Supplementary Figure 1.V-AML Isolation and Purity Analysis

Supplementary Figure Legends

Supplementary Figure 1.V-AML Isolation and purity analysis.

(A) V-AML purification from non-parenchymal liver cells of xenografted mice. Following an initial enrichment sort, mCD45neg mCD31+hCD13/33+ V-AML cells were sorted twice using the gates shown and purity mode settings. Individual thrice sorted cells were sorted directly into 96 well plates for gDNA PCR analysis (Figure 2F), RT-PCR analysis (Figure 4A), or were sorted onto slides for immunostaining and scoring (panel D). (B) Top: flow analysis of donor AML prior to transplant. A very low percentage of AML binds non-specifically to the mouse CD31 antibody; however, sorting using the indicated gate did not enrich this population of hCD33/13+mCD31+cells. Bottom: significant enrichment of V-AML from the NPC from an NSG host mouse transplanted with the same donor. (C) Analysis of twice-sorted AML and mouse EC populations from the liver of AML-engrafted NSG mice (see Figure 3D). The left-most panels show purity analysis by flow cytometry. Following FACS isolation, cells were immunostained with anti-hCD45 (green) and anti-mCD31 (red). Nuclei were stained with DAPI (blue). Representative cells are shown, and single cells co-expressing hCD45 and mCD31 were not detected in multiple independent experiments. (D) Thrice sorted V-AML cells were stained with hCD45 (green), mCD31 (red) and DAPI (blue). Typical examples of IF staining patterns are shown along with the frequency that they were observed for the cells sorted in (A). Note that the hybrid/fused cell shown (yellow) co-expresses circumferential hCD45 and mCD31. Scale bars: 5 microns

Supplementary Figure 2.V-AML is present in bone marrow of engrafted NSG mice.

(A-B) Single 0.2 µm Z planes from AML recipientmouse femur sections. Arrowheads point to integrated V-AML that coexpresses human CD45 (green) and mouse CD31 (red) in bone marrow sinusoids and resembles integrated V-AML cells detected in liver portal vessels (Figure 2A-B). (C) FACS-sorted mCD31+hCD13/33+ V-AML cells isolated from the long bones of AML-recipient mice were subsequently stained with mCD31 (red), hCD45 (green) and DAPI (blue) and imaged. The V-AML phenotype detected in the bone marrow is indistinguishable from the predominant V-AML phenotype detected in the liver (see Figure 2E, Figure S3D). All images show DAPI staining in blue. Scale bars: 10 microns.

Supplementary Figure 3. Imaging ofAML–EC hybrid cells.

A)ConfocalZstack series of the portal vessel of the engrafted liver shown in Figure 3A. Imaging was performed at 0.2 micron intervals and the interval number is indicated on the left. The * highlights a cell in the PV with co-staining of hCD45 and mCD31 throughout the cell membrane. (B) Additional examples of sorted V-AML hybrid cells (also see Figure 2 D&E; Figure 3B). Although hCD45 and mCD31 are detected within the same cell membrane, they are not fully circumferential and their expression pattern is consistent with early cell fusion. A representative Z plane is shown for each cell. Scale bars: 10 microns.

Supplementary Figure 4. Immunofluorescence analysis of L-ECFCs derived from AML patients.

Cells were stained with the hematopoietic and endothelial lineage markers indicated in each panel (green) and DAPI (blue). Consistent with the flow cytometry analysis in Figure 5, expression of the EC marker CD105 is seen throughout L-ECFCs, whereas hematopoietic markers are not detected.