|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2556-2570
Nonobese Diabetic/Severe Combined Immunodeficiency (NOD/SCID)
Mouse as a Model System to Study the Engraftment and Mobilization
of Human Peripheral Blood Stem Cells
By
Johannes C.M. van der Loo,
Helmut Hanenberg,
Ryan J. Cooper,
F.-Y. Luo,
Emmanuel N. Lazaridis, and
David A. Williams
From the Department of Pediatrics, Section of Hematology/Oncology,
Herman B Wells Center for Pediatric Research, the Stem Cell Laboratory,
Cancer Research Building, and the Division of Biostatistics,
Regenstrief Institute for Health Care, Indiana University School of
Medicine, Indianapolis, IN; and Howard Hughes Medical Institute,
Indiana University School of Medicine, Indianapolis, IN.
 |
ABSTRACT |
Mobilized CD34+ cells from human peripheral blood (PB)
are increasingly used for hematopoietic stem-cell transplantation.
However, the mechanisms involved in the mobilization of human
hematopoietic stem and progenitor cells are largely unknown. To study
the mobilization of human progenitor cells in an experimental animal
model in response to different treatment regimens, we injected
intravenously a total of 92 immunodeficient nonobese diabetic/severe
combined immunodeficiency (NOD/SCID) mice with various numbers of
granulocyte colony-stimulating factor (G-CSF) -mobilized
CD34+ PB cells (ranging from 2 to 50 × 106
cells per animal). Engraftment of human cells was detectable for up to
6.5 months after transplantation and, depending on the number of cells
injected, reached as high as 96% in the bone marrow (BM), displaying
an organ-specific maturation pattern of T- and B-lymphoid and myeloid
cells. Among the different mobilization regimens tested, human
clonogenic cells could be mobilized from the BM into the PB (P
= .019) with a high or low dose of human G-CSF, alone or in
combination with human stem-cell factor (SCF), with an average increase
of 4.6-fold over control. Therefore, xenotransplantation of human cells
in NOD/SCID mice will provide a basis to further study the mechanisms
of mobilization and the biology of the mobilized primitive human
hematopoietic cell.
| |
INTRODUCTION |
MOBILIZED HUMAN peripheral blood (PB)
CD34+ cells are increasingly used in clinical protocols as
a source of hematopoietic stem cells for allogeneic and autologous
transplantation.1, 2 More recently, this alternative source
of stem cells is being used as a target for genetic modification in
somatic cell gene therapy trials.3 Compared with bone
marrow (BM), the use of PB stem cells in patients undergoing autologous
transplantation has resulted in a significantly accelerated
engraftment.4-7 However, the mechanisms involved in the
mobilization of these cells, and the quality and biological
characteristics of the stem-cell subsets in the PB products are largely
unknown.8 Other clinically important issues include the
safety of the mobilizing regimens, the timing of mobilization and
collection, the amount of stem and progenitor cells that can be
harvested, and, ultimately, the adequacy of the product with respect to
the speed, level, and stability of engraftment. Moreover, with the
emergence of new candidate molecules for the mobilization of primitive
hematopoietic cells, such as interleukin-8, macrophage inhibitory
protein (MIP)-1 , and Flt3 ligand (as recently reviewed by To et
al8), there is a great need for an animal model in which
the mobilization of human cells can be studied in vivo.
Mouse hematopoietic stem cells have functionally been defined as cells
that have the ability to engraft upon transplantation, proliferate, and
sustain multilineage hematopoiesis in vivo for an extended period. In
contrast, primitive human hematopoietic cells have only been
systematically studied using clonogenic assays or long-term BM
cultures.9,10 These assays have proven to be valuable in
estimating the quality and frequency of human hematopoietic cells with
extensive proliferative capacity. However, evidence from gene-marking
studies in human clinical trials suggests that primitive hematopoietic
cells cannot be adequately studied using in vitro assays, as no
correlation could be found between the expression of transgenes in
vitro and after transplantation in vivo.11-14 Similarly,
when comparing retroviral gene transfer in human long-term
culture-initiating cells (LTC-IC) and cells that are capable of
engrafting immunodeficient mice, LTC-IC but not in vivo repopulating
cells could be readily transduced.15 Although some
investigators have reported significant overlap between LTC-IC and
cells that give rise to engraftment in immunodeficient
mice,16 others have described these subsets to be
biologically distinct; differing in frequency,17
phenotype,18 and the ability to be maintained on human BM
stromal cells in vitro.19
To study the engraftment and differentiation of primitive human
hematopoietic cells in vivo, various models of immunodeficient mice
have been developed (see Wermann et al20 for a recent
review). These models include the classical C.B-17 scid/scid severe
combined immunodeficiency (SCID) mouse,21 the beige athymic
nude X-linked (bnx) immunodeficient mouse,22 the humanized
SCID (SCID-hu) mouse, in which human (fetal) hematopoietic tissues such
as liver, thymus, and bone fragments, are surgically
transplanted,23-25 and a transgenic SCID mouse that
expresses the genes for human IL-3, granulocyte-macrophage
colony-stimulating factor (GM-CSF), and stem-cell factor
(SCF).26 Recently, a new mouse strain
(NOD/LtSz-scid/scid, hereafter referred to as NOD/SCID) was
developed by crossing SCID mice with nonobese diabetic (NOD/Lt)
mice.27 It has been reported that the engraftment level of
human cells in the NOD/SCID model, after transplantation of
splenocytes,28 PB blood mononuclear cells,29
BM, or cord blood cells,15,26,27,30-32 was fivefold to
10-fold higher than could be achieved in the classical SCID mouse.
In the present paper we used the NOD/SCID mouse to provide a detailed
study of the engraftment and maturation of human mobilized PB
CD34+ cells in the mouse and to investigate whether
engrafted animals can be used as a model to study the mobilization of
human hematopoietic progenitor cells in vivo. Our results show that
NOD/SCID mice can be highly engrafted by PB CD34+ cells
with differentiation along multiple lineages in a
microenvironment-specific fashion, without supplemental human
cytokines. In addition, using various regimens that have been
previously shown to induce the mobilization of clonogenic cells in
humans or primates, our data show for the first time that human
progenitor cells can be mobilized from the BM of the NOD/SCID
transplanted with PB stem cells by treatment of the animals for 4 to 6 days with G-CSF, or G-CSF and SCF. This model will enable future
studies directed at investigating the biological mechanisms that
modulate the mobilization of human progenitor cells in vivo.
| |
MATERIALS AND METHODS |
Animals.
A breeding colony of NOD/LtSz-scid/scid (NOD/SCID)
mice27 was established at the Laboratory Animal Research
Center at the Indiana University School of Medicine (Indianapolis, IN)
from breeding pairs kindly provided by Dr Leonard D. Shultz (The Jackson Laboratory, Bar Harbor, ME). Animals were housed in
a positive airflow ventilated rack (Lab Products, Maywood, NJ)
and bred and maintained in microisolators under specific pathogen-free
conditions. Animals were tested for the absence of mouse (CD4 + CD3+) T cells in the PB. Typically, T cells were
undetectable (<1%, in lymphocyte light-scatter gate) in 50% to 60%
of the animals (12-16 weeks of age). About 30% to 40% of the mice had
a low level of circulating T cells (1%-5%), whereas 10% of the mice
contained 14% ± 6% T cells. Before transplantation, 10- to
15-week-old male or female NOD/SCID mice received a sublethal dose of
300 cGy total-body irradiation (TBI) at 86 cGy/minute using a GammaCell
40 (Nordion International Inc, Ontario, Canada) equipped with two
opposing 137Cesium sources. Radiation-associated mortality
was not observed at this dose. In one specific experiment, animals were
splenectomized 4 weeks before transplantation. All animal experiments
were performed in accordance with institutional guidelines approved by
the Animal Care Committee of the Indiana University School of Medicine.
Transplantation of human cells.
Using protocols approved by the Institutional Review Board of the
Indiana University School of Medicine, healthy adult human volunteers
were treated for 5 days with human granulocyte colony-stimulating factor (hG-CSF) (Filgrastim, Neupogen; Amgen, Thousand Oaks, CA; 10 µg/kg/d, subcutaneously). White blood cells were collected by
apheresis and CD34+ cells were isolated by immunomagnetic
methods using the Isolex 300i cell selection device (Baxter
Immunotherapy, Irvine, CA). For these experiments, cell selection kits
and disposables were kindly provided by Baxter Immunotherapy. The
average purity of the CD34+ cells was 89% ± 7% with an overall recovery of 53% ± 19% from a total of 18 apheresis products containing 3.9 ± 1.7 × 1010
nucleated cells (mean ± standard deviation [SD]). After
separation, CD34+ cells were washed twice and injected into
the lateral tail vein of preirradiated NOD/SCID mice at 2 to 50 × 106 cells/animal in 0.5 mL Hanks' Balanced Salt Solution
(HBSS), 25 mmol/L HEPES, 20 U/mL heparin. In one specific experiment
CD34 cells were collected, irradiated with 20 Gy,
depleted of adherent cells by plastic-adherence for 30 minutes at
37°C, and injected intravenously (25 × 106
cells/animal) 4 hours before injection with CD34+ cells.
