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Stem Cells, Vol. 14, No. 6, 651-660, November 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

In Vivo Effects of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor on Hematopoiesis in Normal Mice

Koji Kabaya, Hiromichi Akahori, Kazunori Shibuya, Yuko Nitta, Masumi Ida, Masaru Kusaka, Takashi Kato, Hiroshi Miyazaki

Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Takasaki, Gunma, Japan

Key Words. Megakaryocyte growth and development factor (MGDF) • Thrombopoietin (TPO) • Megakaryocytic progenitor • Megakaryocyte • Platelet

Dr. Hiroshi Miyazaki, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki, Gunma 370-12, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The in vivo effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of recombinant human thrombopoietin modified with polyethylene glycol, were investigated in normal Balb/c mice. PEG-rHuMGDF was more potent in producing platelets and the dose-response curve was steeper compared with the case of the nonpegylated form of this molecule. Five consecutive injections with PEG-rHuMGDF caused a dose-dependent increase in peripheral platelet counts with a peak on day 8. There was a dose-dependent rise in platelet counts on day 8 at daily doses from 0.333 to 30 µg/kg. Intermediate doses of PEG-rHuMGDF (1.111 to 10 µg/kg/day) caused a significant decrease in mean platelet volume, and conversely, higher doses of PEG-rHuMGDF (30 to 270 µg/kg/day) induced a dose-dependent increase in mean platelet volume. There was a dose-dependent decrease in hemoglobin concentration with a minimum on day 8 but no significant reduction in reticulocyte counts following PEG-rHuMGDF administration. White blood cell counts were unchanged by PEG-rHuMGDF treatment. Marrow megakaryocyte size enlarged to 1.5-fold and the number of marrow megakaryocytes increased to sixfold by consecutive administration of PEG-rHuMGDF at 30 µg/kg/day. A twofold increase in the number of marrow megakaryocytic progenitor cells (colony-forming units-megakaryocyte) was also observed. Marrow erythroid progenitor (colony forming units-erythroid) counts decreased by splenic colony-forming units-erythroid, marrow and splenic erythro/myeloid progenitor cell counts, and splenic granulocyte/macrophage progenitor cell counts increased with PEG-rHuMGDF treatment. Marrow and splenic erythroid burst-forming cells were unchanged. These results indicate that PEG-rHuMGDF, a truncated molecule of thrombopoietin, is a potent stimulator for megakaryopoiesis and thrombopoiesis, and also affects the development of other hematopoietic cells in normal mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Megakaryopoiesis leading to platelet production has been thought to be regulated by a lineage-specific humoral factor, termed thrombopoietin (TPO). Recently, several groups including us reported the identification, purification and cloning of TPO also called c-Mpl ligand [1-6]. We purified rat TPO from the plasma of irradiated rats by using a quantitative in vitro assay to measure the production of megakaryocytes from a GPIIb/IIIa+ population of rat megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), and based on the amino acid sequence information, we cloned the rat TPO cDNA [3, 7, 8]. Subsequently, we cloned human TPO cDNA from human liver cDNA library and human genomic DNA [9]. The expression of TPO mRNA was highest in the liver among various rat tissues tested, and TPO activity was produced from the rat hepatoma-derived cell line [10].

Recent in vitro studies regarding the biologic action of TPO have shown that TPO directly supports the formation of megakaryocyte colonies from murine bone marrow cells and human CD34+ cells in semisolid culture [11-14]. TPO also acts as a potent maturation factor, since megakaryocytes generated with TPO in liquid culture exhibit markedly increased ploidy and well-developed demarcation membranes [12-15].

Administration of murine TPO into mice stimulates a sevenfold increase in circulating platelet counts and marked expansion of the marrow megakaryocyte mass and the CFU-MK pool in the hematopoietic tissues [5, 16, 17]. In normal non-human primates, human TPO is also a potent stimulator for platelet production and significantly increases the frequency of marrow CFU-MK and multipotential progenitor cells (colony-forming units-granulocyte/erythrocyte/macrophage /megakaryocyte [CFU-GEMM]) [18]. Moreover, TPO enhances the erythroid-burst colony formation in the presence of erythropoietin (EPO) in vitro [19], and promotes the erythropoietic recovery in myelosuppressed mice [17].

