Functional Heterogeneity of Human CD34+ Cells Isolated in Subcompartments of the G0 /G1 Phase of the Cell Cycle

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Abstract
Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34+ cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34+ cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34+ cells isolated in G0 (G0CD34+ cells) than in those residing in G1 (G1CD34+ cells). However, as MPB CD34+ cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34+ cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34+ cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34+ cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34+ cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2dim population whereby LTHC-IC frequency was higher for CD34+ cells reselected in G0 after in vitro division than for CD34+ cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2bright CD34+ cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.