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Keywords:

  • Stem cells;
  • Umbilical cord blood;
  • Ex vivo expansion;
  • NOD/SCID model;
  • Interleukin 3

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

In umbilical cord blood (UCB) transplantation, the number of nucleated cells per kilogram is a major predictive and critical factor of hematopoietic recovery. Thus, ex vivo expansion of hematopoietic UCB progenitors could potentially accelerate engraftment. Whereas Flt-3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO) are considered indispensable, the role of interleukin 3 (IL-3) is still controversial: it has been reported either to support or abrogate the reconstituting ability of stem cells. By adding IL-3 we aimed to enhance the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells.

Demonstrating a positive impact of IL-3 on the proliferation of all progenitor subsets, the amplification of CD34+ UCB cells was increased 20.9-fold ± 5.4 (mean ± standard error) in serum-free culture with FL, SCF, TPO, and IL-3 as opposed to 9.3-fold ± 3.2 without IL-3 after 7 days. If IL-3 was included, primitive long-term culture-initiating cells and committed colony-forming cells were expanded 16.3-fold ± 5.5 and 18.1-fold ± 2.4, respectively, compared to 12.6-fold ± 5.6 and 9.1-fold ± 2.0 without IL-3.

Analysis of cultured CD34+ UCB cells in sublethally irradiated nonobese diabetic/severe combined immunodeficient mice confirmed that cultured cells had preserved their repopulating potential. After 6 weeks, all mice showed multilineage engraftment with their bone marrow containing an average of 45% human CD45+ cells of the unmanipulated sample, 43% of cells after culture in the presence of IL-3, and 27% of cells after culture without IL-3. In combination with early acting cytokines, IL-3 therefore improves the ex vivo expansion of UCB stem and progenitor cells without impairing their engraftment potential.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Umbilical cord blood (UCB) as a source of hematopoietic stem cells has been used successfully in clinical trials in children. Hence, the number of nucleated UCB cells infused proved to be the major prognostic factor for engraftment and survival [1-3]. As the content of CD34+ progenitor cells was shown to correlate with the speed of neutrophil and platelet recovery [4], the use of UCB for allogeneic transplantation in adults has been hindered by the concern that a single cord blood may contain insufficient numbers of stem and progenitor cells to reconstitute these heavier patients in a timely manner. Ex vivo expansion of primitive multilineage and lineage-committed hematopoietic progenitor cells, which has been extensively evaluated over the past years, could conceivably circumvent this problem.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse is widely accepted as a model to assess the in vitro optimization of human progenitor expansion before proceeding to human trials [5]. A modest amplification of human primitive progenitor cells capable of repopulating irradiated NOD/SCID mice has been achieved using UCB samples [6-8] or human bone marrow (BM) [5, 9, 10]. Nevertheless, reports on the use of expanded UCB cells have been limited to preliminary clinical observations indicating that ex vivo-expanded grafts may accelerate hematopoietic reconstitution [11, 12]. A number of cases of engraftment failure of expanded human BM [13] or peripheral blood stem cells (PBSC) [14, 15] have also been published, indicating that long-term engraftment of ex vivo-expanded cells may be compromised, possibly depending on the specific culture conditions employed.

It is generally accepted that short-term expansion cultures of primitive human progenitor cells should include the cytokines stem cell factor (SCF), thrombopoietin (TPO), and flt-3 ligand (FL) which are particularly effective in preserving stem cell quality. In contrast, the role of interleukin 3 (IL-3), a multipotent cytokine capable of stimulating primitive as well as lineage-committed progenitors [16-18], has been discussed controversially. Data on benefit as well as loss of stem cell potential due to differentiation or altered homing properties have been reported, in particular in the murine system. Thus, the negative impact of IL-3 on the engraftment ability of cultured murine BM cells and a complete abrogation of the B-lymphoid potential indicated that IL-3 may be a stage-specific negative regulator and may suppress the earliest process of hematopoiesis, including self-renewal of the stem cells [19, 20]. In spite of recent data blaming fetal calf serum (FCS)-containing culture conditions for the extensive loss of reconstituting activity [21], IL-3 has been omitted from several protocols for the in vitro expansion of primitive murine BM cells [22, 23].

