1. No category

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1994 84: 2930-2939
A comparative study of the phenotype and proliferative capacity of
peripheral blood (PB) CD34+ cells mobilized by four different
protocols and those of steady-phase PB and bone marrow CD34+
cells
LB To, DN Haylock, T Dowse, PJ Simmons, S Trimboli, LK Ashman and CA Juttner
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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A Comparative Study of the Phenotype and Proliferative Capacity of
Peripheral Blood (PB) CD34+ Cells Mobilized by Four Different Protocols
and Those of Steady-Phase PB and Bone Marrow CD34' Cells
By L.B. To, D.N. Haylock, T. Dowse, P.J. Simmons, S. Trimboli, L.K. Ashman, and C.A. Juttner
Peripheral blood (PB) CD34+cells from four commonlyused
mobilization protocols were studied t o compare their phenotype and proliferative capacity with steady-state PB or
bone marrow (BM) CD34' cells. Mobilized PB CD34+ cells
were collected during hematopoietic recovery after myelosuppressive chemotherapy with or without granulocytemacrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) or during G-CSF
administration alone. The expression of activation and lineage-associated markers and c-kitgene product werestudied by flowcytometry. Proliferative capacity was measured
by generation of nascent myeloid progenitorcells (granulocyte-macrophage colony-stimulating factor; CFU-GM) and
nucleated cells in a stroma-free liquid culture stimulated by
a combination of six hematopoieticgrowth factors (interleukin-l (IL-l), IL-3, IL-6, GM-CSF, G-CSF, and stem cell factor).
G-CSF-mobilized CD34' cells have the highest percentage
of CD38- cells ( P < .0081), but otherwise, CD34+ cells from
different mobilization protocols were
similar t o one another
in theirphenotype and proliferativecapacity. The spectrum
of primitive and mature myeloid progenitors in mobilized
PBCD34+ cells was similar t o their steady-state counterparts, but the percentages of CD34+ cells expressing CD10
or CD19 were lower ( P < .0028). Although steady-state PB
and chemotherapy-mobilized CD34+ cells generated fewer
CFU-GM at day 21 than G-CSF-mobilized and steady-state
BM CD34+cells ( P < .0449), the generation of nucleated cells
and CFU-GM were otherwise comparable. The presence of
increased or comparable numbers of hematopoietic progenitors within PB collections with equivalent proliferative capacity to BMCD34' cells is notunexpected given the rapid
and complete hematopoietic reconstitution observed with
mobilized PB. However, all four types
of mobilized PB CD34'
cells are different from steady-state BM CD34' cells in that
they express less c-kit ( P < .0002) and CD71 ( P < .04) and
retain less rhodamine 123 ( P < .0001). These observations
are novel and suggest that different mobilization protocols
may act via similar pathways involving the down-regulation
of c-kit and may beindependent of cell-cycle status.
0 1994 by The American Societyof Hematology.
M
is the reverse of the process of homing seen during fetal
development or after stem cell infusion in transplantation.
Despite the increasingunderstandingofprogenitor/stroma
interactions, the mechanisms of mobilization remain undefined.
Autotransplantation using mobilized PB isassociated with
more rapidhematopoietic reconstitution (HR), lower requirementforblood
products and parenteralantibiotics,and
shorter
hospitalization
compared with
BM
transplantation.4.7.'I It has further been shown that the number of my-
ARKED INCREASES in the number ofperipheral
blood (PB) CD34+ cells and clonogenic hematopoietic progenitors occur during the
recovery phase after myelosuppressive chemotherapy with or without concomitant
granulocyte-macrophage colony-stimulating factor
(GMCSF) or granulocyte colony-stimulating factor (G-CSF) or
during G-CSF administrationalone."7 GM-CSF alone, interleukin-3 (IL-3) alone, sequential IL-3, and GM-CSF with
or without chemotherapy*"' have also been used for mobilization of CD34' cells with variable efficacy. Whereas some
protocols involvemyelosuppression, othersare associated
with increases in leukocyte counts and bone marrow (BM)
cellularity. Mobilization occurs within 1 week in some protocols andseveralweeksin
others. Hence,thereremains
considerable empiricism in designing peripheral blood (PB)
mobilization protocols. Furthermore, the mobilization phenomenon, ie, the entry of large numbers of progenitors into
PB, does not occur during
steady-state hematopoiesis and
From the Leukaemia Research Unit, Hanson Centre for Cancer
Reseurch, Institute of Medical and Veterinary Science, Adelaide,
Australia.
Submitted November 23, 1993; accepted June 29, 1994.
Supported by grants from the National Health and Medical Research Council and Anti-Cancer Foundation of the Universities of
South Australia, and by Brrxter Healthcare Corp, Deetfeld, IL, and
Amgen. Thousand Ouks, CA.
Address reprint requests toL.B.To,MD,Director,
Leukaemia
Research Unit, Hanson Centre for Cancer Research, Institute of
Medical and Veterinary Science, Frome Rd, Adelaide 5000, Australia.
