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HEMATOPOIESIS
From Developmental Stem Cell Biology and Regenerative
Medicine, John P. Robarts Research Institute; the Department of
Microbiology and Immunology, University of Western Ontario; and the
London Health Sciences Center, London, Ontario; and Hamilton Health
Sciences Center, Ontario.
Using in vitro progenitor assays, serum-free in vitro cultures, and
the nonobese diabetic/severe combined immune-deficient (NOD/SCID)
ecotropic murine virus knockout xenotransplantation model to
detect human SCID repopulating cells (SRCs) with multilineage reconstituting function, we have characterized and compared purified subpopulations harvested from the peripheral blood (PB) of patients receiving granulocyte colony-stimulating factor (G-CSF) alone or in
combination with stem cell factor (SCF). Mobilized G-CSF plus
SCF PB showed a 2-fold increase in total mononuclear cell content and a
5-fold increase in CD34-expressing cells depleted for lineage-marker
expression (CD34+Lin Intensive chemotherapeutic treatment together with
subsequent transplantation of autologous hematopoietic support has
provided beneficial outcomes for several hematopoietic and
nonhematopoietic malignancies such as lymphoma and multiple
myeloma.1-4 Mobilized peripheral blood (MPB) has largely
replaced bone marrow (BM) as a source of hematopoietic repopulating
cells for autologous transplantation, primarily owing to the rapid
hematological recovery and ease of collection associated with
mobilization.2,3,5 The time to hematopoietic recovery can
be correlated with the absolute number of mobilized CD34+
progenitor cells infused, and recent studies have established the
infusion of more than 5 × 106 CD34+ cells
per kilogram in consistent and rapid engraftment in a large proportion
of patients.6,7
A number of hematopoietic growth factors and cytokines, including
granulocyte colony-stimulating factor (G-CSF),8,9
granulocyte-macrophage CSF (GM-CSF),10 and stem cell
factor (SCF),11,12 used for mobilization have been shown
to expand hematopoietic progenitor cells in vitro. Although recombinant
human SCF alone exerts little colony-stimulating activity on human
BM-derived cells in vitro, combination with other recombinant
hematopoietic growth factors results in a synergistic increase in
colony formation.13 In primate and mouse models, in vivo
administration of SCF and G-CSF also has a synergistic effect
that increases peripheral blood (PB) progenitor
cell mobilization as compared with G-CSF
alone.13,14 On the basis of these encouraging results,
several clinical trials have been initiated; they reported the
ability of the combination of SCF with G-CSF to successfully mobilize
PB progenitor cells in patients.15-18 Treatment with a
combination of SCF and G-CSF also resulted in significantly improved
CD34+ cell yield, with a concomitant reduction in the
amount of leukapheresis product required to collect an optimal harvest
of 5 × 106 CD34+ cells per kilogram as
compared with G-CSF mobilization alone.15 In
addition, the combination of SCF and G-CSF was well tolerated and
effective in achieving rapid engraftment even in heavily pretreated lymphoma and multiple myeloma patients at risk of poor
mobilization.15,17
Despite the early success of clinical trials evaluating the
effectiveness of mobilization of progenitor cells with the use of the
combination of SCF and G-CSF, a detailed analysis of the repopulating
capacity of human cells mobilized by G-CSF alone or by SCF in
combination with G-CSF and their respective subsets has not been
examined. With the use of an in vivo assay for primitive human
hematopoietic cells capable of repopulation,19-22 previous studies have used limiting dilution analysis (LDA) to compare the
frequency of repopulating cells harvested from various human hematopoietic sources such as umbilical cord blood, adult BM, MPB, and
fetal tissue.23,24 Verfaillie and
colleagues25 compared the short-term and long-term
repopulating characteristics of G-CSF-mobilized BM and PB cells in the
human-fetal sheep xenotransplantation model; they demonstrated that
mobilized CD34+ cells, when serially transferred into
secondary and tertiary recipients, supported earlier development of
human hematopoiesis and earlier exhaustion of human
hematopoiesis. These data suggested fundamental differences in
the repopulating potential of MPB cells that were independent of cell
phenotype used to identify human repopulating stem
cells.25 Given the increased use of SCF in combination
with G-CSF in patients,15-18 it is imperative to
understand the effects these growth factors have on the biological
function of recently characterized stem/progenitor subsets mobilized.
