Cord blood is better than bone marrow for generating megakaryocytic progenitor cells

Experimental Hematology 27 (1999) 293–301

MEGAKARYOCYTOPOIESIS

Cord blood is better than bone marrow for generating megakaryocytic progenitor cells Helen Taoa, Leonie Gaudrya, Alison Riceb, and Beng Chonga a

UNSW Centre for Thrombosis & Vascular Research, Department of Haematology, Prince Wales of Hospital, Randwick, Australia Blood & Marrow Transplant Laboratories, Children’s Cancer Research Institute, Sydney Children’s Hospital, Sydney, Australia

b

(Received 13 April 1998; revised 1 July 1998; accepted 9 July 1998)

Thrombocytopenia remains an important problem for patients post high-dose chemotherapy and hematopoietic stem cell transplantation. The study of megakaryocytes, the direct precursors of platelets, has been hampered by their relatively low frequency in hematopoietic tissues. In an attempt to obtain a large number of functional megakaryocytic cells, we established a serum-free culture system to grow megakaryocytic progenitor cells derived from normal human bone marrow (BM) and cord blood (CB). Highly purified (purity .95%) CD341 cells were obtained using magnetic cell sorting (MACS) followed by fluorescence activated cell sorting (FACS). The cells were cultured in a serum-free culture system for 3 weeks in the presence of a single dose of MGDF (50 ng/mL). On days 0, 5, 8, 12, 14, 18, and 21 of culture, the cellularity and morphology were examined. Megakaryocytic cells were monitored by detecting the expression of GPIIIa (CD61), GPIIb/IIIa (CD41) and GPIb (CD42b), and the distribution of megakaryocyte (MK) ploidy was analyzed by two-color flow cytometry. MGDF alone induced maximal nucleated cell expansion at day 14, resulting in a 38.20 6 10.47-fold increase in cell number for CB and a 5.08 6 1.30-fold increase in cell number for BM. On day 14 of the culture, the percentage of CD411/CD142 cells derived from CB reached 73.54% 6 6.01% giving an absolute number of CD411/CD142 cells of 27.25 6 2.23 3 104/mL (27,250-fold increase), whilst the percentage of CD411/CD142 cells derived from BM was only 29.21% 6 5.63% with an absolute number of 1.36 6 0.26 3 104/mL (680-fold increase). Increased expression of GPIIIa occurred the earliest in culture, followed by GPIIb/IIIa, and then GPIb. The majority (81.6%– 92.6%) of megakaryocytes (CD411 cells) on day 14 of culture were 2N, although we did detect some 4N, 8N and greater ploidy cells. In conclusion, CD341 cells stimulated by MGDF alone generated highly enriched MK progenitor cells at day 14 of serum-free culture. CB stem and progenitor cells have a greater proliferative response to MGDF alone than those de-

Offprint requests to: Beng Chong, MBBS, Ph.D., UNSW Centre for Thrombosis & Vascular Research, Department of Haematology, Prince Wales of Hospital, High Street, Randwick, NSW 2031 Australia; E-mail: b.h.chong @unsw.edu.au

rived from BM and may, therefore, prove to be a better source of cells for MK expansion. © 1999 International Society for Experimental Hematology. Published by Elseiver Science Inc. Keywords: Cord blood—Megakaryocytes—MGDF—Ex vivo expansion

Introduction The immediate danger following high-dose chemoradiotherapy is severe bone marrow aplasia. Hematopoietic stem and progenitor cell transplantation has been demonstrated to speed up neutrophil reconstitution. However, the time to recover adequate platelet levels is significantly longer. The need for new ways to rapidly accelerate platelet engraftment is required. To achieve sufficient, functional hematopoietic stem and progenitor cells for transplantation, a cytokine induced mobilization strategy has been used since the late 1980s. In recent years, technology for ex vivo expansion of hematopoietic stem cells has been developed [1]. Utilizing synergistic combinations of recombinant human hematopoietic growth factors, significant numbers of hematopoietic progenitor cells can be generated in vitro from stem cell populations. More recently, the ex vivo expansion technique has been used successfully to expand the megakaryocyte (MK) population by culturing CD341 cells derived from human bone marrow (BM) [2,3], CB [4–6] and PB [7,8] in the presence of different combinations of hematopoietic growth factors. Moreover, the expanded PB MKs have been safely administered to autologous transplant recipients [8]. Umbilical cord blood (CB) is rich in hematopoietic stem cells [9,10] and has been successfully used as an alternative source of stem cells to reconstitute hematopoiesis after myeloablative therapy [10]. Ex vivo expansion studies using CB suggest that CB stem cells have a higher proliferative capacity in response to cytokine stimulation [11]. A number of cytokines are responsible for the regulation of megakaryocytopoiesis and platelet production such as

