Murine Megakaryocyte Progenitor Cells and Their Susceptibility to Suppression by - 《stemcells》2004-2005文献干细胞之家



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江边孤钓

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ABSTRACT
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In agar cultures of mouse bone marrow cells, mega-karyocyte colony-forming cells exhibited shorter survival times than granulocyte-macrophage progenitor cells when initially cultured in the absence of stimulating factors. Initiation of cultures with G-CSF improved the survival times of granulocyte-macrophage progenitor cells and those of megakaryocyte progenitor cells. Paradoxically, G-CSF was found to consistently inhibit megakaryocyte colony formation stimulated by erythropoietin or by stem cell factor plus interleukin-3 (IL-3) plus erythropoietin. G-CSF was a less-consistent inhibitor of megakaryocyte colonies stimulated by thrombopoietin or IL-3. Analysis of the response of marrow cells from mice with the deletion of the genes encoding CIS, SOCS-1, SOCS-2, SOCS-3, SOCS-5, SOCS-6, or SOCS-7 indicated that the inhibitory SOCS proteins, with the possible exception of SOCS-3, were not involved in the G-CSF–initiated suppression of megakaryocyte colony formation.
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INTRODUCTION5 l9 Z0 E9 a/ f- g) }% `$ g' d

In the hierarchy of hematopoietic precursor cells, one of the differences between stem cells and committed progenitor cells is the nature of their dependence on extrinsic signaling by hematopoietic regulators. Those stem cells, mature enough to be able to be cultured in vitro as long-term culture-initiating cells or blast colony-forming cells, tend to require simultaneous stimulation by multiple regulators to initiate and sustain cell division [1–4]. In contrast, most lineage-committed progenitor cells can proliferate in vitro when stimulated by a single regulatory factor [5], although certain regulator combinations show synergistic activity on progenitor cells [6, 7], and there are some progenitor cells that seem to require multiple stimuli to exhibit proliferation [6].
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Committed megakaryocyte progenitor cells are somewhat anomalous in that most of these cells, even those that are quite mature and able to form only a few progeny cells, require stimulation by a combination of regulators [7, 8]. For example, the combination of stem cell factor (SCF) plus interleukin-3 (IL-3) plus erythropoietin (EPO) stimulates the formation of more than double the number of colonies stimulated by individual regulators, of which IL-3 and EPO are the most active [8]. Megakaryocyte progenitor cells are also the most clearly separable from other committed progenitor cells by fluorescence-activated cell sorting [9], and these cells provide the strongest evidence against the postulated existence of a single common, uncommitted, progenitor cell pool [10].) a1 i1 q8 @" V) Y/ F" o# n' N
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For several reasons, therefore, megakaryocyte progenitor cells are an intriguing progenitor cell type whose properties and responses to regulatory control warrant more detailed investigation.
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The present studies took advantage of the ease of identifying mouse megakaryocytic cells because of their exclusive content of acetylcholinesterase and were undertaken to establish more complete information on the properties of megakaryocyte progenitor cells. In particular, the previous observation that G-CSF had an unexpected capacity to partially inhibit megakaryocyte colony formation [8] was analyzed in more detail to establish a possible cellular basis for the phenomenon. The present findings have confirmed and extended earlier observations indicating that megakaryocyte progenitor cells have certain unusual characteristics and have eliminated most members of the SOCS family of inducible intracytoplasmic inhibitors of cytokine signaling as being necessary for the selective inhibitory actions of G-CSF.

MATERIALS AND METHODS
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Mice1 T2 O  p1 \$ B( q9 m* [' P

Normal mice used were 6 – 8-week-old mice of the C57BL strain. The CIS–/–, SOCS-2–/–, SOCS-3–/–, SOCS-5–/–, SOCS-6–/–, and SOCS-7–/– mice used were also aged 6 to 8 weeks and were on a C57BL genetic background, but the SOCS-1–/– mice were aged only 2 weeks. The original CIS–/– mice were a kind gift from Dr. J. Ihle of St. Jude’s Hospital, Memphis, and mice with the other gene inactivations were produced in these laboratories using procedures that have been described elsewhere [11–14]. All mice were bred in the Walter and Eliza Hall Institute of Medical Research in protected animal rooms and monitored regularly to detect the possible presence of viral or bacterial pathogens.
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Culture Technique& U" h9 g1 {" x* h
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All cultures were prepared using femoral marrow cells that were harvested and converted to dispersed cell suspensions using Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum.
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Cultures were performed in 35-mm plastic petri dishes, and each contained a 1-ml volume of Dulbecco’s modified Eagle’s medium with a final concentration of 0.3% Bacto-agar and 20% preselected newborn calf serum (HyClone Lab, Inc, Logan, UT). Routinely, the 1-ml cultures contained 25,000 marrow cells and, after gelling, the cultures were incubated for 7 days at 37～C in a fully humidified atmosphere of 10% CO2 in air.
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After 7 days of incubation, provisional total colony counts were performed using an Olympus dissection microscope (Olympus, Tokyo, Japan) and indirect lighting. The cultures were then fixed by the addition of 1 ml of 2.5% glutaraldehyde. Four hours later, the fixed cultures were floated intact onto a water bath and captured on 50 x 75-mm glass slides. After drying, cultures were stained for acetylcholinesterase and then with Luxol Fast Blue and hematoxylin. After drying, the stained cultures were mounted with coverslips, and the entire cultures were analyzed at x 60 and x 120 magnifications, enumerating all colony types and the number and cell content of all clones containing two or more acetylcholinesterase-positive cells.6 {, s. ]6 O5 a* Z
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Stimuli Used in Cultures

Colony formation was stimulated by addition of 0.1 ml of stimulating factor to the empty culture dishes before addition of the marrow cell suspensions in agar-media. With two exceptions, all stimuli used were purified recombinant mouse factors and were purchased from PeproTech (Rocky Hill Rd, NJ) or prepared in this laboratory. Recombinant human G-CSF and human EPO were obtained from Amgen (Thousand Oaks, CA). The final concentrations of stimuli used in the cultures, whether used alone or in combination, were GM-CSF, G-CSF, M-CSF, IL-3 10 ng/ml; EPO 2 IU/ml; IL-6 100 ng/ml; SCF 100 ng/ml; thrombopoietin (TPO) 50 ng/ml.

Delayed Addition of Stimuli* @4 r. V1 z: y' b1 y
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In experiments to determine the consequences of delaying the inclusion of necessary stimuli to cultures, cultures of marrow cells were initiated in agar medium lacking the appropriate stimulus, and then, at daily intervals, 0.1 ml of the stimulus was added gently to the surface of quadruplicate cultures. These cultures were then incubated for an additional 7 days from the time of addition of the stimuli, before being analyzed as above.

Statistical Analyses
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Paired comparison of megakaryocyte colony numbers developing in quadruplicate cultures with different stimuli was made using the Student’s t-test. Analysis of significant differences in survival curves was made using the Graph Pad Prism program to perform a log-rank test. Confirmation of the linearity of megakaryocyte colony formation was established by linear regression analysis.

RESULTS

Linearity of Megakaryocyte Colony Formation In Vitro Versus Cultured Cell Numbers

The growth of typical megakaryocyte colonies from fluorescence-activated cell sorter–enriched populations of mouse bone marrow cells strongly indicated that the action of stimulating factors on megakaryocyte progenitor cells was direct and did not depend on indirect cell-cell interactions [8, 9]. This conclusion was supported by the culture of varying numbers of bone marrow cells with each of the single agents active in stimulating colony formation. Figure 1 is typical of the results from three such experiments and shows strict linearity between cultured cell numbers and the numbers of megakaryocyte colonies developing after stimulation either by EPO, IL-3, or TPO (r2 = 0.999, 0.997, and 0.992, respectively). The higher colony numbers with EPO or IL-3 than with TPO are typical of the response of mouse bone marrow cells in agar culture despite the dominant action of TPO in vivo [15, 16]. A similar linearity was observed when the combined stimulus of SCF plus IL-3 plus EPO was used to stimulate colony formation.

Dependency of Megakaryocyte Progenitor Cells on Regulatory Factors for Survival In Vitro
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Committed progenitor cells progressively die or lose their capacity for proliferation if cultured in the initial absence of appropriate stimulating factors. For adult marrow cells, the overall survival half-life of colony-forming cells in the absence of stimulation is 20 to 24 hours [5]. However, a quite different response was observed with megakaryo-cyte progenitor cell survival. The data shown in Figure 2 are typical of the results obtained in three replicate experiments in which the addition of IL-3 or a mixture of SCF plus IL-3 plus EPO was delayed for varying time periods. The survival of granulocyte-macrophage progenitor cells was 20 to 24 hours, in agreement with previous studies. However, Figure 2 shows that the survival of committed megakaryo-cyte progenitor cells was significantly shorter, varying in the different experiments from 7 to 12 hours. By log-rank analysis, this difference was significantly different (p 6 i+ K. Q; M! b0 O6 R( W1 }

Inhibition of Megakaryocyte Colony Formation by G-CSF5 N9 V% d: y! W
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Previous studies showed that G-CSF had the unusual ability to reduce the number of megakaryocyte colonies developing in cultures stimulated by EPO but less uniformly in cultures stimulated by TPO or by IL-3 [8]./ Q! R4 @9 V1 |- o8 ?

In the present experiments, this inhibition was shown also to occur significantly (p
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In nine replicate experiments, the numbers of ace-tylcholinesterase-positive cells were determined in each megakaryocyte colony, developing in cultures stimulated by SCF plus IL-3 plus EPO with or without added G-CSF. As shown in Table 1, no inhibition by G-CSF was observed of medium-sized (20 to 100 cells) or large-sized (> 100 cells) colonies, and inhibition was consistently confined to small colonies containing fewer than 20 acetylcholinesterase-positive cells. Most such colonies are presumed to have been formed by relatively mature megakaryocytic progenitor cells.
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To test whether G-CSF might have a survival-enhancing action on megakaryocyte progenitor cells despite its inhibitory action for actual colony formation, cultures of 25,000 C57BL bone marrow cells were initiated containing G-CSF or saline, and then at intervals the combined stimulus of SCF plus IL-3 plus EPO was added. Figure 4 shows the pooled data from three representative experiments of this type. Initiation with G-CSF allowed a significant (p 7 s6 e8 z9 c& `2 d3 L* ^: _

The positive effects of G-CSF on the survival and proliferation of granulocyte-macrophage progenitor cells are in agreement with previous studies showing that G-CSF can initiate colony formation by numerous granulocyte-macrophage colony-forming cells but is able to sustain the proliferation of only a small subset of these [17].
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Possible Mediators of the Inhibitory Action of G-CSF

To establish the consistency of the inhibitory action of G-CSF on megakaryocyte colony formation by C57BL bone marrow cells, 26 separate experiments were performed to test the action of G-CSF on EPO, TPO, IL-3, or SCF plus IL-3 plus EPO. The results are summarized in Table 3. G-CSF was inhibitory in all cases for megakaryocyte colony formation stimulated by EPO or SCF plus IL-3 plus EPO but was only active in approximately half the experiments using TPO or IL-3 as the megakaryocyte stimulus.( W, k& [  M, \0 X# v/ K
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With this background, tests were then performed on marrow cells from a variety of genetically manipulated mice where the gene product might have been involved as a mediator of the G-CSF–induced suppression. Of particular interest were the SOCS family of SH2-containing proteins, whose production is induced by cytokine signaling and which act to suppress cytokine signaling. Examples of these tests are shown in Figure 5. Of special interest were CIS–/– mice that exhibit excessive megakaryocyte colony formation in vitro and SOCS-3–/– mice, because the SOCS-3 protein is a major inhibitor of G-CSF signaling [13]. As shown in Table 3, CIS–/– cells behaved in a manner similar to control C57BL marrow cells, but SOCS-3–/– bone marrow cells were slightly more variable in behavior. This was a weakly significant difference that raised the possibility that the SOCS-3 protein might play some role in the G-CSF suppressive action. Less extensive studies with SOCS-1–/–, SOCS-2–/–, SOCS-5–/–, SOCS-6–/–, and SOCS-7–/– marrow cells, all on a C57BL background, failed to reveal any significant differences from the behavior of normal C57BL marrow cells.
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Other studies analyzed marrow cells from mpl–/– [16], max41 [18], IL-6–/–, and Stat-5–/– mice. All behaved as did the control C57BL mice with the interesting anomaly that Stat-5–/– cells were consistently unresponsive to EPO-stimulated megakaryocyte colony formation (data not shown). Stat-5 is known to be an important signal transducer for EPO signaling in erythroid cells [19] and clearly plays the same role in megakaryocytic cells when stimulated by EPO, although the cells were able to generate megakaryo-cyte colonies with other stimuli.! x9 q4 P$ p' Q1 C! Q5 x* I

DISCUSSION

The present experiments have extended earlier studies [8, 9] indicating that megakaryocytic progenitor cells respond directly to the various growth factors able to stimulate their proliferation in vitro–EPO, TPO, IL-3, and SCF plus IL-3 plus EPO. Megakaryocytic progenitor cells were found to be atypical in exhibiting an accelerated death rate when cultured in the initial absence of growth factors. In this, they resembled the less mature blast colony-forming cells, which is of interest because both cell types are also atypical in requiring combined growth factor stimulation to exhibit full proliferative responses [1-4, 8]. This parallel is limited, however, because G-CSF has a strong potentiating action on the proliferation of blast colony-forming cells when stimulated by SCF [20], whereas G-CSF is inhibitory for megakaryocyte colony formation. Curiously, G-CSF had a weak capacity to promote the survival of megakaryocyte colony-forming cells at the same time as inhibiting mega-karyocyte colony formation.. K9 S& O" `9 B
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Inhibitory effects when positive proliferative regulators are combined are unusual but have been encountered previously. For example, combination of GM-CSF with M-CSF potentiates the growth of some granulocyte-macrophage colonies but is also clearly inhibitory for the growth of some macrophage colonies [6]. The inhibitory action of G-CSF on megakaryocyte colony formation was seen with all positive stimulating factors〞EPO, TPO, IL-3, and SCF stimulated by EPO or SCF plus IL-3 plus EPO. No data have been published on whether mega-karyocyte progenitor cells express G-CSF receptors, so it is unclear whether the present inhibitory effects of G-CSF are direct or indirect effects of G-CSF action.( {- C  t  m& p( I- a+ ~1 l

G-CSF had noinhibitory effects on granulocyte-macrophage colony formation in the same cultures in which it was inhibiting megakaryocyte colony formation, and its action was restricted further to colony formation by relatively mature megakaryocyte precursors. It would seem that these cells are particularly susceptible to the consequences of G-CSF-initiated signaling, but the critical molecular events involved remain for further studies to elucidate.
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The possibility was examined that the inhibition by G-CSF might be mediated by one or other of the SOCS family of cytokine-inducible inhibitors of receptor signaling [21]. However, with six of the SOCS molecules tested, deletion of the gene did not prevent the inhibitory action of G-CSF, and deletion of SOCS-3 had only an inconsistent effect in preventing the action of G-CSF.$ Y& n% Y! v# W8 \. |# Q5 Q/ ?
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The present findings may represent a curious in vitro artifact, but it has been a clinical observation that treatment with G-CSF can lead to a reversible, dose-related fall in platelet levels in some humans [22–24], and a similar fall has been noted in hamsters [25]. Partial suppression of megakaryocyte production by G-CSF might be one possible cellular basis for such falls in platelet levels, and the in vitro suppression of megakaryocyte colony formation warrants additional study to establish the intracellular mechanisms responsible.0 L4 p7 _% x. G9 W5 S. o
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ACKNOWLEDGMENTS/ a! ~1 }+ |6 x/ p7 o6 C

The authors are indebted to Dr. Warren S. Alexander for supplying the various genetically manipulated mice and to Drs. Warren S. Alexander and A. Roberts for assistance with statistical analyses. This work was supported by the Carden Fellowship Fund of the Cancer Council Victoria, the National Health and Medical Research Council, Canberra, the Cooperative Research Centre for Cellular Growth Factors, the AMRAD Corporation, Melbourne, and National Institutes of Health grant CA22556.
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