The publication costs of this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8409-0024$3.00/0
2930
eloid progenitor cells (granulocyte-macrophagecolony-stimulating factor; CFU-GM) infused correlates with the rate of
neutrophil and platelet recovery.".'2 However, the CFU-GM
dose infusedis only a surrogate measure of HR capacity
and is not necessarily predictive for individual patients.I3 In
addition, murine transplantation studies suggest thatposttransplant recovery is polyphasic with different progenitors
responsible for different phases of r e ~ 0 v e r y .Early
l~
murine
and human studies suggested that there are few,if any, longterm marrowrepopulating cells(LTMRCs) in circulation
during steadystate,'"I6 whereas recent data in the murine
system suggests that LTMRCs are present in cyclophosphamide or G-CSF-mobilized PB.17"9 Furthermore, the presence ofpreprogenitorsinmobilized
PB CD34' cells has
beenshown by thesustainedproduction of nascent CFUGM in stromal-free liquid culture for at least three weeks.'"
There is alsoincreasing evidence that the HLA-DR, CD33 ,
CD38-, Rhodamine 123d"i',
and c-kit' subsets of human BM
CD34' cells contain
the
most
primitive
hematopoietic
Recently,
long-term
culture initiating
cells
(LTCICs) have been documented atlowlevels in normal
human PB.25,2"
These findings suggest that an assessment of the phenotypeand proliferativecapacity of PBCD34' cells would
describe the spectrumof hematopoietic stedprogenitor cells
in mobilized PB. Furthermore,suchstudies
may provide
Blood, Vol 84, No 9 (November l), 1994: pp 2930-2939
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
2931
CHARACTERIZATION OF MOBILIZED PB CD34+ CELLS
valuable insights into the mechanisms responsible for mobilization of hematopoietic progenitor cells.
This report describes the expression of activation and lineage-associated antigens on mobilized PB CD34+ cells and
their proliferative capacity. Cells from patients treated with
the four most commonly used PB stem cell mobilization
protocols were studied: recovery phase after myelosuppressive chemotherapy alone, recovery phase after chemotherapy
and GM-CSF or G-CSF, and during administration of GCSF alone. Our data show that CD34+ cells mobilized using
different stimuli were remarkably similar to each other and
contained similar proportions of mature and primitive progenitors to those in steady-state CD34+ cells. However, all
four types of mobilized PB CD34+ cells differ from their
steady-state counterparts in their lower expression of c-kit
and CD71 and in their low rhodamine-l23 (Rh123; Molecular Probes Inc, Eugene, OR) retention. The consistent downregulation of c-kit on CD34+ cells in mobilized PB compared
with the levels on steady-state BM CD34+ cells is particularly striking. While the significance of this observation remains to be determined, it is tempting to speculate that this
may play a direct and pivotal role in the different mobilization methods studied.
MATERIALS AND METHODS
PBSC Mobilization Protocols
Myelosuppressive chemotherapy with or without cytokines. Patients received cyclophosphamide 4 or 7 g/m2 as previously reand depending on enrollment into clinical study protocols
received GM-CSF, G-CSF, or no hematopoietic growth factors. GMCSF was administered daily in the form of human recombinant GMCSF (Leucomax; Sandoz, Basel, Switzerland) at 5 pg/kg/d subcutaneously from day 2 onward until a maximum of six aphereses were
completed. G-CSF was administered in the form of human recombinant G-CSF (Filgrastim; Amgen, Thousand Oaks, CA) at 5pg/kg/d
subcutaneously daily from day 2 onward until a maximum of four
aphereses were completed. Leukapheresis was commenced when the
leukocyte count reached 1 X 109/L.
A number of patients underwent mobilization as part of planned
chemotherapy. One patient with lymphoma received DHAP2*and
G-CSF at 5 pg/kg/d by subcutaneous injection. One patient with
breast carcinoma received cyclophosphamide 4 g/m’ with epirubicin
200 mg/m2and G-CSF at 5 pg/kg/d by subcutaneous injection. Three
other patients received the same doses of cyclophosphamide and
epirubicin as well as GM-CSF at 5 pg/kg/d by subcutaneous injection.
G-CSF. Patients received human recombinant G-CSF at 12 pg/
kg/d by continuous subcutaneous infusion over 6 days as previously
reported? Leukapheresis was performed on days 5, 6, and 7 using
a Fenwal CS3000 continuous flow blood cell separator (Baxter
Healthcare Corp, Deerfield, IL) as previously rep~rted.’~
The apheresis product was processed by separation on a Lymphoprep density gradient (1.077 g/dL, Nycomed Pharma As, Oslo, Norway) and washed three times by centrifugation at 400g before cryopreservation in 10% dimethyl sulfoxide and 20% autologous plasma
in liquid nitrogen.”
Normal Subjects
Normal volunteers under the age of 40 years enrolled in a normal
donor program providing BM and PB. This study has been approved
by the Human Ethics Committee of the Royal Adelaide Hospital,
Adelaide.
Preparation of Cells
BM from volunteer donors was collected into preservative-free
heparin and separated on a Lymphoprep density gradient. Lightdensity cells were washed twice in wash buffer: Hanks balanced salt
solution (HBSS) with 5% fetal calf serum (FCS; Commonwealth
Serum Laboratories, Parkville, Victoria, Australia) and resuspended
at 1 x IO7 cells/mL. Similarly, light-density mononuclear cells from
apheresis products were washed and resuspended at 1 X lo7 cells/
mL. Cryopreserved samples were separated over Lymphoprep,
washed, and also resuspended at 1 X lo7 cells/mL. Steady-state
PB mononuclear cells (PBMNCs) were collected from five normal
subjects after 1 hour of apheresis on the Fenwal CS3000. After
Lymphoprep separation, CD34’ cells within the light-density fraction were enriched using a CEPRATE avidin column (Cellpro;
Bothell, Seattle, WA) as previously described.” Without this enrichment step, it was not possible to perform reliable immunophenotyping in steady-state PBMNCs because of the low frequency of CD34+
cells (0.05% to 0.1%).
Immunophenotyping and Fluorescence-Activated Cell
Sorting (FACS)
Enumeration of CD34+ cells in cell suspensions was performed
as described by Sutherland et a1.4’ Briefly, the cell suspension was
stained simultaneously with a fluorescein isothiocyanate (FITC)conjugated CD45 antibody (HLe-1, Becton Dickinson, Mountain
View, CA) and a phycoerythrin (PE)-conjugated CD34 antibody
(HPCA-2-PE). The incidence ofCD34’ cells was determined by
“back gating” for CD45 expression and side scatter where true
CD34’ events were characterized by low-density CD45 expression
and low side scatter.
The expression of activation or lineage-associated antigens on
CD34’ cells was determined by incubation with antibody against
CD34 together with antibody to the specific marker. Thus cells were
incubated for 40 minutes at 4°C with 8G12-FITC (anti CD34: 5 pg/
L X lo6 cells [gift from Dr J. Bender, Baxter Healthcare Corp])
alone or with one of the following PE-conjugated monoclonal antibodies, HLA-DR, LeuM9 (CD33), Leu17 (CD38), Leu12 (CD19),
LeuM7 (CD13), or IgGPE. Cells were also stained with HPCA-2PE (CD34) alone or with one of the following FITC conjugates:
LeuMl (CD15), CDllb (Serotec Ltd, Oxford, UK), CDIO, CD71,
and Leu9 (CD7). Unless otherwise stated, all antibodies were supplied by Becton Dickinson and used as recommended by the manufacturer.
Expression of c-kit was determined by incubating cells with the
IgM CD34 antibody 12.8 (gift from Dr R. Andrews, Seattle, WA)
andthe IgG antibody YB5.B831 followed by the isotype-specific
second antibodies conjugated to FITC and PE, respectively (Southem Biotechnology, Birmingham, AL). When steady-state PB was
processed on the CEPRATE device then CD34’ cells were labeled
by either a FITC- or PE-conjugated goat-antimouse IgM antibody
and expression of other antigens determined by using IgG-specific
antibodies conjugated to either PE or FITC as appropriate.
After immunolabeling, cells were washed twice then resuspended
in FACS Fixative (2% vol/vol formaldehyde in Dulbecco’s phosphate-buffered solution) unless they were to be sorted. Samples were
analyzed within two days using a Coulter Profile I1 flow cytometer
(Coulter Electronics, Miami, FL) with standard optical configuration
and an argon laser emitting 488 nm at 15 mW. Listmode data from
100,000 events was collected and analyzed to determine the proportion of CD34+ cells expressing the relevant marker.
Rh123 staining was performed according to the method described
by Bertoncello.” Briefly, 2 X lo6 light-density cells were incubated
for 45 minutes at 37°C in 5 mL of wash buffer containing Rh123
at 0.1 pg/mL. After two washes, excess internalized Rh123 was
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2932
TO ET AL
removed by incubating the cells with wash buffer for 15 minutes at
37°C. The Rhl23-labeled cells were then incubated with PE-conjugated HPCA-2 as described above. Analysis was performed on a
Coulter Profile I1 flow cytometer within 30 minutes of staining. At
least 100,000 cells were examined and the distribution of Rh123
uptake by CD34' cells was determined by analysis of CD34' cells
discriminated on the PE-side-scatter dot plot.
Pre-Progenitor (Pre-CFU) Assay
This assayhasbeenreportedpreviously2"andis
an adaptionof
the 4-day suspension culture system described by Iscove et a l ? 3 The
generation of nascent CFU-GM by FACS-sorted CD34'
cells was used
as anindex of pre-CFU.Briefly, after immunolabeling, cells were
cells weresorted into
resuspendedin HBSS/5% FCSthenCD34'
a FACSGI?~
' "~
Iscove's minimalessentialmedium(IMDM)using
(Becton Dickinson). Postsort purity checks were performed routinely.
Sorted CD34' cells were resuspended at 1 X lo3 cells/mL in PreCFU medium (IMDM supplemented with 30% FCS, 1% bovine
m o l n pserum albumin, 3 mmol/L L-glutamine and 5 X
mercaptoethanol). Six replicate l-mL suspension cultures were established in 24-well plates (Nunc, Intermed, Denmark) in the presence of six recombinant human hematopoietic growth factors
(HGFs), all at 10 ng/mL: IL-l, IL-3, IL-6, GM-CSF, G-CSF, and
stem cell factor (SCF; Amgen, Thousand Oaks, CA). This combination of HGF was chosen because it gives rise to the highest CFUGM generation compared with any other combinations using any
five or fewer HGFs or single HGF."
The cultures were incubated at 37°C in 5% CO2 ina fully humidified atmosphere. On day 7, three wells were harvested, counted, and
cultured in a CFU-GM assay while the remaining three wells were
refed with IO ng/mL of each of the six HGF. On day 14, the three
remaining wells were harvested for cell counts and CFU-GM assay,
and one fifth of the contents of each well were replated into new
wells together with another 10 ng/mL of each of the six HGF. These
triplicate cultures were again harvested at day 21 with a proportion
of the cells cultured for CFU-GM and a proportion replated in preCFU assay with fresh media and six HGF until day 28. At each
time point of sampling, the total number of nucleated cells and CFUGM present was determined after accounting for culture dilution
and the number of cellslplate in the CFU-GM assay.
CFU-GM Assay
Triplicate l-mL cultures were established in 35-mm plates in 0.9%
methyl cellulose in IMDM supplemented with 30% FCS and 3 mmoV
L L-glutamine as previously reported.% Cultures were stimulated by
the same six-growth-factor combination as in the pre-CFU assay,
and aggregates of 40 or more cells present after 14 days were scored
as CFU-GM.
Statistics
Multiple group comparisons were performed using analysis of
variance (ANOVA) and group-group comparisons were based on
the Fisher PLSD (Fisher Scientific CO, Boston, MA). X' statistics
was used for nominal variables. Mean 2 1 SE was used unless
otherwise specified.
RESULTS
Cells collected by apheresis were studied from five patients (14 apheresis samples) who received myelosuppressive chemotherapy (Cm), three patients (3 apheresis samples) who received Cm + GM-CSF, three patients (3
apheresis samples) who received Cm + G-CSF, and eight
patients (8 apheresis samples) who received G-CSF alone.
Cells from BM and the PB of five normal individuals were
used as steady-state controls. Details of the patients and
normal donors and the percentage of CD34' cells in the cell
suspensions analyzed are listed in Table I .
Phenotype o j CD34' Cells
The immunophenotype of CD34' cells from the four mobilization protocols and from steady-state PBandBM are
shown in Table 2. For the majority of antigens studied, the
proportion of antigen-positive or antigen-negative cells
could be accurately enumerated. This is shown by representative examples in Fig 1 for the expression of CD38, HLADR, and CD10 on CD34' cells. However the expression of
CD33, CD71, and c-kit on PB CD34' cells was continuous
from low to high levels of expression without discrete positive and negative populations (Fig 2). Thus, the percentage
values shown for these are arbitrary values that indicate the
relative number of cells with antigen expression above that
of the 99th percentile of the isotype control. An alternative
parameter of the expression is the peak shift in fluorescence
intensity of cells stained for CD33, CD7 1, and c-kit as compared with the peak fluorescence for cells stained with an
isotype control. This is also reported in Table 2. Cells with
a stronger antigen expression have a higher peak-shift value.
Activation Status (CD38, HLA-DR, Rhodamine, and
CD71)
Eighty-eight percent 2 3.0% ofG-CSF-mobilized CD34'
cells expressed CD38, significantly lower than that of other
CD34+ cells, (means ranged from96.1% to 99.0%; oneway ANOVA, P = ,0032; Fisher's PLSD comparing G-CSF
mobilized CD34' cells with other CD34' cells, P values
ranged from .OW7 to .0081). This lower expression was seen
bothin patients whohadandwhohadnot
received prior
chemotherapy. Eighty-nine percent 2 4.8% of G-CSF-mobilized CD34' cells expressed HLA-DR, lower thanthe
other CD34+ cells (means ranged from 94.3% to 98.4%),
but the difference did not reach statistical significance.
The intensity of Rh123 staining of CD34' cells was expressed as bright, intermediate, or low based on a comparison
with the monocytes (which exhibit high Rh123 staining) in
the CD34- population from the same specimen (Fig 3). BM
CD34' cells were predominantly Rh123bngh'(919) and
steady-state PB CD34' cells were either Rh123'"'ermed'a'e
(21
7) or I"" (5/7), whereas 13/14 of mobilizedCD34'
cells
were Rhodamine 123'"" and 1/14 was Rh123i"'e'm"d'a'e
. These
differences between the groups were statistically significant
( x 2 test, P = .0001). Post-hoc analysis showedthat BM
CD34' cells were significantly more Rh123b"gh',
whereas the
mobilized CD34' cells were significantly more Rh123'"".
The pattern of CD71 expression was quite different between the different types of CD34' cells. BM CD34' cells
consisted of three populations: CD7 1br'gh', CD7 Id'"', and
CD7 1 with the bright population predominant. Steady-state
PB CD34+ cells also consisted of the same three populations,
but the dim population was the predominant population. MObilized CD34' cells were mostly CD71 and most of the
positive population was CD7 1
-
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CHARACTERIZATION OF MOBILIZED PB CD34+ CELLS
2933
Table 1. Details of Six TvDes of CD34+ Cells Studied
UPN
Steady-state BM
NB1
NB2
NB3
NB4
NB5
NB6
Steady-state PB
NPI
N P2
NP3
N P4
N P5
G-CSF mobilization
no. 1523
Apheresis
597
1467
1460
1504
1505
1493
888
Cfl mobilization
939
939
939
939
939
1287
1024
1037
927
cell
927
cell
927
cell
927
cell
927
cell
927
cell
Cfl + GM-CSF mobilization
1444
1548
1287
Cfl .t G-CSF mobilization
no. 889
Apheresis
1020 no.
Apheresis
1156
no.