Here, we have used the nonobese diabetic/severe combined
immune-deficient (NOD/SCID) assay for detection of human SCID
repopulation cells (SRCs) and have compared the engraftment potential
of both mixed and purified populations of cells harvested from patients mobilized with G-CSF alone versus G-CSF in combination with SCF. G-CSF
combined with SCF effectively increased total CD34+
lineage-depleted (Lin Patient samples
Human cells
Purification of primitive cell populations Lin cells were isolated from mobilized MNCs by
means of a standard immunomagnetic protocol.19 Briefly,
MNCs were stained with a cocktail of 9 lineage-specific antibodies
(CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and glycophorin A)
followed by a secondary antibody conjugated to metal colloid. For cell
culture experiments in which
CD34+CD38Lin or
CD34CD38AC133+Lin
cells were to be isolated, CD33 and CD38 antibodies were added to the lineage-depletion cocktail. Lin cells were
purified by negative selection by means of a StemSep device as
described by the manufacturer (Stem Cell Technologies, Vancouver, BC,
Canada).19 Lin cells were stained with
fluoroscein isothiocyanate (FITC)-conjugated human anti-CD34,
allophycocyanin-conjugated human anti-CD38 (Becton Dickinson,
San Jose, CA), and phycoerythrin (PE)-conjugated human anti-AC133
(Miltenyi Biotechnology, Auburn, CA). Primitive
CD34+Lin,
CD34+CD38Lin, or
CD34CD38AC133+Lin
cells were isolated by flow cytometry by means of a Vantage SE cell
sorter (Becton Dickinson).20,28 Sorting gates
were established on Lin cells stained with isotype
immunoglobulin G1 (IgG1) conjugated to the appropriate fluorochromes
(Becton Dickinson) as shown previously.28
Transplantation of human cells into NOD/SCID mice MPB MNCs or selected subpopulations from Lin
populations were transplanted into sublethally irradiated (340 to 375 cGy with the use of a 137Cs  -irradiator) 8- to
10-week-old NOD/LtSz-scid/scid (NOD/SCID) ecotropic murine virus
knockout (EMVnull) (Jackson Laboratories, Bar Harbor, MA)
mice according to our standard protocol.19 Mice that
received transplants of purified cell populations were supplemented
with 105 irradiated (1500 cGy with the use of a cobalt
source) Lin+ accessory cells as shown
previously.29 NOD/SCID EMVnull mice were bred
in the defined flora barrier facility at the Robarts Research Institute
(London, ON, Canada). The Animal Care committee at the Robarts Research
Institute and at the University of Western Ontario (London, ON, Canada)
approved animal experiments. Mice were maintained under sterile
conditions in microisolator cages in a ventilated rack and were killed
6 to 8 weeks after transplantation. BM cells were harvested from
the femurs, tibiae, and iliac crests of animals and suspended in
IMDM according to our standard
protocols.28,30
Flow cytometric analysis of mouse BM To prepare mouse BM cells for flow cytometric analysis, red cells were lysed by means of 0.8% ammonium chloride solution, and the remaining cells were washed in PBS containing 5% FBS. Approximately 106 cells were incubated for 30 minutes at 4°C with human panleukocyte-specific marker anti-CD45-FITC in combination with human anti-CD38-PE (Becton Dickinson) or isotype controls. Cells were then washed 3 times in PBS plus 5% FCS and analyzed by flow cytometry on a FACSCalibur with CellQuest software (Becton Dickinson). Analysis of multilineage engraftment was performed on mouse BM that demonstrated