FUNCTIONAL-HETEROGENEITY OF HUMAN CD34(+) CELLS ISOLATED IN SUBCOMPARTMENTS OF THE G(0) G(1) PHASE OF THE CELL-CYCLE/

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34(+) cel ls were isolated in subcompartments of the G(0)/G(1) phase of the cell cycle by flow cytometric cell sorting, In both bone marrow (BM) and m obilized peripheral blood (WIPE) CD34(+) cells, primitive long-term he matopoietic culture-initiating cell (LTHC-IC) activity was higher in C D34(+) cells isolated in G(0) (G(0)CD34(+) cells) than in those residi ng in G(1) (G(1)CD34(+) cells). However, as MPB CD34(+) cells displaye d a more homogeneous cell-cycle status within the G(0)/G(1) phase and a relative absence of cells in late G(1), DNA/RNA fractionation was le ss effective in segregating LTHC-IC in MPB than in BM, BM CD34(+) cell s belonging to four subcompartments of increasing RNA content within t he G(0)/G(1) phase were evaluated in functional assays, The persistenc e of CD34 expression in suspension culture was inversely correlated wi th the initial RNA content of test cells, Multipotential progenitors w ere present in G(0) or early G(1) subcompartments, while lineage-restr icted granulomonocytic progenitors were more abundant in late G(1). In vitro hematopoiesis was maintained for up to 6 weeks with G(0)CD34(+) cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G(1)CD34(+) cells. To test the hypothesis t hat primitive LTHC-ICs would reenter a state of relative quiescence af ter in vitro division, BM CD34(+) cells proliferating in ex vivo cultu res were identified from their quiescent counterparts by a relative lo ss of membrane intercalating dye PKH2, and were further fractionated w ith Hst and PY. The same functional hierarchy was documented within th e PKH2(dim) population whereby LTHC-IC frequency was higher for CD34() cells reselected in G(0) after in vitro division than for CD34(+) ce lls reisolated in G(1) or in S/G(2) + M. However, the highest LTHC-IC frequency was found in quiescent PKH2(bright) CD34(+) cells. Together, these results support the concept that cells with distinct hematopoie tic capabilities follow different pathways during the G(0)/G(1) phase of the cell cycle both in vivo and during ex vivo culture. (C) 1997 by The American Society of Hematology.