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Thrombopoietin-Stimulated ex Vivo Expansion of Human Bone Marrow Megakaryocytes

^a Atherosclerosis Program, Rehabilitation Institute of Chicago and Northwestern University, Department of Cell, Molecular and Structural Biology;
^b Department of Neonatology;
^c Department of Pathology;
^d Department of Preventive Medicine, Northwestern University, Chicago, Illinois, USA;
^e University of Minnesota, Department of Laboratory Medicine and Pathology, Minneapolis, Minnesota, USA;
^f Zymogenetics Corporation, Seattle, Washington, USA

Key Words. Thrombopoietin • Megakaryocytopoiesis • Mononuclear cells • CD34⁺ cells • Bone marrow • Cytokines

Dr. Isaac Cohen, Northwestern University Medical School, Rehabilitation Institute of Chicago, 345 East Superior, Chicago, IL 60611, USA.

	Abstract

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The effect of thrombopoietin (TPO) on megakaryocytopoiesis (MKP) has been mainly studied using clonogenic assays in murine systems. In this study, we evaluated MKP in liquid culture using human bone marrow cells. While interleukin 3 (IL-3) and stem cell factor (SCF) are potent activators of TPO-stimulated MKP in the murine system, only IL-3 exhibited synergistic activity with TPO in cultures of human bone marrow. The IL-3 effect on TPO-stimulated megakaryocyte (MK) proliferation, expressed as the absolute number of MKs per seeded CD34⁺ cell, was more pronounced with purified CD34⁺ cells (8 ± 1.6 SE versus 2.8 ± 0.7 SE in the presence and absence of IL-3, respectively) than with mononuclear cells (MNC) (16 ± 2.8 SE versus 11 ± 2.0 SE). This effect of IL-3 on TPO-stimulated MK proliferation was due to a general proliferation of all cell types since the relative frequency of MKs (32.1 ± 3 SE and 55.8 ± 3 SE in MNC and CD34⁺ cells, respectively) was not affected by IL-3. The effect of TPO alone, TPO + IL-3, TPO + SCF, and TPO + IL-3 + SCF on MK proliferation was examined in MNC and CD34⁺ cultures. Greater numbers of MK per seeded CD34⁺ were observed in MNC compared to CD34⁺ cultures under all conditions except when TPO was added with both IL-3 and SCF. The enhancing effect of MNC was also observed on MK ploidy in the presence of TPO and IL-3. While proliferation and ploidy increase with TPO concentration in the murine system, they are inversely related in the human system. A significant 2.5-fold enhancement of TPO-induced MK proliferation was observed when purified CD34⁺ cells were cultured in inserts separated from human bone marrow stroma, indicating that soluble stimulatory factors are released from the stroma. These observations will be useful for ex vivo expansion of MKs to treat post-transplant or chemotherapy-associated thrombocytopenia.

	Introduction

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The recently cloned thrombopoietin (TPO) is a megakaryocyte (MK) lineage-specific growth factor which acts at all levels of megakaryocytopoiesis and thrombopoiesis [1-5]. Its availability will be of considerable importance for the treatment of thrombocytopenias following irradiation and chemotherapy. In view of the slow platelet engraftment following transplant of hematopoietic progenitors [6-8], the use of ex vivo expanded MKs as a transplant supplement could reduce the time for platelet recovery. In murine systems it has been demonstrated that interleukin 3 (IL-3) and stem cell factor (SCF) strongly potentiate the effect of TPO on MK colony formation [9, 10]. In this study, we evaluated MK expansion in vitro in liquid cultures of human bone marrow mononuclear cells (MNC) or purified CD34⁺ cells supplemented with TPO alone or in combination with IL-3 and/or SCF. Since megakaryocytopoiesis is regulated by bone marrow stromal cells [11, 12], we also evaluated the effect of human and murine stroma on TPO-treated CD34⁺ cells.

