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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2967-2974
RED CELLS
Selectively increased growth of fetal hemoglobin-expressing
adult erythroid progenitors after brief treatment of early
progenitors with transforming growth factor beta
Ralph M. Bohmer,
Thomas A. Campbell, and
Diana W. Bianchi
From the Division of Genetics, Department of Pediatrics, New England
Medical Center and Tufts University Medical School, Boston, MA, and
Wallac Inc, Akron, OH.
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Abstract |
We have studied the effect of transforming growth factor beta
(TGF ) on erythropoiesis in cultures from adult peripheral blood, using flow cytometric enumeration of fetal hemoglobin (HbF)-containing cells. TGF caused a dramatic increase in the proportions of cells that accumulated HbF together with adult hemoglobin (HbA) (F+A+ cells). This highly significant (P < .0001) increase in
F+ cell proportion was achieved by TGF treatment during the first
4 days of culture and was sustained during further culture expansion in
the absence of TGF. The increase in F+ cell proportions did not
depend on the cytokine combination (EPO+SCF+IL3, EPO+SCF, EPO+IL3, SCF+IL3) used during the phase of TGF treatment.
Increased F+ cell proportions were paralleled by an increased
molecular ratio of HbF/ HbF+ HbA, measured by cation exchange
high-performance liquid chromatography (HPLC). In addition
to the effect on F+ cell proportions, TGF caused a dramatic
increase in overall cell division potential. By the time cultures
reached terminal growth arrest (12-14 days in controls and 18-26 days
after TGF), the overall numbers of F+ cells produced per initially
seeded clonogenic cell was approximately 10 times higher in the
TGF-treated cultures than in the controls. We propose to investigate
whether the TGF-induced increase in relative and absolute numbers of
nucleated F+ cells, as demonstrated in vitro, can be translated into
increased F+ erythrocytes in vivo, allowing therapeutic
application for some beta-hemoglobinopathies.
(Blood. 2000;95:2967-2974)
© 2000 by The American Society of Hematology.
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Introduction |
Peripheral blood contains clonogenic cells that produce
erythroid colonies and bursts in semisolid culture, given the
appropriate combination of growth factors. Individual cells in such
colonies can accumulate fetal hemoglobin (HbF), adult hemoglobin (HbA), or a combination of both. The pattern of hemoglobin expression and
accumulation is different in cultures from fetal and adult blood. Fetal
erythroid cells express only HbF for at least 1 week of culture, which
is followed by low levels of HbA expression only after cells have
reached maximum HbF levels.1,2 In cultures from adult
blood, nucleated red cells accumulate either only HbA (F A+) or a
combination of HbF and HbA (F+A+).2-5 Individual colonies
contain both F+ and F cells, which means that both types are
progeny from the same circulating stem cells.5,6 The proportion of F+ cells developing in cultures from normal adult peripheral blood is not preprogrammed in vivo but depends on culture conditions. A shift into the combined HbF and HbA expression pathway can, for example, be achieved in vitro by high serum
concentrations,2,5-7 based on an unidentified activity that
can be absorbed on activated charcoal. Thus, a mechanism must exist
whereby cells during the early stages of development in a culture
execute an option whether or not to express HbF.
The possibility to manipulate adult erythroid stem and progenitor cell
development toward fetal erythropoiesis is of direct clinical interest
because hemoglobin disorders such as sickle cell anemia and the
beta-thalassemias are ameliorated by increased HbF production
(reviewed by Jane, Cunningham, Olivieri, and
Bunn8-10). Agents that stimulate HbF in vivo include
5-aza-cytidine, 11,12 hydroxyurea,13 and
butyrates.14,15 These agents act via different mechanisms
that are not yet completely understood. Their effectiveness has been
demonstrated in several clinical trials, but is limited by unwanted
side effects and variability in patient responses.8,9 Although even minimal increases in HbF levels are helpful in sickle cell disease, beta-thalassemias require much higher increases that are
not reliably achieved by any of the currently used agents.9 Therefore, there is a need for additional and novel HbF-increasing agents.
We found in erythroid cell cultures from adult peripheral blood that
TGF treatment caused a dramatic increase in the proportions of cells
accumulating HbF, exceeding the effect of the highest usable serum
concentrations. TGF has multiple, mostly inhibitory effects on
hemopoiesis. It causes growth arrest and premature maturation in
erythroid cultures,16-18 induces hemoglobin accumulation in
erythroid cell lines,18-20 and also reversibly suppresses
hemopoiesis in vivo.21-23 A role of TGF in the
regulation of in vivo erythropoiesis might be implicated from a recent
study showing a positive correlation between elevated plasma levels of
TGF and HbF in patients with sickle cell disease.24 We
show here that a relatively brief exposure to TGF during the first
few days of cell culture causes a lasting increase in the proportions
of HbF-expressing cells during continued clonal expansion, as well as
an increase of overall division potential until terminal growth arrest.