0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(98)0 0 0 5 0 - 2

294

H. Tao et al./Experimental Hematology 27 (1999) 293–301

SCF, FLT3-ligand, IL-3, IL-6 and IL-11 [12–14]. Recently cloned and purified thrombopoietin (TPO) or megakaryocyte growth and development factor (MGDF) [15] has been demonstrated to be the major growth factor for the megakaryocytic lineage as it is capable of acting at different levels of megakaryocytopoiesis [16,17]. Furthermore, some investigators have observed accelerated recovery of platelets after BM or PB transplantation in animals administered with PEG-rHuMGDF [18] or who had received hematopoietic stem cells derived from Mp1-ligand pretreated donor animals [19]. The potential of CB stem cells to provide a large number of megakaryocytic cells has not been fully investigated. In this study, we established a serum-free culture system supplemented with a single dose of PEG-rHu MGDF to expand megakaryocytic progenitor cells derived from normal human BM and CB stem cells. The results show that CB stem and progenitor cells have a greater proliferative response to MGDF alone than those derived from BM and could therefore prove to be a better source for megakaryocyte expansion.

Materials and methods Human CB and BM collection Prior to all collections, written informed consent was obtained. CB was collected, following full-term vaginal delivery of the babies, into transfer bags containing Acid Citrate Dextrose (ACD; pediatric collection bags, Baxter), kept at 48C and processed within 24 hours. BM was aspirated at the posterior iliac crest from hematologically normal donors under local anaesthesia. CB and BM collections were approved by both the University of New South Wales Committee on Experimental Procedures Involving Human Subjects and the Research Ethics Committee of the Eastern Sydney Area Health Service. Monoclonal antibodies Fluorescein (FITC) conjugated CD34, and phycoerythrin (PE) conjugated CD3, CD4, CD13, CD14, CD16, CD19, CD33, CD34, CD56, and IgG1 monoclonal antibodies (mAb) were purchased from Becton Dickinson (San Jose, CA). PE conjugated CD71 mAb was purchased from Caltag Laboratories (San Francisco, CA). FITC conjugated CD41, CD61, CD42b and IgG1 mAb were obtained from DAKO corporation (Carpinteria, CA). Isolation of CD341 cells Whole CB (mean vol 5 50 mL) or BM (mean vol 5 30 mL) were diluted with Ca11 and Mg11 free phosphate buffered saline (PBS) supplemented with 0.5% bovine serum albumin (BSA) and 2 mM EDTA, designated MACS buffer. Mononuclear cells (MNC) were obtained by Ficoll-Paque (Pharmacia, Sweden) density (1.077g/ cm3) gradient separation. CB and BM CD341 cells were isolated by a two-step positive selection procedure. CD341 cells were firstly enriched from MNC populations using the magnetic cell sorting system (MACS; Miltenyi Biotec, Germany) according to the manufacturer’s instructions, and then purified by fluorescence activated cell sorting (FACS) using CD34-FITC mAb and a FACStarplus cell sorter (Becton Dickinson). The purities of CD341 cells were 70.19% 6 3.08% in CB (n 5

