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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 481-487
Human Growth Factor-Enhanced Regeneration of Transplantable Human
Hematopoietic Stem Cells in Nonobese Diabetic/Severe
Combined Immunodeficient Mice
By
Johanne D. Cashman and
Connie J. Eaves
From the Terry Fox Laboratory, British Columbia Cancer Agency and
Department of Medical Genetics, University of British Columbia,
Vancouver, BC, Canada.
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ABSTRACT |
Self-renewal is considered to be the essential defining property of
a stem cell. Retroviral marking, in vitro amplification, and serial
transplantation of human cells that can sustain long-term lymphomyelopoiesis in vivo have provided evidence that human
hematopoietic stem cell self-renewal occurs both in vitro and in vivo.
To investigate whether this process can be manipulated by cytokines, we
administered two different combinations of human growth factors to
sublethally irradiated nonobese diabetic/severe combined
immunodeficient (SCID) mice transplanted with
107 light-density human cord blood cells and then performed
secondary transplants to compare the number of transplantable human
lymphomyeloid reconstituting cells present 4 to 6 weeks
post-transplant. A 2-week course of Steel factor + interleukin (IL)-3 + granulocyte-macrophage colony-stimulating factor + erythropoietin
(3 times per week just before sacrifice) specifically and significantly
enhanced the numbers of transplantable human lymphomyeloid stem cells
detectable in the primary mice (by a factor of 10). Steel factor + Flt3-ligand + IL-6 (using either the same schedule or
administered daily until sacrifice 4 weeks post-transplant) gave a
threefold enhancement of this population. These effects were obtained
at a time when the regenerating human progenitor populations in such
primary mice are known to be maximally cycling even in the absence of growth factor administration suggesting that the underlying mechanism may reflect an ability of these growth factors to alter the probability of differentiation of stem cells stimulated to proliferate in vivo.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DISCOVERY OF THE ABILITY OF normal human
cells to engraft highly immunocompromised xenogeneic recipients at
experimentally useful efficiencies1-4 has paved the way for
the development of assays for quantitating transplantable human
hematopoietic stem cell frequencies using limiting-dilution analysis.
This is based on previous extensive validation of the use of this
approach for measuring murine stem cells in which the availability of
congenic donors and recipients allows sensitive, precise, and specific measurements of long-term lymphomyeloid repopulating cell frequencies in a variety of test populations.5-9 Because of the way
such transplantable hematopoietic stem cells are identified, we have proposed the term competitive repopulating unit (CRU) for their designation. A biologically similar type of human hematopoietic cell
(CRU) can be detected by its ability to generate both
lymphoid and myeloid progeny in the marrow of sublethally irradiated
(350 cGy) nonobese diabetic-scid/scid (NOD/SCID) mice after
their intravenous injection 6 to 8 weeks previously. We chose this
period to assess engraftment of the mice in our initial studies based
on the finding that maximum numbers of regenerated human lymphoid and
myeloid cells are detected at that time
post-transplant.3,10 Both DNA-11 and
fluorescence-activated cell sorting (FACS)-based detection
methods12 have been used to identify positive mice containing detectable populations of engrafted human cells with similar
sensitivities. However, FACS offers the additional capability of
identifying the different lineages of human cells produced. This
ensures the specificity of the assay by eliminating the detection of in
vivo repopulating cells that have acquired a distorted or constrained
differentiation potential, a situation that has been shown to occur
under certain circumstances; for example, when murine stem cells are
serially transplanted.13
Both retroviral marking14 and limiting dilution analysis
approaches12 indicate that single human CRU can generate
large numbers of lymphoid and myeloid progeny in immunocompromised
mice, even in the absence of injections of exogenous human cytokines. In vitro, these same cells can also proliferate and amplify their numbers but require stimulation by known growth
factors.12,15 Thus, at least some human stem cells can
execute self-renewal divisions in vitro. Recent studies indicate that
this can also occur in NOD/SCID and SCID mice transplanted with human
hematopoietic cells.10,16,17 However, the factors that may
influence this process in vivo are questions that are just beginning to
be addressed. Our initial results with secondary transplants suggested
that primary mice administered repeated injections of a combination of
human steel factor (SF), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Ep) in the 2 weeks before their sacrifice had greater (human) secondary repopulating
activity than parallel mice administered no human growth factors.