a high level of human engraftment (greater than 10% human). Briefly, 105 cells from mouse BM were stained and gated for human cells (anti-CD45-peridinin chlorophyll protein (PerCP) and analyzed for B-lymphoid cells (anti-CD20-FITC, anti-CD19-PE); myeloid cells (anti-CD33-FITC, anti-CD15-PE); primitive cells (anti-CD34-FITC, anti-CD38-PE); and T-lymphoid cells (anti-CD4-FITC, anti-CD8-PE) (all antibodies from Becton Dickinson).Analysis of human cell engraftment High-molecular-weight DNA was also isolated from the BM of mice that received transplants by means of phenol/chloroform extraction or DNAzol reagent (Gibco BRL) according to the manufacturer's specifications. The proportion of human cells in the mouse BM was determined by Southern blot analysis with the use of a human chromosome 17-specific -satellite probe (p17H8)31 as described previously.22 The level of human cell engraftment was
quantified by analysis of Southern blots with the use of a
phosphoimager and ImageQuant software (Molecular Dynamics, Sunnyvale,
CA) by comparing the characteristic 2.7-kilobase (kb) band
with human-to-mouse DNA mixture controls (limit of detection,
approximately 0.1% human DNA).
Colony-forming unit assays Human clonogenic progenitor cell assays were performed by plating human cells into Methocult H4434 (Stem Cell Technologies) containing 50 ng/mL recombinant human SCF (rhSCF) (Amgen), 10 ng/mL rhGM-CSF, 10 ng/mL rh-interleukin-3 (rhIL-3), and 3 U/mL rh-erythropoietin (all from R&D Systems, Minneapolis, MN). Differential colony counts were assessed following incubation for 10 to 14 days at 37°C and 5% CO2 in a humidified atmosphere as shown previously.20,28Serum-free in vitro cultures De novo isolated CD34+CD38Lin and
CD34CD38AC133+Lin
cell populations (200 to 1500 cells) were directly isolated by FACS
analysis prior to seeding in serum-free liquid culture.
Serum-free cultures contained medium consisting of 9500 bovine serum
albumin, insulin, and transferrin (BIT) (Stem Cell
Technologies) supplemented with 104 M  -mercaptoethanol
and 2 mM L-glutamine (Gibco BRL) in combination 300 ng/mL
rhSCF, 50 ng/mL rhG-SCF (Amgen), 300 ng/mL rhFlt-3, 10 ng/mL rhIL-3,
and 10 ng/mL rhIL-6 (R&D Systems) (complete growth factor media) as
optimized previously for both CD34+ and CD34
primitive human cells.20,28,32 Cells were plated
in fibronectin-coated 96-well plates (Becton Dickinson) at 37°C and
5% CO2. Fresh complete growth factor medium was
replenished every 2 to 3 days. After 10 days of culture, viable cells
were counted and analyzed for CD34, CD38, and AC133 expression by flow
cytometry as described previously.28,30
Statistics Levels of human engraftment were shown as the mean ± SEM for mice grouped according to the number of transplanted cells. The frequency of SRCs was determined by LDA as described previously.19,24 Briefly, a mouse that had received transplants was scored as positive (engrafted) if any human cells were detectable by Southern blot. The data from several transplanted doses were grouped and analyzed by applying Poisson statistics with the use of the single-hit model. The frequency of SRCs in a population of cells was calculated by means of the maximum likelihood estimator, with the assumption of a normal distribution.19,24 Analysis of statistical significance for colony-forming unit (CFU) data (frequency of colonies ± SEM) was performed by a 2-tailed Student t test with the use of Microsoft Excel software.
