Endothelial cells from embryonic stem cells in a chemically defined medium.

Graduate Program in Quantitative and Systems Biology, University of California, Merced, California, USA.
Stem cells and development (Impact Factor: 4.15). 03/2011; 20(12):2153-61. DOI: 10.1089/scd.2010.0432
Source: PubMed

ABSTRACT Endothelial cells (ECs) are desired for their therapeutic potential in a variety of areas including gene therapy, cardiac regeneration, development of tissue-engineered vascular grafts, and prevascularized tissue transplants. Pluripotent embryonic stem cells (ESCs) can be induced to differentiate into ECs in vitro using embryoid bodies, monolayer cultures, or by genetic manipulation and immortalization. However, obtaining homogeneous cultures of proliferating ESC-derived ECs without genetic manipulation is a challenging undertaking and often requires optimization of protocols and rigorous purification techniques. Moreover, current differentiation methods that use medium containing fetal calf or bovine serum components introduce additional challenges because of our limited ability to control the differentiation signals and batch-to-batch variations of serum. We have explored the development of new medium formulations for deriving ECs from murine embryonic stem cells (mESCs) using only chemically defined reagents. We present 2 different medium formulations along with the detailed methodologies, including the optimization of extracellular matrix-derived substrates known to play a role in cell attachment and proliferation as well as cell differentiation. Characterization of the ESC-derived ECs indicate that (1) chemically defined medium formulations reproducibly generate superior ECs compared with previous serum-containing formulations, (2) fibronectin, and not collagen type-IV, is the optimal substrate for EC induction in our chemically defined medium formulations, (3) without additional activation of Notch-signaling, ESC-ECs develop predominantly into venous ECs, and (4) using these medium formulations, a second rigorous selection step is not required to generate proliferating ECs from ESCs, but it does enhance the final purity of the ECs.

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    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 ESC-derived EC subpopulations are generated from outgrowths of Flk-1+ vascular progenitor cells with high levels of vascular endothelial growth factor (VEGF) 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 hours 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 embryonic stem cells, and shows that phalanx-like EC can be purified and maintain in culture separate from the tip/stalk-like containing EC.
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