Ph⁺/VE-cadherin⁺ leukemic cells form hematopoietic colonies/cords on bone marrow niche stromal cells and differentiate into endothelial cells in vitro. (A) Phase-contrast micrographs of Ph⁺ ALL Sup-B15 (top) and Nalm27 (bottom) cells cultured in medium alone (long-term medium culture, LTMC) or long-term cocultured (LTCC) on murine stromal cell line S10 (top and bottom right). Images were collected by using a Leica DMIL microscope and 10×/0.22 objective (Houston, TX). (B) Confocal LSM microphotographs of hematopoietic cords formed on stromal cells. Cells were surface stained with anti-CD34, CD19, VE-cadherin, and CD133, respectively. Nucleic DNA dyes include Sytox (green), PI (red), and TO-PRO-3 (blue). Z stacks were 3-D reconstructed with LSM510 software (version 3.2; Zeiss, Jena, Germany). The turning axis is the y-axis, and turning angle for the displayed static pictures is 45° and confocal images were obtained using 20×/0.75 Fluar objective (rows 1, 3) and 10×/0.50 Fluar objective (rows 2, 4). (C) Confocal micrographs of hematopoietic colonies (“hemospheres”) stained with a panel of early stem cell (ESC) markers. Stromal monolayer cells underneath hemospheres were counterstained with anti–VCAM-1. Cell nuclei were counterstained with DNA dyes PI (red fluorescence) or SYTOX Green (green fluorescence) to be compatible with the fluorochrome tag of the secondary antibodies of anti-ESC antibody. Merged images of 3-channel colors are shown in the final column and images were acquired using a 10×/0.5 Fluar objective. (D) Three-dimensional reconstruction of z-stacks showing the distribution of transcriptional factor Oct-4 within a hematopoietic cord. Cell nuclei were counterstained with PI (red), and localization of Oct-4 was labeled with green fluorescence. The turning axis is the y-axis. The tip of the hematopoietic cord was pressed flat by the cover slip and images were acquired using a 10×/0.5 Fluar objective. (E) DsRed red fluorescent protein-–labeled Sup-B15 cells (top left) or eGFP-labeled Nalm27 cells (bottom left) were cultured in standard medium or in EndoCult endothelial-defined medium (top and bottom right). White arrows denote individual endothelial sprouting observed for individual Ph⁺/VE-cadherin⁺ ALL cells.