14) and 68.22% 6 7.37% in BM (n 5 5) post-MACS, and 98.60% 6 0.18% in CB and 97.48% 6 0.71% in BM after FACS. Cell culture The growth of megakaryocytic progenitor cells derived from BM or CB CD341 cells were evaluated using three different culture conditions. Iscove’s modification of Dulbecco’s medium (IMDM; Trace Biosciences, Australia) was used as a basic culture medium supplemented with 2 3 1023 M glutamine, 1 3 1024 M sodium pyruvate, 1 3 1024 M mercaptoethanol, and 100 U/100 mg penicillin/streptomycin. The culture media were completed by adding either 10% heat inactivated foetal calf serum (FCS; GIBCO-BRL, Gaithersburg, MD), 10% AB human platelet poor serum (PPS) prepared according to a modified method of Mitjavila et al. [20], or serum-free supplement (see below). The serum-free liquid culture medium was modified from the method by Plantier et al. [21] and used IMDM supplemented with 1.5% BSA, 300 mg/mL iron saturated human transferrin, 10 mg/ mL insulin, 0.74 mg/mL cholesterol, 0.614 mg/mL oleic acid, 0.74 mg/mL soybean L-phosphatidylcholine (Sigma, MO) and 28 mg/ mL calcium chloride. Purified CD341 cells were seeded at a concentration of 1 3 104 cells/mL in 24-well Costar tissue culture plates at 1 mL per well. Recombinant human MGDF (Amgen, Thousand Oaks, CA), was added into the cultures to a final concentration of 50 ng/mL. The cultures were incubated at 378C in a fully humidified atmosphere containing 5% CO2 for up to 21 days without further addition of medium or MGDF. An aliquot of sorted CD341 cells (for day 0) and cultured cells were harvested at specified intervals, on days 5, 8, 12, 14, 18, and 21 of culture. Total and viable cell counts were performed by trypan blue dye exclusion. Morphology of the cultured cells was investigated by cytocentrifugation followed by Wright’s staining and cell membrane antigen expression was analyzed by flow cytometry. Phenotype analysis The growth of megakaryocytic cells in liquid culture were monitored using two-color flow cytometric technique by detecting the expression of megakaryocytic lineage specific antigens. The cultured cells were harvested at different time points, washed with a modified megakaryocyte medium (MK medium) [22], which consisted of Ca11 and Mg11-free PBS containing 13.6 mmol/L tri-sodium citrate, 1 mmol/L theophylline, 11 mmol/L glucose, and 1% BSA, adjusted to pH 7.3 and osmolarity 290 mOsm/L. After centrifugation at 100 g for 10 minutes at 48C, cells were suspended in MK medium at a concentration of 1 3 106/mL and incubated with normal human AB plasma at a final dilution of 1:10 at 48C for 30 minutes to block nonspecific binding. After washing, ,1 3 106 cells for each aliquot were resuspended in 50 ml of MK medium. Cells were then labeled with 5 mL of either FITC-anti CD41, CD61 or CD42b mAb to identify megakaryocytic cells and 5 mL of PE-anti CD14 mAb to exclude monocytes with platelets attached [23], labeled with FITC and PE-mouse IgG1 as a negative control. After incubation in the dark for 30 minutes at 48C, cells were washed, resuspended in MK medium, and then analyzed by flow cytometry within 2 hours. Prior to analysis, 200 mL of propidium iodide (PI, Sigma) at a concentration of 5 mg/mL in PBS were added to each sample to exclude nonviable cells. At least 20,000 viable cells for each sample were acquired and analyzed using Winlist for Win 16 software (Verity Software House, Top-

H. Tao et al./Experimental Hematology 27 (1999) 293–301

sham, ME). The CD411/CD142, CD611/CD142, or CD42b1/ CD142 cells were considered to be megakaryocytic cells. Megakaryocyte ploidy measurement Megakaryocyte ploidy was analyzed using a modified two-color flow cytometric technique described by Tomer et al. [22]. After blocking with AB plasma, the cells were incubated with 5 ml of FITC-anti CD41, CD61, CD42b or IgG1 mAb at 48C for 30 minutes in the dark, pelleted, and resuspended in MK medium. The labeled cells were then fixed at 48C for 30 minutes with 1% paraformaldehyde (BDH). The fixed cells were washed and resuspended in 1 mL of PBS containing 0.1% sodium citrate, 0.1% Triton X-100 (Sigma) and 50 mg PI and kept at 48C overnight in the dark. Prior to analysis, 50 mg of RNase (Sigma) were added to the cell suspensions. At least 25,000 cells were analyzed for each sample by FACStarplus. The ploidy distribution of CD411, CD611 or CD42b1 cells were monitored. The proportion of megakaryocytes in each ploidy class was determined by setting markers at the nadirs between peaks. Statistical analysis Results expressed as the mean 6 standard error of the mean (SEM) were obtained from 14 experiments for CB and 5 experiments for BM performed on separate occasions. Statworks software was used to evaluate statistical significance. The significance of differences in the means was determined using the two-tailed Student’s t-test for paired and unpaired samples and values of p , 0.05 were considered to be statistically significant.

