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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 6 (September 15), 1999:
pp. 1926-1932
Rapid Differentiation of a Rare Subset of Adult Human
Lin CD34CD38 Cells
Stimulated by Multiple Growth Factors In Vitro
By
Tomoaki Fujisaki,
Marc G. Berger,
Stefan Rose-John, and
Connie J. Eaves
From the Terry Fox Laboratory, British Columbia Cancer Agency, and
the University of British Columbia, Vancouver, British Columbia,
Canada; and I. Medizinische Klinik, Johannes Gutenberg Universitat
Mainz, Mainz, Germany.
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ABSTRACT |
Recently, several reports of lineage-negative (lin )
CD34 cells with in vivo hematopoietic activity have
focused interest on the properties and growth factor response
characteristics of these cells. We have now identified a combination of
5 growth factors that are necessary and sufficient to stimulate a
marked mitogenic and differentiation response by a subset of human
linCD34CD38 cells present
in normal adult human marrow and granulocyte colony-stimulating factor
(G-CSF)-mobilized blood. Less than 0.1% of the cells in highly
purified (including doubly sorted)
linCD34CD38 cells from
these 2 sources formed colonies directly in semisolid medium or
generated such cells after 6 weeks in long-term culture. Nevertheless,
approximately 1% of the same
linCD34CD38 cells were
able to proliferate rapidly in serum-free liquid suspension cultures
containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper-IL-6 to produce a net 28- ± 8-fold increase in total cells within 10 days. Of the cells present in these
10-day cultures, 5% ± 2% were CD34+ and 2.5% ± 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or
multilineage colony-forming cells (CFC) (13 ± 7 CFC per
linCD34CD38 pre-CFC). In
contrast to linCD34+CD38
cells, this response of
linCD34CD38 cells required
exposure to all of the 5 growth factors used. Up to 1.7 × 105 linCD34 adult marrow
cells failed to engraft sublethally irradiated
NOD/SCID- 2M/ mice. These studies
demonstrate unique properties of a rare subset of
linCD34CD38 cells present
in both adult human marrow and mobilized blood samples that allow their
rapid proliferation and differentiation in vitro within an overall
period of 3 to 4 weeks. The rapidity of this response challenges
current concepts about the normal duration and coordinated control of
these processes in adults.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
PRESENT DAY understanding of
hematopoietic cell differentiation in the normal adult is based on
evidence of an orderly sequence of changes in gene expression occurring
over many cell generations. These are thought to lead first to lineage
restriction and ultimately allow the production of large numbers of
mature blood cells from a relatively small pool of stem cells. Such a hierarchical model is supported by an observed association of changes
in the phenotype, proliferative potential, and differentiation ability
of progenitors defined by various in vitro assays.1 Of
particular interest to investigations of the molecular basis of this
model has been the characterization of cells capable of sustaining the
output of all blood cell lineages for long periods of time. Such cells
are also of considerable practical relevance, because they include
cells responsible for permanent engraftment in transplant recipients
and are also the desired targets for many gene therapy strategies. In
humans, expression of the CD34 antigen has been shown to identify a
small population of hematopoietic cells that include most classes of
multipotent as well as early committed lymphoid and myeloid
progenitors.2 Purified human CD34+ cells have
also been shown to reconstitute both lymphoid and myeloid compartments
in autologous3 as well as allogeneic4 and
xenogeneic5-7 recipients. Thus, expression of CD34 has come to be accepted as a hallmark of transplantable human hematopoietic stem
cells. However, in mice, CD34/lo hematopoietic stem
cells also exist,8-10 and recently evidence of analogous
cells in other species, including humans, has been reported.11-13 Interestingly, in large animals, very few
lineage-negative (lin) CD34 cells
could be detected directly as colony-forming cells (CFC) in semisolid
media or as their stromal cell-responsive precursors in long-term
culture assays (referred to as long-term culture-initiating cells
[LTC-IC]).11,13
In previous studies, we and others have shown that the most primitive
CD34+ cells of human origin have different growth factor
requirements from those able to stimulate their more differentiated
progeny. These include differences both in the types and concentrations of the factors to which the cells are exposed.14-19 These
investigations identified flt-3 ligand (FL), Steel factor (SF),
interleukin-3 (IL-3), thrombopoietin (TPO), and factors that activate
gp130 as potential contributors to the stimulation of very early human hematopoietic cells. In the present study, we used hyper-IL-6 (H-IL-6) to activate gp130. H-IL-6 is a recombinant growth factor fusion protein in which human IL-6 and its soluble receptor (sIL-6R) are linked by a flexible linker peptide.20 On the basis of
preliminary results suggesting that adult human sources of highly
purified linCD34 cells might
proliferate and differentiate in response to a combination of these
five growth factors, additional studies were designed to confirm and
further characterize the cell types produced. The present report
describes the results of these studies.
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MATERIALS AND METHODS |
Cells.
Normal adult human bone marrow cells were obtained either from donors
of allogeneic marrow transplants at our center or cadaveric donors
(Northwest Tissue Center, Seattle WA). Granulocyte colony-stimulating factor (G-CSF)-mobilized blood cells were from patients undergoing leukapheresis at our center for autologous transplantation. All cells
were obtained with informed consent according to institutional guidelines. Low-density cells (<1.077 g/mL) were isolated using Ficoll-Paque (Pharmacia, Uppsala, Sweden) and resuspended in Hank's HEPES-buffered salt solution containing 2% fetal calf serum (HFN; StemCell Technologies, Vancouver, British Columbia, Canada).
Flow cytometry.
Low-density marrow or blood cells were incubated for 30 minutes at
4°C with a cocktail of monoclonal antibodies against the following
lin markers: CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b, and
glycophorin A. These markers were previously conjugated to
antidextran antibodies (StemCell) and then with magnetic dextran-iron particles for another 30 minutes at 4°C. Labeled cells were removed using a StemSep column and magnet (StemCell) and the
lin cells collected in the flow through according to
the manufacturer's directions. These cells were then incubated for 30 minutes with monoclonal antibodies specific for CD34 (8G12) conjugated
with fluorescein isothiocyanate (FITC; kindly provided by Dr P. Lansdorp, Terry Fox Laboratory, Vancouver, British Columbia, Canada)
and CD38 (Leu-17) conjugated with R phycoerythrin (PE; Becton
Dickinson, San Jose, CA) at 10 and 2.5 µg/mL, respectively. Cells
were finally washed once in HFN and once again in HFN with 2 µg/mL
propidium iodide (PI; Sigma Chemical Co, St Louis, MO) before being
sorted on a FACStar Plus (Becton Dickinson) equipped with a 5-W argon laser and a 30-mW helium laser. PI cells with low to
medium forward and low side light scattering characteristics and a
CD34CD38 phenotype were collected
in Iscove's medium supplemented with a serum substitute for immediate
culture (see below) or in HFN if they were first to be
resorted, as indicated. CD34+ cells were
collected simultaneously and the
CD34+CD38 subpopulation was subsequently
isolated. For assessment of in vivo repopulating activity,
lin cells were stained with anti-CD34-PE (Becton
Dickinson) and anti-CD7-FITC (M-T701; Pharmingen, Mississauga, Ontario,
Canada), and CD34CD7+ and
CD34CD7 populations were
separately collected (<5% and >95% of all
linCD34 cells, respectively).
Data acquisition and analysis was performed with PC-lysis software
(Becton Dickinson). Gates defining negative populations were set using
FITC- and PE-conjugated isotype controls and included 99.9% of cells
reactive with these antibodies.
Progenitor assays.
Cells were plated at suitable frequencies in methylcellulose medium
with 30% fetal calf serum (FCS) or a serum substitute (MethoCult H4230
and H4236, respectively; StemCell) supplemented with 40 µg/mL
low-density human serum lipoproteins (Sigma; for H4236 only) and,
unless otherwise indicated, with 3 U/mL human erythropoietin
(StemCell), 50 ng/mL of SF (Terry Fox Laboratory), and 20 ng/mL each of
IL-3 (Novartis, Basel, Switzerland), IL-6 (Cangene, Mississauga, ON),
G-CSF (StemCell), and granulocyte-macrophage colony-stimulating factor
(GM-CSF; Novartis) to assess their direct granulopoietic,
erythropoietic, and multilineage CFC content, as
described.14,15 Additional aliquots were assayed for cells able to generate colonies of CD41+ megakaryocytes in
serum-free collagen cultures (MegaCult-C; StemCell) containing 50 ng/mL
of TPO and 10 ng/mL each of IL-3 and IL-6 as recommended by the
supplier, using a modified procedure originally developed for agarose
cultures.21 For LTC-IC assays, cells were cocultured for 6 weeks at 37°C with pre-established, irradiated feeder layers of
mouse fibroblasts engineered to produce human IL-3, G-CSF, and
SF.22 These LTC were maintained in a medium consisting of
MyeloCult (H5100; StemCell) supplemented with freshly dissolved
106 mol/L hydrocortisone sodium hemisuccinate
(Sigma) with weekly half medium changes.22 At the end of 6 weeks, a single cell suspension was prepared of the whole culture and
the cells were then assayed for CFC.
Serum-free liquid cultures.
Cells were cultured in 100 µL of phenol red-free Iscove's medium
supplemented with 10 mg/mL of bovine serum albumin, 10 µg/mL of
bovine insulin, and 200 µg/mL of human transferrin (BIT 9500; StemCell) plus 40 µg/mL of human low-density lipoprotein (Sigma), 2 mmol/L L-glutamine (Sigma), 104 mol/L
2-mercaptoethanol (Sigma), and growth factors, as indicated, at the
following final concentrations: SF at 100 ng/mL, FL (Immunex Corp,
Seattle, WA) at 100 ng/mL, TPO (Genentech, San Francisco, CA) at 50 ng/mL, IL-3 at 20 ng/mL, and H-IL-6 prepared and purified as
described20 at 10 ng/mL. After 10 days of incubation, cells were assayed for CFC and/or CD34 expression after labeling with anti-CD34-FITC and analysis on a FACScan flow cytometer (Becton Dickinson) using the same controls and gating criteria as for cell sorting.
Limiting dilution assays (LDA).
Multiple serum-free suspension cultures containing all 5 cytokines were
set up with varying numbers of doubly sorted (from 10 to 500)
linCD34CD38
cells in 20 µL of medium in the individual wells of a 60-well Terasaki microwell plate (6 to 12 replicates per cell dose). After 10 days (unless specified otherwise), each well was examined for the
presence of viable (refractile) cells and then assayed individually for
CFC. Wells were scored as positive for growth factor responsiveness when approximately 20 viable cells (or more) could be seen on day 10. All other wells were scored as negative and usually contained no
detectable cells. Wells were scored as positive for pre-CFC whenever
 1 CFC was detected. The frequencies of growth factor-responsive cells
and of pre-CFC in the
linCD34CD38
cells used to initiate the cultures were calculated from the proportion
of negative wells obtained with different input cell numbers based on
Poisson statistics and the method of maximum likelihood23
using the L-Calc software program (StemCell).
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RESULTS |
Frequency of CD34CD38 cells in
normal adult marrow and mobilized blood.
Lin low-density cells isolated from 5 normal adult
marrow and 8 G-CSF-mobilized blood samples were sorted into
CD34CD38, CD34+, and
CD34+CD38 populations as described in
Materials and Methods and shown in Fig 1.
From such analyses, the size of each population was obtained and the
corresponding number of cells present in the original low-density
fraction calculated (assuming 100% recovery of each population in the
lin fraction). As summarized in
Table 1,
CD34CD38 cells were 6- to 9-fold
less numerous than CD34+ cells, similar in frequency to the
CD34+CD38 cells, and constituted
approximately 1 in 300 cells of the low-density population obtained
from either marrow or mobilized blood. However, because the yield of
all cells after removal of the lin+ cells was much higher
for marrow than for the mobilized blood cell populations (5% ± 2%
v 0.5% ± 0.4%), the yield of
linCD34CD38
marrow cells was also higher. Comparison of the frequencies of CD34+CD38 and
CD34CD38 cells in individual
samples showed that the relative sizes of these 2 populations were not
significantly correlated (R = .2, P > .05).
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| Fig 1.
A representative FACS profile of normal adult human
lin bone marrow cells after staining with CD34-FITC and
CD38-PE. Lineage marker-positive cells were removed using a StemSep
column and the flow through (lin) cells were then
labeled. Dead (PI+) cells were excluded from the
analysis. The left panel shows the level of nonspecific staining
obtained with isotype control antibodies. The right panel shows the
result obtained after staining with anti-CD34-FITC and anti-CD38-PE.
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Table 1.
Proportions of Light Density and Lin
Marrow and Mobilized Blood Cells That Are
LinCD34+,
LinCD34+CD38, or
LinCD34CD38
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Progenitor content of
linCD34CD38
cells.
CFC and LTC-IC (6-week) assays were performed on the
linCD34CD38
cells isolated from 2 of the mobilized blood samples and 5 of the
normal marrow samples (Table 2). The
results showed these to be undetectable (<0.1%) in most instances.
Based on the results obtained with the initial samples studied, the
total number of cells assessed from the later samples analyzed was
increased from 1 or 2 × 103 cells to 104
cells. Although a few granulocyte-macrophage colonies were then obtained in direct CFC assays, the frequency of CFC in the
linCD34CD38
fraction was still less than 0.1%. This value is more than 10× lower than the frequency of CFC detectable in the
linCD34+CD38
population present in normal adult human bone marrow24 and is also somewhat lower than that reported by Bhatia et al13 for human cord blood linCD34
CD38 cells, which we have confirmed (unpublished
findings). Because we found that the same
linCD34CD38
marrow cells could proliferate in liquid suspension cultures containing
a serum substitute instead of FCS and a different combination of growth
factors (than what had been added to the methylcellulose cultures used
to detect CFC; see below), cells from 2 of the marrow samples were also
assayed in methylcellulose medium of identical composition to the
medium used in the liquid cultures. However, even under these
conditions, no proliferation by up to 2 × 103
linCD34CD38
cells in semisolid medium was seen. In LTC-IC assays, 6 of 7 samples of
linCD34CD38
cells (2 blood samples and 4 marrow samples) were also negative. However, in 1 case (BM4 in Table 2), several hundred CFC were generated
from 5,000 linCD34CD38
cells. Note that the frequency of LTC-IC in the matching
linCD34+CD38 cells
isolated from this sample was less than 1%, in contrast to BM3, in
which 10% of the
linCD34+CD38 cells
were LTC-IC (see Table 5, below). The large disparity in LTC-IC content
of the CD34+ and CD34 subsets of
linCD38 cells (compare results
shown in Tables 2 and 5) argues strongly against the likelihood
of cross-contamination as an explanation for a low but measurable
frequency of CD34CD38 LTC-IC and
emphasizes the functional differences between the linCD34+CD38 and
linCD34CD38+
populations in adult marrow.
Production of CD34+ cells and CFC in 10-day liquid
cultures.
In serum-free liquid cultures containing FL, SF, TPO, IL-3, and
H-IL-6,
linCD34CD38
cells proliferated extensively to yield (28 ± 8) × 103 PI cells per 103 input
cells (n = 4). When analyzed for CD34 expression, 5.4% ± 1.6% (n = 4) of these 10-day progeny were CD34+
(Fig 2), resulting in a corresponding yield
after 10 days of 1,700 ± 250 CD34+ cells per
103 initial
linCD34CD38
cells.
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| Fig 2.
A representative FACS histogram showing the proportion of
CD34+ cells in the population generated by stimulating
linCD34CD38 marrow cells
for 10 days with FL, SF, TPO, IL-3, and H-IL-6.
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In 2 experiments in which the generation of cells detectable as CFC was
assessed, parallel serum-free cultures were set up with either
103
linCD34CD38
cells or 103
linCD34+CD38 cells
isolated from the same samples. The effect of adding 3 different growth
factor combinations on the progenitors obtained from each of these
cultures 10 days later was then compared. As expected,14,15,18,19 FL + SF + IL-3, FL + TPO + H-IL-6,
and the combination of all 5 of these growth factors stimulated the production of large numbers of CFC from the
linCD34+CD38 cells in
both experiments (Table 3). Readily
detectable numbers of CFC were also present in 10-day cultures
initiated with
linCD34CD38
cells (n = 9), but only when all 5 growth factors were present (n = 2).
In an additional experiment (BM3 in Table 3), it was shown that
omission of IL-3 reduced the number of CFC produced by adult
linCD34CD38
cells approximately 3-fold and that omission of any 1 of the other 4 factors eliminated CFC generation.
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Table 3.
Production of CFC From
LinCD34CD38 Adult Human
Marrow or Mobilized Peripheral Blood Cells in 10-Day Cultures
Containing Various Cytokines
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The colonies obtained in the CFC assay of cells produced in vitro by
linCD34CD38
cells that had been stimulated with the 5 growth factor combination contained either erythroblasts, granulocytes, macrophages, or megakaryocytes exclusively, or mixtures of these. In many cases, but
not all, these colonies grew to a large size within the 2- to 3-week
CFC assay period (Table 4). Thus, complete
maturation along all of the major myeloid lineages could be
reproducibly achieved within an overall period of 3 to 4 weeks of
exposure of
linCD34CD38
cells and their progeny to an appropriate sequence of defined growth
factors.
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Table 4.
Distribution of Different Types of Progenitors Present
in 10-Day Cultures Initiated With
LinCD34CD38 Adult Human
Marrow or Mobilized Peripheral Blood Cells
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Resorting of the initial
linCD34CD38
cells isolated from 4 different marrows showed the purity of the first
sort to be consistently greater than 98%. Moreover, the number (Table
3) and types (Table 4) of CFC generated from resorted samples were
similar to what had been obtained from cells isolated using a single
sort. In 2 experiments, 1 of several replicate cultures of
103
linCD34CD38
cells each was harvested and assayed for CFC after 5 and 7 as well as
10 days of incubation. The results of these assays showed that no CFC
could be detected after 5 days and that near maximum numbers were
already detectable by 7 days.
The frequency of
linCD34CD38
cells able to proliferate in liquid cultures within 10 days in response
to stimulation by FL, SF, TPO, IL-3, and H-IL-6, as well as the
frequency of those with pre-CFC activity, was determined for 4 of the
marrow samples by LDA (see Materials and Methods) of doubly sorted
cells. As shown in Table 5, the frequencies
of such cells varied over a 40-fold range between marrow samples (as
did the frequency of LTC-IC in the
linCD34+CD38 cells
from the same samples), with an average frequency of growth factor-responsive
linCD34CD38
cells and pre-CFC of approximately 1 per 100 and approximately 1 per
200, respectively. (CFC were detected in only ~50% of the wells and
only in wells observed to contain viable cells). The results of a
representative LDA experiment are shown in
Fig 3. The average output of cells (after
10 days) per responsive
linCD34CD38
cell could thus be calculated to be approximately 104 and
the corresponding average output of CFC per pre-CFC (after 10 days) to
be 13 ± 7. Assuming 100% recovery of these pre-CFC in the
linCD34CD38
population means their frequency in normal adult human marrow would be
less than 1 per 5 × 104 light-density cells.
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Table 5.
Frequency of Growth Factor-Responsive Cells in
LinCD34CD38 Marrow Cells
and LTC-IC in the Matching
LinCD34+CD38
Populations
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| Fig 3.
Results of an LDA experiment to determine the frequency
of growth factor-responsive cells ( ) and pre-CFC ( ) in the
linCD34CD38 population of
a representative marrow sample.
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LTC-IC were detected in 10-day cultures initiated with
linCD34CD38
cells from only 2 of 5 samples and, then, only when all 5 growth factors were also added to the LTC supernatants (yielding 10 and 60 CFC, respectively, after 6 weeks from 500 linCD34CD38
cells initially placed in culture). Whether this reflects inadequate conditions in the primary liquid suspension cultures for the generation and amplification or maintenance of LTC-IC from a starting population of linCD34CD38
cells has yet to be determined. Interestingly, in 2 separate experiments, no transplantable human hematopoietic cell activity could
be detected in any of 4 sublethally irradiated (300 to 350 cGy)
NOD/SCID-2M/
mice.25 These mice were injected immediately
postirradiation with 106 irradiated unseparated human
marrow (carrier) cells and either 2,000 or 4,000 doubly sorted
linCD34CD7+ cells
(which represent a subset of the
CD34CD38 cells; Bhatia et
al13 and our own findings) or 3 × 104 or
1.7 × 105 doubly sorted
linCD34CD7
cells per mouse. Two months later, all 4 mice were killed and their
marrow cells were stained with antihuman CD34, CD45, CD71, CD19, CD20,
CD15, and CD66b antibodies or isotype controls as described.26 Fluorescence-activated cell sorting (FACS)
analysis of 20,000 PI events failed to show any
evidence of human cells in any of the 4 mice (<5 positive events
outside the control gates), despite parallel data (Glimm and Eaves,
unpublished observations) indicating that the
NOD/SCID-2M/ mouse allows
engraftment of 10× more transplantable lympho-myeloid stem cells
from unseparated adult human marrow than the NOD/SCID mouse. In the
present experiments, the linCD34
cells were split into CD7+ and CD7
subpopulations based on a recent report that
linCD34CD7+ cord
blood cells may have in vivo lymphopoietic activity.27
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DISCUSSION |
In this study, we describe a growth factor combination that can
initiate the rapid proliferation and differentiation in vitro of a
subset (~1%) of
linCD34CD38
cells that are present in normal adult human marrow. Determination by
limiting dilution analysis of the frequency of those able to generate
CFC in vitro (pre-CFC) showed that these represent a subset (~50%)
of the total growth factor-responsive
linCD34CD38
subpopulation. In confirmation of previous reports,11,13
cells able to proliferate in semisolid media (CFC) or able to generate CFC for 6 weeks in stromal cell-containing cultures (LTC-IC) were not
detected at equivalent frequencies among these cells. Preliminary
studies have also not detected in vivo repopulating activity within the
linCD34 adult marrow population.
This latter finding contrasts with what has been described for
transplants of adult marrow
linCD34 cells using fetal sheep
as recipients12 or for transplants of cord blood
linCD34 cells using irradiated
NOD/SCID mice as recipients.13 In the present experiments,
a more sensitive host than the regular NOD/SCID mouse was used.
Nevertheless, the number of cells transplanted and/or their homing
efficiency28 may still have been limiting. Additional
studies using other culture conditions to influence these
parameters13 may thus be required to ultimately establish the relationship of the multipotent pre-CFC described here with other
types of hematopoietic cells. Interestingly, however,
linCD34CD38
cells with hematopoietic progenitor activity were shown to be mobilized
into the blood by in vivo G-CSF treatment. The distinct growth factor
requirements exhibited by these adult
linCD34CD38
cells, their persistence at the same frequency in resorted, greater than 98% pure
linCD34CD38
cell populations, the failure to detect cells among the
linCD34CD38
cells that display the less restricted in vitro growth requirements of
most CD34+ cells (including
CD34+CD38 cells), and the fact that
CD34+CD38 cells are not more prevalent
in the lin population all argue strongly against the
possibility that the functional properties here assigned to
linCD34CD38
cells reflect contaminating CD34+ cells.
Although the frequencies of growth factor-responsive cells and pre-CFC
within the
linCD34CD38
population isolated from different marrow samples were variable over a
40-fold range, the same variability was seen in the LTC-IC content of
the matching
linCD34+CD38
populations. This degree of variation may reflect in part the fact that
some samples were from allogeneic transplant harvests, whereas others
were from cadaveric vertebral body harvests. Importantly, despite this
variation between samples, cells able to proliferate in liquid
suspension culture were always more numerous than those able to
proliferate directly in semisolid media, even when exposed to otherwise
identical culture conditions. This latter finding focuses attention on
an intriguing feature of the differentiation process that these cells
undergo in liquid cultures containing FL, SL, TPO, IL-3, and H-IL-6;
ie, the acquisition of an ability to proliferate in semisolid media.
Although the molecular mechanisms underlying this latter property are
not presently understood, it may be speculated that growth
factor-induced changes in the structure of the cytoskeleton are
involved. Interestingly, a similar discrepancy in ability to
proliferate in liquid versus semisolid media has been demonstrated for
freshly isolated CD34+CD38 human marrow
cells.24
A second important characteristic of the cells generated within 7 to 10 days from
linCD34CD38
cells stimulated by FL, SF, TPO, IL-3, and H-IL-6 is their acquisition of an ability to rapidly enter the terminal phases of multiple blood
cell differentiation programs when subsequently stimulated by
appropriate late-acting growth factors. Moreover, this appears to occur
without passage through a step in which the cells display LTC-IC
activity. Such behavior contrasts markedly with that expected from a
prolonged multistep process in which changes in responses to soluble or
stromal-bound growth factors, proliferative potential, and
differentiation status are coordinately regulated. Current acceptance
of such a linkage is based on an observed hierarchy of progenitor
subtypes detected during both steady-state and regenerating hematopoiesis in the adult. However, in vitro, wide variations in the
rate of initiation of terminal differentiation have been noted
previously.16 The rapidity of differentiation of
linCD34CD38
cells into terminal blood cells observed here provides further support
for the concept that the speed at which hematopoietic cells
differentiate may be subject to regulation by exogenous growth factor
stimulation.15,29-31
| |
ACKNOWLEDGMENT |
The authors thank their many colleagues in the Division of Hematology
of the University of British Columbia and the Stem Cell Assay
Laboratory of the BC Cancer Agency for assistance in procuring and
initial processing of patient samples. The technical assistance of
Gayle Thornbury, Richard Zapf, and Giovanna Cameron in operating the
FACS and the assistance of Tara Palmater in preparing the manuscript is
also acknowledged. Thanks is also due to Dr P. Lansdorp, Cangene,
Genentech, Novartis, and StemCell for generous gifts of reagents.
| |
FOOTNOTES |
Submitted January 18, 1999; accepted May 11, 1999.
Supported by grants from the National Cancer Institute of Canada (NCIC)
with funds from the Terry Fox Run, the National Institutes of Health
(NHLBI-HL55435), and Novartis, Canada. M.G.B. was supported by a grant
from the Association pour la Recherche Contre le Cancer (France),
S.R.-J. was supported by grants from the Deutsche
Forschungsgemeinschaft (Bonn, Germany) and the Stiftung Rheinland-Pfalz
für Innovation (Mainz, Germany), and 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 reprint requests to Connie J. Eaves, PhD, Terry Fox Laboratory,
601 W 10th Ave, Vancouver, British Columbia, Canada V5Z 1L3; e-mail:
connie{at}terryfox.ubc.ca.
| |
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TOP
ABSTRACT
INTRODUCTION