-AEC and ESC-EC behave similarly in CAM assays. Images of CAM vasculature resulting from the (A) Matrigel vehicle control, (B) undifferentiated R1-ESC, (C) ESC-AEC, (D) ESC-EC, and (E) MCEC. The CAM were then stained for murine cell incorporation (brown), and as expected, the (F) vehicle control stained negative, while the incorporation of murine cells (brown) into the CAM tissue vasculature is observed for all of the cells: (G) R1 mESC, (H) ESC-AEC, (I) ESC-EC, and (J) MCEC. (K) The quantification of vessel lengths for blank, vehicle control, and undifferentiated R1-ESC populations yield comparable amounts of vascular activity, while the ESC-AEC, ESC-EC, and MCEC yield significantly increased vascular activity. Statistical significance differences in average vessel lengths is not seen between the 3 EC populations, but all 3 EC grafts do significantly (*) increase vasculature in vivo compared with the 3 control groups. Error bars=SEM. MCEC, murine cardiac endothelial cells; CAM, chick chorioallantoic membrane. Color images available online at www.liebertpub.com/scd

FIG. 7.

Summary of ESC-AEC and ESC-EC characterization. (A) The table summarizes the results from this article. The ESC-AEC, which are thought to include tip/stalk EC, are more angiogenic, migratory, and proliferative compared with ESC-EC. ESC-AEC also express higher levels of tip/stalk markers: Notch1, CXCR4, HSP27 phosphorylation, and F-actin filaments. Conversely, the ESC-EC exhibit a cobblestone morphology and express higher levels of phalanx-specific markers Flt-1, Tie-1, Tie-2, and extracellular VE-cadherin. (B) The diagram shows these different cell types: the tip, stalk, and phalanx EC within a sprouting blood vessel, together with some of the known phenotypic and functional differences between these 3 specialized EC that are highlighted in the literature discussed in the introduction [–28].