Stem Cells 2004;22:216-224
www.StemCells.com
© 2004 AlphaMed Press
IL-3-Dependent Early Erythropoiesis Is Stimulated by Autocrine Transforming Growth Factor Beta
Ralph M. Böhmer
Genetic Technologies Pty Ltd., Melbourne, Australia
Key Words. AC133 antigen • Adult stem cells • Erythropoiesis • Proliferation • IL-3 • Multiparameter flow cytometry • Stem cell culture
Ralph Bohmer, Ph.D., Genetic Technologies Pty Ltd., P.O. Box 115, Fitzroy, Victoria 3065, Australia. Telephone: 61-3-8412-7020; Fax: 61-3-9417-6863; e-mail: ralph.bohmer{at}genetype.com.au
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ABSTRACT
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Autocrine/paracrine transforming growth factor beta (TGF-ß) is an important regulator of stem cell quiescence and generally suppresses stem cell proliferation. However, we show here that during the first few days of an erythroid cell culture from adult blood stem cells, the presence of neutralizing antibodies against TGF-ß had a suppressive effect on subsequent erythropoiesis, indicating a stimulatory action of autocrine TGF-ß. The suppression occured in the form of a delay in erythroblast proliferation rather than a reduction in final erythroid colony numbers. The inhibitory effect of anti-TGF-ß occured in the presence of interleukin-3 (IL-3) but not in cultures with only stem cell factor and erythropoietin. Erythroblasts expressing gamma-globin (
+) were more strongly suppressed than erythroblasts expressing only beta-globin (-ß+), so that stem cell treatment with anti-TGF-ß caused a decrease in the proportion of + cells. Anti-TGF-ß had an inhibitory effect on erythropoiesis only when administered during the first 4 days of culture, that is, before the onset of globin expression and dependence on erythropoietin. The decreasing effect of anti-TGF-ß with delayed addition coincided with a decreasing dependence on IL-3. CD133+ stem cells were more strongly suppressed by anti-TGF-ß than the complementary CD133-CD34+ stem cells, and the latter were also much less dependent on IL-3. The treatment of very early stem cell cultures with a pulse of added TGF-ß1 in the presence of IL-3 increased the subsequent proliferation of erythroblasts. Taken together, the data suggest that IL-3-driven early erythropoiesis from immature peripheral blood stem cells is stimulated by autocrine TGF-ß.
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INTRODUCTION
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The proliferation and differentiation of cultured peripheral blood hematopoietic stem cells are strongly affected by transforming growth factor beta (TGF-ß) in multiple and seemingly disparate ways, depending on assay conditions [1–3]. The predominant effects of autocrine, paracrine, and added TGF-ß on hematopoietic stem cells are the suppression of proliferation and the maintenance of quiescence [1–16]. The inhibitory effect of autocrine TGF-ß on stem cell proliferation can be overcome by neutralizing TGF-ß or its type II receptor [6, 7, 9, 10, 13]. Once erythropoiesis has begun, the suppressive effect of TGF-ß on proliferation is accompanied by an acceleration of hemoglobin production and maturation to the point of enucleation [15, 16].
However, we showed recently that, in cultures from adult peripheral blood stem cells, a pulse of TGF-ß treatment within the first 4 days of culture increased not only the proportions of fetal hemoglobin-expressing cells but also the overall numbers of developing erythroid cells [17]. A stimulation of subsequent erythropoiesis by brief TGF-ß treatment appears to conflict with the well-established suppression of proliferation by added or autocrine TGF-ß. However, most studies showing the antiproliferative effects of TGF-ß employed primary or secondary colony assays and did not monitor the proliferation of early erythroblasts.
