Lentiviral Rescue of VEGFR-2 Expression in flk1-/- ES Cells Shows Early Priming of Endothelial Precursors

¹ Uppsala University, Dept. Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjöldsv. 20, 751 85 Uppsala, Sweden
² Uppsala University, Dept. Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjöldsv. 20, 751 85 Uppsala, Sweden Karolinska Institute, Dept. Cell and Molecular Biology, Box 285, 171 77 Stockholm, Sweden
³ Karolinska Institute, Dept. Cell and Molecular Biology, Box 285, 171 77 Stockholm, Sweden
⁴ Institute of Pathology, TUD-Faculty of Medicine "Carl Gustav Carus", Fetscherstrasse 74, D-01307 Dresden, Germany

^* To whom correspondence should be addressed. E-mail: Lena.Welsh{at}genpat.uu.se.

	Abstract

The vascular endothelial growth factor (VEGF) family and its receptors are important for vascular development and maintenance of blood vessels, as well as for angiogenesis, the formation of new vessels. Loss of VEGF receptor-2 (VEGFR-2; denoted Flk-1 in mouse) results in arrest of vascular and hematopoietic development in vivo. We used lentiviral transduction to reconstitute VEGFR-2 expression in flk1-/- embryonic stem (ES) cells. VEGF-induced vasculogenesis, as well as sprouting angiogenesis were rescued in transduced ES cultures differentiating in vitro as embryoid bodies (EBs). Although the transgene was expressed in the pluripotent stem cells and lacked linage restriction during differentiation, the extent of endothelial recruitment was similar to that in wild-type EBs. Reconstitution of VEGFR-2 in flk1-/- ES cells allowed only pre-committed precursors to differentiate into functional endothelial cells able to organize into vascular structures. Chimeric EB cultures composed of wild-type ES cells mixed with flk1-/- ES cells or reconstituted VEGFR-2-expressing ES were created. In the chimeric cultures, flk1-/- endothelial precursors were excluded from wild type vessel structures, whereas reconstituted VEGFR-2-expressing precursors became integrated together with wild type endothelial cells to form chimeric vessels. We conclude that maturation of endothelial precursors as well as organization into vascular structures requires expression of VEGFR-2.

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