Flow cytometric analysis.
At 6 to 8 weeks after transplantation, tissues were harvested for
analysis. Blood (0.5 mL-1.0 mL) was collected from the subclavian vessels after the animals were anaesthetized with tri-bromo-ethyl alcohol (Sigma, St Louis, MO) and injected intravenously
with 20 U of heparin sodium (Fujisawa USA, Deerfield, IL). Erythrocytes were depleted from the blood by a 2-minute incubation in 155 mmol/L NH4Cl, 10 mmol/L KHCO3, 0.1 mmol/L EDTA at
4°C. In addition, single-cell suspensions were prepared from the
BM, spleen, and thymus. For flow cytometric analysis, cells were
preincubated for 30 minutes at 4°C in phosphate-buffered saline
(PBS) containing 0.1% (wt/vol) bovine serum albumin (BSA), 10% mouse
serum (Caltag, South San Francisco, CA), and 10% rat serum (Caltag).
Cells were then incubated with monoclonal antibodies (MoAbs) [specific
or isotype controls] conjugated to either fluorescein isothiocyanate
(FITC), phycoerythrin (PE), or PerCP (at 0.5 µg/0.5-1.0 × 106 cells) for 30 minutes at 4°C, washed and analyzed
for three-color fluorescence on a FACScan flow cytometer (Becton
Dickinson, Mountain View, CA). Routinely, 40,000 events per sample were
collected. The lack of cross-reactivity of human-specific antibodies
with mouse cells was confirmed in every experiment by staining BM cells from a nontransplanted irradiated control animal. In addition, every
analysis included isotype control antibodies to assess the level of
background fluorescence. Engraftment of human cells was defined by the
presence of at least 1% nucleated cells showing expression of CD45
over the background. FITC- and PE-conjugated mouse (IgG1 and IgG2b) and
rat (IgG2a) isotype control antibodies were purchased from Caltag, and
PerCP-conjugated mouse IgG1 was obtained from Becton Dickinson. The
IgG2a rat anti-mouse CD18 (mCD18)-FITC antibody was purchased from
Caltag. All other antibodies were specific for human cell-surface
antigens. CD10-FITC, CD19-PE, and CD38-PE (all IgG1 isotype) were
purchased from Caltag; anti-human IgM-FITC (IgG1) and HLA-DR-FITC
(IgG2b) were purchased from Pharmingen (San Diego, CA); goat anti-human
IgD-PE was purchased from Southern Biotechnology (Birmingham, AL);
Kappa-FITC and Lambda-FITC were bought from Kallestad (Redmond, WA);
CD11b-FITC (IgG2b), CD16-FITC, and Glycophorin A (Gly A)-PE (both IgG1)
were purchased from Immunotech (Westbrook, ME); and CD34-FITC,
CD8-FITC, CD4-PE, CD7-PE, CD33-PE, CD20-PerCP, CD3-PerCP, CD45-PerCP
(all IgG1 isotype), were bought from Becton Dickinson.
Mobilization.
In six individual experiments, groups of animals were treated with the
following mobilizing regimen: Cyclophosphamide (Cytoxan; Mead Johnson,
Princeton, NJ) at 200 mg/kg intraperitoneally followed 2 days later by
4 days of hG-CSF (Filgrastim Neupogen, Amgen) at 250 µg/kg/d
subcutaneously33; hG-CSF at 25 µg/kg/d subcutaneously or
250 µg/kg/d subcutaneously for 4 days34; or hG-CSF at 25 µg/kg/d subcutaneously or 250 µg/kg/d subbcutaneously together with
pegylated-human stem-cell factor (PEG-hSCF; Amgen) at 20 µg/kg/d subcutaneously for 4 to 6 days.34,35 Animals were
individually weighed before injection. Cyclophosphamide was diluted in
PBS and administered once intraperitoneally, PEG-hSCF was administered
once-a-day subcutaneously in HBSS containing 25 mmol/L HEPES and 0.1%
BSA, and hG-CSF was admistered twice-a-day subcutaneously in the
morning and evening diluted in 0.9% NaCl with 5% fetal calf serum
(FCS) at pH 4.5 (to ensure stability of the protein). For analysis,
tissues and PB were harvested the day after the last injection.
Cultures.
PB and BM from transplanted animals were tested for the presence of
human progenitor cells in semi-solid tissue culture medium consisting
of Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island,
NY), 25% FCS, 10% human plasma, 2% Pen/Strep, 5 × 10-5 mol/L  -mercapto-ethanol, 11 ng/mL human IL-3
(Peprotech, Rocky Hill, NJ), 100 ng/mL hSCF (Amgen), 4 U/mL human
erythropoietin (Amgen), and 0.8% methylcellulose (Stem Cell
Technologies, Vancouver, BC, Canada). Triplicate plates with 3 × 104 to 3 × 105 cells/dish were cultured
for 14 days at 37°C in a humidified environment with 5%
CO2. The number of colonies was determined by microscope.
Histology.
In selected experiments, tissues from transplanted animals
and controls were fixed and kept in 8% formaldehyde, 15%
(wt/vol) sucrose in PBS. For histopathological examination, mouse
humeri were decalcified, embedded in paraffin, and sectioned. The
slides were stained with hematoxylin and eosin using standard
procedures.
View larger version (19K):
[in this window]
[in a new window]
View larger version (16K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
View larger version (17K):
[in this window]
[in a new window]
| Fig 1.
Engraftment of G-CSF-mobilized human peripheral blood
CD34+ cells in sublethally irradiated NOD/SCID mice. The
percentage of human cells in (A) BM, (B) spleen, and (C) PB was
determined by flow cytometry at 6 to 8 weeks after transplantation.
Open circles represent untreated animals, closed circles represent
animals treated with human G-CSF, triangles indicate animals treated
with G-CSF and SCF (see Materials and Methods for dosage). Number of
animals depicted in (A) through (C) is 81, 38, and 75, respectively.
(D) Mean level of bone marrow engraftment (± SD) in five individual
experiments spanning the range of the number of CD34+
cells injected. Number of animals: in experiments 1 and 2, n = 6; in
experiments 3 through 5, n = 4.
|
|
Statistical analysis.
Statistical variation in the text is indicated by the SD, whereas
differences between groups and data in figures are expressed as the
mean ± standard error of the mean (SEM). Differences between percentages were calculated using the Wilcoxon test, whereas
differences between other groups were compared using either a
Student's t-test or analysis of variance (ANOVA). The
frequency of the NOD/SCID-repopulating cell was estimated using a
generalized additive statistical model36 to describe the
relationships between the number of CD34+ cells injected,
the purity of the CD34+ cells, and engraftment.
Specifically, the logit transformed percentage of human cells was
modeled as the sum of smoothing spline and loess functions of the
number of input CD34+ and non-CD34+ cells,
using the SAS (SAS Institute, Cary, NC) statistical package. Finally,
differences in mobilization between growth-factor treated and control
groups were calculated based on a generalized linear model37 using SAS.
| |
RESULTS |
Conditioning of NOD/SCID mice.
The SCID mutation has been described to affect radiation-induced DNA
repair.38 In initial experiments we monitored the survival of 10- to 15-week old NOD/SCID mice after various doses of TBI. After a
dose of 450 cGy or higher all animals died between 1 and 2 weeks after
irradiation. At 350 cGy to 400 cGy a late mortality (between 35 and 50 days after irradiation) was observed in about 20% of the animals,
whereas after 300 cGy all animals survived for at least 8 weeks. In
addition, in pilot studies we transplanted groups of 300 cGy, 200 cGy,
and 100 cGy irradiated animals and one group of nonirradiated animals
(3 animals per group) with 12 × 106 human
G-CSF-mobilized PB CD34+ cells per mouse. At 6 weeks after
transplantation no human cells could be detected in the BM of the
nonirradiated animals, but chimerism in the 300 cGy, 200 cGy, and 100 cGy groups was 63.9%, 56.0%, and 36.5% (± 11.3%, pooled SD),
respectively. Based on these results we established 300 cGy as a safe
and adequate radiation dose for our subsequent transplantation
experiments.
Engraftment of human hematopoietic cells in BM, spleen, and PB of
NOD/SCID.
