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Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 1966-1976
Transplantation of Human Umbilical Cord Blood Cells in
Macrophage-Depleted SCID Mice: Evidence for Accessory Cell Involvement
in Expansion of Immature CD34+CD38
Cells
By
Monique M.A. Verstegen,
Paula B. van Hennik,
Wim Terpstra,
Cor van
den Bos,
Jenne J. Wielenga,
Nico van Rooijen,
Rob E. Ploemacher,
Gerard Wagemaker, and
Albertus W. Wognum
From the Institute of Hematology, Erasmus University Rotterdam,
Rotterdam, The Netherlands; and the Department of Biochemistry, Free
University Amsterdam, Amsterdam, The Netherlands.
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ABSTRACT |
In vivo expansion and multilineage outgrowth of human immature
hematopoietic cell subsets from umbilical cord blood (UCB) were studied
by transplantation into hereditary immunodeficient (SCID) mice. The
mice were preconditioned with Cl2MDP-liposomes to deplete
macrophages and 3.5 Gy total body irradiation (TBI). As measured by
immunophenotyping, this procedure resulted in high levels of human
CD45+ cells in SCID mouse bone marrow (BM) 5 weeks after
transplantation, similar to the levels of human cells observed in
NOD/SCID mice preconditioned with TBI. Grafts containing approximately
107 unfractionated cells, approximately 105
purified CD34+ cells, or 5 × 103 purified
CD34+CD38 cells yielded equivalent numbers
of human CD45+ cells in the SCID mouse BM, which
contained human CD34+ cells, monocytes, granulocytes,
erythroid cells, and B lymphocytes at different stages of maturation.
Low numbers of human GpA+ erythroid cells and
CD41+ platelets were observed in the peripheral blood of
engrafted mice. CD34+CD38+ cells (5 × 104/mouse) failed to engraft, whereas
CD34 cells (107/mouse) displayed only low
levels of chimerism, mainly due to mature T lymphocytes.
Transplantation of graded numbers of UCB cells resulted in a
proportional increase of the percentages of CD45+ and
CD34+ cells produced in SCID mouse BM. In contrast, the
number of immature, CD34+CD38 cells
produced in vivo showed a second-order relation to CD34+
graft size, and mice engrafted with purified
CD34+CD38 grafts produced 10-fold fewer
CD34+ cells without detectable
CD34+CD38 cells than mice transplanted
with equivalent numbers of unfractionated or purified
CD34+ cells. These results indicate that SCID
repopulating CD34+CD38 cells require
CD34+CD38+ accessory cell support for
survival and expansion of immature cells, but not for production of
mature multilineage progeny in SCID mouse BM. These accessory cells are
present in the purified, nonrepopulating
CD34+CD38+ subset as was directly proven by
the ability of this fraction to restore the maintenance and expansion
of immature CD34+CD38 cells in vivo when
cotransplanted with purified CD34+CD38
grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth
factors, and other stimuli. Such information will be essential to
design efficient stem cell expansion procedures for clinical use.
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INTRODUCTION |
TRADITIONAL SOURCES OF hematopoietic stem
and progenitor cells for transplantation include autologous and
allogeneic bone marrow (BM) and mobilized peripheral blood (PB).
Recently, human umbilical cord blood (UCB) has been shown to be a
realistic alternative source of stem cells.1,2 UCB contains
cells of all of the hematopoietic lineages, including cells that can
produce granulocyte-macrophage colony-forming unit (GM-CFU) after
extended long-term stromal cell-supported culture. Most of these
long-term culture-initiating cells (LTC-IC) are found in the small
subset of CD34+CD38 cells.3
The ability to cryopreserve, select, and expand progenitors without
loss of proliferative capacity4 makes UCB an appropriate model to identify immature hematopoietic cell subsets involved in
hematopoiesis in vivo, select appropriate growth factor (GF) combinations and culture conditions to maintain and expand stem cells
in vitro, and design optimal gene transfer conditions aimed at
efficient and stable transduction of transplantable stem
cells.5
Hereditary immunodeficient SCID and NOD/SCID mice are useful recipients
to assess human stem cell capacities in a transplantation assay and
appear particularly suitable to assess the outgrowth of purified UCB
cell subsets and the effects of ex vivo manipulation on hematopoietic
capacities after transplantation. Several approaches for engrafting
immunodeficient mice with normal or leukemic human hematopoietic cells
have been described. The most frequently used systems involve injection
of mobilized human PB, BM,6 or UCB cells in sublethally
irradiated mice,7,8 electively followed by human GF
treatment9-12 and/or cotransplantation with
nonrepopulating CD34 accessory cells,13
human BM long-term culture-derived stromal cells, or rodent cell lines
that produce human GFs.14 Transgenic SCID mice expressing
the genes for human interleukin-3 (IL-3), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and stem cell factor (SCF) have
also been used to promote human cell engraftment,15 whereas
human fetal liver, thymus,16,17 and/or bone
fragment18 implantation has been used to create a human microenvironment in the mouse.