Moreover, this was observed even though there was no apparent growth
factor effect on the total numbers of human cells, or the numbers of
other types of human hematopoietic progenitors found in the primary
mice.10 We now report the results of additional experiments
designed to test the reproducibility of this preliminary observation
and to quantify the magnitude of the growth factor effects observed.
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MATERIALS AND METHODS |
Preparation of cord blood cells.
Cord blood cells were obtained with informed consent from mothers
undergoing normal full-term cesarean deliveries. The cells were
collected in tubes containing heparin and low-density (< 1.077 g/mL)
cells and were then isolated by centrifugation over Ficoll-Paque
(Pharmacia, Piscataway, NJ). After two washes in phosphate-buffered
saline (PBS), the cells were resuspended in fetal calf serum (FCS;
StemCell Technologies, Vancouver, BC, Canada) and DMSO (Sigma, St
Louis, MO.) added to a final concentration of 10% just before
aliquoting and cryopreservation at  135°C. As required, vials
of frozen cells, usually from two to three donors, were rapidly thawed
at 37°C and the suspensions then diluted slowly with Iscove's
medium containing 10% FCS (StemCell) and 0.25 mg/mL deoxyribonuclease
(type II-S, D4513; Sigma). These cells were then washed twice in
Iscove's medium + 10% FCS, counted, and the majority resuspended in
PBS for injection into irradiated NOD/SCID mice as described below. The
remainder were washed in Hanks' HEPES-buffered salt solution
containing 2% FCS for subsequent antibody staining for phenotype
analyses and progenitor assays.
Animals.
NOD/LtSz-scid/scid were bred and maintained in microisolators
in the animal facility of the British Columbia Cancer Research Center
(Vancouver, BC, Canada) from breeding pairs originally obtained from Dr
L. Schultz (The Jackson Laboratory, Bar Harbor ME). All animals were
kept and handled under sterile conditions, and provided with acidified
water (pH = 3), and sterilized food ad libitum. Six- to 8-week-old mice
were transplanted by intravenous injection within 24 hours after being
administered a sublethal dose of whole-body irradiation (350 cGy of
137Cs  -rays at a dose rate of ~1 cGy per minute).
Human recombinant growth factors, when administered, consisted of a
combination of either 10 U/mouse/injection of Ep (StemCell) +10
µg/mouse/injection of SF (Amgen, Thousand Oaks, CA) + 6 µg/mouse/injection each of IL-3 and GM-CSF (Novartis, Basel,
Switzerland), or 10 µg/mouse/injection of FL, (Immunex, Seattle, WA) + 10 µg/mouse/injection of SF and 2 µg/mouse/injection of IL-6
(Cangene, Mississauga, ON) intraperitoneally in a final volume of less
than 0.5 mL three times per week for the 2 weeks immediately preceding
sacrifice, or FL + SF + IL-6 at a 10-fold lower dose injected daily
from day 1 post-transplant until the mice were sacrificed 4 weeks
later.
Secondary transplants.
Both tibias and femurs of each primary recipient mouse were removed and
the total marrow content then flushed out using a syringe and a
26-gauge needle. After gently resuspending the cells in 2% FCS in
Hanks' balanced salt solution (HBSS; StemCell), in most cases, an
aliquot was removed for FACS analyses and progenitor assays. The
remainder were then pooled and equal aliquots (67% or 100% of the
equivalent of two femurs plus two tibias = 18% or 25%, respectively
of the equivalent of the total bone marrow of a mouse) injected into
secondary irradiated (350 cGy) recipients (usually three or four mice
per group). In a few experiments, the FACS analyses and progenitor
assays were performed on the pooled suspension.
Flow cytometry.
