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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4098-4107
Influence of Cytokines on the Growth Kinetics and Immunophenotype
of Daughter Cells Resulting From the First Division of Single
CD34+Thy-1+lin Cells
By
Julie P. Goff,
Donna S. Shields, and
Joel S. Greenberger
From the Department of Radiation Oncology, University of Pittsburgh
and University of Pittsburgh Cancer Institute, Pittsburgh, PA.
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ABSTRACT |
There is a need to determine whether culture conditions may exist
for ex vivo expansion of hematopoeitic stem cells (HSC), which favor
solely proliferative self-renewal of HSC as opposed to proliferation
with differentiation. Using single cells, we studied the effects of
individual and combinations of cytokines in serum-free medium on the
kinetics of the first cell doubling and the resulting phenotype of each
of individual daughter cell. CD34+Thy-1+lin
cells were plated 1 cell per well in Terasaki plates in
serum-free medium containing cytokines. Each well containing a single
cell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype of
the daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with an
undivided single cell, wells in which the cell had divided, and wells
in which the cell had died were scored. The number of doublets with
conserved phenotype (CD34+lin) was
compared to those wells with one or more differentiated daughter cells
(CD34+lin+). Over 7 days, cells cultured in
single factors showed that between 13% (interleukin-6 [IL-6]) and
29% (thrombopoietin [TPO]) of the cells were undivided, between 13%
(IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) and
greater than 60% (IL-11, IL-1, or hepatocyte growth factor
[HGF]) died. When combinations of cytokines were used
over 7 days, between 5% (FLT-3 ligand [FLT-3L], stem
cell factor [SCF], IL-3, IL-6, granulocyte
colony-stimulating factor [G-CSF],  nerve growth factor
[NGF]) and 22% (FLT-3L + HGF) of the cells
remained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68%
(SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64.6%) of cells with
conserved phenotype (percent conserved doublets + percent with 1 cell
conserved), followed by SCF + TPO, (50%) and the combination of
FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF (53%). These combinations also
produced the highest yield of cells with conserved phenotype after one
division (FLT-3L + TPO  81 cells/100 initial cells, SCF + TPO 68 cells/100 initial cells) (P = .01). Observation of the
time of the initial cell division and phenotype of the daughter cells
allowed us to identify candidate combinations of cytokines that promote
maintenance of lin cells (TPO), or recruit the primitive
cells to divide and undergo phenotypic self-renewal (FLT-3L + TPO,
SCF + TPO).
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE INCREASED clinical indications for
bone marrow transplantation (BMT) and progress in gene therapy have led
to a critical need to expand hematopoietic stem cells in vitro. Recent improvements in the ability to enrich stem cells from human BM, peripheral blood (PB), or umbilical cord blood (CB) do not increase the
number of stem cells available. Progress toward extending the
usefulness of hematopoietic stem cell transplantation has been limited
by lack of sufficient understanding of what is required to induce stem
cell self-renewal or even to maintain stem cells in a
"noncommitted" state in vitro. Studies of human stem cells, in
particular, have been limited by lack of definitive (exclusive) phenotypic characterization, or assays that can be feasibly applied and
agree with other assays. Stem cells in vivo are maintained as a very
minor population of the whole BM and are not amplified except after
severe depletion. This situation in vivo is very different from the
goal of efforts to expand stem cells in vitro, which is to amplify stem
cells while preventing differentiation and then possibly to direct
differentiation to a chosen lineage.
Although the precise cell-surface phenotype of the human hematopoietic
stem cell remains a subject of debate, it is generally agreed that stem
cells are among the CD34+lin fraction of
cells from BM, PB, or CB. Culture of
CD34+lin cells in combinations of
recombinant cytokines has resulted in a significant increase in cell
numbers without loss of the ability to reconstitute myeloablated
hosts.1-3 However, the extent of expansion of true stem
cell numbers in these large pools of expanded cells remains to be
determined. The need to expand and maintain human stem cells in vitro
has led to development of methods for greatly enriching primitive
cells, identification of recombinant cytokines that influence their
behavior, and several assays for primitive cells.
