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Mpl Ligand (MGDF) Alone and in Combination with Stem Cell Factor (SCF) Promotes Proliferation and Survival of Human Megakaryocyte, Erythroid and Granulocyte/Macrophage Progenitors

^a The Walter and Eliza Hall Institute of Medical Research;
^b Department of Medical Oncology and Diagnostic Haematology, Royal Melbourne Hospital, Victoria, Australia

Key Words. MGDF • Thrombopoietin • c-mpl • SCF • Progenitor • Survival • Proliferation • Meg-CFC

Dr. C.G. Begley, The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, 3050 Victoria, Australia.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined cytokine-stimulated proliferation and survival of human megakaryocyte progenitor cells. We used a reliable, immunoenzymatic method of labeling CD41a-, CD42b-stained megakaryocytes in intact agar cultures to specifically identify and enumerate all megakaryocyte-containing colonies. We examined a previously defined population of cells enriched for megakaryocyte progenitors that coexpress CD34 and the megakaryocyte/platelet marker CD61. These CD34⁺61⁺ cells displayed clonogenicity of approximately 30% and contained myeloid, erythroid and megakaryocyte progenitor cells. With single CD34⁺61⁺ cells, megakaryocyte growth and development factor ([MGDF], also known as mpl-ligand or thrombopoietin) stimulated 9% of cells to complete their first cell division by day 2 (versus 21% with stem cell factor [SCF], or 13% with interleukin 3 alone). MGDF showed an additive effect with SCF and interleukin 3 to increase this number at least twofold. In purified CD34⁺61⁺ cells, MGDF stimulated the survival of megakaryocyte-colony forming cells (CFC) when addition of other proliferative factors was delayed for 5, 10 and 15 days (all p < 0.0001 versus saline control). MGDF also promoted survival of BFU-E and granulocyte-macrophage-CFC for at least 10 days (p 0.0013 and p 0.0362, respectively). SCF alone prolonged survival of CD34⁺61⁺ progenitor cells, however, MGDF + SCF was significantly more active. Whereas the action of MGDF on megakaryocyte-CFC was evident both in stimulating proliferation and survival, its ability to promote survival was between two- and fivefold greater than its action to stimulate proliferation. Thus MGDF alone, and in combination with SCF, was active in promoting the survival and proliferation of human progenitor cells of multiple hemopoietic lineages.

	Introduction

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An increasing number of growth factors have been shown to be responsible for the survival, proliferation and enhanced function of many cell types within the hemopoietic system. The action of growth factors to stimulate cell survival applies both to cells within the progenitor compartment and mature end-cells [1, 2]. Moreover, this action of hemopoietic growth factors is evident at concentrations 10- to 100-fold lower than concentrations required to stimulate proliferation [2, 3]. It has even been proposed that the ability to stimulate survival and delay apoptosis may influence differentiation commitment [4].

The receptor c-mpl is a member of the hemopoietin-domain family of cytokine receptors [5]. It is expressed throughout the platelet lineage in humans: from early hemopoietic cells [6-8] through to mature platelets [9-12]. Recently the cognate ligand for c-mpl, also known as thrombopoietin [13-17], has been identified as the most important physiological regulator of megakaryopoiesis and thrombopoiesis [18]. Thrombopoietin has been shown to: A) promote megakaryocyte and erythroid progenitor cell proliferation [19, 20]; B) increase megakaryocyte ploidy, maturation and surface marker expression [21, 22], and C) prime platelets for aggregation and potentiate their attachment to collagen [23, 24].

In contrast to the well-established, proliferative actions of Mpl ligand/megakaryocyte growth and development factor (MGDF), little is currently known regarding the role of this cytokine in promoting cell survival. The aim of this study was to examine the ability of pegylated recombinant human (PEG-rHu)MGDF, a recombinant truncated Mpl ligand conjugated with polyethylene glycol, to directly stimulate cell division and prolong the survival of human bone marrow (BM) progenitor cells of different lineages. In addition, we studied the ability of PEG-rHuMGDF to stimulate directly the first cell division of single progenitor cells. Finally, we sought to establish whether MGDF would cooperate with stem cell factor (SCF) to enhance cell survival.

	Materials and Methods

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Cell Culture
To quantitate the frequency of progenitor cells in human BM, samples from 15 subjects with a variety of solid tumors were examined. Subjects provided written, informed consent for aspiration of BM according to guidelines set by the Institutional Ethics Committee. For the remainder of this study, sterile human BM cells were obtained from the posterior iliac crest of normal donors after informed consent was obtained. BM cells were diluted with an equal volume of Iscove's modified Dulbecco's medium ([IMDM]; GIBCO BRL; Paisley, UK) containing fetal bovine serum ([FBS]; 10% v/v; Multiser, Trace Biosciences; Victoria, Australia), and the mononuclear (MNC) cell fraction was isolated over a Ficoll-Paque density gradient (d = 1.077 g/ml; Pharmacia Biotech; Uppsala, Sweden).