This effect did not depend on the combination of cytokines used to
support erythropoiesis during TGF treatment. We suggest exploring
whether short-term TGF treatment of stem cell preparations ex vivo,
or a pulsed treatment in vivo, could be used for a therapeutic increase
in fetal hemoglobin production.
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Materials and methods |
Blood samples
Peripheral venous blood samples were collected in vacuum glass tubes
containing ethylenediamine tetraacetic acid (EDTA) as an anticoagulant
(Becton-Dickinson, Franklin Lakes, NJ) from adult donors under a
protocol approved by the Institutional Review Board at New England
Medical Center.
Sera
Fetal calf serum (FCS) (dialyzed with 10 000 MW cutoff) was
purchased from Sigma (St Louis, MO). Human umbilical cord blood was
collected without anticoagulant, the clots removed, the remaining blood
centrifuged at 3000 rpm for 20 minutes and the supernatant serum (CHS)
collected. CHS was extracted twice with 3 volumes of chloroform, then
charcoal treated twice by suspending 1 g of acid-washed charcoal (Norit
SX4; Norit, Amsterdam, The Netherlands) in 50 mL serum for 30 minutes
at 4°C. The charcoal was sedimented by centrifugation. Chloroform
extraction removed some growth-inhibitory activities, and charcoal
treatment removed all HbF-stimulating activity.
Cell culture
Blood samples were kept at room temperature and processed as soon as
possible (between 2 and 24 hours after collection). The blood was
diluted 1:4 with phosphate-buffered saline (PBS), the mononuclear cells isolated by density gradient (density 1.077), washed
in PBS with 1% bovine serum albumin (BSA), and cultured without
further processing in standard 6-well plates, 3 mL per well, at a
maximum density of 0.3 million per milliliter. The standard medium
(referred to as "control" medium) was a mixture of two-thirds
Iscoves MDM and one-third RPMI1640, containing methylcellulose (0.9%);
charcoal-treated (C)-CHS (1%); erythropoietin (EPO) (1 U/mL); stem
cell factor (SCF) (20 ng/mL); interleukin 3 (IL3) (10 ng/mL); insulin
(3 µg/mL); iron-saturated transferrin (70 µg/mL); and
mercaptoethanol (0.7 mmol/L). Cytokines were from R&D Systems or
Genzyme (Cambridge, MA). FCS was used at 30%. Recombinant human
TGF1, 2, and 3 were from R&D Systems (Minneapolis, MN). TGF
(rhTGF1) was added at 10 ng/mL, unless indicated otherwise. TGF
or FCS was removed from cultures by washing the cultures twice in
PBS/BSA, then reseeding in fresh control medium. At selected times,
whole cultures containing a large number of colonies were harvested
(ie, the colonies mixed into a single-cell suspension) and the cells
processed for flow cytometric analysis.
Cell labeling
Cells were fixed with 5% formaldehyde in PBS at 37°C for 1 hour, exposed to 100% methanol for 5 minutes at room temperature, then
permeabilized in Solution B of the Caltag Fix & Perm kit (Caltag;
Burlingame, CA) during incubation with phycoerythrin (PE)-conjugated
antibodies to the gamma chain of hemoglobin (HbF) (Cortex, San Leandro,
CA) and fluorescein isothiocyanate (FITC)-conjugated antibodies to the
beta chain of hemoglobin (HbA) (Wallac, Akron, OH) or FITC-conjugated
antibodies specific for sickle cell hemoglobin (HbS) (Wallac). After
labeling, cells were washed twice and suspended in PBS with 1%
formaldehyde and 0.2 µg/mL Hoechst 33342.
Flow cytometry
Cells were processed in a Beckton-Dickinson Vantage flow
cytometer/cell sorter with dual, displaced-beam laser excitation. Hoechst 33324 fluorescence (430 nm) was excited by ultraviolet, and PE
and FITC were excited at 488 nm and measured at 530 and 575 nm,
respectively. Correlated fluorescence values were recorded with color
compensation for PE and FITC. The accuracy of color compensation over 4 logs of fluorescence values was limited.
Intact nucleated cells were selected for all display and numerical
analysis by gating on DNA-specific Hoechst fluorescence. Most
nonproliferative cells that had died (by apoptosis or necrosis) during
the time between seeding and cell harvest were not included in the
DNA-gated profiles because they disintegrated in culture and/or during
the multiple preparation steps from harvest to analysis. This was
confirmed by tests comparing viable cell counts before fixation with
Hoechst-gated cell counts at the time of hemoglobin analysis.
