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Synergistic and Inhibitory Interactions in the In Vitro Control of Murine Megakaryocyte Colony Formation

Division of Cancer and Hematology, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia

Key Words. Megakaryocyte colonies • Synergy • Interleukin-3 • Erythropoietin • Thrombopoietin

Donald Metcalf, M.D., Division of Cancer and Hematology, The Walter and Eliza Hall Institute of Medical Research, Post Office Royal Melbourne Hospital, Victoria 3050 Australia. Telephone: 61-3-9345-2555; Fax: 61-3-9347-0852; e-mail: metcalf{at}wehi.edu.au

	ABSTRACT

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The formation of megakaryocyte colonies in agar cultures of murine bone marrow or spleen cells can be stimulated by the addition of interleukin-3 (IL-3), erythropoietin (EPO), thrombopoietin (TPO), or IL-6. However, greater numbers of colonies developed if combinations of two or more of these stimuli were used, particularly combinations including stem cell factor, with maximal numbers of colonies developing with the combination of stem cell factor plus IL-3 plus EPO. The data indicate that most committed progenitor cells in the megakaryocyte lineage were unusual in that they required stimulation by two or more hematopoietic growth factors. In tests using a range of growth factors, G-CSF was exceptional in that it consistently specifically inhibited megakaryocyte colony formation stimulated by EPO, TPO, or IL-6 but not that stimulated by IL-3. The mechanisms involved in this inhibitory action of G-CSF are unknown, but the inhibitory action could be of relevance for the dose-dependent lowering of platelet levels observed in some subjects injected with G-CSF.

	INTRODUCTION

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In subpopulations of murine marrow cells that have been highly enriched for stem cells, as assessed by repopulating capacity or the ability to form day-14 spleen colonies, a high proportion of the cells can be clonogenic, but the cells characteristically require simultaneous stimulation by multiple growth factors to exhibit proliferation [1–3]. In contrast, single growth factors, such as the colony stimulating factors, are able to stimulate committed progenitor cells to form colonies of single- or double-lineage cells [4, 5]. This has led to the provisional interpretation that very immature hematopoietic cells ("stem cells") require multiple growth stimuli to proliferate, but that, as commitment and maturation occur, the resulting progenitor cells become responsive to single growth factors.

However, progenitor cells and their maturing progeny continue to coexpress receptors for more than one growth factor, and this can allow the occurrence of enhanced or synergistic responses in progenitor-cell-derived colonies when certain combinations of growth factors are used [6, 7]. Such interactions are not always enhancing and, for example, with the interaction of GM-CSF and macrophage-CSF (M-CSF) on murine bone marrow cells, inhibition of some macrophage clonogenic cells was also observed [6].

It has also become evident that certain factors, such as stem cell factor (SCF), Flk-2-ligand (FL), and interleukin-3 (IL-3), appear to be particularly important for the proliferation of the least mature clonogenic cells [1–3, 8–9], while other factors seem most active on more mature precursors [10]. This has given rise to the additional notion of the possible sequential action of early-acting then late-acting growth factors—a commonly repeated and satisfying scheme that, unfortunately, has little convincing supporting data.

The present studies on the control of mouse megakaryocyte colony formation in vitro were initiated with the practical objective of developing a growth stimulus combination that would allow maximal numbers of clonogenic megakaryocyte precursors to be detected, particularly when analyzing mice with genetic manipulations affecting megakaryocyte or platelet formation.

While it is clear that, in vivo, thrombopoietin (TPO) is the most active agent in regulating megakaryocyte and platelet formation [11–13], this is not what is observed in clonal cultures in vitro, where other regulators have much stronger proliferative actions than TPO. The reasons for this discrepancy remain obscure, but the phenomenon is similar to that encountered earlier with weak in vitro responses to G-CSF contrasting with strong in vivo responses [10]. While these differences may make the significance of some in vitro data difficult to interpret, the in vitro data remain of importance for developing methods to detect maximal numbers of clonogenic cells in genetically manipulated mice. The data from the present study document an improved method for detecting maximal numbers of megakaryocyte progenitors but cast doubt on the general applicability of the distinction between the growth factor requirements of stem and progenitor cells. Most megakaryocyte progenitor cells were, in fact, found to require proliferative stimulation by two or more growth factors.