Therefore, we investigated the potential role of autocrine or added TGF-ß during the early development of cultured peripheral blood stem cells toward erythropoiesis. We focused on CD133+ stem cells with erythroid potential, most of which represent a large immature subpopulation of CD34+ cells [18–24], because our preliminary tests showed them to be more responsive than the remaining CD133-CD34+ population to neutralizing anti-TGF-ß antibodies as well as to the early-acting cytokine interleukin-3 (IL-3). Using a sensitive flow cytometric method that can detect very low levels of cellular globin contents [25, 26], we monitored the effects of neutralizing anti-TGF-ß antibodies, applied during the first few days of stem cell development, on the subsequent proliferation and globin expression of erythroid progenitors. We show here that, in the presence of IL-3 but not in its absence, neutralizing anti-TGF-ß antibodies delayed the proliferation of erythroblasts and decreased the occurrence of gamma-globin-expressing cells. Since both effects are the reverse of the effects of added TGF-ß, they indicate a stimulatory activity of autocrine TGF-ß during IL-3-dependent early erythroid development.
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MATERIALS AND METHODS
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Cell Culture
Peripheral venous blood samples were collected from adult donors under a protocol approved by the Institutional Review Board at the New England Medical Center, Boston, MA. Mononuclear cells were isolated by density gradient (1.077), and CD133+ cells were isolated using magnetic beads with CD133-specific antibodies (Miltenyi Biotech; Auburn, CA; http://www.miltenyibiotec.com). CD133-CD34+ cells were isolated by magnetic CD34 beads from the unattached fraction of the CD133+ separation. Stem cell preparations were mixed into medium and aliquots were set into wells of multi-well plates, aiming at cell concentrations that would give rise to 30–50 colonies per culture condition and time point. The medium consisted of a mixture of two-thirds Iscove’s-modified Dulbecco’s medium and one-third RPMI 1640, methylcellulose (0.9%), insulin (3 mg/ml), iron-saturated transferrin (70 µg/ml), mercaptoethanol (0.7 mM), recombinant human erythropoietin (rhEPO, 1 U/ml), and stem cell factor (rhSCF, 20 ng/ml). IL-3 (rh, 10 ng/ml) and TGF-ß1 (rh, 10 ng/ml) were added as required. The base methylcellulose medium was from Stem Cell Technologies (Vancouver, Canada; http://www.stemcell.com). All cytokines were from R&D Systems (Minneapolis, MN; http://www.rndsystems.com). The medium was further supplemented with 1% of chloroform-extracted and charcoal-stripped pooled human umbilical cord serum, as described previously [16]. This supplement did not contain active TGF-ß at levels detectable by enzyme-linked immunosorbent assay, and immunodepletion of serum with bead-bound anti-TGF-ß antibodies did not affect the experimental results. Neutralizing antibodies against TGF-ß (pan-specific polyclonal anti-TGF-ß; mAb anti-TGF-ß1, mAb anti-TGF-ßII), the TGF-ß receptor II (mAb anti-TGF-ßRII), and latency-associated peptide (LAP TGF-ß1) were from R&D Systems.
At selected times, whole cultures containing a large number (>30) of colonies were harvested (i.e., the colonies mixed into a single cell suspension) and the cells were processed for flow cytometric analysis.
Flow Cytometry
Cells were fixed with 5% formaldehyde in phosphate-buffered saline (PBS) at 37°C for 1 hour, exposed to 100% methanol for 5 minutes at room temperature, then permeabilized in reagent B of the Caltag Fix & Perm permeabilization kit (Burlingame, CA; http://www.caltag.com) during incubation with phycoerythrin (PE)-conjugated antibodies to the gamma chain of hemoglobin (-globin; Cortex; San Leandro, CA; http://www.cortex-biochem.com) and fluorescein isothiocyanate (FITC)-conjugated antibodies to the beta chain of hemoglobin (ß-globin; Wallac; Perkin Elmer; Boston, MA; http://www.perkinelmer.com). After labeling, cells were washed and suspended in PBS with 1% formaldehyde and 0.2 mg/ml Hoechst 33342.
