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Stem Cells, Vol. 17, No. 2, 92-99, March 1999
© 1999 AlphaMed Press

Cytokine-Induced Expansion of Human CD34+ Stem/Progenitor and CD34+CD41+ Early Megakaryocytic Marrow Cells Cultured on Normal Osteoblasts

N. Ahmed, M.A. Khokher, H.T. Hassan

Division of Biomedical Sciences, School of Health Sciences, University of Wolverhampton, Wolverhampton, United Kingdom

Key Words. CD34 • CD41 • Interleukin • Osteoblast • Stem cell factor • Thrombopoietin

Dr. H.T. Hassan, Division of Biomedical Sciences, School of Health Sciences, University of Wolverhampton, 62-68 Lichfield Street, Wolverhampton WV1 1DJ, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic bone marrow transplantation (BMT), which consumes millions each year for frequent platelet transfusions. Using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts, we investigated the expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor marrow cells in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia. After seven days of culture, normal human osteoblasts alone without cytokines significantly increased the number of CD34+ and CD34+CD41+ marrow cells. Among the various cytokine combinations tested, both stem cell factor (SCF), interleukin 3 (IL-3)+IL-11 and SCF+IL-3+IL-11+thrombopoietin (TPO) emerged as the most effective in expanding early CD34+CD41+ megakaryocytic cells. Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures, and by 62- and 94-fold, respectively, compared with day 0 input, respectively. Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same two combinations. In addition, the same cytokine combinations achieved 17.6- and 13.3-fold increases in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. The combination (SCF+IL-3+IL-11+TPO) achieved the highest expansion of CD34+CD41+ early megakaryocytic cells from human marrow CD34+ cells reported so far in the literature. Recently, transplantation of SCF+IL-1+IL-3+TPO ex vivo expanded megakaryocytic progenitor cells as a supplement has been shown to accelerate platelet recovery by three to five days in mice. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ and megakaryocytic progenitor cells from a portion of the donor's marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT. The present study has confirmed the role of human osteoblasts in supporting the proliferation and maintenance of human CD34+ stem/progenitor marrow cells. Given the facilitating role of osteoblasts shown previously in several allogeneic BMT studies in mice, it is possible to envisage a future role for donor osteoblasts in clinical BMT. Transplantation of the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as a supplement might not only accelerate platelet recovery but also prevent acute graft-versus-host disease in allogeneic BMT. The present novel culture system should have useful clinical application in allogeneic BMT.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human bone marrow stroma is responsible for supporting both the daily production of billions of mature blood cells [1] and the generation of osteoblasts [2, 3], the major contributor to bone formation from CD34+ marrow stem/progenitor cells [4, 5]. Ultrastructural and developmental studies have also demonstrated the close physical association between endosteal osteoblasts and hemopoietic marrow cells in the bone marrow (BM) cavity [6-8]. Moreover, normal human osteoblasts produce several hemopoietic cytokines, including G-CSF [9], macrophage colony-stimulating factor (M-CSF) [10], interleukin 6 (IL-6) [11], tumor necrosis factor-alpha (TNF-{alpha}) [12], leukemia inhibitory factor (LIF) [13] and osteoclast differentiation factor (ODF) [14] as well as GM-CSF and IL-8 upon IL-1 stimulation [15]. These cytokines are required for both hemopoiesis [1] and the generation of osteoclasts for bone modulation from peripheral blood [14, 16, 17], cord blood [18], and BM [19] cells. Also, allogeneic transplantation of a donor-derived bone graft along with BM stem/progenitor cells in mice has promoted complete hemopoietic reconstitution and improved immune reconstitution after transplants in a chimeric-resistant combination [20-23], indicating a clear facilitating role for osteoblasts in allogeneic bone marrow transplantation (BMT).