Past interest has been on identifying which growth factors are able to
stimulate proliferation and differentiation, whereas current focus is
on what cytokines allow primitive and progenitor cells to remain
viable. In particular, FLT-3 ligand (FLT-3L) has been
shown to have a direct effect on primitive hematopoietic cells from
both mice and humans,4-14 is synergistic with
other growth factors, and the yield of primitive cells can be increased with use of FLT-3L in cultures. Studies by Haylock et al10
showed that FLT-3L recruited more
CD34+CD38 cells into division, and that
transduction of these cells was enhanced by FLT-3L. Stem cell factor
(SCF) has been shown to be effective in promoting
viability of progenitor cells in culture,11,15-17 but to
varying degrees. Borge et al18 showed that thrombopoietin (TPO) was more efficient in promoting viability of primitive cells than
SCF, and that it was effective in suppressing apoptosis. Other recent
studies19-22 support the fact that TPO plays role in
supporting the survival of primitive cells, in addition to its effects
on megakaryocyte development.23 When TPO is used in
combination with other early acting growth factors, such as SCF or
FLT-3L, the effect is more prolonged, in vitro,
maintenance and expansion of primitive populations of
cells from mice4,24,25 and humans.26-32
Piacibello et al9 showed that with the combination of
FLT-3L and TPO, CB CD34+ cells could be expanded and
continue to produce committed progenitors for more than 6 months. Using
the combination of FLT-3L, TPO, and SCF, Solar et al33
reported an increase in total cells over 8 weeks in culture and that
28% of these cells were CD34+CD38. With
the addition of interleukin-6 (IL-6) to TPO, SCF, and flk2/flt3 ligand,
Murray et al34 reported less expansion of BM
CD34+Thy-1+lin cells over 6 days than without IL-6, but a higher percent of cells retaining Thy-1.
In a recent study by Luens et al,35 it was shown that the
combination of TPO, flk2/flt3 ligand (FL), and
c-kit ligand (KL) stimulated the majority of the
CD34+Thy-1+lin cells in
their cultures to divide with little loss of CD34 or Thy-1 expression.
Although these questions are provocative, the question remains as to
whether the numbers of true stem cells can be expanded significantly in
vitro. A key to answering this question is the ability to detect stem
cell self-renewal in vitro. Because daughter cells from the first
doubling can produce other cytokines with negative regulatory capacity
and can, therefore, limit stem cell self-renewal, we have studied the
fate of single human umbilical CB
CD34+Thy-1+lin cells in
serum-free medium containing different combinations of recombinant
cytokines. We investigated how selected cytokines affect the kinetics
of proliferation of single CD34+Thy
1+lin cells and the ability of these
cells to self-renew in vitro. In this study we show that TPO as a
single factor can support the survival of
CD34+lin cells, and that it can interact
with FLT-3L and SCF to promote doubling and phenotypic self-renewal of
primitive cells. Our data provide evidence of phenotypic self-renewal
or differentiation that can be detected as early as the first cell
division. Significant variation in the frequencies of phenotypic
self-renewal of single cells is obtained depending on the cytokine
combination used. These studies provide basic information on both the
heterogeneity of human cord blood
CD34+Thy-1+lin cells after
one division and support use of single cell culture methods for
identifying conditions that support/induce the self-renewal of a subset
of these cells.
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MATERIALS AND METHODS |
Hematopoietic Growth Factors
Recombinant human hepatocyte growth factor (HGF) was generously
supplied by Dr G.K. Michalopoulos (University of Pittsburgh). All other
recombinant growth factors were purchased from PeproTech, Inc (Rocky
Hill, NJ). Unless indicated otherwise, growth factors were used at the
following concentrations: FLT-3L 50 ng/mL; TPO, 10 U/mL; IL-1, 10 ng/mL; SCF, 50 and 100 ng/mL; HGF, 1 ng/mL; IL-6, 20 ng/mL; IL-11, 10 ng/mL when used singly. When multifactor combinations were used the
concentrations were as follows: the combination of FLT-3L 100 ng/mL,
SCF 100 ng/mL, IL-3 20 ng/mL, IL-6 20 ng/mL, granulocyte
colony-stimulating factor (G-CSF) 20 ng/mL, nerve growth factor
(NGF) 5 ng/mL; the combination of SCF 100 ng/mL, IL-3
100 ng/mL, IL-6 10 ng/mL, G-CSF 30 ng/mL, granulocyte-macrophage
(GM)-CSF 20 ng/mL; the combination of HGF 1 ng/mL, IL-1 10 ng/mL, IL-6
or IL-11 10 ng/mL, G-CSF 20 ng/mL; FLT-3L 50 ng/mL + TPO 10 U/mL;
FLT-3L 50 ng/mL + HGF 1 ng/mL; SCF 50 ng/mL + TPO 10 U/mL.
