ASSESSMENT OF PROLIFERATIVE AND COLONY-FORMING CAPACITY AFTER SUCCESSIVE IN-VITRO DIVISIONS OF SINGLE HUMAN CD34(+) CELLS INITIALLY ISOLATED IN G(0)

Exit of primitive hematopoietic progenitor cells (HPCs) from the G(0) phase of the cell cycle in response to in vitro cytokine stimulation i s a limiting step in successful ex vivo expansion. Simultaneous DNA/RN A staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34(+) cells residing in G(0) (G(0)CD34(+)) from those c ycling in G(1) and S/G(2)+M. Compared with CD34(+) cells isolated in G (1), G(0)CD34(+) cells were characterized by a delayed response to cyt okine stimulation and were enriched for long-term hematopoietic cultur e-initiating cells. We next compared the activation kinetics of indivi dually sorted G(0)CD34(+) cells stimulated with stem cell factor (SCF) , flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a no vel clonal proliferation assay, the functional status of cells that ha d remained quiescent after an initial 7-day period and of those that h ad completed successive division cycles under each of these three fact ors was evaluated by assessment of subsequent proliferative capacity a nd maintenance of colony-forming cell precursor (pre-CFC) activity. Al l three cytokines were equally able to support the survival of primiti ve HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after ea ch division cycle. However, G(0)CD34(+) cells displayed a heterogeneou s response pattern to cytokine stimulation whereby SCF appeared to hav e a superior ability to promote the cycling of cells with high prolife rative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationship s between cellular division in response to different stimuli and maint enance of hematopoietic function.