	MATERIALS AND METHODS

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Mice
The mice used in most experiments were 6- to 8-week old, male or female C57BL mice that were bred and maintained in protected animal quarters and were regularly monitored for infectious microorganisms. Additional experiments were performed using cells from CBA or BALB/c mice.

Cultures
All cultures were prepared in 1-ml volumes in 35-ml plastic petri dishes using Dulbecco’s modified Eagle’s medium with a final concentration of 20% preselected newborn calf serum (HyClone Lab, Inc.; Logan, UT; http://www.hyclone.com) and 0.3% agar. In most cultures, dispersed marrow cells were cultured at a concentration of 25,000 or 50,000 cells per ml. Stimuli were added in 0.1-ml volumes to the empty petri dishes before addition of the marrow cell suspension in agar medium. After mixing with the stimulus and allowing gel formation to occur, cultures were incubated at 37°C in a fully humidified atmosphere of 10% CO₂ in air. After 7 days of incubation, colonies were provisionally counted using an Olympus dissection microscope (Olympus; Tokyo, Japan; http://www.olympus.com), then 1 ml of 2.5% glutaraldehyde was added gently to each culture. After 4 hours of fixation, intact cultures were floated onto glass slides, allowed to dry, then stained in sequence for acetylcholinesterase (megakaryocytes), Luxol Fast Blue (eosinophils), and hematoxylin (granulocytes, macrophages, and erythroid cells). The stained cultures were then mounted with cover slips, and the number and composition of all colonies in the culture were determined at 50x to 200x magnifications. Included in this analysis were counts on the number and type of individual cells in each megakaryocyte colony. Three types of megakaryocyte-containing colonies were encountered where not all colony cells were acetylcholinesterase positive. These were A) blast colonies containing megakaryocytes; B) large colonies where there was a mixture of megakaryocytes of different sizes (maturation stages), ranging down to small acetylcholinesterase-negative cells, and C) colonies containing both megakaryocytes and erythroid cells. Acetyl-cholinesterase-negative cells were not included in cell counts for each of these three types of mixed colonies.

The analysis of each culture, therefore, generated two basic sets of megakaryocyte data from the stained cultures—total megakaryocyte colony numbers and total colony megakaryocyte cell counts per culture—in addition to information on the numbers and composition of other colonies in the cultures.

Stimuli
The stimuli used in the cultures were all purified recombinant molecules—murine IL-3 and IL-7 were purchased from PeproTech (Rocky Hill Road, NJ; http://www.peprotech.com); murine IL-1ß was from Genzyme (Cambridge, MA), and human IL-2 was from Cetus (Emeryville, CA). Murine IL-6 was a kind gift of Dr. Richard Simpson (The Ludwig Institute for Cancer Research and The Walter and Eliza Hall Institute; Melbourne, Australia), and human G-CSF and erythropoietin (EPO) were obtained from Amgen (Thousand Oaks, CA; http://www.amgen.com). Recombinant murine GM-CSF, IL-5, SCF, FL, and TPO were prepared and purified in our laboratory. Recombinant murine leukemia inhibitory factor (LIF) was supplied by the Amrad Corporation (Melbourne, Australia). The concentration of each stimulus used in culture was shown by titration to be supramaximal for colony formation, and final concentrations for the relevant factors were: GM-CSF, G-CSF, IL-3, M-CSF, and LIF 10 ng/ml; SCF and IL-6 100 ng/ml; EPO 2 IU/ml; FL 500 ng/ml, and TPO 50 ng/ml.

