Hematopoietic and endothelial potential of CD34⁺ subsets. (A) CFC potential of MACS-sorted CD34⁺ subsets with indicated phenotype (+/– chart) was tested using MethoCult GF+ assay. Results are the mean ± SD from 9 independent experiments with H1 (n = 6) and H9 (n = 3) cells. NA indicates not applicable (subset was not detected/sorted). (B) qRT-PCR analysis of CD34⁺ subsets on day 6 of H1/OP9 coculture. The stacked bar graph shows expression levels of indicated transcripts represented by relative units (see “Materials and methods” for details). qPCR results are the means from 3 independent experiments. Representative agarose gel electrophoresis of qPCR products is shown. (C) Endothelial culture of CD34⁺ subsets isolated on day 6 of H1/OP9 coculture. Photographs show 5-day culture of indicated CD34⁺ subsets in endothelial conditions (scale bar represents 50 μm). Insets show VE-cadherin expression (scale bar represents 50 μm). VE-cadherin was stained by anti–VE-cadherin antibody (goat IgG; R&D Systems, Minneapolis, MN) followed by anti–goat IgG–Alexa Fluor-488 conjugate (green fluorescence; Molecular Probes). Cell nuclei were visualized by DAPI staining (blue fluorescence). Images were captured with an inverted DMIRB microscope (Leica Microsystems) equipped with a 20×/0.3 objective lens, and were acquired through MagnaFire camera and software (Optronics). Fluorescent images were composed using Adobe Photoshop software (Adobe Systems, San Jose, CA). The representative experiment is shown. Identical results were obtained with CD34⁺ subsets isolated on day 9 (H1, H9) and day 6 (H9). (D) Wright-stained cytospins of CD34⁺ subsets isolated on day 9 of H1/OP9 coculture (scale bar represents 10 μm). Images were captured with a Microphot-SA microscope (Nikon, Melville, NY) equipped with a 100×/1.40 oil-immersion objective lens, and were acquired through a DFC320 camera and Firecam 1.3 software (Leica Microsystems). (E) CD34⁺CD43^– KDR⁺ cells isolated on day 5 of H1/OP9 coculture were cultured 6 days with fresh OP9 cells (i) or without feeder cells (ii) in StemSpan serum-free medium (StemCell Technologies) supplemented with 2% FBS (HyClone Laboratories), Ex-Cyte (1/500; Serological, Norcross, GA), 10 ng/mL bFGF, 50 ng/mL SCF, 10 ng/mL TPO, 20 ng/mL IL-6. Central endothelial clusters (ECs) surrounded by proliferating hematopoietic clusters (HCs) were observed in coculture with OP9 cells (i; scale bar represents 100 μm). Similar hematoendothelial colonies were formed in feeder-free cultures (ii; scale bar represents 50 μm); bright-field (left panel) and fluorescent (right panel) photographs show the same colony stained with anti-CD43 mAb (BD Pharmingen, San Diego, CA) and anti–VE-cadherin antibody (goat IgG; R&D Systems) followed by anti–mouse IgG–Alexa Fluor-488 (green fluorescence) and anti–goat IgG–Alexa Fluor-555 conjugates (red florescence; Molecular Probes). Images captured with an inverted DMIRB microscope (Leica Microsystems) equipped with a 10×/0.25 (i) or a 20×/0.3 (ii) objective lens, and were acquired with a MagnaFire camera and software (Optronics). Note a clear separation of the hematopoietic and endothelial cells by CD43 staining: all rounded hematopoietic cells are CD43⁺, but adherent VE-cadherin⁺ endothelial cells are CD43^–.