	Materials and Methods

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Preparation of Low Density Nonadherent MNC
Bone marrow (BM) samples were collected in accordance with the guidelines of the Institutional Review Board on Human Subjects. BM, obtained from the femur of hematologically normal patients having total hip arthroplasty, was collected in a special anticoagulant mixture designed to prevent platelet activation and containing final concentrations of 50 U/ml preservative-free heparin, 1 mM Na₂EDTA, 1 mM adenosine, 2 mM theophylline, 2.2 µM prostaglandin E₁ and 0.1 mg/ml DNase I. Marrow cells were repeatedly extracted from bone fragments with a modified MK medium [13] which consists of Ca²⁺-Mg²⁺-free phosphate-buffered saline (Dulbecco's PBS, GIBCO; Grand Island, NY) containing 13.6 mM Na citrate, 11 mM dextrose, 1 mM theophylline, 1% bovine serum albumin, 2.2 µM PGE₁ and 0.1 mg/ml DNase I. Following homogenization by passage through a 21 gauge needle, low density cells were extracted with the use of Ficoll-Paque as described [14]. Cells resuspended in MK medium were centrifuged at 380 x g through a 10% human serum albumin (HSA) cushion in PBS to reduce platelet contamination. Residual red cells were lysed with NH₄Cl as described [15], and the remaining cells were recovered by centrifugation through a 10% HSA cushion. Adherent cells were discarded following overnight incubation in -thioglycerol-free Iscove's modified Dulbecco's medium (IMDM) containing 10% fetal bovine serum (FBS). All culture media were supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin and incubation was carried out at 37°C in a 5% CO₂ fully humidified atmosphere.

Purification of CD34⁺ Cells
CD34⁺ cells were purified by positive selection using the CD34 magnetic cell sorting Mini-MACS kit (Myltenyi Biotec; Auburn, CA) in accordance with the manufacturer's recommendation.

BM Stroma
Human BM stroma was prepared according to Koller et al. [16]. Essentially, BM MNC were plated in either collagen-free or collagen-coated 6-well plates in McCoy's 5A medium which was supplemented with FBS, horse serum, amino acids, vitamins and hydrocortisone. At confluency, the plates were irradiated with a dose of 12 Gy from a ¹³⁷Cs source (Gammacell 40, Nordion International Inc.; Kanata, Ontario, Canada). The M2-10B4 murine stromal cell line was kindly provided by Dr. Connie Eaves (Terry Fox Laboratory; Vancouver, BC, Canada) and grown over collagen-coated plates. At confluency, the plates were irradiated at 80 Gy. Collagen-coated plates were from Collaborative Research (Becton Dickinson; San Jose, CA).

Culture Conditions
Low density nonadherent MNC and purified CD34⁺ cells were cultured for 12-14 days at 37°C at concentrations of 10⁶ and 6 x 10⁴ cells/ml, respectively, in an IMDM-based medium with 1% HSA and 2.5% normal human serum which was found to be necessary for MK cultures. The concentration of MNC and purified CD34⁺ cells seeded contained equivalent concentrations of CD34⁺ cells. In order to prevent the inhibitory effects on MK growth of transforming growth factor-ß, ß-thromboglobulin and platelet factor 4 released from activated platelets [17-19], the serum was obtained by recalcification of citrated platelet-free plasma. TPO (Zymogenetics Corporation; Seattle, WA) was used at a concentration of 50 U/ml (10 U = TPO quantity which stimulates one-half maximal proliferation of BaF3/mpl cells) which yielded the maximal concentration of MKs. Concentrations of IL-3 and SCF (R&D Systems; Minneapolis, MN), were 0.16 ng/ml (0.4-1.6 units based on one unit = ED50 of TF1 cell line proliferation) and 50 ng/ml, respectively.

