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Endothelial cells genetically selected from differentiating mouse embryonic stem cells incorporate at sites of neovascularization in vivo

Sandrine Marchetti^1,*, Clotilde Gimond^1,*, Kristiina Iljin², Christine Bourcier¹, Kari Alitalo², Jacques Pouysségur¹ and Gilles Pagès^1,

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

View larger version (10K): [in a new window]   Fig. 1. Activities of tie-1 and VE-cadherin promoters in cultured cells. 600 ng of each luciferase reporter construct were transfected into BAE (bovine aortic endothelial cell), CCL39 and 293 cells (non-endothelial cells). In each assay, 300 ng of the ß-galactosidase expression vector were co-transfected in order to normalize for transfection efficiency. 48 hours after transfection, luciferase activity was measured. The relative activity of each construct was expressed as fold induction over the pGL2 basic vector. The results shown in this figure are representative of three independent experiments. Each set of data represents the mean of quadruplicate determinations.

View larger version (41K): [in a new window]   Fig. 2. Expression of vascular markers in embryoid bodies (EBs). ES cells were differentiated into EBs, fixed on day 5 or 8 and stained with antibodies to CD31, VEGFR-2 and VE-cadherin. When indicated, differentiation medium was supplemented with 10 ng/ml human rVEGF-165. (A) While the CD31 antibody stained cell clumps in untreated 5- and 8-day-old EBs, the addition of 10 ng/ml rVEGF promoted the organization of CD31+ cells into pseudo-vascular structures. (B) Vascular endothelial networks in 8-day-old EBs were co-stained for CD31 (red) and VEGFR-2 (green), and similar structures also expressed VE-cadherin (green). Bar, 100 µM.

View larger version (64K): [in a new window]   Fig. 3. Analysis of the tie-1 promoter for endothelial specificity in EBs and 2D culture. (A) tie-1-EGFP ES cells were aggregated into EBs and plated on gelatin in the presence of 10 ng/ml of rVEGF. Whole 10-day-old EBs were stained for CD31 (red) to visualize pseudovascular structures. Analysis of EGFP fluorescence showed localization of EGFP+ cells within the CD31-stained vascular network in EBs derived from the A1TG clone (top panel). The two photographs on the bottom represent staining of EBs derived from a neomycin-selected control ES-cell clone, which does not carry the tie-1-EGFP transgene. Bar, 100 µM. (B) 10-day-old EBs were enzymatically dissociated and single cells were plated on gelatin for 1 day. Immunofluorescence staining for CD31 confirmed that all EGFP+ cells were also CD31+. Note the presence of CD31+EGFP- cell clumps, that possibly represent endothelial cell progenitor colonies. Bar, 50 µM. (C) tie-1-EGFP ES cells were plated on gelatin-coated slides in the presence of 10 ng/ml rVEGF. After 10 days of differentiation, slides were fixed and stained for CD31. In addition, cell nuclei were stained with DAPI. Bar, 50 µM.

View larger version (57K): [in a new window]   Fig. 4. Selection of endothelial cells from a population of differentiating tie-1-EGFP/tie-1-puror ES cells by puromycin. (A) Analysis of tie-1-puror transgene integration into genomic DNA. PCR products were amplified using oligonucleotides for the tie-1 promoter and either puror gene or the EGFP cDNA. PCR products are shown for two resistant clones, B9TP and C4TP, and the parental clone A1TG. Amplification of a 500 bp fragment of the tie-1-puror transgene in B9TP (lane 2), C4TP (lane 3) and with the tie-1-puror control plasmid (lane 4). Amplification of a 310 bp fragment of the tie-1-EGFP transgene in the parental clone A1TG (lane 5), B9TP (lane 6), C4TP (lane 7) and with the tie-1-EGFP control plasmid (lane 8). Lane 1, molecular weight markers. (B) Puromycin selection of ES-derived endothelial cells from 2D plane cultures of B9TP cells. B9TP cells were allowed to differentiate for 7 days in the presence of 10 ng/ml rVEGF and subjected (right panel) or not (left panel) to a 4-day puromycin selection (1 µg/ml). While in the absence of puromycin, CD31+EGFP+ cells are present in an heterogenous population, after puromycin selection, almost all remaining cells are CD31+EGFP+. DAPI staining allows visualization of all cells present in the field. Bar, 100 µM.

View larger version (78K): [in a new window]   Fig. 5. The puromycin-selected population expresses endothelial markers. (A) Puromycin-selected cells were cultured as spheroids in the presence of 10 ng/ml VEGF and stained for endothelial cell markers, CD31, VEGFR-2, CD34 and VE-cadherin. Note that all EGFP+ cells expressed VE-cadherin, CD31, VEGFR-2 and CD34. Note the presence of cell clusters that were negative for EGFP, but positive for CD31, VEGFR-2 and CD34. These clusters are likely to be endothelial progenitors. Bar, 50 µM. (B) Semi-quantitative RT-PCR analysis of undifferentiated B9TP (lane 1) and purified cells (lane 2) for the expression of the vegfr-2, vegfr-1, tie-1, icam-2 and hprt genes.

View larger version (57K): [in a new window]   Fig. 6. The puromycin-selected population contains smooth muscle cells. (A) TGF-ß1 and laminin-1 increase the number of -SMA+ cells. Puromycin-selected cells were cultured on either gelatin or laminin-1, in the presence of 10 ng/ml rVEGF or 5 ng/ml TGF-ß1, and stained for -smooth muscle actin (FITC, actin filaments in green) and CD31 (red). The diffuse green staining reveals the expression of EGFP in CD31+ cells that do not contain -SMA. Bar, 50 µM. (B) CD31/-SMA double staining. While some cells only expressed -SMA (arrows), others expressed both -SMA and CD31 (arrowheads). Bar, 30 µM.

View larger version (101K): [in a new window]   Fig. 7. Puromycin-selected endothelial cells are incorporated into neovessels in vivo. (A) Localization of EGFP+ in cryo-sections of co-injected (left) and control (right) tumors. EGFP+ cells are incorporated into the tumor neovasculature, as visualized by CD31 staining. Arrows indicate EGFP+CD31+ cells present in sections of `co-injected' tumors. Note that the extent of neovascularization was similar in both types of tumors, but that the EGFP expression was observed only in co-injected tumors. (B) Analysis of -SMA expression in tumors. Tumor sections were co-stained for -SMA (red) and CD31 (green). Bar, 100 µM.

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