BMT has been successfully used during the last two decades to restore hemopoiesis after myeloablative therapy in cancer patients. However, compatible donors are in short supply, and autologous marrow has often been either damaged from previous cycles of chemotherapy or contaminated with malignant cells [24]. Therefore, there has been great interest recently in ex vivo expansion of marrow stem/progenitor CD34+ cells that could replace whole marrow for a long-lasting restoration of normal hemopoiesis. These ex vivo expanded CD34+ cells could offer a graft devoid of malignant cells for autologous BMT and may not require immune suppression as is currently needed for preventing graft-versus-host disease (GVHD) in allogeneic BMT [25].

Recently, normal human osteoblasts have been shown to support the proliferation and maintenance of early hemopoietic CD34+ stem/progenitor marrow cells to the same extent as BM stroma for two weeks [26], probably due to the augmented production of IL-6 by osteoblasts in response to CD34+ marrow cells [27]. Interestingly, CD34+ marrow cells cultured on normal human osteoblasts have maintained their immature phenotype better than on BM stroma [26]. Also, in the presence of IL-3, GM-CSF, and erythropoietin, cultures of CD34+ marrow cells on normal human osteoblasts have produced a significantly higher percentage of erythroid colonies formed than those cultured in cytokine-treated controls and/or on BM stroma [26]. Given the close overlapping relationship between erythropoiesis and megakaryopoiesis [28], the present study was carried out to evaluate the role of normal human osteoblasts in supporting cytokine-induced expansion of megakaryocytic progenitor marrow cells. Thrombocytopenia remains a significant cause of morbidity in cancer patients undergoing allogeneic BMT [29], which consumes millions each year for frequent platelet transfusions [30]. Thus, the aim of the present study was to achieve a maximum expansion of early megakaryocytic progenitor cells while maintaining the number of CD34+ stem/progenitor cells using a novel culture system containing appropriate cytokine(s) on a layer of normal human osteoblasts in an attempt to provide an effective solution for the problem of post-transplant thrombocytopenia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human BM CD34+ and Bone Cells
Human BM and bone cells were obtained after informed consent from normal adults undergoing hip replacement operation. The bone cells were prepared as previously described [31]. Briefly, bone pieces were rinsed with phosphate-buffered saline (PBS), cleaned from any fibrous tissue, and cut into small pieces. These small pieces were placed in 35-mm tissue culture petri dishes containing {alpha}-minimal essential medium ({alpha}-MEM) (Life Technologies; Glasgow, UK) supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin, and amphotericin.

The BM mononuclear cells were separated by Ficoll-Paque density-gradient centrifugation at 400 g for 30 min at room temperature and the low-density interface cells (<1.077 g/cm3) were collected, washed three times with {alpha}-MEM, and counted on a Coulter counter. The CD34+ cells were selected from BM mononuclear cells as previously described [32]. Briefly, mononuclear cells were washed twice with PBS and resuspended in PBS with 0.1% human antibody (AB) serum albumin at a concentration of 2 x 107 cells per ml and then incubated with 20 µg/ml of the biotinylated anti-CD34 monoclonal antibody (mAb) for 25 min at room temperature. After washing with PBS to remove any excess AB, this AB-sensitized mononuclear cell fraction was filtered through the Ceprate avidin column (CellPro, Inc.; Bothell, WA). In this system, the CD34+ cells link with the avidin-coated polyacrylamide beads through an "avidin-biotin-anti-CD34 antibody-CD34 cell" complex. After washing the column with PBS to remove nonspecifically bound cells, the CD34+ cells were released from the avidin beads by mechanical agitation and resuspended in {alpha}-MEM. Aliquots were taken from the mononuclear, CD34+ and CD34– fractions for both flow cytometry analysis and clonogenic cell culture assays. The purity of selected CD34+ cells from the five BM samples used in the present study was 80%, 85%, 91%, 94%, and 95%.