Fluorescence-Activated Cell Sorting (FACS) Fractionation
C57BL marrow cell suspensions were sorted for lin^-kit⁺Sca-1^- cells using a MoFlo Cell Sorter (Dayko Cytomation; Fort Collins, CO) and labeled antibodies, as described previously [14].

	RESULTS

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In cultures of 25,000 or 50,000 C57BL marrow cells, the following individual growth factors were tested: IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, LIF, FL, SCF, GM-CSF, G-CSF, M-CSF, EPO, and TPO and, of these, only three—IL-3, EPO, and TPO—had the capacity, when acting alone at the concentrations used, to stimulate megakaryocyte colony formation. The resulting megakaryocyte colonies differed characteristically. TPO-stimulated colonies contained few cells, but these were large and polyploid. EPO-stimulated colonies also often contained erythroid cells, and the megakaryocytic cells ranged in size from large mature cells to small immature cells. Thus, these colonies were characteristically composed of cells of variable sizes not all of which were acetylcholinesterase positive. Colonies stimulated by IL-3 resembled those stimulated by EPO but also included sizable colonies of large mature megakaryocytes.

As shown by typical data in Figure 1, as assessed by megakaryocyte colony numbers, IL-3 was usually slightly more potent than EPO, and both stimulated the formation of more colonies than TPO. When total colony megakaryocyte numbers were considered, IL-3 was the most active, and TPO was the weakest of the three stimuli (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Figure 1. Megakaryocyte colony formation is enhanced by using multiple stimuli. Mean numbers of megakaryocyte colonies developing in duplicate cultures of 50,000 C57BL marrow cells stimulated by IL-3, EPO, TPO, SCF, or combinations of these factors.

View larger version (12K): [in this window] [in a new window]   Figure 2. Total megakaryocyte colony cell numbers are enhanced by using multiple stimuli. Mean total numbers of colony megakaryocytes developing in duplicate cultures of 25,000 C57BL marrow cells stimulated by various single factors or combinations of factors.

The low frequency of megakaryocyte colonies and the difficulty in identifying them before the colony cells became polyploid made it impractical to attempt colony transfer experiments to determine whether cells responding, for example, to IL-3 or EPO also responded to SCF plus TPO. The experiments were not able, therefore, to exclude the possible existence of multiple discrete subsets of progenitor cells, but it is more likely that there was overlap in many of the populations responding to the various factors or factor combinations.

Not shown in Figure 1 is the fact that the combination of TPO with IL-3 and EPO resulted in colony numbers not much lower than those stimulated by SCF plus IL-3 plus EPO, although TPO tended to dominate the type of colonies developing by inducing maturation and size increases in the colony megakaryocytes. Colonies stimulated only by the two-factor combination of TPO plus IL-3 or EPO tended to be small and to be composed of large mature cells.

Parallel cultures of CBA or BALB/c marrow cells showed identical response patterns of megakaryocyte colony formation to that observed with C57BL marrow cells.

A factor of potential interest, but found to have no synergistic action on megakaryocyte colony formation stimulated by IL-3, TPO or EPO, was FL.

Size Analysis of Megakaryocyte Colonies
As shown in a typical example in Figure 3, the three single-acting stimuli of megakaryocyte colony formation led to the formation of colonies with distinct differences in cell numbers—uniformly low for TPO-stimulated colonies and variable in number for both EPO- and IL-3-stimulated colonies. It is noteworthy that the combined stimulus not only resulted in greater numbers of colonies that contained very large cell numbers (some above 400 cells) but also resulted in greater numbers of small colonies that were, presumably, the progeny of relatively mature progenitor cells. Thus, the action of the combined stimuli was not confined to immature progenitor cells.

View larger version (18K): [in this window] [in a new window]   Figure 3. Use of multiple stimuli results in greater numbers both of large and small colonies. Analysis of the numbers of acetylcholinesterase-positive cells present in individual colonies in entire cultures of 50,000 C57BL bone marrow cells stimulated by TPO, EPO, IL-3, or SCF plus IL-3 plus EPO. Numbers in circles are the total numbers of megakaryocyte colonies in the cultures. Solid bars indicate colonies that also contained additional acetylcholinesterase-negative cells.