For megakaryocyte progenitor assays, we established an Alkaline Phosphatase Anti-Alkaline Phosphatase method of staining whole agar cultures in situ. Cultures were established using 0.3% agar, IMDM, 25% pretested FBS and 10⁴ or 10⁵ MNC cells per milliliter. In cultures using purified CD34⁺61⁺ cells (see below) 6,000 and 10,000 cells were examined per milliliter. To achieve maximal stimulation of progenitor cells, cultures were stimulated with purified recombinant growth factors; G-CSF (500 U/ml), GM-CSF (100 ng/ml), SCF (100 ng/ml), interleukin 3 (IL-3) (100 ng/ml), IL-6 (100 ng/ml), erythropoietin ([EPO]; 4 U/ml), PEG-rHuMGDF (200 ng/ml), provided by Amgen (Thousand Oaks, CA). After incubation for 14 days at 37°C in 5% CO₂ in air, cultures were dehydrated in the 35-mm petri dish. This was achieved using Whatman Filter paper No. 1. One piece of filter paper was placed onto each unfixed culture, air bubbles gently removed and a second piece of filter paper placed on top. Cultures were allowed to dry overnight at room temperature. Filter papers were then removed; it was frequently necessary to moisten the filter paper in direct contact with the agar and the culture allowed to dry again overnight. Cultures were then digested with ß-agarose-1 (1 U/ml in phosphate-buffered saline pH 6.5, 37°C, 1 h; Calbiochem; La Jolla, CA).

In some cases, cultures were fixed with 1 ml methanol:acetone (2:1), 10 min at room temperature prior to digestion with ß-agarose. This allowed unstained cultures to be stored for 1 month. Subsequently 1 ml of 2% human AB serum (in Tris 0.05 M buffered saline [TBS], pH 7.6) was added for 20 min at room temperature. Antibody solutions (anti-CD41a:SP2/0-Ag.14; Biodesign; Kennebunk, ME, and anti-CD42b:63.Ag.8; Biodesign) were pretitrated in TBS pH 7.6 and used at a final concentration of 1:100 and 1:250, respectively. A volume of 0.75 ml/culture was added for 40 min at room temperature on an orbital mixer. Cultures were rinsed three times with TBS pH 7.6. The staining with the DAKO Alkaline Phosphatase Anti-Alkaline Phosphatase Kit (Glostrup, Denmark), was performed according to the manufacturer's instructions. Cultures were counter-stained using 1 ml of 1:10 dilution of 2% Methyl Green (BDH-Gurr) in tap water. Cultures were washed in tap water, air-dried and mounted using aqueous mounting solution. Stained megakaryocyte colonies were identified using an Olympus CK2 inverted microscope at 40x and 100x magnifications. Colonies of three or more megakaryocytes, including mixed colonies that contained three or more megakaryocytes, were enumerated.

For erythroid and granulocyte/macrophage progenitor assays, we used our previously published method [25]. The cytokines used for stimulation of day 14 granulocyte/ macrophage-colony forming cell (GM-CFC) cultures were G-CSF, GM-CSF and SCF; and for BFU-E were G-CSF, GM-CSF, SCF, IL-3, IL-6, and EPO (at the concentrations listed above). All BM samples were cultured in triplicate at both 10⁴ and 10⁵ MNC cells per milliliter. In assays using purified cells, 10³ cells were examined per milliliter. Colonies were enumerated from cultures that allowed accurate quantitation as previously described [26].

Analysis of the effect of G-CSF on megakaryocyte progenitor cells was performed in triplicate agar cultures of CD34⁺61⁺ cells (see below) stimulated by PEG-rHuMGDF (200 ng/ml). Cultures were also stimulated with G-CSF added in twofold dilutions from a maximum final concentration of 2000 ng/ml. Megakaryocyte colonies were scored as described above following incubation for 14 days.

Purified Cell Populations
Following centrifugation through a density gradient, cellular aggregates were minimized by passing the cell suspension through a 63 µm nylon mesh and through a 21 gauge needle several times. To reduce cell sorting time, washed MNC were first enriched for CD34⁺ cells using the MiniMACS magnetic cell sorting kit (Miltenyi Biotec; Sunnyvale, CA), according to the manufacturer's instructions. Populations enriched for CD34-expressing cells were used immediately, or after no greater than 14 h storage in IMDM containing 10% FBS at 4°C.