Apheresis
Disease
%CD34+
Cells
Sample
Mean ? SEM
BM
BM
BM
BM
BM
BM
1.40
2.20
3.00
2.00
3.30
3.00
0.3
2.48
ss PB
ss PB
ss PB
ss PB
ss PB
0.30
0.02
0.10
0.20
0.10
0.14 ? 0.05
1.30
0.20
1.60
4.20
0.90
0.90
2.75
0.40
1.53 ? 0.47
NHL
NHL
Ca Br I1
MM
Ca ovary
Germ
Germ
Germ
Germ
Germ
Germ
no.Apheresis
1
no.Apheresis
3
no.Apheresis
4
NHL
no.Apheresis
5
no.Apheresis
6
no. Apheresis
1
no.Apheresis
1
Apheresis no. 4
no. Apheresis
1
no.Apheresis
2
no.Apheresis
3
no.Apheresis
4
no.Apheresis
5
no.Apheresis
6
1.40
2.60
2.20
1.70
1.50
1.30
16.40
1.10
0.90
1.90
2.00
2.00
3.10
2.20
2.88 ? 1.05
Ca Br IV
Ca Br
Ca Br
Apheresis no. 9
Apheresis no. 9
Apheresis no. 11
4.10
10.50
0.40
lr 2.95
0.40
1.oo
1.70
2 0.38
SS
SS
SS
SS
SS
SS
NHL
HD no.
Ca Br II
Ca Br II
Ca Br II
Ca Br 111
Ca Br II
NHL
NHL
NHL
Apheresis
no.Apheresis
no.Apheresis
no.Apheresis
no.Apheresis
no.Apheresis
no.Apheresis
NHL
NHL
NHL
3
3
1
2
1
3
3
3
3
2
3 2.14
5.00
The frequency of CD34' cells in each sample is expressed as a proportion of the total number of CD45+ events collected.
Abbreviations: UPN, unique patient number; NB1, normal BM donor 1; SSBM, steady-state BM; NP1, normal PB donor 1; SSPB, steady-state
PB; HD, Hodgkin's disease; NHL, non-Hodgkin's lymphoma; Ca Br 11, carcinoma breast stage 2; MM, multiple myeloma.
Myeloid and Lymphoid Antigens
A number of Cm-mobilized CD34+ cells (92.4% 5 4.5%)
expressed CD33, significantlyhigher thanthat
of other
CD34+ cells (means ranged from 28.2% to 44.1%; one-way
ANOVA, P = .0009; Fisher's PLSD comparing Cm-mobilized CD34' cells with others, P values ranged from <.OW1
to .0048). The difference in peak shift was also significant
(one-way ANOVA, P = .0232). Therewasno significant
difference inthe expression of CD13 (meansrangedfrom
59.1%to 82.7%),CD15 (means ranged from 2.2%to 6.9%),
and CD1 lb (means ranged from 0.9% to 5.8%) among the
six types of CD34+ cells.
The percentages of mobilized or steady-state PB CD34'
cells expressing CD19 were significantly lower than that of
BM (means range from 0.1% to 0.9% c.f. 16.1%; one-way
ANOVA, P = .0025; Fisher's PLSD P values ranged from
.0004to .0027). The percentages of mobilized CD34+ cells
expressing CD10 were significantlylower thanthat of BM
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
TO ET AL
2934
Table 2. Comparison of Phenotype of Six Types of CD34' Cells
SSBM (n
Activation
HLA-DR%
CD38%
CD71%
CD71 peak shift
Myeloid
CD33%
CD33 peak shift
CD13%
CD15%
CD1 1b%
Lymphoid
CD10%
CD19%
CD7%
Adhesion
c-kit %
c-kit peak shift
=
5)
SSP6 ( n = 5)
G-CSF (n
=
8)
94.8 i 1.5
(88.9-97.0)
96.1 i 0.8
(93.0-97.6)
61.6 t 4.9
(42.4-69.4)
5.1 i 0.3
(3.8-7.1)
94.3 i 2.0
(87.4-98.4)
96.7 t- 0.5
(95.4-97.8)
39.0 z 6.6
(15.7-53.0)
5.6 i- 4.3
(1.0-18.4)
89.0 C 4.8
(67.0-99.8)
88.0 i- 3.0*
(74.7-96.1)
44.5 ? 7.8
(7.4-76.4)
1.3 2 0.5*
(0.3-3.9)
38.5 2 9.4
(11.3-59.5)
3.7 i 2.0
(0.0-1 1.0)
59.1 2 4.1
(45.0-66.5)
6.9 2 0.5
(5.4-8.3)
2.3 2 0.7
(0.7-4.3)
37.8 2 13.9
(6.2-88.6)
2.1 t- 1.4
(0.0-6.2)
75.7 t 6.7
(55.4-96.8)
2.9 t 0.8
(1.5-5.9)
5.8 2 1.3
(4.0-9.6)
18.8 2 6.9
(1.9-34.8)
16.1 i 6.1
(2.6-32.7)
4.0 t- 1.7
(0.1-10.1)
46.2 t- 5.2
(34.8-68.8)
5.2 i 0.6
(3.2-7.4)
HDC (n = 14)
CR
+ GM-CSF (n = 3)
Cn + G-CSF (n
=
98.4 i 0.2
(96.9-99.0)
99.1 2 0.2
(97.4-99.81
27.7 t- 3.2
(9.6-51.5)
0.6 2 0.1*
(0.2-1.2)
96.3 C 2.6
(91.2-98.9)
96.4 5 2.1
(92.5-99.6)
23.9 2 6.4
(0.0-0.5)
0.3 ? 0.2*
(0.0-0.5)
96.7 !: 0.4
(95.9-97.3)
97.0 i 1.1
(95.0-98.9)
29.2 2 9.5
(12.0-44.9)
1.1 t 0.3*
(0.6-1.5)
28.2 C 7.6
(4.6-53.3)
1.2 t 0.3
(0.0-1.9)
67.9 +- 9.7
(11 .O-94.0)
3.4 2 1.2
(0.4-11.1)
5.7 2 4.1
(0.7-30.1)
96.2 ? 1.6*
(75.5-99.3)
14.7 i 2.1*
(1.8-30.6)
86.5 t 5.0
(23.4-96.8)
3.7 i- 0.8
(0.8-10.4)
2.7 i 0.5
(1.4-3.5)
44.1 2 21.2
(12.8-84.4)
2.1 t 0.8
(1.2-3.8)
82.7 i- 6.7
(69.6-91.6)
3.4 t 1.6
(1.2-6.6)
2.6 I1.0
(0.9-4.5)
39.9 'c 17.9
(9.3-71.2)
2.2 -t 1.6
