Single CD34⁻Lin⁻CD45⁻CD133⁻ cells give rise to hematopoietic and endothelial lineage cells on LTC-IC-HE culture. (A) Strategy used to sort human CD34⁻Lin⁻CD45⁻CD133⁻ (1), CD34⁻Lin⁻CD45⁻CD133⁺ (2), CD34⁻Lin⁻CD45⁺CD133⁻ (3), and CD34⁻Lin⁻CD45⁺CD133⁺ (4) cells. (B) Flow cytometric analysis showing that after 6 weeks in LTC-IC-HEs only CD34⁻Lin⁻CD45⁻CD133⁻ cells gave rise, starting from a single cell, to both CD45⁻KDR⁺ (endothelial) and CD45⁺KDR⁻ (hematopoietic) cells. (C) Poisson analysis of 5 independent experiments, showing the frequency of wells containing CD45⁺KDR⁻ only, CD45⁻KDR⁺ only, or CD45⁺KDR⁻ and CD45⁻KDR⁺ cells, obtained after LTC-IC-HE of the 4 distinct sorted cell populations. (D) Confocal images of cells derived from single CD34⁻Lin⁻CD45⁻CD133⁻ cells after 6-week culture (under LTC-IC-HE conditions). Round CD45⁺ (hematopoietic-like) and spindle-shaped VE-cadherin⁺ (endothelial-like) cells in intimate contact with MS-5 murine stromal cells are shown. (E) May-Grunwald-Giemsa staining showing typical myeloid cells generated in CFU assay of LTC-IC-HEs started from single CD34⁻Lin⁻CD45⁻CD133⁻ cells. Original magnification 40×. (F) RT-PCR showing the expression of the hematopoietic CD45 marker and the endothelial VE-cadherin marker in the 4 sorted cell populations at day 0 and after 6 weeks in LTC-IC-HE. Three representative LTC-IC-HE wells (a-c) per cell population after 6 weeks are shown. Only the endothelial-positive control HUVEC line and LTC-IC-HE wells derived from CD34⁻Lin⁻CD45⁻CD133⁻ contained cells expressing VE-cadherin. The concomitant expression of VE-cadherin and CD45 in a single well indicated that, starting from a single cell, CD34⁻Lin⁻CD45⁻CD133⁻ cells gave rise to cells of both endothelial and hematopoietic lineages. (G) Representative flow cytometric histogram showing the expression of VE-cadherin (green line) by cells derived from CD34⁻Lin⁻CD45⁻CD133⁻ single cells (gray line indicates isotype control). At the end of the culture, 11 of 60 seeded wells contained a sufficient number of cells for analysis. Of these 11 wells, 3 tested positive for VE-cadherin expression. APC indicates allophycocyanin.