The CD34⁺ enriched cells were then stained with anti-CD34 antibody (HPCA-2; Becton Dickinson; San Jose, CA) conjugated to phycoerythrin, and one of anti-c-mpl (Genzyme; Cambridge, MA), anti-CD61 (anti-IIIa complex; Y2/51; DAKO), or anti-CD41 (anti-IIb complex; SP2/O-Ag.14; Immunotech; Marseille, France) antibodies conjugated to fluorescein isothiocyanate. Background fluorescence was established using an IgG₁ isotype control antibody (Coulter; Hialeah, FL). All antibody staining was performed at 22°C for 15 min. Staining with propidium iodide (1 µg/ml; Calbiochem) was used to exclude nonviable cells. Cell sorting was performed on an unmodified dual laser FACStar^Plus (Becton Dickinson) configured with primary beam tuned at 488 nm and secondary beam tuned at 605 nm. The probability of doublets or absorbed platelets was minimized using the width parameters of the instrument's real-time pulse processing doublet discriminator. The absence of absorbed platelets was confirmed by microscopic examination of purified cell populations. All cell-sorted samples were also reanalyzed to confirm at least 95% purity of cell populations, allowing for fluorescence quenching. In the experiments indicated, single CD34⁺61⁺ cells were deposited into microtiter plates (Lux; Nunc; Naperville, IL) containing 15 µl of IMDM, 105 FBS and cytokine(s) using an automatic cell deposition unit. After visual confirmation of one cell per microwell, cultures were incubated at 37°C in a fully humidified atmosphere of 5% CO₂ in air. Viable cells in each well were quantified based on their refractile appearance using an Olympus inverted microscope (100x-200x magnification). The number of wells in which 1 cell division had occurred was recorded daily by visual inspection.

Delayed Addition Cultures
Delayed addition experiments were performed as described [27], with the following modifications: Agar cultures of CD34⁺61⁺ cells were established at 37°C in a fully humidified atmosphere of 5% CO₂ in air with primary stimuli of PEG-rHuMGDF (200 ng/ml), SCF (100 ng/ml), PEG-rHuMGDF + SCF or saline in triplicate. In all cultures, a selected batch of bovine calf serum (HyClone Laboratories; Logan, UT) was used. A combination of seven cytokines including G-CSF, GM-CSF, SCF, IL-3, IL-6, EPO, PEG-rHuMGDF (hereafter referred to throughout the text as MGDF), all at the concentrations listed above, was added to the cultures on indicated days to stimulate colony formation by surviving clonogenic cells. Separate cultures were established for assay of megakaryocyte, erythroid and granulocyte/macrophage progenitor cells. Erythroid, granulocyte/macrophage and megakaryocyte colony formations were quantified 14 days following the delayed addition. In assays for megakaryocyte progenitor (megakaryocyte [Meg]-CFC) survival, the number of CD34⁺61⁺ cells plated per 1 ml culture dish was either 6,000 or 10,000, and for erythroid (BFU-E) and granulocyte/macrophage (GM-CFC) cultures, 1,000 cells were used.

Statistical analyses of delayed addition experiments were performed by first normalizing mean colony data at day 0 to 100% for each stimulus (although statistical significance was similar without normalization). Post-hoc analysis of variance between paired stimuli was tested for each day using Scheffé's F procedure on the StatView 4.51 data analysis and presentation program. Where appropriate, the less stringent Bonferroni/Dunn and Fisher's protected least significant difference procedures were performed. All tests of statistical significance were two-way with significance defined as p < 0.05.

	Results

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Clonogenicity of Megakaryocyte Progenitors in Human BM
In order to consistently identify megakaryocytes in semi-solid medium, we established and validated an immunoenzymatic method of labeling cells in situ. An example of a stained megakaryocyte colony grown in agar is shown in Figure 1. We found that specific staining of megakaryocytes was best achieved by dual labeling with anti-CD41 and anti-CD42. The system exhibited excellent reproducibility and, due to low background staining, readily enabled identification of megakaryocyte-containing colonies.

View larger version (142K): [in this window] [in a new window]   Figure 1. Megakaryocyte colony formed in agar. A discrete megakaryocyte colony, labeled with anti-CD41a + anti-CD42b and stained red using an alkaline phosphatase anti-alkaline phosphatase method. Distinctive morphological features include large cell size, characteristic cell spacing and the appearance of surrounding cell fragments (platelets). A nearby myeloid colony stained with methyl green is shown for comparison.

View larger version (12K): [in this window] [in a new window]   Figure 2. Linearity of megakaryocyte colony formation in cultures of enriched progenitor cells. Increasing numbers of bone marrow mononuclear cells were added to 1 ml agar cultures containing a mixture of seven cytokines. Results are mean (± standard deviation) number of megakaryocyte colonies from triplicate cultures.

