|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4325-4335
Most Acute Myeloid Leukemia Progenitor Cells With Long-Term
Proliferative Ability In Vitro and In Vivo Have the Phenotype
CD34+/CD71 /HLA-DR
By
A. Blair,
D.E. Hogge, and
H.J. Sutherland
From the Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver Hospital and Health Sciences Centre, and the Department of
Medicine, University of British Columbia, Vancouver, BC, Canada.
 |
ABSTRACT |
Acute myeloid leukemia (AML) occurs as the result of malignant
transformation in a hematopoietic progenitor cell, which proliferates to form an accumulation of AML blasts. Only a minority of these AML
cells are capable of proliferation in vitro, suggesting that AML cells
may be organized in a hierarchy, with only the most primitive of these
cells capable of maintaining the leukemic clone. To further investigate
this hypothesis, we have evaluated a strategy for purifying these
primitive cells based on surface antigen expression. As an in vitro
endpoint, we have determined the phenotype of AML progenitor cells
which are capable of producing AML colony-forming cells (CFU) for up to
8 weeks in suspension culture (SC) and compared the phenotype with that
of cells which reproduce AML in nonobese diabetic/severe combined
immunodeficiency (NOD/SCID) mice. AML cells were fluorescence-activated
cell sorted (FACS) for coexpression of CD34 and CD71, CD38,
and/or HLA-DR and the subfractions were assayed in vitro and in
vivo at various cell doses to estimate purification. While the majority
of primary AML CFU lacked expression of CD34, most cells capable of
producing CFU after 2 to 8 weeks in SC were
CD34+/CD71 . HLA-DR expression was
heterogeneous on cells producing CFU after 2 to 4 weeks.
However, after 6 to 8 weeks in SC, the majority of CFU were derived
from CD34+/HLA-DR cells. Similarly, the
majority of cells capable of long-term CFU production from SC were
CD34+/CD38. Most cells that were capable
of engrafting NOD/SCID mice were also
CD34+/CD71 and
CD34+/HLA-DR. Engraftment was not achieved
with CD34+/CD71+ or HLA-DR+
subfractions, however, in two patients, both the CD34+
and CD34 subfractions were capable of engrafting the
NOD/SCID mice. A three-color sorting strategy combining these antigens
allowed approximately a 2-log purification of these NOD/SCID leukemia initiating cells, with engraftment achieved using as few as 400 cells
in one experiment. Phenotyping studies suggest even higher purification
could be achieved by combining lack of CD38 expression with the
CD34+/CD71 or CD34+/HLA
DR phenotype. These results suggest that most AML cells
capable of long-term proliferation in vitro and in vivo share the
CD34+/CD71/HLA-DR phenotype
with normal stem cells. Our data suggests that in this group of
patients the leukemic transformation has occurred in a primitive
progenitor, as defined by phenotype, with some degree of subsequent
differentiation as defined by functional assays.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
HUMAN ACUTE MYELOID leukemia (AML) is a
clonal disorder characterized by the accumulation of aberrant myeloid
blasts, defective in their maturation and function, in the blood and
bone marrow of affected individuals. The cell of origin of AML has been
the subject of much investigation, as AML patients with both multilineage and single lineage involvement within the leukemic clone
have been identified.1-4 It has been proposed in the past that the morphologic appearance and phenotype of the bulk of the AML
blasts would provide evidence for the cell of origin, with French-American-British (FAB) M1 being more primitive, while FAB M4,
which express monocytic surface antigens showing evidence of
differentiation.2 Other studies have demonstrated
considerable heterogeneity in both the proliferative and self-renewal
abilities of the transformed cells within patients,5,6
suggesting that AML cell populations within individual patients are
organized in a hierarchy. Thus, it is possible that only the most
primitive of these AML cells is responsible for maintenance of the
disease.
In recent years, it has been accepted that normal primitive long-term
culture initiating cells (LTC-IC)7 can be distinguished and
separated from more mature cells on the basis of their expression/lack of expression of specific cell surface markers. LTC-IC represent a rare
population in the bone marrow, they have low forward light scatter,
express CD34 and Thy-1, most lack HLA-DR, CD71, CD38, and CD45RA
expression and they are Rhodamine123dull.7-13
CD71 antibody is specific for the human transferrin
receptor.14 It is thought to be essential for transporting
iron into proliferating cells and is expressed on erythroid
progenitors. HLA-DR is a human class II major histocompatibility
complex (MHC) antigen, which controls responsiveness to soluble
antigens.15 It is expressed on B lymphocytes, activated T
lymphocytes, and natural killer cells, monocytes and
macrophages,16,17 as well as multipotential clonogenic
progenitor cells (colony-forming unit-granulocyte, erythroid, monocyte,
megakaryocyte [CFU-GEMM]).18 The phenotype of AML cells, which are capable of forming colonies in semisolid medium
has been reported.2,3,19 However, these assay systems, at
least in normal hematopoiesis, detect progenitors that have limited
self-renewal and proliferative capacities. We have previously described
a suspension culture assay,20,21 which detects cells with
long-term proliferative ability in vitro. AML progenitor cells, which
are capable of long-term proliferation in vitro in this system, are
less frequent than primary CFU20 and differ from the
majority of AML cells in that they are enriched within the
CD34+/CD38/Thy-1
subfraction.20,21 More recently, work by our group and that of others has shown that AML cells, which are capable of engrafting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice are
CD34+/Thy-121 and
CD34+/CD38.22
To further phenotypically characterize and purify the cells that have
long-term proliferative ability and may be responsible for maintenance
of the disease, we have evaluated the coexpression of CD34 and CD71,
HLA-DR, or CD38 on cells, which are capable of producing colonies for
up to 8 weeks in suspension culture and on cells which are capable of
engrafting NOD/SCID mice. We have also evaluated the ability of a
multiparameter sorting strategy using CD34, CD71, and HLA-DR to enrich
primitive AML cells with NOD/SCID engrafting capacity. Finally, a
phenotyping study using a variety of monoclonal antibodies was
performed to provide further information on which antibody combinations
would be most useful to enrich for primitive AML cells.
| |
MATERIALS AND METHODS |
Patient cells.
Peripheral blood cells were obtained from patients at diagnosis of AML
after informed consent and with approval of the Clinical Research
Ethics Board of the University of British Columbia. Blood cells were
Ficoll separated to obtain a mononuclear cell population and then
frozen in Dulbecco's modified Eagle's medium (DMEM; StemCell Technologies, Inc, Vancouver, BC, Canada) with 30% fetal calf serum
(FCS; StemCell Technologies, Inc) and 10% dimethyl sulfoxide (DMSO)
and stored at  135°C.
AML cell phenotyping and sorting.