CD34+ Cell Isolation
Human umbilical CB samples were obtained immediately after delivery in
accordance with institutional guidelines, and placed in 50-mL tubes
containing ACD-A (Cytosol Labs, Braintree, MA). Low-density mononuclear
cells were isolated by Ficoll-Paque (1.077 g/mL) density gradient
centrifugation (Pharmacia Biochem, Piscataway, NJ). CD34+
progenitor cells were isolated by immunomagnetic selection techniques, as previously described.36 Briefly, cells were incubated
with blocking reagent and anti-CD34 antibody at 4°C, then washed in phosphate-buffered saline/anticoagulant citrate dextrose solution-A (PBS/ACD-A) followed by incubation with a secondary
antibody-magnetic microbead conjugate at 4°C. The unlabeled
fraction of CD34 cells were separated from the
labeled CD34+ fraction on a high gradient magnetic
separation column (Miltenyi Biotec, Sunnyvale, CA). The percentage of
CD34+ cells was determined by flow cytometric analysis.
Enrichment of
CD34+Thy-1+lin Cells
CD34+ cells initially isolated by immunomagnetic selection
were labeled with a mixture of fluorescein isothiocyanate
(FITC)-conjugated anti-CD34 (HPCA-2) (Becton Dickinson, San Jose, CA),
Thy-1(anti-CDw90) (Pharmingen, San Diego, CA), and phycoerythrin
(PE)-conjugated antilineage markers, including the following: anti-CD3,
CD11b, CD19, CD33, CD38, CD56, and HLA-DR (Becton Dickinson); and
anti-glycophorin A antibodies (Dako Corp, Carpenteria, CA). FITC and
PE isotype-matched mouse IgG1 and IgG2a were
used as controls. Labeled cells were washed and resuspended in PBS + 2% fetal calf serum (FCS) for sorting. Cells were sorted using a
Becton Dickinson FACStar Plus flow cytometer. Viability of the
CD34+Thy-1+lin cells after
sorting as determined by Trypan blue dye exclusion was greater than
95%.
Single-Cell Culture and Growth Kinetics
CD34+Thy-1+lin cells were
plated at a density of 1 cell/well in Terasaki plates in 15 µL
serum-free medium. Iscove's medium was supplemented with 1% bovine
serum albumin (BSA), 10 µg/mL insulin, 200 µg/mL transferrin (Stem
Cell Technologies, Vancouver, BC, Canada), 104 mol/L
2-mercaptoethanol (Sigma, St Louis, MO), 40 µg/mL low-density lipoproteins (Bayer Corp, Kankakee, IL), 2 mmol/L glutamine, 50 µg/mL
streptomycin, 50 U/mL penicillin (GIBCO, Gaithersburg, MD), and
selected recombinant growth factors as described above. The initial
number of cells, in each well, in all plates was verified by light
microscopy, and only those wells that contained one viable cell were
included. Each well containing a single cell was observed daily for up
to 7 days for evidence of maintenance (cell is viable, but undivided),
cell doubling, or cell death. Characteristic morphology using phase
contrast microscopy was used for identification of viable cells on days
1-7. CD34+ cells were plated in parallel, monitored daily,
and stained for phenotype as were the lin cells;
these cells served as a positive control for fluorescence detection
(data not shown). Cells that remained undivided over the 7 days were
tested for viability using Trypan blue dye exclusion.
Phenotypic Analysis of Individual Daughter Cells
The wells in which a single cell had divided were stained with
FITC-anti-CD34 and PE antilineage antibodies for phenotype assessment
of the daughter cells. The media were removed from each well containing
a cell doublet and replaced with 7 µL of the FITC/PE antibody
cocktail. The antibodies were used at a predetermined optimal
concentration for each individual antibody (range of protein concentration, 0.2 µg/mL to 1 µg/mL). After incubation for 15 to 20 minutes at 4°C, each well was washed three times with
13 µL of PBS + 2% FCS. The phenotype of each individual daughter cell of the cell doublet was scored, by the same observer each day,
based on detection (by eye) using epifluorescence microscopy immediately after staining. Each of four different phenotypes possible
for each cell in the doublet was observed: 1, FITC+
(34+lin); 2, PE+
(34lin+); 3, FITC+/PE+ (34+lin+); 4, FITC/PE
(34lin). The frequency of each
phenotype (conserved doublet, both daughter cells
CD34+lin [phenotypic self-renewal] or
differentiated, one or two differentiated cells) was determined.