The effect of stroma from either human BM or M2-10B4 murine cell line was evaluated by culturing purified CD34⁺ cells in the presence of TPO (100 U/ml) either in direct contact with stroma (contact) or in inserts (Becton Dickinson; Franklin Lakes, NJ) separated from stroma by a 0.4 µm pore membrane (noncontact). In control experiments CD34⁺ cells were cultured in inserts placed over stroma-free wells (stroma-free).

In all cultures, MK proliferation was expressed by the absolute number of CD41a⁺ cells obtained per seeded CD34⁺ cell. The latter was calculated from the relative frequency of CD34⁺ cells determined in MNC and purified CD34⁺ cells.

Cell Labeling and Flow Cytometric Analysis
The relative frequency of MK progenitors was determined at day 1 on MNC and purified CD34⁺ cells. Cell aliquots were treated with 200 pkat chymopapain (Knoll Pharmaceutical Co.; Lincolnshire, IL). This treatment detaches most platelets and platelet fragments from cells which otherwise would stain with the anti-CD41a antibody (anti-GPIIb/IIIa) [20]. After washing, the cells were double-stained with phycoerythrin (PE) conjugated-anti-CD34 (HPCA-2, Becton Dickinson) and fluorescein isothiocyanate (FITC) conjugated anti-CD41a (Immunotech-Amac; Westbrook, ME) and analyzed by flow cytometry. Negative controls were PE-anti-mouse IgG₁ and FITC-anti-mouse IgG₁ used at equivalent IgG₁ concentrations. The relative frequency of mature MKs was determined following a 12-14 day culture by flow cytometric analysis of cells stained with FITC-anti-CD41a.

Flow cytometric analysis was performed using a Coulter Cytometry XL flow cytometer (Coulter; Hialeah, FL). Fluorescence attributable to FITC- and PE-labeled antibodies was excited by an argon laser operating at 488 nm. Emission from fluorescein and PE was measured using bandpass filters of 525 nm and 585 nm, respectively. The percentage of positive cells was corrected by subtracting the percentage of positive cells in the isotope-stained control within the same integration region.

Ploidy was determined by a modification of a procedure described by Tomer et al. [13]. Essentially, cells were labeled with FITC-anti-CD41a then fixed in 0.5% paraformaldehyde. DNA was then stained with propidium iodide in the presence of Triton X-100 for cell permeabilization. This was followed by RNA digestion. Samples were analyzed gating on peak versus integral red fluorescence (488 nm laser blocking filter, 600 nm dichroic, 635 nm bandpass filter) to exclude doublets. Green fluorescence was defined by a 525 nm bandpass filter. Ploidy was expressed by the ploidy index, defined as %4N + %8N + %16N + %32N/%2N.

In some experiments, aliquots of cultured cells were cytospun onto slides, fixed with methanol and stained with a mixture of anti-Ib and anti-IIb (Immunotech-Amac). Antibody binding was revealed with rhodamine-conjugated anti-mouse IgG F(ab)'₂ fragments (Immunotech-Amac).

Electron Microscopy
MK suspensions, washed with MK medium, were fixed in 3% glutaraldehyde in White's buffer [21]. Following washing in phosphate buffer, pellets were suspended in 1% osmium tetroxide in phosphate buffer for 30 min at 0°C. The samples were then dehydrated in a graded series of ethanol concentrations, then treated with propylene oxide and embedded in Epon 812. The sections were stained with uranyl acetate and lead citrate to enhance contrast. Examination was carried out in a Philips (Mahxah, NJ) 301 electron microscope.

Statistical Analysis
The results are expressed as the mean ± SE. Differences were evaluated using the Student's paired t-test, and p values <0.05 were considered statistically significant.