The initial cord blood cells and the cells harvested from the marrows
of both primary and secondary mice were stained at 4°C
with various murine antihuman monoclonal antibodies (MoAb) directly conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE)
as described10 at  107 cells/mL after lysis of
the red cells and pretreatment of the suspension with human serum and
an antimouse IgG receptor antibody (2.4G218) to block human
and mouse Fc receptors. After staining, the cells were washed once in
2% HBSS and then once again in the same medium containing 2 µg/mL
propidium iodide (PI) to identify nonviable cells. Cells were analyzed
and sorted on a FACStar Plus (Becton Dickinson) also as
described.3 Controls consisted of staining additional
aliquots of the same cells with irrelevant isotype-matched control
antibodies directly labeled with the identical fluorochromes. When
mouse-human cell mixtures were being analyzed, marrow cells from
untreated NOD/SCID mice were stained with the same antibodies in
parallel, to ensure nonreactivity of the antibodies used with murine
cells. A minimum of five positive events from a viable
(PI ) population of 5000 cells at settings excluding
greater than 99.9% of all negative controls was adopted as the minimum
criterion for deriving positive values for any particular human
phenotype. Total numbers of human cells were determined as those
staining positive with a combination of anti-CD45 and anti-CD71
antibodies, human B cells were identified as
CD34CD19+ cells, and human myeloid cells
were identified as CD19CD15+ cells. From
the CD45/71+ population, the CD34+ subset was
also sorted and used to initiate colony-forming cell (CFC)
and long-term culture-initiating cell (LTC-IC) assays, as described
below.
Progenitor assays.
Myeloid (CFU-GM), erythroid (BFU-E and CFU-E), and multilineage
(CFU-GEMM) progenitor numbers were determined by plating cells in 0.8%
methylcellulose medium containing 30% FCS, 1% bovine serum albumin
(BSA), 104 mol/L  -mercaptoethanol in Iscove's
medium (Methocult 4435; StemCell), supplemented with 3 U/mL human Ep,
50 ng/mL SF, and 20 ng/mL each of IL-3, IL-6, GM-CSF, and G-CSF (Terry
Fox Laboratory, Vancouver, BC, Canada), and counting the colonies
obtained after 16 days of incubation at 37°C.19
LTC-IC numbers were determined by plating another aliquot of cells in
long-term culture medium (Myelocult; StemCell), to which 106 mol/L of freshly dissolved hydrocortisone sodium
hemisuccinate (Sigma) was added just before use, on top of preformed
feeder layers of murine fibroblasts previously engineered to produce SF, G-CSF, and IL-3.19 These cultures were maintained for 6 weeks at 37°C with weekly half-medium changes and were then
harvested and assayed for their CFC content. LTC-IC numbers were
calculated from the CFC numbers detected in these assays assuming each
LTC-IC (of cord blood origin) will produce, on average, ~30
CFC.19
General experimental design.
The effect of injecting different combinations (and schedules) of human
growth factors on the ability of human cells from primary NOD/SCID mice
to engraft secondary NOD/SCID recipients was studied in a total of six
experiments. (Table 1, experiments one to
four; Table 2, experiments one and two.) In
each experiment, groups of from 4 to 12 primary, sublethally irradiated
NOD/SCID mice were injected intravenously with 107
light-density cells from a pool of several human cord blood collections and then half of the mice were injected with growth factors (and the
rest not), as indicated. All primary mice in each experiment were
sacrificed at the same time between 4 and 6 weeks post-transplant (when
human LTC-IC numbers have been shown to reach maximum and subsequently
stable values in this model10) and their marrow cells were
then isolated and injected into groups of secondary recipients as
described above. The total level of repopulation of the marrow of the
primary mice with human (CD45/71+) cells in these six
experiments was 42% ± 4%, which gave similar absolute numbers of
human cells as reported previously.10
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Table 1.
Increased Numbers of Human Progenitors Detected in
Secondary Recipients of Cells from Primary Recipients Administered
Human SF, IL-3, GM-CSF, and Ep
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Table 2.
