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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2406-2414
By
From the Department of Genetics, Research Institute, Hospital for
Sick Children; the Department of Molecular and Medical Genetics,
University of Toronto; the Department of Medicine, Princess Margaret
Hospital; the Department of Hematology/Oncology, the Toronto Hospital,
Toronto, Ontario; and the Terry Fox Laboratory, British Columbia Cancer
Agency, Vancouver, British Columbia, Canada.
We have previously shown that intravenously injected peripheral
blood (PB) or bone marrow (BM) cells from newly diagnosed chronic
myeloid leukemia (CML) patients can engraft the BM of sublethally
irradiated severe combined immunodeficient (SCID) mice. We now report
engraftment results for chronic phase CML cells in nonobese diabetic
(NOD)/SCID recipients which show the superiority of this latter model.
Transplantation of NOD/SCID mice with 7 to 10 × 107 patient PB or BM cells resulted in the continuing
presence of human cells in the BM of the mice for up to 7 months, and
primitive human CD34+ cells, including those detectable
as colony-forming cells (CFC), as long-term culture-initiating cells,
or by their coexpression of Thy-1, were found in a higher proportion of
the NOD/SCID recipients analyzed, and at higher levels than were seen
previously in SCID recipients. The human CFC and total human cells
present in the BM of the NOD/SCID mice transplanted with CML cells also
contained higher proportions of leukemic cells than were obtained in
the SCID model, and NOD/SCID mice could be repopulated with transplants of enriched CD34+ cells from patients with CML. These
results suggest that the NOD/SCID mouse may allow greater engraftment
and amplification of both normal and leukemic (Ph+)
cells sufficient for the quantitation and characterization of the
normal and leukemic stem cells present in patients with CML. In
addition, this model should make practical the investigation of
mechanisms underlying progression of the disease and the development of
more effective in vivo therapies.
CHRONIC MYELOID LEUKEMIA (CML) is a
clonal multilineage myeloproliferative disorder. It is now defined by
the presence in the clonal cells of a unique genetic abnormality. In
most cases, this is caused by a characteristic reciprocal exchange of
genetic material, manifested cytogenetically as the Philadelphia
chromosome (Ph),1 and involving the translocation of the
c-abl proto-oncogene from chromosome 9 to a new position on
chromosome 222 within the BCR-1 gene.3
This BCR-ABL fusion gene encodes a 210-kD chimeric protein
product which has increased tyrosine kinase activity compared with the
normal c-abl protein,4 and its presence in the
cytoplasm5 is also believed to be key to its transforming activity. CML follows a biphasic or triphasic clinical course: an
initial chronic phase characterized by the presence of a dominant clone
of BCR-ABL+/Ph+ cells which differentiate
normally, followed inevitably a few years later by a blastic phase
resembling acute leukemia.6 In some patients, the emergence
of the blastic phase may be preceded by clinical signs of disease
progression, referred to as an accelerated phase.
Much of our understanding of the biological changes underlying the
pathophysiology of chronic phase CML has come from in vitro studies of
the properties and behavior of lineage-restricted and multipotent
colony-forming cells (CFC),7 and more recently, long-term
culture-initiating cells (LTC-IC) present in the peripheral blood
(PB) and bone marrow (BM) of patients with CML.8 Early studies of long-term marrow cultures of cells from patients with CML
together with clinical experience demonstrating the ability of patients
to achieve cytogenetic remissions after treatment with high-dose
chemotherapy9,10 or interferon- Early attempts by other groups to establish an in vivo transplantation
model of chronic phase CML by using severe combined immunodeficient
(SCID) mice as recipients of human cells were not
successful.15,16 Even blast crisis CML cells, when injected into SCID mice intraperitoneally, under the renal capsule, or into
subcutaneously implanted human fetal bone fragments, were found to
disseminate poorly to the BM of the mice.15-17 More
recently, we reported that reproducible and sustained engraftment of
SCID mice with intravenously transplanted chronic phase cells could be
obtained if large enough numbers of cells from patients with high white
blood cell (WBC) counts were injected and the mice were conditioned by
a near-lethal dose of irradiation,18 based on our prior
success with this approach using transplants of normal human adult BM
or umbilical cord blood cells.19,20 However, we also found
that the level of human hematopoiesis obtained in SCID mice was
generally low, regardless of the source of the injected cells, and even
with very large transplants of CML cells, only a minority of the cells
later found to be present in the mice were leukemic. Subsequently we
discovered that nonobese diabetic (NOD)/SCID mice, which have
additional defects in natural killer (NK) cell activity as well as
defective macrophage and complement function,21 allow
superior engraftment of normal22-24 and
leukemic25 human hematopoietic cells. It therefore seemed
likely that these mice might also prove to be better recipients of CML
cells. We now describe an improved in vivo model for CML, using the
NOD/SCID mouse as a recipient. By comparison with the SCID model,
engraftment of NOD/SCID mice with multiple types of normal and leukemic
human cells was higher and could be achieved with lower numbers of
CD34+ cell-enriched populations. These experiments provide
a foundation for the future characterization of the phenotype and
properties of normal and Ph+ cells that have long-term in
vivo repopulating activity, as well as for the development of
strategies to selectively manipulate normal and Ph+
stem cell populations in vivo.