	Results

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Determination of MK Progenitors in Purified CD34⁺ Cells
A recovery of about 60% CD34⁺ cells was obtained with a purity of 86.4% ± 1.5 SE (n = 6) on the basis of flow cytometric analysis following staining with PE-anti-CD34 (HPCA-2). The relative frequency of MK progenitors staining with both anti-CD34 and anti-CD41a was 5.8% ± 0.4 SE (n = 6) (Fig. 1). In Figure 1, a representative flow cytometric pattern showed co-expression of CD34 and CD41a antigens in 4.3% of cells.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Representative flow cytometric analysis of coexpression of CD34 and CD41a in purified CD34+ cells. Left, CD34-PE versus side scatter (SS) of the cell population, over 90% of the gated cells express CD34+; Middle, CD34-PE versus isotypic control; Right, two-color analysis of the gated CD34+ population (CD34-PE versus CD41a-FITC) showing 4.3% double-stained cells.

View larger version (209K): [in this window] [in a new window]   Fig. 2. Ultrastructure of bone marrow megakaryocytes cultured with TPO. Normal distribution of -granules and demarcation membranes is observed. Magnified x 640.

View larger version (168K): [in this window] [in a new window]   Fig. 3. A) Phase contrast microscopy of cultured megakaryocytes showing production of platelet-like elements; B) same field after staining with a mixture of anti-Ib and anti-IIb/rhodamine.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effect of different TPO concentrations on MK proliferation and ploidy in MNC cultures. () proliferation expressed by absolute number of MKs/seeded CD34+ cells; () ploidy index (%4N + %8N + %16N + %32N/%2N). Inverse relationship is observed between proliferation and ploidy. Each value represents the mean ± SE of seven to nine separate experiments. p < 0.005 for 1 and 10 U/ml of TPO versus 50 U/ml TPO for both MK/CD34 and ploidy.

View larger version (13K): [in this window] [in a new window]   Fig. 5. Effect of TPO, IL-3 and SCF on MK proliferation in cultures of MNC and CD34+ cells. Black bars, MNC; Empty bars, CD34+ cells. Each value represents the mean ± SE of seven separate experiments. *p < 0.05 when comparing MNC with CD34+ cells.

The potentiating effect of IL-3 and SCF on TPO-stimulated nuclear maturation of MKs was analyzed by ploidy measurements. IL-3 and SCF did not alter TPO-stimulated ploidy of MNC or CD34⁺ cells. On the other hand, a statistically significant higher ploidy was obtained in MNC than in purified CD34⁺ cells when IL-3 was used in combination with either TPO or TPO and SCF (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effect of TPO, IL-3 and SCF on MK ploidy in cultures of MNC and CD34+ cells. Black bars, MNC; Empty bars, CD34+ cells. Mean (± SE) number of ploidy index (%4N + %8N + %16N + %32N/%2N). Each value represents the mean ± SE of eight separate experiments. *p < 0.05 when comparing MNC with CD34+ cells.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Effect of human and murine stroma on TPO-stimulated CD34+ cells. Black bars, human stroma; Empty bars, M2-10B4 murine stroma cell line. Mean (± SE) number of CD41a+ cells per seeded CD34+ cell by study condition. For human stroma, p = 0.04 for "stroma-free" versus "noncontact."

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The effect of IL-3 and SCF on TPO-stimulated human megakaryocytopoiesis in BM cultures of MNC and CD34⁺ cells was different than in the murine system [9, 10]. In the human system, while IL-3 synergized with TPO to stimulate megakaryocytopoiesis in cultures of both MNC and purified CD34⁺ cells, the effect of IL-3 was more potent in purified CD34⁺ cells than in MNC. Higher numbers of MKs per seeded CD34⁺ cells in cultured MNC compared to cultured CD34⁺ cells were obtained with TPO alone or supplemented with IL-3 or SCF but not when TPO was added with both IL-3 and SCF. Since MNC and purified CD34⁺ cells were seeded at equivalent concentrations of CD34⁺ cells, the enhancing effect of MNC could be due to the contribution of additional growth factors released by accessory cells. The relative frequency of MKs in TPO-containing cultures was not affected by IL-3. Therefore, the increase of the absolute number of MKs per seeded CD34⁺ cells in the presence of IL-3 was due to a general proliferation of all cell types. No effect of SCF was observed when used in combination with TPO in cultures of either MNC or purified CD34⁺ cells. A possible explanation is that TPO targets the same population of early progenitors as SCF. In this respect, Methia et al. [23] reported the expression of c-mpl receptor mRNA in the early CD34⁺/CD38^– stem cell population. When SCF was used in combination with TPO and IL-3 in MNC cultures, the potentiating effect of IL-3 on TPO-stimulated megakaryocytopoiesis was abolished. This effect, which was not observed in cultures of purified CD34⁺ cells, may be due to an as yet undetermined action of SCF on accessory cells present in the MNC population. In the murine system, unlike the human system, SCF synergized with TPO to stimulate proliferation of early and late MK progenitors whereas IL-3 and TPO had additive and synergistic effects when tested on late and early MK progenitors, respectively [9, 10].

