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Stem Cells 2002;20:73-79 www.StemCells.com
© 2002 AlphaMed Press

Decrease in Apoptosis and Increase in Polyploidization of Megakaryocytes by Stem Cell Factor During Ex Vivo Expansion of Human Cord Blood CD34+ Cells Using Thrombopoietin

Jeong-Hae Kiea, Woo-Ick Yanga, Mi-Kyung Leeb, Tae-Jung Kwonc, Yoo-Hong Mind, Hyun-Ok Kime, Hyo-Seop Ahnf, Seock-Ah Img, Hyung-Lae Kimh, Hae-Young Parkh, Kyung-Ha Ryui, Wha-Soon Chungj, Myeong-Heon Shink, Yu-Jin Jungl, So-Youn Wool, Hae-Kyung Parkl, Ju-Young Seohl

a Department of Pathology,
d Internal Medicine,
e Clinical Pathology, College of Medicine, Yonsei University, Seoul, Korea;
b Department of Pathology, National Health Insurance Cooperation Ilsan Hospital, Koyang, Korea;
c Department of Forensic Pathology, National Institute of Scientific Investigation, Seoul, Korea;
f Department of Pediatrics, Seoul National University College of Medicine, Seoul, Korea;
l Department of Microbiology,
g Hematology-Oncology,
h Biochemistry,
i Pediatrics,
j Clinical Pathology,
k Parasitology, Medical Research Center, College of Medicine, Ewha Womans University, Seoul, Korea

Key Words. Cord blood • Megakaryocyte • Thrombopoietin • Stem cell factor • Ex vivo expansion • Apoptosis

Ju-Young Seoh, M.D., Ph.D., Department of Microbiology, College of Medicine, Ewha Womans University, Mok-6-Dong 911-1, Yangchon-Gu, Seoul 158-710, Korea. Telephone: 822-650-5738; Fax: 822-653-8891; e-mail: jyseoh{at}mm.ewha.ac.kr


   ABSTRACT
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thrombopoietin (TPO) is widely used for ex vivo expansion of hematopoietic stem cells. Previously, we have reported that TPO induces a characteristic pattern of apoptosis, and the TPO-induced apoptosis is closely associated with megakaryocyte (MK) differentiation. In the present study, several cytokines, flt3-ligand, stem cell factor (SCF), interleukin-3 (IL-3), IL-6, IL-11, leukemia inhibitory factor, G-CSF, and erythropoietin, which are known to affect megakaryocytopoiesis, have been evaluated to elucidate their effects on the TPO-induced apoptosis.

Measurement of apoptosis by flow cytometry revealed that only SCF absolutely reduced the TPO-induced apoptosis in MK fractions, particularly in the late phase of ex vivo expansion. Platelet production was demonstrated by electron microscopy in a later phase when SCF was added. Simultaneous measurement of DNA contents with immunophenotyping demonstrated a significant increase in polyploidization in the CD41+ cell fraction when cultured with SCF. These results suggested that SCF not only inhibited premature senescence but also enhanced maturation of the differentiating cells of MK lineage during ex vivo expansion using TPO.


   INTRODUCTION
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thrombopoietin (TPO) is the primary regulator of megakaryocytopoiesis and thrombopoiesis. TPO supports the differentiation and proliferation of megakaryocyte (MK) progenitor cells and has an essential role for the complete maturation of MKs [1–5]. TPO also supports the formation and function of platelets in vitro [6,7]. Beside its MK-lineage specific effects, several lines of evidence have demonstrated that TPO has an important role in early hematopoiesis. TPO alone or in combination with other early-acting cytokines, such as stem cell factor (SCF), flt-3 ligand (FL), or interleukin-3 (IL-3), supports the proliferation of primitive hematopoietic progenitor cells (HPCs) in vitro [8–10]. The receptor for TPO, c-Mpl, is expressed on the primitive hematopoietic stem cells, and stem cell activity has been found to segregate with c-Mpl expression [11]. More direct evidence for the regulatory role of TPO in early hematopoiesis has been provided by TPO-deficient mice and c-mpl-deficient mice [12,13]. In these mice, multilineage deficiencies were observed as well as the megakaryocytic lineage-specific deficiency. However, results from clinical trials of TPO in human subjects have raised several concerns including the potential of TPO to induce neutralizing antibodies [14] and to give rise to such untoward reactions as induction of leukemic cell proliferation [15] or myelofibrosis [16].