The proportion (given in percentage) of F+ cells is defined as the
number of all cells that label with HbF antibody (F+A plus F+A+,
as indicated in the first profile of Figure
1), divided by the number of all cells
labeling with any of the 2 hemoglobin antibodies (ie, the sum of
F+A, F+A+, and FA+ cells). Cells negative for both
antibodies (eg, nonerythroid cells) were not included in the
calculation.
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| Fig 1.
Profiles of correlated cellular hemoglobin contents after
treatment with 30% FCS or 10 ng/ mL rhTGF1.
Comparison between continuous exposure (days 0-7, left column) and
exposure for the first 4 days of culture (days 0-4, right column).
Analysis on day 7 of culture. Cultures were mixed into single-cell
suspensions and the correlated contents of HbF and HbA for each
individual cell measured by 2-color flow cytometry. Each analyzed cell
sample results from a mixture of about 100 colonies. Each profile
results from 10 000 intact nucleated cells with normal DNA content,
gated by Hoechst fluorescence (see "Materials and methods"). The
proportions of F+ cells (percentage of all Hb+ cells, see "Materials
and methods") are indicated in the upper right corner of each
profile.
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To determine absolute cell numbers in any area of the hemoglobin
profiles, known amounts of fluorescent plastic beads (Immunobrite Level
IV; Coulter, Miami, FL) were added to each cell suspension. The
absolute cell numbers per sample could then be calculated from the
ratio of cells to beads, multiplied by the number of beads added to the
sample.25 For the display of Hb profiles, the beads were
gated out. The flow cytometric study of erythropoiesis as used here is
further described and discussed in Bohmer.26
Statistics
Each analyzed cell sample resulted from a mixture of large numbers
of colonies. Experiments in which cultures turned out to contain far
less than 100 colonies (per condition analyzed) were not used for this
presentation, because the variation of profiles between individual
colonies is significant.27 From each prepared cell sample,
at least 20 000 particles (intact nucleated cells plus added beads)
were analyzed in the flow cytometer, and each hemoglobin profile is
presented with 10 000 cells. The standard error of measurement between
replicate identical cultures within the same experiment, or between
replicate flow cytometry runs from the same harvested culture, was
found to be reliably less than ±5% of the measured value. Between
cultures derived from different donors, the variability of numerical
results was more substantial, and time course data from separate
experiments could not be pooled with meaningful normalization.
Therefore, some kinetic results are demonstrated in form of an
individual representative experiment, and the statistics between
independent experiments are shown separately.
High-performance liquid chromatographic analysis of hemoglobins
Hemoglobins present in the cell lysates were separated by
high-performance liquid chromatography (HPLC) using a PolyCAT A cation
exchange column (PolyLC, Columbia, MD), 200 × 4.6 mm using the
buffer system described in Ou and Rognerud.28 The buffer gradient was delivered via a Rainin HPX HPLC system (Rainin Instrument Corp, Woburn, MA) and hemoglobins detected at 415 nm using a
Perkin-Elmer 785A UV/Vis detector (Perkin-Elmer, Norwalk, CT).
Hemoglobins in the lysates were identified by comparing retention times
with known hemoglobin standards used for isoelectric focusing (Wallac).
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Results |
Relative increase in fetal hemoglobin-containing cells by serum
or transforming growth factor beta
To compare the well-known effect of high serum concentrations with
the effect of TGF, we cultured mononuclear cells from adult blood
with 30% FCS or 10 ng/mL rhTGF-1 (TGF). The control (CON) contained
1% charcoal-treated human cord serum, a supplementation that allows
fast colony growth while minimizing the proportions of HbF-containing
cells.27 On day 4, part of the cultures were washed and
reseeded in control medium without FCS and TGF, the other part
remaining unmodified until the time of harvest. On day 7, cultures
containing about 100 colonies were turned into single-cell suspensions
and the correlated contents of HbA and HbF measured by flow cytometry
(Figure 1). In the control cultures, the large majority of nucleated
red cells were clustered with a range of HbA levels but with little or
no HbF (FA+). A small proportion contained HbF, together with
HbA (F+A+). Some cells were F+A, and some were spread over other
areas of the profile. The subdivision chosen for numerical evaluations
is indicated in the first profile, and the percentage of F+ cells (see
"Material and methods" for definition) is indicated in each
profile. More data on the time course of hemoglobin accumulation in
erythroid cultures are shown elsewhere.2,26,27 TGF
increased the proportions of F+ cells more strongly than 30% FCS. Four
days of exposure to the HbF-inducing agents (right column of Figure 1)
increased the proportions of F+ cells nearly as effectively as
continuous exposure up to the day of analysis.