To study the level of engraftment of human PB cells in various
hematopoietic organs of the NOD/SCID mouse, sublethally (300 cGy)
irradiated animals were intravenously injected with increasing numbers
of CD34+ cells. At 6 to 8 weeks after transplantation, PB,
BM, and spleen were harvested and analyzed by flow cytometry for the
presence of human cells (>1% of all nucleated cells) using
antibodies against human pan-leukocyte marker CD45 and mouse CD18
(Fig 1A, B, C). Open circles indicate untreated animals,
solid circles and triangles indicate animals that were treated for 4 to
6 days with G-CSF or G-CSF and SCF, respectively, which was part of a
mobilization study that will be described below. The level of
engraftment in BM and spleen (as determined by the percentage
CD45+ mCD18 cells among all nucleated cells)
showed a dose response with respect to the number of cells infused, and
reached as high as 95% in the BM and 90% in the spleen (Fig 1A and
1B). No statistically significant change in engraftment was observed
when animals were either preinjected with irradiated
CD34 cells (25 × 106
cells/animal), or were splenectomized 2 weeks before transplantation (Table 1). In contrast to the gradual
dose-dependent increase in engraftment in BM and spleen (Fig 1A and
1B), there appeared to be a higher level of human cells in the PB only
when high numbers of cells were infused (Fig 1C). More specifically,
the level of human cells in the PB in animals with less than 60% human
chimerism in the BM, stayed below 25%, whereas a high level of
chimerism could only be observed in highly (>80% in BM) engrafted
animals. In contrast to the overall variation in engraftment as
observed in Fig 1A, the engraftment between animals in individual
experiments (Fig 1D) has been shown to be highly reproducible.
View this table:
[in this window]
[in a new window]
|
Table 1.
Engraftment of CD34+ Cells in Animals
Preinjected With Irradiated CD34 Cells and in
Splenectomized Animals as Compared to Controls
|
|
To calculate the frequency of the NOD/SCID-repopulating cell
(SRC)17 in mobilized PB cells and to study the relationship between the number of human cells injected and level of engraftment in
various organs, the data in Fig 1A were analyzed using a generalized additive statistical model.36 Using a logit-transformation, we found a linear relationship between the engraftment in the BM
(expressed as logit-transformed percentage of human cells) and the log
of the number of CD34+ cells injected (P < .0001;
Fig 2A). The logit transformation of
percentages of human cells is used to transform the S-shaped curve
shown in Fig 1A to linearity, which is required for the statistical
model to assure a constant variance over the range of numbers of cells
injected. Based on these data, the frequency of the
NOD/SCID-repopulating cell (SRC) in mobilized PB cells could be
calculated, and was estimated to be one in 1.7 × 106
CD34+ cells, with a 95% confidence interval ranging from
0.9 to 3.1 × 106 CD34+ cells. The
calculation was based on the level of engraftment in animals that were
not treated with growth factors.
View larger version (19K):
[in this window]
[in a new window]
View larger version (53K):
[in this window]
[in a new window]
| Fig 2.
(A) Mean linear relationship between the natural log of
the number of CD34+ cells injected (in millions) and the
logit-transformed percentage of human cells in the BM (n = 63); and
(B) mean estimated relationship (in 50 animals) between the number of
CD34+ cells and CD34 cells injected
(linear scale, horizontal axes) and the logit-transformed percentage of
human cells in the BM (vertical axis). Data are based on a generalized
additive statistical model calculated by the SAS statistical package.
Both (A) and (B) only included animals that were not treated with
growth factors. In (B), not every combination of CD34+
and CD34 cells was available for analysis. The areas
outside the data range are indicated by zero engraftment in the
far-left and front-right corners of the graph. The graph was plotted
using the S-Plus (Mathsoft) statistics package.
|
|
In addition to the relationship between engraftment and the number of
CD34+ cells injected, the statistical model also showed a
linear relationship between engraftment and the number of coinjected
CD34 cells (P = .0027; not shown). This
contrasts with the data noted above on preinjection of irradiated
CD34 cells. The numbers of CD34+ and
CD34 cells were deducted from the purity of the
apheresis products used for transplantation in each individual
experiment. The estimated 3-dimensional response surface for the
relationship between the level of engraftment and the number of
CD34+ and CD34 cells injected, is
plotted in Fig 2B. The engraftment in the BM is shown on the vertical
axis (again expressed as the logit-transformed percentage of human
cells) as a function of the number of CD34+ and
CD34 cells injected per animal (on a linear scale on
the horizontal axes). The areas in the far-left and front-right corners
of the graph show no engraftment due to the absence of data as not
every combination of CD34+ and CD34
cells was available for analysis. The plot shows that the level of
engraftment in the BM correlates with both the number of
CD34+ and CD34 cells injected (P < .0001). Although the variables involved in the effect of
CD34 cells on engraftment are unknown, the plot does
show that these cells affect engraftment in the BM to a greater extent
when low numbers of CD34+ are injected.
In transplanted NOD/SCID mice, mature human hematopoietic cells, as
well as colony-forming cells (2091 ± 902 [SEM] colony-forming unit-granulocyte-macrophage [CFU-GM] per femur, 555 ± 397 erythroid burst forming unit [BFU-E] per femur) could be
detected as long as 6.5 months after transplantation. Specifically,
three animals were analyzed after 5 months, showing 60%, 63%, and
48% CD45+ cells in BM, whereas in another experiment three
mice were analyzed after 6.5 months, showing 21%, 33%, and 74%
CD45+ cells, respectively. No animals were found to be
negative for human cells. The extent of hematopoietic differentiation
found in these animals was similar to the differentiation described below. The longevity of hematopoietic chimerism, and the observed multilineage differentiation, are both indicative of the engraftment of
a primitive human hematopoietic cell.
One highly engrafted animal was chosen for histological examination
(Fig 3). Flow cytometric analysis showed
high levels of human (CD45+ mCD18) cells in
both BM (88%, Fig 3A) and PB (92%, Fig 3C), whereas engraftment in
the spleen was remarkably lower (15%, Fig 3B). Interestingly, the
humerus of this animal appeared pale as compared with the humerus of an
irradiated nontransplanted animal (Fig 3D). Confirming this observation
at a histological level, examination of the humerus showed the presence
of a solid mass of mostly undifferentiated cells and the apparent
absence of erythropoiesis (Fig 3F) as compared with an irradiated
nontransplanted control (Fig 3E). Despite the marked reduction in BM
erythropoiesis, the animal was not anemic (not shown) but did have an
enlarged spleen (with a cellularity of 17 × 106
cells; control spleens contained 7.4 × 106 cells). As
detected by flow cytometry, the spleen contained mouse as well as human
(glycophorin A+) erythrocytes. In the five animals studied
that had more than 80% human cells in the BM, the average spleen
cellularity was 2.3 ± 0.6-fold higher (P < .05) than in
control animals. In other human-mouse xenotransplants, the presence of
an enlarged spleen has been attributed to compensatory mouse
hematopoiesis39,40 due to the replacement of endogenous
hematopoiesis in the BM by the xenotransplant.
View larger version (22K):
[in this window]
[in a new window]
View larger version (75K):
[in this window]
[in a new window]
| Fig 3.
Flow cytometric analysis of (A) BM, (B) spleen, and (C)
PB of an animal transplanted with 25 × 106
G-CSF-mobilized human peripheral blood CD34+ cells. The
percentage of human cells (CD45+ and
mCD18) is indicated in the graphs. The spleen of this
animal was enlarged as compared with control (17 × 106 v 5.4 × 106 cells). In (D),
humerus of this animal (top) as compared with the humerus of an
irradiated nontransplanted control animal (bottom). In (E) and (F),
histology (hematoxylin and eosin stain) of the bone marrow of control
and transplanted animal, respectively.
|
|
Overall distribution and maturation of myeloid and B-lymphoid
lineages in the hematopoietic organs.
To investigate the capacity of human PB stem cells in NOD/SCID mice to
differentiate into multiple lineages, and to compare the
differentiation of human cells in different hematopoietic organs, cells
in BM, spleen, and PB of transplanted animals were tested for the
expression of the human myeloid-specific marker CD33 and B
lymphoid-specific marker CD19 by flow cytometry.41 The
distribution of CD33+ and CD19+ cells showed
that the majority of human cells in the BM (n = 50) and spleen (n = 10)
had differentiated into the B-lymphoid lineage
(Fig 4A). On the other hand, in the PB (n = 50) myeloid and lymphoid cells were equally represented. When comparing
BM and spleen, the percentage of myeloid cells in the BM (28.2 ± 16.4, mean ± SD, n = 50) was significantly higher (P = 0.3)
than in the spleen (16.0 ± 10.3, n = 10), indicating that
myelopoiesis preferentially took place in the BM. In animals that were
treated with either G-CSF or G-CSF and SCF in our mobilization study
(described below), the percentage of human myeloid cells in the BM had
increased to 39.1% ± 17.0% (n = 18). This is significantly higher
than the percentage in untreated animals (P = .02),
which indicated that the treatment had triggered human myelopoiesis. In
addition, in highly engrafted animals (with more than 70% human cells)
the percentage of myeloid cells in the BM (56.1% ± 9.2%
CD33+, n = 9) was significantly higher than in animals
engrafted at lower levels (32.8% ± 18.8% CD33+, n = 41; P< .001), suggesting that the graft itself contributed to
the microenvironment in supporting myelopoiesis. A more detailed flow
cytometric analysis of the cells engrafted in the BM, which was based
on the expression of CD33 and HLA-DR for myeloid cells and CD10 and
CD20 for B-lymphoid cells (Fig 4B), showed that the majority of both
myeloid and B-lymphoid cells had an immature phenotype. Although in the
myeloid lineage mature monocytes and granulocytes could be identified
based on the level of expression of CD33 and HLA-DR, maturation in the
B-lymphoid lineage in the BM appeared to be incomplete as judged by the
absence of the more mature CD20+ CD10
cells.