Cells were processed in a Beckton-Dickinson (Franklin Lakes, NJ; http://www.bd.com) Vantage flow cytometer/ cell sorter with dual, displaced-beam laser excitation. Hoechst 33342 fluorescence (430 nm) was excited by UV, and PE and FITC were excited at 488 nm and measured at 530 nm and 575 nm, respectively.
Intact nucleated cells were selected for all display and numerical analyses by gating on DNA-specific Hoechst fluorescence. To determine absolute numbers of + and -ß+ cells per culture from the hemoglobin profiles, known amounts of fluorescent plastic beads (Coulter Immunobrite Level IV; Fullerton, CA; http://www.coulter.com) were added to each cell suspension, and absolute cell numbers per sample were calculated from the ratio of cells to beads. For the display of hemoglobin profiles, the beads were gated out. The flow cytometric study of erythropoiesis as employed here is further described and discussed in [26].
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RESULTS AND DISCUSSION
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IL3-Dependent Suppression of Erythroblast Proliferation by Anti-TGF-ß
To study the potential role of autocrine TGF-ß on erythroid stem cell development, we cultured CD133+ peripheral blood stem cells in the presence of neutralizing antibodies to TGF-ß. After 8 days, colonies were pooled, and the cell suspensions were analyzed by flow cytometry, recording two-parameter globin profiles and calculating absolute cell numbers per culture. Figure 1
compares the effect of anti-TGF-ß (pan-specific polyclonal antibody) in cultures with different cytokine combinations (SCF + IL-3, SCF only, and IL-3 only). Representative examples of two-parameter globin profiles (ß-globin versus -globin) are shown in Figure 1A. The profiles show cells with -globin (region 1 indicated in the first profile, +), ß-globin only (region 2, -ß+), and some cells with none of the two measured types of globin. Under all conditions, the large majority of nucleated cells in the cultures contained some combination of globins, which are summarily called erythroblasts in this study. A comparison of cluster densities in the profiles suggests that the proportions of + cells are reduced by anti-TGF-ß (Fig. 1B).
The control profiles with only IL-3 showed a much higher level of globin content than the profiles with IL-3 + SCF, which may confirm a maturation-retarding effect of SCF [27]. Colonies with only IL-3 were very small and looked necrotic/apoptotic, but were equal in number to the large colonies in cultures with IL-3 + SCF (not shown).
Figure 1B
provides a numerical evaluation and statistics from a large number of independent experiments with stem cells from different blood donors. The effect is shown as day-8 erythroblast numbers in cultures with anti-TGF-ß, divided by erythroblast numbers of untreated controls (set to 100%). Gamma-positive and -ß+ cell numbers are shown separately, and values corresponding to the same experiment are identified by a connecting line in order to show the statistical reliability of the differential effect on + and -ß+ cells. The data show that, in the presence of IL-3, erythroblast numbers were substantially suppressed by anti-TGF-ß. The suppressive effect was consistently stronger for + cells than for -ß+ cells, which implies that the proportion of + cells in the culture was reduced by anti-TGF-ß. In contrast, there was no suppressive effect of anti-TGF-ß in the absence of IL-3 (SCF + EPO only), and occasionally, but not consistently, a stimulatory effect was observed.
Comparison of TGF-ß-Neutralizing Agents
We also tested the effect of neutralizing monoclonal antibodies to the TGF-ß receptor II (TGF-ß RII), TGF-ß1, TGF-ß2, as well as TGF-ß1-neutralizing LAP (Fig. 2A). Anti-TGF-ß2 had no inhibitory effect, which may help to rule out a nonspecific toxicity of the antibody preparations (anti-TGF-ß2 serving as an irrelevant isotype control). The other agents suppressed erythroblast numbers approximately as effectively as the pan-specific anti-TGF-ß. The combination of pan-specific anti-TGF-ß and anti-TGF-ßRII was only slightly more potent than each agent alone. Therefore, further experiments were carried out with pan-specific anti-TGF-ß alone.
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Figure 2. Comparison of TGF-ß-neutralizing agents and effect on colony numbers. A) CD133+ cells from many different blood donors were cultured in methylcellulose medium with Epo, SCF, and IL-3 and exposed from day 0 to different potentially TGF-ß-neutralizing agents, specifically pan-antiTGF-ß, anti-TGF-ß RII, anti-TGF-ß1, anti-TGF-ß2, and LAP (TGF-ß1), as indicated in the graph. After 8 days, whole cultures were harvested into a single cell suspension and the number of + cells was determined by flow cytometry. Cell numbers were normalized to the values in control cultures, set to 100%. Data are shown individually for each experiment, and the averages for each condition are shown as horizontal bars. B) CD133+ cells from four different blood donors were grown as in (A), with and without anti-TGF-ß (pan-specific, 10 mg/ml). Erythroid colonies (sum of multifocal, single-focal, and mixed erythroid) were counted by microscope after 2.5–3 weeks. The values are normalized as in A. Results from individual experiments are shown and the average is indicated by a horizontal bar.
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No Effect of Anti-TGF-ß on Final Erythroid Colony Numbers
The effect of anti-TGF-ß on erythroid colony growth from CD133 stem/progenitor cells was examined by counting erythroid and mixed erythroid colonies after 2.5–3 weeks of culture in methylcellulose medium with Epo, SCF, and IL-3. The data were normalized with values of control conditions set to 100% (Fig. 2B). Anti-TGF-ß did not significantly affect the numbers of erythroid colonies. Thus, the suppression of erythroblast numbers observed after 1 week of culture must be based on a suppression or delay of proliferation within the nascent erythroid colonies, rather than a reduction in colony numbers.
Dose-Response Curves for Anti-TGF-ß
The dose-response effect of anti-TGF-ß on the growth of erythroblasts from CD133 stem/progenitor cells was analyzed in methylcellulose medium containing Epo, SCF, and IL-3. Colonies were pooled after 8 days to evaluate in each condition the production of + and -ß+ cells, as well as total cell proliferation. The dose-response curves from a representative experiment are shown in Figure 3, separately plotted for the numbers of + cells and -ß+ cells, as well as the sum of all cells, including those not containing any globin. The curves of + and -ß+ cells diverge, showing the relatively stronger suppression of + cells. The total numbers of nucleated cells in the culture, including those not showing any globin label, are suppressed as well, which is to be expected since the vast majority of cells on day 8 were erythroblast (Fig. 1A). This rules out the possibility that the lower counts of globin-containing cells were due merely to a suppression of globin accumulation that would leave part of the proliferating cells uncounted on the basis of the globin label.
No saturation was apparent within the range of antibody concentrations examined (up to 20 µg/ml). The lack of saturation does not indicate a nonspecific toxicity, but can be explained by a situation that differs from one where a fixed amount of TGF-ß is to be neutralized by a fixed amount of antibody in a concentrated solution: A) gradual sequestration of antibody by the continuously produced autocrine TGF-ß, and B) antibody is evenly distributed in the volume of viscous methylcellulose medium that may limit the rate of diffusion, so that increasing antibody concentration does not only affect the overall ratio of antibody to antigen, but also shortens the diffusion distance and time to the target. In contrast, little diffusion is needed for autocrine TGF-ß to act back on its source.
The available data fit the model of a temporary effect of neutralizing antibody. The temporary nature of the inhibition could be based on the gradual sequestration of antibody or on cells bypassing the TGF-ß requirement. This needs to be explored further.
Delayed Exposure to Anti-TGF-ß
To explore the kinetics of the inhibitory effect, we added anti-TGF-ß at different times after the beginning of culture. Figure 4A shows that the inhibitory potential decreased rapidly with delayed addition and that by day 4, the addition of anti-TGF-ß had no inhibitory effect, as measured on day 8. The separate plot of + and -ß+ cell numbers confirms the stronger suppression of + cells. The data suggest that the activity of autocrine TGF-ß occurs only within the first 4 days of culture. The inset in Figure 4A further confirms the absence of inhibition by anti-TGF-ß at a later culture phase, and demonstrates that treatment with anti-TGF-ß at a later culture interval (day 7-day 11) had a slight and variable stimulatory effect on erythropoiesis. Anti-TGF-ß may become stimulatory at this stage of culture because of a continued production of autocrine TGF-ß at a phase of erythroid development where the well-known inhibitory effect of TGF-ß has set in [15, 16] (also see Fig. 6). The proportions of + cells were not substantially affected by anti-TGF-ß treatment beginning after more than 4 days. The lack of inhibitory effect of anti-TGF-ß at later culture stages may also confirm that the antibody preparations did not act via nonspecific toxic activity.
This 4-day theme of cytokine action was also encountered in other kinetic experiments on early erythropoiesis. Figure 4B shows the kinetics of IL-3 and Epo dependence, explored by withdrawal/addition of these cytokines at different times after the beginning of culture. IL-3 withdrawal (in the continuous presence of Epo and SCF) after increasing culture times reveals a rapid decrease of the initially dramatic IL-3 dependence, with none left after 4 days (Fig. 4B, diamonds). Delayed Epo addition (in the continuous presence of SCF and IL-3) demonstrates that Epo requirement sets in at around day 4 (Fig. 4B, squares). Recently, we demonstrated that only during the first four culture days can TGF-ß treatment increase the proportions of subsequently developing + cells [17]. Finally, sensitive flow cytometric globin measurement shows that the first significant cohort of globin-containing cells appears between day 4 and 5 [25] (see also the globin profiles of Fig. 6). Thus, the first 4 days appear to mark a distinct phase of early stem cell development toward erythropoiesis.
Effects of IL-3 and Anti-TGF-ß on Different Stem Cell Subpopulations
CD133+ stem cells with erythroid potential represent mostly an immature subpopulation of CD34+ stem cells with erythroid potential [18–24]. Figure 5 compares the differential effects of IL-3 and anti-TGF-ß on these subpopulations. The kinetics of erythroblast proliferation show that CD133+ cells are dramatically more dependent on IL-3 than the balance of CD133-CD34+ cells (Fig. 5A). The reproducibility of this effect is confirmed by statistics from different blood donors (Fig. 5A, last graph).
A comparison between the effects of TGF-ß-neutralizing antibodies on CD133+ and CD133-CD34+ cells is shown in Fig. 5B. The suppression by anti-TGF-ß of early erythropoiesis was stronger for CD133+ cells than for the more mature CD133-CD34+ cells. The differential effect on + and -ß+ cells applied for both cell types. Together with the data in Figures 1 and 4, this correlation appears to support the concept of an interdependence between the stimulatory effects of IL-3 and autocrine TGF-ß on very early erythropoiesis.
Effect of Added TGF-ß1
To test the interpretation that the negative effect of TGF-ß-neutralizing antibodies reflects a stimulatory activity of autocrine TGF-ß, we explored the effects of TGF-ß1 added to the cultures during the early stages of stem cell development (Fig. 6). The data shown in Figure 6 are based on purified CD34+ cells, but test experiments confirmed that added TGF-ß1 had the same effect on the CD133+ subpopulation.
Figure 6A shows examples of globin profiles after 4 and 6 days of continuous exposure to TGF-ß. On day 4, there are very few globin-containing cells in the controls but a significant number are seen in cultures with TGF-ß. In day-6 controls, the bulk of cells has begun to express globin and contains low levels of mostly -ß+ globin. In contrast, the TGF-ß-treated cultures show not only a larger proportion of + cells, but also much higher levels of globin, confirming previous reports that TGF-ß accelerates maturation [15, 16], perhaps by downmodulating the c-kit receptor and thus interfering with the maturation-retarding effect of SCF [27–29].
Figure 6B shows, in a representative experiment, the kinetics of cell proliferation during the first few days of continuous TGF-ß treatment, separately displayed for + and -ß+ erythroblasts, as well as for the sum of all nucleated cells in the culture, including those not containing any globin. In concordance with the globin profiles, the numbers of globin-containing cells are initially substantially increased by TGF-ß. Later, after about day 4–5, the controls catch up and proliferate unhindered, while the growth curves in cells treated with TGF-ß begin to show inhibition. However, the growth curves of all cells, including those without globin, do not show any initial stimulatory effect of TGF-ß but only the later inhibition. The lack of overall proliferative stimulation during continuous TGF-ß was confirmed by the observation that cell counts per colony in the budding day 3 and day 4 colonies were not affected by TGF-ß1 (not shown). In summary, the data of Figure 6 suggest that the increased numbers of globin-containing cells during the first few days of culture are due only to an accelerated globin expression that shifts cells over the globin detection threshold set for flow cytometric quantitation.
Figure 6C explores the effect of TGF-ß treatment for the first 24 hours of stem cell culture followed by a further 7 days of culture without TGF-ß. The brief TGF-ß pulse caused a substantial increase in subsequently developing + and -ß+ erythroblasts. The same increase was seen for the sum of all cells (last column), as expected since the overwhelming majority of cells on day 8 were erythroblasts under our culture conditions (Fig. 1A). This implies that the stimulation of erythropoiesis did not occur as a shift from (at the expense of) other cell lineages growing in the culture.
The graph further shows that the stimulatory effect occured only when stem cells were stimulated with IL-3. In the presence of only SCF, the same timing of TGF-ß treatment caused an inhibitory effect.
The apparent discrepancy between the data of Figure 6B (no increased proliferation during TGF-ß treatment) and Figure 6C (increased proliferation after TGF-ß treatment) can be resolved by the following hypothetic mechanism: in many differentiating cell systems where proliferation and maturation are coupled, the cell cycle shortens with every division toward terminal differentiation. An increasing S-phase fraction (i.e., shorter cell cycle) with increasing globin content was demonstrated in erythroid cultures from blood stem cells [26]. Brief TGF-ß treatment advances the maturation levels, and when TGF-ß is removed before its inhibitory effects set in (i.e., before day 4), the advanced maturation leads to a proliferative advantage of TGF-ß-treated cultures over control. A similar situation may apply for autocrine TGF-ß, where the maturation-advancing activity is not compensated for by inhibitory activity that may require much higher concentrations.
In summary, the data presented in this study suggest that IL-3-dependent early stem cell development toward erythropoietic maturation is stimulated by autocrine TGF-ß. The ability of anti-TGF-ß antibodies to decrease the proportions of + cells also suggests that autocrine TGF-ß may contribute to the residual -globin reactivation in serum-free stem cell cultures from adult peripheral blood.
A stimulatory effect of TGF-ß on subsequent erythropoiesis was reported earlier, based on different assay techniques [30, 31]. We used a novel method to study early erythropoiesis, based on the early detection and quantitation of cells containing globin at low levels. Therefore, our data are not easily comparable with the results of other assays showing the pleiotropic effects of TGF-ß on erythropoietic stem/progenitor cells. Further work will reconcile apparent discrepancies between data obtained with the different methods and will establish flow cytometric, two-parameter globin analysis as a valuable and complementary tool for the study of erythropoiesis.
The results presented here may be relevant for the design of approaches to collect and expand stem cells for transplantation. While TGF-ß is thought to promote the maintenance of CD34+ stem cell populations during stem cell expansion for transplantation [12, 32], our data suggest the opposite effect on erythropoiesis. This shows the importance of further research into the effects and implications of autocrine/paracrine and added TGF-ß in the preparation of stem cells for clinical purposes.
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ACKNOWLEDGMENT
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This work was in part carried out at the Tufts New England Medical Center and was supported by the National Institutes of Health contract NIH HD N01 3204.
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Received August 31, 2003;
accepted for publication October 30, 2003.
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Abstract
Introduction