Culture and Characterization of Normal Human Osteoblasts
The small bone pieces placed in 35-mm tissue culture petri dishes containing {alpha}-MEM supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, and amphotericin were cultured at 37°C in a humidified incubator with 5% CO2. The osteoblast-like cells started growing after 7-10 days and were fed weekly with fresh supplemented {alpha}-MEM until they reached confluence after four to five weeks. The nature of these cultured osteoblast cells was determined as previously described [33] by their typical morphology after Giemsa-Wright staining ( Fig. 1), as well as alkaline phosphatase, collagen, and osteocalcin synthesis. More than 90% of the osteoblast cells had cytoplasm positive for alkaline phosphatase (ALP) after cytochemical staining (Fig.1). The cell viability was more than 88% by trypan blue dye exclusion. In addition to staining the cells for ALP, we measured bone-specific ALP as previously described [33] to exclude the possibility that the observed ALP production is due to fibroblasts.



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  		Figure 1. A) Morphology of human osteoblasts after four weeks of culture. Magnification x 800. B) Alkaline phosphatase staining of human osteoblasts after four weeks of culture. Magnification x 800.

 
Culture of Normal Human BM CD34+ Cells on Osteoblasts
Normal human BM CD34+ cells were cultured at a concentration of 104 cells per ml in {alpha}-MEM supplemented with 10% FBS, L-glutamine, penicillin, streptomycin, and amphotericin in 35-mm culture petri dishes containing a confluent osteoblast layer at 37°C for 14 days in a humidified incubator with 5% CO2. The following cytokines were purchased from R & D Systems (Oxon, UK): stem cell factor ([SCF], specific activity of 50 U/µg), IL-3 (specific activity of 5 MU/mg), IL-11, (specific activity of 2 MU/mg), and thrombopoietin (TPO). They were added twice weekly at a concentration of 100 ng/ml except for the IL-3 at concentration of 50 ng/ml to the CD34+ cell cultures. We have previously shown these concentrations to be the most effective in human marrow cultures [34]. Control cultures containing no cytokines were also set up in conditions otherwise identical. Medium exchange was performed by removing half the medium and adding instead fresh medium with or without the cytokines to restore the original culture volume every second day.

Flow Cytometry Measurement of CD34+, CD34+CD41+, and CD41+ Cells
The CD34+, CD34+CD41+ and CD41+ cells were determined as previously described [34, 35]. Briefly, 1 x 106 cells from each sample on days 0, 7, and 14 were incubated for 30 min at 4°C in darkness with the phycoerythrin (PE)-conjugated mAb anti-CD34 or anti-CD41 and fluorescein isothiocyanate (FITC)-conjugated anti-CD45 obtained from Coulter (Bedford, UK) then washed twice with PBS/1% bovine serum albumin. For the CD34+CD41+ measurement, the Cy5-conjugated anti-CD34, PE-conjugated anti-CD41, and FITC-conjugated anti-CD45 mAb were used in the same way. The cells were analyzed on a FACScan flow cytometry (EPICS, Coulter-Immunotech; Bedford, UK). A minimum of 20,000 events were acquired for both control and positive samples in order to determine a resolvable CD34+ population. The total numbers of CD34+, CD34+CD41+ and CD41+ cells were calculated by multiplying the number of cells per sample by the percentage of either CD34+, CD34+CD41+, or CD41+ cells in the samples tested.

Statistical Analysis
Results represent the mean ± standard deviation of determinations from three separate experiments performed in triplicate with five normal human BM and bone samples. Statistical significance was evaluated using the two-tailed Student's t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Hemopoietic Cytokines on Normal Human Marrow CD34+ Cells Cultured on Normal Human Osteoblasts
After seven days of culture, normal human osteoblasts alone in the absence of cytokines have significantly increased the number of human CD34+ cells from 1 x 104 per ml input on day 0 to 6.1 ± 1.2 x 104 per ml, p < 0.01 ( Fig. 2). In the presence of single cytokines, only SCF and IL-3 could further increase the number of human CD34+ cells to 8.5 ± 1.7 and 11.3 ± 2.2 x 104 per ml on day 7, respectively ( Fig. 2), whereas in the presence of TPO, the number of human CD34+ cells was maintained at 6.4 ± 1.3 x 104 per ml on day 7 ( Fig. 2). Adding IL-11 decreased the number of human CD34+ cells to only 2.6 ± 0.5 x 104 per ml on day 7 ( Fig. 2). The combinations of SCF with IL-3, IL-11, or TPO did not increase the number of human CD34+ cells on day 7 above that achieved by SCF alone ( Fig. 2). The triple combination of SCF+IL-3+IL-11 markedly increased the number of human CD34+ cells to 17.6 ± 3.5 x 104 per ml on day 7 ( Fig. 2). Adding TPO to this triple combination maintained the profound increase in the number of human CD34+ cells at 13.3 ± 2.7 x 104 per ml on day 7 ( Fig. 2).