G-CSF plus SCF mobilization increased the frequency and total number of CD34+ cells Clinical trials have reported the ability of the combination of SCF with G-CSF to successfully mobilize PB, resulting in significantly improved CD34+ cell yield with a concomitant reduction in the number of leukapheresis procedures required to collect a harvest of 5 × 106 CD34+ cells per kilogram as compared with G-CSF mobilization alone.15 Direct comparison of MPB cells from 4 patients for each mobilization regime confirmed that G-CSF plus SCF mobilization was more effective at mobilizing leukocytes into the periphery and that it improved leukapheresis cell harvests for autologous transplantation (Table 1), results that are consistent with those shown in previous studies.3,15,33 The total number of leukocytes collected per leukapheresis were increased in G-CSF plus SCF-mobilized PB (12.6 ± 4.3 × 1010 cells) when compared with G-CSF-mobilized PB (8.8 ± 2.2 × 1010 cells). The increase in total cellularity was observed despite a decrease in the mean volume of leukapheresis product collected from G-CSF plus SCF-mobilized PB (Table 1). G-CSF plus SCF leukapheresis product contained increased total MNCs (P < .05) and higher numbers of MNCs per milliliter of leukapheresis product (P < .01) when compared with G-CSF treatment (Table 1). G-CSF plus SCF leukapheresis product also contained an increased frequency of total CD34+ cells (4.1% ± 1.2%) when compared with G-CSF-mobilized MNCs (1.2% ± 0.5%) (P < .05) (Table 1), resulting in a significant increase in the total number of CD34+ cells per milliliter of leukapheresis product (P < .05). The increase in overall cellularity and increased frequency of CD34+ cells resulted in a 5-fold increase in the total number of CD34+ cells obtained for reinfusion after G-CSF plus SCF mobilization as compared with G-CSF alone (Table 1).
SRC function of transplanted MNCs obtained from G-CSF plus SCF-mobilized PB is similar to that of G-CSF-mobilized PB Although total CD34+ cell content has been suggested as a predictor for short-term engraftment and hematologic recovery,6,15,34 this measure is unable to quantify the long-term repopulating function of human stem cell populations mobilized under different conditions. Accordingly, we performed a quantitative analysis of human stem cells capable of multilineage reconstitution in NOD/SCID EMVnull mice and compared the frequency and total number of these repopulating cells within the G-CSF- and G-CSF plus SCF-mobilized PB samples. In this context, transplantation of human sources of hematopoietic stem cells into the NOD/SCID EMVnull mouse provides an in vivo assay for long-term human hematopoietic stem cell repopulating function.19,21,22,24 To quantify the frequency of SRCs isolated from patients mobilized with G-CSF plus SCF or with G-CSF alone, we employed the NOD/SCID EMVnull mouse and LDA. The two mobilization regimes demonstrated remarkably similar reconstituting capacity when dose ranges between 5 × 106 and 25 × 106 MNCs were intravenously transplanted into NOD/SCID EMVnull mice. Transplantation of fewer than 10 × 106 cells allowed 7 out of 10 mice to engraft, at an average of 0.4% ± 0.2% for G-CSF-mobilized PB cells; 5 out of 9 mice engrafted at an average level of 0.7% ± 0.3% G-CSF plus SCF-mobilized PB cells (Figure 1A-B). Higher doses of cells led to increased average levels of engraftment (3.1% ± 1.6% and 6.1% ± 3.3% for G-CSF- or G-CSF plus SCF-mobilized PB, respectively) with populations from either mobilization regime. Statistical analysis of in vivo repopulating data demonstrated comparable frequencies of SRCs for both mobilization regimes, with 1 SRC in 8.2 × 106 (range, 4.3-14.8 × 106 MNCs) G-CSF-mobilized MNCs, and 1 SRC in 8.1 × 106 (range, 4.6-14.0 × 106 MNCs) G-CSF plus SCF-mobilized MNCs (Figure 1A-B).
Repopulating human (CD45+) stem cells from either
mobilization regime were analyzed for mature leukocyte markers; they
demonstrated multilineage potential in vivo (Figure 1C-D) with similar
representation of human B-cells (Figure 1Ciii,Diii), human myeloid
cells (Figure 1Civ,Div), and primitive cells (Figure Cv,Dv). Quadrant
frequencies were established with the use of isotype controls (Figure
Cii,Dii) and are representative of human cell maturation observed
previously in the NOD/SCID mouse.35 High levels of myeloid
and B-lymphoid development were observed under both mobilization
regimes with a concurrent absence of human T-lymphocytes (Figure
Cvi,Dvi); these results are not supported in the NOD/SCID
model.35 In addition, analysis of primitive human
hematopoietic markers demonstrated the presence of primitive
repopulating CD34+CD38 Total harvested SRCs from G-CSF plus SCF-mobilized PB were greater than 2-fold higher than the total number of SRCs derived from the use of G-CSF alone (Table 1). A total of 7220 ± 1320 SRCs within G-CSF plus SCF-mobilized PB could be isolated in comparison with 3130 ± 936 SRCs mobilized with the use of G-CSF alone (P < .05). The increase in total SRCs from G-CSF plus SCF-mobilized PB samples was also accompanied by a significant increase in the number of CD34+ cells per milliliter of leukapheresis product (Table 1; P < .01) when compared with G-CSF-mobilized PB. However, the frequency of SRCs calculated per 1 million CD34+ cells of leukapheresis product was approximately 4-fold lower in G-CSF plus SCF-mobilized PB MNCs as compared with G-CSF-mobilized PB (Table 1). Although combined G-CSF and SCF mobilization provided a phenotypic increase in the frequency of CD34+ cells per milliliter leukapheresis (Table 1), this increase did not correlate with an enhanced frequency of cells with NOD/SCID repopulating function. In comparison with G-CSF-mobilized PB, the inability to correlate enhanced CD34+ cell number with SRC function in G-CSF plus SCF-mobilized PB samples, prompted the purification of subsets from G-CSF- and G-CSF plus SCF treated-mobilized PB for direct comparison of in vivo repopulating capacity and in vitro progenitor activity. CD34 expression correlates with increased in vitro progenitor
content of Lin BM
and umbilical cord blood (CB) samples for the isolation of primitive CD34+ and CD34 subpopulations with primitive
hematopoietic function.20,28,36 Using a similar isolation
strategy, we have purified primitive hematopoietic cells from growth
factor-mobilized PB samples. Comparison of Lin cells
from G-CSF- or G-CSF plus SCF-mobilized PB analyzed for the primitive
stem cell markers CD34 and CD38 are represented in Figure
2A-B. Live cells were selected with the
use of forward- and side-scatter properties and 7-AAD staining (data
not shown); quadrant statistics (based on isotype controls [Figure
inset] performed on the day of analysis) were used to isolate
CD34+CD38Lin cells for
functional analysis (gated R1, Figure 2A-B). The frequency of cells
expressing CD34 (Figure 2A-B) from G-CSF plus SCF-mobilized PB were
increased as compared with samples mobilized with G-CSF alone.
Upon analysis of G-CSF (n = 4) and G-CSF plus SCF (n = 5)
Lin samples, the average frequency of primitive
CD34+CD38Lin cells was
5.2% ± 1.2% for G-CSF plus SCF-mobilized and 1.1% ± 0.6%
G-CSF-mobilized samples (P < .05). Therefore, the
increase in the frequency of CD34+ cells from G-CSF plus
SCF-mobilized PB leukapheresis products (Table 1) was reflected in an
increased frequency of primitive CD34+CD38Lin cells as compared
with G-CSF-mobilized PB. These results indicate that G-CSF plus
SCF-mobilized PB samples contain a higher frequency of primitive
CD34+ and
CD34+CD38Lin, as compared with
G-CSF-mobilized PB.
To characterize the function of purified cell populations from MPB
sources, purified subsets were assayed for in vitro progenitor and in
vivo repopulating function.
CD34+CD38 Long-term assessment of CFUs was also performed both on
CD34+CD38 Characterization of human SRCs derived from Lin cells and increased clonogenic
progenitor capacity seen with G-CSF plus SCF-mobilized PB (Figure 2C),
the NOD/SCID repopulating capacity of MPB MNCs isolated following G-CSF
plus SCF mobilization was equivalent to that of MNCs isolated from
G-CSF mobilization alone. To further examine the repopulating
characteristics of purified cells derived from a total of 11 independent G-CSF-mobilized PB samples (n = 35) and 10 independent
G-CSF plus SCF-mobilized PB samples (n = 32), purified
CD34+CD38Lin cells were
transplanted into recipient NOD/SCID EMVnull mice (Figure
3). Human engraftment was observed with
as few as 1000 G-CSF-mobilized
CD34+CD38Lin cells (Figure
3Ai-Aii). LDA revealed a frequency of 1 SRC in 7200 (range, 1 SRC in
3800-13 900 cells) G-CSF-mobilized
CD34+CD38Lin cells. Although
human engraftment was consistently observed after transplantation of
fewer than 10 000 CD34+CD38Lin
cells isolated from G-CSF-mobilized PB, human SRC function could not
be observed from the purified G-CSF plus SCF
CD34+CD38Lin cells at doses up
to 500 000 cells (Figure 3Bi). Remarkably, the addition of SCF to the
mobilization regime increased the number of
CD34+CD38Lin progenitor cells
harvested by leukapheresis (Figure 2C); however, the in vivo
repopulating function of these cells was reduced in comparison with
G-CSF-mobilized PB. Furthermore, both G-CSF- and G-CSF plus
SCF-mobilized PB CD34+CD38Lin
cells expressed CXCR4 (data not shown), the receptor for the chemokine
stromal derived factor-1 implicated in the migration of primitive
human hematopoietic cells.39 Taken together, these data
demonstrate that the addition of SCF during mobilization produced
altered functional characteristics in the
CD34+CD38Lin subset that
enhanced in vitro proliferation of progenitors (Figure 2C) at the
potential expense of SRC function (Figure 3).
On the basis of the detection of SRC function with G-CSF plus
SCF-mobilized PB MNCs (Figure 1B) and the lack of SRC function upon
transplantation of CD34+CD38
G-CSF plus SCF-mobilized
CD34 Lin
cells.20,40-42 Our laboratory has demonstrated enhanced
primitive hematopoietic function in a subfraction of the
CD34Lin population in cells that express
the cell surface receptor AC133 derived from full-term
CB.28 CB-derived
CD34CD38AC133+Lin
cells were shown to proliferate in serum-free culture with a percentage
of the cells acquiring CD34 expression, whereas
CD34CD38AC133Lin
cells possessed limited differentiating potential over 4 days of culture.28 In addition, CB
CD34+CD38Lin cells have been
previously shown to proliferate under identical culture conditions,
with the majority of cells retaining CD34 expression with a
concomitant up-regulation of CD38 on the cell surface.32 By means of a similar purification strategy
optimized for the selection of primitive populations, adult
CD34+CD38Lin and
CD34CD38AC133+Lin
cells were isolated from G-CSF- and G-CSF plus SCF-mobilized samples.
Figure 5Ai,Bi shows a representative
example and the average frequencies of these subsets after depletion of
lineage marker and CD38-expressing cells. In addition to an increase in the percentage CD34+CD38Lin
within the G-CSF plus SCF-mobilized PB cells, this mobilization regime
also showed a relative increase in the proportion of
CD34CD38AC133+Lin
human cells (3.2% ± 1.2%) when compared with G-CSF-mobilized PB
(0.7% ± 0.6%; P = 0.17). The frequency of
CD34CD38AC133+Lin
cells from G-CSF plus SCF samples was also increased more than 5-fold
in comparison with the frequency detected in identically processed CB
sources.28 It is important to note that
G-CSF-mobilized PB provided such low numbers of primitive CD34 cells
that, in the majority of samples, the number of
CD34CD38AC133+Lin
cells that could be isolated from G-CSF-mobilized PB was insufficient (fewer than 300 cells) to analyze cell surface phenotype of cultured cells after 10 days.
We examined and compared the survival and the proliferative and
developmental potential of
CD34+CD38 Within G-CSF-mobilized PB samples, both
CD34 In contrast to cultured
CD34
Hematopoietic cells treated in vivo with growth factors (G-CSF
alone or G-CSF plus SCF) administered to mobilize stem/progenitor cells
into the periphery produced several fundamental differences in the
repopulating properties and progenitor capacity of treated adults.
Phenotypic analysis of MPB depleted for mature cell lineage markers
(ie, Lin Purified CD34+CD38 Autologous transplantation protocols for MBP were designed to employ
the transfer of the entire leukapheresis product. Our data confirm that
MNCs from the two mobilization protocols were equally capable of
multilineage repopulation and suggested that the injected mixed-cell
populations contained functional SRCs. Given the absence of
repopulating capacity with
CD34+CD38 Our study indicates that G-CSF versus G-CSF plus SCF administration in
humans causes phenotypically and functionally distinct cellular subsets to be mobilized into the circulation. Although the
cellular basis for the inability to enrich for repopulating cells in
G-CSF plus SCF-mobilized PB is unknown, this property seems to be
distinct from G-CSF-mobilized products. We favor the hypothesis that
G-CSF plus SCF-mobilized PB is predisposed toward differentiation.
This was supported by analysis of the maintenance of primitive cell
phenotypes when directly comparing G-CSF versus G-CSF plus
SCF-mobilized cells in identical serum-free cultures. Primitive CD34+CD38
Submitted October 12, 2001; accepted April 1, 2002.
Supported by funding from Amgen Inc, Thousand Oaks, CA, and Mississauga, Ontario; an operating grant from the Multi-organ Transplant Group, London Health Sciences Center, Ontario; and the Canadian Institutes for Health Research, Ottawa, Ontario; and by scientist scholarship award no. MSH-35681 (M.B.), the Canadian Research Chair in Stem Cell Biology (M.B.), and a fellowship award (D.H) provided by the Canadian Institutes for Health Research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mickie Bhatia, The John P. Robarts Research Institute, 100 Perth Dr, London N6A 5K8, ON, Canada; e-mail: mbhatia{at}rri.on.ca.
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Top
Abstract
Introduction
) represents a single mouse recipient of transplanted MNCs (5 to
50 × 106 cells) derived from a total of 6 independent
G-CSF-mobilized samples (n = 18) and 9 independent G-CSF plus
SCF-mobilized samples (n = 27). Horizontal lines represent the
average level of engraftment for each cell dose and did not show a
statistical difference between mobilization regimes. LDA showed a
similar frequency of SCID repopulating cells (1 SRC in approximately
8 × 106 MNCs) for both G-CSF- and G-CSF plus
SCF-mobilized PB. (C-D) Representative analysis of multilineage stem
cell repopulation assessed in human-engrafted mice receiving
transplants of 25 × 106 G-CSF- (C) or G-CSF plus
SCF-mobilized PB MNCs (D). Mouse BM cells were stained with monoclonal
antibody combinations for human-specific antibody against the
panleukocyte marker CD45 (gated R1) (Ci,Di), and human cells were
assayed for presence of mature B lymphoid cells (CD20, CD19)
(Ciii,Diii), mature myeloid cells (CD33, CD15) (Civ,Div), primitive
cells (CD34, CD38) (Cv,Dv), and mature T-lymphoid cells (CD4 and CD38)
(Cvi,Dvi). Limits for quadrants were established with the use of FITC
isotype and PE isotype controls (Cii,Dii), and statistics display the
frequency of subsets representative of human hematopoietic
differentiation in the BM of NOD/SCID recipients of transplants of
either G-CSF- or G-CSF plus SCF-mobilized PB MNCs.
) and CD34(+) cells from mobilized blood differs from that of CD34(