View larger version (21K):
[in this window]
[in a new window]
View larger version (21K):
[in this window]
[in a new window]
| Fig 4.
Analysis of myelopoiesis and B lymphopoiesis in
transplanted NOD/SCID. (A) Overall distribution of myeloid
(CD33+) and B-lymphoid (CD19+) lineages
among human cells in spleen (n = 10), bone marrow (n = 50), and
peripheral blood (n = 50). Differences between SPL and BM were
significant (P < .05), differences between SPL and
BM, and BM and PB were highly significant (P < .001). Bars
indicate mean ± SEM. (B) Relative distribution of various myeloid and
B-lymphoid subsets in the bone marrow of four representative animals
(containing 84.5%, 76.6%, 78.9%, and 73.5% CD45+
cells in the BM, respectively). Immature cells and monocytes both
express HLA-DR+, whereas monocytes also express high
levels of CD33. Granulocytes express CD33 but are negative for HLA-DR
(left panel). All B cells express CD19 of which the most immature B
cells lack both CD10 and CD20, with differentiation expression of CD10
is followed by expression of CD20 and, ultimately, loss of CD10 on the
mature B cell (right panel). Animals included in this analysis had not
been treated with a mobilizing regimen. Bars represent mean ± SEM (n
= 4).
|
|
Flow cytometric analysis of various lineages in bone marrow and
spleen.
A detailed flow cytometric analysis of the human hematopoietic cells in
BM and spleen of a representative animal is shown in
Fig 5A through E. The
differentiation of human B lymphocytes can be characterized by an
increase in expression of CD20 on CD10+ cells, followed by
a subsequent loss of CD10 from the cells expressing high levels of
CD20.41 We show that both BM and spleen contained immature
CD10+20 and
CD10+20+ cells, whereas mature
CD1020+ cells were present only in the
spleen (Fig 5A). Consistent with this observation, expression of IgM
and IgD heavy chain as well as kappa and lambda light-chain
immunoglobulins on CD19+ B cells was substantially greater
in the spleen than the BM (Fig 5B). In contrast, differentiation into
the granulocytic lineage could be observed in the BM but not in the
spleen. Granulocytes could be identified by the intermediate expression
of CD33 and low or medium expression of activation-marker HLA-DR (Fig
5C, circled subsets). This observation was confirmed by analyzing BM
and spleen for the expression of CD11b (alpha subunit of beta 2-integrin Mac-1) and CD16 (Fc gamma receptor IIIb), of which the level
of CD16 on CD11b+ cells increases with granulocyte
maturation (Fig 5D). Again, granulocytes could only be identified in
the BM but not in the spleen. In contrast, monocytes (as defined by a
high expression of CD33) could be found in both BM and spleen (Fig 5C),
although monocytes in the spleen were mostly nonactivated as indicated by the low expression of HLA-DR.42 CD34 could be detected
on human cells in both BM and spleen. On average, 14.2% ± 4.9%
(mean ± SD, n=39) of human cells in the BM expressed
CD34, whereas the percentage in the spleen was found to be
approximately threefold lower (not shown). Finally, human erythroid
cells could also be detected in both BM and spleen but only at a
relatively low level (ranging from 4% to less than 1% of all cells).
When gated for CD45 cells, a clear population of
human Glycophorin A (Gly A)-positive (mouse CD18)
cells could be identified in the BM (18% of gated cells) and the
spleen (3% of gated cells) (Fig 5E). The vast majority of Gly
A+ cells had a moderate to high forward light scatter (not
shown), supporting the notion that the in vivo maturation of erythroid progenitor cells into erythrocytes (which have a low forward light scatter) may have been impaired. Finally, a low level of platelet antigen (CD41 or CD61) could be detected in the BM, but was always associated with a high expression of CD33 (not shown), indicating the
possible presence of human platelets on the surface of monocytes. In
the PB, platelets could not be detected.
View larger version (32K):
[in this window]
[in a new window]
View larger version (15K):
[in this window]
[in a new window]
View larger version (33K):
[in this window]
[in a new window]
View larger version (27K):
[in this window]
[in a new window]
View larger version (26K):
[in this window]
[in a new window]
| Fig 5.
Detailed flow cytometric analysis of the various
hematopoietic lineages in bone marrow and spleen of engrafted NOD/SCID.
Plots are from a representative experiment in which animals were
transplanted with 15 × 106 CD34+ cells and
were analyzed after 6 to 8 weeks. (A) Distribution of B-cell markers
CD10 and CD20 on all nucleated cells. (B) Distribution of heavy and
light-chain cell-surface immunoglobulin on CD19+ human B
cells (mean ± SEM of n = 4 to n = 7). All differences between BM
and SPL were highly significant (P < .001). (C) Distribution
of HLA-DR and CD33 (circles indicate activated HLA-DR+
and resting HLA-DR granulocytes) among
CD45+ cells, (D) expression of CD11b and CD16 on
CD45+ cells, and (E) expression of human Glycophorin A
(Gly A) on CD45 nucleated and enucleated cells. 40,000 events per analysis were recorded.
|
|
Flow cytometric analysis of human cells in the thymus.
Human T lymphocytes, as defined by the expression of CD4 and CD3, could
never be detected in BM, spleen, or PB of engrafted animals. However,
human thymocytes could be detected in the thymus of highly engrafted
animals (Fig 6A and B). Statistical
analysis showed a positive correlation between the level of engraftment in the BM and the presence of human cells in the thymus (Fig 6A, left
panel; P < .05). Of the 13 animals analyzed, thymocytes could be detected in 5 out of 6 animals that had been engrafted in the BM
with at least 40% human cells. Interestingly, human thymocytes could
also be detected in the thymus of animals that had only 25% human
cells in the BM, but which had previously been treated for 6 days with
human G-CSF and PEG-SCF (as part of a mobilization experiment), whereas
the thymus of untreated animals contained virtually no human cells (Fig
6A, right panel). Phenotypic analysis of these thymocytes, as shown for
a representative animal in Fig 6B, showed a large percentage of
CD4+ CD8+ double-positive cells and cells that
coexpress CD8 and CD7. In addition, the majority of
CD4+8 single-positive cells expressed
high levels of CD3 (not shown), whereas some CD7+ cells
also expressed the activation marker CD69. In the engrafted thymus,
expression of CD33, CD20, or HLA-DR could not be detected. This
phenotype was similar in all animals examined and is consistent with
normal human T-cell lymphopoiesis.
View larger version (14K):
[in this window]
[in a new window]
View larger version (35K):
[in this window]
[in a new window]
| Fig 6.
Percentage and phenotype of human cells in the thymus of
transplanted NOD/SCID. (A, left panel) Relationship between the
percentage of human thymocytes in the thymus and level of engraftment
in the BM (n = 13). Animals in individual experiments are indicated
by the same symbol. (A, right panel) Mean engraftment in BM and thymus
(THY) of animals that were either untreated (n=3) or treated (n=4)
for 6 days with human G-CSF (250 µg/kg/d, subcutaneously) and
pegylated human SCF (20 µg/kg/d, subcutaneously). Bars represent mean ± SEM. Differences between untreated and G-CSF/SCF-treated animals in
the thymus were significant (P < .05) whereas chimerism in
the BM was not significantly different. (B) Phenotypic analysis of the
thymus of a representative animal. This animal was engrafted with human
PB CD34+ cells and was treated with G-CSF and SCF. The
left top panel shows the distribution of chimerism in various organs.
Cells were analyzed for the expression of the human T-cell markers CD3,
CD7, CD4, CD8, and activation marker CD69, or were incubated with
isotype control antibodies.
|
|
Mobilization of mouse progenitors in nontransplanted NOD/SCID.
To study mobilization, we first established that mouse progenitor cells
could be mobilized in a normal nontransplanted NOD/SCID. To test
whether preirradiation interfered with this ability we also included a
group of animals that had been sublethally irradiated 6-weeks before
treatment. Both nonirradiated and sublethally irradiated animals were
administered a single injection of cyclophosphamide (at 200 mg/kg
interperoneally) followed 2-days later by 4 days of G-CSF (at 250 µg/kg/d). This mobilization protocol has previously been used by
Neben et al33 to mobilize cells in C57BL/6 mice. Animals
were sacrificed on day 6 after cyclophosphamide treatment and
progenitor cells in both PB and BM were enumerated in methylcellulose cultures (Fig 7A). The results show that
mouse progenitor cells could be efficiently mobilized in unirradiated
as well as irradiated animals (1,120- and 300-fold increase in PB
progenitors, respectively). The difference between unirradiated and
irradiated animals was not statistically significant (P = .07).
As expected, the increase in progenitor cells was accompanied by an
increase in spleen weight (Fig 7B) and number of white cells in the
blood (Fig 7C). These results indicate that the NOD/SCID mouse allowed
murine progenitor cells to be mobilized into the periphery after an
appropriate stimulus, and that circulating progenitor cells were
present in sufficient numbers to be detected by standard colony assays
even though the animals had not been splenectomized.