Statistics
Statistical significance was determined using the Student's
t-test or Exact Confidence Limits for binomial distributions.
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RESULTS |
To study the influence of growth factors on the doubling kinetics and
immunophenotype of individual daughter cells resulting from the first
division of single cells, CB
CD34+Thy-1+lin cells were
cultured in the presence of single and various combinations of
recombinant human cytokines. Single
CD34+Thy-1+lin cells were
plated at a density of 1 cell/well in Terasaki plates in serum-free
medium containing cytokines. Each well containing a single cell was
observed daily for 7 days for evidence of maintenance (cell remains
viable, but undivided), cell death, or cell division. The wells in
which division was observed (Fig 1) were
then stained with anti-CD34 and antilineage antibodies to determine the
phenotype of the daughter cells. The growth kinetics (percent of wells
with maintenance, division, death) for each day (days 1-7) in culture, for each of the single and combinations of factors used, are shown in
Tables 1 through
15,
and the cumulative percent over 7 days and the phenotype data are shown
in Table 16.
Figures 2 and 3 show the number (yield) of cells with conserved (Fig 2) or
differentiated (Fig 3) phenotype resulting from the first cell division
of 100 initial
CD34+Thy-1+lin cells.
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| Fig 1.
Phase contrast images of a single
CD34+Thy-1+lin cell
undergoing its first cell division. The time elapsed from when the
beginning of division was observed (upper left) until division was
complete (lower right) was approximately 25 minutes. When a doublet was
observed in a well, the daughter cells were stained for phenotype
assessment by fluorescence microscopy.
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Table 12.
Growth Kinetics of Single
CD34+lin Cells in Cultures Supplemented
With FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF
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Table 16.
Cumulative Growth Kinetics Over 7 Days and Phenotype of
Daughter Cells After the First Division of Single
CD34+Thy-1+lin Cells
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| Fig 2.
The graph represents the yield of cells (number) with the
CD34+lin phenotype obtained from 100 initial individual cells that have undergone one cell division based on
the percent of cells that doubled and the percent of daughter cells
that retained the CD34+lin phenotype
(phenotypic self-renewal). Although the yields obtained with the
combination of FLT-3L + TPO and SCF + TPO were not significantly
different from each other, they were significantly different (P  .01) from all other cytokine combinations tested.
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| Fig 3.
The graph represent the yield of cells (number) with
differentiated phenotype (CD34+lin+)
obtained from 100 initial individual cells that have undergone one cell
division based on the percent of cells that doubled and the percent of
daughter cells that expressed lineage markers (did not phenotypically
self-renew). The yields obtained with the combination of SCF + TPO
were significantly different (P .01) from all other
cytokine combinations tested.
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Effects of Single Cytokine Stimulation on the First Doubling
Kinetics of Single
CD34+Thy-1+lin Cells
TPO promotes viability of 34+lin cells.
Individual 34+lin cells were cultured in
the presence of single growth factor stimulation or no added factors,
and the kinetics of cell growth over 7 days in TPO, FLT-3L, SCF (50 and
100 ng/mL), IL-6, IL-11, IL-1, and HGF was examined (Tables 1 through
9). Cultures supplemented with TPO, SCF (50 ng/mL), and FLT-3L had the
most single cells that remained undivided and viable (maintained) cumulatively over 7 days, with 29.4%, 26%, and 22.1% of the wells, respectively, with one live cell (Table 16) (P .05). The least number of single cells maintained undivided and viable
cumulatively over 7 days were in cultures with IL-6 and IL-11, where
only 13.1% and 13.6% of the wells, respectively, had 1 live cell.
Tables 1 through 9 show the growth kinetics for each individual day in
culture, for days 1-7. On day 7, with FLT-3L (Table 3), no added
factors (Table 4), SCF 100 ng/mL (Table 5), IL-6 (Table 6), IL-11
(Table 7), IL-1 (Table 8), and HGF (Table 9), less than 24% of the
starting number of wells had one undivided cell. In contrast, on day 7 with TPO (Table 1), 30.9% and with SCF (50 ng/mL) 27.7% of the
starting number of wells had one undivided cell. Of all
the single factors tested, the largest total percent of viable cells
(cells that remained undivided + cells that had doubled), 64.7% were
in cultures supplemented with TPO. These cultures had 15.7% more
viable cells cumulatively over 7 days than did cultures supplemented
with SCF (50 ng/mL) and 18% more than with FLT-3L.
