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Cycling status of CD34+ cells mobilized into peripheral blood of healthy
donors by recombinant human granulocyte colony-stimulating factor
RM Lemoli, A Tafuri, A Fortuna, MT Petrucci, MR Ricciardi, L Catani, D Rondelli, M Fogli, G Leopardi, C Ariola and S Tura
Institute of Hematology L. & A. Seragnoli, University of Bologna,
Italy.
In this study, we assessed the functional and kinetic characteristics of
highly purified hematopoietic CD34+ cells from the apheresis products of 16
normal donors undergoing glycosylated granulocyte colony- stimulating
factor (G-CSF) treatment for peripheral blood stem cells (PBSC)
mobilization and transplantation in allogeneic recipients. Mobilized CD34+
cells were evaluated for their colony-forming capacity and trilineage
proliferative response to selected recombinant human (rh) CSF in vitro and
the content of very primitive long-term culture initiating cells (LTC-IC).
In addition, the cycling status of circulating CD34+ cells, including
committed clonogenic progenitor cells and the more immature LTC-IC, was
determined by the cytosine arabinoside (Ara-C) suicide test and the
acridine orange flow cytometric technique. By comparison, bone marrow (BM)
CD34+ cells from the same individuals were studied under steady-state
conditions and during G-CSF administration. 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 medium (PHA-LCM). However,
mobilized CD34+ cells were significantly more 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 presence of erythropoietin (Epo). In
cultures added with SCF, IL-3, and Epo, we found a mean increase of 1.5-
+/- 1-fold (standard error of the mean [SEM]) of PB CFU-granulocyte-
macrophage and erythroid progenitors (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-state BM, respectively (P = not significant). The Ara-C suicide
assay showed that 4% +/- 5% (standard deviation [SD]) of committed
precursors and 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 cells.
Furthermore, studies of cell-cycle distribution on PB and BM CD34+ cells
confirmed the low number of circulating progenitor cells in S- and
G2M-phase, whereas simultaneous DNA/RNA analysis showed that the majority
of PB CD34+ cells are not quiescent (ie, in G0-phase), being in G1-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 prevented apoptosis in a small but
significant proportion of mobilized CD34+ cells. Thus, our results indicate
that mobilized and BM CD34+ cells can be considered equivalent for the
frequency of both committed and more immature hematopoietic progenitor
cells, although they show different kinetic and functional profiles. In
contrast with previous reports, 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 of PBSC transplantation.
Volume 89,
Issue 4,
pp. 1189-1196,
02/15/1997
Copyright © 1997 by The American Society of Hematology
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