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Collection of Cord Blood Samples and Enrichment

UCB was collected immediately after delivery in a sterile tube containing 5,000 I.E. heparin. Informed consent of the mother was obtained. Mononuclear cells (MNC) were isolated prior to processing of the cord blood sample over Ficoll/Hypaque density cushion. The CD34+ selection was performed with the Miltenyi MiniMACS column according to the manufacturer's instruction. Briefly, UCB MNC were incubated for 15 min at 4°C with human IgG to block the Fc receptors and an anti-CD34 antibody (QBEND10, Miltenyi Biotec; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com) modified with a hapten. Cells were washed with phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) (Sigma; Taufkirchen, Germany; http://www.sigma-aldrich.com) and 0.1% EDTA (Titriplex III, Merck; Darmstadt, Germany) (referred to as MACS buffer) and incubated for 15 min at 4°C with an anti-hapten mouse monoclonal antibody conjugated to colloidal superparamagnetic beads (Miltenyi Biotec). Labeled cells were applied to magnetic column (MS column, Miltenyi Biotec), unbound cells washed out, and CD34+ cells eluted from the column with MACS buffer. To improve the purity of the CD34+ cells, a second purification cycle was performed using a smaller column (VS column, Miltenyi Biotec).

Short-Term Suspension Cultures

5 × 103 CD34+ cells/ml were seeded in 12-well-plates or 5 × 104 CD34+ cells/ml in culture bags using X-VIVO 10 (BioWhittaker; Verviers, Belgium; http://www.biowhittaker.com) plus 1% BSA (Sigma) and 1% glutamin (200 mM, GIBCO BRL; Paisley, Scotland) supplemented with recombinant human IL-3 (10 ng/ml), TPO (20 ng/ml), SCF, and FL (50 ng/ml each, R&D; Wiesbaden, Germany; http://www.rndsystems.com/) and incubated at 37°C in humidified atmosphere of 5% CO2. Without medium exchange or cytokine readdition, cells were harvested after 7 days, counted and assayed for colony-forming cells (CFC), long-term culture-initiating cells (LTC-IC), and expression of CD34 and CXCR4.

Clonogenic Progenitor Cell Assay Prior to and After a 7-Day Liquid Culture

Cells were plated at 50 cells/ml on day 0 and 200 cells/ml on day 7, respectively, in 0.9% semisolid methylcellulose-containing Iscove's modified Dulbecco's medium (IMDM) (Biochrom; Berlin, Germany; http://www.biochrom.de/) supplemented with 30% FCS (Hyclone; Logan, UT; http://www.hyclone.com), 1% BSA, human erythropoietin (3 U/ml) and IL-3, G-CSF, GM-CSF (20 ng/ml each), and incubated at 37°C for 12-14 days in humidified atmosphere of 5% CO2. Each assay was plated in triplicate. After 14 days of incubation, colonies derived from granuloid, erythroid, and multilineage progenitors (colony-forming units-granulocyte macrophage, BFU-E, CFU-granulocyte, erythroid, macrophage, megakaryocyte) were counted.

Limiting Dilution Assays for LTC-IC

Cells were seeded onto irradiated M2-10B4 and Sl/Sl stromal cell feeders (kindly provided by Donna Hogge, Terry Fox Labroatory; Vancouver, British Columbia, Canada) in limiting dilutions (10 replicates, 243, 81, 27, 9, 3, and 1 cells/well on day 0 and 2,430, 810, 270, 90, 30, and 10 cells/well on day 7) in IMDM supplemented with 12.5% FCS and 12.5% horse serum. Cultures were maintained at 37°C in a fully humidified atmosphere at 5% CO2 for 6 weeks with weekly exchanging of half of the medium. Cultures were then overlaid with methycellulose medium. Evaluation of wells for the presence or absence of secondary clonogenic cells was performed 2 weeks later. The absolute number of LTC-IC was calculated as the reciprocal of the concentration of test cells that yielded 37% negative wells.

Flow Cytometry of Cord Blood Cells

For fluorescence-activated cell sorter (FACS) analysis fresh or cultured cells were washed in PBS containing 1% FCS and 0.1% NaN3. Five μl of the phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated antibody were added, followed by incubation for 15 minutes at 4°C. After washing, the cells were fixed in 300 μl PBS with 2% formaldehyde. The antibodies were labeled as follow: FITC-conjugated antibodies (CD34, CD45, Becton Dickinson; Heidelberg, Germany; http://www.bd.com), PE-conjugated antibodies (CD34, Becton Dickinson and CXCR4, PharMingen; San Diego, CA; http://www.pharmingen.com), and Percep-conjugated antibody (CD34, Becton Dickinson). As negative control we used PE-, FITC-, and Percep-conjugated anti-huIgG1 (Becton Dickinson). Five thousand to 10,000 events were counted. Analysis was performed at a FACScan (Becton Dickinson) using CellQuest and PC-Lysis software.