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  		Figure 2. Values represent the mean ± standard deviation of nine determinations of triplicate cultures at day 7 from three separate experiments. SCF = 100 ng/ml; IL-3 = 50 ng/ml; IL-11 = 100 ng/ml; TPO = 100 ng/ml. compared to control cultures using t-distribution test.

 
On the other hand, after 14 days of culture on normal human osteoblasts alone, the percentage of human CD34+ cells declined sharply to 0.5% in the absence of cytokines ( Table 1). In the presence of single cytokines, only SCF and IL-3 could maintain the percentages of human CD34+ cells at 10.3% and 9.0% on day 14, respectively ( Table 1). The combinations of SCF with IL-3, or TPO but not IL-11, increased the percentage of human CD34+ cells on day 14 to 24.3% and 21.3%, respectively ( Table 1). In the cultures containing SCF+IL-3+IL-11 and SCF+IL-3+IL-11+TPO, the percentage of human CD34+ cells on day 14 was maintained at the high level of more than 40% ( Table 1). Also, only these two combinations (SCF+IL-3+IL-11 and SCF+IL-3+IL-11+TPO) could achieve a CD34+ cell recovery of more than two-thirds (77% and 62.6%, respectively) after 14 days of culture ( Table 1).


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  		Table 1. Percentage and recovery of normal human BM CD34+ cells after 14 days of culture on human osteoblasts
 
Effect of Hemopoietic Cytokines on Normal Human Marrow CD34+CD41+ Early Megakaryocytic Cells Cultured on Normal Human Osteoblasts
After seven days of culture, normal human osteoblasts alone in the absence of cytokines significantly increased the number of human CD34+CD41+ cells from only 0.1 x 104 per ml input on day 0 to 2.0 ± 1.2 x 104 per ml, p < 0.01 ( Fig. 2). In the presence of single cytokines, only SCF or IL-3 could further increase the number of human CD34+CD41+ cells to 4.1 ± 2.1 and 5.5 ± 2.8 x 104 per ml on day 7, respectively ( Fig. 2). In the presence of TPO or IL-11, however, the number of human CD34+CD41+ cells was maintained at 2.5 ± 1.2 and 1.8 ± 0.4 x 104 per ml on day 7 ( Fig. 2). The combinations of SCF with IL-3, IL-11 or TPO did not increase the number of human CD34+CD41+ cells on day 7 above that achieved by SCF alone ( Fig. 2). The triple combination of SCF+IL-3+IL-11 has significantly increased the number of human CD34+CD41+ cells to 6.2 ± 3.1 x 104 per ml on day 7 ( Fig. 2). Adding TPO to this triple combination profoundly increased the number of human CD34+CD41+ cells to 9.4 ± 4.7 x 104 per ml on day 7 ( Fig. 2).

On the other hand, after 14 days of culture, the number of human CD34+CD41+ cells has decreased below the 2 x 104 per ml achieved in control cultures on day 7 except in the presence of the triple combination of SCF+IL-3+IL-11 with or without TPO ( Table 2).


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  		Table 2. Number of normal human BM CD34+CD41+ early megakaryocytic cells after 14 days of culture on human osteoblasts
 
Effect of Hemopoietic Cytokines on Normal Human Marrow CD41+ Late Megakaryocytic Cells Cultured on Normal Human Osteoblasts
After seven days of culture, normal human osteoblasts alone in the absence of cytokines produced only a minimal number of human CD41+ megakaryocytic cells ( Table 3). In the presence of single cytokines, only TPO increased the percentage of human CD41+ megakaryocytic cells to 27.6% ( Table 3). Also, only the combination of SCF + TPO increased the percentage of human CD41+ megakaryocytic cells on day 7 to 33.5% ( Table 3). The triple combination of SCF+IL-3+IL-11 significantly increased the percentage of human CD41+ megakaryocytic cells on day 7 to 40%, p < 0.01 ( Table 3). Adding TPO to this triple combination profoundly increased the percentage of human CD41+ megakaryocytic cells on day 7 to 50.7% ( Table 3).