Injection with human cytokines or other additional treatment is not
required to establish high-level human cell engraftment after
transplantation of human UCB cells in immunodeficient mice, which
suggested that neonatal cells either respond differently to the murine
microenvironment or provide their own cytokines in a paracrine
fashion.7,8 However, analysis of the hematopoietic potential of UCB cells in SCID is limited by the large number of cells
required to achieve significant engraftment levels, possibly because of
low seeding efficiencies of stem cells or elimination of transplanted
cells by natural killer (NK) cells or the mononuclear phagocytic
system, which are intact in SCID mice. More reproducible and higher
levels of engraftment with smaller graft sizes have been achieved with
NOD/SCID mice, which has been attributed to the lack of functional
macrophages, NK cells, and complement activity in this mouse
strain.19 Specific elimination of phagocytic cells in
spleen and liver of SCID mice can be achieved within 24 hours after a
single intravenous (IV) injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP).20-22
As shown recently for human acute myeloid leukemia (AML) and UCB cells,
macrophage-depleted SCID mice supported the production of similar
levels of human cells from 10-fold fewer transplanted cells as compared
with SCID mice conditioned with total body irradiation (TBI) alone. For AML cells, preconditioning of SCID mice with liposomes led to similar
levels of engraftment as observed in NOD/SCID mice, which suggested
that macrophages have a prominent role in eliminating injected human
cells in SCID mice.22
The present study was undertaken to quantitatively analyze the
maintenance and outgrowth of distinct UCB immature cell subsets in
macrophage-depleted SCID mice and to assess the hematopoietic cell
lineages produced.
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MATERIALS AND METHODS |
Human UCB cells.
UCB samples were obtained after informed consent in conformity with
legal regulations in The Netherlands from placentas of full-term normal
pregnancies. Mononucleated cells were isolated by Ficoll density
gradient centrifugation (1.077 g/mL; Nycomed Pharma AS, Oslo, Norway)
and were cryopreserved in 10% dimethylsulphoxide, 20%
heat-inactivated fetal calf serum (FCS), and 70% Hank's Balanced Salt
Solution (HBSS; GIBCO, Breda, The Netherlands) at  196°C as
described.23 After thawing by stepwise dilution in HBSS
containing 2% FCS, the cells were washed with HBSS containing 1% FCS
and used for flow cytometric analysis, transplantation into SCID mice (unfractionated grafts), or subset purification.
Subset purification.
Purification of CD34+ cells was performed by positive
selection using Variomacs Immunomagnetic Separation System as
described24 (CLB, Amsterdam, The Netherlands). The
percentage CD34+ cells in the unseparated population
(unfractionated UCB) and in the purified CD34+ and
CD34 fractions was determined by
fluorescence-activated cell sorting (FACS) analysis. For isolation of
CD34+CD38+,
CD34+CD38+/, and
CD34+CD38 subsets, purified
CD34+ cells were stained with fluorescein isothiocyanate
(FITC)- and R-phycoerythrin (PE)-conjugated antibodies against human
CD34 and CD38 (CD34-FITC, CD38-PE; Becton Dickinson, San Jose,
CA) for 30 minutes on ice in HFN (HBBS, 2% [wt/vol]
FCS, 0.05% [wt/vol] sodium-azide) containing 2% (vol/vol) normal
human serum (NHS). After incubation, the cells were washed twice,
resuspended in HBSS, and sorted using a FACS Vantage flow cytometer
(Becton Dickinson).
Transplantation of UCB cells in immunodeficient mice.
Female, specified pathogen-free (SPF) CB-17-scid/scid (SCID) mice, 6 to
9 weeks of age, were obtained from Harlan (CPB, Austerlitz, The
Netherlands). NOD/LtSz-scid/scid mice (NOD/SCID) were obtained from The
Jackson Laboratory (Bar Harbor, ME). The mice were housed under SPF
conditions in a laminar air flow unit and supplied with sterile food
and acidified drinking water containing 100 mg/L ciprofloxacine (Bayer
AG, Leverkusen, Germany) ad libitum. The plasma Ig levels of the mice
were determined with an enzyme-linked immunosorbent assay using a sheep
antimouse antibody reacting with mouse IgG (Boehringer Mannheim
Biochemica, Penzberg, Germany), and animals with plasma Ig levels
greater than 40 µg/mL were excluded.25 To deplete
macrophages, the SCID-mice were injected IV into a lateral tail vein
with 200 µL liposome stock solution containing di-chloromethylene
di-phosphonate (Cl2MDP; a gift of Boehringer Mannheim GmbH,
Mannheim, Germany) 1 day before transplantation of
hematopoietic cells.26 In previous studies22
with human acute leukemia and UCB cells, this approach required 10-fold
fewer cells for uniform engraftment than in SCID mice conditioned with TBI alone. All mice received TBI at 3.5 Gy, delivered by a
137Cs source adapted for the irradiation of mice
(Gammacell; Atomic Energy of Canada, Ottawa, Ontario, Canada) 2 to 4 hours before transplantation. The transplants were suspended in 200 µL HBSS containing 0.1% bovine serum albumin (BSA; Sigma, St Louis,
MO) and injected IV into a lateral tail vein. Transplanted
cell numbers were 107 (unfractionated and
CD34 cells), 105 (CD34+
cells), 5 × 104 (CD34+CD38+
cells), and 5 × 103
(CD34+CD38 cells), unless stated
otherwise in the results.