Reconstitution of NOD/SCID Mice with Human Hematopoietic Cells

Aliquots consisting of 1 × 105 cord blood CD34+ cells and the corresponding progeny generated after ex vivo expansion were transplanted into NOD/SCID mice aged 4 to 8 weeks. NOD/SCID breeding pairs were obtained from Jackson Laboratories (Bar Harbor, ME; http://www.jax.org). The mice were bred and maintained in the animal facility of the ASTA MEDICA (Frankfurt, Germany). Prior to transplantation of the cord blood cells, the mice were sublethally irradiated with 100 cGy and were cotransplanted with irradiated rat fibroblasts in Matrigel genetically modified to produce human IL-3. Six weeks after transplantation the mice were sacrificed. Both femurs were prepared, and the presence of the human hematopoietic cells in the BM of the NOD/SCID recipients was analyzed by flow cytometry. Aliquots of the murine BM cells were incubated with antibodies directed against the human common leukocyte antigen CD45 or double-stained with anti-human CD45-FITC in combination with anti-human CD34 PE for 20 min at 4°C. For analysis of multilineage engraftment the cells were incubated with anti-human CD33 PE and antihuman CD19-FITC (both Becton Dickinson). Thereafter, red cells were lysed by adding 2 ml FACS lysing solution (Becton Dickinson) and incubated for 30 minutes at room temperature. Cells were then washed in FACS wash (Becton Dickinson), resuspended, and analyzed by flow cytometry. BM cells from NOD/SCID mice having undergone transplantation labeled with conjugated-nonspecific isotype-matched antibodies and BM cells from NOD/SCID mice not transplanted were stained with anti-human-CD45 FITC and CD34 PE antibodies, as controls for nonspecific staining of human and mouse cells. Ten thousand to 50,000 events were counted.

Statistical Analysis

Statistical significance was tested with the Wilcoxon test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

NOD/SCID Repopulating Ability of Unmanipulated and Ex Vivo-Expanded Grafts

To compare the engraftment potential of ex vivo-expanded and unmanipulated fresh UCB samples, 7-day suspension cultures were initiated with 5 × 104 CD34+ cells (purity 90.4 ± 1.4%) in the presence of SCF, FL, and TPO with or without IL-3. Aliquots of 1 × 105 freshly thawed CD34+ cells (control group) or the corresponding progeny obtained after culture were injected in sublethally irradiated NOD/SCID mice. Six weeks after transplantation human donor cells were readily detected in the BM and PB of all recipients by FACS analysis. Supplementation of IL-3 led to the same relative content of human CD45+ cells in the murine BM as found in the unmanipulated control group (43% and 45%, respectively), whereas a lower proportion of CD45+ cells (27%) was shown after transplantation of cells cultured with SCF, TPO, and FL alone (Fig. 1A). Analysis of the level of CD45+/CD34+ double-positive cells revealed no difference between these groups (Fig. 1B). Our results demonstrate that these IL-3-containing culture conditions maintain the repopulating capacity of human UCB stem cells. Multilineage engraftment was assessed by FACS analysis of human CD33+ myeloid and CD19+ B cells (Fig. 2). Although cultured in the presence of IL-3, lymphoid (19%), and myeloid (35%) cells in the murine BM were only slightly reduced compared to the unmanipulated cells (30% and 46%, respectively).

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Figure Figure 1.. Engraftment of human CD34+UCB cells in NOD/SCID mice transplanted immediately after thawing (C) or after 7-day culture with SCF, TPO, FL in the absence (A) or presence (B) of IL-3.A) represents the proportion of human CD45+and B) the proportion of human CD45+/CD34+cells in the BM of 10 mice transplanted in two individual experiments (white and black dots). Mean percentages are also indicated (not statistically significant).

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Figure Figure 2.. Multilineage engraftment of NOD/SCID mice of CD34+UCB transplanted without (A) or after ex vivo expansion for 7 days with SCF, TPO, FL, and IL-3 (B).A) shows the proportion of human CD33+myeloid cells, B) the proportion of human CD19+lymphoid cells in the murine BM of two individual experiments (white and black dots). Mean percentage is also indicated (not statistically significant).