The effect of IL-3 and SCF on TPO-stimulated MK differentiation has not been reported in the murine system. In human BM, IL-3 used in combination with TPO, in the presence or absence of SCF, induced a higher ploidy in MNC than in purified CD34⁺ cells. This points to a potentiating activity of accessory cells not only on MK proliferation but also on their differentiation.

The effect of marrow environment on megakaryocytopoiesis has been recently reported. Although macrophages exhibited an overall stimulatory effect on megakaryocytopoiesis due to their secretion of numerous cytokines [24], they inhibited colony-forming unit-MK growth in amegakaryocytic thrombocytopenia [25]. While unactivated human BM microvascular endothelial cells induced adherence of human CD34⁺ cells and MKs [26], activated human umbilical vein endothelial cells induced adherence of human MKs and increased MK maturation without affecting proliferation [11]. On the other hand, human fibroblasts induce MK proliferation without affecting their maturation [12]. In our study, the overall effect of marrow environment from human stroma on cultures of purified CD34⁺ cells pointed to a synergistic proliferative effect with TPO due to the release of soluble factor(s). When CD34⁺ cells were cocultured in physical contact with human BM stroma a large proportion of MKs adhered to stroma cells. The effect of stroma on proliferation and differentiation of these adherent cells is yet to be determined. As another possible source of stroma we evaluated the effect of a murine stromal cell line. Although four out of five cultures of CD34⁺ cells elicited more MKs in the "noncontact" as compared to the "stroma-free" conditions, no statistical difference in MK growth was found upon comparing the two conditions. This could be due to the great variability found from one marrow sample to another.

In conclusion, our studies show that a different pattern emerges when comparing murine and human megakaryocytopoiesis. This may be related to different frequencies of progenitor populations as well as different architecture of cytokine receptors. Due to the presence of accessory cells, MNC appear more efficient in producing MKs than purified CD34⁺ cells. Nonetheless, purified CD34⁺ cells should be considered for ex vivo expansion in breast cancer or neuroblastoma clinical trials since malignant cells in these disorders may be readily separated from CD34⁺ cells [27-29]. The potentiating effect of IL-3 on MK progenitors is of particular interest in view of its effect in supporting a basal level of megakaryocytopoiesis in the absence of TPO [22]. Furthermore, the efficiency of CD34⁺ cells in producing MKs might be enhanced with the use of conditioned medium obtained from autologous irradiated stroma. In summary, the results reported herein should facilitate the development of ex vivo MK expansion for providing platelet support for transplant and chemotherapy-related thrombocytopenic patients.

	Acknowledgments

 
This work was supported in part by a grant from the U.S. Army Medical Research and Materiel Command (DAMD17-94-J-4465) and the Rehabilitation Institute of Chicago. We thank Dr. Richard L. Wixson for the availability of bone marrow samples and Ms. Linda Pessis for administrative help.

	Footnotes

 
Provisionally accepted September 28, 1995.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schattner, M. Articles by Cohen, I. Search for Related Content PubMed PubMed Citation Articles by Schattner, M. Articles by Cohen, I.