(0.2-5.3)
81.3 t 3.0
(77.3-87.2)
3.8 t- 1.0
(1.8-5.3)
0.9 t- 0.4
(0.0-1.3)
8.9 t 2.7*
(3.2-15.3)
0.7 t- 0.6*
(0.0-2.4)
3.3 t 1.2
(1.3-6.3)
1.2 t 0.4'
(0.0-3.7)
0.9 i 0.3*
(0.0-2.8)
1.9 t 0.5
(0.0-3.8)
0.8 5 0.2"
(0.1-2.2)
0.5 t- 0.3*
(0.0-1.1)
2.1 -t 0.2
(1.1-3.1)
0.1 k 0.1*
(0.0-0.2)
0.3 ? 0.3*
(0.0-0.9)
2.6 t 1.3
(0.9-5.1)
0.2 i 0.2*
(0.0-0.6)
0.1 i 0.1*
0.5 -t 0.3
(0.1-1.1)
44.8 2 7.8
(34.3-60.1)
2.1 f 0.7*
(1.0-3.3)
10.1 z 2.7
(0.0-15.3)
0.6 C 0.2*
(0.2-1.3)
27.0 i 3.5
(3.5-46.8)
0.9 i 0.2'
(0.1-2.5)
34.9 2 17.4
(8.0-67.4)
1.0 2 0.5*
(0.1-1.9)
25.5 i- 9.3
(7.5-38.4)
1.1 i 0.5*
(0.3-2.0)
+
3)
(0.0-0.2)
+
The immunophenotype of CD34' cells from the four mobilization protocols (G-CSF, HDC, C/"
GM-CSF, andl C
/
G-CSF) and from SSPB
and BM are shown as the mean i SEM (and range) percentage of CD34' cells coexpressing the relevant antigen. The peak shift in fluorescence
for CD71, CD33, and c-kit was determined by comparing the peak fluorescence intensity of the test monoclonal antibody with that of isotypecontrol stained cells. For P values, refer to text.
Abbreviations: SSBM, steady-state BM; SSPB, steady-stae PB; HDC, high-dose cyclophosphamide.
* Indicates antigens that are significantly different from SSBM.
(means ranged from 0.1% to 1.2% compared with 18.8%;
one-way ANOVA, P = .0085; Fisher's PLSD P values
ranged from .W15 to .0028), but no different from that of
steady-state PB (8.9% ? 2.7%). In the steady-state PB most
CD34' CD10+ cells had high side-scatter characteristics suggesting that they were mostly monocytes. There was no significant difference in the expression of CD7 among the six
types of CD34+ cells (means range from 0.5% to 4%).
er's PLSD P values ranged from <.WO1 to .0002). Although
the differences did not reach statistical significance, steadyphase PB CD34+ cells had the highest mean peak shift (2.1
t 0.7) among the various PB CD34+ cells. These changes
were confirmed by using three other c-kit antibodies, 17F11,
SR-1, and 1DC3, which identify distinct epitopes of c-kit
from YB5.B8 (data not shown).
Proliferative Capacity of CD34' Cells
c-kit
The expression of c-kit on PB CD34+ cells was significantly different from that on BMCD34+ cells (Fig 2). Instead
of well-defined c-kit' and c-kit- populations as in BM, most
PB CD34' cells expressed low levels of c-kit. As a population, the intensity of expression was a continuum from very
low to intermediate levels. The peak shift in c-kit expression
was significantly higher on CD34+ cells from BM (5.2 2
0.6) than those on PB, steady phase or mobilized (means
ranged from 0.6 to 2.1; one-way ANOVA, P < .Owl;Fish-
The incidence of CFU-GM in CD34' cells from BM,
steady-state PB, G-CSF, Cm, C/T + G-CSF, and C/T +
GM-CSF-mobilized PB (209 40, 105 2 50, 183 2 56,
176 ? 62, 271 ? 85, 277 2 43 CFU-GM/1,000 CD34+
cells, respectively) were not significantly different from one
another (one-way ANOVA).
The proliferative capacity of CD34+ cells as measured by
the generation of nucleated cells and CFU-GM in the preCFU assay for up to 21 days in culture is shown in Fig 4,
A and B, respectively. The actual number of cells present,
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
2935
CHARACTERIZATION OF MOBILIZED PBCD34'CELLS
ssau
88pa
GCBF
Fig 1. Representativefluorescence histogramsof CD34' cells analyzed for their expression of CD38, HLA-DR, and CD10. Results for
CD34+ cells from steady-state BM (SSBM), steady-state PB (SSPBI,
and after mobilizationwith granulocyte-colony stimulating factor (GCSF) are shown. The isotype control is represented by the nontilled
histogram, whereas the blackened histogram shows staining by the
test antibody. Fluorescence histograms are plotted in a logarithmic
scale.
allowing for cells previously removed rather than cumulative
production, is shown at each time point.
The number of nucleated cells increased exponentially for
3 weeks, but started to plateau at 4 weeks (data not shown)
in all six types of CD34+ cells. The maximum-fold increases
in nucleated cell number ranged from 2,520 t 885 (steadystate PB) to 14,333 ? 1,2314 (Cm + GM-CSF) at day 21.
There was no significant difference in the number of nucleated cells generated among the six types of CD34+ cells at
each of the time points studied.
The number of CFU-GM increased exponentially in the
first 2 weeks and then leveled off at 3 weeks, although CFUGM were still present at 4 weeks (data not shown). The
number of CFU-GM present at day 21 varied from 1,427 -f
643 (steady-phase PB) to 15,532 2 6,119 (G-CSF) per 1,000
CD34' cells plated. Despite the difference at the start of
culture, there was no significant difference in the number of
CFU-GM present on days 7, 14, and 21between the six
types of CD34+ cells. On a group-to-group comparison using
the Fisher's PLSD, steady-state PB and Cm-mobilized PB
showed a significantly lower number of CFU-GM at day 21
than G-CSF-mobilized PB and steady-phase BM (Pvalues
range from .0234 to .0449).
ever, some important distinctions exist. Firstly, a higher proportion of G-CSF-mobilized CD34+ cells are CD38- than
other CD34+ cells. G-CSF-mobilized CD34+ cells also generated more CFU-GM than steady-state PB. Secondly, all
four types of mobilized CD34+ cells differ from BM CD34+
cells in their lower expression of c-kit and CD71 and decreased retention of Rh123.
CD34+ cells expressing lineage-associated markers such
as CD33 have been shown to be committed progenitors2'
and are most probably the cells responsible for the first phase
of HR.'4 In contrast, CD34' cells that lack lineage-associated
(eg, CD33) or activation antigens (HLA-DR, CD38) are considered tobemore primitive hematopoietic cells that are
precursors to clonogenic cells and contribute to the late
phases of HR. The present study allows us to compare the
absolute numbers of CD34+ cell subsets in BM and mobilized PB harvests. In our institution, the mean CD34+ cell
yield from a BM harvest is 0.97 t 0.12 X 106kg body
weight (1 l allogeneic BM, 23 autologous BM) and based
on
the
findings of this study, 38.5%
and
3.9%
are
CD34'CD33' , CD34'CD38-, respectively. Thus, on average, a BM transplant would contain 0.37 X lo6
CD34+CD33+ cellskg body
weight
and 0.04 X lo6
CD34+CD38- cellskg body weight. In comparison, the
mean CD34+ cell yield in a G-CSF-mobilized PB harvest
is 6.3 5 1.4 X 106kgbody weight (n = 10) and 28.2% and
12.0% of CD34+ cells are CD33+ and CD38-, respectively.
Thus, transplantation with G-CSF-mobilized blood would
on average provide 1.77 X lo6 CD34+CD33+cellskg body
weight and 0.76 X IO6 CD34+CD38- cellskg body weight.
Similarly, in chemotherapy-mobilized PB, the mean CD34+
CD71
c-klt
b
SSBM
SSPB
DISCUSSION
This is a study of mobilized PB CD34+ cells from four
most commonly used PB stem cell mobilization protocols:
Cm, Cm + GM-CSF, Cm + G-CSF, and G-CSF alone. By
comparing their phenotype and proliferative capacity with
those of steady-phase cells, it provides new information
about the nature of mobilized CD34+ cells. This study shows
that mobilized CD34' cells are quite similar to steady-state
CD34' cells in their expression of activation and lineage
markers and in their ability to generate nucleated cells and
CFU-GM in a stroma-free HGF-driven liquid culture. How-
G-CSF
Fig 2. Representativefluorescence histogramsof CD34+cells analyzed for their expression of CD71 and c-kit. The isotype control is
shown by the nonfilled histogram, whereas stainingwith either CD71
or c-kitantibody is shown by the blackened histogram. Fluorescence
histograms are plotted ina logarithrmic scale.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
TO ET AL
2936
Steady state BM
;
!
Mobilised PB
IT
W
Rh 123 retention
Rh 123 retention
cell yield i \ 3.62 2 2.48 X IO"/kg bodyweight ( n = 10)
with 3.3.5 X 10" CD34'CD33- cells/kg bodyweight and
0.04 X 10" C D 3 4 T D 3 8 - cells/kgbody weight available
for transplantation. Hence, both the
lineage-committed
(CD34'CD33-) and primitive(CD34'CD3X ) subpopulations are either of equal or increased abundance in mobilized
PB compared with steady-state BM.
Bender et a1'5 dcscribcd incrcascdnumbers of CD34lineage' cells in cyclophosphamide mobilized PB. but the
presentstudy shows that CD34' cells mobilized by other
protocols are also similar. Although the similar expression
of other myeloid markers suggests this may not he physiolog-
SSBM
SSP5
G-CSF
C m
n=6
n-4
n=3
n=4
CTT+GM
n=3
Cm+G
n=3
Fig 4.Thegeneration
of nucleated cells (A) and CFU-GM (B) in
stroma-free culture of the six types of CD34' cells. Each column represents the number (mean and standard error)
of nucleated cells
and CFU-GM present initially (H) and generated after 7 days (B), 14
days (B),and 21days (E)from 1,000 CD34+ cells.