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  		Table 3. Percentage and fold increase of normal human BM CD41+ megakaryocytic cells after seven days of culture on human osteoblasts
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Three main factors have been hypothesized for the pathogenesis of post-transplant thrombocytopenia: inadequate production of megakaryocytic growth factors, release of inhibitory cytokines, and an inadequate number of megakaryocytic progenitor cells in the graft [25]. In allogeneic transplantation, acute GVHD has an additional adverse effect [29]. Transplantation of peripheral blood stem/progenitor cells harvested after cytokine ± chemotherapy has not led to a consistent shortening of the post-transplant thrombocytopenia period [25]. Attempts to accelerate platelet recovery with megakaryocytic stimulating factors such as IL-3, IL-6, and IL-11 after chemotherapy in cancer patients have been associated with limited efficiency and sometimes unacceptable toxicity, and TPO is currently undergoing clinical investigations [36]. Also, reducing the number of platelet transfusions in the post-transplant period should decrease the exposure of megakaryocytic progenitor cells to several inhibitory cytokines released from transfused platelets [36]. However, platelet transfusions cannot be withheld during the critical post-transplant period until platelet recovery occurs. Therefore, the feasible approach to this problem is expanding early megakaryocytic progenitor cells in sufficient numbers without compromising the number of CD34+ stem/progenitor cells in a portion of the graft to be transplanted. This approach could significantly reduce the number of platelet transfusions required and also accelerate platelet recovery.

Among the three sources of human hemopoietic stem/progenitor cells, both BM and G-CSF-mobilized peripheral blood CD34+ cells have similar potential for ex vivo expansion [37], and they contain significantly more CD34+CD41+ megakaryocytic progenitor cells than cord blood [38, 39]. Therefore, we have used the BM CD34+ cells for ex vivo expansion of both CD34+CD41+ early and CD41+ late megakaryocytic cells using a novel system cultured with appropriate cytokines on osteoblasts. The four cytokines used in the present study were chosen due to their key involvement in megakaryocytopoiesis [36] as well as being early-acting cytokines on human BM CD34+ cells [40]. The main aim of the present study was to achieve maximum expansion of early and late human megakaryocytic cells and, at the same time, maintain the number of CD34+ marrow cells.