Increased Numbers of Human Progenitors Detected in
Secondary Recipients of Cells From Primary Recipients Adminsitered
Human FL, SF, and IL-6
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RESULTS |
Enhanced engraftment of secondary NOD/SCID mice with cells from primary
mice administered human SF, IL-3, GM-CSF, and Ep.
Figure 1 shows a comparison of the FACS
profiles obtained from the marrows of individual secondary mice
injected 6 weeks previously with marrow cells from primary mice that
had been administered either no growth factors or a 2-week course of
six injections of SF, IL-3, GM-CSF, and Ep during the 2-week period
just before sacrifice. To illustrate the enhancing effect of the growth
factor treatment of the primary mice, what is shown in Fig 1 is the
result obtained in the secondary mouse that contained the highest
frequency of human cells in each of the two groups of secondary mice in a representative experiment. In the experiment shown (Experiment 4 in
Table 1), all four of the secondary recipients of cells from the growth
factor-injected primary mice contained human lymphoid (CD34CD19+) cells. Three also contained
human myeloid (CD19CD15+) cells and all
four contained CD34+ CFC. In contrast, only three of the
four secondary recipients of an equivalent portion of the marrow from
the corresponding control primary mice contained detectable numbers of
human lymphoid cells and only one contained detectable numbers of human
myeloid cells or progenitors.
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| Fig 1.
FACS dot plots showing the frequencies of human
CD45/71+ (total) and human CD34+
(primitive) hematopoietic cells (left panels) and the frequencies of
human CD19+ (B-lymphoid) and CD15+
(granulopoietic) cells (right panels) in the most-highly engrafted
secondary mice injected with equivalent transplants of cells obtained
from primary mice engrafted with human cord blood cells and
administered a 2-week course of SF, IL-3, GM-CSF, and Ep (Mouse 2) or
not (Mouse 5) in a representative experiment.
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Similar results were obtained in another three experiments in which the
effect of this course of growth-factor treatment of the
primary mice was investigated. The combined data from all four experiments are summarized in Fig
2. Table 1 shows the numbers of human CFC and LTC-IC detected in the
CD34+ cell populations isolated from the marrow
of the same secondary mice. CD34+ CFC were found in all but
1 of the 13 mice transplanted with marrow from the growth
factor-injected primary mice. In 5 of these, LTC-IC were also readily
detected. In contrast, none of the 13 secondary recipients of marrow
from the control group of primary mice contained detectable numbers
of human LTC-IC and in only 2 of these were human CFC
identified. Interestingly, the growth factor enhancement of
the secondary transplants was not associated with any difference
in the total cellularity of the bone marrow of the primary
recipients: (1.8 ± 0.2) × 107 versus (2.3 ± 0.3) × 107 cells per four long bones were measured in
the two groups. There was also no difference per primary mouse in the
total numbers of human CD45+ CD71+ cells, or
human B cells, or CD34+ cells, or CFC or LTC-IC
present (data not shown), as expected.10
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| Fig 2.
Comparison of the total number of human cells of the
phenotypes shown found to be present in the tibias and femurs of mice
transplanted with cells obtained from the marrow of primary mice
transplanted with human cord blood cells and administered a 2-week
course of SF, IL-3, GM-CSF, and Ep (solid symbols) or not (open
symbols). Data pooled from the four experiments described in Table 1.
Each symbol denotes an individual secondary mouse. The corresponding % human (CD45/71+) cell repopulation values for the marrows
of these secondary mice that contained greater than 0.1% human cells
ranged from 0.3% to 12%.
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Enhanced engraftment of secondary NOD/SCID mice with cells from
primary mice administered human FL, SF, and IL-6.
Two additional experiments were performed to investigate the potential
effect of a different combination of growth factors (ie, FL, SF, and
IL-6) on the same endpoints. These cytokines were administered either
according to the same 2-week schedule of three injections per week (n = 1) or daily throughout the post-transplant period at a 10-fold lower
dose per injection (n = 2). In these experiments, the primary mice were
sacrificed after 4 weeks, by which time they had received approximately
half the overall dose of growth factors injected into the mice that
were administered during the 2-week course of injections. This daily
schedule was explored based on the observation that serum levels of
human SF, IL-3, and GM-CSF (particularly the latter two) measured in
the previous experiments, were found to decline precipitously after injection and, in all cases, to less than 5 ng/mL within 24 hours (Table 3).
