IL-3-Dependent Early Erythropoiesis Is Stimulated by Autocrine Transforming Grow - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:38 |只看该作者 |倒序浏览 |打印

Genetic Technologies Pty Ltd., Melbourne, Australia
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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@genetype.com.au

ABSTRACT

The proliferation and differentiation of cultured peripheral blood hematopoietic stem cells are strongly affected by transforming growth factor beta (TGF-?) in multiple and seemingly disparate ways, depending on assay conditions . The predominant effects of autocrine, paracrine, and added TGF-? on hematopoietic stem cells are the suppression of proliferation and the maintenance of quiescence . The inhibitory effect of autocrine TGF-? on stem cell proliferation can be overcome by neutralizing TGF-? or its type II receptor . Once erythropoiesis has begun, the suppressive effect of TGF-? on proliferation is accompanied by an acceleration of hemoglobin production and maturation to the point of enucleation .

However, we showed recently that, in cultures from adult peripheral blood stem cells, a pulse of TGF-? 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 . A stimulation of subsequent erythropoiesis by brief TGF-? treatment appears to conflict with the well-established suppression of proliferation by added or autocrine TGF-?. However, most studies showing the antiproliferative effects of TGF-? employed primary or secondary colony assays and did not monitor the proliferation of early erythroblasts." Y: R/ p* _# O
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Therefore, we investigated the potential role of autocrine or added TGF-? 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 , because our preliminary tests showed them to be more responsive than the remaining CD133-CD34  population to neutralizing anti-TGF-? 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 , we monitored the effects of neutralizing anti-TGF-? 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-? 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-?, they indicate a stimulatory activity of autocrine TGF-? during IL-3-dependent early erythroid development." `, }8 `; P0 E) R

MATERIALS AND METHODS. q* D, F% q: K+ h1 L

IL3-Dependent Suppression of Erythroblast Proliferation by Anti-TGF-?4 f; C( M- `1 p$ K# N: U
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To study the potential role of autocrine TGF-? on erythroid stem cell development, we cultured CD133  peripheral blood stem cells in the presence of neutralizing antibodies to TGF-?. 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-? (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 (?-globin versus -globin) are shown in Figure 1A. The profiles show cells with -globin (region 1 indicated in the first profile,  ), ?-globin only (region 2, -? ), 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-? (Fig. 1B).
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Figure 1. IL-3-dependent suppression by anti-TGF-?. CD133  stem cells from adult peripheral blood were grown in methylcellulose medium with EPO, SCF, and IL-3 (as indicated). Anti-TGF-? (pan-specific, 10 μg/ml) was present from day 0. After 8 days, whole cultures were harvested into a single cell suspension and the correlated cellular contents of -globin and ?-globin were monitored by flow cytometry. A) Examples of flow cytometric profiles for the three different cytokine conditions, with and without anti-TGF-?, all from the same stem cell sample. The region settings for the quantitation of (1) and -?  (2) cells are shown in the upper left profile. B) Numerical data: globin profiles were analyzed to quantitate the absolute numbers of (triangles) and -?  (circles) cells. Cell numbers in anti-TGF-? cultures were normalized to the corresponding cell numbers in parallel control cultures without anti-TGF-?, set to 100%. The results from different experiments with different blood donors are shown individually, and the values for and -?  cells from individual experiments are connected to show their correlation.+ o  m3 P/ D# X/ v  Q

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 . 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).( T, V, _/ T  K! |5 d2 D& Q

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-?, divided by erythroblast numbers of untreated controls (set to 100%). Gamma-positive and -?  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 -?  cells. The data show that, in the presence of IL-3, erythroblast numbers were substantially suppressed by anti-TGF-?. The suppressive effect was consistently stronger for cells than for -?  cells, which implies that the proportion of cells in the culture was reduced by anti-TGF-?. In contrast, there was no suppressive effect of anti-TGF-? in the absence of IL-3 (SCF EPO only), and occasionally, but not consistently, a stimulatory effect was observed.+ o& ^7 g5 t9 E% J6 _' L: t
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Comparison of TGF-?-Neutralizing Agents. ]: G' d5 S  z) y6 r2 k5 c6 F' S, s

We also tested the effect of neutralizing monoclonal antibodies to the TGF-? receptor II (TGF-? RII), TGF-?1, TGF-?2, as well as TGF-?1-neutralizing LAP (Fig. 2A). Anti-TGF-?2 had no inhibitory effect, which may help to rule out a nonspecific toxicity of the antibody preparations (anti-TGF-?2 serving as an irrelevant isotype control). The other agents suppressed erythroblast numbers approximately as effectively as the pan-specific anti-TGF-?. The combination of pan-specific anti-TGF-? and anti-TGF-?RII was only slightly more potent than each agent alone. Therefore, further experiments were carried out with pan-specific anti-TGF-? alone.
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Figure 2. Comparison of TGF-?-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-?-neutralizing agents, specifically pan-antiTGF-?, anti-TGF-? RII, anti-TGF-?1, anti-TGF-?2, and LAP (TGF-?1), 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-? (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-? on Final Erythroid Colony Numbers1 r, E/ s' S1 D1 n. A6 q
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The effect of anti-TGF-? 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-? 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.! n& R# s& [" }
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Dose-Response Curves for Anti-TGF-?