In vitro colony assay.
Unfractionated and purified CD34+ and
CD34 grafts as well as chimeric mouse BM samples
were assayed for the presence of GM-CFU and erythroid burst-forming
units (BFU-E) by in vitro colony formation in viscous methylcellulose
culture medium as previously described.27-29 Briefly,
unfractionated and CD34 cells were plated at a
concentration of 25,000 per 35-mm Petri dish (Becton Dickinson),
CD34+ purified grafts at 1,000 per dish, and chimeric mouse
BM at 50,000 per dish. Culture medium consisted of 1 mL Dulbecco's
medium (GIBCO, Gaithersburg, MD), containing 0.8% (wt/vol)
methylcellulose, 5% (vol/vol) FCS, and further supplemented with 1.5%
(wt/vol) BSA, 10 mg/mL insulin, 0.3 mg/mL human transferrin, 15 mmol/L
 -mercaptoethanol, 0.1 mmol/L sodium selenite, 1 mg/mL nucleosides,
15 µmol/L linoleic acid, 15 µmol/L cholesterol, 100 U/mL
penicillin, and 50 mg/mL streptomycin. For BFU-E, cultures were
supplemented with 0.2 mmol/L bovine hemin (Sigma), 200 ng/mL human SCF,
and 4 U/mL (25 µg/mL) human recombinant Epo (Behringwerke AG,
Marburg, Germany). For CFU-GM, cultures were supplemented with 5 ng/mL
human recombinant GM-CSF (Behringwerke AG), 200 ng/mL SCF, and 30 ng/mL
human recombinant IL-3. The cultures were maintained in a humidified
atmosphere of 10% CO2 at 37°C for 14 days, after which
the colonies were counted. Data of duplicate dishes were expressed as
average number of colonies per 105 cells plated.
Tissue collections and analysis.
Mice were examined at a single time point, 35 days after
transplantation, to enable meaningful comparisons between experiments, because individual hematopoietic subsets show differences in
engraftment kinetics in immunodeficient mice.12 Mice were
killed by CO2 inhalation followed by cervical dislocation
in accordance with institutional animal research regulations. From each
mouse, both femurs were collected and BM cell suspensions were prepared
by flushing. After counting, the cells were cultured in colony assays and analyzed by flow cytometry to determine the percentage of human
cells in the mouse BM. Cells were suspended in HBSS containing 2%
(vol/vol) FCS, 0.05% (wt/vol) sodium azide, 2% (vol/vol) human serum,
and 2% (vol/vol) mouse serum and stained for 30 minutes at 40°C
with the pan-leukocyte surface marker CD45-FITC antibody and with
CD33-PE antibody. Positive samples were further analyzed by incubation
with FITC- and PE-labeled mouse monoclonal antibodies to human CD34,
CD19, CD16, CD15, CD38, CD33, CD56, CD4, and CD8 (Becton Dickinson
Immunocytometry Systems, San Jose, CA) and glycophorin A (GpA), CD3,
and CD71 (Dako A/S, Copenhagen, Denmark). Parallel samples
were incubated with isotype-matched control antibodies. Cell samples of
nontransplanted mice were stained as negative controls. Fluorescence
was measured using a FACScan flow cytometer and Lysis II software
(Becton Dickinson). Dead cells were excluded by adding 1 µg/mL
propidium iodide (PI) and gating for PI cells in the
FL3 (PI) channel. For all samples, 10,000 events were collected in a
gate for PI cells. To quantitate CD34+
subsets in selected samples, 1,000 to 10,000 events were also collected
in a gate that included all viable human CD34+ cells.
CD34+ and CD34+CD38
expansion were calculated on the assumption that one femur contains 8.5% of all BM cells.30
In a number of experiments, PB was collected weekly from the tail vein
and analyzed for the presence of human GpA+ erythrocytes
and CD41+ platelets by flow cytometry. Blood samples were
collected in EDTA-coated tubes and stained with CD41-FITC (PharMingen,
San Diego, CA) and GpA-FITC, respectively (Dako A/S) in HBSS with 2%
(vol/vol) FCS, 0.05% (wt/vol) sodium azide, 2% (vol/vol) human serum,
2% (vol/vol) mouse serum, and 2 g/L EDTA for 30 minutes at 40°C.
Cell samples of nontransplanted mice and human blood cells were stained
as controls.
