High‐resolution cell division tracking demonstrates the Flt3‐ligand‐dependence of human marrow CD34+CD38− cell production in vitro

Abstract

Investigation of primitive human haemopoietic cell behaviour requires methodologies for monitoring asynchronously activated cells over several generations. We describe a high-resolution procedure for tracking 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)- labelled human haemopoietic cells through six cell cycles based on the precise halving of their CFSE-fluorescence at each mitosis. Using this approach in combination with DNA or surface antigen staining, we show that 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 cells initially recruited into division in vitro, shortened the overall cycle time of their progeny, and enhanced the production of a derivative CD34+CD38- population through several (up to four) cell generations. These studies also showed that during the first 4d there was no detectable apoptosis among the progeny of the CD34+(CD45RA/CD71)- cells generated in the presence of this four-cytokine cocktail, regardless of the presence of FL. The availability of a technique for monitoring changes in the properties of individual cells as a function of their mitotic history and under conditions where they are asynchronously recruited to divide provides a new and powerful approach for studies of the regulation of primitive human haemopoietic cell proliferation and differentiation.