Results Optimal starting cell population for megakaryocyte growth To choose the optimal cell type for maximally generating megakaryocytic cells, CB mononuclear cells obtained after ficoll density centrifugation, CD342 cells obtained from

Figure 1. Comparison of starting cell types. CB mononuclear, CD342 and CD341 cells were stimulated with a single dose of MGDF at 50 ng/mL in serum-free culture medium for 8, 12, and 16 days. CD411/CD142 cells were monitored by flow cytometry. The values shown are the typical results from one of three experiments.

295

MACS and CD341 cells obtained from MACS and FACS were cultured in serum-free medium in the presence of MGDF at a concentration of 50 ng/mL. The cultured cells were sampled at days 8, 12, and 16 of culture. Megakaryocyte growth was monitored by detection of CD411/CD142 cells. Optimal growth of megakaryocytes in vitro was observed in the CD341 cell population (Fig. 1). All subsequent experiments were carried out with CD341 cells. Optimal culture medium for megakaryocyte growth Three different culture media were tested for their ability to support megakaryocyte growth. Purified CB CD341 cells were cultured in either serum-free medium or IMDM supplemented with 10% FCS or 10% PPS in the presence of MGDF at a concentration of 50 ng/mL. Megakaryocyte growth was monitored by detection of CD411/CD142 cells. Optimal growth of megakaryocytes in vitro was observed in serum-free medium (Fig. 2). All subsequent experiments were carried out using serum-free medium. Changes in viability and cellularity during in vitro culture Highly viable CB and BM CD341 cells were seeded at 0.97 6 0.02 3 104 cells per mL for CB and 0.92 6 0.06 3 104 cells per mL for BM. The cultures were sampled on days 5, 8, 12, 14, 18, and 21 to monitor viability and cellularity. Cell viability remained high (.90%) until day 12 of culture for CB and day 14 for BM. After day 14, viability declined more rapidly in CB than in BM samples. Viability dropped to 39.8% 6 5.48% in CB and 65.7% 6 5.70% in BM by day 21 of culture (p 5 0.05). MGDF (50 ng/mL) alone induced maximal growth of total viable nucleated cells on day 14 of culture in both CB (37.05 6 10.16 3 104/mL) and BM

Figure 2. Comparison of megakaryocyte culture medium. CB CD341 cells were stimulated with a single dose of MGDF at 50 ng/mL in three different culture medium for 8 and 12 days. CD411/CD142 cells were monitored by flow cytometry. The values shown are the typeical results from one of three experiments.

296

H. Tao et al./Experimental Hematology 27 (1999) 293–301

Figure 3. Growth of CD411/CD142 cells. CD CD341 cells were stimulated with a single dose of MGDF at 50 ng/mL in serum-free medium for 21 days. Cells were sampled at specified intervals of culture and labeled with CD41-FITC plus CD14-PE. CD411/CD142 cells were monitored.

(4.67 6 1.20 3 104/mL) samples, resulting in a 38.20 6 10.47-fold increase in cell number for CB and 5.08 6 1.30fold increase for BM over input numbers. There was a significant difference between the two populations (p 5 0.046). Growth of megakaryocytic cells during in vitro culture Growth of megakaryocytic cells was observed by monitoring CD41 expression by flow cytometry. Prior to culture (day 0), the percentage of CD411/CD142 cells in CB was 0.13% 6 0.05% with an absolute number of 0.001 6 0.0005 3 104/mL. The percentage of CD411/CD142 cells in BM was 0.17% 6 0.05% with an absolute number of 0.002 6 0.0005 3 104/mL. Data showed that an increase in expression of CD41 on cells derived from CB and BM CD341 cells started from day 5 of culture, rose steadily and peaked at day 14, before gradually decreasing (Fig. 3). When expansion of CD411/CD142 cells derived from CB and BM CD341 cells was compared, significant differences were observed between the two samples at day 14 of culture (Fig. 4). The percentage of CD411/CD142 cells derived from CB reached 73.54% 6 6.01% giving an absolute num-

Figure 4. CD411/CD142 cell expansion. The CD341 cells derived from either CB or BM were stimulated with a single dose of MGDF at 50 ng/mL in serum-free medium for 21 days. Cells were sampled at specified intervals of culture. CD411/CD142 cells were monitored. (A) Shows the percentage of CD411/CD142 cells detected: *p 5 0.034 (D8), 0.004 (D12), 0.003 (D14). (B) Shows the absolute number of CD411/CD142 cells in 1 mL culture: *p 5 0.020 (D12), 0.045 (D14). The values shown are the mean 6 SEM of 14 and 5 experiments from CB and BM samples, respectively.

ber of 27.25 6 2.23 3 104/mL (27,250-fold increase over the input number), while the percentage of CD411/CD142 cells derived from BM was only 29.21% 6 5.63% with an absolute number of 1.36 6 0.26 3 104/mL (680-fold increase). A statistical significance (p , 0.05) was observed on day 12 and day 14 of culture between CB and BM. Distinction between different megakaryocytic lineage markers We monitored the antigenic expression of different megakaryocytic lineage markers (CD41, CD61 and CD42b) on days 0, 5, 8, 12, 14, 18, and 21 of culture. Data showed increased expression of CD61 occurred earliest in culture, followed by CD41, then CD42b (Fig. 5). There was a statistically significant difference between the expression levels of CD61 and CD41 on days 8 (p 5 0.005), 12 (p 5 0.004), and 14 (p 5 0.001).

H. Tao et al./Experimental Hematology 27 (1999) 293–301

297

CD41 and simultaneously with either CD34, CD33, CD3, CD4, CD13, CD16, CD19, CD56 or CD71, respectively. Figure 8 shows the changes in the different antigenic markers over the 3-week culture period. Data showed that concomitant with the expansion of megakaryocytic (CD411/ CD142) cells, CD341 (CD341/CD412) cells declined. Cells expressing the CD33, CD13 or CD71 antigen (CD331/ CD412, CD131/CD412, and CD711/CD412 cells) were present in the culture, whereas cells expressing the CD4 antigen (CD41/CD412 cells) were only present in the early stage of culture and cells expressing CD3, CD16, CD19 or CD56 antigen (CD31/CD412, CD161/CD412, CD191/ CD412, and CD561/CD412 cells) never appeared in our culture system.

Figure 5. Expression of megakaryocytic lineage markers. CB CD341 cells were stimulated with a single dose of MGDF at 50 ng/mL in serum-free medium for 21 days. Cells were sampled at specified intervals of culture. CD611/CD142, CD411/CD142, and CD42b1/CD142 cells were analyzed. (A) Shows the percentage of positive cells detected. (B) Shows the absolute number of positive cells in 1 mL culture. The values shown are the mean 6 SEM of 14 experiments from CB samples.

Effect of MGDF on megakaryocyte ploidy distribution We investigated the effect of MGDF on megakaryocyte ploidy distribution. On days 0, 5, 8, 12, 14, 18, and 21 of culture, the DNA content of CD411 cells was measured. Figure 6 shows the ploidy distribution of CD411 megakaryocytes derived from CB CD341 cells. Figure 7 shows the changes in megakaryocyte ploidy distribution derived from CB CD341 cells. The 2N and 4N cells make up the majority of cells present over the 3-week culture period. Changes in antigenic expression following culture with MGDF We investigated the kinetics of nonmegakaryocytic lineage cells derived from CB CD341 cells during in vitro culture. At the specified intervals of cultures, cells were labeled with

Discussion Thrombocytopenia remains a significant problem for patients post-high-dose chemotherapy and hematopoietic stem cell transplantation. To accelerate slow platelet engraftment, two strategies have been considered: 1) administration of MGDF alone or in combination with G-CSF to patients to directly stimulate hematopoiesis in vivo [24]; and 2) transfusion of ex vivo expanded megakaryocytic cells into patients to shorten the time to platelet recovery [8]. In vitro manipulation of megakaryocytic cells for transplant use requires a culture system that can provide a large number of functional megakaryocytic cells at different stages of maturation. This study has addressed some of these issues. To obtain a large number of functional megakaryocytes in a controlled manner, the starting cell population, culture medium, and cytokines used are significant issues. It has been reported that pure CD341 cells have greater cytokine mediated expansion potential and are essential for optimal ex vivo expansion of hematopoietic cells [25]. We found that highly purified (purity .95%) CD341 cell populations can give rise to larger numbers of megakaryocytes than CD342 cells and mononuclear cells, suggesting that CD341 cells are suitable targets for stimulating megakaryocytopoiesis. We have shown that optimal proliferation and differentiation of megakaryocytic progenitor cells was obtained in serum-free culture medium. This result is consistant with other studies [8,26], indicating that megakaryocyte growth inhibitors may be present in serum or plasma. Some studies have shown that to grow megakaryocyte progenitor cells in vitro, the culture system needs to be refed with fresh medium and cytokines, and split twice weekly [4,7,8]. Our study showed that cellular viability remained high until day 12 for CB and day 14 for BM, suggesting that our culture system supplemented with a single dose of MGDF is satisfactory to support the growth of CD341 cell progeny for at least a 2-week period. MGDF alone or in combination with other cytokines has been reported to mediate ex vivo expansion of megakaryo-

298

H. Tao et al./Experimental Hematology 27 (1999) 293–301

Figure 6. Measurement of megakaryocyte ploidy distribution. CD341 cells derived from CB were stimulated with a single dose of MGDF at 50 ng/mL in serum-free medium for 8 days. Cells were sampled and megakaryocyte ploidy distribution was analyzed by two-color flow cytrometry. The left panel shows the control sample labeled with IgG and PI. The right panel shows the test sample labeled with CD41 antibody and PI.

Figure 7. Effect of MGDF on megakaryocyte ploidy distribution. CD341 cells derived from CB were stimulated with a single dose of MGDF at 50 ng/mL in serum-free medium for 21 days. Cells were sampled at specified intervals of culture and labeled with CD41 antibody and PI. Megakaryocyte ploidy distribution was analyzed.

H. Tao et al./Experimental Hematology 27 (1999) 293–301

299

Figure 8. Detection of nonmegakaryocytic lineage cells in culture. CB CD341 cells were stimulated with a single dose of MGDF at 50 ng/mL in serum-free medium for 21 days. Cells were sampled at specified intervals of culture. Cells were labeled with CD41-FITC and either CD34, 3, 4, 13, 16, 19, 33, 56, or 71 PE. The values shown are the percentage of positive cells detected.

cyte progenitor cells [2–8]. Guerriero and his colleagues [7] demonstrated that using a culture system refed with fresh medium and cytokines, MGDF alone induced a highly enriched (97%–99%) megakaryocyte population from PB hematopoietic progenitor cells at day 12 of culture. Our results using a single dose of MGDF alone and serum-free culture medium without refeeding enabled the production of a satisfactory number of highly enriched megakaryocyte progenitors from both CB and BM CD341 cells by 14 days. Analysis of megakaryocyte ploidy distribution showed that the majority of the megakaryocytes generated by a single dose of MGDF at day 14 were 2N, and this has been confirmed by morphologic studies. These results suggest that a single dose of MGDF is satisfactory for supporting the proliferation and differentiation of megakaryocytic cells but not their maturation. Our observation is consistent with other studies using colony assay in which thrombopoietin alone stimulated the early proliferation and differentiation of megakaryocyte progenitor cells [6]. Evaluation of megakaryocytic lineage markers showed that the antigenic expression of lineage markers are distinctive in the developmental sequence of megakaryocytic cells. GPIIIa is an early lineage marker and its expression pre-

ceded both GPIIb/IIIa and GPIb. This is consistent with some previous observations [27], but inconsistent with Ayala et al. [4] who showed that GPIIb/IIIa appeared before GPIIIa and GPIb. Our study showed that the cells expressing GPIIIa are the most prevalent, followed by GPIIb/IIIa cells and then GPIb cells. GPIIb/IIIa has been thought to be a specific marker restricted to the megakaryocytic lineage and can be used to identify megakaryocytes [28]. Furthermore, GPIIIa has been detected on endothelial cells, monocytes, and fibroblasts as well as megakaryocytes [29,30]. Our data suggests that GPIIIa is a sensitive, but not specific marker for the megakaryocytic lineage. We monitored the growth of cells from other lineages in the MGDF stimulated serum-free culture system. While megakaryocytes expanded, CD341 cells declined, a finding consistant with other reports [4,7]. Some proliferating erythroid cells (CD711) and myeloid cells (CD331 and CD131) were present in the culture system. Except for a small portion of CD41 cells seen in the early stages of culture, most other lymphoid cells were not detected. These results suggest that the serum-free culture medium we used is specific for the growth of megakaryocytes in vitro. It has been reported that CB contains higher proportions

300

H. Tao et al./Experimental Hematology 27 (1999) 293–301

of CD341/CD382 and CD341/CD332 cells than BM [31] and that CD341/CD382 cells proliferate more rapidly in response to cytokine stimulation and generate more progeny than their BM counterparts [32]. In this study, CB and BM derived CD341 cells were compared for their ability to generate megakaryocytes and striking differences were observed between the two CD341 cell populations. Our data clearly indicate that at day 14 of serum-free stromal-free culture, CB CD341 cells generated significantly greater numbers of total nucleated cells and megakaryocytic cells as compared with those derived from BM. This indicates that CB stem and progenitor cells have a greater proliferative response to MGDF than BM CD341 cells and could, therefore, prove to be a better source of cells for megakaryocyte expansion. In conclusion, CD341 cells stimulated by MGDF alone generated highly enriched megakaryocyte progenitors at day 14 of serum-free culture. CB stem and progenitor cells have a greater proliferative response to MGDF alone than those derived from BM. CB is, therefore, a better source for providing a large number of functional megakaryocytes.

9.

10.

11.

12.

13.

14.

15.

Acknowledgments

16.

The authors would like to acknowledge the Australian Cord Blood Bank for providing cord blood and Amgen Inc for supplying PEGrHuMGDF. This study is supported in part by a NHMRC program grant.

17.

References 1. Haylock DN, To LB, Dowse TL, Juttner CA, Simmons PJ (1992) Ex vivo expansion and maturation of peripheral blood CD341 cells into the myeloid lineage. Blood 80:1405 2. Nagler A, Eldor A, Naparstek E, Mumcuoglu M, Slavin S, Deutsch VR (1995) Ex vivo expansion of megakaryocyte precursors by preincubation of marrow allografts with interleukin-3 and granulocyte-macrophage colony-stimulating factor in vitro. Exp Hematol 23:1268 3. Schattner M, Lefebvre P, Mingolelli SS, Goolsby CL, Rademaker A, White JG, Foster D, Green D, Cohen I (1996) Thrombopoietin-stimulated ex vivo expansion of human bone marrow megakaryocytes. Stem Cells 14:207 4. Ayala IA, Tomer A, Kellar KL (1996) Flow cytometric analysis of megakaryocyte-associated antigens on CD34 cells and their progeny in liquid culture. Stem Cells 14:320 5. Ohmizono Y, Sakabe H, Kimura T, Tanimukai S, Matsumura T, Miyazaki H, Lyman SD, Sonoda Y (1997) Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand. Leukemia 11:524 6. Higuchi T, Koike K, Sawai N, Koike T (1997) Proliferative and differentiative potential of thrombopoietin-responsive precursors: expression of megakaryocytic and erythroid lineages. Exp Hematol 25:463 7. Guerriero R, Testa U, Gabbianelli M, Mattia G, Montesoro E, Macioce G, Pace A, Ziegler B, Hassan HJ, Peschle C (1995) Unilineage megakaryocytic proliferation and differentiation of purified hematopoietic progenitors in serum-free liquid culture. Blood 86:3725 8. Bertolini F, Battaglia M, Pedrazzoli P, Da Prada GA, Lanza A, Soligo D, Caneva L, Sarina B, Murphy S, Thomas T, della Cuna GR (1997) Megakaryocytic progenitors can be generated ex vivo and safely ad-

18.

19.

20.

21.

22. 23.

24.

25.

ministered to autologous peripheral blood progenitor cell transplant recipients. Blood 89:2679 Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D, Arny M, Thomas L, Boyse EA (1989) Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 86:3828 Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE, Gluckman E (1995) Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 346:214 Emerson SG (1996) Ex vivo expansion of hematopoietic precursors, progenitors, and stem cells: the next generation of cellular therapeutics. Blood 87:3082 Avraham H, Vannier E, Cowley S, Jiang S, Chi S, Dinarello CA, Zsebo KM, Groopman JE (1992) Effects of the stem cell factor, c-kit ligand, on human megakaryocytic cells. Blood 79:365 Piacibello W, Garetto L, Sanavio F, Severino A, Fubini L, Stacchini A, Dragonetti G, Aglietta M (1996) The effects of human FLT3 ligand on in vitro human megakaryocytopoiesis. Exp Hematol 24:340 Orazi A, Cooper RJ, Tong J, Gordon MS, Battiato L, Sledge GW, Kaye JA, Kahsai M, Hoffman R (1996) Effects of recombinant human interleukin-11 (Neumega™ rhIL-11 growth factor) on megakaryocytopoiesis in human bone marrow. Exp Hematol 24:1289 de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang W, Oles KJ, Hultgren B, Solberg LAJ, Goeddel DV, Eaton DL (1994) Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand. Nature 369:533 Debili N, Wendling F, Katz A, Guichard J, Breton-Gorius J, Hunt P, Vainchenker W (1995) The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 86:2516 Choi ES, Hokom M, Bartley T, Li YS, Ohashi H, Kato T, Nichol JL, Skrine J, Knudten A, Chen J, Hornkohl A, Grampp G, Sleeman L, Cole S, Trail G, Hunt P (1995) Recombinant human megakaryocyte growth and development factor (rHuMGDF), a ligand for c-Mpl, produces functional platelets in vitro. Stem Cells 13:317 Molineux G, Hartley C, McElroy P, McCrea C, McNiece IK (1996) Megakaryocyte growth and development factor accelerates platelet recovery in peripheral blood progenitor cell transplant recipients. Blood 86:366 Fibbe WE, Heemskerk DPM, Laterveer L, Pruijt JFM, Foster D, Kaushansky K, Willemze R (1995) Accelerated reconstitution of platelets and erythrocytes after syngenetic transplantation of bone marrow cells derived from thrombopoietin pretreated donor mice. Blood 86:3308 Mitjavila MT, Vinci G, Villeval JL, Kieffer N, Henri A, Testa U, Breton-Gorius J, Vainchenker W (1988) Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formation: its relationship to type b transforming growth factor (TGF-b). J Cell Physio 134:93 Plantier JL, Berthier R, Rival Y, Schweitzer A, Rabiet MJ (1994) Evidence for a selective inhibitory effect of thrombin on megakaryocyte progenitor growth mediated by the thrombin receptor. Br J Haematol 87:755 Tomer A, Harker LA, Burstein SA (1988) Flow cytometric analysis of normal human megakaryocytes. Blood 71:1244 Dolzhanskiy A, Basch RS, Karpatkin S (1996) Development of human megakaryocytes: I. Hematopoietic progenitors (CD341 bone marrow cells) are enriched with megakaryocytes expressing CD4. Blood 87:1353 Basser RL, Rasko JEJ, Clarke K, Cebon J, Green MD, Hussein S, Alt C, Menchaca D, Tomita D, Marty J, Fox RM, Begley CG (1996) Thrombopoietic effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) in patients with advanced cancer. Lancet 348:1279 Case J, Rice A, Vowels M (1997) Cytokine mediated expansion of hu-

H. Tao et al./Experimental Hematology 27 (1999) 293–301 man umibilical cord blood CD341 cells: comparison of the use of partially purified and pure CD341 target cells. Hematol Cell Ther 39:193 26. Berthier R, Valiron O, Schweitzer A, Marguerie G (1993) Serum-free medium allows the optimal growth of human megakaryocyte progenitors compared with human plasma supplemented cultures:role of TGFb. Stem Cells 11:120 27. Vinci G, Tabilio A, Deschamps JF, Van Haeke D, Henri A, Guichard J, Tetteroo P, Lansdorp PM, Hercend T, Vainchenker W, BretonGorius J (1984) Immunological study of in vitro maturation of human megakaryocytes. Br J Haematol 56:589 28. Vainchenker W, Debili N, Mouthon MA, Wendling F (1995) Megakaryocytopoiesis: cellular aspects and regulation. Crit Rev Oncol Hematol 20:165

301

29. Thiagarajan P, Shapiro SS, Levine E, DeMarco L, Yalcin A (1985) A monoclonal antibody to human platelet glycoprotein IIIa detects a related protein in cultured human endothelial cells. J Clin Invest 75:896 30. Bai Y, Durbin H, Hogg N (1984) Monoclonal antibodies specific for platelet glycoproteins react with human monocytes. Blood 64:139 31. De Bruyn C, Delforge A, Bron D, Bernier M, Massy M, Ley P, de Hemptinne D, Strykmans P (1995) Comparison of the coexpression of CD38, CD33, and HLA-DR antigens on CD341 purified cells from human cord blood and bone marrow. Stem Cells 13:281 32. Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM (1995) A functional comparison of CD341 CD382 cells in cord blood and bone marrow. Blood 86:3745