View larger version (20K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
| Fig 7.
Mobilization of mouse progenitor cells in NOD/SCID. (A)
Progenitor cells in femur and blood of animals unirradiated or
preirradiated with 300 cGy, untreated or after treatment with
cyclophosphamide (200 mg/kg interperitoneally at day 6) followed by 4 days of G-CSF (250 µg/kg/d, subcutaneously). Bars indicate mean ± SEM (n = 2 per group). The difference between irradiated and
nonirradiated animals was not significant (P = .07). (Note:
left axis has a log scale, right axis has a linear scale.) (B) Spleen
weight and (C) white blood cell (WBC) counts of the same animals (mean ± SEM, n = 2). In nonirradiated animals, the increase in both
spleen weight and WBC counts is highly significant
(P < .01). Increase of WBC in irradiated animals as
compared with control is also highly significant (P < .01)
whereas spleen weight did not change.
|
|
Mobilization of human progenitors in transplanted NOD/SCID.
To detect mobilization of human progenitor cells we used a tissue
culture medium containing the human-specific growth factors SCF and
IL-3 plus Epo, that selectively supported the growth of human but
not mouse progenitor cells (data not shown). For mobilization, NOD/SCID
mice were treated with various regimens that have previously been shown
to mobilize primitive hematopoietic cells (see Materials and Methods).
Treatment was initiated 6 to 8 weeks after transplantation and PB and
BM cells were harvested the morning after the last treatment day. The
distribution of human cells among PB, spleen, and BM of engrafted
animals, in treated and untreated groups of five individual
experiments, is shown in Table 2.
Figure 8 (A through E) shows
the average percentage of human cells in the BM (left panel), and the
number of human progenitor cells detected in BM and PB (right panel,
log scale, and linear scale, respectively) of the same experiments. In
animals treated with G-CSF, the average number of human cells in the PB
was significantly increased as compared with control (P < .01 and P< .05, respectively; Table 2, B and C). In addition,
human progenitor cells were found to be mobilized in animals treated
with a low or high dose of G-CSF (Fig 8B and 8C, respectively), alone
or in combination with SCF (Fig 8D and 8E, respectively). More
specifically, the number of progenitor cells in the PB increased from
1.3-fold in an animal treated with a high-dose G-CSF and SCF (Fig 8E)
to 7.9-fold in two animals treated with a high dose of G-CSF (Fig 8C).
For statistical analysis a generalized linear model37 was
used to account for the volume of blood collected, the number of cells
injected per animal, the number of colonies counted, and whether or not
animals had been treated with hematopoietic growth factors. When
combining the results of individual experiments, an estimated 4.6-fold
increase in progenitors could be found in the PB of animals treated
with either G-CSF or G-CSF and SCF as compared with control
(Table 3; P = .019), with a 95%
confidence interval of (1.24, 17.0). The total number of nucleated
cells and number of mouse cells in the BM or PB of animals treated with
G-CSF or G-CSF and SCF had not changed (P > .05, not shown).
The mobilized PB progenitor cells, as well as the progenitor cells in
the BM of untreated and growth-factor treated animals, were
predominantly CFU-GM (92.0% ± 4.2% (SEM; n = 9); 84.1% ± 4.3% (n = 17); and 85.6% ± 7.1% (n = 15), respectively), with
only a minority being BFU-E (10%-20%) and less than 1% being
CFU-Mix. In contrast to the mobilization described above, no progenitor
cells could be detected in the PB of animals treated with
cyclophosphamide and G-CSF (Fig 8A; Table 3, group I). In addition to
the lack of mobilization, the average number of human cells in PB and
BM had decreased significantly (Table 2; experiment A). In summary, our
experiments show that human hematopoietic progenitor cells can be
induced to egress from the BM space into the PB after stimulation with
human G-CSF or a combination of human G-CSF and pegylated-SCF, albeit
with a low frequency.
View larger version (22K):
[in this window]
[in a new window]
View larger version (25K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
View larger version (24K):
[in this window]
[in a new window]
View larger version (20K):
[in this window]
[in a new window]
| Fig 8.
Mobilization of human hematopoietic progenitors.
Untransplanted and engrafted animals (at 6-8 weeks
post-transplantation) were treated with various regimens to mobilize
hematopoietic cells. In each case, the left hand panel shows the
percentage of human cells in the BM, whereas the right hand panel shows
the number of human progenitor cells per femur and per mL of peripheral
blood. Bars indicate mean ± SEM. (A) Control (n = 4) and treated (n
= 5) with a single injection of cyclophosphamide (200 mg/kg) followed
by 4 days of hG-CSF (250 µg/kg/d); (B) control (n = 3) and treated
(n = 3) with hG-CSF (25 µg/kg/d) for 4 days; (C) control (n = 2)
and treated (n = 3) with hG-CSF (250 µg/kg/d for 4 days); (D)
control (n = 2) and treated with hG-CSF (25 µg/kg/d) with or
without pegylated-hSCF (20 µg/kg/d) for 4 days (both n = 2); and
(E) control (n = 3) and treated (n = 4) with hG-CSF (250 µg/kg/d)
and pegylated-hSCF (20 µg/kg/d) for 6 days.
|
|
| |
DISCUSSION |
The mechanisms of mobilization of progenitor cells from the BM into the
blood are still poorly understood. A systematic study of the
physiologic or therapeutic stimuli that induce mobilization, as well as
the components that modulate the extent and timing of mobilization,
requires the development of an in vivo transplantation model. In the
present study we developed such a model by transplanting a total of 92 sublethally irradiated NOD/SCID mice with increasing numbers of
CD34+ cells harvested from adult G-CSF-mobilized healthy
donors. At 6 to 8 weeks after transplantation, the hematopoietic organs
were analyzed for the presence of differentiated human hematopoietic cells using multiparameter flow cytometry, and groups of engrafted animals were treated with various regimens to study the level of
mobilization of human hematopoietic progenitors into the circulation.
Using statistical modeling, a dose-response relationship could be shown
between the number of cells infused and the level of engraftment in BM
and spleen. In contrast, the level of human cells in the PB generally
stayed below 25%, except in highly engrafted animals, whereas
engraftment in the spleen was always lower than the engraftment in the
BM, indicating the complex interactions between the endogenous mouse
hematopoietic compartment and the xenotransplanted human cells. The
model also showed a positive correlation between the impurity of the
graft (number of CD34 cells injected) and the level
of engraftment in BM, spleen, and PB, suggesting the presence of an
NOD/SCID engraftment-facilitating cell among human-mobilized PB cells.
Interestingly, other investigators have proposed the existence of a
hematopoietic stem cell that lacks the expression of
CD34.43 Whether the positive effect of
CD34 cells on the engraftment in this model is
related to the presence of a facilitating cell or a true
CD34 engrafting cell remains to be investigated.
The frequency of the SRC was estimated to be one in 1.7 × 106 CD34+ cells, or approximately one in 2.2 × 108 total cells (based on an average of 0.76%
CD34+ cells among the nucleated cells in the apheresis
product). This result contrasts with a previously published estimate of
the frequency of the SRC in human-mobilized PB cells of one in 6 × 106 total cells.17 In these
experiments, the SRC frequency was estimated using Poisson statistics
by limiting dilution analysis, in which as little as 0.1% of human
cells could be detected by Southern hybridization. Apart from the
method used for the estimate, and the difference in sensitivity between
flow cytometry and Southern hybridization, a number of other factors
may have played a role in the difference between our estimate and the
previously reported SRC frequency. These may include: the health status
of the NOD/SCID colony; the radiation dose, 375 cGy to 400 cGy as
opposed to 300 cGy in our experiments, allowing higher levels of
engraftment; the addition of human SCF, IL-3, and GM-CSF in the
reported experiments whereas our animals did not receive additional
human cytokines; and finally, the coinjection of
CD34 cells, as our current data suggest that these
cells enhance engraftment. This illustrates that the reported frequency
should only be used for relative comparisons as the variables involved
in the engraftment of human hematopoietic cells in NOD/SCID mice have
not sufficiently been characterized.
As has previously been reported, the frequency of the SRC in
CD34+ cells purified from G-CSF-mobilized PB is much lower
than in umbilical cord blood.17,44 When our results are
compared with a study in which recipients of umbilical cord blood
CD34+ cells were conditioned with 350 cGy to 400 cGy,
approximately 50-fold less cord blood cells were needed to reach the
same level of engraftment in the BM.45 In NOD/SCID
mice, it has been shown that both the engraftment of umbilical
cord blood CD34+ cells, as well as mobilized PB
CD34+ cells, is independent of the addition of human growth
factors.40,45 The size of the graft necessary for the
engraftment of PB cells in NOD/SCID mice, as compared with that of
umbilical cord blood, may be caused by an intrinsic difference in SRC
frequency. Alternatively, this may be due to a difference in the
biological properties of CD34+ cells derived from fetal or
adult hematopoietic tissue.