The present results have shown seven days of culture as the optimum duration required for maximum expansion of human CD34+ stem/progenitors and CD34+CD41+ early megakaryocytic marrow cells. Any longer period of culture has led to a significant decline in the number of CD34+ stem/progenitor cells in all control and cytokine-treated cultures ( Table 2). Among the various cytokine combinations tested, both SCF+IL-3+IL-11 and SCF+IL-3+IL-11+TPO have emerged as the most effective in expanding both early CD34+CD41+ and late CD41+ megakaryocytic cells ( Fig. 2, Table 2). Early CD34+CD41+ megakaryocytic cells have increased by 3.1- and 4.7-fold compared with day 7 control cultures and by 62- and 94-fold compared with day 0 input in the presence of SCF+IL-3+IL-11 and SCF+IL-3+IL-11+TPO, respectively ( Fig. 2). Also, late CD41+ megakaryocytic cells have increased by 15.4- and 27.5-fold compared with day 7 control cultures in the presence of the same cytokine combinations ( Table 2). Two recent in vitro studies have also demonstrated the essential role of early-acting cytokines such as SCF, IL-1, IL-3, and/or IL-11 for optimal ex vivo expansion of human megakaryocytic progenitor cells [41, 42]. In the first study, the combination of SCF+IL-1+IL-3+TPO has increased clonogenic megakaryocytic cells by 15- and 50-fold on days 7 and 14, respectively, of serum-free culture [41]. Also, transplantation of these ex vivo expanded megakaryocytic progenitor cells as a supplement has accelerated the platelet recovery by three to five days in mice [41]. In the second study, the combination of SCF+IL-1+IL-6+IL-11+TPO has induced the highest expansion of late CD41+ cells from human CD34+ marrow cells [42]. Similarly, Schattner et al. [43] reported a maximum of 16 megakaryocytes per CD34+ cell generated ex vivo from normal human BM. Although an in vivo study has demonstrated a significantly faster platelet recovery after syngeneic BMT from TPO-treated donor mice [44], the first clinical study of transplanting additional ex vivo-generated late megakaryocytic cells from mobilized CD34+ cells has shown no effect on the post-transplant thrombocytopenia [45]. Certainly, an optimal ex vivo expansion of both early and late megakaryocytic progenitor marrow cells is essential in a clinical setting. In the present study, the cytokine combination (SCF+IL-3+IL-11+TPO) has achieved the highest expansion of early CD34+CD41+ megakaryocytic cells (94-fold increase) from human marrow CD34+ cells reported so far in the literature. In addition, the same combination has achieved a 13.3-fold increase in the number of CD34+ marrow cells after the same seven days of culture on a layer of human osteoblasts. Interestingly, we have previously shown a similar cytokine combination (SCF+IL-3+IL-11+erythropoietin) to induce the highest expansion of human clonogenic erythroid marrow cells (7.7-fold increase) after seven days of culture [34]. Also, the same combination without erythropoietin (SCF+IL-3+IL-11) has induced the highest expansion of human clonogenic myeloid marrow cells after seven days of culture in the same previous study [34].

Therefore, it seems that all three stroma-derived cytokines (SCF, IL-3, and IL-11) are imperative for any effective ex vivo expansion system of human marrow cells for transplantation. Clinically, all three cytokines have been used in several phase I/II trials to accelerate hematological recovery after chemotherapy in cancer patients [36]. Also, TPO is currently undergoing clinical investigations [36]. Therefore, the clinical use of the combination (SCF+IL-3+IL-11+TPO) for ex vivo expansion of CD34+ stem/progenitor and megakaryocytic progenitor cells from a portion of the donor marrow harvest is warranted in allogeneic BMT. Such a protocol would accelerate platelet recovery and shorten the period of hospitalization after allogeneic BMT and may prove to be cost effective.

The present study has confirmed the role of human osteoblasts in supporting the proliferation of human CD34+ stem/progenitor marrow cells and has also confirmed their unique ability to maintain and even expand the immature phenotype of CD34+ marrow cells [26]. Results of several previous work groups using only cytokine combinations without osteoblast or marrow feeder cells clearly demonstrated a decline in the number of CD34+ cells with the maximum expansion of megakaryocytic progenitor cells achieved [41-43]. Since human osteoblasts produce several additional cytokines, including IL-6, GM-CSF, G-CSF, M-CSF, TNF-{alpha}, LIF, and ODF [9-15], the question of replacing the osteoblasts by these additional cytokines needs to be tested. Also, whether the use of serum-free cultures would alter the present maximum expansion achieved remains to be examined.

Given the facilitating role of osteoblasts demonstrated in several allogeneic BMT studies in mice [20-23], it is possible to envisage a future role for donor osteoblasts in clinical BMT. Since acute GVHD has been shown to delay platelet recovery and contribute to the mortality rate after allogeneic BMT [29], using the cultured donor osteoblasts together with the ex vivo expanded CD34+ marrow cells as supplement might not only accelerate platelet recovery but also reduce acute GVHD in allogeneic BMT. In vivo transplantation studies testing this promising approach in primate models are warranted. The present novel culture system should have useful clinical application in allogeneic BMT.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  39. Slone V, Abu-Ghosh A, Goldman S et al. Deficient number of CD34+CD41+ cells in cord blood in comparison with G-CSF-mobilized peripheral blood. Blood 1996;88(suppl 1):446.

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accepted for publication January 19, 1999.



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