Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo
Author:
Marchetti Sandrine12, Gimond Clotilde12, Iljin Kristiina3, Bourcier Christine1, Alitalo Kari3, Pouysségur Jacques1, Pagès Gilles1
Affiliation:
1. Institute of Signaling, Developmental Biology and Cancer Research, CNRS UMR 6543, Centre Antoine Lacassagne, 33 avenue de Valombrose, Nice, France 2. These authors contributed equally to this work 3. Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research,Haartman Institute, University of Helsinki, Helsinki, Finland
Abstract
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 α-smooth muscle actin-positive cells. Such cells became more numerous when the population was cultured on laminin-1 or in the presence of TGF-β1, 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 andα-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.
Publisher
The Company of Biologists
Reference38 articles.
1. Arciniegas, E., Sutton, A. B., Allen, T. D. and Schor, A. M.(1992). Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.J. Cell Sci.103, 521-529. 2. Asahara, T., Murohara, T., Sullivan, A., Silver, M., van der Zee, R., Li, T., Witzenbichler, B., Schatteman, G. and Isner, J. M.(1997). Isolation of putative progenitor endothelial cells for angiogenesis. Science275, 964-967. 3. Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C.,Silver, M., Kearne, M., Magner, M. and Isner, J. M. (1999a). Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ. Res.85, 221-228. 4. Asahara, T., Takahashi, T., Masuda, H., Kalka, C., Chen, D.,Iwaguro, H., Inai, Y., Silver, M. and Isner, J. M. (1999b). VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J.18, 3964-3972. 5. Bautch, V. L., Redick, S. D., Scalia, A., Harmaty, M.,Carmeliet, P. and Rapoport, R. (2000). Characterization of the vasculogenic block in the absence of vascular endothelial growth factor-A.Blood95, 1979-1987.
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