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We have developed a novel protocol for analysis of cell cycle status within specific subsets of primitive human hematopoietic cells. The technique, referred to as SID (surface, intracellular, and DNA) analysis, allows for the simultaneous characterization of cell surface and intracellular antigens, as well as quantitation of DNA content. To evaluate the technique, early human hematopoietic cells were examined using surface staining for the CD34 and CD38 antigens to identify primitive cells. Relative cell cycle status within defined phenotypic subsets (CD34+ and CD34+/CD38-) was determined by simultaneous two-parameter analysis using DNA content vs. antibody staining for the Ki-67 antigen. Ki-67 is not expressed in quiescent cells, but is quickly up-regulated as cells are induced to cycle. Consequently, expression of Ki-67, in combination with DNA content can be used to delineate all phases of the cell cycle (G0, G1, S, and GZ/M). We demonstrate that cycle induction of CD34+ cells, using IL-3, IL-6, and stem cell factor (SCF), does not correlate with activation of the CD34+CD38- subpopulation during ex vivo culture. Rather, CD34+/CD38- cells are much more refractory to cycle activation, requiring at least 72 hours to show significant levels of induction. In addition, primitive cells derived from bone marrow (BM) vs. mobilized peripheral blood (PB) show differing degrees of responsiveness to conventional ex vivo culture conditions. Finally, the effect of IL-3, IL-6, SCF, and Flt3 ligand (FL) on cycle induction was examined. It was observed that IL-3 synergized strongly with IL6+SCF to activate quiescent CD34+/CD38- cells. Moreover, when FL was combined with IL-3+IL-6+SCF, there was a small but reproducible increase in activation of CD34+/CD38- cells from G0 to G1. These data suggest that ex vivo behavior of primitive human stem cell populations is amenable to comprehensive flow cytometric analysis, and that such studies can provide detailed information on the biological response of stem cells to ex vivo culture and manipulation. |
