HIGH-RESOLUTION CELL-DIVISION TRACKING DEMONSTRATES THE FLT3-LIGAND-DEPENDENCE OF HUMAN MARROW CD34(-) CELL PRODUCTION IN-VITRO()CD38()

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over sever al generations. We describe a high-resolution procedure for tracking 5 - (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labe lled human haemopoietic cells through six cell cycles based on the pre cise halving of their CFSE-fluorescence at each mitosis. Using this ap proach in combination with DNA or surface antigen staining, we show th at the addition of Flt3-ligand (FL) to a cytokine cocktail consisting of Steel factor, IL-3, IL-6 and G-CSF increased the proportion of CD34 (+) (CD45RA/CD71)(-), but not CD34(+)(CD45RA/CD71)(+), human marrow ce lls initially recruited into division in vitro, shortened the overall cycle time of their progeny, and enhanced the production of a derivati ve CD34(+)CD38(-) population through several (up to four) cell generat ions. These studies also showed that during the first 4 d there was no detectable apoptosis among the progeny of the CD34(+)(CD45RA/CD71)(-) cells generated in the presence of this four-cytokine cocktail, regar dless of the presence of FL. The availability of a technique for monit oring changes in the properties of individual cells as a function of t heir mitotic history and under conditions where they are asynchronousl y recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.