In contrast to the 4-day treatment, continuous exposure to TGF (day
0-7) resulted in small colonies that appeared to consist mostly of
apoptotic/necrotic cells. Therefore, continuous TGF treatment was
not considered of further interest for the purpose of this study.
Near-maximum F+ proportions by brief transforming growth factor
beta treatment in the early culture phase
Cultures were treated with TGF or FCS for 0, 1, 2, 3, and 4 days,
then reseeded without TGF, and the proportions of F+ cells were
determined on day 7. Figure 2A shows the
time dependence using a representative experiment (Figure 2B). A small
increase in F+ cells could be seen after a 2-day treatment, and the
effect was nearly maximal with a 4-day treatment. The time course was similar for FCS and TGF. The proportions of F+ cells reached a
plateau at approximately 50% with 30% FCS and at approximately 80%
with TGF. Baseline and induced plateau levels varied between experiments from different blood donors. Further experiments focused on
TGF as the more powerful inducer of F+ cells.
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| Fig 2.
Short-term incubation with FCS or TGF1.
(A) Cultures initiated in the presence of TGF1 or FCS were washed
and reseeded in control medium after 0, 1, 3, and 4 days. The
percentage of F+ cells was determined on day 7 of culture. The data are
from 1 representative experiment. (B) Statistics of the TGF1 effect
in cultures from different donors, comparing various treatment timings
between days 0 and 5. Measurements on day 7. The values from all
individual cases (open circles) are shown to indicate range and
distribution, and the median values are shown as horizontal bars. On
the x-axis label, the upper row of numbers (from) indicates the
starting day of treatment, and the lower row (to) the ending day of
treatment. In the case of 4-day treatment, data pairs are connected.
There is a strong positive correlation (r = +0.84) between
the variations of TGF-induced F+ cell proportions and the variations of
the corresponding baseline F+ cell proportions. The effect of 4-day
treatment is statistically highly significant with
P < .0001.
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Figure 2B further explores the timing of TGF treatment, showing the
statistics of experiments with the blood from many different donors.
Treatment from day 0 to 4 is compared with treatments using narrower
intervals (days 2 to 4 and 1-day intervals within the first 5 days of
culture). The difference between baseline and F+ proportions induced by
4-day TGF treatment was statistically highly significant, with
P < .0001. The value pairs are connected and demonstrate a
positive correlation between the variations of baseline values and
inducible F+ proportions (r = +0.84). Treatment from day 2 to
day 4 was as effective as treatment from day 0 to day 4. Although the
treatment between day 0 and day 1 had no significant effect, a
treatment for as little as 1 day between day 2 and day 4 caused a
substantial increase in the proportions of F+ cells. After day 4, the
potential of TGF to induce F+ cells decreased (see also Figure 4).
The effects of TGF treatments for less than one cell cycle duration
(ie, less than 18 hours) will be the subject of a separate study.
Relative effectiveness of different forms of transforming growth
factor beta
To investigate the relative potencies of different forms of TGF
to increase the proportions of F+ cells, we titrated TGF 1, 2, and 3 over a wide range of concentrations. The averaged data of 2 experiments
with blood cells from different donors are shown in Figure
3. For the titrations, we chose a
relatively brief (38 hours) TGF exposure between days 2 and 4, during the TGF-sensitive culture phase (Figure 2B), because we observed
that, in this case, the titration curves shifted to lower
concentrations, compared with treatments from day 0 to day 4. This may
be due to some TGF degradation from the beginning of its presence in
culture. TGF-1 was approximately 100 times more potent than
TGF-2, and approximately 50 times more potent than TGF-3, with
potency defined by the concentration required to achieve half-maximal
effect. However, the maximum achievable (plateau) levels of F+ cell
proportions were the same with all 3 forms of TGF, indicating that
this level was determined by the treatment schedule.
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| Fig 3.
Relative potency of different forms of TGF.
Cultures were treated with a wide range of concentrations of TGF 1, 2, and 3 between day 2 and day 4 (hour 52  hour 90), then
reseeded and grown in TGF-free medium. The percentage of F+ cells
was determined on day 8. Data are average values from 2 independent
experiments with blood from different donors. The range of values is
indicated by vertical bars. Some bars are not visible because they fall
within the extension of the symbols.