The highest cumulative percent of wells in which the single cell
doubled over 7 days were seen in cultures stimulated with TPO, FLT-3L,
and SCF (50 ng/mL) with 35.3%, 24.6%, and 23%, respectively (Table
16). The cumulative percent of wells in which the single cell doubled
over 7 days ranged from a low of 13.0% (IL-1) to a high of 35.3% for
TPO (P .05). When no factors were added, doubling occurred
in only 2.2% of wells cumulatively over 7 days. The responses with SCF
(100 ng/mL) and HGF were similar to each other, with 21.0% and 20.5%
of the wells doubling, as were responses with IL-6 and IL-11
stimulation, both with 18.2%.
Single cells underwent their first division beginning on day 2, and
most of the doubling occurred by day 4 (Tables 3, and 5 through 9) in
all single-factor-stimulated cultures, except those with TPO (Table 1)
and SCF (50 ng/mL) (Table 2), where initial doubling began on day 3 and
most of the doubling occurred by day 5. Doubling only occurred on days
4 and 5 when no factors were added (Table 4). In cultures supplemented
with TPO (Table 1), SCF (50 ng/mL) (Table 2), FLT-3L (Table 3), and SCF
(100 ng/mL) (Table 5), there were still wells in which the single cell
doubled on day 7. We did observe wells that contained a single cell,
which proliferated to more than 2 cells (either 3 or 4 cells) in the
interval between observations. Although these wells were assessed for
phenotype as were the wells containing only 2 cells, the results are
not included in this study. Cells were also observed to divide during
daily observation. In other experiments with TPO, SCF, FLT-3L, and
FLT-3L + TPO, some of the cells that remained undivided and viable at 7 days were maintained as long as 35 days without dividing. In some
cultures, up to 50% (range, 27% to 50%) of the cells that were
undivided at 7 days were still undivided at 30 days.
With all single-factor stimulation or with no added factors, there were
wells in which the single cell died without dividing on days 1-7 (Tables 1 through 9). The majority of the cell death occurred during
the first 4 days in cultures with SCF (50 and 100 ng/mL) and FLT-3L,
IL-6, and IL-1. In contrast, the majority of cell death in cultures
supplemented with TPO was seen on days 5-7 and on days 4-7 in cultures
supplemented with HGF or IL-11. The IL-6, IL-11, HGF, and IL-1 cultures
had the highest cumulative percent of wells in which the cell died over
7 days with 68.7%, 68.2%, 64.1%, and 66.3%, respectively (Table
16). In contrast, the cumulative percent of wells in which the cell
died over 7 days in the presence of TPO was only 35.3%.
Effects of Combinations of Multiple Cytokines on the First Doubling
Kinetics of Single
CD34+Thy-1+lin Cells
FLT-3L + TPO and SCF + TPO promote viability of
34+lin cells.
Individual 34+lin cells were cultured in
the presence of multiple growth factors and the kinetics of cell growth
over 7 days in FLT-3L + TPO; SCF + TPO; FLT-3L + HGF; the combination
of FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF; the combination of SCF,
IL-3, IL-6, G-CSF, GM-CSF; and the combination of HGF, IL-1, IL-6 or
IL-11 1, G-CSF was examined. The results are presented in
Tables 10 through 15. Cumulatively over 7 days there were fewer wells
in which the cell was maintained undivided than with single factors alone. With combinations of growth factors, the majority of the cells
either doubled or died, depending on the culture stimulation (Table
16).
The percent of wells in which the cell was still undivided on day 7 ranged from 1.2% for SCF 50 ng/mL + TPO to 13.5% for the combination
of HGF, IL-1, IL-6, or IL-11 G-CSF. The largest total percent of wells with viable cells (cells that remained undivided + cells that had doubled) were in cultures supplemented with FLT-3L + TPO, with 73%. These cultures had 4% more viable cells
cumulatively over 7 days than did cultures supplemented with SCF (50 ng/mL) + TPO (69.1%) and 23% more than with FLT-3L + HGF with 50%.
