HIGH-RESOLUTION CELL-CYCLE ANALYSIS OF DEFINED PHENOTYPIC SUBSETS WITHIN PRIMITIVE HUMAN HEMATOPOIETIC-CELL POPULATIONS

We have developed a novel protocol for analysis of cell cycle status w ithin specific subsets of primitive human hematopoietic cells. The tec hnique, referred to as SID (surface, intracellular, and DNA) analysis, allows for the simultaneous characterization of cell surface and intr acellular antigens, as well as quantitation of DNA content. To evaluat e the technique, early human hematopoietic cells were examined using s urface staining for the CD34 and CD38 antigens to identify primitive c ells. Relative cell cycle status within defined phenotypic subsets (CD 34(+) and CD34(+)/CD38(-)) was determined by simultaneous two-paramete r analysis using DNA content vs. antibody staining for the Ki-67 antig en. Ki-67 is not expressed in quiescent cells, but is quickly up-regul ated as cells are induced to cycle. Consequently, expression of Ki-67, in combination with DNA content can be used to delineate all phases o f the cell cycle (G(0), G(1), S, and G(2)/M). We demonstrate that cycl e induction of CD34(+) cells, using IL-3, IL-6, and stem cell factor ( SCF), does not correlate with activation of the CD34(+)/CD38(-) subpop ulation during ex vivo culture. Rather, CD34(+)/CD38(-) cells are much more refractory to cycle activation, requiring at least 72 hours to s how significant levels of induction. In addition, primitive cells deri ved from bone marrow (BM) vs. mobilized peripheral blood (PB) show dif fering degrees of responsiveness to conventional ex vivo culture condi tions. 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 st rongly with IL6+SCF to activate quiescent CD34(+)/CD38(-) cells. Moreo ver, when FL was combined with IL-3+IL-6+SCF, there was a small but re producible increase in activation of CD34(+)/CD38(-) cells from G(0) t o G(1). These data suggest that ex vivo behavior of primitive human st em cell populations is amenable to comprehensive flow cytometric analy sis, and that such studies can provide detailed information on the bio logical response of stem cells to ex vivo culture and manipulation.