View larger version (18K): [in this window] [in a new window]   Figure 3. Enumeration of day 14 progenitor cells in human bone marrow mononuclear cell samples (n = 15). Each point represents the mean of triplicate agar cultures from individual subjects. GM-CFC, BFU-E and Meg-CFC assays were performed in independent cultures. Progenitor cell data from individual subjects are connected by solid lines.

View larger version (27K): [in this window] [in a new window]   Figure 4. Expression of megakaryocyte lineage markers on CD34+ cells. Bone marrow CD34+ cells ( 95% purity following immunomagnetic bead selection) were stained with CD34-phycoerythrin shown on the Y-axis and one of CD61-FITC (fluorescein isothiocyanate), CD41-FITC or c-mpl-FITC. The percentage of cells positive for both markers is indicated in the top-right quadrant of each contour plot. The forward (FSC) and orthogonal (SSC) light scatter properties of these double-positive cells are shown in the arrowed histograms above. Cells positive for both markers (solid line) were larger and more complex than the ungated population (broken line).

Latency of First Cell Division for CD34⁺61⁺ Cells
Experiments were performed to determine the completion of the first cell division in clonal culture. Single fluorescence-activated cell sorter-purified progenitor cells were deposited into microwell cultures containing combinations or single stimuli. Immediately after completion of cell deposition into microwells and daily thereafter, the number of cells was confirmed visually. Any well containing greater than one cell on day 0 (seen in < 0.1% of wells) was excluded from further analysis. As shown in Figure 5, the ability of various cytokines to initiate the first cell division varied considerably: SCF as a single agent was most active. Despite these cells showing a marker of megakaryocytic differentiation as assessed by CD61 expression, MGDF was relatively inactive in inducing cell division and was comparable with IL-3.

View larger version (20K): [in this window] [in a new window]   Figure 5. Rate of accumulation of CD34+61+ cells that had completed their first cell division. Single, purified CD34+61+ cells were deposited in microwells containing the cytokine(s) indicated and monitored daily for evidence of cell division (n = 120 wells for each stimulus). The data presented are the cumulative percentage of cells having divided. Single cytokine stimuli are shown with open symbols and paired stimuli are shown with solid symbols, as indicated. This experiment was performed on three occasions, with similar results.

In addition to documenting the first cell division, the results presented in Figure 5 provide an additional means of determining the clonogenic potential of CD34⁺61⁺ cells following cytokine stimulation. Hence, by day 5 of suspension culture, MGDF stimulated division of less than 15% of cells. In contrast, 34% were stimulated by SCF alone and 50% stimulated by MGDF + SCF. The maximum clonogenic potential of CD34⁺61⁺ cells stimulated by two cytokines at day 5 was achieved by MGDF + SCF or IL-3 + SCF (Fig. 5). Clonogenic potential was thus comparable in this suspension culture assay to clonogenic potential in agar cultures which were examined after 14 days and with multiple factors.

Effect of MGDF on Survival of Progenitor Cells
The ability of MGDF to promote the survival of megakaryocyte progenitor cells was examined using the CD34⁺61⁺ cells. Experiments were performed in which the addition of growth factors to promote colony formation by surviving progenitor cells was delayed for varying intervals. As shown in Figure 6, MGDF promoted a striking enhancement of Meg-CFC survival compared to the saline control at days 5, 10 and 15 (all p < 0.0001). Although this assay potentially measured both cell survival and proliferation stimulated by MGDF alone, the latter seemed less important. This was because of the duration of the assay (maximum 15 days + 14 days) and because significant numbers of clones of megakaryocytes were not detected in those MGDF-alone cultures examined prior to addition of multiple factors.

View larger version (17K): [in this window] [in a new window]   Figure 6. Survival of CD34+61+ progenitor cells in delayed addition experiments. Incubation of purified CD34+61+ cells was initiated in the presence of megakaryocyte growth and development factor (MGDF) (: 200 ng/ml), stem cell factor (SCF) (: 100 ng/ml), MGDF + SCF () or saline (•) in triplicate cultures. Colonies were quantified 14 days following the addition of a mixture of seven cytokines at supramaximal concentrations on the day indicated. Results are mean (± standard deviation) number of colonies from triplicate cultures. Both with and without SCF, MGDF exhibited a marked survival-enhancing effect on Meg-CFC in addition to lesser effects on BFU-E and GM-CFC. This experiment was performed on three occasions, with similar results.

The effect on survival of BFU-E and GM-CFC derived from CD34⁺61⁺ cells was also examined (Fig. 6, lower panels). Although all tests confirmed significance at day 10 (p 0.0013 for BFU-E and p 0.0362 for GM-CFC), the trend of MGDF to promote survival of BFU-E and GM-CFC was of borderline or no significance at days 5 and 15. Further evidence supporting an action of MGDF on GM-CFC and BFU-E was provided in experiments examining MGDF + SCF. Compared to saline, the combination of SCF and MGDF promoted the survival of both BFU-E and GM-CFC at all timepoints (all p < 0.007) and the combination was more active than either factor alone. Although results for GM-CFC at day 5 were of lesser significance (e.g., combination versus saline: Fisher's protected least significant difference, p = 0.0198; and Bonferroni/Dunn, p = 0.0198; whereas Scheffé's F test, p = 0.0990), results at days 10 and 15 for GM-CFC were clearly significant (e.g., combination versus SCF alone at day 15, p = 0.0138 Scheffé's F test). For BFU-E, the combination was more active than either factor alone at all three timepoints (all p 0.03). Despite this evidence that MGDF promoted the survival of myeloid and erythroid progenitor cells, the percentage of surviving clonogenic cells at day 10 following stimulation by MGDF was markedly less for erythroid (13%) and myeloid (19%) than that of megakaryocyte progenitor cells (55%).

Effect of G-CSF on Cloning Efficiency of Megakaryocyte Progenitors
To examine the possible interaction between G-CSF and MGDF, we investigated the effect of G-CSF + MGDF on megakaryocyte colony formation. Figure 7 presents the results of titrating G-CSF in the presence of maximal MGDF stimulation. The clonogenicity of Meg-CFC derived from CD34⁺61⁺ cells stimulated by MGDF alone in this assay was approximately 2%. As evident in the figure, there was no impact of G-CSF, even at final concentrations up to 2,000 ng/ml, on the number of megakaryocyte colonies generated by CD34⁺61⁺ cells stimulated by MGDF alone.

View larger version (15K): [in this window] [in a new window]   Figure 7. Effect of G-CSF on MGDF-stimulated megakaryocyte progenitors. G-CSF at the final concentrations shown was used to stimulate cultures of CD34+61+ cells (4,500 cells/ml) stimulated with MGDF. Megakaryocyte colonies were quantified after incubation for 14 days. Bars indicate 95% confidence intervals.

	Discussion

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Before embarking upon studies of megakaryocyte survival, it was important to establish the linearity of response of megakaryocyte precursor cells in our assay. Primary human megakaryocytes are known to produce biologically active amounts of IL-1ß, IL-6 and GM-CSF [36, 37]. Hence the possible existence of either a constitutively active autocrine loop involving these cytokines or a secondary loop induced by other cytokines (such as transforming growth factor-ß) may act to promote megakaryopoiesis in a non-linear manner [36, 38]. Physiological autocrine loops have been suggested to function in erythroid and myeloid lineages as well [39-41]. In addition, it is well recognized that accessory cells present in cultures are capable of producing cytokines [27, 42]. As shown in Figure 2, cell crowding did not alter the clonogenicity of megakaryocyte progenitor cells in this assay.

Consistent with our previously published observations on progenitor cells of the erythroid and myeloid lineages [26, 43], there was a wide inter-individual variation in numbers of Meg-CFC, BFU-E and GM-CFC. In general, subjects with progenitor cell numbers at the upper and lower limits of the range were consistently placed in assays performed in parallel for all three progenitor cell types. The average myeloid:erythroid:megakaryocyte potential of BM cells was 47:31:1, although, as noted, the range of ratios was considerable (9-254 for myeloid, 4-108 for erythroid, and 1 for megakaryocyte progenitors).

To investigate aspects of clonogenic cell survival, we examined the CD34⁺61⁺ subset of human BM progenitors expressing known surface markers of the megakaryocyte lineage (Fig. 4). Short-term megakaryocyte progenitors have been shown by several groups to express high levels of CD34, some of which express CD41 and/or CD61 [11, 29, 30]. Our aim in using CD34⁺61⁺ cells was to study a defined population of cells of high clonogenic potential which were enriched for Meg-CFC. However CD34⁺61⁺ cells were not homogenous, as evidenced by the presence of progenitor cells of multiple lineages (Fig. 6) [31]. Consequently, we were able to obtain data regarding survival of myeloid, erythroid and megakaryocyte progenitor cells using this enriched population. Indeed, the myeloid- and erythroid-lineage clonogenic potential of CD34⁺61⁺ cells was consistent with similar purified human BM populations studied by other groups [28].

The purified CD34⁺61⁺ cells used in our assays did not require ‘pre-culture’ for six days in suspension cultures to demonstrate megakaryocyte progenitor cell enrichment as has been indicated previously [9]. The ability of CD34⁺61⁺ cells to give rise to erythroid and granulocyte/macrophage colonies, as well as megakaryocytes, suggests maintenance of progenitor cells committed to different cell lineages within a population clearly expressing a megakaryocyte marker (Fig. 6). Although preculture of these cells in MGDF and use of serum-free cultures [44] might increase the number of megakaryocyte progenitors, Meg-CFC were enriched several-fold above both the published concentration found in purified CD34⁺ BM cells and that in unfractionated BM [19, 45]. The myeloid:erythroid:megakaryocyte potential of purified CD34⁺61⁺ cells was 12:11:1 whereas BM cells exhibited a progenitor cell ratio of 47:31:1 (Fig. 3).

A variant of the plate mapping experiments used to determine clonal cell divisions in semi-solid medium was adopted in these experiments. MGDF acting alone induced less than 15% of single CD34⁺61⁺ cells to divide by the fifth day of culture (Fig. 5). Somewhat surprisingly, this was comparable to the action of IL-3 on this subset of cells. In contrast, MGDF promoted the survival of 70% of clonogenic Meg-CFC (Fig. 6). Moreover, by day 5 only 50% of CD34⁺61⁺ cells had been stimulated to divide by the combination of SCF and MGDF (Fig. 5), whereas 100% of clonogenic cells remained viable (Fig. 6). Thus MGDF, alone or in combination, promoted survival of 2- to 5.5-fold more clonogenic cells than the approximately 15% of cells in which it stimulated proliferation. Thus, approximately 15% of cells potentially represented progenitor cells committed to granulocyte-macrophage or erythroid as well as megakaryocyte differentiation. While these data apply only to the enriched CD34⁺61⁺ population examined, they raise the possibility that MGDF may have comparable actions on a broad range of progenitor cells.

Since maximal concentrations of MGDF and SCF exhibited collaboration in promoting both the survival and first division of single human progenitors beyond that seen with either factor alone, it is possible that they may act via different signaling pathways (Figs. 5 and 6). Alternatively MGDF and SCF may be acting on different subsets within the population of CD34⁺61⁺ cells. Although some signal transduction molecules are activated both by SCF and MGDF [45-47], in several megakaryocytic cell lines in which signal transduction via c-mpl was shown to affect Stat5 phosphorylation, SCF failed to activate Stat5 [48]. Hence there is a biochemical basis for a possibly complementary signaling pathway between MGDF and SCF in which both factors are required for maximal effect.

Cells of the megakaryocyte lineage have been shown to express functional receptor for G-CSF, and the possibility that G-CSF may impair the ability of MGDF to stimulate Meg-CFC in culture has been raised [49-51]. Also, cytokine combinations including both G-CSF and MGDF are currently being tested in Phase I/II clinical protocols [52, 53]. It was therefore of interest to determine whether the clonogenicity of maximally stimulated Meg-CFC would be modified by G-CSF. In these studies using purified CD34⁺61⁺ human cells, we demonstrated that over a wide concentration range of G-CSF no significant change occurred in megakaryocyte colony formation. If this system is relevant to the situation in vivo, it is likely that G-CSF administration to patients or normal donors receiving MGDF will not impact on the behavior of megakaryocyte precursors.

In conclusion, the direct action of MGDF to promote both progenitor cell proliferation and survival was documented. This was evident alone and in combination with other cytokines. Furthermore, the survival-promoting effects of MGDF were evident in a clonogenic delayed addition assay. MGDF collaborated with both SCF and IL-3 to reduce the latency of first cell division in single CD34⁺61⁺ cells. The presence of both survival and proliferative effects mediated by this single receptor/ligand axis highlights the subtleties required in signaling in order to distinguish these two effects.

	Acknowledgments

 
We thank Ms. J. Boyd, Ms. H. Zogos and Ms. R. Mansfield for assistance; Drs. J. Szer, A. Grigg and J. Marty for facilitating the study; and Ms. Robyn Muir, Ms. Dora Constantinou and Dr. Francis Battye of the WEHI Flow Systems Laboratory.

This work was supported in part by The Anti-Cancer Council of Victoria, The Rotary Bone Marrow Research Laboratory, The Cooperative Research Centre for Cellular Growth Factors and the National Health and Medical Research Council, Canberra.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Morstyn G, Nicola NA, Metcalf D. Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation. J Cell Physiol 1981;109:133-142.[Medline]

2. Begley CG, Lopez AF, Nicola NA et al. Purified colony-stimulating factors enhance the survival of human neutrophils and eosinophils in vitro: a rapid and sensitive microassay for colony-stimulating factors. Blood 1986;68:162-166.[Abstract/Free Full Text]

3. Williams GT, Smith CA, Spooncer E et al. Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 1990;343:76-79.[Medline]

4. Fairbairn LJ, Cowling GJ, Reipert BM et al. Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell 1993;74:823-832.[Medline]

5. Vigon I, Mornon JP, Cocault L et al. Molecular cloning and characterization of MPL, the human homolog of the v-mpl oncogene: identification of a member of the hematopoietic growth factor receptor superfamily. Proc Natl Acad Sci USA 1992;89:5640-5644.[Abstract/Free Full Text]

6. Zeigler FC, de Sauvage F, Widmer HR et al. In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells. Blood 1994;84:4045-4052.[Abstract/Free Full Text]

7. Berardi AC, Wang AL, Levine JD et al. Functional isolation and characterization of human hematopoietic stem cells. Science 1995;267:104-108.[Abstract/Free Full Text]

8. Kobayashi M, Laver JH, Kato T et al. Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with Steel factor and/or interleukin 3. Blood 1996;88:429-436.[Abstract/Free Full Text]

9. Debili N, Issaad C, Masse JM et al. Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation. Blood 1992;80:3022-3035.[Abstract/Free Full Text]

10. Methia N, Louache F, Vainchenker W et al. Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoeisis. Blood 1993;82:1395-1401.[Abstract/Free Full Text]

11. Nichol JL, Hornkohl AC, Choi ES et al. Enrichment and characterization of peripheral blood-derived megakaryocyte progenitors that mature in short-term liquid culture. STEM CELLS 1994;12:494-505.[Abstract]

12. Debili N, Wendling F, Cosman D et al. The mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. Blood 1995;85:391-401.[Abstract/Free Full Text]

13. Bartley TD, Bogenberger J, Hunt P et al. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994;77:1117-1124.[Medline]

14. de Sauvage FJ, Hass PE, Spencer SD et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994;369:533-538.[Medline]

15. Lok S, Kaushansky K, Holly RD et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994;369:565-568.[Medline]

16. Wendling F, Maraskovsky E, Debili N et al. c-mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994;369:571-574.[Medline]

17. Sohma Y, Akahori H, Seki N et al. Molecular cloning and chromosomal localization of the human thrombopoietin gene. FEBS Lett 1994;353:57-61.[Medline]

18. Kaushansky K. Thrombopoietin: the primary regulator of platelet production. Blood 1995;86:419-431.[Free Full Text]

19. Kobayashi M, Laver JH, Kato T et al. Recombinant human thrombopoietin (mpl ligand) enhances proliferation of erythroid progenitors. Blood 1995;86:2494-2499.[Abstract/Free Full Text]

20. Kaushansky K, Broudy VC, Grossmann A et al. Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy. J Clin Invest 1995;96:1683-1687.

21. Choi ES, Hokom M, Bartley T et al. Recombinant human megakaryocyte growth and development factor (rHuMGDF), a ligand for c-Mpl, produces functional human platelets in vitro. STEM CELLS 1995;13:317-322.

22. Broudy VC, Lin NL, Kaushansky K. Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood 1995;85:1719-1726.[Abstract/Free Full Text]

23. Chen J, Herceg-Harjacek L, Groopman JE et al. Regulation of platelet activation in vitro by the c-mpl ligand, thrombopoietin. Blood 1995;86:4054-4062.[Abstract/Free Full Text]

24. Toombs CF, Young CH, Glaspy JA et al. Megakaryocyte growth and development factor (MGDF) moderately enhances in-vitro platelet aggregation. Thromb Res 1995;80:23-33.[Medline]

25. DeLuca E, Sheridan WP, Watson D et al. Prior chemotherapy does not prevent effective mobilisation by G-CSF of peripheral blood progenitor cells. Br J Cancer 1992;66:893-899.[Medline]

26. Begley CG. Human progenitor cell assays. In: Morstyn G, Sheridan WP, eds. Cell Therapy. Cambridge: Cambridge University Press, 1996:59-74.

27. Rasko JEJ, Metcalf D, Rossner MT et al. The flt3/flk-2 ligand: receptor distribution and action on murine haemopoietic cell survival and proliferation. Leukemia 1995;9:2058-2066.[Medline]

28. De Bruyn C, Delforge A, Bron D et al. Comparison of the coexpression of CD38, CD33 and HLA-DR antigens on CD34⁺ purified cells from human cord blood and bone marrow. STEM CELLS 1995;13:281-288.[Abstract]

29. Macedo A, Orfao A, Ciudad J et al. Phenotypic analysis of CD34 subpopulations in normal human bone marrow and its application for the detection of minimal residual disease. Leukemia 1995;9:1896-1901.[Medline]

30. Zauli G, Valvassori L, Capitani S. Presence and characteristics of circulating megakaryocyte progenitor cells in human fetal blood. Blood 1993;81:385-390.[Abstract/Free Full Text]

31. Miyazaki H, Inoue H, Yanagida M et al. Purification of rat megakaryocyte colony-forming cells using a monoclonal antibody against rat platelet glycoprotein IIb/IIIa. Exp Hematol 1992;20:855-861.[Medline]

32. Metcalf D. Clonal Culture of Hemopoietic Cells: Techniques and Applications. Amsterdam, The Netherlands: Elsevier, 1984.

33. Zhang JL, Stenberg PE, Baker G et al. Immunocytochemical identification of murine and human megakaryocyte colonies in soft-agar cultures. Histochemical J 1994;26:170-178.[Medline]

34. Mazur EM, Hoffman R, Chasis J et al. Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum. Blood 1981;57:277-286.[Abstract/Free Full Text]

35. Teramura M, Katahira J, Hoshino S et al. Effect of recombinant hemopoietic growth factors on human megakaryocyte colony formation in serum-free cultures. Exp Hematol 1989;17:1011-1016.[Medline]

36. Jiang S, Levine JD, Fu Y et al. Cytokine production by primary bone marrow megakaryocytes. Blood 1994;84:4151-4156.[Abstract/Free Full Text]

37. Navarro S, Debili N, Le Couedic JP et al. Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication. Blood 1991;77:461-471.[Abstract/Free Full Text]

38. Kaushansky K, Broudy VC, Lin N et al. Thrombopoietin, the mpl ligand, is essential for full megakaryocyte development. Proc Natl Acad Sci USA 1995;92:3234-3238.[Abstract/Free Full Text]

39. Ratajczak MZ, Kuczynski WI, Sokol DL et al. Expression and physiologic significance of kit ligand and stem cell tyrosine kinase-1 receptor ligand in normal human CD34⁺, c-kit⁺ marrow cells. Blood 1995;86:2161-2167.[Abstract/Free Full Text]

40. Pech N, Hermine O, Goldwasser E. Further study of internal autocrine regulation of multipotent hematopoietic cells. Blood 1993;82:1502-1506.[Abstract/Free Full Text]

41. Hermine O, Beru N, Pech N et al. An autocrine role for erythropoietin in mouse hematopoietic cell differentiation. Blood 1991;78:2253-2260.[Abstract/Free Full Text]

42. Metcalf D, MacDonald HR. Heterogeneity of in vitro colony- and cluster-forming cells in the mouse marrow: segregation by velocity sedimentation. J Cell Physiol 1975;85:643-654.[Medline]

43. Roberts AW, Deluca E, Begley CG et al. Broad inter-individual variations in circulating progenitor cell numbers induced by granulocyte colony-stimulating factor therapy. STEM CELLS 1995;13:512-516.[Abstract]

44. Debili N, Wendling F, Katz A et al. The mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 1995;86:2516-2525.[Abstract/Free Full Text]

45. Berthier R, Valiron O, Schweitzer A et al. Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures: role of TGFß. STEM CELLS 1993;11:120-129.[Abstract]

46. Matsuguchi T, Salgia R, Hallek M et al. Shc phosphorylation in myeloid cells is regulated by granulocyte macrophage colony-stimulating factor, interleukin-3, and steel factor and is constitutively increased by p210(BCR/ABL). J Biol Chem 1994;269:5016-5021.[Abstract/Free Full Text]

47. Blume-Jensen P, Ronnstrand L, Gout I et al. Modulation of Kit/stem cell factor receptor-induced signaling by protein kinase C. J Biol Chem 1994;269:21793-21802.[Abstract/Free Full Text]

48. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995;80:179-185.[Medline]

49. Pallard C, Gouilleux F, Benit L et al. Thrombopoietin activates a STAT5-like factor in hematopoietic cells. EMBO J 1995;14:2847-2856.[Medline]

50. Kanaji T, Okamura T, Nagafuji K et al. Megakaryocytes produce the receptor for granulocyte colony-stimulating factor. Blood 1995;85:3359-3360.[Free Full Text]

51. Shimoda K, Okamura S, Harada N et al. Identification of a functional receptor for granulocyte colony-stimulating factor on platelets. J Clin Invest 1993;91:1310-1313.

52. Basser RL, Rasko JEJ, Clarke K et al. Thrombopoietin effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF). Lancet 1996;348:1279-1281.[Medline]

53. O'Malley CJ, Rasko JEJ, Basser RL et al. Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation. Blood 1996;88:3288-3298.[Abstract/Free Full Text]

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