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F+ cell proportions persist during further culture expansion
The proportions of F+ cells during further culture development were
monitored in separate experiments (Figure
4). After a 4-day treatment with TGF or
serum, cultures were grown in control medium and further subcultivated
on days 7 and 10 to minimize the exhaustion of medium components or the
potential accumulation of inhibitory factors by the rapidly expanding
cell mass. The time course is shown as a representative experiment in
which all conditions were investigated simultaneously on cells from the same donor. The data from 3 further independent experiments are given
as vertical bars, indicating the range of measured values. The
increased proportions of F+ cells, as introduced by brief TGF or
serum treatment, were maintained during further culture growth without
TGF or serum, indicating that F+ and FA+ cells were expanding
at approximately the same rate. A gradual small decline in F+
proportions was observed in the TGF-treated cultures, but the
proportions did not approach the levels of untreated cultures (see also
Figure 7A).
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| Fig 4.
Persistence of F+ cell proportions during the later
culture phase.
Cultures were incubated with TGF or FCS for 4 days, then washed and
reseeded in fresh control medium. On days 7 and 10, the cultures were
further diluted (1:10 and 1:5, respectively) in fresh control medium
(full symbols) or medium supplemented with TGF1 (open symbols).
Percentage of F+ cells was determined at selected times between day 7 and day 13. The time course is shown from 1 individual experiment in
which all conditions were investigated together on the cells from the
same donor. The vertical bars on days 7, 10, and 13 show the ranges of
results obtained in 4 independent experiments with blood from 4 different donors.
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To assess the effect of TGF at this later culture phase, parts of
the cultures in the same experiment were newly supplied with TGF
beginning on day 7 (data included with open symbols in Figure 4). Added
at this later stage of culture, TGF had no effect on F+ proportions
but caused near-complete growth arrest after 3 to 4 days (not shown).
Thus, growth inhibition by TGF occurred with the same kinetics in F+
and FA+ cells. In parallel, TGF caused an accelerated
accumulation of HbA in both F+ and F cells. These data are not
within the scope of this presentation and will be shown in a separate
study. The findings are in agreement with a previous report
demonstrating that TGF caused growth arrest, combined with
accelerated maturation and hemoglobin accumulation.17
Stimulation of F+A cells and transition to F+A+
The hemoglobin profiles of TGF-treated cultures showed not only
increased proportions of F+ cells, but also a change in the distribution within the F+ cell population, with increased proportions of cells containing high levels of HbF but little HbA (F+A) (see profiles in Figures 1 and 8). This population of F+A cells is reminiscent of nucleated red cells in cultures from fetal blood, in
which all cells initially accumulate only HbF for at least 1 week, and
do not begin any HbA accumulation before they have reached maximum
HbF.2,27 The data from many independent experiments are
compiled in Figure 5. On day 7, the
proportions of F+A cells (given as a percentage of F+ cells, not
of all hemoglobin-containing cells) averaged approximately 10% in
controls and approximately 40% in TGF-treated cultures. The
difference in F+A proportions between TGF-treated and control
cultures is statistically significant with P < .0001. With
increasing culture time, the proportions of F+A cells within the
F+ population declined gradually. This decline suggests that the
TGF-induced F+A population moved into the F+A+ compartment
during further culture growth.
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| Fig 5.
Progression from F+A to F+A+ with time in
culture.
F+A cells (defined in Figure 1) were quantitated at different
culture times (days 7, 10, 13, and 16) and expressed as a percentage of
all F+ cells (note: not of all Hb+ cells). Results from all individual
cases are shown (open circles) to indicate range and distribution of
values. Averages are shown by horizontal bars. C = control;
T = TGF treatment. The difference in F+A proportions
between TGF-treated and control cultures on day 7 is significant
with P < .0001.
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Molecular ratios of HbF/HbA+HbF in whole-culture lysates
For reasons elaborated in "Discussion," flow cytometry cannot
provide a calibrated measurement of absolute hemoglobin contents. However, the molecular proportions of HbF (HbF/HbA+HbF) or the relative
synthesis rates of gamma and beta chains are the gold standard to
assess the efficacy of agents to modify the balance between fetal and
adult hemoglobin expression from adult progenitors. Therefore, we used
cation exchange HPLC to measure the molecular ratios of HbF/HbA+HbF in
whole-culture lysates at different times after a 4-day TGF
treatment. Figure 6 shows the data from 7 independent experiments, with cultures from individual blood donors
harvested at different times between day 8 and day 19. The value pairs
from each experiment are connected. TGF treatment caused a
significant (P < .01) increase in the molecular ratios of
HbF/HbF+HbA in whole-culture lysates, in accordance with the increased
proportions of F+ cells in those cultures.
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| Fig 6.
Molecular ratios of HbF/HbF+HbA in whole-culture
lysates.
Cultures were lysed and the relative amounts of fetal hemoglobin
measured by cation exchange HPLC. The ratios HbF/HbF+HbA are multiplied
by 100 to give the percentage of HbF. The 2 values plotted at the 0.1%
level are from samples in which the HbF peak was too small for
quantitation. Each value pair (connected) represents a separate,
independent experiment in which all cells were harvested at 1 time
point between day 8 and day 19. Different culture times are
distinguished by symbols: Diamonds: day 8; crosses: day 11; circles:
day 13 (3 different experiments); triangles: day 16; squares: day 19. The effect of TGF is significant with P < .01.
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Effect of TGF on absolute cell numbers and long-term
proliferation
A change in relative F+ cell numbers could be based on different
mechanisms that might be distinguishable by comparing absolute cell
counts per culture. Cultures that were treated with TGF from days 0 to 4 were subcultivated again on days 7, 10, and 13, and the total
production of F+ and FA+ cells per culture were determined on
days 7, 10, 13, and 16. Figure 7A shows the
time course from a representative experiment: Between days 7 and 10, the total numbers of F+ cells (full symbols) in TGF-treated and control cultures were approximately equal, whereas FA+ cells (open symbols) were dramatically reduced in the TGF-treated
cultures, suggesting a selective inhibition or deletion of FA+
cells during or shortly after the treatment phase. Between days 7 and
10, both cell types in both cultures proliferated rapidly at
approximately the same rate. After day 10, in the control culture, the
proliferation decreased strongly and equally for both F+ and FA+
cells, maintaining the ratio. No secondary colonies were seen after the
subcultivation of control cultures on day 13, in any experiment. In
contrast, the proliferation in the TGF-treated culture continued for
much longer for both F+ and FA+ cells, leading to a much higher
overall production of F+ cells during the entire culture life span.
This conclusion from cell counts is supported by photographs of
cultures on day 18, after reseeding on day 13, showing the growth of
large cell clusters in TGF-treated cultures but not in the controls (Figure 7A insert). The FA+ cell numbers were gradually reducing the gap with F+ cells, but F+ cell proportions remained above 50%. The
number of days for which the proliferation of TGF-treated cultures
could be maintained was highly variable between donors (between 18 and
26 days), whereas the proliferative life spans of untreated control
cultures were less variable (12-14 days). We define the proliferative
life span as the time after which further subcultivation does not lead
to any detectable growth of cell clusters of any size.
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| Fig 7.
Absolute numbers of F+ and FA+ cells.
Cultures were treated with TGF from day 0 to day 4, then propagated
without TGF, with further subcultivation and dilution on days 7 (1:10), 10 (1:5), and 13 (1:5). Absolute numbers of F+ and FA+
cells per culture were determined on days 7, 10, 13, and 16. (A) Time
course of proliferation. Representative example from one experiment.
Overall cell production is calculated by multiplying total cell numbers
per culture with all preceding culture dilution factors. Open symbols:
FA+ cells; full symbols: F+ cells. Triangles: TGF-treated
cultures; circles: controls. Inset: photographs of day 18 cultures,
after reseeding on day 13. Left: control; right: TGF-treated. (B)
Ratios of cell numbers in TGF-treated and control cultures (N[TGF]
/ N[CON]), as a function of culture time. Pairs of F+ and FA+
values from individual experiments are identified by connecting lines.
Median values are indicated by horizontal bars, and individual values
are shown (open circles) to indicate range and distribution. The
increase in the ratios (N[TGF]/N[CON]) between day 7 and day 16 is
statistically significant, with P < .03 for F+ cells and
P < .02 for FA+ cells. The difference in the ratios
N(TGF)/N(CON) between F+ and FA+ cells is significant with
P < .001 at all time points.
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Despite substantial variations in the time course and life span (which
made it impossible to pool the growth curves from different experiments), both the initial selective reduction of FA+ cells and the increase in overall cell production was strictly reproduced in
4 experiments from different donors. The data are summarized in Figure
7B. The ratios of absolute cell numbers in TGF-treated to control
cultures (N[TGF] / N[CON]) are shown on different days, with pairs
of F+ and FA+ values for individual samples identified by
connecting lines, and medians indicated as horizontal bars. Measured on
day 7, absolute F+ cell numbers were little affected by TGF
(ratio = 1), whereas the FA+ cell numbers were reduced approximately 10-fold (ratio = 0.1). This difference in the effect of
TGF on F+ and FA+ cells was highly significant with
P < .0001. The day 7 values are shown from 12 experiments,
and 4 experiments were extended until terminal growth arrest in the
control cultures. With increasing culture time, the ratios increased
for both F+ and FA+ cells, indicating that both types of cells
in TGF-treated cultures were able to outproliferate the controls, as
exemplified in Figure 7A. On average, the F+ population of
TGF-treated cultures grew to nearly 10-fold higher levels than the
controls. The increase from day 7 to day 16 was significant with
P < .03. In 2 experiments, the TGF-treated cultures kept
proliferating and producing secondary colonies beyond 3 weeks, with the
ratios N(TGF)/N(CON) for F+ cells exceeding 100. Data beyond day 16 are
not shown because cell counts in control cultures decreased because of
cellular disintegration or enucleation, making the ratio appear
meaningless during this phase. Under our general culture conditions, a
proliferative life span beyond 3 weeks is observed only in cultures
from fetal blood at an early gestational age.
Transforming growth factor effect does not depend on the
combination of cytokines
As a first attempt to explore the basis of TGF action during the
time of treatment, we examined how it would be affected by the choice
of cytokine cocktail, supporting erythropoiesis during the phase of
TGF treatment. We initiated cultures in EPO+SCF+IL3, EPO+SCF,
EPO+IL3, and SCF+IL3, and treated them with TGF for the first 4 days. The cultures were then reseeded, without TGF, in the full
cytokine cocktail (EPO+SCF+IL3) and analyzed between days 7 and 9 of
culture. Figure 8 shows examples of the
resulting profiles, and Table 1 shows the
numerical evaluations, with proportions of F+ cells, F+A cells,
absolute cell numbers, as well as the numbers of secondary colonies. In
all cytokine combinations, TGF increased the proportions of F+
cells, based mostly on a strong reduction of FA+ cells (ratio
approximately 0.1) and accompanied by a moderate (approximately
2×) decrease in secondary colonies.
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| Fig 8.
Effect of TGF under different cytokine combinations.
Cultures were initiated in media with different combinations of
cytokines (SCF + IL3 + EPO, SCF + EPO, IL3 + EPO) in the
presence or absence of TGF (as indicated in the figure).
After 4 days, all cultures were reseeded without TGF in fresh
medium with the full complement of SCF, IL3, and EPO.
Examples of hemoglobin profiles from day 8 cultures (4 days after
reseeding). See Table 1 for numerical evaluations.
|
|
It must be pointed out that suboptimal cytokine combinations lead to a
reduced cell cycle rate during the first few days of culture, which is
reflected by the reduced secondary colony numbers (in control cultures
without TGF) (Table 1). Therefore, a TGF treatment timing that is
optimal for one cytokine cocktail may not be optimal for another, so
that a quantitative comparison of the magnitudes of TGF effect
between cytokine conditions has limited significance in this
experiment. (Further kinetic exploration will be presented in a
separate study.) Nevertheless, the data of Figure 8 and Table 1
indicate that the 4-day TGF treatment had a similar relative effect
in all cytokine combinations, indicating that it may not be based on an
interference with the support of survival, proliferation, or maturation
by those cytokines.
| |
Discussion |
We have shown that TGF treatment leads to dramatically increased
proportions of HbF-containing cells in erythroid cultures from adult
peripheral blood. This effect was achieved by TGF treatment within
the first 4 days of culture. Both the baseline and TGF-induced
proportions of F+ cells remained nearly constant during further culture
expansion, with F+ and FA+ cells proliferating at approximately
the same rate.
An increase in the proportions of F+ cells, initiated early in culture
and measured after 1 week or later, could be due to different
mechanisms: a reversal of the hemoglobin switch, a selective proliferative boost to F+ cells, or a selective inhibition of FA+ cells. A pure reversal of the hemoglobin switch would lead to increased numbers of F+ cells at the numerically equal expense of
FA+ cells. A selective increase in the rate of F+ cell
proliferation would leave FA+ cell numbers unchanged. Finally, a
selective suppression of FA+ cells would leave F+ cell numbers
unchanged. None of the above is seen in our experiments. The data
indicate a complex situation that may be explained by the superposition of many effects. A selective deletion of progenitors that are programmed to develop into cells expressing only HbA would explain the
strongly and selectively decreased FA+ numbers measured between day 7 and day 10. However, FA+ cells of treated cultures
eventually catch up with controls, and F+ cell numbers of treated
cultures far exceed those of controls during the late stages of
culture. The increased overall cell production excludes mechanisms in
which TGF merely deletes any progenitor subpopulation, or
transiently delays their proliferation. The initial shift to F+A
cells within the compartment of F+ cells appears to fit better with a
reversal of the hemoglobin switch than with a selective deletion of
FA+ cells, because the latter would leave the distribution of
cells within the F+ compartment unchanged. One could speculate that a
reversal of the hemoglobin switch might be coupled with an increased expansion potential of newly initiated F+ cells. However, that would
leave the high division potential of initially suppressed FA+
cells unexplained. Alternatively, TGF might suppress negative growth
control by autocrine/paracrine signaling, similar to the transforming
effects of TGF in some other cell systems.
We emphasize that our flow cytometric data and conclusions deal with
the distinction and enumeration of F+ and FA+ cells, without
quantitating absolute hemoglobin contents in cells. In the lower ranges
of cellular hemoglobin content, which prevail up to approximately day
8, the fluorescent antibody signal is likely to be approximately
proportional to hemoglobin content. However, at later stages of
erythroid maturation, the large amounts of hemoglobin per cell lead to
saturation effects that prevent the resolution of quantitative
differences by fluorescent antibody label.26 Therefore,
although the distinction between F+ and F cells remains reliable
throughout the erythroid development, the use of a complementary method
of hemoglobin measurement was required. Unsurprisingly, increased
proportion of F+ cells in a culture were reflected by an increased
ratio of HbF/HbF+HbA in that culture. The numerical relationship
between F+ cell proportions and the molecular HbF proportions in whole
cultures is complex because F+ cells also accumulate HbA, so that
numerical comparisons between the data from the different assays are
not possible. However, the HPLC results in Figure 6 support our
conclusion that brief TGF treatment increased the relative HbF
levels during further culture development.
Apart from the relative and absolute numbers of F+A+ erythrocytes in a
patient, an important question is whether the hemoglobin ratios within
individual F+A+ erythrocytes are affected by TGF treatment of their
early progenitors. Considering that the final stages of erythroid
maturation in vitro may not reflect the situation in vivo, the terminal
maturation of TGF-treated erythroid progenitors will have to be
studied in animal models.
The effects of TGF were similar in different cytokine combinations,
suggesting that this effect is not based on interference with the
survival- and division-supporting activity of cytokines. This result
appears surprising, considering the reported effects of TGF on the
signaling of SCF and IL3.29,30 A regulatory role of TGF
on HbF levels via the SCF-signaling mechanism has been suggested to
explain a negative correlation between SCF and TGF plasma levels in
patients with sickle cell.24 However, our data may suggest
a more direct causal link between high levels of TGF and high HbF
levels in these patients.
Serum was less effective than TGF in increasing the proportions of
F+ cells, but was also less inhibitory to cell proliferation (data not
shown). The similar kinetics of the serum effect suggest that the
mechanism of action may be the same. However, different experiments
will be needed for quantitative comparisons, because of the
superimposed effects of serum-contained additional growth factors.
Various tests (TGF quantitation in sera by enzyme-linking immunosorbent assay [ELISA], neutralizing antibodies, and
latency-associated peptide [LAP]) indicated that the serum activity
cannot be explained by its content of TGF (unpublished data).
Different forms of TGF (beta 1, 2, and 3) had different potencies to
increase F+ cell proportions, with potency defined as the concentration
required to achieve half-maximal effect. The maximum achievable effect
(ie, the plateau level of F+ proportions at saturating TGF
concentration) was the same for TGF 1, 2, and 3. It will be
interesting to explore if and how the potencies of different TGF
forms to increase F+ cell proportions correlate with their potencies to
cause growth inhibition and acceleration of erythroid
maturation.17
Flow cytometric measurement of the correlated cellular contents of
different hemoglobin types provides a novel tool for the study of
erythropoiesis in culture, permitting the detection of novel features,
as well as the quick screening of the effects of cytokines and drugs.
After substituting HbS for HbA antibodies, the hemoglobin profiles of
nucleated red cells are also suitable to study sickle cell
erythropoiesis, and to monitor the condition and responses of patients
with sickle cell disease.
We were able to demonstrate the same effect of TGF in a culture from
a patient with sickle cell disease (data not shown). We now propose to
study whether our results have any potential application for the
treatment of hemoglobin disorders that are ameliorated by an increase
in the relative amounts of cellular HbF or the relative numbers of F+
cells. Potential clinical avenues may include the repeated
transplantation from a bank of TGF-treated autologous progenitor
cells (eg, from preserved cord blood), or in vivo treatment by brief
TGF pulses at intervals that avoid overall suppression of hemopoiesis.
| |
Footnotes |
Submitted March 4, 1999; accepted January 4, 2000.
Supported by a research grant from Genzyme Genetics.
Reprints: Ralph M. Bohmer, New England Medical Center, Box 394, 750 Washington St, Boston, MA 02111; e-mail: ralph.bohmer{at}es.nemc.org.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction