Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4446-4449
CORRESPONDENCE
Does Recombinant Human Granulocyte Colony-Stimulating
Factor Really Prime Marrow Stem Cells in Mice and
Humans?
 |
LETTER |
To the Editor:
Based on experimental evidence from studies in the murine model, Gyger
et al1 have stated that this is not the case; on the
contrary, granulocyte colony-stimulating factor (G-CSF) most probably
leads to significant early depletion of stem cells from their marrow niches.
However, in the human system (ie, patients), there are several
observations2-5 indicating that the effect of stimulation of hematopoiesis may differ in mouse and human! First, after 5 days of
treatment with recombinant human G-CSF (rhG-CSF) the bone marrow cellularity increases 10- to 100-fold without any change in
CD34+, colony-forming units (CFU), long-term
culture-initiating cells (LTCIC), and pre-CFU frequencies,
corresponding to a 1 to 2 log increase in total numbers per
volume.2-4 Second, the expansion was confirmed by magnetic
resonance imaging, documenting a moderate increase on days 5 to 7 that
surprisingly continued to expand on days 12 to 14.5 Based
on these findings, it is very unlikely that the marrow was in a
negative stem/progenitor cell balance on day 4 postcytokine therapy,
which is the time when marrow harvest was performed in most
studies.6-12 Third, several clinical studies have, until
now, documented cytokine-primed marrow as a sufficient autograft
without any reported graft failures in more than 100 patients.2,4,6-12
The reason for the putative discrepancy seen in humans compared with
mice may be differences in the hematopoietic system. One important
difference is that spleen hematopoiesis in mice13,14 differs from humans as blood circulating progenitors/stem cells in mice
lodge in the spleen and grow as colonies that can be readily counted 7 to 12 days later. It may be that spleen homing progenitors are the
reason for the early depletion of stem cells from their marrow niches
after G-CSF priming.1,13-16
The hypothetical interpretation from Gyger et al1 on
peripheral blood contamination at the time of marrow harvest to explain engraftment capability cannot be supported by data from our center. We
have performed analysis of stem/progenitor cell numbers, CD4/CD8, lactate dehydrogenase (LDH), and growth factor
concentration without documenting any significant blood contamination
in marrow aspirates up to a size of 10 to 20 mL. On the contrary, our
impression is an altered and enhanced microcirculation in the marrow in
parallel with G-CSF-mediated expansion of hematopoiesis.17
This is reflected by a change of bone marrow punctures into easy and
fast aspirations of a sufficient number of stem cells.18
In conclusion, there seems to be sound evidence from studies in humans
suggesting that G-CSF can indirectly expand human stem/progenitor cell
numbers after short-term priming.2-5 Consequently, it may be feasible in some poor mobilizers to perform stem cell harvest after
rhG-CSF priming and expansion with the aim to obtain a safe autograft
with three-lineage engraftment. It is our experience that such a
harvest can be performed with success 5 to 7 days after stopping
rhG-CSF administration to allow maximal expansion and harvest of
greater than 1 × 106 CD34+ marrow cells/kg
during one or two procedures.
In consequence of the results published by Damiami et al,9
we now need to set the clock back and prospectively evaluate the
outcome of high-dose therapy and autografting of quality-assessed primed marrow or mobilized peripheral blood stem/progenitor cells, taking into consideration not only time to three-lineage recovery, but
also the risk of severe infections and relapse. It is our hypothesis
that such primary endpoints may validate the use of rhG-CSF-primed
bone marrow quality assessed in accordance with actual practice.
Hans E. Johnsen
Stem Cell Laboratory
Department of
Haematology
Herlev Hospital
University of Copenhagen
Herlev,
Denmark
| |
REFERENCES |
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Randomized trial of autologous filgrastim-primed bone marrow transplantation versus filgrastim-mobilized peripheral blood stem cell transplantation in lymphoma patients.
Blood
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Johnsen HE, Hansen PB, Plesner T, Jensen L, Gaarsdal E, Andersen H, Birgens H, Jacobsen GK, Kjærsgård E, Hansen NE:
Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with rhG-CSF.
Bone Marrow Transplant
10:229, 1992[Medline]
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Johnsen HE, Jensen L, Gaarsdal E, Hansen PB, Ersbøll J, Hansen NE:
Priming with recombinant human hematopoietic cytokines before bone marrow harvest expands in vivo and enhances ex vivo recovery of myeloid progenitors in short-term liquid cultures.
Exp Hematology
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Hansen PB, Knudsen H, Gaarsdal E, Jensen L, Ralfkier E, Johnsen HE:
Short-term in vivo priming of bone marrow haematopoiesis with rhG-CSF, rhGM-CSF or rhIL-3 before marrow harvest expands myelopoiesis but does not improve engraftment capability.
Bone Marrow Transplant
16:373, 1995[Medline]
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Jensen KE, Hansen PB, Andreé V, Johnsen HE, Ralfkiaer E, Karle H, Henriksen O:
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Knudsen LM, Hansen SW, Daugård G, Jarlbæk L, Agerbæk M, Jensen L, Skovsgård T, Hansen F, Johnsen HE:
Comparison of rhG-CSF primed bone marrow and blood stem cell autografts: A analysis of engraftment in malignant lymphomas and solid tumours.
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Randomized trial of autologous filgrastim-primed bone marrow transplantation versus filgrastim-mobilized peripheral blood stem cell transplantation in lymphoma patients.
Blood
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Cytokine-primed bone marrow stem cells vs. peripheral blood stem cells for autologous transplantation: A randomized comparison of GM-CSF vs. G-CSF.
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Knudsen LM, Johnsen HE, Gaarsdal E, Jensen L:
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Response
We thank Dr Johnsen for his reply to our recent letter to the editor.
The purpose of our reply to Damiani et al1 was to address
concerns about the capacity of G-CSF alone, as given for peripheral
blood stem cell mobilization, to prime the most primitive stem cell
compartment of the bone marrow (CD34+, CD38
,
Lin cells).
We have raised these concerns for two reasons.
(1) The presently proposed molecular mechanisms for stem cell
mobilization do not favor a clear potential for G-CSF-mediated expansion of the uncommitted marrow stem cell pool. Indeed, some studies demonstrate that the rapid stem cell mobilization after G-CSF
administration is potentially mediated by modulation of adhesion
molecules on CD34+ cells, mostly very late antigen-4
(VLA-4), leukocyte function-associated molecule-1 (LFA-1), and
L-selectin (CD62L).2,3 This could lead to a modification of
the adhesion capacity to the stroma. Furthermore, Papayannopoulou et
al4 recently suggested that growth factors such as G-CSF
could initiate mobilization in neutrophils. In support of this theory,
the metalloproteinase gelatinase-B (MMP-9) secreted by neutrophils has
been shown to be involved in the cleaving of the extracellular matrix
molecules to which stem cells are attached.5 G-CSF induced
injury to the extracellular matrix and/or downregulation of adhesion
molecules could lead to a breakdown in the stem-stromal cell
homeostasis and promote egress and shift of the primitive stem cell
compartment to the peripheral blood. In concordance with these
observations, we have summarized data from the murine model that argue
that the true stem cell compartment (uncommitted stem cells) was indeed
in a negative balance after a short course of G-CSF.6
(2) Marrow harvest is invariably associated with hemodilution,
and the timing of G-CSF-primed bone marrow harvest coincides with the
peak incidence of peripheral blood progenitors.1 We have
thus raised the hypothesis that this hemodilution, rather than
G-CSF-induced stem cell expansion, could at least be partly responsible for the enhanced engraftment reported in Damiani et al.1 Is G-CSF bone marrow priming a misconception?
In their reply, the investigators state that human and murine stem cell
physiology are quite different and conclude that there seems to be
sound evidence in humans suggesting that G-CSF can indirectly expand
human/stem progenitor cell numbers after short-term administration,
based on the following evidence that they report. (1) There is an
increase in bone marrow cellularity after 5 days of G-CSF treatment of
candidates for autologous transplantation. (2) This can be confirmed by
magnetic resonance. (3) Several clinical studies have documented
cytokine primed marrow as a sufficient autograft without any reported
graft failures in more than 100 patients. Lastly, the investigators
rule out any significant peripheral blood stem cell contamination that
could have partly contributed to enhanced engraftment.
We fully agree that a short-course administration of G-CSF will result
in increased marrow cellularity by triggering differentiation along the
myeloid lineage of committed progenitors such as colony-forming unit-granulocyte-macrophage (CFU-GM). However, what is
at stake here is the primitive CD34+, CD38,
Lin stem cell compartment that accounts for
approximately 1 in 1 × 105 marrow cells. This compartment
is obviously difficult to assess through magnetic resonance. At least
two studies show that, in humans, a short course of G-CSF
administration can lead to a reduction of the primitive marrow stem
cell compartment. Mandelam et al7 documented a lower
incidence of the primitive CD34+, Lin subset
after 3 days of G-CSF administration in the bone marrow of normal
volunteers. Likewise, Martinez et al8 noted that, although
after 4 days of G-CSF administration to normal donors, there was an
increase in the total number of nucleated BM cells from a median of
41.5 × 109/L before G-CSF to 56 × 109/L,
the proportion of primitive BM hematopoietic progenitors
(CD34+ CD38, CD34+
HLDR, and CD34+ CD117)
decreased during G-CSF administration, as well as the total number of
CD34+ cells. Interestingly, Dicke et al9 have
shown that the administration of G-CSF for 2 days to stage IV breast
cancer patients shifted the stem cell balance in favor of
differentiation over self-renewal. The primitive HLA-DR+
component, although slightly increased after the initial G-CSF injections, returned to baseline values after the second day of cytokine therapy. Concern about further decreases and possible exhaustion of the CD34+ HLA-DR cells with
further G-CSF therapy was even raised by the investigators.
We question how the present investigators can harvest 1 L of marrow
during a 1.0- to 2.5-hour procedure of 200 to 300 aspirations without
documenting any significant contamination of peripheral blood with
aspirates up to a size of 10 to 20 mL.10 Contamination of
peripheral blood during marrow harvest has been a well-documented phenomenon since the early days of BM transplantation and has been the
object of an excellent study by Batinic et al11
demonstrating significant contamination with peripheral blood. This is
a crucial point in this discussion, because it constitutes a potential
mechanism for G-CSF primed marrow enhanced recovery. The study of
lymphoid subsets in the marrow is useful for evaluating the degree of
blood contamination.12 Moreover, mobilized peripheral blood
stem cells have a markedly distinct immunophenotype characterized by a
reduced expression of c-kit, CD11a, CD18, CD49d, and
CD62L.2,3 It would be interesting to assess the incidence
of these immunophenotypically distinct stem cell subsets in a normal
versus G-CSF-primed marrow.
In summary, most of the presently proposed molecular mechanisms of
G-CSF peripheral blood stem cell mobilization and the results of some
murine/human data suggest that a short course of G-CSF administration
(4 days) has the potential to decrease the primitive marrow stem
cell pool. We would like to see more studies focusing on the biology
and immunology of G-CSF primed marrow to evaluate its clinical utility
and advantages, if any, over mobilized peripheral blood stem cells.
Martin Gyger
Entezam Sahovic
Mohamed Aslam
King
Faisal Specialist Hospital and Research Centre
Section of Adult
Hematology/BMT
Riyadh, Kingdom of Saudia Arabia
| |
REFERENCES |
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Randomized trial of autologous filgrastim-primed bone marrow transplantation versus filgrastim-mobilized peripheral blood stem cell transplantation in lymphoma patients.
Blood
90:36, 1997
2.
Mohle R, Murea S, Kirsh M, Haas R:
Differential expression of L-selectin, VIA-4, and LFA-1 on CD34+ progenitor cells from bone marrow and peripheral blood during G-CSF enhanced recovery.
Exp Hematol
23:1535, 1995[Medline]
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3.
Bellucci R, De Propis MS, Buccisano F, Lisci A, Leone G, Tabilio A, de Fabritis P:
Modulation of VLA-4 and L-selectin expression on normal CD34+ cells during mobilization with G-CSF.
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4.
Papayannopoulou T, Priestly G, Nakamoto B:
Anti VLA-4/VCAM-1 induced mobilization requires cooperative signaling through the kit/mkit ligand pathway.
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Pruijt JFM, Fibbe WE, Laterveer L, Willemze R, Masure S, Paemen L, Opdenakker G:
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6.
Gyger M, Sahovic E, Aslam M:
Randomized trial of autologous filgrastim-primed bone marrow versus filgrastim-mobilized peripheral blood stem cell transplantation in lymphoma patients.
Blood
92:3489, 1998
7.
Mandelam RK, Vento CA, Stiff PJ, Brott DA, Smith AK:
Stem and progenitor cell content and ex vivo expansion potential of human bone marrow primed in vivo with G-CSF.
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90:344b, 1997 (abstr, suppl 1, part 2)
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Effects of short-term in vivo administration of G-CSF on bone marrow prior to harvesting.
Exp Hematol
25:34, 1997
10.
Johnsen HE, Hansen PB, Plesner T, Jensen L, Gaarsdal E, Andersen H, Birgens H, Jacobsen GK, Kjaersgard E, Hansen NE:
Increased yield of myeloid progenitor cells in bone marrow harvested for autologous transplantation by pretreatment with rhG-CSF.
Bone Marrow Transplant
10:229, 1992
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Relationship between differing volumes of bone marrow aspirates and their cellular composition.
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