The dose-response effect of anti-TGF-? 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 -?  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 -?  cells, as well as the sum of all cells, including those not containing any globin. The curves of and -?  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.

Figure 3. Anti-TGF-? antibody dose response. Stem cells were cultured in different concentration of anti-TGF-? antibodies, and absolute cell numbers per culture were determined on day 8, normalized with values of untreated cultures set to 100%. Triangles: cells; circles: -?  cells; open diamonds: all nucleated cells including those with no detectable globin content; open squares: number of erythroid colonies.$ k* |  k. f9 j+ f% Y( N4 P
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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-? 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-?, 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-? to act back on its source.
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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-? requirement. This needs to be explored further.+ @2 I: C- G$ R. l

Delayed Exposure to Anti-TGF-?5 y- M% i# v! i
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To explore the kinetics of the inhibitory effect, we added anti-TGF-? 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-? had no inhibitory effect, as measured on day 8. The separate plot of and -?  cell numbers confirms the stronger suppression of cells. The data suggest that the activity of autocrine TGF-? occurs only within the first 4 days of culture. The inset in Figure 4A further confirms the absence of inhibition by anti-TGF-? at a later culture phase, and demonstrates that treatment with anti-TGF-? at a later culture interval (day 7-day 11) had a slight and variable stimulatory effect on erythropoiesis. Anti-TGF-? may become stimulatory at this stage of culture because of a continued production of autocrine TGF-? at a phase of erythroid development where the well-known inhibitory effect of TGF-? has set in  (also see Fig. 6). The proportions of cells were not substantially affected by anti-TGF-? treatment beginning after more than 4 days. The lack of inhibitory effect of anti-TGF-? at later culture stages may also confirm that the antibody preparations did not act via nonspecific toxic activity.
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Figure 4. Kinetics of antibody and cytokine action. A) Anti-TGF-? (10 μg/ml) was added to stem cell cultures at the indicated times. The numbers of erythroblasts were determined after 8 days. Data are averages from two independent experiments and are normalized with control values (no anti-TGF-?) set to 100%. Inset in A: Stem cells were cultured for 7 days then reseeded in the presence of pan-specific anti-TGF-? (10 μg/ml) anti-TGF-? RII (10 μg/ml). The numbers of erythroblasts were determined 4 days later (day 11 of total culture time). Data from three independent experiments are shown, with individual experiments identified by connecting the values of and -?  values from each experiment. Averages are indicated by horizontal bars. B) Epo (squares): stem cells were cultured in the presence of IL-3 and SCF but in the absence of Epo, and Epo was added at selected times. The numbers of erythroblasts (sum of and -?  cells per culture) were determined on day 8 and normalized with control values (Epo from day 0) set to 100%. IL-3 (diamonds): stem cells were cultured with Epo, SCF, and IL-3, and at selected times, IL-3 was withdrawn by washing and reseeding in only Epo and SCF. The numbers of erythroblasts (sum of and -?  cells per culture) were determined on day 8 and normalized with control values (no IL-3 withdrawal) set to 100%.) g$ a6 \1 `  [6 M+ j/ q

Figure 6. Effect of added TGF-?. Stem cells were cultured in the presence of added TGF-?1 (10 ng/ml) for selected times. A) Examples of globin profiles after 4 and 6 days (top and bottom row, as indicated) of continuous TGF-? exposure (right column) and control without TGF-? (left column). B) Kinetics of cell growth: cell numbers per culture as a function of culture time during continuous incubation with TGF-?1. Left: cells, middle: -?  cells, right: all cells (with and without globin). The dramatic increase in and -?  cells on day 3–4, as well as the lack of effect on the sum of all cells on day 3–4, were reproduced more than five times. C) Effect of 1-day TGF-? treatment on subsequent cell growth. Stem cells were cultured in medium with/without added TGF-?. After 1 day (24 hours), the cultures were washed and reseeded without TGF-?. The graph comprises a comparison between cultures with and without IL-3. The numbers of and -?  as well as all cells (with and without globin content) are normalized with controls (without TGF-? treatment) set to 100%. The data are averages from three independent experiments with different blood donors.

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-? treatment increase the proportions of subsequently developing cells . Finally, sensitive flow cytometric globin measurement shows that the first significant cohort of globin-containing cells appears between day 4 and 5  (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-? on Different Stem Cell Subpopulations' C& m, s: i: c/ W3 K4 ^) m1 }" T( I
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CD133  stem cells with erythroid potential represent mostly an immature subpopulation of CD34  stem cells with erythroid potential . Figure 5 compares the differential effects of IL-3 and anti-TGF-? 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).

Figure 5. Differential effects on CD133  and CD133-CD34  stem cells. CD133  and CD133-CD34  cells were derived from peripheral blood using sequential isolation with specific magnetic beads. The effects of IL-3 and anti-TGF-? on the two stem cell types were compared. A) Effect of IL-3: cultures in medium with IL-3 (full squares) and without (open squares). Numbers of hemoglobinized erythroblasts (sum of cells with and -?  globin) per culture were determined as a function of culture time. The kinetic data are from one representative experiment. The third graph shows the statistics from multiple experiments from different blood donors. Day-10 erythroblast numbers with IL-3 are shown as a percent of erythroblast numbers in controls without IL-3. n = 10 for CD133  cultures (black column) and n = 5 for CD133-CD34  cultures (white column). B) Effect of anti-TGF?: Stem cells (black columns: CD133 ; white columns: CD133-CD34 ) were grown in the presence or absence of anti-TGF-?, and the numbers of and -?  erythroblasts (as indicated) were determined. Cell numbers are normalized with controls (no antibody) set to 100%. Data are averages from two independent experiments from two different blood donors, with range of values indicated.% ~& C; b" N& S% U4 D
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A comparison between the effects of TGF-?-neutralizing antibodies on CD133  and CD133-CD34  cells is shown in Fig. 5B. The suppression by anti-TGF-? of early erythropoiesis was stronger for CD133  cells than for the more mature CD133-CD34  cells. The differential effect on and -?  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-? on very early erythropoiesis.

Effect of Added TGF-?1

To test the interpretation that the negative effect of TGF-?-neutralizing antibodies reflects a stimulatory activity of autocrine TGF-?, we explored the effects of TGF-?1 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-?1 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-?. On day 4, there are very few globin-containing cells in the controls but a significant number are seen in cultures with TGF-?. In day-6 controls, the bulk of cells has begun to express globin and contains low levels of mostly -?  globin. In contrast, the TGF-?-treated cultures show not only a larger proportion of cells, but also much higher levels of globin, confirming previous reports that TGF-? accelerates maturation , perhaps by downmodulating the c-kit receptor and thus interfering with the maturation-retarding effect of SCF .0 C* I; L& {3 I9 \

Figure 6B shows, in a representative experiment, the kinetics of cell proliferation during the first few days of continuous TGF-? treatment, separately displayed for and -?  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-?. Later, after about day 4–5, the controls catch up and proliferate unhindered, while the growth curves in cells treated with TGF-? begin to show inhibition. However, the growth curves of all cells, including those without globin, do not show any initial stimulatory effect of TGF-? but only the later inhibition. The lack of overall proliferative stimulation during continuous TGF-? was confirmed by the observation that cell counts per colony in the budding day 3 and day 4 colonies were not affected by TGF-?1 (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.
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Figure 6C explores the effect of TGF-? treatment for the first 24 hours of stem cell culture followed by a further 7 days of culture without TGF-?. The brief TGF-? pulse caused a substantial increase in subsequently developing and -?  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.
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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-? treatment caused an inhibitory effect.9 D5 V3 d) F! z  r

The apparent discrepancy between the data of Figure 6B (no increased proliferation during TGF-? treatment) and Figure 6C (increased proliferation after TGF-? 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 . Brief TGF-? treatment advances the maturation levels, and when TGF-? is removed before its inhibitory effects set in (i.e., before day 4), the advanced maturation leads to a proliferative advantage of TGF-?-treated cultures over control. A similar situation may apply for autocrine TGF-?, where the maturation-advancing activity is not compensated for by inhibitory activity that may require much higher concentrations.
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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-?. The ability of anti-TGF-? antibodies to decrease the proportions of cells also suggests that autocrine TGF-? may contribute to the residual -globin reactivation in serum-free stem cell cultures from adult peripheral blood.) c  f  s  y4 o0 ]
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A stimulatory effect of TGF-? on subsequent erythropoiesis was reported earlier, based on different assay techniques . 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-? 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.7 X% Y6 v) O0 m3 H
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The results presented here may be relevant for the design of approaches to collect and expand stem cells for transplantation. While TGF-? is thought to promote the maintenance of CD34  stem cell populations during stem cell expansion for transplantation , 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-? in the preparation of stem cells for clinical purposes.

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