In the present study, we further evaluated the action of TPO on normal hematopoiesis including megakaryocyte and platelet production in mice using pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated TPO modified with polyethylene glycol.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Mice
Male Balb/c mice, 9 weeks old, were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were housed in autoclaved cages and were maintained in an air-conditioned, specific pathogen-free animal room regulated at a temperature of 21°-23°C and relative humidity of 50%-60%. The lighting cycle was 12/12 hours beginning from 8:00 a.m. The mice were given sterilized commercial rodent chow and water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of our laboratory.

Administration of PEG-rHuMGDF
A truncated molecule of human TPO [1, 3] expressed in E. coli was purified to homogeneity, and this truncated peptide was modified by the covalent attachment of polyethylene glycol and purified further. This derivative is designated as PEG-rHuMGDF. PEG-rHuMGDF was diluted with acetate buffered solution containing 0.02% syngeneic mouse serum (vehicle) and was injected into mice s.c. in a volume of 4 ml/kg for five consecutive days (days 1 through 5). The same volume of vehicle solution alone was injected into mice as a control.

Hematological Analysis
About 70 µl of peripheral blood was obtained from the retro-orbital plexus of an individual mouse using 75 mm heparinized capillary tubes (Funakoshi Pharmaceutical Co.; Tokyo, Japan) for measuring the hematological parameters. Platelet count, mean platelet volume (MPV), hemoglobin concentration, and white blood cell (WBC) count were determined using an automatic microcell counter K-2000 (Toa Medical Electronics; Kobe, Japan). Smears of blood cells stained with New Methylene Blue were prepared and the percentage of reticulocytes in the total red blood cell population determined using a blood cell analyzer (MICROX HEG-70A, Omuron; Tokyo, Japan).

Progenitor Cell Assay
Bone marrow cells were collected aseptically from the femurs of mice and suspended in the {alpha}-medium (Flow Laboratories; McLean, VA). The spleen was also removed aseptically and then dissociated to give single-cell suspensions. CFU-MK cultures were performed according to the method previously described by Miyazaki et al. [20] with minor modifications. Briefly, 2 x 105 bone marrow cells were cultured in 1 ml of 0.3% Noble agar (Difco; Detroit, MI) containing Iscove's modified Dulbecco's medium (Sigma; St. Louis, MO) supplemented 10% fetal calf serum (GIBCO; Grand Island, NY), 2 mM glutamine, 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol (MERCK; Darmstadt, Germany) in the presence of 50 ng recombinant mouse interleukin 3 (mIL-3; Kirin Brewery Co., Ltd.; Tokyo, Japan), in a 35 mm tissue culture dish (Nunc; Naperville, IL). After seven days of culture, agar disks were detached from the culture dishes and placed onto glass slides and stained with acetylcholinesterase according to the method described by Jackson [21]. Megakaryocyte colonies comprising three or more cells were counted as CFU-MK-derived colonies.

Cultures of colony-forming units of erythroid (CFU-E), BFU-E, mixed erythroid colony-forming cells (E-Mix), and colony-forming units of granulocyte/macrophage (CFU-GM) were performed by a modified methylcellulose method as reported by Iscove et al. [22]. Briefly, constant numbers of bone marrow cells or spleen cells were added to the {alpha}-medium containing 0.88% methylcellulose (Shinetsu Scientific Inc.; Tokyo, Japan), 30% fetal calf-serum, 1% bovine serum albumin (Sigma), 5 x 10–5 M 2-mercaptoethanol and 2 IU rHuEPO (ESPO®, Kirin Brewery Co., Ltd.) to support CFU-E colony formation. To support E-Mix and CFU-GM colony formation, 16 IU rHuEPO and 10 ng mIL-3 were added to the culture. Colony counts were performed using an inverted microscope. After two days of culture in an incubator, hemoglobinized colonies of eight or more cells were counted as CFU-E. After seven days of culture, mixed erythroid colonies and pure erythroid burst colonies (BFU-E) were counted as E-Mixes, and colonies of more than 50 cells consisting of granulocytes and/or macrophages were scored as CFU-GM.