Effects of IL-3 on Ex Vivo Expansion of Primitive and Committed Progenitor Cells

To uncover a correlation between the engraftment level of recipients and the progenitor cell dose transplanted, the amplification of CD34+ cell, CFC, and LTC-IC numbers was analyzed (Fig. 3). We found a significant difference in the expansion of CD34+ cells (p = 0.05) and CFC (p = 0.008) after a 7-day culture of the UCB cells depending on supplemented IL-3. In the absence of IL-3 we achieved an average amplification of CD34+ cells and CFC of 9.3 ± 3.2-fold and 9.1 ± 2-fold, respectively. Addition of IL-3 led to a higher average amplification of CD34+ cells and CFC of 20.9 ± 5.4-fold and 18.1 ± 2.4-fold, respectively. The mean cloning efficiency was 28.6 ± 7% on day 0 and 15.7 ± 2.4% with IL-3 and 20.3 ± 2.9% without IL-3. LTC-IC expansion with or without IL-3 showed the same trend but was not statistically significant (p = 0.17). Nevertheless, addition of IL-3 led to a slightly increased amplification compared to culture conditions without IL-3 (16.3 ± 5.5-fold and 12.6 ± 5.6-fold, respectively). The frequency of LTC-IC after cultivation in the absence or presence of IL-3 was 1:141 and 1:542, respectively. In fresh cells we detected one LTC-IC of 68 cells. In summary, inclusion of IL-3 into the growth factor cocktail of SCF, FL, and TPO enhanced the ex vivo expansion of all progenitor populations without impairing their engraftment potential.

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Figure Figure 3.. Ex vivo expansion of primitive and committed progenitor cells.UCB CD34+cells were cultured with SCF, TPO, FL (light columns), and IL-3 (dark columns) for 7 days. Data are shown as mean amplification ± standard error (n = 6).

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Effects of IL-3 on CXCR4+ CD34+ Cells

The chemokine receptor CXCR4-binding stromal derived factor-1 was recently found to contribute to the homing of hematopoietic stem cells to the BM. Therefore, CXCR4 expressing CD34+ cells were determined by FACS analysis (Fig. 4). On day 0 we detected 24 ± 5.2% CXCR4+CD34+ cells. After culture we found 8.6% CXCR4+CD34+ cells in the absence of IL-3, in contrast to 4.4% CXCR4+CD34+ cells with supplemented IL-3. However, due to the higher cell count in IL-3-containing cultures, the total number of CXCR4+CD34+ cells obtained was increased by adding IL-3 (3.4 × 104 versus 1.5 × 104 per ml) (Fig. 5). These results confirm that in conjunction with SCF, FL, and TPO, IL-3 enhances the ex vivo expansion of primitive cell subsets without abrogating the expression of the CXCR4 receptor considered essential for the engraftment potential of transplanted cells.

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Figure Figure 4.. Flow cytometry of CD34+/CXCR4+CB cells before expansion (D0), on the second day of culture (D2), and after 7 days of culture (D7) with SCF, TPO, FL plus/minus IL-3.Also shown is the isotype control.

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Figure Figure 5.. Amplification of CD34+CXCR4+CB cells during 7-day culture with SCF, TPO, FL (), and IL-3 (

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) shown as average of total cell numbers ± standard error (n= 2).

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Kinetics of CXCR4 Expression During Ex Vivo Culture; Effects of IL-3

Modulation of CXCR4 by cytokines was demonstrated to influence the homing efficiency of stem and progenitor cells. We therefore investigated the kinetics of development of the CXCR4+CD34+ population. The culture was initiated with 2.5 × 104 CD34+ cells/ml in the absence or presence of IL-3. After the first day of culture, the output of CXCR4+CD34+ cells was more than 1.5-fold higher with IL-3 than under conditions lacking this cytokine, as shown in Figure 5. By day 2 we observed an increase in CXCR4+CD34+ cells under both culture conditions which may result from upregulation of the CXCR4 expression. The difference between both culture conditions became even more pronounced in the further course of the culture: in contrast to a remarkable increase of the CXCR4+CD34+ cell numbers after 5 days in the presence of IL-3, the CXCR4+CD34+ cell numbers decreased in IL-3-lacking cultures.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The present study was specifically directed at investigating the impact of IL-3 on ex vivo expansion of human UCB stem and progenitor cells. We considered the cytokines SCF, TPO, and FL to be generally accepted as promoting the growth of primitive hematopoietic cells [24-28], and thus routinely included them into all cultures. Conflicting data on beneficial or detrimental effects of IL-3 on stem cells in vitro have been published. In primitive murine hematopoietic cells with lympho-myeloid potential, IL-3 impaired the self-renewal capacity of these cells and blocked the cytokine-stimulated generation of lymphoid precursors [29]. Impairment [30, 31] or abrogation [32] of NOD/SCID repopulating capacity by IL-3-containing culture media was also described for human UCB cells. Conversely, several investigators have included IL-3 in cytokine cocktails used for ex vivo expansion of human UCB cells with retention of their long-term repopulating ability in sheep [33] or NOD/SCID mice [7, 10, 34, 35]. However, these reports did not directly compare the effects of adding or omitting IL-3.

