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Research Article |
1 Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR
6543, Centre Antoine Lacassagne, 33 avenue de Valombrose, Nice, France
2 Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research,
Haartman Institute, University of Helsinki, Helsinki, Finland
* These authors contributed equally to this work
Author for correspondence (e-mail:
gpages{at}unice.fr
)
Accepted 7 March 2002
Large scale purification of endothelial cells is of great interest as it
could improve tissue transplantation, reperfusion of ischemic tissues and
treatment of pathologies in which an endothelial cell dysfunction exists. In
this study, we describe a novel genetic approach that selects for endothelial
cells from differentiating embryonic stem (ES) cells. Our strategy is based on
the establishment of ES-cell clones that carry an integrated puromycin
resistance gene under the control of a vascular endothelium-specific promoter,
tie-1. Using EGFP as a reporter gene, we first confirmed the
endothelial specificity of the tie-1 promoter in the embryoid body
model and in cells differentiated in 2D cultures. Subsequently,
tie-1-EGFP ES cells were used as recipients for the
tie-1-driven puror transgene. The resulting stable clones
were expanded and differentiated for seven days in the presence of VEGF before
puromycin selection. As expected, puromycin-resistant cells were positive for
EGFP and also expressed several endothelial markers, including CD31, CD34,
VEGFR-1, VEGFR-2, Tie-1, VE-cadherin and ICAM-2. Release from the puromycin
selection resulted in the appearance of 
-smooth muscle actin-positive
cells. Such cells became more numerous when the population was cultured on
laminin-1 or in the presence of TGF-ß1, two known inducers of smooth
muscle cell differentiation. The hypothesis that endothelial cells or their
progenitors may differentiate towards a smooth muscle cell phenotype was
further supported by the presence of cells expressing both CD31 and
-smooth muscle actin markers. Finally, we show that purified
endothelial cells can incorporate into the neovasculature of transplanted
tumors in nude mice. Taken together, these results suggest that application of
endothelial lineage selection to differentiating ES cells may become a useful
approach for future pro-angiogenic and endothelial cell replacement
therapies.
Key words: Embryonic stem cells, Endothelial cells, Differentiation, Selection, Angiogenesis
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