Cells doubled on days 3-6, with most doubling occurring by day 4 for
all combinations tested, and only the combination of HGF, IL-1, IL-6,
G-CSF still had doubling that occurred on day 7. The highest cumulative
percent (67.9%) of wells in which the cell doubled over 7 days was in
cultures with SCF + TPO. With FLT-3L + TPO culture stimulation, the
cumulative percent of wells in which the cell doubled over 7 days was
less, 64%, but in these cultures more cells remained undivided (10%
v 1.2%) and fewer died over the 7 days. For all but the FLT-3L + TPO combination, cells died without dividing on days 3-7 (Tables 11
through 16). With FLT-3L + TPO, cell death was observed on days 2-7 (Table 10). In all cultures, most death was seen between days 3 and 5. The cumulative percent, over 7 days, of wells in which the a cell died
without dividing ranged from 27% for FLT-3L + TPO to 70.3% for the
combination of HGF, IL-1, IL-6 or IL-11 G-CSF (Table 16). A larger total percent of viable cells (cells that remained undivided + cells that had doubled) were observed with the combinations of FLT-3L + TPO and SCF + TPO than with the best single factor, TPO.
Phenotype of Daughter Cells After the Initial Cell Division. FLT-3L + TPO Promotes Phenotypic Self-Renewal
To determine whether cells undergo a change in phenotype after only one
cell division and whether the influence of cytokines could affect the
frequency of phenotypic self-renewal after the first cell doubling of
CD34+Thy-1+ lin cells in
vitro, wells containing single cells were monitored daily for evidence
of division. The wells in which a single cell had divided (Fig 1) were
stained with FITC-anti-CD34 and PE antilineage antibodies for
phenotype assessment of the daughter cells. The phenotype of each
individual daughter cell of the cell doublet was scored using
fluorescence microscopy and the percent of cells undergoing a conserved
doubling (phenotypic self-renewal) to two like daughter cells
(CD34+lin) or the percent of doublets
with one or two differentiated cells were determined. Each of four
different phenotypes possible for each cell in the doublet was
observed: 1, FITC+ (34+lin);
2, PE+ (34lin+); 3, FITC+/PE+ (34+lin+); 4, FITC/PE
(34lin). The frequency of each phenotype outcome was
determined and results are shown in Table 16, and the yield (number) of
cells obtained with conserved or differentiated phenotype/100 initial cells is shown in Figs 2 and 3 for each combination of growth factors.
A population of CD34+ cells were plated in parallel,
monitored, and stained as the lin population; these
served as a positive control for the staining (data not shown).
Conserved doublets were defined as
CD34+lin in both daughter cells and
differentiated doublets as those where both cells were
lin+.
There were no doublets with conserved phenotype (both daughter cells
CD34+lin) detected in cultures
supplemented with IL-11, HGF, or no added factors. Doublets with
conserved phenotype were detected with all other cytokines used. With
the combination of FLT-3L + TPO, 57% of the doublets were of conserved
phenotype (CD34+lin), the highest
percent of phenotypic self-renewal of all the combinations tested. SCF + TPO; the combination of FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF; and
IL-6 used alone had the next highest percent of doublets with conserved
phenotype, with 39%, 39%, and 38%, respectively. There
was no correlation between when the cell divided and the phenotype of
the daughter cells.
When looking at the total percent of cells with conserved phenotype
(percent conserved doublets + percent with 1 cell conserved), FLT-3L + TPO was still best overall, with 64%. Although in cultures with IL-1,
IL-6, no added factors; the combination of HGF, IL-1, IL-6, G-CSF; or
the combination of SCF, IL-3, IL-6, G-CSF, GM-CSF there were more
doublets with one daughter cell conserved, over the 7 days, as much as
55% more cells died than doubled or than remained
undivided. Therefore, the number of cells with the
34+lin phenotype was less. Other
combinations in which the total percent of cells with conserved
phenotype was high were FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF; the
combination of SCF, IL-3, IL-6, G-CSF, GM-CSF; and SCF + TPO with 53%,
53%, and 43%, respectively. The yield (number) of cells with
conserved phenotype after the first division of 100 initial cells was
determined for each cytokine combination (Fig 2). The combinations of
FLT-3L + TPO and SCF + TPO produced the highest yield of cells with
34+lin phenotype after one division
(FLT-3L + TPO 81 cells/100 initial cells and SCF + TPO 68 cells/100
initial cells). Although the yields are not significantly different
from each other, they are significantly (P .01) different
from all the other cytokines tested. Cultures with no added factors and
IL-11 had the lowest yield of cells with conserved phenotype (2 cells/100 initial cells, and 7 cells/100 initial cells, respectively).