We demonstrate that repopulation of NOD/SCID mice by freshly isolated as well as cultured CD34+ CB cells is highly efficient: all mice had engrafted by 6 weeks after transplantation. No decline in the in vivo repopulation capacity was noticed between unmanipulated CD34+ CB cells and those expanded in the presence of SCF, TPO, FL, and IL-3; but without IL-3, the proportion of human cells was slightly reduced. Despite the presence of IL-3, multilineage engraftment of the cultured UCB cells was achieved. In conclusion, our data do not show detrimental effects of IL-3 on the ex vivo expansion of primitive hematopoietic progenitors.

Recently, the concept of a significant persisting quiescent population of transplantable stem cells in cultures of human UCB cells was challenged by Glimm et al. [34] demonstrating that most CD34+ repopulating UCB cells are stimulated to undergo multiple divisions under IL-3-containing culture conditions. On the other hand, results of previously published in vitro tests suggested a direct and negative impact of high levels of IL-3 on BM LTC-IC expansion [36]. Our data show a proportionate increase of the 7-day expanded CD34+ cell population as well as the primitive (LTC-IC) and committed (CFC) subsets with a trend towards higher amplification of primitive cells in the presence of SCF, TPO, and FL. The addition of IL-3 enhanced the proliferation of all cell populations analyzed, i.e., a 20-fold expansion of CD34+ and CFC and 16-fold of LTC-IC, surprisingly without accompanying extensive amplification of mature progenitors at the expense of progenitors and repopulating primitive stem cells.

After the first four days of our culture, IL-3 and TPO levels declined rapidly and became undetectable without cytokine replenishment, whereas SCF and FL concentrations decreased more slowly and remained detectable throughout the 7-day culture period (data not shown). SCF, FL, TPO, and IL-3 are known to initiate the proliferation of G0CD34+ human BM cells, but only IL-3 sustained maximal proliferation of SCF and FL prestimulated G0CD34+ cells [26, 27, 37]. Therefore, IL-3 may be useful for optimal amplification of progenitor cells during the first days of culture. In the face of persisting high levels of SCF and FL, low levels of IL-3 after the fourth day of culture may have prevented a conceivable impairment of the self-renewal of primitive cells. Such an orderly activation of G0CD34+ cells may be suitable for maintenance of proliferation of primitive hematopoietic stem and progenitor cells. Our results are consistent with the hypothesis that the sequential cytokine exposure may be critical for optimal numerical stem cell expansion with concomitant maintenance of their functional properties.

In preclinical animal models as well as in the clinical transplant setting, homing of the transfused stem cells to the appropriate microenvironmental niches is one of the earliest essential steps preceding hematopoietic reconstitution. The CXCR4 receptor, which is more highly expressed on the most primitive PB CD34+ cells (the CD34+CD38 subset) than on the CD34+ population overall [38], appears to play a particularly prominent role in the homing and engraftment of stem cells. Upregulation of CXCR4 expression on human CD34+ cells following their exposure to SCF and IL-6 increased the effective repopulating activity of these cells within a frame too short to be readily accounted for by stem cell divisions [39]. On the other hand, it has been suggested that prolonged culture of CD34+ cells, particularly in the presence of IL-3, may impair the chemotactic responsiveness of stem and progenitor cells, possibly by downmodulation of CXCR4. We therefore compared CXCR4 expression in cultured cells as a function of IL-3 and, consistent with the results of Jo et al. [40], indeed observed a reduced percentage of CD34+CXCR4+ cells among the 7-day expanded cells. Nevertheless, according to our data engraftment of expanded CD34+ cells is comparable to fresh cells. We hypothesize that in the presence of IL-3, the simultaneous increase of CD34+CXCR4+ cell numbers by more than one log prevented the loss of repopulating ability.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

To clarify whether IL-3 leads to a loss of human stem cell characteristics and engraftment potential of CD34+ UCB cells in short-term cultures designed for clinical applicability, we investigated the effect of IL-3 in conjunction with SCF, TPO, and FL. We show that engraftment of human UCB-derived CD34+ cells in NOD/SCID mice was not compromised by the presence of IL-3. Concurrently, IL-3 increased the total number of clonogenic progenitor and LTC-IC, and amplified the overall number of CD34+ cells as well as CD34+CXCR4+ cells, which are considered to have a homing advantage. We therefore conclude that the incorporation of IL-3 in expansion cultures is warranted in studies designed to confirm the engraftment benefit in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

We gratefully thank Dr. Grimminger and the staff of midwives at the St. Vincenz- and Elisabeth-Hospital Mainz for UCB collection and Dr. Henschler for critical reading of the manuscript. This work was supported by the Deutsche Knochenmarkspenderdatei, Germany.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
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