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Table 3.
Levels of Human Growth Factors Present in the Plasma
of Cord Blood Transplanted Mice at Varying Intervals After
Injection
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An increased level of repopulation of every type of human hematopoietic
cell examined was consistently observed in the secondary recipients of
cells from all primary mice administered FL, SF, and IL-6, regardless
of the dose administered or schedule used (Fig 3 and Table 2). As was found with the
other growth factor combination, there was not a comparable difference
(P > .05, Student's t-test) in the total number of
human cells or human CD34CD19+
(B-lineage) cells observed in the marrows of the primary mice from
which the secondary transplants were obtained. On the other hand, in
this case the total number of CD34+ cells in the primary
mice administered FL, SF, and IL-6 was consistently lower (P < .05) than in the controls administered no human growth factors, and
the primary mice administered the higher dose of FL, SF, and IL-6 over
a 2-week period contained fewer human CFC (~fivefold reduced,
P < .05) and more human LTC-IC (~fourfold higher than the
primary mice administered no growth factors [P = .3], Fig
4). However, because these represent data
from a single experiment, additional studies will be required to
confirm their reproducibility. Comparison of the total numbers of human
cells, or human B cells or CD34+ cells regenerated in
secondary recipients of cells from mice administered the two different
cytokine combinations (Fig 2 versus Fig 3) suggests that the
combination of FL, SF, and IL-6 may be slightly less effective (given
that the cells from the control groups gave similar outcomes). However,
comparison of the CFC and LTC-IC populations regenerated in the same
groups of secondary mice suggests that injected human FL, SF, and IL-6
injections may selectively enhance the subsequent output of these
earlier cell types.
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| Fig 3.
Comparison of the total number of human cells of the
phenotypes shown found to be present in the tibias and femurs of mice
transplanted with cells obtained from the marrow of primary mice
transplanted with human cord blood cells and administered either no
further treatment (open symbols) or FL, SF, and IL-6 (solid symbols) at
a low dose, daily (A), or at a high dose three times per week for 2 weeks (B). Data pooled from the two experiments described in Table 2.
Each symbol denotes an individual secondary mouse. The corresponding % human (CD45/71+) cell repopulation values for the marrows
of these secondary mice that contained greater than 0.1% human cells
ranged from 0.1% to 12%.
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| Fig 4.
Comparison of the number of different phenotypically and
functionally defined human hematopoietic cells detected in the tibias
and femurs of mice 4 weeks after being transplanted with
107 light-density human cord blood cells and then
administered no further treatment ( ) or FL, SF, and IL-6 injected
daily at a low dose ( ), or three times per week for the 2 weeks
before sacrifice at a high dose ( ). Values are the mean ± standard
error of mean (SEM) of results obtained from three to seven individual
mice in the three experiments described in Table 2.
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Limiting dilution analysis shows a significant effect of human growth
factors in vivo on the numbers of transplantable human lymphomyeloid
repopulating cells regenerated in primary NOD/SCID mice.
Most of the groups of secondary recipients analyzed in these
experiments included some mice that did not contain any detectable (ie, < 0.1%) human cells plus others that did. Therefore it was possible to use these data to derive repopulating cell
frequencies by applying Poisson statistics.20 To determine
the incidence of transplantable human cells with lymphomyeloid
repopulating ability, ie, CRU,12 the same endpoints as
previously validated for quantifying these cells in freshly isolated
suspensions of human cord blood were adopted. These require that both
human CD34+ CFC (either BFU-E and/or CFU-GM
and/or CFU-GEMM) and human
CD34CD19+ (B-lymphoid) cells (> 5 per 5000 viable cells analyzed) be present for a mouse to be
considered as positive.12 Using these criteria, human CRU
frequencies and, hence, total human CRU numbers in the marrow of the
various groups of primary mice evaluated in the present study could be
calculated. As indicated in Table 4, the marrow of mice administered a 2-week course of SF, IL-3, GM-CSF, and Ep
had a 10-fold higher content of human CRU than those administered no
growth factors (P < .001). The effect of the FL, SF, and IL-6 injections (assuming no effect of the delivery protocol) on human CRU
output was less (threefold increase) and did not quite
reach statistical significance (P = .1).
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Table 4.
Frequency of Human CRU in Secondary Recipients of Marrow
from Control and Growth Factor-Injected Primary Mice
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DISCUSSION |
Current investigations of the molecular mechanisms regulating human
hematopoietic stem cell populations face three challenges. The first is
the development of quantitative assays for the cells of interest with
sufficient sensitivity, specificity, precision, and convenience to be
experimentally useful. Available evidence from the murine studies
suggest that the CRU assay, originally developed for determining murine
stem cell frequencies5,21 meets this first requirement.
This assay has recently been adapted for human cells using sublethally
irradiated NOD/SCID mice11,12 or C12
MDP-liposome-treated SCID mice17 as recipients.
Nevertheless, improved sensitivity of these procedures and further
characterization of the cellular phenotypes they detect4,22
is still needed.
A second challenge is to identify those external factors that can
influence (either positively or negatively) the viability and/or proliferative activity of human hematopoietic cells with stem cell properties. Again, such studies are well advanced in the
murine system in which retroviral marking of highly purified CRU
populations has provided the most convincing evidence of their proliferation in response to specific growth factors in
vitro,23,24 although factors capable of either
amplifying9 or inhibiting25 murine CRU in
cultures of highly purified input populations have also been reported.
Similar retroviral marking data now exists for human
cells.14,26-29 However, such approaches are not well suited
to systematic analyses of the roles of specific factors (or factor
combinations) on human stem cell proliferation and self-renewal.
A third and major challenge is the delineation of the intracellular
targets and sequence of events that influence whether a hematopoietic
stem cell will begin to differentiate (irreversibly) or not.
Ultimately, this process is believed to involve a complex series of
changes in gene transcription, whose regulation in turn depends on key
transcription factors and their activities. Although some of these have
now been identified,30,31 very little is as yet known about
the types of interactions they engage in or how these are regulated.
However, it is interesting to note that the types and concentrations of
growth factors to which a stem cell is exposed can influence its
probability of self-renewal independent of its mitogenic
stimulation.32 On the other hand, no set of in vitro
conditions has yet been found to reproduce the extent of hematopoietic
stem cell expansion achieved in vivo either during
ontogeny,33 or after the transplantation of stem cells into
irradiated recipients.6,34-36 Thus, delineation of in vivo
mechanisms that sustain hematopoietic stem cell proliferation and
self-renewal remain of great interest.
The present studies have confirmed that this process can take place
when human hematopoietic stem cells (identified as CRU) engraft the
marrow of xenogeneic recipients, in this case, irradiated NOD/SCID
mice. The number of CRU injected into the primary mice analyzed here
would have been approximately 10, based on our previous estimates of
CRU numbers in human cord blood.12 This means that, in the
absence of human growth factor injections, this number of injected
human CRU resulted in the regeneration of at least 10% of the number
initially injected (assuming a 100% detection efficiency). With
appropriate growth factor injections this number could be increased a
further 10-fold (Table 4), ie, to a level at least comparable to the
input value. In murine recipients of syngeneic cells, the detection
efficiency of CRU can be estimated to be on the order of 10% to 20%
because CRU purities of this level can be achieved.13,37-39
The frequency of CRU in the CD34+CD38
population isolated from human cord blood, as determined by assaying the cells in NOD/SCID mice, is 0.1% to 0.2%.12,40
Although it is unlikely that CD34+CD38
cord blood cells are functionally homogeneous, factors inherent in the
assessment of human stem cells in mice probably also reduce considerably their efficiency of detection. We have previously measured
the actual proportion of various subpopulations of injected human cells
that are present in the marrow of NOD/SCID mice 2 to 3 days
post-transplant, including human LTC-IC.10 This value was
consistently found to be less than 1%. Thus, the numbers of CRU
measured 4 to 6 weeks later are likely to represent a greater than 10- to 100-fold regeneration of the injected CRU population. Secondary
transplants performed early after transplantation of primary mice with
human cord blood cells would provide more precise information about the
seeding efficiency of human CRU in NOD/SCID mice and hence allow the
overall expansion that occurs in the 4- to 6-week post-transplant
period to be more accurately determined.
Although the present studies clearly show an enhancing effect of
certain human growth factors on CRU expansion in vivo (in NOD/SCID
mice), they do not address the underlying mechanism or even whether the
effects observed are directly or indirectly mediated. Interestingly,
Verstegen et al17 have recently reported that the
production of human CD34+CD38 cells from
CD34+CD38 cells transplanted into a
similar mouse model is dependent on the cotransfer of other human
CD34+ cells. These authors suggest that the coinjected
human CD34+ cells are most likely to act by serving as a
source of essential growth factors. In the present studies, potential
effects of repeated growth factor injections on the number of human
CD34+CD38 cells regenerated in primary
recipients of unfractionated human cord blood cells were not
investigated. However, it should be noted that the effects observed on
CRU numbers did not extend to any other human cell type assessed,
including LTC-IC. This reinforces the concept that the CRU and LTC-IC
assays do not measure the same functions and hence may not necessarily
detect the same cell populations according to their extent of overlap
at the single-cell level. The fact that a 2-week course of SF, IL-3,
GM-CSF, and Ep seemed to be more effective than the combination of FL,
SF, and IL-6 administered according to the same protocol was also not
anticipated. A recent analysis of the effects of different growth
factor combinations on adult marrow and cord blood LTC-IC amplification41 led us to anticipate that the first
combination of growth factors would be suboptimal, and better results
were expected with FL, SF, and IL-6. However, the effects of these on
human cord blood CRU amplification in vivo were reversed (Table 2).
Enhanced regeneration of transplanted murine CRU has also been obtained
in recipients of cells transduced with a HOX B4-encoding retroviral
vector,35 in mice transplanted with low numbers of normal
CRU,6 in mice transplanted with CRU of fetal
origin,6,7 and in recipients of normal adult marrow cells
followed by repeated injections of SF and IL-11.42
Moreover, in none of these situations was the mature blood cell output
affected and, in the latter case, the effects obtained with the
injections of SF and IL-11 were attributed to a selective ability of
these two growth factors to promote stem cell self-renewal. This
conclusion was based on evidence that the stem cells were proliferating
maximally at the time of assessment in the absence of the growth factor
injections. It is, therefore, inviting to speculate that a similar
mechanism may underlie the growth factor effects observed here on human stem-cell regeneration in vivo. Further experiments to test this hypothesis are now underway. If confirmed, these would indicate a
previously unappreciated and clinically significant use of growth factors to selectively modulate hematopoietic stem cell proliferation and self-renewal in vivo as well as in vitro.32
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ACKNOWLEDGMENT |
The authors thank Jessyca Maltman and Maya Sinclaire for assistance
with the animal work, Gayle Thornbury and Giovanna Cameron for
assistance in cell sorting, the staff of the Stem Cell Assay Service
for initial processing of the cord bloods, and Bernadine Fox for
manuscript preparation. The authors also thank Amgen, Cangene, Immunex,
Novartis, StemCell, and Dr Peter Lansdorp (Terry Fox Laboratory) for
generous gifts of reagents.
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FOOTNOTES |
Submitted June 4, 1998;
accepted August 27, 1998.
Supported by grants from the National Cancer Institute of Canada
(NCIC, with funds from the Terry Fox Run), from the NIH (NHLBI POI-5545), and from Novartis. C.J.E. is a Terry Fox Cancer Research Scientist of the NCIC.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Connie J. Eaves, PhD, Terry Fox Laboratory,
601 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada; email:
connie{at}terryfox.ubc.ca.
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REFERENCES |
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Abstract
Introduction