Before sorting, thawed AML cells were suspended in HFN (Hanks' medium + 2% FCS and 0.1% sodium azide) at 107 cells/mL. Cells
were stained for 30 minutes on ice with monoclonal antibodies directly
coupled to the fluorochromes fluorescein isothiocyanate (FITC),
phycoerythrin (PE), or cyanine5-succinimidylester (Cy5). CD34-Cy5/PE,
CD71-PE/FITC were kind gifts from Dr Peter Lansdorp (Terry Fox
Laboratory, Vancouver, Canada); CD38-PE, HLA-DR-FITC were obtained from
Becton Dickinson (San Jose, CA); and CD33-FITC was obtained from
Immunotech (Westbrook, ME). Antibodies were used at 1 µg/mL with the
exception of CD34 and CD33, which were used at 4 µg/mL. Cells were
then washed twice in HFN at 4°C, propridium iodide (PI) at 2 µg/mL was added to the cells before the second wash, and the cells
were maintained on ice before sorting.
Cells were analyzed and sorted on a dual laser FACStarplus
(Becton Dickinson) on the basis of fluorescence intensity after gating out nonviable (PI+) cells and gating on low side scatter as
shown in Fig 1. Sort gates were set up to
exclude 100% of the cells in an irrelevant isotype control. A 10 channel separation was used to discriminate positive from negative
fractions to ensure purity. Fractions were sorted into Iscove's
modified Dulbecco's medium (StemCell Technologies, Inc) with 50% FCS
in microcentrifuge tubes at 4°C. Sort windows used to isolate the
specific subfractions are shown in Fig 1. Sorted fractions were washed,
resuspended at known cell concentrations, and used to initiate primary
CFU and suspension culture assays or used in the NOD/SCID assay.
View larger version (38K):
[in this window]
[in a new window]
| Fig 1.
Purification strategy for isolating cells stained with
CD34/CD71 and HLA-DR. Live cells were gated on the basis of PI staining
(A), and these cells were then gated on the basis of low side scatter
(B). Subsequently, cells were gated for expression of CD34-Cy5, into
CD34+ (R3) or CD34 (R4) windows (C). The
CD34+ cells in R3 were then sorted on the basis of their
expression of CD71-PE and HLA-DR-FITC (D). Sort gates for C and D were
set up to exclude 100% of the cells in an isotype control.
|
|
AML colony assays (CFU).
Unsorted AML mononuclear blood cells or FACS-sorted cells immediately
following sorting or cells removed during media changes after at least
2 weeks in suspension culture were plated in  -methylcellulose culture medium (Methocult M3230, StemCell Technologies, Inc), containing 3 U/mL recombinant human (rh) erythropoietin (StemCell Technologies, Inc), 20 ng/mL rh interleukin-3 (IL-3) (Sandoz, Basel,
Switzerland), 20 ng/mL rhIL-6 (Terry Fox Laboratory), 20 ng/mL rhG-CSF
(Amgen Canada Inc, Mississauga, Ontario), 20 ng/mL rh
granulocyte-macrophage colony-stimulating factor (GM-CSF) (Sandoz), and
50 ng/mL rhSteel Factor (SF) (Terry Fox Laboratory). After 14 days of
incubation at 37°C in a 5% CO2 humidified incubator, AML blast clusters (10 to 20 cells) or colonies (>20 cells) were counted and the numbers were pooled to obtain CFU counts. Most colonies
appeared abnormal (blastlike or monocytic). These abnormal appearing
colonies comprised 55% of the CFU counts from the primary CFU assays
in the 19 patients used for the in vitro studies, while clusters
represented 33%, and the remainder were of erythroid origin. Secondary
CFU assays using cells derived from SC or from NOD/SCID mice contained
similar colony types to those seen in the primary assays from the
individual patients.
Suspension culture assays (SC).
Suspension cultures were initiated and maintained as previously
described.21 Briefly, unsorted cells or FACS-sorted AML cells were suspended at up to 106 cells/mL in serum-free
medium (SFM) consisting of Iscove's modified Dulbecco's medium
containing 10 µg/mL insulin, 200 µg/mL transferrin, 2% bovine
serum albumin, 0.9% NaHCO3 (StemCell Technologies, Inc) and the recombinant growth factors IL-3, IL-6, G-CSF, GM-CSF, and SF in
the concentrations described above for the CFU assay. Cultures were
maintained at 37°C in a 5% CO2 humidified incubator and were demi-depopulated weekly by removal of half the cells plus
media and replacing it with fresh media. Every second week the cells,
which were removed, were cultured in -methylcellulose to determine
the CFU content of the suspension culture. Suspension cultures were
maintained for 8 weeks and the entire contents of the wells were
procured and assessed for CFU content. To allow comparisons between
experiments, the proportion of progenitors derived from each sorted
fraction at each time point was determined by comparison to a
progenitor recovery of 100% from all fractions at that time point.
Transplantation of leukemic cells into NOD/SCID mice.
NOD/SCID mice (Jackson Laboratory, Bar Harbor, ME) were bred and
maintained in sterile microisolator cages. Twenty-four hours before
transplantation, mice were irradiated with 3.5 Gy  irradiation from
a 137Cs source at a dose rate of 1.25 cGy/minute. Unsorted
AML cells and sorted subfractions were suspended in 0.3 mL minimal
essential medium (StemCell Technologies, Inc) with 5% FCS and injected
intravenously into the lateral tail vein of 6- to 8-week-old NOD/SCID
mice. Whenever possible, various cell concentrations were injected as a
semiquantitative assessment of the number of cells required for
engraftment. Normal human bone marrow (NBM) cells were given 15 Gy
irradiation using 250 kVp x-rays (Philips RT-250, HVL 1.5 mm Cu) at a
dose rate of 5.1 Gy/min. In each case, 106 irradiated NBM
cells were coinjected with the unsorted cells and sorted AML
subfractions to enhance engraftment potential. Animals were given
subcutaneous injections of 6 µg human IL-3 and 10 µg human Steel
Factor twice per week and were maintained until 8 weeks postinjection.
After killing, the gross anatomy of each mouse was inspected, and
femoral bone marrow samples were removed for flow cytometry,
fluorescent in situ hybridization (FISH), and histologic analysis.
Flow cytometry analysis of murine tissues.
Cell suspensions from femoral bone marrow were lysed in ammonium
chloride for 20 minutes and then washed in HFN with 5% human serum.
Cells were stained with a human pan leukocyte antibody, 9.4 (anti-CD45)-FITC (Dr P. Lansdorp) for 30 minutes on ice.
Separate aliquots were stained with an irrelevant IgG1-FITC
antibody (Becton Dickinson) as an isotype control. Aliquots of normal
human peripheral blood cells and bone marrow cells from a noninjected
NOD/SCID mouse were also stained with CD45-FITC and the isotype control to serve as positive and negative controls for antibody specificity. After staining, the cells were washed in HFN containing PI (2 µg/mL)
and resuspended in 300 µL HFN. Samples were analyzed using a FACScan
(Becton Dickinson), nonviable cells were gated out based on PI uptake.
Isotype controls were run for each tissue sample and used to set the
gates. Cells were defined as positive using gate settings, which
excluded  99.9% of the cells in the matched isotype control, any
positive value for the isotype control was then subtracted from the
percentage positive in samples stained with CD45-FITC. The expression
of CD45-FITC is very specific, in 55 control (noninjected) mice,
expression of CD45 was 0.002% ± 0.002% and levels of expression
above 0.06% have never been observed in these control animals. We
defined human engraftment as expression of 0.1% CD45+
cells in a sample. Whenever possible, the CD45+ cells
derived from murine tissue were sorted and set up in suspension culture
or plated in -methylcellulose with recombinant growth factors. The
derived colonies were plucked onto slides and cells from the suspension
cultures were transferred directly onto slides for subsequent
cytogenetic analysis.
Cytogenetic analysis.
Colonies from primary CFU or from CFU, which were derived from SC were
evaluated for the leukemia-specific cytogenetic change by either
standard cytogenetics or by FISH whenever possible. Likewise, sorted
CD45+ cells from bone marrow removed from NOD/SCID mice and
CFU derived from these cells were evaluated for the leukemic
transformation. For analysis, a random selection of single colonies or
clusters from methylcellulose dishes were plucked and plated onto
slides after colony synchronization with colcemid.23
Colonies derived from these assays had a uniform morphology and
appeared abnormal. Analysis was not restricted to colonies with a
blastlike appearance or to only colonies and clusters of the myeloid
lineage, those of erythroid origin were also evaluated. In one case
only (patient 5), pools of 3 to 5 colonies were analyzed, rather than
individual colonies. Whole chromosome 9 painting probe directly labeled
with spectrum green (BRL Life Technologies, Gaithersburg, MD) was used to detect the t(9;11) in one patient. The inversion (inv) 16 probe directly labeled with digoxigenin (Oncor, Gaithersburg, MD) was used to
detect inv16 in two other patients. The centromere-specific chromosome
8 probe directly labeled to digoxigenin (Oncor) was used to highlight
at least one additional chromosome 8 in four other patients. The +8
probe and inv16 probes were hybridized in Hybrisol VI (Oncor) with
amplification of the signal by rabbit antisheep FITC (Jackson Immuno
Research Laboratories, West Grove, PA) and detection by sheep
antidigoxigenin-FITC (Boehringer Mannheim, Quebec, Canada).
Counterstaining was in PI at 200 ng/mL in Vectashield (Vector
Laboratories, Inc, Burlingome, CA). The chromosome 9 probe was
hybridized by the manufacturer's instructions with counter-staining as
above. Due to the technical difficulty in obtaining only cells derived
from the small colonies, while excluding cells contained in surrounding
methylcellulose by manual plucking, colonies were defined as positive
whenever 60% of cells contained the respective leukemic karyotype of
that patient. Colonies containing only 40% to 60% of cells with the
leukemic change were classed as undetermined and those with <40%
leukemic cells were defined as negative. In the three patients where
both metaphase and interphase cells could be scored, due to the
presence of an additional chromosome 8, approximately 200 cells (mean ± standard error [SE] 210 ± 3 cells) were counted per colony.
Statistics.
Statistical analysis was performed using analysis of variance (ANOVA)
with Tukey's posthoc analysis of means.
| |
RESULTS |
Expression of CD34 and CD71 or HLA-DR or CD38 on AML cells with long
term proliferative ability in vitro.
Ficoll-separated blood mononuclear cells from 19 AML patients at
diagnosis were used for the present investigation, the clinical characteristics of these patients are shown in
Table 1. Because of limitations in
available cell numbers, only subsets of this group, as indicated, could
be analyzed with each sorting strategy. Peripheral blood cells from 14 of these patients were sorted for coexpression of CD34 and CD71
(Fig 2). Eight of these patient samples had
a high number of CD34+ nucleated cells (range, 10% to 87%
CD34+), the remaining six samples had only a very small
proportion of CD34+ cells (0.11% to 4%). Overall, the
CD34+/CD71+ subfraction represented a small
fraction of nucleated cells in the blood in these patients (mean ± SEM 12% ± 4%), the CD34+/CD71
subfraction represented 20% ± 7% and the majority of nucleated cells overall were CD34 (68% ± 9%). The
majority of primary CFU were also derived from the
CD34 fraction (54% ± 13%). However, after 2 weeks in suspension culture, the majority of detectable CFU (56% ± 10%) were derived from the CD34+/CD71
subfraction. Only 22% ± 7% and 22% ± 8% of CFU at week 2 were derived from the CD34+/CD71+ and
CD34 subfractions, respectively. After 4 weeks in
suspension culture (SC), 62% ± 11% of CFU were derived from the
CD34+/CD71subfraction. The proportion of
CFU derived from the CD34+/CD71
subfraction increased significantly with length of time in SC to 66% ± 10% at week 6 and 83% ± 8% at week 8 (F = 5.77**, degrees of freedom [df] = 5, P < .0002). Of the six
patients with very low numbers of CD34+ cells in their
blast population (mean ± SEM 1.7% ± 0.7%), in four patient
samples, all of the detectable CFU after at least 4 weeks in suspension
culture were derived from the CD34+/CD71
subfraction. In one of these four samples, cells with this phenotype represented only 0.1% of nucleated cells, but produced 100% of the
CFU in SC at week 4. In the two remaining patient samples, the majority
of primary CFU (93% ± 6%) and CFU derived from SC at
week 2 (99%) were derived from the CD34
subfraction. In one of these two samples, CFU could not be detected from any subfraction after 6 weeks in culture. In the other sample, the
majority of CFU detected at week 4 (70%), at week 6 (64%), and week 8 (52%) were derived from the CD34 subfraction.
View larger version (25K):
[in this window]
[in a new window]
| Fig 2.
Proportion of AML peripheral blood cells and progenitors
expressing CD34 and CD71 antigens in 14 patients (patients 2 to 5, 8 to
16, and 18). ( ), CD34+/CD71+; ( ),
CD34+/CD71; ( ), CD34.
|
|
Peripheral blood cells from 11 patients were sorted for coexpression of
CD34 and HLA-DR (Fig 3). The majority of
nucleated cells at diagnosis were CD34 (74% ± 7%), while 13% ± 4% and 13% ± 5% of cells were
CD34+/HLA-DR+ and
CD34+/HLA-DR, respectively. Cells, which
were capable of producing CFU after 2 weeks and 4 weeks in SC, were
heterogeneous in their HLA-DR expression and some were in the
CD34 subfraction. However, after 6 weeks and 8 weeks
in SC, significantly higher numbers of CFU (71% ± 9% and
86% ± 9%, respectively) were derived from the
CD34+/HLA-DR subfraction, compared with
earlier time points (F = 10.82**, df = 4, P < .00003).
View larger version (21K):
[in this window]
[in a new window]
| Fig 3.
Proportion of AML peripheral blood cells and progenitors
expressing CD34 and HLA-DR antigens in 11 patients (patients 1, 2, 4, 5, 7, 8, 10, 11, 13, 15, and 19). ),
CD34+/HLA-DR+; (),
CD34+/HLA-DR; (),
CD34.
|
|
Cells from seven patients were sorted for coexpression of CD34 and CD38
and evaluated in the suspension culture assay
(Fig 4). The
CD34+/CD38+ subfraction represented 28% ± 15% of nucleated cells at diagnosis, 17% ± 12% (range, 0.03 to
45%) were CD34+/CD38 and the majority
were CD34 (55% ± 15%). However, the
majority of CFU detected after 2 weeks (55% ± 12%) and 4 weeks
(96% ± 4%) in suspension culture were derived from the
CD34+/CD38 subfraction. Subsequently, at
weeks 6 to 8, in the four patient samples where CFU were still
detectable, all were derived from the
CD34+/CD38 subfraction.
View larger version (18K):
[in this window]
[in a new window]
| Fig 4.
Proportion of AML peripheral blood cells and progenitors
expressing CD34 and CD38 antigens in seven patients (patients 2, 3, 5, 6, 8, 10, and 13). (), CD34+/CD38+;
(), CD34+/CD38; (),
CD34.
|
|
Cytogenetic analysis of in vitro studies.
Standard cytogenetic or FISH analysis was performed on primary CFU
derived from unsorted and sorted subfractions and on CFU derived from
suspension cultures initiated with these subfractions (Table 2). In experiments with seven
patient samples, all primary CFU colonies analyzed had the respective
leukemic change. The majority (40 of 41) of CFU derived from SC of
unsorted cells at weeks 2 to 8 were found to be derived from the
leukemic clone, with only one undetermined colony. All primary CFU
derived from sorted subfractions were found to have the expected
chromosomal abnormality. CFU derived from SC of
CD34+/CD71,
CD34+/HLA-DR+, and
CD34+/HLA-DR subfractions were analyzed
in 18 cases (eight patients). The majority of colonies analyzed were
found to be positive for the leukemic karyotype (77 of 84), while five
colonies were undetermined and two were negative.
In vivo NOD/SCID assay.
Unsorted and sorted AML cells from 14 patients were evaluated for their
ability to engraft NOD/SCID mice after sorting for coexpression of CD34
and CD71 and/or HLA-DR. However, engraftment with any sorted or
unsorted fraction was achieved using cells from only 12 of these
patients, with injections of up to 5 × 106 unsorted
cells per mouse. Unsorted cells from 11 of the 12 patients engrafted in
the NOD/SCIDs (Table 3). Engraftment was
achieved using 106 cells from nine of 11 patients evaluated
(mean ± SEM, 27% ± 11% CD45+) using 5 × 105 unsorted cells from six of nine patients evaluated
(26% ± 15% CD45+) and using 105 unsorted
cells from four of eight patients evaluated (20% ± 15% CD45+). One patient, of eight tested, engrafted with 5 × 104 cells (26% CD45+) and no
engraftment was achieved using  104 unsorted
cells.
View this table:
[in this window]
[in a new window]
|
Table 3.
Percentage of CD45+ Cells Detected in the
BM of NOD/SCID Mice Injected With Unsorted AML Peripheral Blood
Cells
|
|
Cells from nine AML patients were sorted for CD34 and CD71 expression
and the subfractions were evaluated in NOD/SCID mice. In
five patients who had high numbers of CD34+ nucleated cells
(28% to 75%) (Table 4), engraftment was
achieved using the CD34+/CD71 sorted
subfraction from all patients, although there was considerable heterogeneity in the degree of engraftment achieved (range, 0.16% to
74% CD45+ using 104 to 106 cells).
Engraftment was not obtained when CD34+/CD71+
or CD34 subfractions from those patients were
injected. Four patients who had very low numbers (0.11% to 1.3%) of
CD34+ cells (Table
5) were also evaluated using this strategy. Engraftment was achieved using small numbers of
CD34+/CD71 cells from all of these
patients (range, 0.13% to 27% CD45+ with 103
to 104 cells). Interestingly, engraftment was achieved
using the CD34 fraction from patient 2 (37%
CD45+ with 4 ×105 cells) and patient 8 (11% CD45+) with 105 cells, (54%) with 5 × 105 and (31%) with 106 cells. The
majority of CFU in suspension culture at 2 to 8 weeks was derived from
the CD34 subfraction in these two patients. However,
considerably higher numbers of CD34 cells
(105) had to be injected to achieve engraftment
equivalent to that achieved with 4 × 103
CD34+/CD71 cells. No engraftment was
achieved with 106 CD34 cells from the
other two patients. Engraftment was not achieved with
CD34+/CD71+ cells from any patient.
View this table:
[in this window]
[in a new window]
|
Table 4.
Expression of Human CD45 Antigen in NOD/SCID Recipients
of AML Cells Sorted for Coexpression of CD34 and CD71
(CD34+ patients)
|
|
View this table:
[in this window]
[in a new window]
|
Table 5.
Expression of Human CD45 Antigen in NOD/SCID Recipients
of AML Cells Sorted for Coexpression of CD34 and CD71
(CD34 patients)
|
|
To determine the HLA-DR phenotype of NOD/SCID engrafting cells and to
evaluate the possible enrichment of these cells with three-color FACS
sorting, cells from six patients, which included three evaluated in
Table 4 or 5, were sorted for
CD34, CD71, and HLA-DR combinations (Table 6). The number of
CD34+ nucleated cells was low (0.11% to 28%) in these
patients. In five patients, both CD34+/HLA-DR+
and CD34+/HLA-DR fractions were evaluated
and engraftment was achieved with
CD34+/HLA-DR cells from four of these
patients (4% to 38% CD45+). However, engraftment was not
achieved with CD34+/HLA-DR+ subfraction, where
approximately equivalent numbers of cells were transplanted, or with
the CD34 subfraction, with the exception of patient
8 (98% CD45+ with 106 cells). Cells from four
of these five patients, plus one other, were sorted for expression of
CD34, lack of CD71, and expression/lack of HLA-DR to further define
HLA-DR phenotype and determine enrichment. In all cases, engraftment
was achieved using very low numbers of
CD34+/CD71/HLA-DR
cells (1% to 40% CD45+ using 4 × 102 to
3.7 × 104 cells), but was not seen with equivalent or
greater numbers of CD34+/CD71/HLA-DR+ cells.
This triple sorting strategy achieved  2 log enrichment of NOD/SCID
engrafting cells in the four patients where this could be evaluated and
improved on the enrichment observed with
CD34+/CD71 or
CD34+/HLA-DR sorting alone
(Fig 5).
View this table:
[in this window]
[in a new window]
|
Table 6.
Expression of Human CD45 Antigen in NOD/SCID Recipients
of AML Cells Sorted for Coexpression of CD34, HLA-DR, and CD71
|
|
View larger version (19K):
[in this window]
[in a new window]
| Fig 5.
Enrichment of NOD/SCID leukemia initiating cells. Graphs
depict the engraftment achieved with variable numbers of number of
unsorted and sorted cells from four patients (patients 4, 8, 13, and
15). All points represent at least 0.1% engraftment in a single
NOD/SCID mouse femoral bone marrow.
|
|
Cytogenetic analysis of transplanted cells.
It was possible to analyze colonies derived from sorted
CD45+ cells from NOD/SCID recipients injected with AML
cells in seven experiments (five patients). In six cases, the majority
of CFU derived from recipients of unsorted cells contained 60%
leukemic cells (25 of 29) and four were negative. Plucked colonies from patient 4 were heterogeneous in detection of the +13 change by FISH
from both sorted and unsorted cells. However, the cytogenetic analysis
of this patient at presentation reported that only half of the
metaphases analyzed had trisomy 13. Our results are therefore consistent with this finding. In all other cases, the majority of CFU
derived from recipients of either the
CD34+/71 (6 of 8),
CD34+/HLA-DR (8 of 8), or
CD34+/CD71/HLA-DR (14 of 14) subfractions were defined as being positive for the specific
karyotype of that patient. CFU derived from recipients of the
CD34 subfraction were analyzed in one patient and
all were found to be leukemic (19 of 19 positive colonies).
Cell phenotyping study.
To determine which combinations of antibodies might be optimal to
obtain purification of primitive AML progenitors, peripheral blood
mononuclear cells from 13 AML patients at diagnosis were stained with
combinations of three monoclonal antibodies
(Table 7). These AML patients
were FAB M0 (n = 1), M1 (n = 2), M2 (n = 2), M4 (n = 4), M5 (n = 3),
and one patient had an undetermined subtype. Expression of CD34 was
high in most patients of this set (44% ± 10%
CD34+). The
CD34+/CD38/HLA-DR
subfraction represented only 0.3% ± 0.1% of the total nucleated cell population, thus a sorting strategy using this combination of
monoclonal antibodies could potentially enrich for primitive cells with
this phenotype over 300-fold. Using CD34 in combination with CD38/CD71
or CD71/HLA-DR could produce enrichments of >100-fold and 67-fold,
respectively, of primitive cells, which expressed CD34 and lacked
expression of the other markers. The other antibody combinations that
were evaluated would also enrich for primitive cells, although to a
lesser extent, and may be less useful in a purification strategy.
| |
DISCUSSION |
The finding that AML cells with different phenotypes differ in their
proliferative ability in functional assays suggests that AML cells may
exist as a hierarchy in most patients. We have previously shown that
only a minority of AML blasts have long-term proliferative ability,
with cells that have longer term proliferative ability being less
frequent than those with shorter term proliferative potential.20 In this study, we have compared the phenotype
of cells that grow long-term in vitro with those that engraft NOD/SCID mice. AML cells capable of producing human AML in immunodeficient NOD/SCID mice may be similar to the AML sustaining cells in the patients. The possibility that only a small minority of AML tumor cells
have the ability to act as stem cells in vivo to maintain the malignant
population has important therapeutic significance, as these cells may
be the only relevant cells to target with treatment regimens. The main
objectives in our study were to further phenotypically characterize and
purify the cells which are responsible for maintenance of AML. Here we
investigated the coexpression of CD34 and CD71, HLA-DR, or CD38
antigens on AML cells with long-term proliferative ability in vitro.
Subsequently, we evaluated whether the phenotype of AML cells capable
of long-term proliferation in vitro matched that of cells, which were
capable of engrafting sublethally-irradiated NOD/SCID mice.
Our findings here indicate that among AML cells the
CD34+/CD71 subfraction contained a
relatively small proportion of nucleated cells (20% ± 7%) and
primary CFU (34% ± 12%), but the majority of cells capable of
producing colonies after 2 to 8 weeks in suspension culture. The
CD34+/HLA-DR phenotype selected 13% ± 5% of leukemic cells at diagnosis. However, after 8 weeks in
suspension culture, the majority of CFU (86% ± 9%) were derived
from cells with the CD34+/HLA-DR
phenotype. To further confirm the CD34+,
CD71, HLA-DR phenotype of
primitive AML cells, we evaluated the ability of the sorted
subfractions to engraft NOD/SCID mice. Engraftment (0.13% to 74%
CD45+) was achieved using 103 to
106 CD34+/CD71 cells from
nine patient samples. However, there was no engraftment (<0.1%
CD45+) with as many as 106 cells when the
CD34+/CD71+ subfraction was used. Among five
patients, a mean of 2% of the leukemic cell population was
CD34+/HLA-DR. Engraftment was achieved
using this subfraction (range, 4% to 38% CD45+ with 3 × 103 to 5 × 105 cells) from four
of these samples. In contrast, engraftment could not be attained using
equal or greater numbers of CD34+/HLA-DR+ cells
from the same patients. These findings show that the majority of AML
cells, which are capable of engrafting NOD/SCID mice, as well as the
majority of those generating CFU in long-term suspension culture, have
the phenotype CD34+/CD71 and
HLA-DR.
In addition, AML progenitors detected after 4 to 8 weeks in the SC were
also shown to be CD34+/CD38, although
only 17% ± 12% of the starting cell population expressed this
phenotype. The CD34+/CD38 phenotype of
NOD/SCID leukemia initiating cells has been previously described by
other investigators22 and in one experiment, we confirmed
this result with 48% CD45+ cells in the mouse transplanted
with 106 CD34+/CD38 cells
and no engraftment in the mouse injected with 106
CD34+/CD38+ cells. Thus, AML cells capable of
long-term proliferation in vitro and in vivo repopulation share a
phenotype, also seen on primitive normal cells (LTC-IC), which express
CD34 and lack expression of CD71, HLA-DR, and CD38.
The majority of nucleated cells in six patients were
CD34 (<4% CD34+). In two of these
patients, the majority of detectable CFU were always derived from the
CD34 subfraction. CFU produced by the
CD34 cells in these two patients were subsequently
confirmed to have the leukemic karyotype by FISH. Engraftment in
NOD/SCID mice was also achieved using CD34 cells in
these two patients. This finding raises the possibility that in these
two patients, the leukemic transformation may have occurred in a
CD34 cell. However, in both cases, engraftment could
also be achieved using considerably smaller numbers of
CD34+/CD71 cells (103 and 4 × 103, respectively) and in patient 8, an equivalent
degree of engraftment was attained with as few as 4 × 102
CD34+/CD71/HLA-DR
cells. These results suggest that in these patients, the leukemic transformation may have occurred in a primitive cell, which expressed CD34, with subsequent clonal evolution so that cells which no longer
express the antigen, nevertheless retain their ability to proliferate
and engraft NOD/SCID mice. These findings are consistent with our
previous report21 and that of Terpstra et al,24
demonstrating the presence of cells with high proliferative ability in
the CD34 subfraction of some AML patients. The
CD34 cells from the remainder of the patients
analyzed in the present study were not capable of long-term
proliferation in vitro and had no engraftment potential, suggesting
that loss of expression of CD34 in these cases was associated with loss
of proliferative ability. This finding provides evidence that
differentiation, as defined by this phenotypic change, is usually, but
not always, associated with loss of long-term proliferative ability in
AML.
As a semiquantitative assessment of the number of AML cells, which are
required to engraft the mice, a number of cell doses were evaluated
whenever possible. The frequency of NOD/SCID leukemia initiating cells
has previously estimated to be 0.2 to 100/106 AML
cells,22 while other groups have reported a minimum of 1.1 × 106 unsorted cells were required for engraftment of
SCID mice.25 Our data using the NOD/SCID model suggests
that in these patients, the frequency of the NOD/SCID leukemic
initiating cells ranges from <1 to 20/106 unsorted cells
and further confirms the considerable heterogeneity between individual
patients.
The CD34+/CD71/HLA-DR
subfraction from five patients, representing on average 0.35% of
nucleated cells, was capable of engrafting NOD/SCID mice (1% to 40%
CD45+) using very small numbers of cells (4 × 102 to 105), while engraftment could not be
achieved using equivalent numbers of
CD34+/CD71 or
CD34+/HLA-DR cells. As shown in Fig 5,
the combined use of all three antigens in a sorting strategy lead to
approximately a 2 log enrichment of NOD/SCID leukemia initiating cells.
Furthermore, as illustrated in Table 7, using combinations of CD34 and
CD38 plus CD71 or HLA-DR may make it possible to achieve even greater
(up to 300-fold) enrichment of primitive AML cells. The ability to
enrich for NOD/SCID engrafting activity in some subfractions and
correspondingly deplete this activity from other subfractions provides
additional evidence for the stem cell model in AML.
Our results support the notion that the leukemic transformation occurs
in a primitive cell, which has a
CD34+/CD71/HLA-DR/CD38
phenotype similar to primitive normal hematopoietic cells. Other groups
have also found the primitive AML phenotype to be
CD34+/CD3822,26,27 or
CD34+/CD38/CD33.28
We have previously shown these primitive AML cells differ from normal
primitive progenitor cells in their lack of expression of
Thy-1.21 Understanding of the phenotypic similarities and differences between normal and leukemic stem cells may enable the
development of new purging strategies for use in autologous transplantation. The ability to isolate purified populations of primitive AML progenitors and to study their functional characteristics in biological assays will facilitate studies designed to investigate the molecular basis of human leukemogenesis.
| |
ACKNOWLEDGMENT |
The authors thank R. Zapf, B. Gerhard, G. Thornbury, and G. Shaw for
excellent technical assistance.
| |
FOOTNOTES |
Submitted January 29, 1998;
accepted August 3, 1998.
Supported by a grant from the National Cancer Institute of Canada. A.B.
is a fellow of the Leukaemia Research Fund of Canada.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to H.J. Sutherland, MD, PhD,
Terry Fox Laboratory, 601 W 10th Ave, Vancouver, BC, Canada V5Z 1L3.
| |
REFERENCES |
1.
Fialkow PJ, Singer JW, Adamson JW, Vaidya K, Dow LW, Ochs J, Moohr JW:
Acute nonlymphocytic leukemia: Heterogeneity of stem cell origin.
Blood
57:1068, 1981[Abstract/Free Full Text]
2.
Sabbath KD, Ball ED, Larcom P, Davis RB, Griffin JD:
Heterogeneity of clonogenic cells in acute myeloblastic leukemia.
J Clin Invest
75:746, 1985
3.
Kabral A, Bradstock KF, Grimsley P, Favaloro EJ:
Immunophenotype of clonogenic cells in myeloid leukaemia.
Leuk Res
12:51, 1988[Medline]
[Order article via Infotrieve]
4.
Lim HS, Culligan D, Couzens S, Fisher J, John S, Pollard P, Burnett A, Whittaker J:
Molecular evidence for a common leukaemic progenitor in acute mixed lymphoid and myeloid leukaemia.
Br J Haematol
92:131, 1996[Medline]
[Order article via Infotrieve]
5.
Moore MAS, Williams N, Metcalf D:
In vitro colony formation by normal and leukemic human hematopoietic cells: Characterization of the colony-forming cells.
J Natl Cancer Inst
50:603, 1973
6.
Minden MD, Buick RN, McCulloch EA:
Separation of blast cell and T-lymphocyte progenitors in the blood of patients with acute myeloblastic leukemia.
Blood
54:186, 1979[Abstract/Free Full Text]
7.
Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM:
Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro.
Blood
74:1563, 1989[Abstract/Free Full Text]
8.
Sutherland HJ, Eaves CJ, Eaves AC, Lansdorp PM:
Differential expression of antigens on cells that initiate haemopoiesis in long-term human marrow culture, in
Knapp W,
Dorken B,
Gilks WR,
Rieber EP,
Schmidt RE,
Stein H,
von dem Borne AEG
(eds):
Leucocyte Typing IV. White Cell Differentiation Antigens. New York, NY, Oxford, 1989, p 910.
9.
Terstappen LWMM, Huang S, Safford M, Lansdorp PM, Loken MR:
Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38 progenitor cells.
Blood
77:1218, 1991[Abstract/Free Full Text]
10.
Mayani H, Lansdorp PM:
Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood.
Blood
83:2410, 1994[Abstract/Free Full Text]
11.
Andrews RG, Singer JW, Bernstein ID:
Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties.
J Exp Med
169:1721, 1989[Abstract/Free Full Text]
12.
Lansdorp PM, Sutherland HJ, Eaves CJ:
Selective expression of CD45 isoforms on functional subpopulations of CD34+ hemopoietic cells from human bone marrow.
J Exp Med
172:363, 1990[Abstract/Free Full Text]
13.
Udomsakdi C, Eaves CJ, Sutherland HJ, Lansdorp PM:
Separation of functionally distinct subpopulations of primitive human hematopoietic cells using rhodamine-123.
Exp Hematol
19:338, 1991[Medline]
[Order article via Infotrieve]
14.
Sutherland R, Delia D, Schneider C, Newman R, Kemshead J, Greaves M:
Ubiquitous cell-surface glycoprotein on tumor cells is proliferation-associated receptor for transferrin.
Proc Natl Acad Sci USA
78:4515, 1981[Abstract/Free Full Text]
15.
Joventino LP, Stock W, Lane NJ, Daly KM, Mick R, Le Beau MM, Larson RA:
Certain HLA antigens are associated with specific morphologic and cytogenetic subsets of acute myeloid leukemia.
Leukemia
9:443, 1995
16.
Engleman EG, Warnke R, Fox RI, Dilley J, Benike CJ, Levy R:
Studies of a human T lymphocyte antigen recognized by a monoclonal antibody.
Proc Natl Acad Sci USA
78:1791, 1981[Abstract/Free Full Text]
17.
Warnke R, Levy R:
Detection of T and B cell antigens with hybridoma monoclonal antibodies.
J Histochem Cytochem
28:771, 1980[Abstract]
18.
Brandt J, Baird N, Lu L, Srour E, Hoffman R:
Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro.
J Clin Invest
82:1017, 1988
19.
Griffin JD, Larcom P, Schlossman SF:
Use of surface markers to identify a subset of acute myelomonocytic leukemia cells with progenitor cell properties.
Blood
62:1300, 1983[Abstract/Free Full Text]
20.
Sutherland HJ, Blair A, Zapf RW:
Characterization of a hierarchy in human acute myeloid leukemia progenitor cells.
Blood
87:4754, 1996[Abstract/Free Full Text]
21.
Blair A, Hogge DE, Ailles LE, Lansdorp PM, Sutherland HJ:
Lack of expression of Thy-1 (CD90) on acute myeloid leukaemia cells with long-term proliferative ability in vitro and in vivo.
Blood
89:3104, 1997[Abstract/Free Full Text]
22.
Bonnet D, Dick JE:
Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.
Nature Med
3:730, 1997[Medline]
[Order article via Infotrieve]
23.
Fraser C, Eaves CJ, Kalousek DK:
Fluorodeoxyuridine synchronization of hemopoietic colonies.
Cancer Genet Cytogenet
24:1, 1987[Medline]
[Order article via Infotrieve]
24.
Terpstra W, Prins A, Ploemacher RE, Wognum BW, Wagemaker G, Lowenberg B, Wielenga JJ:
Long-term leukemia-initiating capacity of a CD34- subpopulation of acute myeloid leukemia.
Blood
87:2187, 1996[Abstract/Free Full Text]
25.
Terpstra W, Prins A, Visser T, Wognum B, Wagemaker G, Lowenberg B, Wielenga J:
Conditions for engraftment of human acute myeloid leukemia (AML) in SCID mice.
Leukemia
9:1573, 1995[Medline]
[Order article via Infotrieve]
26.
Haase D, Feuring-Buske M, Konemann S, Fonatsch C, Troff C, Verbeek W, Pekrun A, Hiddemann W, Wormann B:
Evidence for malignant transformation in acute myeloid leukemia at the level of early hematopoietic stem cells by cytogenetic analysis of CD34+ subpopulations.
Blood
86:2906, 1995[Abstract/Free Full Text]
27.
Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE:
A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.
Nature
367:645, 1994[Medline]
[Order article via Infotrieve]
28.
Mehrotra B, George TI, Kavanau K, Avet-Loiseau H, Moore D II, Willman CL, Slovak ML, Atwater S, Head DR, Pallavicini MG:
Cytogenetically aberrant cells in the stem cell compartment (CD34+lin) in acute myeloid leukemia.
Blood
86:1139, 1995[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
C. V. Cox, P. Diamanti, R. S. Evely, P. R. Kearns, and A. Blair
Expression of CD133 on leukemia-initiating cells in childhood ALL
Blood,
April 2, 2009;
113(14):
3287 - 3296.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Fukushima, I. Matsumura, S. Ezoe, M. Tokunaga, M. Yasumi, Y. Satoh, H. Shibayama, H. Tanaka, A. Iwama, and Y. Kanakura
FIP1L1-PDGFR{alpha} Imposes Eosinophil Lineage Commitment on Hematopoietic Stem/Progenitor Cells
J. Biol. Chem.,
March 20, 2009;
284(12):
7719 - 7732.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Argiropoulos, E. Yung, and R. K. Humphries
Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis
Genes & Dev.,
November 15, 2007;
21(22):
2845 - 2849.
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Flynn and D. S. Kaufman
Donor cell leukemia: insight into cancer stem cells and the stem cell niche
Blood,
April 1, 2007;
109(7):
2688 - 2692.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. V. Cox, H. M. Martin, P. R. Kearns, P. Virgo, R. S. Evely, and A. Blair
Characterization of a progenitor cell population in childhood T-cell acute lymphoblastic leukemia
Blood,
January 15, 2007;
109(2):
674 - 682.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Yalcintepe, A. E. Frankel, and D. E. Hogge
Expression of interleukin-3 receptor subunits on defined subpopulations of acute myeloid leukemia blasts predicts the cytotoxicity of diphtheria toxin interleukin-3 fusion protein against malignant progenitors that engraft in immunodeficient mice
Blood,
November 15, 2006;
108(10):
3530 - 3537.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. E. Hogge, L. Yalcintepe, S.-H. Wong, B. Gerhard, and A. E. Frankel
Variant Diphtheria Toxin-Interleukin-3 Fusion Proteins with Increased Receptor Affinity Have Enhanced Cytotoxicity against Acute Myeloid Leukemia Progenitors
Clin. Cancer Res.,
February 15, 2006;
12(4):
1284 - 1291.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. J. Pearce, D. Taussig, K. Zibara, L.-L. Smith, C. M. Ridler, C. Preudhomme, B. D. Young, A. Z. Rohatiner, T. A. Lister, and D. Bonnet
AML engraftment in the NOD/SCID assay reflects the outcome of AML: implications for our understanding of the heterogeneity of AML
Blood,
February 1, 2006;
107(3):
1166 - 1173.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Huff, W. Matsui, B. D. Smith, and R. J. Jones
The paradox of response and survival in cancer therapeutics
Blood,
January 15, 2006;
107(2):
431 - 434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Levis, K. M. Murphy, R. Pham, K.-T. Kim, A. Stine, L. Li, I. McNiece, B. D. Smith, and D. Small
Internal tandem duplications of the FLT3 gene are present in leukemia stem cells
Blood,
July 15, 2005;
106(2):
673 - 680.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. G. Wang, M. P. Pasillas, and M. P. Kamps
Meis1 programs transcription of FLT3 and cancer stem cell character, using a mechanism that requires interaction with Pbx and a novel function of the Meis1 C-terminus
Blood,
July 1, 2005;
106(1):
254 - 264.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Guzman, R. M. Rossi, L. Karnischky, X. Li, D. R. Peterson, D. S. Howard, and C. T. Jordan
The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells
Blood,
June 1, 2005;
105(11):
4163 - 4169.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. H.G.P. Raaijmakers, E. P.L.M. de Grouw, L. H.H. Heuver, B. A. van der Reijden, J. H. Jansen, R. J. Scheper, G. L. Scheffer, T. J.M. de Witte, and R. A.P. Raymakers
Breast Cancer Resistance Protein in Drug Resistance of Primitive CD34+38- Cells in Acute Myeloid Leukemia
Clin. Cancer Res.,
March 15, 2005;
11(6):
2436 - 2444.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. V. Cox, R. S. Evely, A. Oakhill, D. H. Pamphilon, N. J. Goulden, and A. Blair
Characterization of acute lymphoblastic leukemia progenitor cells
Blood,
November 1, 2004;
104(9):
2919 - 2925.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Vercauteren and H. J. Sutherland
Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo
Blood,
October 15, 2004;
104(8):
2315 - 2322.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. L. Liesveld, C. T. Jordan, and G. L. Phillips II
The Hematopoietic Stem Cell in Myelodysplasia
Stem Cells,
July 1, 2004;
22(4):
590 - 599.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. J. Jones, W. H. Matsui, and B. D. Smith
Cancer Stem Cells: Are We Missing the Target?
J Natl Cancer Inst,
April 21, 2004;
96(8):
583 - 585.
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Monaco, M. Konopleva, M. Munsell, C. Leysath, R.-Y. Wang, C. E. Jackson, M. Korbling, E. Estey, J. Belmont, and M. Andreeff
Engraftment of Acute Myeloid Leukemia in NOD/SCID Mice Is Independent of CXCR4 and Predicts Poor Patient Survival
Stem Cells,
March 1, 2004;
22(2):
188 - 201.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Guzman, C. F. Swiderski, D. S. Howard, B. A. Grimes, R. M. Rossi, S. J. Szilvassy, and C. T. Jordan
Preferential induction of apoptosis for primary human leukemic stem cells
PNAS,
December 10, 2002;
99(25):
16220 - 16225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Testa, R. Riccioni, S. Militi, E. Coccia, E. Stellacci, P. Samoggia, R. Latagliata, G. Mariani, A. Rossini, A. Battistini, et al.
Elevated expression of IL-3Ralpha in acute myelogenous leukemia is associated with enhanced blast proliferation, increased cellularity, and poor prognosis
Blood,
September 26, 2002;
100(8):
2980 - 2988.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Feuring-Buske, A. E. Frankel, R. L. Alexander, B. Gerhard, and D. E. Hogge
A Diphtheria Toxin-Interleukin 3 Fusion Protein Is Cytotoxic to Primitive Acute Myeloid Leukemia Progenitors But Spares Normal Progenitors
Cancer Res.,
March 1, 2002;
62(6):
1730 - 1736.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Bazin, E. Aubin, L. Boyer, I. St-Amour, C. Roberge, and R. Lemieux
Functional in vivo characterization of human monoclonal anti-D in NOD-scid mice
Blood,
February 15, 2002;
99(4):
1267 - 1272.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Guzman, S. J. Neering, D. Upchurch, B. Grimes, D. S. Howard, D. A. Rizzieri, S. M. Luger, and C. T. Jordan
Nuclear factor-{kappa}B is constitutively activated in primitive human acute myelogenous leukemia cells
Blood,
October 15, 2001;
98(8):
2301 - 2307.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. G. Wulf, R.-Y. Wang, I. Kuehnle, D. Weidner, F. Marini, M. K. Brenner, M. Andreeff, and M. A. Goodell
A leukemic stem cell with intrinsic drug efflux capacity in acute myeloid leukemia
Blood,
August 15, 2001;
98(4):
1166 - 1173.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Feuring-Buske and D. E. Hogge
Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34+CD38{-} progenitor cells from patients with acute myeloid leukemia
Blood,
June 15, 2001;
97(12):
3882 - 3889.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Hirai, D. Radisky, R. Boudreau, M. Simian, M. E. Stevens, Y. Oka, K. Takebe, S. Niwa, and M. J. Bissell
Epimorphin Mediates Mammary Luminal Morphogenesis through Control of C/EBP{beta}
J. Cell Biol.,
May 14, 2001;
153(4):
785 - 794.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. L. Guzman, D. Upchurch, B. Grimes, D. S. Howard, D. A. Rizzieri, S. M. Luger, G. L. Phillips, and C. T. Jordan
Expression of tumor-suppressor genes interferon regulatory factor 1 and death-associated protein kinase in primitive acute myelogenous leukemia cells
Blood,
April 1, 2001;
97(7):
2177 - 2179.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Bruserud, B. T. Gjertsen, B. Foss, and T.-S. Huang
New Strategies in the Treatment of Acute Myelogenous Leukemia (AML): In Vitro Culture of AML Cells--The Present Use in Experimental Studies and the Possible Importance for Future Therapeutic Approaches
Stem Cells,
January 1, 2001;
19(1):
1 - 11.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
F. R. Appelbaum, J. M. Rowe, J. Radich, and J. E. Dick
Acute Myeloid Leukemia
Hematology,
January 1, 2001;
2001(1):
62 - 86.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Glimm, I.-H. Oh, and C. J. Eaves
Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0
Blood,
December 15, 2000;
96(13):
4185 - 4193.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Bruserud, G. Tjønnfjord, B. T. Gjertsen, B. Foss, and P. Ernst
New Strategies in the Treatment of Acute Myelogenous Leukemia: Mobilization and Transplantation of Autologous Peripheral Blood Stem Cells in Adult Patients
Stem Cells,
September 1, 2000;
18(5):
343 - 351.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
R. T. Costello, F. Mallet, B. Gaugler, D. Sainty, C. Arnoulet, J.-A. Gastaut, and D. Olive
Human Acute Myeloid Leukemia CD34+/CD38- Progenitor Cells Have Decreased Sensitivity to Chemotherapy and Fas-induced Apoptosis, Reduced Immunogenicity, and Impaired Dendritic Cell Transformation Capacities
Cancer Res.,
August 1, 2000;
60(16):
4403 - 4411.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
L. E. Ailles, B. Gerhard, H. Kawagoe, and D. E. Hogge
Growth Characteristics of Acute Myelogenous Leukemia Progenitors That Initiate Malignant Hematopoiesis in Nonobese Diabetic/Severe Combined Immunodeficient Mice
Blood,
September 1, 1999;
94(5):
1761 - 1772.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction