Stem Cells, Vol. 14, No. 1, 124-131,
January 1996
© 1996 AlphaMed Press
Overexpression of Granulocyte Colony-Stimulating Factor In Vivo Decreases the Level of Polyploidization of Mouse Bone Marrow Megakaryocytes
Masahiro Saitoa,b,
Koji Takadaa,b,
Taketo Yamadac,
Junichiro Fujimotoa
a Department of Pathology, National Children's Medical Research Center, Tokyo, Japan;
b Department of Pediatrics, Juntendo University School of Medicine, Tokyo, Japan;
c Department of Pathology, Keio University School of Medicine, Tokyo, Japan
Key Words. G-CSF • TPO • Megakaryocyte • Megakaryocytopoiesis • DNA • Ploidy
Dr. Junichiro Fujimoto, Department of Pathology, National Children's Medical Research Center 3-35-31, Taishido, Setagaya-ku, Tokyo, 154, Japan.
 |
Abstract
|
|---|
The in vivo effect of G-CSF on the maturation of mouse bone marrow megakaryocytes was studied by monitoring the DNA contents. Megakaryocytes were first identified by a specific 1C2 monoclonal antibody against mouse platelets and megakaryocytes and DNA contents of these cells were measured by propidium iodine. Megakaryocytes of mice transgenic for human G-CSF had a modal DNA class of 8N, showing a striking contrast to the previous reports that normal mouse megakaryocytes from most strains have 16N DNA content as a modal class. Daily 10 µg administration of G-CSF to mice for three to five days affected the DNA distribution pattern of bone marrow megakaryocytes, with a higher proportion of cells having 8N DNA contents. This G-CSF treatment, however, did not influence the peripheral blood platelet count or bone marrow megakaryocyte number. Administration of G-CSF along with thrombopoietin (TPO) reduced the proportion of megakaryocytes, with 32N DNA, the DNA class that was increased by TPO. Finally, the presence of mRNA for the mouse G-CSF receptor was demonstrated in two megakaryoblastic cell lines by reverse transcriptase polymerase chain reaction. These results indicated that G-CSF may have a suppressive effect on the maturation of mouse bone marrow megakaryocytes when monitored by the DNA polyploidy. Although further study is clearly necessary, the presence of mRNA for the G-CSF receptor in megakaryocytic lineage strongly suggests the direct action of G-CSF on this cell lineage.
|
Introduction
|
|---|
The action of G-CSF has mainly been investigated in myeloid cell lineages as well as at the progenitor level [1–3]. A binding assay utilizing tracer-labeled recombinant G-CSF, as well as a sensitive reverse transcriptase polymerase chain reaction (RT-PCR) assay, have revealed the wider range of expression of the receptor for G-CSF than had been expected [4–6]. In vivo as well as in vitro administration of G-CSF has not been shown to play any significant role on megakaryocytopoiesis and platelet production [1, 7, 8]. Recent reports describing the G-CSF receptor expression on human platelets and in vitro-induced human megakaryocytes [1, 5], however, may imply a role for this cytokine in megakaryocytopoiesis.
In our transgenic mice that constitutively express human G-CSF [9, 10], no apparent change of platelet count was observed, although the megakaryocyte number in the spleen increased by 20- to 30-fold compared to control animals (Yamada et al., submitted).
Under such circumstances, this study was undertaken to evaluate the role of G-CSF on megakaryocytopoiesis. We found that the in vivo expression of G-CSF via transgene or exogenous administration altered the DNA polyploidy distribution of mouse bone marrow megakaryocytes leading to a higher proportion of these cells with 8N DNA content. We also show that G-CSF exerts a suppressive effect on the DNA ploidy maturation of megakaryocytes induced by the recently identified thrombopoietic factor, c-mpl ligand, namely, thrombopoietin (TPO) [11–13].
|
Materials and Methods
|
|---|
Mice
Female C57BL/6, (C57BL/6 x DBA/2)F1, namely, BDF1, and C3H/HEJ mice at 8 to 10 weeks old were purchased from Japan Clea Co., Ltd. (Tokyo, Japan). Female 8- to 10-week-old mice transgenic for human G-CSF described below were also used in this study. All animals were housed in an approved facility at the National Children's Medical Research Center.
Reagents
All reagents were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise indicated.
Preparation of Bone Marrow Cells, Splenocytes and Peripheral Blood
Bone marrow cells were flushed out from two femora and tibiae using a buffer consisting of 10 mM sodium phosphate, pH 7.2, 13.6 mM sodium citrate, 133 mM NaCl, 11.1 mM glucose and 1% bovine serum albumin (BSA) (MEG buffer). The cell number was counted by hemocytometer and 2 x 105 cells were attached to slide glass using a cytospin apparatus. On the slide, acetylcholine esterase (AChE) staining [14] was performed and the number of cells positive for AChE was counted as number of megakaryocytes. The number of total bone marrow cells and megakaryocytes was calculated based on the data of cell counting and AChE staining. Bone marrow cells were further fractionated by 45% Percoll gradient centrifugation and were used to study the DNA content of megakaryocytes. Splenocytes were also used after fractionating by 45% Percoll gradient centrifugation as described above. Peripheral blood was collected from orbital plexus into pipettes containing EDTA and the platelet number was counted by phase-contrast microscopy using a hemocytometer.
Labeling of Megakaryocytes by a Monoclonal Antibody and Propidium Iodine
Bone marrow cells and splenocytes enriched for megakaryocytes were stained by fluorescein isothiocyanate (FITC)-labeled monoclonal antibody 1C2 (FITC-1C2) reactive with mouse platelets and megakaryocytes. Details of 1C2 antibody are described elsewhere [15]. FITC labeling of antibody was performed using purified 1C2 isolated by Protein A agarose column from culture supernatant containing antibody. After reacting with FITC-1C2 for 30 min at 4°C and washing twice with MEG buffer, the DNA of cells was stained by incubating for 3 h at 4°C, with 50 µg/ml of propidium iodine in hypotonic buffer consisting of 0.1% sodium citrate and 1% BSA. DNase-free RNase was added to cells to make a final concentration of 50 µg/ml and incubated for 30 min at 25°C. Using an EPICS-PROFILE flow cytometer (Coulter Instruments; Hialeah, FL), FITC-1C2+ cells were selected and DNA histograms of these cells were generated. The proportion of cells in each ploidy class was determined by integrating the number of cells under each DNA peak. The Mann-Whitney rank sum test was used to compare the statistical difference between megakaryocyte DNA distributions.
In Vivo Treatment of Mice with G-CSF or G-CSF Plus TPO
Recombinant cytokines including human G-CSF and human TPO were generously supplied by Kirin Brewery Co., Ltd. (Tokyo, Japan). BDF1 mice (three animals per group) were injected i.p. with 10 µg of G-CSF, 100 ng of TPO or equivalent doses of G-CSF plus TPO once a day for three or five days. The cytokine used for injection was diluted with phosphate-buffered saline (PBS) containing 1% of syngeneic mouse serum and 0.2 ml per animal was injected. As controls, groups of BDF1 mice injected with PBS containing 1% syngeneic mouse serum alone were set up.
Mice Transgenic for Human G-CSF
Generation and characterization of the mice transgenic for human G-CSF (designated as granulocyte-thioguanine [G-TG]) are described elsewhere [9, 10]. Briefly, human G-CSF cDNA under the control of SR
promoter [16] was introduced into fertilized eggs obtained from female BDF1 mice mated with male BDF1 mice. A male offspring carrying the transgene was used as a founder and mated with female BDF1 to generate mice carrying human G-CSF cDNA. Female transgenic mice were used as G-TG and their litter mates carrying no transgene were used as controls for G-TG. In our preliminary study, the following results have been obtained [9]. G-TG mice produced approximately one ng/ml of biologically active human G-CSF in the sera. High white blood cell count consisting of mainly mature granulocytes was noticed in G-TG but platelet count was not altered. In the enlarged spleen of G-TG, the number of megakaryocytes increased by 20- to 30-fold compared to control animals.
RT-PCR Followed by Southern Blot Hybridization
From mouse megakaryoblastic cell lines (C2 and L8057) [17, 18] and the mouse myelogenous leukemia line NFS60 [19], total RNA was extracted and complimentary DNA was generated by using a cDNA generation kit (Pharmacia Biotech; Uppsala, Sweden). A set of primers, 5'-TTCCTGGAGAACGAAGGTCCA (sense) and 5'-CAGGGTCTTCAAGATACAAGG (antisense), was used to amplify a 1.2 kb fragment of mouse G-CSF receptor cDNA [20]. PCR reaction was repeated for 30 cycles with heating at 94°C for 45 sec, annealing at 55°C for 45 sec, elongation at 72°C for 2 min and the products were separated on 2% agarose gel, transferred to filter, and hybridized with 32P-labeled mouse G-CSF receptor cDNA fragment [21]. A mouse G-CSF receptor cDNA fragment used as a probe was a 1.2 kb of RT-PCR product obtained from NFS60 subcloned in pUC18 plasmid. Sequencing analysis revealed that this 1.2 kb fragment corresponded to a portion of cDNA reported for the mouse G-CSF receptor (data not shown).
|
Results
|
|---|
DNA Polyploidy Distribution of Bone Marrow Megakaryocytes of G-TG
DNA contents of bone marrow megakaryocytes of G-TG are shown in Figure 1
. As is evident, the modal DNA ploidy class of megakaryocytes of G-TG was 8N (Figs. 1A and 1B, top panel). This was in sharp contrast to the DNA distribution of bone marrow megakaryocytes obtained from litter mates (Figs. 1A and 1B, upper middle), C57BL/6 mice (Figs. 1A and 1B, lower middle) or BDF1 (Figs. 3A and 3B, top) where 16N was the modal DNA ploidy level. In order to know whether the same mechanism affects the megakaryocytopoiesis in the spleen, splenic megakaryocytes of G-TG were analyzed. As shown by the DNA histogram (Fig. 2), the proportion of megakaryocytes with 8N DNA class greatly increased, reaching to the level of 16N peak (Fig. 2, upper panel), while the splenic megakaryocytes of a litter mate (Fig. 2, lower panel) exhibited a DNA histogram comparable to that of bone marrow (Fig. 1B, upper middle). For comparison and to monitor the reliability of our assay system, C3H/HEJ mice whose megakaryocytes have a modal DNA content of 32N [22, 23] were analyzed (Figs. 1A and 1B, bottom).
View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1. Bone marrow megakaryocyte DNA content distribution of mice transgenic for human G-CSF. A) DNA contents of 1C2+ mouse bone marrow cells obtained from mice transgenic for human G-CSF (G-TG), litter mates (LM), C57BL/6 and C3H/HEJ are shown. The Y-axis represents the frequency (%) of cells belonging to each DNA content. Data of two experiments were analyzed. Bars represent means ± SD. B) Representative DNA histograms of bone marrow megakaryocytes of G-TG, LM, C57BL/6 and C3H/HEJ are shown.
|
|
View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3. Alteration of DNA polyploidy distribution of megakaryocytes of BDF1 mice treated with G-CSF. A) The proportion of bone marrow megakaryocytes with 8N DNA content increased in BDF1 mice treated with G-CSF. Data from three experiments were analyzed and megakaryocytes with DNA contents  8N are shown. B) Representative DNA histograms of BDF1 mice treated with G-CSF are shown. Note the increase in the proportion of megakaryocytes with 8N DNA contents as G-CSF treatment was prolonged.
|
|
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2. Splenic megakaryocyte DNA content distribution of human G-CSF (G-TG). DNA histograms of splenic megakaryocytes of G-TG and litter mates (LM) are shown.
|
|
Alteration of DNA Polyploidy Distribution of Bone Marrow Megakaryocytes Following In Vivo G-CSF Administration
Results obtained in G-TG prompted us to analyze whether in vivo administration of G-TG has a similar effect on the DNA ploidy class of megakaryocytes. As shown in Figure 3A
, the proportion of bone marrow megakaryocytes having an 8N DNA class increased in BDF1 mice injected with G-CSF. In mice treated with G-CSF for five days, the frequency of megakaryocytes with 8N versus 32N reversed (Fig. 3A, lower panel), compared to control animals (upper panel) who received PBS with syngeneic serum alone (p < 0.01). Even in mice treated with G-CSF for three days (Fig. 3A, middle panel), a weak but significant difference in distributions was detected when compared to controls (p < 0.05). Histograms representative of each group clearly show the increase in the proportion of megakaryocytes having 8N DNA content (Fig. 3B).
Although DNA distribution of megakaryocytes changed in mice treated with G-CSF as described above, no obvious alteration in platelet count, bone marrow cell number or total bone marrow megakaryocyte number as defined by AChE staining was observed in these animals (Table 1, not significant by Student's t-test). The absolute number of megakaryocytes in each DNA class was calculated based on the data of megakaryocyte counts and the frequency of cells in each DNA class. As shown in Table 2, the number of megakaryocytes with 8N DNA class significantly increased (p < 0.01), and that of 32N DNA class decreased (p < 0.01) in BDF1 mice treated with G-CSF for five days compared to controls.
Suppressive Effect of G-CSF on the DNA Maturation of Megakaryocytes Induced by TPO
We next questioned whether the above-mentioned effect of G-CSF could also be demonstrated in mice receiving a cytokine that upregulates megakaryocytopoiesis. As shown in Figure 4A, administration of TPO to BDF1 mice increased the proportion of megakaryocytes having a 32N DNA class. When G-CSF was administered to BDF1 mice together with TPO, a weak but significant (p < 0.05) decrease in the proportion of megakaryocytes with 32N was observed. Figure 4B demonstrates a typical example in which the proportion of megakaryocytes with 32N in a BDF1 mouse treated with G-CSF and TPO was reduced.
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4. The suppressive effect of G-CSF on the DNA polyploidy maturation of megakaryocytes induced by TPO. A) Data from five days of treatment of BDF1 mice with TPO or TPO plus G-CSF are shown. TPO treatment for five days increased the proportion of bone marrow megakaryocytes with 32N DNA contents, but the reduction of this class was apparent by TPO plus G-CSF treatment. Data from three experiments were analyzed and megakaryocytes with DNA contents 8N are shown. B) Representative DNA histograms of BDF1 mice treated with cytokines for five days are shown. Note the increase of 32N peak in TPO-treated BDF1 mice and the decrease of 32N peak in mice treated with TPO plus G-CSF.
|
|
Identification of the G-CSF Receptor mRNA in Mouse Megakaryoblastic Cell Lines
Expression of the mRNA for the G-CSF receptor in mouse megakaryoblastic cell lines was examined by RT-PCR, followed by Southern blot hybridization. As shown in Figure 5, an amplified 1.2 kb fragment specific for mouse G-CSF receptor mRNA was detected in C2 and L8057 lines, both of which contain predominantly the immature megakaryoblasts [17, 18]. A fragment having an identical size was obtained from the NFS60 cell line, which is known to express the G-CSF receptor. In order to distinguish the 1.2 kb fragment of the G-CSF receptor cDNA from the same sized band possibly amplified from the pseudogene, PCR products were digested with Apa I endonuclease, whose site is present in the amplified fragment from the G-CSF receptor cDNA, but absent from that of the pseudogene [24]. Apa I digestion of all three PCR products (C2, L8057 and NFS60) generated 1.0 and 0.2 kb bands, indicating the presence of mRNA for the G-CSF receptor (data not shown).
View larger version (36K):
[in this window]
[in a new window]
|
Fig. 5. Southern blot hybridization of RT-PCR products of G-CSF receptor mRNA RT-PCR products obtained from NFS60 (lane 1), C2 (lane 2) and L8057 (lane 3) was applied. The mouse G-CSF receptor cDNA probe was hybridized with a 1.2 kb band in all samples.
|
|
|
Discussion
|
|---|
Megakaryocytopoiesis and thrombopoiesis are regulated in part by various cytokines including CSFs, interleukins (ILs) and a newly recognized molecule called c-mpl ligand, namely, TPO [7, 8, 11–13, 25–27]. With regard to the effect of G-CSF, a stimulatory effect was demonstrated in an in vitro system. G-CSF alone does not induce a megakaryocyte colony but augments the appearance of these colonies induced by other cytokines such as IL-3 and IL-6 [7, 8]. Although in vivo administration of G-CSF exerts a minimum effect on platelet count, dramatic changes occur at the precursor level [28, 29]. Injection of G-CSF decreased the number of colony-forming units of megakaryocyte (CFU-Meg) in bone marrow but increased it in the spleen [29]. Since the accumulation of megakaryocyte precursors in the spleen does not seem to contribute to the platelet production [30–32], an effective production of platelets is thought to be operated in the bone marrow even under the condition of a decreased number of CFU-Meg. If this is the case, it is conceivable that acceleration of megakaryocyte maturation would be expected in the bone marrow. Transient decrease and gradual recovery of platelet numbers during G-CSF administration [1] seem to support this idea. Our results, however, suggest that this may not be the case when bone marrow megakaryocytopoiesis is monitored by DNA polyploidy distribution.
We demonstrated in this study that DNA polyploidy distribution of bone marrow megakaryocytes of mice expressing G-CSF was significantly different from that of control mice. As has been reported and was shown in this study, the modal DNA class of bone marrow megakaryocytes of most mouse strains is 16N except for a strain of C3H background that has 32N model DNA class [22, 23]. In mice expressing G-CSF as a transgene (G-TG), however, the modal DNA class of megakaryocytes shifted to 8N. A high proportion of megakaryocytes with 8N DNA class was also shown in the spleen of G-TG. These observations indicate that a shift to the lower ploidy class is not the result of the migration of highly polyploid megakaryocytes to the spleen, but that G-CSF affects the splenic megakaryocytopoiesis by the same mechanism. A similar effect of G-CSF was also shown by a short-term exogenous G-CSF administration. In addition, G-CSF reduced the proportion of megakaryocytes with 32N DNA class in a state of upregulated megakaryopoiesis induced by TPO. No obvious change of platelet count, bone marrow cell number and megakaryocyte number was observed in G-CSF-treated animals. As several reports described the transient decrease and recovery of bone marrow cell number and platelet count during G-CSF treatment [1, 29, 33], platelet and bone marrow cell counts in our study seem to reflect the recovery phase.
We therefore speculate that G-CSF exerts a suppressive effect on the maturation of megakaryocytes, but other possibilities must be ruled out. That the absolute number of bone marrow megakaryocytes with 8N DNA class increased following G-CSF treatment may reflect the stimulatory effect and may indicate the induction of low ploidy megakaryocytes. However, we think this is not likely because G-CSF significantly decreased the number of megakaryocytes with 32N DNA class, and G-CSF reduced the proportion of megakaryocytes with 32N DNA class in a condition of accelerated megakaryocytopoiesis under the influence of TPO, a strong promoter for induction and maturation of megakaryocytes.
A unique maturational process of this particular cell lineage should also be considered. Maturation of megakaryocytes involves two unique phenomena: DNA polyploidy and cytoplasmic maturation [34]. These two phenotypically distinct processes can be induced by a single cytokine like TPO, although whether these two pathways are inseparably or independently regulated remains to be solved. Generally, it is conceivable that the larger the cell size of megakaryocytes, the better platelet production is expected, and enlargement of megakaryocyte size usually accompanies the hyper DNA polyploidy and the full maturation of cytoplasm as is reported in the case of TPO administration [25]. In this regard, our preliminary observation is interesting. By electron microscopic analysis on G-TG, we could frequently identify the megakaryocytes having fully matured cytoplasm but less matured nuclei (Yamada, manuscript in preparation). Thus, we speculate that G-CSF may suppress the process of DNA polyploidy, namely, endomitosis, while exhibiting no influence on the cytoplasmic maturation. We are currently investigating this possibility on the megakaryocytes obtained from G-CSF-expressing animals and from an in vitro induction system.
G-CSF was originally reported as a cytokine that induced differentiation of a myelomonocytic cell line and stimulated formation of granulocytic colonies from normal bone marrow cells, but recent studies have shown a wider range of activity of G-CSF than expected. Mobilization of hematopoietic stem cells into circulation is one example and this phenomenon is particularly important when the harvest of such cells for transplantation is taken into consideration [35]. A binding assay utilizing tracer-labeled recombinant G-CSF and a sensitive PCR method of detecting a specific message for the G-CSF receptor have revealed the expression of the G-CSF receptor in cells other than granulocytic cells. In humans, in vitro-induced megakaryocytes as well as peripheral blood platelets have been shown to express mRNA for the G-CSF receptor [4, 5]. G-CSF receptors on platelets seem to be functioning because G-CSF augments the secondary aggregation of platelets induced by a low concentration of adenosine diphosphate [4]. Identification of the G-CSF receptor mRNA in two megakaryoblastic cell lines as shown in this study further supports that the G-CSF receptor is indeed expressed in the megakaryocytic lineage. The fact that the addition of G-CSF into culture, in combination with other megakaryocytopoietic factors, increases the number of megakaryocyte colonies indicates the possibility of the existence of the G-CSF receptor in megakaryocyte precursors [7, 8]. Although the official conclusion that the functioning receptor for G-CSF is expressed in megakaryocytic lineage must await the biological and the biochemical analysis on the pure population of cells at various stages of differentiation, our results described herein as well as others strongly indicate that G-CSF acts directly on megakaryocytic cells. The mechanism by which G-CSF exerts its effect on megakaryocytic lineage remains unclear at this moment. It is conceivable that G-CSF generates certain signals through the binding to its receptor, but it is possible that G-CSF may bind to the TPO receptor thereby blocking the effect of TPO. A binding inhibition study should disclose this possibility and this assay is now under investigation.
In conclusion, G-CSF alters the DNA polyploidy distribution of mouse bone marrow megakaryocytes when this cytokine is expressed in vivo, and this effect is also reproducible in the state of upregulated megakaryocytopoiesis induced by TPO. Identification of the receptor, for G-CSF in mouse megakaryoblastic cell lines suggests the direct action of G-CSF on megakaryocytic lineage. Further study is clearly necessary to determine the mechanism of these phenomena, but our results demonstrate the potential importance of G-CSF as a regulatory molecule in megakaryocytopoiesis. We are currently investigating whether G-CSF shows similar effects on a megakaryocyte maturation in semisolid culture assay in which other complicated elements can be eliminated. This experiment will disclose whether G-CSF indeed acts on megakaryocyte maturation, especially on the process of megakaryocyte endomitosis.
|
Acknowledgments
|
|---|
We thank Ms. Mie Sone for her excellent secretarial work and Mr. Junzo Sato for his skillful animal care. We also thank Kirin Brewery Co., Ltd. for the distribution of human G-CSF and human TPO, and Dr. H. Sasaki at Yokohama City University School of Medicine for providing us with the L8057 cell line.
This work was supported in part by a Grant for Pediatric Research (6C-01, 6C-05) and a Grant-in-Aid for Cancer Research (5-24), from the Ministry of Health and Welfare, Japan.
|
References
|
|---|
-
Tamura M, Hattori K, Nomura H et al. Induction of neutrophilic granulocytosis in mice by administration of purified human native granulocyte colony-stimulating factor (G-CSF). Biochem Biophys Res Commun 1987;142:454–460.[Medline]
-
Welte K, Bonilla M, Gillio A et al. Recombinant human granulocyte colony-stimulating factor: effects on hematopoiesis in normal and cyclophosphamide-treated primates. J Exp Med 1987;165:941–948.[Abstract/Free Full Text]
-
Duhrsen U, Villeval JL, Boyd J et al. Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 1988;72:2074–2081.[Abstract/Free Full Text]
-
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.
-
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]
-
Bocchietto E, Guglielmetti A, Silvagno F et al. Proliferative and migratory responses of murine microvascular endothelial cells to granulocyte-colony-stimulating factor. J Cell Physiol 1993;155:89–95.[Medline]
-
McNiece I, McGrath H, Quesenberry P. Granulocyte colony-stimulating factor augments in vitro megakaryocyte colony formation by interleukin 3. Exp Hematol 1988;16:807–810.[Medline]
-
Quesenberry P, McGrath H, Williams M et al. Multifactor stimulation of megakaryocytopoiesis: effects of interleukin 6. Exp Hematol 1991;19:35–41.[Medline]
-
Yamada T, Kaneko H, Iizuka K et al. Elevation of lymphocyte and hematopoietic stem cell numbers in mice transgenic for human granulocyte colony-stimulating factor. Lab Invest (in press).
-
Kaneko H, Fujimoto J, Iizuka K et al. Engraftment of rat bone marrow cells in mice transgenic for granulocyte-colony stimulating factor. Transplant Proc 1995;27:1551–1552.[Medline]
-
de Sauvage F, Hass P, Spencer S et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand. Nature 1994;369:533–538.[Medline]
-
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]
-
Bartley T, 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]
-
Jackson C. Cholinestrase as a possible marker for early cells of the megakaryocytic series. Blood 1973;42:413–421.[Abstract/Free Full Text]
-
Takada K, Saito M, Kaneko H et al. Novel monoclonal antibody reactive with thrombin-sensitive 74-kDa glycoproteins present on platelets and megakaryocytes both from mouse and rat. Hybridoma 1995;14:361–367.[Medline]
-
Takebe Y, Seiki M, Fujisawa J et al. SR promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol 1988;8:466–472.[Abstract/Free Full Text]
-
Kajigaya S, Suda T, Suda J et al. Establishment of megakaryoblastic cell lines by coinfection of abelson murine leukemia virus and recombinant SV40-retrovirus. Jpn J Cancer Res 1988;79:726–733.[Medline]
-
Ishida Y, Levin J, Baker G et al. Biological and biochemical characteristics of murine megakaryoblastic cell line L8057. Exp Hematol 1993;21:289–298.[Medline]
-
Hara K, Suda T, Suda J et al. Bipotential murine hematopoietic cell line (NFS-60) that is responsive to IL-3, GM-CSF, G-CSF, and erythropoietin. Exp Hematol 1988;16:256–261.[Medline]
-
Steinman RA, Tweardy DJ. Granulocyte colony-stimulating factor receptor mRNA upregulation is an immediate early marker of myeloid differentiation and exhibits dysfunctional regulation in leukemic cells. Blood 1994;83:119–127.[Abstract/Free Full Text]
-
Maniatis T, Fritsch E, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.
-
Jackson C, Steward S, Chenaille P et al. An analysis of megakaryocytopoiesis in the C3H mouse: an animal model whose megakaryocytes have 32N as the modal DNA class. Blood 1990;76:690–696.[Abstract/Free Full Text]
-
Corash L, Levin J. The relationship between megakaryocyte ploidy and platelet volume in normal and thrombocytopenic C3H mice. Exp Hematol 1990;18:985–989.[Medline]
-
Ito Y, Seto Y, Brannan CI et al. Structural analysis of the functional gene and pseudogene encoding the murine granulocyte colony-stimulating-factor receptor. Eur J Biochem 1994;220:881–891.[Medline]
-
Kaushansky K, Lok S, Holly RD et al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-mpl ligand thrombopoietin. Nature 1994;369:568–571.[Medline]
-
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 vivo. Blood 1995;85:1719–1726.[Abstract/Free Full Text]
-
Metcalf D. Hematopoietic regulators: redundancy or subtlety? Blood 1993;82:3515–3523.[Free Full Text]
-
Chang JM, Metcalf D, Gonda TJ et al. Long-term exposure to retrovirally expressed granulocyte colony-stimulating factor induces a nonneoplastic granulocytic and progenitor cell hyperplasia without tissue damage in mice. J Clin Invest 1989;84:1488–1496.
-
Pojda Z, Tanaka K, Aoki Y et al. In vivo and in vitro interaction between interleukin 6 and granulocyte colony-stimulating factor in the regulation of murine hematopoiesis. Exp Hematol 1992;20:874–878.[Medline]
-
Davis E, Corash L, Baker G et al. Splenic thrombopoiesis after bone marrow ablation with radiostrontium: a murine model. J Lab Clin Med 1990;116:879–888.[Medline]
-
Layendecker S, McDonald T. The relative roles of the spleen and bone marrow in platelet production in mice. Exp Hematol 1982;10:332–342.[Medline]
-
Tanum G, Sonstevold A, Jakobson E. The effect of splenectomy on platelet formation and megakaryocyte DNA content in rats. Blood 1984;63:593–597.[Abstract/Free Full Text]
-
Molineux G, Pojda Z, Dexter TM. A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor. Blood 1990;75:563–569.[Abstract/Free Full Text]
-
Harker L. Kinetics of thrombopoiesis. J Clin Invest 1968;47:458–465.
-
Molineux G, Pojda Z, Hampson IN et al. Transplantation potential of peripheral blood stem cells induced by granulocyte colony-stimulating factor. Blood 1990;76:2153–2158.[Abstract/Free Full Text]
Received August 9, 1995;
Revision received September 11, 1995.
accepted for publication October 23, 1995.
Top
Abstract
Introduction
