Freshly isolated CD34⁻Lin⁻CD45⁻CD133⁻ cells have a stem cell profile compatible with hematopoietic and endothelial potential. (A) May-Grunwald-Giemsa staining showing that freshly isolated CD34⁻Lin⁻CD45⁻CD133⁻ cells have blastoid structure typical of stem cells. (B) Flow cytometric analysis showing that freshly isolated CD34⁻Lin⁻CD45⁻CD133⁻ cells express c-Kit and CXCR4, according to their progenitor nature. (C) RT-PCR showing that freshly isolated CD34⁻Lin⁻CD45⁻CD133⁻ cells express selected stem cell markers as well as markers shared by progenitors of hematopoietic and endothelial lineages. HSCs and primary human endothelial cells (HUVECs) were used as positive controls. One representative of 8 independent experiments is shown. (D) Failure to detect expression of Oct4 (red) and Nanog (green) in freshly isolated CD34⁻Lin⁻CD45⁻CD133⁻ cells and CD34⁺ HSCs, as assessed by confocal microscopy after immunostaining with specific Abs. Nuclei (blue) are stained with DAPI. iPSC served as a positive control (results not shown). FSC indicates forward scatter. Original magnification in panels A and D is 20×.