The engraftment pattern of PB CD34+ cells in NOD/SCID mice
was multilineage and could be observed for at least 6.5 months after transplantation. When comparing the different hematopoietic organs, distinct differences were observed in the differentiation and maturation of human hematopoietic cells. Human myelopoiesis was favored
in the BM, in which different maturation stages of granulopoiesis could
be identified by flow cytometry. In contrast, whereas resting human
monocytes could be detected in the spleen, granulocytes appeared to
be absent. As has been reported earlier,44,45 our data show
that the microenvironment of BM and spleen in NOD/SCID mice differ
in their support of human B-cell maturation. Although immature B cells
were present in large quantities in the BM, human B-cell maturation, as
defined by the loss of CD10 expression on CD19+CD20+ and expression of cell-surface
immunoglobulin (IgM, IgD, kappa and lambda), took place primarily in
the spleen. Finally, our data confirm a previous report44
that the BM of the NOD/SCID was the preferred site of human
hematopoiesis because it contained the highest number of
CD34+ cells as well as the highest number of in vitro
clonable myelo-erythroid progenitors (J.C.M.v.d.L. and D.A.W.,
unpublished data, January 1998).
The engraftment of human T cells in the thymus of SCID or NOD/SCID mice
transplanted with umbilical cord blood CD34+ cells has been
reported to be inconsistent.31,45 In the present study we
have found a dose-response relationship between the number of cells
transplanted and the presence of human thymocytes. Because human
thymocytes could only be found in moderately to highly engrafted animals, we speculate that the thymus was seeded with a T-cell precursor from the BM. This may be a rare event, explaining the lack of
thymocytes in animals showing lower levels of human engraftment. On the other hand, the data may also suggest a defect in the
recruitment and mobilization of human T-cell precursors from the BM or
a defect in the seeding of the murine thymus. It has been shown that
human T-cell precursors could egress from the BM of SCID mice
transplanted with human fetal BM or fetal liver cells, migrate to the
implanted human thymus, and give rise to mature T cells that populated
the PB.46
In our mobilization studies, we could also detect human thymocytes in
animals that were engrafted at low levels but only if animals had been
treated with G-CSF and SCF. This finding supports the notion of
mobilization of a T-cell precursor from the BM that might have been
induced by G-CSF or SCF. Alternatively, treatment with G-CSF or SCF
could also have stimulated a dormant T-cell precursor that had seeded
the thymus directly at the time of transplantation as SCF is a growth
factor that has been described to play an important role in the
expansion of primitive thymocytes in vivo.47 As has been
elegantly shown by gene marking of transplanted cells in bnx
mice,48 and in SCID mice implanted with a human
thymus,46 a shared retroviral integration pattern in both
lymphocytic and myelocytic lineages would ultimately provide proof of a
multilineage precursor in this model. Our results are in agreement with
the finding that human thymocytes can be generated in the mouse thymus, as was shown in vitro using fetal thymus organ cultures seeded with
human BM or umbilical cord blood cells.49 The fact that no
human T cells could be detected in either the BM, spleen, or PB
suggests that there may be a defect in either the maturation of human T
cells grown in the xenograft microenvironment in vivo, or in their
ability to egress from the thymus.
In both patients and experimental animals it has been shown that
progenitor cells as well as primitive hematopoietic cells can be
induced to mobilize from the BM into the blood stream by a variety of
different stimuli (see review8). These stimuli include
treatment with cytostatics, such as cyclophosphamide, with or without a
subsequent course of hematopoietic growth factors33;
hematopoietic growth factors alone or in combination, of which G-CSF or
a combination of G-CSF and SCF have been most widely studied1,35,50-52; and infusion of antibodies that
interfere with the binding of hematopoietic cells to either the
endothelium or components of the BM extracellular matrix.53
However, despite the potential for clinical application, the mechanisms
involved in the mobilization of hematopoietic cells are still poorly
understood. Some stimuli, such as IL-8, have been reported to mobilize
cells within 30 minutes wheras G-CSF needs to be administered for
several days.8 Moreover, the emergence of new regulators of
hematopoiesis such as chemokines underscore the need for a model in
which mobilization of human hematopoietic cells can be studied. In this
study we present such a model and show that human hematopoietic cells
can be induced to mobilize from the BM into the PB.
We have investigated several methods that have been reported to induce
mobilization by presumably distinct mechanisms. Engrafted NOD/SCID mice
were treated with either cyclophosphamide followed by high doses of
human G-CSF,33 or G-CSF alone or G-CSF in combination with
human (pegylated) SCF. Our results show for the first time that human
progenitor cells can be mobilized from the BM of the NOD/SCID mouse
into the PB after treatment with G-CSF or G-CSF and SCF, albeit at a
low level. These results, and the generally low level of circulating
human cells in the blood of nonmobilized animals, both seem to indicate
that human cells do not egress normally from the BM into the blood
stream. The reasons for this may be related to the lack of the
appropriate human stimuli in the microenvironment, or the inability of
human hematopoietic cells to physically negotiate the mouse endothelial
and adventitial cell barrier.54
In summary, we have shown that human G-CSF-mobilized PB stem cells are
able to induce long-term engraftment of NOD/SCID mice without
exogenously added human cytokines. Together with previously published
data,44,45 our observations show that the engraftment of
human cells in NOD/SCID mice recapitulates the anatomical distribution of normal hematopoiesis in humans as well as in mice. Our results show
that human G-CSF mobilized PB CD34+ cells can reliably
engraft sublethally irradiated NOD/SCID mice, home to the appropriate
sites of hematopoiesis, differentiate into multiple lineages, and
mature in specific hematopoietic organs. In engrafted animals, all
human hematopoietic lineages were represented, including cells of the
T-cell lineage, and the data show that the mouse hematopoietic
microenvironment played an important role in the maturation of specific
lineages. In addition, we report for the first time that human
hematopoietic cells can be induced to mobilize from the BM into the PB
of NOD/SCID mice transplanted with human PB CD34+ cells.
Therefore, we postulate that the xenotransplanted NOD/SCID mouse may be
an excellent model to further study the mechanisms involved in the
mobilization of human hematopoietic cells.
| |
FOOTNOTES |
Submitted March 5, 1998;
accepted June 2, 1998.
The Wells Center for Pediatric Research is a Center of Excellence in
Molecular Hematology funded by the National Institute of Diabetes and
Digestive and Kidney Diseases (P50 DK 49218) which supports the
NOD/SCID colony and the Stem Cell laboratory.
Address correspondence to David A. Williams, MD, Herman B Wells Center
for Pediatric Research, James Whitcomb Riley Hospital for Children,
Cancer Research Building, 1044 W Walnut St, Rm 406C, Indianapolis, IN
46202; email: dwilliam{at}iupui.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank the members of the Stem Cell Laboratory of Indiana
University Medical School for the processing, purification, and
analyses of the apheresis products and Dr Attilio Orazi for processing
and reviewing the histology. We acknowledge Dr Leonard D. Shultz for
providing the founders of our NOD/LtSz-scid/scid colony, Amgen
for the gift of pegylated human SCF, and Baxter Immunotherapy (Irvine,
CA) for providing us with preclinical Isolex immunomagnetic
CD34-selection kits. In addition, we thank Dr John E. Dick and the
members of the Wells Center for critically reviewing the manuscript.
| |
REFERENCES |
1.
Duhrsen U,
Villeval JL,
Boyd J,
Kannourakis G,
Morstyn G,
Metcalf D:
Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients.
Blood
72:2074,
1988[Abstract/Free Full Text]
2.
Anderlini P,
Korbling M:
The use of mobilized peripheral blood stem cells from normal donors for allografting.
Stem Cells
15:9,
1997[Abstract/Free Full Text]
3.
Kohn DB:
Gene therapy for haematopoietic and lymphoid disorders.
Clin Exp Immunol
107:54,
1997
4.
Sheridan WP,
Begley CG,
To LB,
Grigg A,
Szer J,
Maher D,
Green MD,
Rowlings PA,
McGrath KM,
Cebon J,
Dyson P,
Watson D,
Bayly J,
de Luca E,
Tomita D,
Hoffman E,
Morstyn G,
Juttner CA,
Fox RM:
Phase II study of autologous filgrastim (G-CSF)-mobilized peripheral blood progenitor cells to restore hemopoiesis after high-dose chemotherapy for lymphoid malignancies [published erratum appears in Bone Marrow Transplant 15:654, 1995].
Bone Marrow Transplant
14:105,
1994[Medline]
[Order article via Infotrieve]
5.
Bensinger W,
Singer J,
Appelbaum F,
Lilleby K,
Longin K,
Rowley S,
Clarke E,
Clift R,
Hansen J,
Shields T,
Storb R,
Weaver C,
Weiden P,
Buckner CD:
Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor.
Blood
81:3158,
1993[Abstract/Free Full Text]
6.
Chao NJ,
Schriber JR,
Grimes K,
Long GD,
Negrin RS,
Raimondi CM,
Horning SJ,
Brown SL,
Miller L,
Blume KG:
Granulocyte colony-stimulating factor "mobilized" peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy.
Blood
81:2031,
1993[Abstract/Free Full Text]
7.
Grigg A,
Begley CG,
Juttner CA,
Szer J,
To LB,
Maher D,
McGrath KM,
Morstyn G,
Fox RM,
Sheridan WP:
Effect of peripheral blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy.
Bone Marrow Transplant
11:23,
1993
8.
To LB,
Haylock DN,
Simmons PJ,
Juttner CA:
The biology and clinical uses of blood stem cells.
Blood
89:2233,
1997[Free Full Text]
9.
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]
10.
Breems DA,
Blokland EAW,
Neben S,
Ploemacher RE:
Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay.
Leukemia
8:1095,
1994[Medline]
[Order article via Infotrieve]
11.
Kohn DB,
Weinberg KI,
Nolta JA,
Heiss LN,
Lenarsky C,
Crooks GM,
Hanley ME,
Annett G,
Brooks JS,
El-Khoureiy A,
Lawrence K,
Wells S,
Moen RC,
Bastian J,
Williams-Herman DE,
Elder M,
Wara D,
Bowen T,
Hershfield MS,
Mullen CA,
Blaese RM,
Parkman R:
Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency.
Nat Med
1:1017,
1995[Medline]
[Order article via Infotrieve]
12.
Dunbar,
CE,
Cottler-Fox M,
O'Shaughnessy JA,
Doren S,
Carter C,
Berenson R,
Brown S,
Moen RC,
Greenblatt J,
Stewart FM,
Leitman SF,
Wilson WH,
Cowan K,
Young NS,
Nienhuis AW:
Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation.
Blood
85:3048,
1995[Abstract/Free Full Text]
13.
Emmons RV,
Doren S,
Zujewski J,
Cottler-Fox M,
Carter CS,
Hines K,
O'Shaughnessy JA,
Leitman SF,
Greenblatt JJ,
Cowan K,
Dunbar CE:
Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.
Blood
89:4040,
1997[Abstract/Free Full Text]
14.
Malech HL,
Maples PB,
Whiting-Theobald N,
Linton GF,
Sekhsaria S,
Vowells SJ,
Li F,
Miller JA,
DeCarlo E,
Holland SM,
Leitman SF,
Carter CS,
Butz RE,
Read EJ,
Fleisher TA,
Schneiderman RD,
Van Epps DE,
Spratt SK,
Maack CA,
Rokovich JA,
Cohen LK,
Gallin JI:
Prolonged induction of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease.
Proc Natl Acad Sci USA
94:12133,
1997[Abstract/Free Full Text]
15.
Larochelle A,
Vormoor J,
Hanenberg H,
Wang JCY,
Bhatia M,
Lapidot T,
Moritz T,
Murdoch B,
Xiao XL,
Kato I,
Williams DA,
Dick JE:
Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy.
Nat Med
2:1329,
1996[Medline]
[Order article via Infotrieve]
16.
Conneally E,
Cashman J,
Petzer A,
Eaves C:
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci USA
94:9836,
1997[Abstract/Free Full Text]
17.
Wang JCY,
Doedens M,
Dick JE:
Primitive human hematopoietic cells are enriched in cord blood compared to adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell (SRC) assay.
Blood
89:3919,
1997[Abstract/Free Full Text]
18.
Bhatia M,
Bonnet D,
Kapp U,
Wang JC,
Murdoch B,
Dick JE:
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med
186:619,
1997[Abstract/Free Full Text]
19.
Gan OI,
Murdoch B,
Larochelle A,
Dick JE:
Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.
Blood
90:641,
1997[Abstract/Free Full Text]
20.
Wermann K,
Fruehauf S,
Haas R,
Zeller WJ:
Human-mouse xenografts in stem cell research.
J Hematother
5:379,
1996[Medline]
[Order article via Infotrieve]
21.
Bosma GC,
Custer RP,
Bosma MJ:
A severe combined immunodeficiency mutation in the mouse.
Nature
301:527,
1983[Medline]
[Order article via Infotrieve]
22.
Nolta JA,
Hanley MB,
Kohn DB:
Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: Analysis of gene transduction of long-lived progenitors.
Blood
83:3041,
1994[Abstract/Free Full Text]
23.
McCune JM,
Namikawa R,
Kaneshima H,
Shultz LD,
Lieberman M,
Weissman IL:
The SCID-hu mouse: Murine model for the analysis of human hematolymphoid differentiation and function.
Science
241:1632,
1988[Abstract/Free Full Text]
24.
Namikawa R,
Weilbaecher KN,
Kaneshima H,
Yee EJ,
McCune JM:
Long-term human hematopoiesis in the SCID-hu mouse.
J Exp Med
172:1055,
1990[Abstract/Free Full Text]
25.
Kyoizumi S,
Baum CM,
Kaneshima H,
McCune JM,
Yee EJ,
Namikawa R:
Implantation and maintenance of functional human bone marrow in SCID-hu mice.
Blood
79:1704,
1992[Abstract/Free Full Text]
26.
Bock TA,
Orlic D,
Dunbar CE,
Broxmeyer HE,
Bodine DM:
Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.
J Exp Med
182:2037,
1995[Abstract/Free Full Text]
27.
Shultz LD,
Schweitzer PA,
Christianson SW,
Gott B,
Schweitzer IB,
Tennent L,
McKenna S,
Mobraaten L,
Rajan TV,
Greiner DL,
Leiter EH:
Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.
J Immunol
154:180,
1995[Abstract]
28.
Greiner DL,
Shultz LD,
Yates J,
Appel MC,
Perdrizet G,
Hesselton RM,
Schweitzer I,
Beamer WG,
Shultz KL,
Pelsue SC,
Leif JH,
Hesselton RM,
Rajan TV:
Improved engraftment of human spleen cells in NOD/LtSz-scid/scid mice as compared with C.B-17-scid/scid mice.
Am J Pathol
146:888,
1995[Abstract]
29.
Hesselton RM,
Greiner DL,
Mordes JP,
Rajan TV,
Sullivan JL,
Shultz LD:
High levels of human peripheral blood mononuclear cell engraftment and enhanced susceptibility to human immunodeficiency virus type 1 infection in NOD/LtSz-scid/scid mice.
J Infect Dis
172:974,
1995[Medline]
[Order article via Infotrieve]
30.
Lowry PA,
Shultz LD,
Greiner DL,
Hesselton RM,
Kittler ELW,
Tiarks CY,
Rao SS,
Reilly J,
Leif JH,
Ramshaw H,
Stewart FM,
Quesenberry PJ:
Improved engraftment of human cord blood stem cells in NOD/LtSz-scid/scid mice after irradiation or multiple-day injections into unirradiated recipients.
Biol Blood Marrow Transplant
2:15,
1996[Medline]
[Order article via Infotrieve]
31.
Pflumio F,
Izac B,
Katz A,
Shultz LD,
Vainchenker W,
Coulombel L:
Phenotype and function of human hematopoietic cells engrafting immune-deficient CB17-severe combined immunodeficiency mice and nonobese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells.
Blood
88:3731,
1996[Abstract/Free Full Text]
32.
Cashman JD,
Lapidot T,
Wang JC,
Doedens M,
Shultz LD,
Lansdorp P,
Dick JE,
Eaves CJ:
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood
89:4307,
1997[Abstract/Free Full Text]
33.
Neben S,
Marcus K,
Mauch P:
Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor.
Blood
81:1960,
1993[Abstract/Free Full Text]
34. .
McNiece IK,
Briddell RA,
Hartley CA,
Andrews RG:
The role of stem cell factor in mobilization of peripheral blood progenitor cells: synergy with G-CSF.
Stem Cells
11:83,
1993
35.
Yan XQ,
Hartley C,
McElroy P,
Chang A,
McCrea C,
McNiece I:
Peripheral blood progenitor cells mobilized by recombinant human granulocyte colony-stimulating factor plus recombinant rat stem cell factor contain long-term engrafting cells capable of cellular proliferation for more than two years as shown by serial transplantation in mice.
Blood
85:2303,
1995[Abstract/Free Full Text]
36. Hastie TJ, Tibshirani RJ: Generalized Additive Models. Chapman & Hall, London, UK, 1990
37. McCullagh P, Nelder JA: Generalized Linear Models.
2nd Edition ed. Chapman & Hall, London, 1989
38.
Phillips RA,
Fulop GM:
Pleiotropic effects of the scid mutation: Effects on lymphoid differentiation and on repair of radiation damage.
Curr Top Microbiol Immunol
152:11,
1989[Medline]
[Order article via Infotrieve]
39.
Lapidot T,
Pflumio F,
Doedens M,
Murdoch B,
Williams DE,
Dick JE:
Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice.
Science
255:1137,
1992[Abstract/Free Full Text]
40.
Vormoor J,
Lapidot T,
Pflumio F,
Risdon G,
Patterson B,
Broxmeyer HE,
Dick JE:
Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice.
Blood
83:2489,
1994[Abstract/Free Full Text]
41.
Rothe G,
Schmitz G:
Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis.
Leukemia
10:877,
1996[Medline]
[Order article via Infotrieve]
42.
Owen CA,
Campbell MA,
Boukedes SS,
Stockley RA,
Campbell,
EJ:
A discrete subpopulation of human monocytes expresses a neutrophil-like proinflammatory (P) phenotype.
Am J Physiol
267:L775,
1994[Abstract/Free Full Text]
43.
Goodell MA,
Rosenzweig M,
Kim H,
Marks DF,
DeMaria M,
Paradis G,
Grupp SA,
Sieff CA,
Mulligan RC,
Johnson RP:
Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.
Nat Med
3:1337,
1997[Medline]
[Order article via Infotrieve]
44.
Hogan CJ,
Shpall EJ,
McNiece I,
Keller G:
Multilineage engraftment in NOD/LtSz-scid/scid mice from mobilized human CD34+ peripheral blood progenitor cells.
Biol Blood Marrow Transplant
3:236,
1997[Medline]
[Order article via Infotrieve]
45.
Hogan CJ,
Shpall EJ,
McNulty O,
McNiece I,
Dick JE,
Shultz LD,
Keller G:
Engraftment and development of human CD34(+)-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice.
Blood
90:85,
1997[Abstract/Free Full Text]
46.
Yurasov S,
Kollmann TR,
Kim A,
Raker CA,
Hachamovitch M,
Wong-Staal F,
Goldstein H:
Severe combined immunodeficiency mice engrafted with human T cells, B cells, and myeloid cells after transplantation with human fetal bone marrow or liver cells and implanted with human fetal thymus: A model for studying human gene therapy.
Blood
89:1800,
1997[Abstract/Free Full Text]
47.
Rodewald HR,
Kretzschmar K,
Swat W,
Takeda S:
Intrathymically expressed c-kit ligand (stem cell factor) is a major factor driving expansion of very immature thymocytes in vivo.
Immunity
3:313,
1995[Medline]
[Order article via Infotrieve]
48.
Nolta JA,
Dao MA,
Wells S,
Smogorzewska EM,
Kohn DB:
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after engraftment in immune-deficient mice.
Proc Natl Acad Sci USA
93:2414,
1996[Abstract/Free Full Text]
49.
Yeoman H,
Gress RE,
Bare CV,
Leary AG,
Boyse EA,
Bard J,
Shultz LD,
Harris DT,
DeLuca D:
Human bone marrow and umbilical cord blood cells generate CD4+ and CD8+ single-positive T cells in murine fetal thymus organ culture.
Proc Natl Acad Sci USA
90:10778,
1993[Abstract/Free Full Text]
50.
Roberts AW,
Metcalf D:
Noncycling state of peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor and other cytokines.
Blood
86:1600,
1995[Abstract/Free Full Text]
51.
Andrews RG,
Briddell RA,
Knitter GH,
Rowley SD,
Appelbaum FR,
McNiece IK:
Rapid engraftment by peripheral blood progenitor cells mobilized by recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in nonhuman primates.
Blood
85:15,
1995[Abstract/Free Full Text]
52.
Nemunaitis J:
Cytokine-mobilized peripheral blood progenitor cells.
Semin Oncol
23:9,
1996[Medline]
[Order article via Infotrieve]
53.
Craddock CF,
Nakamoto B,
Andrews RG,
Priestley GV,
Papayannopoulou T:
Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice.
Blood
90:4779,
1997[Abstract/Free Full Text]
54.
Petrides PE,
Dittmann KH:
How do normal and leukemic white blood cells egress from the bone marrow? Morphological facts and biochemical riddles.
Blut
61:3,
1990[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
K. Yamamura, K. Ohishi, M. Masuya, E. Miyata, Y. Sugimoto, S. Nakamura, A. Fujieda, H. Araki, and N. Katayama
Ex Vivo Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Adversely Influences Their Distribution to Other Bone Marrow Compartments After Intra-Bone Marrow Transplantation
Stem Cells,
February 1, 2008;
26(2):
543 - 549.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Wu, J. Vieira, N. Fiore, P. Banerjee, M. Sieburg, R. Rochford, W. Harrington Jr, and G. Feuer
KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo
Blood,
July 1, 2006;
108(1):
141 - 151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. D. Shultz, B. L. Lyons, L. M. Burzenski, B. Gott, X. Chen, S. Chaleff, M. Kotb, S. D. Gillies, M. King, J. Mangada, et al.
Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2R{gamma}null Mice Engrafted with Mobilized Human Hemopoietic Stem Cells
J. Immunol.,
May 15, 2005;
174(10):
6477 - 6489.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Xu, E. Bruno, J. Chao, H. Ni, V. Lindgren, R. Nunez, N. Mahmud, G. Finazzi, S. M. Fruchtman, U. Popat, et al.
The constitutive mobilization of bone marrow-repopulating cells into the peripheral blood in idiopathic myelofibrosis
Blood,
February 15, 2005;
105(4):
1699 - 1705.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Samira, C. Ferrand, A. Peled, A. Nagler, Y. Tovbin, H. Ben-Hur, N. Taylor, A. Globerson, and T. Lapidot
Tumor Necrosis Factor Promotes Human T-Cell Development in Nonobese Diabetic/Severe Combined Immunodeficient Mice
Stem Cells,
November 1, 2004;
22(6):
1085 - 1100.
[Abstract]
[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]
|
|
|
|
|
|
|
S. Brenner, N. L. Whiting-Theobald, G. F. Linton, K. L. Holmes, M. Anderson-Cohen, P. F. Kelly, E. F. Vanin, A. M. Pilon, D. M. Bodine, M. E. Horwitz, et al.
Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/{beta}2-microglobulin-/- repopulating mobilized human peripheral blood CD34+ cells
Blood,
October 15, 2003;
102(8):
2789 - 2797.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. P. Kalberer, U. Siegler, and A. Wodnar-Filipowicz
Human NK cell development in NOD/SCID mice receiving grafts of cord blood CD34+ cells
Blood,
July 1, 2003;
102(1):
127 - 135.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Saito, Y. Kametani, K. Hozumi, N. Mochida, K. Ando, M. Ito, T. Nomura, Y. Tokuda, H. Makuuchi, T. Tajima, et al.
The in vivo development of human T cells from CD34+ cells in the murine thymic environment
Int. Immunol.,
October 1, 2002;
14(10):
1113 - 1124.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. M. Wagner, S. M. Guichard, R. A. Burger, C. L. Morton, C. M. Straign, R. A. Ashmun, L. C. Harris, P. J. Houghton, P. M. Potter, and M. K. Danks
Efficacy and Toxicity of a Virus-directed Enzyme Prodrug Therapy Purging Method: Preclinical Assessment and Application to Bone Marrow Samples from Neuroblastoma Patients
Cancer Res.,
September 1, 2002;
62(17):
5001 - 5007.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Yahata, K. Ando, Y. Nakamura, Y. Ueyama, K. Shimamura, N. Tamaoki, S. Kato, and T. Hotta
Functional Human T Lymphocyte Development from Cord Blood CD34+ Cells in Nonobese Diabetic/Shi-scid, IL-2 Receptor {gamma} Null Mice
J. Immunol.,
July 1, 2002;
169(1):
204 - 209.
[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]
|
|
|
|
|
|
|
I. D. Lewis, G. Almeida-Porada, J. Du, I. R. Lemischka, K. A. Moore, E. D. Zanjani, and C. M. Verfaillie
Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system
Blood,
June 1, 2001;
97(11):
3441 - 3449.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. E. Perez, H. M. Rinder, C. Wang, J. B. Tracey, N. Maun, and D. S. Krause
Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production
Blood,
March 15, 2001;
97(6):
1635 - 1643.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Wilpshaar, J. H. F. Falkenburg, X. Tong, W. A. Noort, R. Breese, D. Heilman, H. Kanhai, C. M. Orschell-Traycoff, and E. F. Srour
Similar repopulating capacity of mitotically active and resting umbilical cord blood CD34+ cells in NOD/SCID mice
Blood,
September 15, 2000;
96(6):
2100 - 2107.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Kollet, A. Peled, T. Byk, H. Ben-Hur, D. Greiner, L. Shultz, and T. Lapidot
beta 2 Microglobulin-deficient (B2mnull) NOD/SCID mice are excellent recipients for studying human stem cell function
Blood,
May 15, 2000;
95(10):
3102 - 3105.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Schiedlmeier, K. Kuhlcke, H. G. Eckert, C. Baum, W. J. Zeller, and S. Fruehauf
Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice
Blood,
February 15, 2000;
95(4):
1237 - 1248.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Robin, F. Pflumio, W. Vainchenker, and L. Coulombel
Identification of Lymphomyeloid Primitive Progenitor Cells in Fresh Human Cord Blood and in the Marrow of Nonobese Diabetic-Severe Combined Immunodeficient (NOD-SCID) Mice Transplanted with Human CD34+ Cord Blood Cells
J. Exp. Med.,
May 17, 1999;
189(10):
1601 - 1610.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract