The AWE vector tag hemogenic and hematopoietic progenitors.

<p>(<b>A</b>) CFU potential of eGFP+ cells. AWE- transduced H9 cells were incubated in EB hematopoietic differentiation media. 10–12 days later, eGFP⁺ and CD34⁺ cells were sorted and seeded in methylcellulose H4434 (Stem Cell Technologies, Vancouver, Canada). Left graph shows CFU efficiency obtained from eGFP+ sorted cells compared to CD34⁺. Data are shown as average from three independent experiments +/− SEM. Right graphs shows relative frequency of the different types of colonies (G = granulocytes; GM = Granulocytes/macrophages; M = Macrophages) obtained from eGFP⁺ (left) and CD34⁺ (right) sorted cells. (<b>B</b>) Hemogenic potential of CD45⁻CD31⁺ cells expressing eGFP. AWE- transduced H9 cells were incubated in EB hematopoietic differentiation media. 10–12 days later the cells were analyzed for eGFP (top left plot) as well as CD45 and CD31 expression (top right plot). CD31⁺eGFP⁻ (left panels) and CD31⁺eGFP⁺ (right panels) were sorted and induced in StemSpan with Flt-3L, SCF, IL-3, IL-6 and G-CSF(see M&M). Expression of CD45 and eGFP was analyzed 10 days later (bottom-black plots). (<b>C</b>) Hemogenic potential of CD45⁻CD31⁻ cells expressing eGFP. AWE- transduced H9 cells were incubated in EB hematopoietic differentiation media. 10–12 days later differentiated cells were analyzed as above. CD31⁻eGFP⁻ (left panels) and CD31⁻eGFP⁺ (right panels) were sorted and induced to differentiate toward haematopoiesis as above. Note that the CD45⁻CD31⁻eGFP⁺ cells (but not the CD45⁻CD31⁻eGFP⁻) generate CD45⁺ cells. (<b>D</b>) Hemogenic efficiency of AWE-labelled CD45⁻ populations. Graph showing the fold enrichment in hemogenic progenitors of the eGFP⁺ versus the eGFP⁻ cells on CD31⁺ and CD31⁻ cells. Data were obtained by dividing the percentages of CD45⁺ cells derived from the eGFP⁺CD31⁺ sorted cells with the values obtained with the eGFP⁻CD31⁺ sorted cells (left column) or eGFP⁺CD31⁻ sorted cells with eGFP⁻CD31⁻ sorted cells (right column).