title: 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.
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada.
authors.forename: R E
authors.initials: RE
authors.lastname: Nordon
authors.forename: S S
authors.initials: SS
authors.lastname: Ginsberg
authors.forename: C J
authors.initials: CJ
authors.lastname: Eaves
mesh_terms: D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000378:metabolism; D000943:Antigens, Differentiation / Q000378:metabolism; D002455:Cell Division; D002460:Cell Line; D019070:Cell Lineage; D005452:Fluoresceins / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000378:metabolism; D010641:Phenotype; D013388:Succinimides / Q000378:metabolism
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006133:Growth Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D013388:Succinimides; C084881:flt3 ligand protein; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1