Patient cells.
BM and PB samples were obtained from patients with informed consent
according to procedures approved by the Human Experimentation Committee
at the Princess Margaret Hospital, Toronto; the Toronto Hospital; and
the Vancouver Health Sciences Hospital. All patients had
Ph+ CML and were in chronic phase at the time the sample
was taken (Table 1). Fresh PB or BM was
diluted 1:2 with Iscove's modified Dulbecco's medium (IMDM;
GIBCO-BRL, Burlington, Ontario, Canada) containing 10% fetal calf
serum (FCS) and enriched for mononuclear cells by Ficoll
density gradient centrifugation. CD34+ cells were then
selected from some of these samples by using QBEnd10, an antibody that
detects a class II CD34 epitope (generously provided by Dr Dinesh
Jacob, Quantum Biosystems, Cambridge, UK), and anti-mouse Ig-coated
magnetic beads (Dynabeads M-450, Dynal, Great Neck, NY) as described
previously.26-28 CD34+ cells were detached from
the beads with Pasteurella haemolytica glycoprotease
(Cedarlane, Hornby, Ontario, Canada) under conditions that efficiently
removed class I and class II CD34 epitopes (as detected by using
QBEnd10-phycoerythrin) from 106 KGIa cells suspended in 50 µl of RPMI within 20 minutes at 37°C. The percentage of
CD34+ cells in the glycoprotease-selected fraction was
determined by flow cytometry using antibodies to
glycoprotease-resistant epitopes of CD34 and CD45, and light scatter,
as described.29 In some experiments, unseparated or
CD34-enriched cells were suspended in 10% dimethyl sulfoxide, frozen
at
Analysis of mice.
NOD/SCID mice were bred and maintained in the defined flora animal
colony at the Ontario Cancer Institute, Toronto. Unless otherwise
stated, 7.5 to 10 × 107 light density cells or various
numbers of enriched CD34+ cells were injected into the tail
vein of sublethally irradiated (400 cGy using a 137Cs
BCR Southern analyses.
DNA was extracted from cells obtained from the BM of transplanted mice,
digested, and separated by gel electrophoresis. Southern blot analysis
was then performed using a BglII/HindIII probe that contains the human BCR exon 1.31 The relative
intensities of the germline and rearranged bands were determined by
scanning the developed film on a Computing Densitometer (Model 300A;
Molecular Dynamics, Sunnyvale, CA) followed by analysis
using ImageQuant software (Version 3.3; Molecular Dynamics). The
percentage of leukemic cells was calculated assuming that normal cells
contribute two copies to the germline band and leukemic cells
contribute one copy each to the germline and rearranged bands. The
limit of detection for this assay is approximately 0.1 µg of DNA.
Statistical analysis.
Comparisons of the level of engraftment in NOD/SCID versus SCID mice
were performed using the Mann-Whitney Rank Sum Test (SigmaStat version
1.0; Jandel Software; Labtronics, Inc, Guelph, Ontario, Canada). Results are expressed as mean ± SEM.
Engraftment of NOD/SCID mice by BM and PB cells from patients with
chronic phase CML.
