Additional file 1:

Heterogeneity and pattern formation of mouse EPCs after isolation and CD31 cell sorting shown at lower magnification. a) Pattern formation of almost confluent cells after the first CD31 selection shown by phase contrast microscopy. b) The same cells as in a). Heterogeneity of cells is demonstrated by immunofluorescence several days after Lyve1-positive selection. Note CD31-positive BECs surrounding Lyve1-positive LECs, which express variable amounts of CD31 (yellow). 100-fold.

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Additional file 2:

Isolation of MLMVECs from mouse lungs by FACS sorting. Low passage (passage 3) MLMVECs were sorted by FACS into CD31⁺/Lyve1⁺ (gate R1) and CD31⁺/Lyve1^- (gate R2) cells for further subculturing and immunophenotyping. The percentage of cells in the different regions is indicated (%).

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Additional file 3:

Subculturing of Lyve1⁺ and Lyve1^- cells after CD31/Lyve1 cell sorting. Early passage MLMVEC (passage 3) were FACS sorted (same cells as in supplementary figure 2) and separately cultured. Ten days after sub-culturing (passage 4) cells were studied again by FACS analysis with anti-Lyve1 antibodies. The CD31⁺/Lyve1⁺ cells indicated a stable high Lyve1 expression (a) whereas CD31⁺/Lyve^- cells now expressed Lyve1 at a comparably high rate (c). One passage later (passage 5) some CD31⁺/Lyve⁺ cells were negative for Lyve1 (b) whereas CD31⁺/Lyve1^- cells were getting even more positive for Lyve1 (d).

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Additional file 4:

Sprout formation of MLMVEC spheroids on Matrigel in the presence of different growth factor combinations. Quantitative sprout formation of MLMVECs (in μm) in the presence of different growth factors 5 days after plating together with Matrigel. Growth factor concentrations in Matrigel: VEGF-C (500 ng/ml); VEGF-A (50 ng/ml); bFGF (20 ng/ml); TNF-α (10 ng/ml); HGF (20 ng/ml); ECGF= endothelial cell growth factor supplement (50 μg/ml + heparin). Sprouts were measured with an inverted stereo microscope (Zeiss) using specific software (AxioVision Rel. 4.5) for the determination of sprout lengths. Cells from A were isolated from C57Bl/6 mice and cells from B were isolated from Balb/c mice. Values= means ± SD.

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Additional file 5:

Double labelling of lung EPCs with Lyve1, MECA32 and LA5. Immunophenotyping of cells was performed with cytometric analysis. Mouse lung EPCs are double positive for Lyve1 and MECA32. Only one part of the Lyve1⁺ cells are also positive for LA5. Cells from two isolations have an almost an identical pattern of the three markers.

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Additional file 6:

Mouse lung EPCs have the capacity to produce NO in vitro. Fluorescence detection of NO production was done with a practical FACS assay with living cells. Membrane-permeable DAF-2 diacetate has been used for indirect detection of NO production.

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Additional file 7:

Extended in vivo studies of mouse lung EPCs after lentiviral transduction with GFP marker gene. De novo formation of lymphatic vessels as revealed by double staining with anti-GFP and anti-LA102 (which is LEC-specific) in combination with nuclear Dapi staining. (Upper 4 figures). Note that staining with anti-CD45 (leukocyte common antigen) resulted in the staining of some scattered cells in the interstitium but no double staining with LA102 could be observed. (Lower 4 figures) 200-fold.

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Additional file 8:

In vivo studies of mouse lung EPCs to detect blood and lymphatic networks in the implanted Matrigel. Formation of blood and lymphatic networks in the same Matrigel by triple staining with anti-GFP, anti-podoplanin (LEC-specific) and anti-CD31. Note that the merged figure contained GFP-tube forming cells which are CD31 and podoplanin positive (LEC) and podoplanin negative cells (BEC). 200-fold.

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