At present, TPO is one of the most popular cytokines used for ex vivo expansion of cord blood (CB) HPCs [17]. In particular, Piacibello et al. demonstrated that the simple combination of TPO and FL was capable of sustaining proliferation of CB HPCs for more than 6 months [18], and that the expanded cells were capable of repopulating nonobese diabetic/severe combined immunodeficient mice [19]. Using TPO and FL, we have also investigated myeloid differentiation from CB CD34+ cells [20]. We have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human CB CD34+ cells [21] and that the TPO-induced apoptotic cells belong to MK lineage [22]. In the present study, several cytokines, including FL, SCF, IL-3, IL-6, IL-11, leukemia inhibitory factor (LIF), G-CSF, and erythropoietin (EPO), which are known to affect megakaryocytopoiesis [23,24], have been evaluated to elucidate their effects on the TPO-induced apoptosis. Of the cytokines evaluated, FL, IL-3, G-CSF, and EPO significantly reduced the TPO-induced apoptotic fractions, albeit due to a relative increase in all cell fractions other than those of MK lineage. By contrast, SCF reduced the TPO-induced apoptotic fractions absolutely. Furthermore, SCF significantly increased polyploidization of MK fractions, implying the enhancing effect of SCF for MK maturation.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification of CB CD34+ Cells and Ex Vivo Expansion
CD34+ cells were purified using Ficoll-Hypaque (density, 1.077; Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com) density centrifugation and immunomagnetic (Miltenyi Biotec; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com) selection, as previously described [20]. The efficiency of purification, as verified by flow cytometry counterstaining with an anti-human CD34 antibody conjugated with fluoroscein isothiocyanate (FITC; HPCA-2; Becton Dickinson; Mountain View, CA; http://www.bd.com) was consistently more than 95%.

The purified cells were cultured at a density of 1.0 x 105 cells/ml in serum-free essential media supplemented with bovine serum albumin, insulin, and transferrin (StemCell Technologies; Vancouver, Canada; http://www.stemcell.com). Cultures were stimulated with TPO (50 ng/ml) alone or in combination with FL (50 ng/ml), SCF (50 ng/ml), IL-3 (100 ng/ml), IL-6 (10 ng/ml), IL-11 (20 ng/ml), G-CSF (100 ng/ml), LIF (10 ng/ml), and EPO (2 U/ml). TPO was pegylated truncated E. coli-produced form, and all of the above cytokines were recombinant human cytokines. TPO, SCF, and EPO were gifts from Kirin Brewery (Maebashi, Japan;http://www1.kirin.co.jp/english/r_d/pha/index.html). FL, IL-3, IL-11, and G-CSF were purchased from Chemicon (Temecula, CA; http://www.chemicon.com) while IL-6 and LIF were purchased from Endogen (Woburn, MA; http://www.endogen.com). Twice a week, the cells were demipopulated by removing half of the culture volume, which was replaced by fresh medium and growth factors. The cells were cultured for up to 4 weeks.

Measurement of Apoptosis
Multidimensional flow cytometry was employed for simultaneous measurement of apoptosis and concurrent immunophenotyping. Apoptosis was measured by staining cells with FITC- or phycoerythrin (PE)-conjugated annexin V (Pharmingen; San Diego, CA; http://www.pharmingen.com). FITC-anti-human CD41 (DAKO; Copenhagen, Denmark; http://www.dako.dk) or peridinin chlorophyll protein (PerCP)-anti-human CD61 (Becton Dickinson) antibodies were used for staining MK markers. The negative control consisted of incubation with isotype-matched irrelevant antibodies. Samples were analyzed on a FACSCalibur flow cytometer (Becton Dickinson). Ten thousand events were acquired for each analysis, and data were analyzed using CellQuest (Becton Dickinson) software.

