CYCLING STATUS OF CD34(-BLOOD OF HEALTHY DONORS BY RECOMBINANT HUMAN GRANULOCYTE-COLONY-STIMULATING FACTOR() CELLS MOBILIZED INTO PERIPHERAL)

In this study, we assessed the functional and kinetic characteristics of highly purified hematopoietic CD34(+) cells from the apheresis prod ucts of 16 normal donors undergoing glycosylated granulocyte colony-st imulating factor (G-CSF) treatment for peripheral blood stem cells (PB SC) mobilization and transplantation in allogeneic recipients. Mobiliz ed CD34(+) cells were evaluated for their colony-forming capacity and trilineage proliferative response to selected recombinant human (rh) C SF in vitro and the content of very primitive long-term culture initia ting cells (LTC-IC). In addition, the cycling status of circulating CD 34(+) cells, including committed clonogenic progenitor cells and the m ore immature LTC-IC, was determined by the cytosine arabinoside (Ara-C ) suicide test and the acridine orange flow cytometric technique. By c omparison, bone marrow (BM) CD34(+) cells from the same individuals we re studied under steady-state conditions and during G-CSF administrati on. Clonogenic assays in methylcellulose showed the same frequency of colony-forming unit cells (CFU-C) when PB-primed CD34(+) cells and BM cells were stimulated with phytohemagglutinin-lymphocyte-conditioned m edium (PHA-LCM). However, mobilized CD34(+) cells were significantly m ore responsive than their steady-state BM counterparts to interleukin- 3 (IL-3) and stem cell factor (SCF) combined with G-CSF or IL-3 in pre sence of erythropoietin (Epo). In cultures added with SCF, IL-3, and E po, we found a mean increase of 1.5- +/- 1-fold (standard error of the mean [SEM]) of PB CFU-granulocyte-macrophage and erythroid progenitor s (burst-forming units-erythroid) as compared with BM CD34(+) cells (P < .05). Conversely, circulating and BM megakaryocyte precursors (CFU- megakaryocyte) showed the same clonogenic efficiency in response to IL -3, granulocyte-macrophage-CSF and IL-3, IL-6, and Epo. After 5 weeks of liquid culture supported by the engineered murine stromal cell line M2-10B4 to produce G-CSF and IL-3, we reported 48.2 +/- 35 (SEM) and 62.5 +/- 54 (SEM) LTC-IC per 10(4) CD34(+) cells in PB and steady-stat e BM, respectively (P = not significant). The Ara-C suicide assay show ed that 4% +/- 5% (standard deviation [SD]) of committed precursors an d 1% +/- 3% (SEM) of LTC-IC in PB are in S-phase as compared with 25.5 % +/- 12% (SD) and 21% +/- 8% (SEM) of baseline BM, respectively (P < .001). However, longer incubation with Ara-C (16 to 18 hours), in the presence of SCF, IL-3 and G-CSF, or IL-6, showed that more than 60% of LTC-IC are actually cycling, with no difference being found with BM c ells. Furthermore, studies of cell-cycle distribution on PB and BM CD3 4(+) cells confirmed the low number of circulating progenitor cells in S- and G(2)M-phase, whereas simultaneous DNA/RNA analysis showed that the majority of PB CD34(+) cells are not quiescent tie, in G(0)-phase ), being in G(1)-phase with a significant difference with baseline and G-CSF-treated BM (80% +/- 5% [SEM] v 61.9% +/- 6% [SEM] and 48% +/- 4 % [SEM], respectively; P < .05). Moreover, G-CSF administration preven ted apoptosis in a small but significant proportion of mobilized CD34( +) cells. Thus, our results indicate that mobilized and BM CD34(+) cel ls can be considered equivalent for the frequency of both committed an d more immature hematopoietic progenitor cells, although they show dif ferent kinetic and functional profiles. In contrast with previous repo rts, we found that PB CD34(+) cells, including very primitive LTC-IC, are cycling and ready to progress into S-phase under CSF stimulation. This finding should be taken into account for a better understanding o f PBSC transplantation. (C) 1997 by The American Society of Hematology .