Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sawai, N.
Right arrow Articles by Komiyama, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sawai, N.
Right arrow Articles by Komiyama, A.
Stem Cells, Vol. 17, No. 1, 45-54, January 1999
© 1999 AlphaMed Press

Apoptosis of Erythroid Precursors under Stimulation with Thrombopoietin: Contribution to Megakaryocytic Lineage Choice

Nobukuni Sawaia, Kenichi Koikea, Susumu Itob, Yumi Kurokawaa, Hadija Hemed Mwamtemia, Tatsuya Kinoshitaa, Kazuo Sakashitaa, Tsukasa Higuchia, Kouichi Takeuchia, Masaaki Shioharaa, Kinya Ogamic, Atsushi Komiyamaa

a Department of Pediatrics, Shinshu University School of Medicine, Matsumoto, Japan;
b Blood Transfusion Service, Shinshu University Hospital, Matsumoto, Japan;
c Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Takasaki, Japan

Key Words. Thrombopoietin • Apoptosis • CD34–/CD41– cells • c-Mpl • Erythroid differentiation • Cord blood cells

Dr. Kenichi Koike, Department of Pediatrics, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the effect of thrombopoietin (TPO) on megakaryocyte production is well established, its role in the commitment of multipotential hematopoietic progenitors to the megakaryocytic lineage remains to be determined. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO. Day 7 cultured cells grown by TPO derived from cord blood CD34+ cells were divided into four subpopulations on the basis of CD34 and CD41 expression. The CD34–/CD41– cells showed the labeling pattern of anti-CD42b and anti-CD9 antibodies closer to that of the CD34+/CD41– cells than the CD34+/CD41+ cells. Replating experiments revealed that approximately 40% of the CD34–/CD41– cells proliferated in response to a combination of growth factors, and more than 80% of them were pure erythroid precursors. However, this subpopulation failed to grow/survive and fell into apoptosis in the presence of TPO alone. In contrast, the CD34+/CD41+ cells, which predominantly contained megakaryocytic precursors, exerted a low but significant proliferative potential in the presence of TPO. The insufficient response to TPO of the CD34–/CD41– cells may result from the apparently low expression of c-Mpl, as determined by flow cytometric analysis and reverse transcription-polymerase chain reaction analysis. Therefore, these results suggest that the apoptosis of hematopoietic precursors other than megakaryocytic precursors is related to the determination of the terminal differentiation under the influence of TPO.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Erythropoiesis involves a great variety and number of cells at different stages of maturation, starting with the stem cell progeny committed to erythroid differentiation and ending with the mature circulating red cell. The growth of erythroid burst-forming units (BFU-E) requires interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) as well as erythropoietin (EPO). Erythroid colony-forming units (CFU-E) are highly responsive to low concentrations of EPO. The spectrum of maturing megakaryocytes is produced by megakaryocytic progenitors. This process is believed to be regulated by several growth factors. Among them, thrombopoietin (TPO), c-mpl ligand, was cloned by several independent groups [1-5] and demonstrated to stimulate the proliferation and differentiation of megakaryocytic progenitors in vitro. In addition, the administration of TPO causes an increase in the peripheral blood platelet count but not in erythrocyte and leukocyte counts in normal mice [6].

Although numerous experiments showed a close relationship between erythropoiesis and megakaryocytopoiesis, the process of the commitment of stem cell progeny to the erythroid or megakaryocytic pathway is poorly understood. Our recent study revealed that TPO alone generates precursors with the potential to differentiate into erythroid and megakaryocytic lineages [7]. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO, by means of kinetic study of hematopoietic precursors generated by TPO from cord blood CD34+ cells in a serum-deprived liquid culture system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Factors and Antibodies
Human recombinant TPO, SCF, IL-3, GM-CSF, and EPO were generously provided by Kirin Brewery Co. Ltd. (Takasaki, Japan). Human recombinant granulocyte colony-stimulating factor (G-CSF) was provided by Chugai Pharmaceutical Co. (Tokyo, Japan).

For the flow cytometric analysis, the monoclonal antibodies (mAbs) for CD34 (HPCA-2, fluorescein isothiocyanate [FITC], phycoerythrin [PE]), CD11b (Leu15, PE), HLA-DR (L243, PE), and CD38 (Leu17, PE) were purchased from Becton Dickinson Immunocytometry Systems (Mountain View, CA). The mAbs for CD41 (SZ.22, Biotin), c-kit (95C3, PE), CD36 (FA6 152, FITC), and CD9 (ALB-6 FITC) were purchased from Immunotech S.A. (Marseilles, France). The mAbs for CD42b (AN51, PE) and glycophorin A (GPA; JC159, FITC) were from DAKO (Glostrup, Denmark). The mAb for CD41 (TP80, FITC) was from Nichirei (Tokyo, Japan). For the multicolor analysis, Cy-Chrome-labeled streptavidin was purchased from PharMingen (San Diego, CA). An mAb against human c-Mpl domain 1, M1 [8] was obtained from Genzyme Co. (Cambridge, MA). In our preliminary experiments, the Western blot analysis revealed that M1 mAb recognized the c-Mpl with the molecular weight of 82 kDa in a platelet lysate.

For the immunocytochemical analysis, a purified mAb for CD2 (T11) was purchased from Coulter (Miami, FL); the mAbs for CD11b (2LPM19c), CD14 (TÜK4), CD15 (C3D-1), CD19 (HD37), and GPA (JC159) were from DAKO.

Cell Preparation
Cord blood samples were aspirated in heparinized plastic syringes from the umbilical vein at normal delivery. Fully informed consent was obtained from the mothers of all neonates before the specimens were harvested. Mononuclear cells were separated by density centrifugation over Ficoll-Paque (Pharmacia Fine Chemicals; Piscataway, NJ), washed twice, and suspended in Ca2+- and Mg2+-free cold phosphate-buffered saline (PBS) containing 1 mmol/L EDTA-2 Na, and 2.5% fetal bovine serum (Hyclone; Logan, UT). After treatment with Silica (Immuno Biological Laboratories; Fujioka, Japan) for 30 min at 37°C, the CD34+ cells were enriched using a Dynal CD34 Progenitor Cell Selection System (Dynal A.S.; Oslo, Norway), as described previously [7]. Briefly, 1.0 x 107 cells were mixed with the same number of polystyrene beads coated with an mAb specific for CD34 (Dynabeads M-450 CD34), and incubated for 30 min at 4°C. Bead-rosetted cells were separated by a magnet. For the detachment of the beads from the cells, affinity-purified polyclonal antibodies against the Fab portion of anti-CD34 antibody (Detach-a-bead CD34) were added, and incubation was carried out for 45 min at room temperature. The detached beads were removed by the magnet, and the cells were collected as CD34+ cells. More than 90% of the isolated cells were CD34+, as determined by FACScan flow cytometry (Becton Dickinson).

Liquid Culture System
The serum-deprived liquid culture was carried out in a 24-well culture plate (#3047; Becton Dickinson & Company; Lincoln Park, NJ) using a modification of the technique described previously [9-11]. Two x 104 CD34+ cells were cultured in each well containing 2 ml of alpha medium (Flow Laboratories Inc.; Rockville, MD) supplemented with 1% deionized bovine serum albumin (Sigma Chemical Co.; St Louis, MO), 600 µg/ml fully iron-saturated human transferrin (approximately 98% pure; Sigma), 16 µg/ml soybean lecithin (Sigma), 9.6 µg/ml cholesterol (Nacalai Tesque; Kyoto, Japan), and 10 ng/ml of TPO, or a combination of 10 ng/ml of TPO, 2 U/ml of EPO, 10 ng/ml of GM-CSF, 100 U/ml of IL-3, 10 ng/ml of G-CSF, and 10 ng/ml of SCF (growth factors [GF]). TPO at higher than 5 ng/ml was the optimal concentration, as described previously [7]. The plates were incubated at 37°C in a humidified atmosphere flushed with a mixture of 5% CO2, 5% O2, and 90% N2. On days 4-14, the number of viable cells was determined by a trypan blue exclusion test, using a hemocytometer.

Flow Cytometric Analysis of Cell Surface Markers
To analyze the surface markers on the cells grown by TPO, 1-2 x 106 cells were collected in plastic tubes and incubated with appropriately diluted FITC- or PE-conjugated mAbs. The cells were washed twice, after which their phenotype was analyzed with a FACScan flow cytometer equipped with a 15-mW coherent argon ion laser tuned at 488 nm, using the LYSIS 2 software program (Becton Dickinson). Viable cells were gated according to their forward light scatter characteristics (FSC) and side scatter characteristics (SSC). The proportion of positive cells was determined by comparison with cells stained with FITC- or PE-conjugated mouse IgG (DAKO).

For the multicolor analysis, the cells were stained with a mixture of FITC-conjugated anti-CD34 mAb, PE-conjugated anti-CD38, CD42b, HLA-DR, or c-kit mAb, and biotinized anti-CD41 mAb, or a mixture of PE-conjugated anti-CD34 mAb, FITC-conjugated anti-CD9, CD36 or GPA mAb, and biotinized anti-CD41 mAb, followed by Cy-Chrome-labeled streptavidin. For the analysis of the surface expression of c-Mpl on the cultured cells, 1 x 105 cells were incubated with 20 µl anti-c-Mpl mAb for 30 min at 4°C. Isotype mAb was used as a control. The cells were washed three times, and stained with FITC-conjugated goat anti-mouse immunoglobulin (Becton Dickinson) for 15 min. After the three washings and treatment with mouse serum for 15 min, the cells were stained with PE-conjugated anti-CD34 mAb and biotinized anti-CD41 mAb, followed by Cy-Chrome-labeled streptavidin. PE-conjugated and biotinized isotype antibodies were used as controls. The cells were washed twice and analyzed with a flow cytometer. The FITC, PE, and Cy-Chrome emissions were collected after 525/25, 575/26, and 680/30 nm band pass filters, respectively. Compensation was set using individual cells labeled with FITC, PE, or Cy-Chrome alone. The analysis of the multivariate data was also performed with the LYSIS 2 software.

Cell Sorting
To determine the proliferation and differentiation of the cells grown by TPO, the cells were harvested on day 7 and incubated with PE-conjugated anti-CD34 mAb and FITC-conjugated anti-CD41 mAb at 4°C for 30 min. The cells were divided into four groups: CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ cells. The four subpopulations were individually sorted by a FACStar plus flow cytometer (Becton Dickinson), as described previously [7, 11]. Each group of cells was collected in 5-ml plastic tubes containing the medium. More than 90% of the sorted cells were viable according to a trypan blue exclusion test. After the completion of the liquid cultures with TPO or GF, the cells were collected and analyzed by inverted microscopy and flow cytometry.

Single-Cell Culture
Single-cell sorting was performed by two-step sorting, as described previously [11]. The cells grown by TPO were individually collected in 5 ml-tubes, and were resorted into the individual wells of a 96-well U-bottomed tissue culture plate (#3077; Becton Dickinson) containing GF, using a FACStar plus flow cytometer equipped with an automatic cell deposition unit. Ninety-nine percent of the wells contained a single cell on the first day of culture.

Determination of Colony Types
To identify the colony type, we lifted a colony on day 12, and divided it into two aliquots. One-half of the sample spread on glass slides was stained with May-Grünwald-Giemsa to identify erythroblasts, granulocytes, macrophages, and blasts. The other half was stained using the LSAB Kit with the anti-CD41 mAb for the identification of megakaryocytes. The colony type was classified according to the criteria described previously [12, 13]. The abbreviations used here for the colony types are as follows: M, megakaryocyte; E, erythrocyte; EM, erythrocyte-megakaryocyte; GM, granulocyte-macrophage; Blast, blast cell colonies. Discrete aggregates of four or more cells were scored as M colonies, and the other types of colonies were defined as aggregates of more than 50 cells.

Immunocytochemical Staining
To examine the generation of T cells, B cells, neutrophils, macrophages, and erythroblasts in the culture of CD34+ cord blood cells with TPO, 5 x 103 cultured cells were spread on glass slides using a Cytospin II (Shandon Southern; Sewickly, PA). The samples were fixed with buffered paraformaldehyde-acetone for 30 sec. The immunocytochemical staining was performed with the DAKO LSAB Kit (DAKO), as described previously [10]. Briefly, after treatment with blocking reagents, the specimens were incubated with the cell lineage-specific mAbs for 60 min at room temperature. Next, they were incubated with biotinized goat anti-mouse Ab for 30 min, followed by alkaline phosphatase-conjugated streptavidin for 10 min and substrate-chromogen solution for 15 min. The specimens were counterstained with hematoxylin. Three hundred cells were examined.

Detection of Cellular Apoptosis
The detection of apoptotic cells was achieved by May-Grünwald-Giemsa staining and an ApopDETEK System (Enzo Diagnostics; Farmingdale, NY), as described in the manufacturer's protocol. The latter identified the apoptotic cells by the internucleosomal fragmentation of DNA. A flow cytometric analysis was also carried out using propidium iodide ([PI]; Sigma) according to the procedure described previously [14]. Briefly, cells (1 x 105) were washed with PBS and fixed overnight in 70% ethanol at -20°C. The cells were then washed twice with PBS and resuspended in 1 ml PBS containing 20 µg/ml of PI and 250 µg/ml RNase (Sigma), and incubated for 30 min at room temperature under agitation. The DNA content of 2 x 104 cells was monitored with a flow cytometer.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed according to a modification of the procedure described previously [15]. Total RNA was isolated from day 7 cultured cells grown by TPO, using Isogen (Wako Pure Chemical Industries; Osaka, Japan). Next, 500 ng of RNA was reverse-transcribed in 200 U SuperScriptTM II (Life Technologies; Gaithersburg, MD), 10–7 M oligo dT primer (Takara Shuzo Co.; Ohtsu, Japan), and 10 U RNase inhibitor (Boehringer Mannheim; Mannheim, Germany) in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, and 3 mM MgCl2. The prepared solution was incubated at 37°C for 1 h. PCR amplification was performed using the cDNA corresponding to 25 ng of total RNA, Taq polymerase (Takara Shuzo Co.), and c-Mpl-specific primers according to the manufacturer's instructions. The primers for amplification were 5'-CTTGGTGACCGCTCTGCATCT-3' (nt 1479-1499) and 5'-GAGGATTTCAAGGAGGCTGGG-3' (nt 1713-1693) for P-form of c-Mpl; 5'-CTTGGTGACCGCTCTGCATCT (nt 1479-1499) and 5'-AGAGGTGACGTGCAGGAAGTG-3' (nt 1711-1691) for K-form of c-Mpl. Thirty-five cycles consisting of denaturation at 95°C for 20 seconds, annealing at 61°C for 20 sec, and polymerization at 72°C for 1 min were performed in a GeneAmpTM PCR System 9600 (Perkin-Elmer Cetus; Norwalk, CT). As a control, RT-PCR was performed with the primers specific for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, 5'-AGAAGGCTGGGGCTCATTTG (nt 377-396) and 5'-GCATCCTGGGCTACACTGAG (nt 875-894).

Statistical Analysis
All experiments were carried out at least three times and were shown to be reproducible. Values are expressed as the mean of three experiments. The statistical analysis of four independent groups was performed using the one-way analysis of variance, followed by post hoc contrasts with the Bonferroni limitation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of CD34–/CD41– Cells by Cord Blood CD34+ Cells under Stimulation with TPO
To examine the generation of hematopoietic precursors by cord blood CD34+ cells under stimulation with TPO, we cultured 2 x 104 CD34+ cells in a well containing 10 ng/ml of TPO. The total number of viable cells increased until day 12, with a peak of approximately 18 times the input quantity (the mean of three experiments); the number of viable cells decreased after day 14. No cell growth was seen in the absence of TPO. The expression of CD34 and CD41 antigens on the cultured cells was serially examined by flow cytometry. In addition to the production of CD34+/CD41–, CD34+/CD41+ and CD34–/CD41+ cells, a significant number of CD34–/CD41– cells were generated between day 6 and day 12. As presented in Figure 1, the frequency of CD34–/CD41– cells was 50.3% among all cultured cells on day 7.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 1. Generation of CD34–/CD41– cells by cord blood CD34+ cells under stimulation with TPO. Cord blood CD34+ cells (2 x104/ well) were cultured with 10 ng/ml of TPO for seven days. The expressions of CD34 and CD41 were examined by the flow cytometer. Viable cells were gated according to the FSC and SSC (R1). R2,R3, R4, and R5 correspond to CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ cells, respectively. The results shown are derived from one representative experiment of three.Similar results were obtained in the other two experiments.

 
We carried out the phenotypical analysis of each subpopulation. The results are presented in Figure 2. The comparison of the CD34+/CD41– and CD34–/CD41+ cells showed that CD42b, CD9, and CD36 were maturation markers, whereas the mean fluorescence intensity of the HLA DR+ cells was decreased with the differentiation. Most of the CD34–/CD41– cells were negative for CD42b and CD9. This pattern was similar to that of the CD34+/CD41– cells. The CD34+/CD41+ cells showed intermediate expressions of CD42b and CD9. A wide spectrum of CD36 expression and high HLA-DR antigen density were seen in both the CD34–/CD41– cells and the CD34+/CD41+ cells. None of the four cell subsets expressed T cell, B cell, neutrophil, macrophage, or erythroid lineage markers (CD2, CD19, CD15, CD14, CD11b, and GPA) according to the immunocytochemical studies.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Phenotypic analysis of day 7 CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ cells grown by TPO. The surface expressions of CD42b, CD9, CD36, and HLA-DR were determined by three-coloranalysis. (-) = labeled with FITC- or PE-conjugated anti-CD42b, CD9, CD36, and HLA-DR mAbs;(—-) = labeled with FITC- or PE-conjugated control mouse IgG1. The abscissa shows thefluorescence intensity of each surface marker, and the ordinate shows the number of cells. R2, R3,R4, and R5 correspond to CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, CD34–/CD41+ cells, respectively.

 
Proliferative and Differentiative Potential of CD34+/CD41–, CD34–/CD41–, CD34+/CD41+ and CD34–/CD41+ Cells Grown by TPO
To compare the proliferative and differentiative abilities of each subpopulation, the CD34+/CD41–, CD34–/CD41–, CD34+/CD41+ and CD34–/CD41+ cells were individually sorted by the FACStar Plus flow cytometer and cultured in wells containing TPO or a combination of GF on day 7. The accuracy of each subpopulation after sorting was 98.3%, 93.1%, 87.3%, and 99.7%, respectively, as compared with that before sorting. More than 90% of the cells in each subset were shown to be viable by a trypan blue exclusion test. The results are presented in Figures 3 and 4. The CD34+/CD41– cells generated the greatest number of progeny in the presence of TPO (p < .0001). On day 9, most of the cells were still small and round, and the frequency of proplatelet-like formation was rare. The CD34+/CD41+ cells exerted a low proliferative potential; approximately 15% of the progeny developed proplatelet-like formation on day 7. The CD34–/CD41+ cells did not have a significant proliferative ability, and apparent proplatelet-like formation was observed on day 3. A different maturation potential was also found regarding the CD42b expression. Only 43% of the cells grown from CD34+/CD41– cells were positive for CD42b on day 9, whereas 96%-98% of the cells from CD34+/CD41+ cells or CD34–/CD41+ cells expressed CD42b at earlier culture periods. The most notable observation was that the CD34–/CD41– cells failed to proliferate substantially and eventually degenerated.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Proliferative potential of CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ cells in the presence of TPO or a combination of GF. Day 7 cultured cells were sorted according to gates as shown in Figure1 and replated into TPO (A) or the combination of GF (B). The values expressed are the means ofthree separate experiments. CD34+/CD41– cells = open circles; CD34–/CD41– cells = closed circles; CD34+/CD41+ cells = open squares; CD34–/CD41+ cells = closed squares.

 


View larger version (93K):
[in this window]
[in a new window]
  		Figure 4. In situ appearance of the cells generated by the replating of four subpopulations into TPO. The photographs (original magnification x 200) of cells generated by CD34+/CD41– cells (A), CD34–/CD41– cells (B), CD34+/CD41+ cells (C), and CD34–/CD41+ cells (D) were taken on day 9, day 5, day 5, and day 3 after the replating, respectively.

 
In the presence of GF, the CD34–/CD41– cells and CD34+/CD41– cells generated progeny to a significantly greater extent than did the CD34+/CD41+ cells (p < .0001). The CD34–/CD41+ cells had the lowest proliferative ability (p < .0001). The majority of progeny were GPA+ cells, and some of the progeny were CD42b+ cells in the cultures of all four of the subpopulations. A significant number of CD11b+ cells were grown from only the CD34+/CD41– cells.

Number and Type of Precursors among CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ Cells Grown by TPO
We examined the number and type of precursors in the four subpopulations using the single-cell culture system. Individual cells were sorted from each group and incubated with GF. The results are shown in Table 1. Approximately 60% of the CD34+/CD41– cells multiplied, and most of the precursors generated erythroid and/or megakaryocytic cells. The frequency of precursors differentiating into granulocytes-macrophages with or without other lineages was 31%, the largest percentage among the four subpopulations (p < .0001). Approximately 40% of the CD34–/CD41– cells proliferated, and 83% of them were pure erythroid precursors. EPO alone could support the growth of erythroid colonies, but their number was half the value obtained by GF. The frequency of megakaryocytic precursors was the lowest in the CD34–/CD41– subpopulation (p < .0001). On the other hand, the frequencies of GF-responsive precursors in the CD34+/CD41+ and CD34–/CD41+ cells were both less than 10%; most of the precursors were committed to the megakaryocytic lineage.


View this table:
[in this window]
[in a new window]
  		Table 1. Number and type of hematopoietic progenitors in CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ cells generated by TPO
 
Apoptosis of CD34–/CD41– Cells in the Presence of TPO
We examined the mechanism underlying the cell lysis of the CD34–/CD41– subset in the presence of TPO. As shown in Figure 5, the flow cytometric analysis revealed the appearance of a sub-G1 peak of the sorted CD34–/CD41– cells in the replating into TPO. Half the cells exhibited marked nuclear fragmentation and chromatin condensation on the cytospin preparations stained with May-Grünwald-Giemsa. In addition, two-thirds of the cells were positive for the terminal deoxynucleotidyl transferase labeling of fragmented DNA. Thus, these results suggest that the sub-G1 peak was due to the induction of apoptosis.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. Appearance of apoptotic cells in the culture of CD34–/CD41– cells with TPO. The CD34–/CD41– cells were sorted on day 7, and replated into 10 ng/ml TPO. The surface marker expression and theDNA content of the CD34–/CD41– cells were serially analyzed with a flow cytometer. The percentage of the sub-G1 peak was calculated using LYSIS 2 software.

 
To elucidate whether the failure of the CD34–/CD41– cells to proliferate in response to TPO was based on the receptor level, we examined the expression of c-Mpl on the cultured cells using a three-color FACScan analysis and RT-PCR analysis. As shown in Figure 6, both the CD34+/CD41+ cells and the CD34–/CD41+ cells reacted with the anti-c-Mpl mAb, whereas the c-Mpl expression was not detectable on the surface of the CD34–/CD41– or CD34+/CD41– cells. Next, we performed RT-PCR analysis. Thirty-five cycles were used for amplification to ensure that the amount of amplified DNA was linear in the cycle numbers and correlated with the amount of specific mRNA in the original sample. The molecular sizes of RT-PCR products obtained with the primers were compatible with the expected molecular size: 232 bp for P-form of c-Mpl, 234 bp for K-form of c-Mpl, and 517 bp for GAPDH. As presented in Figure 7, the expression of c-Mpl mRNA (both P-form and K-form) was apparently lower in the CD34–/CD41– cells compared with the level of the CD34–/CD41+ cells.



View larger version (38K):
[in this window]
[in a new window]
  		Figure 6. c-Mpl expression on day 7 CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34 /CD41+ cells. Cord blood CD34+ cells (2 x 104/well) were cultured with 10 ng/ml TPO for seven days. A) The viable cells (R1) were gated on theFSC and SSC screen. B) Isotype-matched immunoglobulin was used as a negative control. C) R2,R3, R4, and R5 correspond to CD34+/CD41–, CD34–/CD41–, CD34+/CD41+, and CD34–/CD41+ cells, respectively. The cells were labeled with anti-c-Mpl mAb (-) or with mouse IgG1,(   ), and then stained with FITC-conjugated goat anti-mouse immunoglobulin.After treatment with mouse serum, the cells were stained with a PE-conjugated CD34 mAb andbiotinized anti-CD41 mAb, followed by Cy chrome-labeled streptavidin.

 


View larger version (20K):
[in this window]
[in a new window]
  		Figure 7. c-Mpl mRNA expression in day 7 CD34–/CD41– and CD34–/CD41+ cells. One x 105 day 7 CD34–/CD41– and CD34–/CD41+ cells were sorted by flow cytometry, and total RNA was isolated from the cells. The c-Mpl andGAPDH cDNA of the cells were amplified by PCR at 35 cycles. Lane 1 contains CD34–/CD41– cells; lane 2 contains CD34–/CD41+ cells; and lane 3 contains no RNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Debili et al. [16] have demonstrated, using human bone marrow cells, that the cells grown on day 7 by aplastic anemia plasma are divided into three subpopulations on the basis of CD34 and glycoprotein (Gp) IIIa expression: CD34+/Gp IIIa–, CD34+/Gp IIIa+, and CD34–/Gp IIIa+ cells, and that the coexpressed cells are at the transitional stage of megakaryocytic differentiation. In the present study, day 7 CD34+/CD41– cells expressed high HLA-DR antigen but were virtually negative for CD42b and CD9. They were also highly positive for c-kit and CD38 (data not shown). This subpopulation generated the greatest number of progeny in response to TPO among the four cell subpopulations. Most of them did not display proplatelet-like formation, and half of them were negative for CD42b antigen, even nine days after the replating into TPO. In the single-cell culture study, approximately 60% of this cell population responded to GF and yielded single or a mixture of erythrocytes, megakaryocytes, and granulocytes macrophages, indicating that day 7 CD34+/CD41– cells possessed the potential to differentiate into multiple lineages. The CD34+/CD41+ cells showed intermediate expressions of CD42b and CD9 compared with the CD34+/CD41– and CD34–/CD41+ cells. The CD34+/CD41+ cells generated progeny exclusively positive for CD42b in the presence of TPO. The replating experiments into GF revealed that a majority of the precursors in this subset were committed to the megakaryocytic lineage. Thus, CD34+/CD41+ cells are presumably at the transitional stage of megakaryocytic differentiation, corresponding to the CD34+/Gp IIIa+ cells described above. In addition, our results may be related to the evidence described by Papayannopoulou et al. [17] that the presence of CD41 on human bone marrow CD34+ cells segregates late erythroid and late megakaryocytic progenitors from early erythroid and early megakaryocytic progenitors, which are CD41–. It is of interest that a new subset, the CD34–/CD41– cells, were generated in addition to the CD34+/CD41– cells, CD34+/CD41+, cells and CD34/CD41+ cells in our TPO culture conditions. The production of this subpopulation was not due to contamination with cells other than megakaryocytic cells, because the CD34–/CD41– cells reacted at negligible levels with mAbs against the other lineage markers. The flow cytometric analysis showed that the CD34–/CD41– cells had a phenotype closer to that of the CD34+/CD41– cells than the CD34+/CD41+ cells. The single-cell culture study revealed that the main precursors in the CD34–/CD41– cells were pure erythroid lineage. In replating of the CD34–/CD41– cells into either GF or EPO, half of these erythroid precursors could proliferate in response to EPO alone. Coupled with our previous evidence [7], TPO alone may support the generation of both megakaryocytic precursors and erythroid precursors which encompass at least CFU-E.

The terminal differentiation pathway supported by TPO is a megakaryocyte-platelet lineage [1-6, 18], although TPO exerts stimulatory effects on the growth of various types of hematopoietic progenitors, including erythroid progenitors [7, 19, 20]. Actually, in the present study, the great majority of progeny generated by TPO were positive for CD41 at the end of the culture period. Our kinetic study of hematopoietic precursors appearing at the intermediate culture period showed that the CD34+/CD41+ cells expressed c-Mpl and exerted a low but significant proliferative potential by stimulation with TPO. In contrast, the CD34–/CD41– cells failed to grow/survive and fell into apoptosis in the replating into TPO. This may result from the apparently low expression of c-Mpl, as determined by the flow cytometric analysis and RT-PCR analysis. Although it is well known that apoptotic death occurs in hematopoietic progenitors after they are deprived of GF, the present study may support that the receptor expression rather than the GF is an important determinant in the commitment process of hematopoietic progenitors.


   Acknowledgments
 
This work was supported by Grants-in-Aid Numbers 09670796 and 09770537 from the Ministry of Education of Japan.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bartley TD, Bogenberger J, Hunt P et al. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994;77:1117-1124.[Medline]

  2. deSauvage FJ, Hass PE, Spencer SD et al. Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-Mpl ligand. Nature 1994;369:533-538.[Medline]

  3. Lok S, Kaushansky K, Holly RD et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994;369:565-568.[Medline]

  4. Wendling F, Maraskovsky E, Debili N et al. c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994;369:571-574.[Medline]

  5. Kato T, Ogami K, Shimada Y et al. Purification and characterization of thrombopoietin. J Biochem 1995;118:229-236.[Abstract/Free Full Text]

  6. Kaushansky K, Lin N, Grossmann A et al. Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice. Exp Hematol 1996;24:265-269.[Medline]

  7. Higuchi T, Koike K, Sawai N et al. Proliferative and differentiative potential of thrombopoietin-responsive precursors: expression of megakaryocytic and erythroid lineages. Exp Hematol 1997;25:463-470.[Medline]

  8. Debili N, Wendling F, Cosman D et al. The Mpl receptor expressed in the megakaryocytic lineage from late progenitors to platelets. Blood 1995;85:391-401.[Abstract/Free Full Text]

  9. Koike K, Stanley ER, Ihle JN et al. Macrophage colony formation supported by purified CSF-1 and/or interleukin 3 in serum-free culture: evidence for hierarchical difference in macrophage colony-forming cells. Blood 1986;67:859-864.[Abstract/Free Full Text]

  10. Higuchi T, Koike K, Sawai N et al. Megakaryocytes derived from CD34-positive cord blood cells produce interleukin-8. Br J Haematol 1997;99:509-516.[Medline]

  11. Sawai N, Koike K, Higuchi T et al. Thrombopoietin enhances the production of myeloid cells, but not megakaryocytes in juvenile chronic myelogenous leukemia. Blood 1998;91:4065-4073.[Abstract/Free Full Text]

  12. Koike K, Nakahata T, Takagi M et al. Synergism of BSF-2/interleukin 6 and interleukin 3 on development of multi-potential hemopoietic progenitors in serum-free culture. J Exp Med 1988;168:879-890.[Abstract/Free Full Text]

  13. Amano Y, Koike K, Nakahata T. Stem cell factor enhances the growth of primitive erythroid progenitors to a greater extent than interleukin-3 in patients with aplastic anaemia. Br J Haematol 1993;85:663-669.[Medline]

  14. Mollereau B, Deckert M, Deas O et al. CD2-induced apoptosis in activated human peripheral T cells: a Fas-independent pathway that requires early protein tyrosine phosphorylation. J Immunol 1996;156:3184-3190.[Abstract]

  15. Shimada Y, Kato T, Ogami K et al. Production of thrombopoietin (TPO) by rat hepatocytes and hepatoma cell lines. Exp Hematol 1995;23:1388-1396.[Medline]

  16. Debili N, Issaad C, Masse J-M et al. Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation. Blood 1992;80:3022-3035.[Abstract/Free Full Text]

  17. Papayannopoulou T, Brice M, Farrer D et al. Insights into the cellular mechanisms of erythropoietin-thrombopoietin synergy. Exp Hematol 1996;24:660-669.[Medline]

  18. Methia N, Louache F, Vainchenker W et al. Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 1993;82:1395-1401.[Abstract/Free Full Text]

  19. Kobayashi M, Laver JH, Kato T et al. Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors. Blood 1995;86:2494-2499.[Abstract/Free Full Text]

  20. Kobayashi M, Laver JH, Kato T et al. Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with Steel factor and/or interleukin-3. Blood 1996;88:429-436.[Abstract/Free Full Text]

accepted for publication December 3, 1998.



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
K. Takeuchi, K. Koike, T. Kamijo, S. Ishida, Y. Nakazawa, Y. Kurokawa, K. Sakashita, T. Kinoshita, S. Matsuzawa, M. Shiohara, et al.
STI571 inhibits growth and adhesion of human mast cells in culture
J. Leukoc. Biol., December 1, 2003; 74(6): 1026 - 1034.
[Abstract] [Full Text]

Home page
 Am. J. Pathol.Home page
A. N. Goldfarb, D. Wong, and F. K. Racke
Induction of Megakaryocytic Differentiation in Primary Human Erythroblasts : A Physiological Basis for Leukemic Lineage Plasticity
Am. J. Pathol., April 1, 2001; 158(4): 1191 - 1198.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sawai, N.
Right arrow Articles by Komiyama, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sawai, N.
Right arrow Articles by Komiyama, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS
http://www.stemcellsportal.com/