Megakaryocyte Ploidy
Simultaneous measurement of DNA contents and surface immunophenotyping was employed to investigate DNA ploidy in the cells differentiating into MK lineage [25]. For the identification of MK fractions, cells were labeled with an FITC-anti-CD41 antibody (DAKO). The surface-stained cells were washed in cold phosphate-buffered saline (PBS), fixed and permeabilized in PBS containing 1% paraformaldehyde (PFA; Merck; Darmstadt, Germany; http://www.merck.com) and 0.05% Nonidet P40 (NP40; British Drug House Ltd.; Dorset, UK) by overnight incubation on a rocking platform at 4°C. On the following day, PFA and NP40 were removed by washing the cells in PBS containing 1% glycine. Then, DNA was stained by resuspending the cells in 10 µg/ml 7-amino-actinomycin D (Sigma; St. Louis, MO; http://www.sigma-aldrich.com) in PBS containing 100 µg/ml RNase (Sigma) for 90 minutes at 4°C in the dark. Samples were analyzed within 2 hours using a FACSCalibur flow cytometer.

Electron Microscopy
Cells were quickly washed in 0.1 M PBS (pH 7.3), sedimented at 1,200 rpm, and immediately fixed in 0.125 M HEPES buffer (pH 7.2) containing 4% PFA and 2% glutaraldehyde for 2 hours at room temperature. After being washed in HEPES buffer, the cells were post-fixed with 1% OsO4 (pH 7.2 in HEPES buffer) for 2 hours, alcohol dehydrated, and embedded in Embed. Thin sections were collected on nickel grids and stained with uranyl acetate and lead citrate. The prepared samples were observed under a Philips CM 10 transmission electron microscope.

Statistical Analysis
Data are presented as mean ± standard deviation of four to six separate experiments. Statistical significance was determined by using the Student's t-test. All comparisons were two-tailed, and a p value of less than 0.05 was considered significant.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Cytokines on the TPO-Induced Apoptosis
Of the cytokines tested, FL, SCF, IL-3, EPO, and G-CSF significantly reduced the proportions of TPO-induced apoptotic fractions (Table 1Go). However, this decrease in the apoptotic fractions was due to an increase in the cell fractions other than those of MK lineage as well as an increase in total cell numbers. Except SCF, the TPO-induced apoptosis in the MK fractions was not influenced by any of the above-mentioned cytokines. Only SCF significantly reduced the proportions of apoptotic cells within MK fractions, particularly at the late phase of ex vivo expansion (Fig. 1Go).


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  		Table 1. Apoptosis, cellularity, and megakaryocytic differentiation according to the type of cytokines during ex vivo expansion of human cord blood CD34+ cells
 


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  		Figure 1. Proportions of apoptotic cells within the megakaryocyte (MK) fractions. CD34+cells purified from cord blood were cultured in serum-free conditions with thrombopoietin (T; 50 ng/ml) alone or in combination with stem cell factor (S; 50 ng/ml). The cells were stained with PerCP-anti-CD61 antibody and FITC-annexin V. CD61+fractions were gated to analyze apoptotic fraction determined by staining with annexin V within MK fractions. The proportions of apoptotic cells within MK fractions were significantly different (*p <0.05) at late phase of ex vivo expansion, from day 12 to day 18, between the cultures in the presence and absence of SCF.

 
Effects of the Cytokines on the Polyploidization in MK Fraction
Simultaneous measurement of DNA contents and immunophenotyping revealed that the majority of the MK fractions induced by TPO remained within low ploidy (<=8N) until day 14 (Table 2Go). When SCF was added, the polyploidization of the MK fractions was significantly increased. However, the other cytokines induced no significant change in the polyploidization of the MK fractions (data not shown).


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  		Table 2. Ploid distribution of CD41+ fractions derived from cord blood CD34+ cells by stimulation with TPO alone or in combination with SCF
 
Ultrastructural Analysis
In electron microscopic observation of the expanded cells with TPO, platelet demarcation channels and electron-dense granules began to be observed from day 7 (data not shown). On day 11, the cellularity was sparse and morphologic features of apoptosis, such as peripheral chromatin margination, cytoplasmic vacuolation, and hydration, were observed in those cells showing the morphologic characteristics of MKs (Fig. 2AGo). Apoptotic changes in MKs became more intense from day 14 (Fig. 2BGo), and almost all cells with the morphologic features of MKs had disappeared by day 18 (data not shown).







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  		Figure 2. Electron micrographs of the ex vivo expanded cells. (A, B) Cord blood CD34+cells were cultured in serum-free conditions with thrombopoietin (TPO; 50 ng/ml) for 11 days. A) The cellularity is sparse and some cells with demarcation membranes, dense core granules, and blocked glycogen granules show apoptotic features such as segregated chromatin, condensed chromatin, and cytoplasmic hydration (bar = 10 µm). B) An advanced apoptotic megakaryocyte (MK) with residual demarcation membrane is observed on day 14 (bar = 5 µm). C-E) Cord blood CD34+cells cultured for 11 days with TPO (50 ng/ml) and stem cell factor (50 ng/ml). C) The cellularity is dense and many cells showing multinucleation with demarcation membranes, dense core granules, and blocked glycogen granules are observed (bar = 10 µm). D) An MK showing well-developed demarcation membranes, abundant dense core granules, and blocked glycogen granules is observed on day 18 (bar = 5 µm). E) A platelet with microfilament, dense core granules, and open canalicular system is also observed on day 18 (bar = 1 µm).

 
In the cultures where SCF was added, the cellularity was dense on and beyond day 11, and many cells showing multinucleation with demarcation membranes, dense core granules, and blocked glycogen granules, were observed (Fig. 2CGo). Platelet demarcation systems and produced platelets, in which microfilaments, granules, and open cisternae were identified, could still be demonstrated until day 18 (Figs. 2D, 2EGo).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
During ex vivo expansion of CB CD34+ cells, TPO induces a characteristic pattern of apoptosis in MK lineage, which is closely associated with downregulation of CD44 and very late activation antigen [21,26]. In the present study, addition of FL, SCF, IL-3, IL-6, IL-11, G-CSF, LIF, and/or EPO did not change the characteristic pattern of apoptosis induced by TPO (data not shown). The quantitatively measured values of apoptotic fractions were somewhat different according to the methods of measurement [27]. When the TPO-induced apoptosis was measured by flow cytometry after staining the cells with annexin V, it reached a peak level at day 14 [22]. Although FL, IL-3, EPO, or G-CSF significantly reduced the TPO-induced apoptotic fractions, the apoptotic fractions within MK fractions were not significantly changed, implying that the decrease in apoptosis was due to the relative increase in cells other than MK lineage (Table 1Go). Only SCF significantly reduced the apoptotic fractions within MK fractions, particularly at the late phase of MK maturation (Fig. 1Go), suggesting that SCF delayed the TPO-induced apoptosis in MK fractions. Furthermore, SCF increased polyploidization of MKs, suggesting an enhancing effect of SCF on MK maturation (Table 2Go). Electron microscopic examination also supported the idea that SCF inhibited apoptosis and enhanced maturation of MKs by showing that live MKs with platelet demarcation systems (Fig. 2DGo) and morphologically intact platelets (Fig. 2EGo) could be demonstrated later in the cultures with SCF. These results may explain the synergistic effect of SCF with TPO for expansion of MKs [28].

By day 14, when MK development as well as apoptosis exceeded their peaks during ex vivo expansion of CB CD34+ cells using TPO, the DNA contents of MK fractions remained within low ploidy (<=8N). This result is comparable with the findings noted by others [28,29], suggesting that when stimulated by TPO, MK progenitors in CB undergo high proliferative responses instead of being matured to hyperploid MKs. Miyazaki et al. also demonstrated inferior capability of CB to generate mature MKs, compared with bone marrow [30]. In this regard, the major limit for in vitro production of platelets by ex vivo expansion of CB CD34+ cells may be the maturation stage of MKs. The results of the present study suggest that addition of SCF would be a means of enhancing maturation of MKs derived from CB CD34+ cells.

Our results suggest that SCF not only inhibits premature senescence but also enhances maturation of MKs during ex vivo expansion of human CB CD34+ cells. Taken together, SCF may be useful for in vitro production of MKs and platelets by ex vivo expansion of human CB CD34+ cells using TPO.


   ACKNOWLEDGMENT
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors appreciate Kirin Brewery Co. for its donation of the cytokines, TPO, SCF, and EPO. This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (HMP-00-CH-04-0004).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received September 6, 2001; accepted for publication October 26, 2001.




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