Measurements of the Number and Size of Megakaryocytes in the Femur
Formalin-fixed paraffin-embedded decalcified femurs were sectioned longitudinally and stained with hematoxylin-eosin. The number of megakaryocytes per six randomly chosen 400x field in the femur section was counted using a light microscope. To measure the megakaryocyte size, morphometric analysis was performed with a VIDAS image analyzer composed of a light microscope unit (Carl Zeiss; Oberkochen, Germany). A total of 150 megakaryocytes in each group was analyzed.

Statistical Analysis
Measured values are shown as the mean ± SE. Statistical significance of differences in hematological values between the vehicle-and PEG rHuMGDF-treated groups was assessed by Dunnett's test. The statistical significance of differences in progenitor cell counts examined in this study was assessed by Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Effects of PEG-rHuMGDF on Peripheral Blood Cell Counts
We first compared the effects of pegylated and nonpegylated forms of rHuMGDF on peripheral platelet counts (Fig. 1Go). Administration of PEG-rHuMGDF yielded a large number of platelets than the nonpegylated molecule at the same respective doses. Linear regression formulas were Y = 2254.1 x log X + 1913.4 (r = 0.964 for PEG-rHuMGDF and Y= 691.9 log X + 1078.5 (r = 0.985) for rHuMGDF. These data indicate that PEG-rHuMGDF has more potent platelet increasing effect than rHuMGDF. Five consecutive s.c. administrations with PEG-rHuMGDF into normal mice induced a dose-dependent thrombocytosis with a peak on day 8 (Fig. 2Go). Intravenous injections of PEG-rHuMGDF also had dose-dependent platelet-increasing effects, but they were less potent than s.c. treatment (data not shown). In mice injected with PEG-rHuMGDF at daily doses from 0.0046 to 270 µg/kg/day (increasing in a common ratio of three) for five consecutive days, there was a linear relationship between platelet counts and doses of PEG-rHuMGDF in the range of 0.333 to 30 µg/kg/day (Fig. 3AGo). Treatment with PEG-rHuMGDF at intermediate doses ranging from 1.111 to 10 µg/kg/day caused a decrease in MPV but stimulated a dose-dependent increase in MPV at higher doses ranging from 30 to 270 µg/kg/day. (Fig. 3BGo).



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  		Figure 1. Comparison of the nonpegylated recombinant human megakaryocyte growth and development factor (rHuMGDF) (•) and pegylated (PEG) rHuMGDF ({bigcirc}) on platelet counts in mice. The formulas of two regression lines are Y = 2254.1 log X + 1913.4 (r = 0.964) and Y = 691.9 log X + 1078.5 (r = 0.985) for mice treated with rHuMGDF and PEG-rHuMGDF, respectively. Mice were injected s.c. daily for five consecutive days with various dosages of each form of rHuMGDF. Blood samples were obtained three days after the last injection of each form of rHuMGDF. Each point represents the mean ± SE from six mice. The significance of differences (**p < 0.01) as compared with mice infected with vehicle solution was tested by Dunnett's test, and differences between rHuMGDF and PEG-rHuMGDF at the same dosage were represented as #p < 0.05: ##p < 0.01 determined by Student's t-test.

 


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  		Figure 2. Effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet counts in mice. Mice received s.c. PEG-rHuMGDF at doses of 1 ({blacktriangleup}), 5({blacksquare}) and 30 µg/kg/day ({square}) or vehicle solution ({bigcirc}) for five consecutive days. Each point represents the mean ± SE from six mice. The significance of differences (*p < 0.01) as compared with mice injected with vehicle solution was tested by Dunnett's test.

 


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  		Figure 3. Dose-response curve for platelet counts [A] and mean platelet volume [B] in PEG-rHuMGDF-treated mice. Mice were administered s.c. with PEG-rHuMGDF at doses from 0.0046 to 270 µg/kg/day or vehicle for five consecutive days. Blood samples were obtained three days after the last injection. Each point represents the mean ± SE from six mice. The significance of differences (*p < 0.05: **p < 0.01) as compared with mice injected with vehicle solution was tested by Dunnett's test.

 
There was no significant change in reticulocyte counts (Fig.4AGo) but there was a dose-dependent decrease in hemoglobin concentration with a minimum on day 8 following the treatment with PEG-rHuMGDF (Fig. 4BGo) No significant change in WBC counts occurred in these mice (data not shown).



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  		Figure 4. Variations in reticulocyte counts and hemoglobin concentration in mice treated with PEG-rHuMGDF. PEG-rHuMGDF was administered s.c. at doses of 1 ({blacksquare}), 3 ({Delta}), 10 ({blacktriangleup}), and 30 µg/kg/day (•) or vehicle ({bigcirc}) for five consecutive days. Each point represents the mean ± SE from six mice. The significance of differences (*p < 0.05: **p < 0.01) as compared with mice injected with vehicle solution was tested by Dunnett's test.

 
Effects of PEG-rHuMGDF on the Size and Number of Megakaryocytes in the Femur
When mice were treated with PEG-rHuMGDF at 30 µg/kg/day for five consecutive days, marrow megakaryocyte size increased by day 3 and reached a maximum of 1.5-fold on days 3 and 6, and conversely, slightly decreased on day 12, compared with vehicle-treated mice (Fig. 5AGo). The number of marrow megakaryocyte counts increased by day 3, markedly on days 6 (around sixfold) and 8 (around fourfold), and returned to a normal level on day 12 (Fig. 5BGo).



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  		Figure 5. Variations in marrow megakaryocyte size (A) and number (B) in mice treated with PEG-rHuMGDF. Mice received s.c. PEG-rHuMGDF at a dose of 30 µg/kg/day ({square}) or vehicle ({blacksquare}) for five consecutive days (days 1 through 5). Each bar represents the mean ± SE from four or five mice. The significance of differences (*p < 0.05: **p < 0.01) as compared with mice infected with vehicle solution was tested by Dunnett's test.

 
Effects of PEG-rHuMGDF on CFU-MK Counts in Hematopoietic Tissues
Following treatment with PEG-rHuMGDF at a dose of 30 µg/kg/day, marrow CFU-MK counts significantly increased by day 3 with a peak on day 6, and decreased thereafter (Fig. 6Go). Splenic CFU-MK numbers increased to 36-and 30-fold on days 6 and 8, respectively (Table 1Go).



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  		Figure 6. Variations in marrow CFU-MK counts of PEG-rHuMGDF-treated mice. Mice received s.c. PEG-rHuMGDF at a dose of 30 µg/kg/day ({square}) or vehicle ({blacksquare}) for five consecutive days (days 1 through 5). Each bar represents the mean ± SE from five mice. The significance of differences (*p < 0.05: **p < 0.01) as compared with mice injected with vehicle solution was tested by Dunnett's test.

 

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  		Table 1. Effects of PEG-rHuMGDF on hematopoietic progenitor cells in the femur and spleen in mice
 
Influence of PEG-rHuMGDF on Other Hematopoietic Progenitor Cells
Marrow CFU-E counts decreased to around 60% of the control on day 6 following daily administration with rHuMGDF, while splenic CFU-E counts increased ninefold (Table 1Go). On day 8, marrow CFU-E counts almost returned to normal value and splenic CFU-E counts were still slightly higher than control (at 1.4-fold) (Table 1Go). Marrow E-Mix counts increased to twofold on day 6. Splenic E-Mixes also increased to 2.5-and 3-fold on days 6 and 8, respectively. However, no change in BFU-E counts was observed in the femur, but a slight decrease in BFU-E counts in the spleen on day 8 was seen. Although marrow CFU-GM counts slightly increased on day 6, splenic CFU-GM counts increased to 5.5-and 4-fold on days 6 and 8, respectively (Table 1Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
PEG treatment techniques have been applied to reduce in antigenicity and/or in total body clearance rate of the proteins in vivo. Previous works showed that PEG-rHuIL-6 [23] and PEG-rHuG-CSF [24-27] were more potent than unmodified molecules due to prolongation of circulating cytokine levels. Therefore, it was expected that pegylation of recombinant human truncated TPO enhanced the in vivo thrombopoietic activity. Actually, PEG-rHuMGDF possessed more potent platelet-increasing effect compared with nonpegylated rHuMGDF in normal mice. Pegylation enhanced the thrombopoietic effect of rHuMGDF to a level at least 30-fold more potent (Fig. 1Go), similarly reported by Hokom et al. [28]. In the present study, we used PEG-rHuMGDF to further clarify the effects of TPO on in vivo hematopoiesis in normal mice.

Consecutive administration of PEG-rHuMGDF induced a dose-dependent thrombocytosis; especially, circulating platelet counts increased fivefold at a daily dose of 30 µg/kg (Fig.2Go). The dosage over 30 µg/kg/day attenuated platelet-increasing effect (Fig. 3AGo). Similar phenomena were observed in nonhuman primates receiving daily consecutive administration of rHuMGDF [18]. Treatment with the higher doses of PEG-rHuMGDF may cause the downregulation of the TPO receptor, c-Mpl, to suppress the progress of megakaryopoiesis, and may also give rise to some negative feedback regulatory mechanisms.

A relationship between the dosage of PEG-rHuMGDF and MPV is of interest. Lower dosages of PEG-rHuMGDF, capable of increasing platelet counts, decreased MPV. A previous report showed that single injection of nonpegylated rHuMGDF to normal mice induced a dose-dependent reduction in MPV [29]. The dose of rHuMGDF that caused a significant decrease in MPV was slightly higher than the total dose of the lower dosage of PEG-rHuMGDF in this study. However, since rHuMGDF is less active than the pegylated from, the total activity of rHuMGDF injected may be similar in both cases. On the other hand, high dosages of this factor increased MPV and the MPV kept rising with dosages over 30 µg/kg/day which gave maximal platelet counts (Fig. 3BGo). The explanation for PEG-rHuMGDF-induced alterations in MPV is unclear at present. However, one possibility is that a moderate stimulation of megakaryopoiesis by low doses of PEG-rHuMGDF may dominantly facilitate the release of platelets from megakaryocytes rather than megakaryocyte maturation in the bone marrow, leading to generation of smaller platelets. High doses of PEG-rHuMGDF may result in progressive maturation of megakaryocytes, including markedly increased nuclear ploidy and cytoplasmic volume with subsequent production of larger platelets. It has been shown that experimentally induced severe thrombocytopenia caused a significant increase in MPV due to stimulated megakaryopoiesis in mice [30].

Marrow megakaryocyte size enlarged to a maximum of 1.5-fold by day 3 with PEG-rHuMGDF injections (Fig 5AGo), and the number of megakaryocytes increased to sixfold on day 6 (Fig 5BGo), indicating that administration of PEG-rHuMGDF increased the marrow megakaryocyte size prior to a promotion of proliferation of megakaryocyte precursors. Recent in vitro studies have shown that TPO markedly increased megakaryocyte ploidy [14, 16, 31]. The mean ploidy of megakaryocytes increased up to threefold in liquid culture containing TPO [13]. A 1.5-fold increase in megakaryocyte size observed with PEG-rHuMGDF treatment may be reflected by progressive nuclear polyploidization of megakaryocytes. These changes in marrow megakaryocytes by the treatment with PEG-rHuMGDF preceded a maximal increase in circulating platelet counts (Fig. 2Go), indicating that these platelets were newly released from megakaryocytes stimulated by PEG-rHuMGDF.

Kobayashi et al. [19] and Kaushansky et al. [17] have shown that TPO enhances proliferation of erythroid progenitors in vitro and in vivo. In the present study, treatment with PEG-rHuMGDF induced a dose-dependent reduction in hemoglobin concentration on day 8, peak day of platelet counts (Fig. 4BGo). Despite a significant decrease in hemoglobin concentration, no reduction in reticulocyte counts was noted (Fig. 4AGo). A decrease in marrow CFU-E number, but an increase in splenic CFU-E number, was observed on day 6, simultaneously (Table 1Go). To determine whether administration of PEG-rHuMGDF suppresses the development of erythroid precursors, we used actual counts of CFU-E in the femur and spleen to compare the calculated total number of CFU-E between control and PEG-rHuMGDF-treated mice, assuming that one femur represents 6% of the total marrow volume [32]. The results showed that there was no statistically significant difference in the calculated mean total number of CFU-E, which are 181,241 ± 15,810 (SE) for control mice and 169,453 ± 18,007 (SE) for PEG-rHuMGDF-treated mice (p = 0.636 determined by Student's t-test). In addition, PEG-rHuMGDF did not affect in vitro CFU-E colony formation stimulated by rHuEPO (data not shown). Taken together, the present results indicate that administration of PEG-rHuMGDF does not suppress the early erythropoiesis involving the development of CFU-E. Further experiments are needed to clarify the mechanism of reduction in hemoglobin concentration.

Previous works have shown that a marked stimulation of granulopoiesis following rHuG-CSF treatment depresses marrow erythropoiesis, such as a decrease in CFU-E counts, and promoted splenic erythropoiesis in mice [33-35]. Similarly, a marked increase in megakaryocyte size and number induced by PEG-rHuMGDF may result in a decrease in marrow CFU-E counts and mobilization of CFU-E to the spleen. It has been reported that administration of rHuG-CSF and rHuEPO mobilized hematopoietic stem and progenitor cells into the circulation and that those cells were applied to a substitute for marrow cell transplantation [36-38]. In the present study, not only megakaryocytic progenitors but also other lineage progenitor cells significantly increased in the spleen following PEG-rHuMGDF treatment (Table 1Go), suggesting that PEG-rHuMGDF may be useful for peripheral blood progenitor cell transplantation.

Administration of PEG-rHuMGDF into normal mice also increased the number of E-Mixes and CFU-GM in the bone marrow and spleen (Table 1Go). Farese et al. have shown that rHuMGDF increases marrow CFU-GEMM population in a nonhuman primate [18]. Recently, Zeigler et al. have reported that cocultivation of a population of stem cells with stromal cells in the presence of TPO stimulates megakaryopoiesis as well as myelopoiesis [39], and Ku et al. have shown that TPO stimulates the proliferation of primitive multipotential progenitors in a combination with stem cell factor and IL-3 [40]. These reports possibly suggest that exogenous PEG-rHuMGDF acts in concert with endogenous IL-3 or stem cell factor to stimulate the expansion of erythroid and granulocyte progenitors. According to a stochastic theory described by Ogawa [41], an increase in CFU-MK by PEG-rHuMGDF administration may induce an expansion of more early progenitor cells, such as CFU-EM [42] and CFU-GMM [43]. An increase in CFU-EM and CFU-GMM may result in an increase in BFU-E and/or CFU-GM. At present, it remains unclear why the peripheral WBC count failed to rise in spite of a marked increase in splenic CFU-GM.

In summary, we have demonstrated that PEG-rHuMGDF, a truncated molecule of human TPO, is a potent stimulator for megakaryocyte development and platelet production, and also influences the development of hematopoietic cells other than megakaryocytic cells in normal mice in vivo.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr. Sinichiro Kato for designation of morphometric analysis program. We also thank Ms. Masako Obuchi, Ms. Miyuki Kato, and Ms. Kazumi Takahashi for their excellent assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received March 11, 1996; accepted for publication July 5, 1996.




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