Fig 3.Representativetwo-dimension contour plots of CD34cells analyzed for their Rh123
content.
The
cells were incubated with Rh123, washed, and
thenincubatedwith
PE-conjugated HPCA-2 (CD34). Analysis
was performedonthe
Coulter
Profile II within 30 minutes after
staining and 100,000 cells examined.
ically signilicant, C/T mobilization yields a higher percentage of CD34'CDM' cclls. The expression of T and B murkers is similar to thosepreviouslyreported
and so is the
expression of CD71 in steady-state PB CD34 cell\."' We
have also measured the levels o f CD34'CD61' cclls as an
indicator of megakaryocytic progenitors. Such a population
was identified by flow cytomctry, but sorting based on CD61
expressiondid not lead to any enrichment of clonogenic
megakaryocytic progenitors.Furtherstudiesshowed
that a
largenumber of CD34'CD61' events on flow cytomctry
were an artifact caused by adherent platclcts rather than true
positivity. Unless sample preparation involves washing with
theophyllineand adenosine to inhibit platelet aggregation.
cautionshould be exercisedwheninterpreting
f o w cytometric data on CD61 expression or other platelet markcrs
on CD34' cells.
The question of whether there are LTMRCs in mobili~ed
PB rclnains unanswered as there are no in vitro assays folLTMRCs. Recently. the LTClC has been proposed as a candidate LTMRC" and its presence has been described in cyclophosphamide, G-CSF, and C/T + G-CSF-mobilized PB
stern
in man.
However.
the
levels
of LTClC i n
these mobilized PB have not been cpantitatcd. In a murine
PB mobilization model, primitive stem cells capable o f selrrenewaland competitive repopulationhave been shown in
mobilizedblood at levels approaching that seen in normal
BM.'" Phenotypic studies suggest that LTClC arc found in
the CD33-, HLA-DR-, and Rh123""" subsets of CD34'
This study shows that suchcells are present a t a
similar incidence in mobilized PB CD34' cells a s i n \teadyphasc BM and PB.
A recent modification of a stroma-free liquid-culturc system stimulated by a six cytokinc combination has led to the
concept of preprogenitorsasmeasured
by nucleated cells
and CFU-GM generation in CLI~LI~~.'~).''.''
Data presented in
this report suggest that CD34' cclls from m o h i l i d PB are
capable of generating as many nucleated cells and CFU-GM
as those from steady-phase BM and PB. This suggests that
there are similar levels and/or proliferative capacity of pre-
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
CHARACTERIZATION OF MOBILIZED PB CD34' CELLS
2937
mobilization:' the disparate patterns of mobilization of granprogenitors in mobilized PB as in BM.In particular, Gulocytes and primitive cells make it mostlikely that primitive
CSF-mobilized CD34+ cells have a significantly higher percell mobilization occurs via a separate mechanism." The
centage of CD38- cells and their generation of C m - G M
highefficacy of SCF as a mobilization agent in baboon
after 21 days is as high as BM CD34+ cells, consistent with
provides further circumstantial e~idence.4~
Craddock et all8
the sustained long-term HR seen in patients autotransplanted
with G-CSF-mobilized PB.38 Hence, the presence of
also reported that cyclophosphamide-mobilized murine stem
LTMRC in mobilized PB is supported by clinical observacells did not bind to cultured stroma, whereas repopulating
tions and by studies in this report. The proof ofLTh4RC
stem cells in BM do. Studies comparing mobilized CD34'
cells and BM CD34' cells in adhesion mediated specifically
will have to await transplantation using genetically marked
by the c-kit/SCF interaction are in progress to test this specustem cells or allogeneic PB.
lation. Murine studies suggested that IL-3 and GM-CSF may
Quiescent cell-cycle status is another characteristic often
down-regulate c-kit,- so further studies to determine
attributed to primitive stem cells. The low CD71 expression
and the Rh123d""status in mobilized PB CD34' cells suggest
whether the reduced c-kit expression is caused by blocking
that there is a high proportion of quiescent cells among them.
or down-regulation by circulating SCF need to be performed.
This requires formal testing of cell-cycle status using tritiated
Nevertheless, it is possible that mobilization after C/T and/
thymidine suicide or BrDU labeling, and such studies are in
or HGF administration may be mediated via a common cyprogress. Steady-state PB C m - G M were ascribed a high
tokine network that ultimately alters the expression of c-kit
cycling status based on a hydroxyurea killing technique,39 on CD34' cells, thus inducing their egress from the BM. In
but no such studies on mobilized PB have been reported.
view of the increasing evidence that mobilized PB containing
The low c-kit expression of mobilized PB CD34+ cells
large number of progenitors leadto faster HR and safer
stands out against the findings on other lineage and activation
transplantation, these studies have major significance in developing more effective mobilization protocols.
markers that are essentially the same as steady-state CD34+
cells. Because the expression of c-kit on mobilized CD34+
ACKNOWLEDGMENT
cells is more or less continuous, the low expression represents an overall reduction rather than the presence of a large
We thank A. Bishop, B. Swart, P. Dyson, andT. Rawling for
c-kit negative population. We have previously reported that
technical assistance and M. Huxtable for stenographic.
the majority of CD34+CD19+cells that are putative B-lymphocyte progenitors are c-kit-." However, in steady-state
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