Although cultures with IL-1, IL-6, no added factors; the combination of
HGF, IL-1, IL-6, G-CSF; or the combination of SCF, IL-3, IL-6, G-CSF,
GM-CSF had more doublets with one daughter cell conserved, over the 7 days, more cells died than doubled or were maintained. Therefore, the
actual yield of cells with the 34+lin
phenotype was significantly (P .01) less than with FLT-3L + TPO; SCF + TPO; or FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF.
The highest percent of doublets with both daughter cells having a
differentiated phenotype (CD34+lin+,
CD34lin+) were seen in cultures with
IL-11 and HGF with 63% each, and the lowest percent when single cells
were cultured in the absence of added growth factors (Table 16). The
combination of SCF + TPO produced the highest (P .01) yield
(number) of cells with CD34+lin+ differentiated
phenotype after one division (68 cells/100 initial cells) (Fig 3). With
SCF + TPO the yield of conserved cells was equal to the yield of
differentiated cells. The combinations of FLT-3L + TPO and SCF + TPO
allowed the most single cells to undergo a conserved doubling, due to
the fact that more cells divided than died in these cultures, combined
with the total percent of cells that were still the
CD34+lin phenotype.
| |
DISCUSSION |
In this study we have investigated the phenotype of cells resulting
from the first division of single CD34+Thy
1+lin cells, whether changes in
phenotype occurred with the first cell division, and the influence of
growth factors on the survival and doubling kinetics of individual
cells. We report that cells self-renew or differentiate with respect to
phenotype after the first division, and this is influenced by different
growth factors. The question of what cytokines are able to maintain
most viable cells and selectively enhance maintenance rather than
proliferation or death is still unanswered, although recent studies
illustrate the ability of SCF, TPO, and FLT-3L to stimulate growth and
survival of primitive hematopoietic cells from CB and BM.
Cultures supplemented with TPO, SCF, and FLT-3L had the most single
cells that remained undivided and viable (maintained) cumulatively over
7 days (P .05). Of all the single factors tested, the
largest total percent of viable cells (cells that remained undivided + cells that had doubled) were in cultures supplemented with TPO. These
cultures had more viable cells cumulatively over 7 days than did
cultures supplemented with SCF alone or with FLT-3L alone. The
combination of FLT-3L + TPO produced the highest total percent of wells
with viable cells (cells that divided + percent of cells viable and
undivided) of all the cytokines evaluated. These cultures had more
viable cells cumulatively over 7 days than did cultures supplemented
with SCF + TPO or FLT-3L + HGF, both of which were better that TPO
alone. In some cultures, 27% to 50% of the cells that remained
undivided on day 7 were still undivided and viable on day 30. With
combinations of growth factors, the majority of the cells either
doubled or died depending on the culture stimulation. As expected, all
cytokines studied induced some division and death. In some cultures,
more cells remained viable (cells that were maintained + cells that
divided) cumulatively over 7 days than died.
There was less doubling with single factors, and this was consistent
with the fact that for proliferation of more primitive cells
multifactor stimulation is required. SCF + TPO and FLT-3L + TPO induced
more doubling than death, whereas the percent of cells that doubled in
cultures supplemented with single factors was only slightly higher than
the percent than remained undivided. The cumulative percent of wells in
which the single cell doubled over 7 days varied with the culture
stimulation.
Culture wells with the lowest percentage of single cell death over 7 days were those with FLT-3L + TPO and SCF + TPO, consistent with the
fact that with these combinations induced more than 60% of the cells
to divide and, therefore, had a larger total percent of viable cells
(cells that remained undivided + cells that had doubled) than those
with the best single factor, TPO. These observations are in agreement
with other studies showing TPO is more effective with the addition of
other early acting cytokines, such as SCF and FLT-3L.
Phenotypic self-renewal of both daughter cells (both
CD34+lin) was not observed in cultures
supplemented with IL-11, HGF, or no added factors. Doublets with
conserved phenotype were detected with all other cytokines used. The
combination of FLT-3L + TPO induced the highest percent of phenotypic
self-renewal and yield (P .01) of all the combinations
tested. Cultures with SCF + TPO, the combination of FLT-3L, SCF, IL-3,
IL-6, G-CSF, NGF, were also effective. FLT-3L + TPO was still best
overall when looking at the total percent of cells with conserved
phenotype (percent conserved doublets + percent with one cell
conserved). Although in cultures with IL-1, IL-6, no added factors; the
combination of HGF, IL-1, IL-6, G-CSF; or the combination of SCF, IL-3,
IL-6, G-CSF, GM-CSF had more doublets with one daughter cell conserved over the 7 days, more cells died than doubled or than remained undivided. Therefore, the number of cells with the
34+lin phenotype was less than with
FLT-3L + TPO, which may be important for obtaining
expanded populations of cells with primitive phenotype. In addition,
approximately 50% of the cells that remained undivided after 30 days
in culture still retained the CD34+lin
phenotype. The present studies do not address which particular lineage
marker is being expressed. The daughter cells that are lineage positive
could be expressing any one of the markers present in the cocktail. As
the feasibility of determining this on individual cells increases,
future studies will look at which specific markers are being expressed
as soon as the first division and which others are picked up or lost
with subsequent divisions.
Analysis of individual wells shows heterogeneity among individual CB
samples in the rate of doubling, maintenance, and death, and daughter
cell phenotype. A study by Gothot et al37 showed that the
position of cells in the G0/G1 phase of the cell cycle could account
for the functional differences (heterogeneity) in CD34+
cells.
Other single cell studies have relied on allowing the single cells to
grow to large numbers over time (looking at total fold expansion) and
analyzing phenotype on large populations of cells. We have used this
single-cell system to look at the concept of possible proliferation
without differentiation and to track this occurrence after the initial
cell division of primitive population containing stem
cells. These observations are in agreement with other
studies using with FLT-3L, TPO, and SCF, and follow what has been
observed by examining larger numbers of cells or single cells that
proliferate to large numbers and phenotype assessment by FACS. TPO as a
single factor can support the survival of
34+lin cells and acts with FLT-3L and
SCF to induce doubling and viability. Although stem cell phenotype does
not necessarily correlate with function, our studies use phenotype as a
starting point, because long-term culture-initiating
cells and in vivo repopulating cells are in the
population with conserved phenotype. Retention of phenotype after division does not mean certain loss of function, but there is certain loss of stem cell function if there is not
retention of phenotype. Recent studies35 using
104
CD34+Thy-1+lin
cells/well cultured with TPO, FL, and KL show similar
retention of primitive phenotype and that engraftment potential was
retained.
These present data show, on a single-cell basis, that early acting
cytokines which recruit cells to divide stimulate phenotypic self-renewal, and that cell fate decisions (self-renew or
differentiate) are seen as soon as the initial cell division of
CD34+Thy 1+lin cells, and
that this can be influenced by culture conditions. These
results may have implications for targeting primitive cells for gene
therapy, by knowing which day cells are most likely to divide and which
cytokines stimulate phenotypic conserved or differentiated division,
and the yield of cells retaining the
CD34+lin phenotype that could be
obtained within a population. These data may allow the study of the
factors that control quiescence and self-renewal of expanded
homogeneous populations of primitive cells. Although others have looked
at the effects of these cytokines on single-cell proliferation and
assessment of phenotype on large numbers of the progeny of those single
cells, this is the first study to show the effects of cytokines on the
potential of single primitive cells to retain or change phenotype after
one cell division.
Our data clearly show the effects of the synergy between FLT-3L +TPO
and SCF + TPO in affecting proliferation and maintenance of stem cell
phenotype and the ability of these factors to induce division with less
differentiation to increase the numbers of primitive cells that have
undergone phenotypic self-renewal, and that cell fate/phenotype
decisions are made as early as the first division.
| |
ACKNOWLEDGMENT |
The authors express appreciation to Christine Contis of Magee Womens
Research Institute for her efforts in the collection of cord blood
samples, and to Bob Lakomy and Alex Styche of the University of
Pittsburgh Cancer Institute Flow Cytometry Facility for
the cell sorting. We also thank Dr Sallie Boggs for her helpful discussions.
| |
FOOTNOTES |
Submitted April 17, 1998;
accepted July 16, 1998.
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 Joel S. Greenberger, MD, Professor and
Chairman, Department of Radiation Oncology, University of Pittsburgh,
200 Lothrop St, Pittsburgh, PA 15213.
| |
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Abstract
Introduction