Statistical and regression analysis.
Results are expressed as individual data or as the arithmetic mean ± standard deviation. The regression analysis of the percentage of
human CD45+, CD34+, and
CD34+CD38 cells in the chimeric BM as a
function of the number of CD34+ cells transplanted was
performed by plotting the data on a double logarithmic scale and
calculating the regression using the general formula y = axb. By this method, an exponent b = 1 proves first order
(single-hit) kinetics, ie, direct proportionality (linearity) of
chimeric cell numbers and cells transplanted, whereas an exponent b = 2 demonstrates second order (two-hit) kinetics. The frequency of
repopulating cells in the SCID mice was approximated using Poisson
statistics.
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RESULTS |
Chimeric BM analysis.
Chimerism in SCID mouse BM was assessed by flow cytometric analysis 35 days after UCB transplantation. Typical results of chimeric BM stained
with CD45-FITC versus CD33-PE and CD45-FITC versus CD34-PE are shown in
Fig 1A and B, respectively. The percentage of CD45+ cells was used as a measure for engraftment levels
of human cells in the mouse BM. Only mice with percentages larger than
1% CD45+ cells were considered to be engrafted. Positive
staining for any of these markers was not found in nontransplanted mice
(Fig 1C and data not shown), demonstrating the specificity of the
antibodies for human cells. As shown in Fig 1A and B, the
CD45+ cells were heterogeneous with respect to CD33 and
CD34 expression.
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| Fig 1.
Flow cytometric analysis of chimeric mouse BM stained
with CD45-FITC versus CD33-PE (A) and CD45-FITC versus CD34-PE (B). BM
of nontransplanted mice showed no staining with the CD45-FITC or
CD33-PE antibody (C).
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Parallel groups of mice were injected with unfractionated mononucleated
UCB cells or with purified CD34+ or CD34
cells (Fig 2) in SCID mice conditioned with either TBI or TBI and
macrophage depletion or in TBI-conditioned NOD/SCID mice. Transplantation of unfractionated mononucleated UCB cells into macrophage-depleted SCID mice resulted in more prominent engraftment levels compared with SCID mice conditioned with TBI alone. After transplantation with 107 unfractionated or 105
purified CD34+ cells, the macrophage-depleted SCID mice
showed similar levels of chimerism as NOD/SCID mice preconditioned with
TBI. CD34 cells (107 cells transplanted)
did not result in high levels of chimerism in either mouse strain
(Fig 2).
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| Fig 2.
Engraftment levels of 4 different UCB samples in SCID and
SCID/NOD mice. The percentages CD45+ cells are shown for
individual SCID mice preconditioned with TBI and Cl2MDP
( ), SCID mice preconditioned by TBI alone ( ), and NOD/SCID mice
preconditioned by TBI alone ( ).
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As shown in Table 1, transplantation of
107 unfractionated cells from 5 different UCB samples
resulted in high levels of chimerism in all mice (n = 22) transplanted.
Transplantation of 105 purified CD34+ cells
also resulted in high levels of human cells in 35 of 38 mice, whereas
mice transplanted with 107 CD34 cells
showed only low levels of engraftment in 5 of 18 mice transplanted. These results show that relatively low numbers of purified
CD34+ UCB cells are capable of proliferation in the
macrophage-depleted SCID mouse microenvironment without the support of
accessory cells or addition of hematopoietic growth factors. The
CD34+ cells were further separated into a CD38+
subset (~50% of CD34+ cells containing >90% of
clonogenic progenitors) and a CD38 subset (~5% of
the CD34+ population, enriched for immature, multipotent
progenitors31) and transplanted into preconditioned SCID
mice; cell numbers were 5 × 104 and 5 × 103, respectively. The
CD34+CD38 subset showed high levels of
engraftment in 4 of 6 mice with chimerism levels similar to those
obtained with 20-fold larger numbers of CD34+ cells and
200-fold larger numbers of unfractionated UCB (Table 1). Despite the
10-fold larger cell numbers, only 1 of 4 mice engrafted with sorted
CD34+CD38+ cells at the low level of 1.7%.
These results show that the ability to repopulate SCID mice resides
exclusively in the CD34+CD38 immature
population.
Multilineage outgrowth of UCB cells.
BM cells of chimeric mice were cultured in standard methylcellulose
culture under conditions of stimulation with recombinant human GF that
selectively favor the outgrowth of human monomyeloid and erythroid
progenitors and failed to stimulate mouse progenitors. Comparison of
clonogenic cell numbers in 15 chimeric mice with the numbers of
colony-forming cells in the grafts showed a median expansion of
2.7-fold (range, 0 to 11) and 1.7-fold (range, 0 to 13) for CFU-GM and
BFU-E numbers, respectively, as measured 35 days after transplantation.
Because these progenitor cell populations have a high turnover rate,
this observation demonstrates that monomyelocytic and erythroid
progenitors are produced from more immature progenitors in the mouse
hematopoietic environment.
The composition of the human cell population in the BM of chimeric mice
was assessed by flow cytometry using a panel of lineage-specific markers (Fig 3). The percentage of cells in
each subset identified was expressed relative to the percentage cells
stained with the panleukocyte marker CD45
(Fig 4). Mice transplanted with
107 unfractionated UCB cells showed multilineage outgrowth
(Fig 4A). The most prominent population (25% to 50% of the human
CD45+ cells) consisted of B-lymphoid cells, which contained
immature CD19+CD20 as well as mature
CD19+CD20+ cells (Figs 3F and 4).
CD15+CD33+ monocytes,
CD15+CD33+/ granulocytes, and
CD15CD33+ immature myelomonocytic cells
were present at percentages ranging between 6% and 16% of the human
cells (Figs 3B and 4). GpA+CD71++ erythroblasts
and, occasionally, GpA+CD71 mature red
blood cells (not visible in Fig 3D) were present in low numbers. In
keeping with the presence of CD71 on activated nonerythroid cells, the
large population of CD71+GpA cells (Fig
3D) contained cells of multiple lineages.32 The composition
of the BM of mice transplanted with CD34+ (Fig 4B) or
CD34+CD38 cells (Fig 4C) was similar to
that of mice transplanted with unfractionated UCB. The few mice that
showed detectable chimerism after transplantation of
CD34 cells also had outgrowth of low numbers of
myeloid, erythroid, and B-lymphoid cells, which were possibly derived
from the low numbers of CD34+ cells (0.1% to 0.6%) still
present in the fraction. However, greater than 50% of the cells
growing in these mice consisted of mature CD3+ T
lymphocytes, which also expressed CD4 or CD8. CD3+ cells
were also identified in mice transplanted with unfractionated, CD34+, or CD34+CD38 cell
subsets, but these CD3+ cells expressed neither CD4 nor CD8
(Figs 3 and 4A through C). These cells may represent a subset of NK
cells, because CD3 is expressed on some CD56+
cells33 and CD56+ cells were also identified in
low numbers in chimeric mice, including those transplanted with
purified CD34+CD38 cells (Figs 3C and
4C). The large population of CD3 cells that
expressed CD4 or CD8 (Fig 3E) most likely consisted of CD4+
monocytes.
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| Fig 3.
Immunophenotyping of chimeric mouse BM. BM (>10%
CD45+) was stained with a panel of antibodies specific
against different human blood cell lineages. FACS profiles of a
representative mouse are shown for CD34 versus CD38 (A), CD15 versus
CD33 (B), CD16 versus CD56 (C), GpA versus CD71 (D), CD3 versus CD4 and
CD8 (E), and CD19 versus CD20 (F) expression, respectively.
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| Fig 4.
Composition of the human CD45+ cell
population in chimeric SCID mice stained for the human markers shown in
Fig 3. Results (average ± SD) are those of 23 mice in total,
transplanted with unfractionated UCB (A), purified CD34+
(B), CD34+CD38 (C), and
CD34 (D) grafts derived from 5 UCB
samples. The percentage of cells in each subset was expressed relative
to the percentage of CD45+ cells present in the BM of
each mouse. The percentage of chimerism ranged between 10% and 40%
for the data shown in (A) through (C) and between 1% and 15% for the
data shown in (D).
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Despite large numbers of human cells in the BM of SCID mice, very few
human cells were detected in the leukocyte fraction of PB, spleen, and
thymus (data not shown). Whole tail vein blood samples of
CD34+ transplanted mice collected at various time points
after transplantation contained human GpA+ erythrocytes at
very low levels (~0.1%) that could only be detected if very large
cell numbers (>105) were analyzed
(Fig 5C). The largest quantities (0.1% to
0.2%) were found 2 weeks after transplantation. From week 3 on, the level decreased and became undetectable in week 5. Human
CD41+ platelets could also be detected in the mouse PB and
followed a similar time course as the erythroid cells, with peak levels of 0.5% in week 2 (Fig 5B).
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| Fig 5.
Circulating CD41+ platelets and
GpA+ erythrocytes in the PB of CD34+
transplanted SCID mice. Blood was collected in the presence of 2 g/L
EDTA and stained immediately with CD41-FITC (B) and GpA-FITC (C). (A)
shows the blood of a nontransplanted mouse stained with CD41-FITC.
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Evidence for accessory cell requirement for immature cell expansion
but not for outgrowth of human UCB cells in SCID mice.
The UCB cell number required for engraftment was analyzed by injection
of graded numbers of unfractionated or CD34+ cells.
Transplantation of 2 × 103 CD34+ cells
resulted in a low, but measurable level of chimerism of 1.4%
CD45+ cells (Fig 6). The level
of chimerism increased proportionally with cell dose, reaching
approximately 60% human CD45+ cells after injection of 2 × 105 purified CD34+ cells. Engraftment
after transplantation of unfractionated mononuclear UCB cells and
purified CD34+ cells followed similar proportional
patterns, with exponents of 0.8 and 1, respectively (Fig 6). Also the
percentage of human CD34+ cells detected in SCID mouse BM
after 35 days showed a linear relation with graft size (Fig 6). These
results demonstrate that the outgrowth of human UCB cells in the SCID
mouse BM does not require the support from accessory cells present in
either the CD34+ or CD34 UCB fractions.
Figure 3A shows that the CD34+ cells produced in SCID mouse
BM were heterogeneous with respect to CD38 expression and included
cells with low CD38 expression, which suggested that very immature
cells were maintained and/or expanded in the mouse
microenvironment. As shown in Fig 6, the production of cells with an
immature CD34+CD38 phenotype showed a
much steeper dependence on the number of CD34+
transplanted, with a exponent of 2, demonstrating second order (two-hit) kinetics. CD34+CD38 cells were
not detected in BM of mice transplanted with purified CD34+CD38 cells (Table 1 and
Fig 7B), whereas, in addition,
the numbers of CD34+ cells in these mice were 10-fold lower
than in mice transplanted with unfractionated or CD34+
grafts, despite similar levels of CD45+ cells (Table 1).
Taken together, the nonlinear relation between graft size and the
percentage of CD34+CD38 cells after 35 days, the lower number of CD34+ cells, and the absence of
CD34+CD38 cells in mice transplanted
with purified CD34+CD38 grafts show that
immature CD34+CD38 cells can be
maintained in the mouse microenvironment, but only with the support of
accessory cells.
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| Fig 6.
Relationship between the number of CD34+
cells transplanted and percentage of human CD45+ (),
CD34+ ( ), and immature
CD34+CD38 () cells detected in SCID
mouse BM after 5 weeks. Results show the mean ± SD for 3 mice per
data point. For comparison, the numbers of CD45+ cells
detected in BM of mice transplanted with graded numbers of
unfractionated cells are also shown ( ).
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| Fig 7.
Distribution of human CD34 and CD38 in
chimeric mouse BM after transplantation of CD34+ subsets,
sorted as defined by the windows in (A). (B) through (E) provide the
results 35 days after transplantation of 5 × 103
CD34+CD38 cells (B), 5 × 103
CD34+CD38 + 25 × 103 CD34+CD38+ cells (C),
105 CD34+ cells (D), or 25 × 103 CD34+CD38+/ (E). One
thousand to 10,000 events were collected in a window containing
CD34+ cells only. Quadrants were set to indicate
CD34+CD38+ and
CD34+CD38 cells. The percentages indicate
the frequency of human CD34+ CD38 cells in
mouse BM. CD34+CD38+ cells did not engraft
(data not shown). The dissociation of outgrowth of CD45+
cells and maintenance or expansion of
CD34+CD38 cells is also in this experiment
indicated by the CD45 percentages, ie, 25.1% for (B) without
CD34+CD38 cells and 5.5% for (C), 46.7%
for (D), and 2.9% for (E) with similar frequencies of
CD34+CD38 cells.
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Figure 8 shows the actual expansion of
CD34+CD38 cells in BM of the 32 (from
69) chimeric mice in which such cells were detectable. The expansion
ranged between 0.2 and 22.1, with a median expansion of threefold for
unfractionated mononucleated UCB cells, and between 1.6 and 63.1, with
a median value of sevenfold after transplantation of CD34+
grafts. This difference is statistically not significant.
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| Fig 8.
Expansion of CD34+CD38 cells
after transplantation of unfractionated or CD34+ cells.
Data for 32 mice that showed detectable
CD34+CD38 cells from a group of 69 chimeric mice (>1% CD45+ cells). Twenty-eight mice
were transplanted with unfractionated (unfract.) cells and 41 mice were
transplanted with CD34+ grafts from 5 different UCB
samples. The arrow shows the median expansion factor of
CD34+CD38 cells in each group.
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Direct proof of an accessory role of CD34+CD38+
cells in the maintenance of the transplanted
CD34+CD38 population in vivo was
obtained by cotransplantation of
CD34+CD38 cells and
CD34+CD38+ cells. Dot plots of CD38 versus CD34
expression, collected in a gate for CD34+ cells, show that
transplantation of 5 × 103
CD34+CD38 cells results in production of
CD34+ cells, which are all CD38+ (Fig 7B).
After transplantation of 5 × 103
CD34+CD38 cells to which 25 × 103 CD34+CD38+ cells were added,
CD34+CD38 cells were clearly produced in
the mouse BM (Fig 7C) at frequencies similar to those observed in mice
transplanted with 105 CD34+ cells (Fig 7D).
Also in this experiment, transplantation of 5 × 104
CD34+CD38+ cells alone did not result in human
cell engraftment (similar to the data presented in Table 1). Sorted
CD34+CD38+/ cells (corresponding to 20%
of the CD34+ cells) also repopulated transplanted SCID mice
with propagation of immature CD34+CD38
cells, which can be explained by the presence of repopulating and
accessory cells in this subset (Fig 7E).
Repopulating cell frequency.
The maintenance or expansion of
CD34+CD38 cells in SCID mice might be
considered as a more significant characteristic of the capacity of
repopulating stem cells than the ability to produce mature progeny.
Taking into account that the seeding efficiency of repopulating cells
in transplanted SCID mice is unknown and that the support provided by
accessory cells may be suboptimal, a lower limit for the frequency of
cells with the ability to maintain or expand the numbers of
CD34+CD38 cells was estimated using the
pooled data of 69 mice engrafted with graded doses of unfractionated or
purified CD34+ cells from 5 different UCB samples
(Table 2). By using Poisson statistics, a
value of 1 repopulating cell per 70,000 CD34+ cells was
estimated (95% confidence limits, 54,000 to 102,000). This would
correspond to 1 repopulating cell per approximately 7 × 106 unfractionated UCB cells and 1 per 3,500 CD34+CD38 cells.
| |
DISCUSSION |
Engraftment of UCB in SCID mice preconditioned by 3.5 Gy TBI and
injection of CL2MDP liposomes was more prominent than in SCID mice conditioned with TBI alone and similar to that observed in
NOD/SCID mice. The macrophage-depleted SCID mice supported the
multilineage outgrowth of unfractionated UCB, purified
CD34+ cells and the immature subset of
CD34+CD38 UCB cells, with production of
B lymphocytes, monocytes, granulocytes, erythroid cells, NK cells, and
platelets as well as production of CD34+ cells, including
phenotypically immature CD34+CD38 cells.
Small numbers of purified CD34+CD38
cells also engrafted efficiently with chimerism levels similar to those
observed in accessory cell and/or GF-supported NOD/SCID mice,13 whereas CD34+CD38
cells did not engraft, indicating that the SCID repopulating potential
resides exclusively in the CD34+CD38
subset.
The detection of CD34+CD38 cells in SCID
mouse BM is consistent with the finding that
CD34+CD38 cells recovered from the BM of
engrafted SCID and NOD/SCID mice have retained the capacity to produce
clonogenic progeny in long-term culture and to differentiate into
myeloid and lymphoid cells in single-cell/well
cultures.34,35 Results showing that purified human cells
from NOD/SCID mouse BM may engraft secondary recipients also suggest
that repopulating stem cells are maintained in the BM of
immunodeficient mice.36 Taken together, these data show that immature CD34+CD38 UCB cells can
survive and expand in transplanted immunodeficient mice.
The level of expansion of the immature
CD34+CD38 subset in chimeric mouse BM,
but not the multilineage production of more differentiated progeny,
appeared to be dependent on accessory cells. This is most clearly
demonstrated by the second order (two-hit) kinetics of the relation
between graft size and the numbers of immature CD34+CD38 cells produced in the SCID
mouse BM in contrast to the directly proportional relation between
graft size and the numbers of mature CD45+ cells and the
CD34+ population as a whole (Fig 6). Additional data show
that engraftment levels and types of human cells produced in the BM of
mice transplanted with 5 × 103
CD34+CD38 cell were similar to those
obtained with 20-fold more CD34+ cells or 2,000-fold larger
numbers of unfractionated mononucleated UCB cells, which also showed
that accessory cells or exogenous GFs are not needed for multilineage
outgrowth of immature human cells in immunodeficient mice. In contrast,
the observation that SCID mice transplanted with
CD34+CD38 grafts produced 10-fold fewer
CD34+ cells and no detectable
CD34+CD38 cells, despite equal numbers
of CD45+ cells, than mice transplanted with unfractionated
or CD34+ grafts with equivalent numbers of
CD34+CD38 cells (Table 1), provides
additional evidence for an involvement of accessory cells in the
maintenance and expansion of immature UCB cells in the SCID mouse
microenvironment. Because mice transplanted with unfractionated
mononucleated UCB cells did not show larger numbers of
CD34+ cells (Table 1) or more extensive expansion of
CD34+CD38 cells (Fig 8) than mice
transplanted with purified CD34+ cells, we postulated that
the accessory cells needed for the support of immature UCB cells are
present in the CD34+ population. Formal proof was obtained
by cotransplantation of CD34+CD38 cells
and CD34+CD38+ cells (Fig 7). Whereas
transplantation of CD34+CD38 cells alone
did not result in the maintenance of these cells, the addition of 50%
CD34+CD38+, a fraction that by itself did not
result in substantial chimerism, restored the propagation of
CD34+CD38 cells in engrafted mice (Fig
7).
One possible function of the accessory cells UCB cells might be to
prevent elimination of the small numbers of
CD34+CD38 cells by residual
immune-reactivity in the SCID mouse by providing an excess of human
cells. However, because small numbers of
CD34+CD38 cells produced equal numbers
of mature progeny in the macrophage-depleted SCID mice than much larger
unfractionated or CD34+ grafts (Table 1), it is unlikely
that such a mechanism plays a prominent role in promoting
CD34+CD38 cell engraftment. It is more
likely that accessory cells provide essential GFs or other stimuli
needed for the self-renewal of immature human cells that are not
provided by the mouse microenvironment. CD34+ UCB cells and
their immediate progeny have been shown to produce various GF,
including IL-3, G-CSF, and GM-CSF, which stimulate in vitro colony
formation by UCB in an autocrine or paracrine fashion.37 A
role of accessory cell-derived GF in the maintenance of immature cells
is also suggested by the supportive role of a cocktail of
erythropoietin, Steel factor, IL-3, and GM-CSF for expansion of human cells in NOD/SCID mice transplanted with high numbers of unfractionated human BM cells, which was only observed late
after transplantation, when the number of human cells were reduced.12 Further studies are required to examine to what
extent optimal combinations of these or other GFs can replace accessory cells in maintaining and expanding
CD34+CD38 cells in immunodeficient mice.
Estimation, by Poisson statistics, of the frequency of original UCB
cells that can maintain or expand
CD34+CD38 cell numbers during the 5 weeks of engraftment period yielded a value of 1 in 70,000 CD34+ cells (corresponding to 1 in 3,500 CD34+CD38 cells). This value is lower,
but in the same order of magnitude, than the 1 in 600 SCID repopulating
CD34+CD38 cells that has been calculated
on the basis of the frequency of transplanted NOD/SCID mice with
detectable numbers of human cells in the BM as assessed by Southern
blots.38 The difference is most likely due to the criteria
chosen in that the ability to expand
CD34+CD38 cells is a more stringent
parameter for engraftment of immature cells than the production of
mature progeny at a level of as low as 0.05% human cells detected by
DNA blotting analysis.38 Such low engraftment levels can in
principle be derived from contaminating mature cells, as shown in our
study by the low, but detectable (>0.5% of mouse BM) engraftment
with mature T cells in some mice transplanted with purified
CD34 cells. The ability to maintain or expand
CD34+CD38 cell numbers in SCID mouse BM
is probably more characteristic for repopulating stem cells than
production of mature progeny per se, because it may reflect an
essential hematopoietic stem cells feature, ie, the ability to maintain
its own numbers in vivo.
The differences in repopulating cell frequencies might also be due to
the cotransplantation of accessory cells and/or administration of growth factors in the NOD/SCID mouse model that may have promoted human cell engraftment.38 Although it is clear that
CD34+CD38 cells still represent a
heterogeneous cell population with only a minority of cells capable of
hematopoietic reconstitution, all frequency estimates of the SCID mouse
repopulating human cells likely underestimate the frequency of human
repopulating cells and should be treated with caution. In particular,
the seeding efficiency of these cells has not been assessed yet,
whereas the efficacy of the growth stimuli provided by the xenogeneic
environment, accessory cells in the transplant or exogenous growth
factor administration might very well be suboptimal. Studies into the
kinetics of human BM cell engraftment in immunodeficient mice have
shown that the number of immature, CD34+Thy1+
cells that can be detected in the mouse BM 2 days after transplantation is at least 2 logs lower than input numbers, suggesting that only a
very small fraction of the immature human cells develop in these mice.12
The present study provides evidence for differential regulation of the
expansion as opposed to multilineage outgrowth of immature human
hematopoietic stem cells in transplanted SCID mice. The possibility to
distinguish experimentally between these essential functions in the
SCID mouse transplantation assay now opens an experimental approach to
examine the effects of various GFs, cell subsets, and other agonists on
the self-renewal of human immature stem cell subsets. This information
will be essential to design and test conditions for ex vivo activation
and expansion of immature hematopoietic cells and for various
experimental purposes, such as required for the development of
efficient gene transfer protocols into hematopoietic cells with
retention of repopulating ability.
| |
FOOTNOTES |
Submitted July 24, 1997;
accepted November 7, 1997.
Supported in part by grants of the Netherlands Organization for
Scientific Research NWO, the Netherlands Cancer Foundation Koningin
Wilhelmina Fonds, the Royal Netherlands Academy of Arts and Sciences,
and contracts of the Commission of the European Communities.
Address reprint requests to Gerard Wagemaker, PhD, Institute of
Hematology, Room H Ee1314, Erasmus University Rotterdam, Dr. Molewaterplein 50, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr F.K. Lotgering and the staff of the Obstetrics
Department at the Sophia Children's Hospital (Rotterdam, The
Netherlands) and Dr A.Th. Alberda and the staff of the St Fransiscus
Hospital (Rotterdam, The Netherlands) for the collection of cord blood
samples used in this study. We acknowledge Dr W.A.M. Loenen for the
initial cord blood work in our lab and Els van Bodegom for taking care
of the SCID mice.
| |
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Abstract
Introduction
Abstract