Fresh or previously frozen light density cells from the BM or PB of 11 patients with chronic phase CML (Table 1) were transplanted by tail
vein injection into sublethally irradiated NOD/SCID mice. The extent of
human cell engraftment in the BM of these mice was determined by
Southern blotting using a human-specific DNA probe, or by flow
cytometric detection of cells expressing the human-specific markers
CD45 and CD71. DNA results from one experiment are shown in Fig
1A. In several of the mice in this
experiment, more than 50% of all the cells in the BM were human,
although there was considerable variability in the level of
engraftment, even among mice treated identically. The three lanes
marked by an asterisk indicate mice that were killed more than 5 months
after transplantation, showing the durability of the engraftment. Fig
1B shows the proportion of human cells detected in the BM of all 65 mice transplanted with chronic phase cells that were analyzed. In 56 of
these (86%), at least 1% of the cells in the BM were human, and in 31 (48%), at least 10% of the cells were human, including two mice
transplanted with only 8 × 106 PB cells from patient 5. Very high levels of human cells (40% to 80%) could be detected in 16 of the 65 mice (25%) for up to 7 weeks posttransplant. All 9 of 9 mice
that were analyzed between 3 and 6.5 months posttransplant were
engrafted and 6 of these mice contained between 1% to 10% human
cells, indicating that the graft persists for a long period of time.
Human cell engraftment (>1% human cells) was observed in mice
transplanted with cells from 10 of the 11 patients. The degree of
engraftment tended to be higher after transplantation of the same
number of PB as compared with BM cells. (The BM of all 39 mice
transplanted with CML PB cells contained at least 1% human cells,
whereas this was true for only 18 of 26 mice (69%) transplanted with
CML BM cells.) Treatment of transplanted mice with various human
cytokines on alternate days did not obviously enhance the level of
human cell engraftment (data not shown).
Presence of primitive human hematopoietic cells in the BM of
engrafted mice.
To determine whether primitive human hematopoietic cells could be
detected in the transplanted mice, BM cells from 33 engrafted mice (1%
to 80% human cells) were examined by flow cytometry for expression of
human Thy-1 and/or CD34. All but one of the mice were analyzed
between 2 and 7 weeks; one mouse was sacrificed after 6.5 months. The
example shown in Fig 2 is from a mouse that was sacrificed 4 weeks posttransplant; 49% of the viable
(PI
Both normal and leukemic cells engraft the BM of NOD/SCID mice
transplanted with CML cells.
We previously found that the majority of human progenitors present in
the BM of SCID mice that have been engrafted with PB or BM cells from
patients with chronic phase CML were not leukemic.18 A
similar analysis was therefore undertaken in the present study. From a
total of 17 mice, 109 colonies were obtained and analyzed cytogenetically and 69 of these (71% ± 8%, Table
2) were found to contain the Ph chromosome.
However, there was significant inter-mouse variability in the
proportion of human progenitors present that were leukemic. In the one
mouse where metaphases could be obtained from human LTC-IC-derived
colonies, 15 of 17 were normal but the other 2 were Ph+
(Table 2).
Populations enriched in CD34+ cells from patients
with chronic phase CML can engraft NOD/SCID mice.
To assess whether CD34+ cells could engraft NOD/SCID mice,
we transplanted mice with 105 to 2 × 106
fresh or thawed populations of enriched CD34+ cells from
three chronic phase patients (purity, 63% to 90%). Cells from 6 of
these mice were analyzed between 2 and 3 weeks because of morbidity.
The rest were analyzed after 5 weeks, including 2 mice that were killed
after 19 weeks. Fig 4A shows a Southern blot analysis of 7 mice transplanted with enriched CD34+
cells from one patient. In total, 13 of the 15 mice analyzed had
detectable human cells in their BM by Southern blot, at levels ranging
from less than 0.1% to 70%. The 2 mice with no detectable engraftment
had received the lowest dose of enriched CD34+ cells
(105). Two mice that received thawed cells had
sufficiently high levels of engraftment with human cells to be assessed
by BCR Southern analysis. In these, the percentage of leukemic cells
was found to be 90% and 99% (Fig 4B).
Engraftment of CML cells is superior in NOD/SCID mice compared with
SCID mice.
To compare the level of human cell engraftment obtained using NOD/SCID
and SCID mice as recipients, the levels of human cell engraftment in
the 65 NOD/SCID mice transplanted from 11 donors (data from Fig 1) were
compared with those previously observed in 66 SCID mice transplanted
from 10 donors using the same transplantation system.18 In
each study, all patients were newly diagnosed and untreated (with the
few exceptions noted). Engraftment in NOD/SCID mice was significantly
higher than in SCID mice, both when data from all of the transplanted
mice were pooled (5% ± 1% v 1% ± 1%, P < .0001) and when the mean levels of engraftment from
individual patients were compared (12% ± 3% v
2% ± 1%, P = .015; Fig 5).For a more direct comparison, cells from an additional patient with chronic phase CML were transplanted into mice of both strains. One
NOD/SCID mouse transplanted with 10 × 107 unseparated BM
cells was killed after 18 days because it appeared to be sick. Very
high numbers of human myeloerythroid CFC were found in the murine BM,
as well as a small number of multilineage CFC. Human CD34+
cells and LTC-IC were also detected. A second NOD/SCID mouse transplanted with 1.5 × 106 enriched CD34+
cells from the same donor and killed after 14 days had similarly high
numbers of human CFC in the BM. Flow cytometric and LTC studies were
not performed in this mouse. In contrast, two SCID mice transplanted with 10 × 107 unseparated cells remained healthy and had
30- to 60-fold fewer human CFC in the BM after 6 weeks. No human
CD34+ cells were detectable. The vast majority (>90%) of
human progenitors present in the BM of both NOD/SCID mice were
Ph+ by cytogenetic analysis.
In this study we describe an experimental in vivo model of human CML
which involves the intravenous injection of patient PB or BM cells into
sublethally irradiated NOD/SCID mice. The BM of these mice was
routinely found to contain at least 1% human cells for up to 6.5 months after transplantation of 108 light density cells
from 10 of the 11 high WBC count chronic phase patients studied.
Although the highest levels of human cells (up to 80%) were detected
within 7 weeks posttransplant, the detection of human cells at the 1%
to 10% levels for up to 6.5 months indicates the graft persists for
long periods of time. Similar engraftment kinetics have recently been
reported for normal human BM.23 CML PB cells engrafted as
well as, or better than, BM cells, as was noted for similarly
transplanted SCID mice.18 Although marked variability
(>10-fold) in levels of engraftment were again seen, both between
individual recipients of the same cells and between recipients of cells
from different patients, the levels of engraftment seen here in
NOD/SCID recipients were much higher than those noted previously using
SCID hosts.18 For example, 25% of NOD/SCID recipients had
40% to 80% human cells, whereas only 3% of SCID recipients contained
similarly high levels. Intermediate stages of human hematopoietic cell
development detectable as in vitro CFC were found in a similar
proportion of NOD/SCID and SCID recipients of CML cells (82% v
77%), but more primitive cell types were found more frequently in the
NOD/SCID mice (LTC-IC, 52% v 13%; CD34+ cells,
100% v 44%; CD34+Thy-1+ cells, 48%
v 0%).
Submitted July 17, 1997;
accepted November 10, 1997.
We thank I. McNiece (Amgen), D. Williams (Immunex), and Novartis for
providing cytokines; StemCell Technologies (Vancouver, BC) for media;
P. Lansdorp for antibodies; N. Jamal, H. Messner, M. Baker, D. Roy, and
M. Barnett for providing patient samples; and members of the Dick lab
for critically reviewing the manuscript.
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|
Abstract
Introduction
Abstract
11 showed the
common persistence of a suppressed but functionally intact reservoir of
normal (Ph
) stem cells despite dominance of the
Ph+ clone in the CFC and more mature compartments of PB and
BM cells. Ph+ LTC-IC have also been shown, but conditions
that would allow the sustained growth of leukemic cells from either the
PB or BM of patients with CML have not yet been
identified.
70°C, stored in liquid nitrogen until required, and then thawed
before transplantation into mice.
-irradiator) 8-week-old NOD/SCID mice. Some mice also received various combinations of intraperitoneally injected human cytokines on
alternate days as indicated. Cytokines used included mast cell growth
factor (MGF, 10 µg), PIXY321 (7 µg), interleukin-3 (IL-3, 6 µg),
granulocyte-macrophage colony-stimulating factor (GM-CSF, 6 µg),
G-CSF (6 µg), and Flt-3 ligand (FL, 6 to 10 µg) (MGF, PIXY, and FL
from Immunex; IL-3, GM-CSF, and G-CSF from Amgen; for the last two
experiments, stem cell factor from Amgen was used instead of MGF). Mice
were killed 1 to 7 months after transplantation, or as early as 2 weeks
posttransplant if they appeared to be sick. The cells present in eight
bones (ie, both femurs, tibiae, iliac crests, and humeri) were obtained
in IMDM containing 10% FCS. The proportion of all human cells in the
BM of transplanted mice was quantified by Southern blot analysis using
a human-specific probe as previously described,
and 
) or
PB (
and 
) cells from the 11 patients with chronic phase CML
studied, as determined by Southern blot analysis (
) Indicate geometric means.
BCR probe, was found to range from 8% to 100% (66% ± 7%,
mean ± SEM, n = 24 mice). A representative blot is shown in Fig