View larger version (16K): [in this window] [in a new window]   Figure 4. Multiple stimuli result in greater numbers of large and small colonies developing from enriched populations. Analysis of the numbers of acetylcholinesterase-positive cells present in individual colonies in entire cultures of 1,000 lin-kit+Sca-1- cells stimulated by TPO, EPO, or IL-3, or SCF plus IL-3 plus EPO. Numbers in circles are the total numbers of megakaryocyte colonies in the cultures. Solid bars indicate colonies that also contained additional acetylcholinesterase-negative cells.

Inhibitory Action of G-CSF
In an effort to detect further examples of synergistic action, other hematopoietic factors were included in cultures stimulated by IL-3, EPO, or TPO. Of particular interest were cultures to which GM-CSF had been added, because GM-CSF, at much higher concentrations than used in the present experiments, is able, when acting alone, to stimulate megakaryocyte colony formation [4]. Figures 5 and 6 show results that are typical of those obtained in five replicate experiments. Addition of GM-CSF or M-CSF had no effect on megakaryocyte colony formation stimulated by IL-3, EPO, or TPO. In contrast, the combination of G-CSF with either EPO or TPO resulted in lower megakaryocyte colony numbers and cellular content, often with those colony cells that did develop being small and unhealthy. However, as shown in Figure 7, G-CSF had no suppressive action on megakaryocyte colonies stimulated to develop by IL-3. Titration of G-CSF showed that its inhibitory action, particularly in terms of total colony cell numbers, was detectable with concentration as low as 100 pg/ml (Fig. 8). This is the same concentrations range over which the proliferative effects of G-CSF on granulocyte colony formation are observable [15], and granulocyte colony formation was not inhibited in the present cultures by the combination of G-CSF with either EPO or TPO.

View larger version (15K): [in this window] [in a new window]   Figure 5. Inhibition by G-CSF of megakaryocyte colonies stimulated by TPO. Total megakaryocyte colonies and colony megakaryocytes in cultures of 50,000 C57BL marrow cells stimulated by TPO alone or in combination with other factors. Note the inhibition caused by addition of G-CSF.

View larger version (17K): [in this window] [in a new window]   Figure 6. Inhibition by G-CSF of megakaryocyte colonies stimulated by EPO. Total megakaryocyte colonies and colony megakaryocytes in cultures of 50,000 C57BL marrow cells stimulated by EPO alone or in combination with other factors. Note the inhibition caused by addition of G-CSF.

View larger version (9K): [in this window] [in a new window]   Figure 7. No suppression by G-CSF of megakaryocyte colonies stimulated by IL-3. In cultures of 50,000 C57BL bone marrow cells, G-CSF inhibited megakaryocyte colony formation stimulated by EPO or TPO but not that stimulated by IL-3. Mean colony numbers from duplicate cultures.

View larger version (13K): [in this window] [in a new window]   Figure 8. Titration of inhibition of megakaryocyte colony formation by G-CSF. Titration of the ability of G-CSF to inhibit megakaryocyte colony formation in cultures of 50,000 C57BL marrow cells stimulated by EPO.

View larger version (21K): [in this window] [in a new window]   Figure 9. Inhibition by G-CSF of megakaryocyte colony formation stimulated by IL-6. Inhibition by G-CSF of megakaryocyte colony formation stimulated by 100 ng/ml IL-6 in cultures of 100,000 C57BL bone marrow cells.

	DISCUSSION

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It was not practicable to use transfer studies to establish whether cells responding to individual single stimuli were distinct subsets of megakaryocyte progenitor cells or belonged to a common set. Most of the large colonies required two or more stimuli to develop, suggesting that the initiating cells for these large colonies were less mature and had more proliferative potential—properties placing them closer to the stem cell category. However, the combined stimuli also led to the formation of greater numbers of mature colonies containing only a few cells. This strongly suggests that, at all stages in megakaryocyte progenitor maturation, most cells require double or multiple stimuli for proliferation. Prior studies using micromanipulated single cells [19] indicated that the synergistic proliferative actions resulted from direct actions on the responding cells.

The general findings cast doubt on the validity of the notion that stem cells and progenitor cells differ because of a differing responsiveness to single versus multiple regulatory factors [21] and, at least in the megakaryocytic lineage, this parameter no longer serves to distinguish stem cells from progenitor cells.

The findings also raise the further question of whether there might be functional differences between megakaryocytes generated using a single versus multiple stimuli. There was a striking effect on colony megakaryocyte size when TPO was included in the mixture. Together with earlier data [18] on the effects of TPO on megakaryocyte ploidy, the data suggest strongly that TPO can influence endomitosis, and possibly, the maturation of cells that are responding, in part at least, to other proliferative stimuli. Extension of this notion raises the possibility that each active factor in a combined stimulus might be able to induce particular characteristics in the megakaryocytes being generated, and these cells may then differ in some manner from those generated by the use of single stimuli.

Spleen and bone marrow populations were found to differ somewhat in their content of megakaryocyte progenitor cells dependent on multiple stimuli. Whereas, in the marrow, multiple stimuli only resulted in a fourfold greater number of megakaryocyte colonies versus single factor-responsive colonies, for the spleen, combined stimuli resulted in tenfold more detectable megakaryocyte colony-forming cells. The data provide further evidence that spleen hematopoietic populations do differ from those in marrow and are not merely an additional increment of marrow-type cells.

The inhibitory action of G-CSF on megakaryocyte colony formation stimulated by EPO, TPO, or IL-6 was unexpected although an inhibitory interaction has been reported with the combination of IL-6 and G-CSF on granulocyte-macrophage colony formation [22]. G-CSF appears unlikely to have any toxic effect on colony megakaryocytes, since no inhibition was observed if IL-3 was used to generate colony formation. The clinical use of G-CSF can result in a reversible dose-related reduction in platelet numbers in normal subjects [23], but the mechanism involved has not been determined. Such subjects do develop minor enlargement of the spleen, and hypersplenism may be a possible cause of reduced platelet numbers, but it is equally possible that what is being observed in vivo is similar to the inhibitory action noted in cultures of megakaryocyte colonies. The mechanism responsible for the in vitro suppression could be examined further by analyzing signaling pathway changes in continuous cell lines engineered to express sets of receptors both for G-CSF, and EPO or TPO.

SCF, acting alone, has only an inconsistent and very weak megakaryocyte colony-stimulating activity. It is curious, therefore, that its strong synergistic actions noted here and in earlier studies [18–20] contrast strongly with the failure of another early-active factor, FL, to exhibit synergistic effects. The data support an earlier conclusion that within the stem cell/early progenitor cell compartment there are two major subsets of cells, with some cells being responsive to FL and others to SCF [24]. As previously shown, these subsets of preprogenitor cells are distinct, and the present data indicate that the progeny of preprogenitor cells able to form megakaryocyte colonies fall into the SCF-responsive group.

	ACKNOWLEDGMENT

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The authors are indebted to Craig Hyland for his assistance in the FACS experiments.

This work was supported by the Carden Fellowship Fund of the Cancer Council, Victoria, Australia, the National Health and Medical Research Council, Canberra, Australia, the Cooperative Research Centre for Cellular Growth Factors and the National Institutes of Health, Bethesda, Grant No. CA22556.

	REFERENCES

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This Article Abstract Full Text (PDF) An erratum has been published Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Metcalf, D. Articles by Mifsud, S. Search for Related Content PubMed PubMed Citation Articles by Metcalf, D. Articles by Mifsud, S.