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HEMATOPOIESIS
From INSERM U.506, Hôpital Paul Brousse, 14 avenue Paul Vaillant Couturier, F-94800 Villejuif, France; and INSERM
U.311, Etablissement Français du Sang-Alsace, 10 rue Spielmann,
B.P. 36, F-67065 Strasbourg Cedex, France.
Megakaryocytopoiesis is a complex multistep process involving cell
division, endoreplication, and maturation and resulting in the release
of platelets into the blood circulation. Megakaryocytes (MK)
progressively express lineage-restricted proteins, some of which play
essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42)
and GPIIb (CD41) are examples of MK-specific proteins having receptor
properties essential for platelet adhesion and aggregation. This study
defined the progressive expression of the GPIb-V-IX complex during in
vitro MK maturation and compared it to that of GPIIb, an early MK
marker. Human cord blood CD34+ progenitor cells were
cultured in the presence of cytokines inducing megakaryocytic
differentiation. GPIb-V-IX expression appeared at day 3 of culture and
was strictly dependent on MK cytokine induction, whereas GPIIb was
already present in immature CD34+ cells. Analysis by flow
cytometry and of the messenger RNA level both showed that GPV appeared
1 day later than GPIb-IX. Microscopy studies confirmed the late
appearance of GPV, which was principally localized in the cytoplasm
when GPIb-IX was found on the cell surface, suggesting a delayed
program of GPV synthesis and trafficking. Cell sorting studies
revealed that the CD41+GPV+ population
contained 4N and 8N cells at day 7, and was less effective than
CD41+GPV Megakaryocytopoiesis is the cellular
differentiation process that leads to the release of platelets into the
circulation. Megakaryocyte (MK) progenitor cells proliferate,
polyploidize, increase in size, and develop lineage-specific cell
surface and cytoplasmic markers. The regulation of megakaryocytopoiesis
occurs in the bone marrow within 2 major compartments. The first one contains different populations of proliferative bipotent
erythromegakaryocytic progenitor cells (burst-forming units of
erythrocytes and megakaryocytes [BFU-E/MK]).1 In the
second compartment, terminal maturation is accompanied by a decrease in
proliferation, an increase in cell ploidy, and cytoplasmic
maturation.2 The most immature MK progenitors are the
burst-forming units of megakaryocytes (BFU-MK), made of primitive cells
expressing the CD34 antigen and HLA-DR Proliferation and maturation of MK precursors are controlled by several
pleiotropic cytokines.4,5 Although thrombopoietin (TPO)
plays a major role in regulating MK and platelet
production,6-8 many studies point to additional
contributions from stem cell factor (SCF), interleukin-3 (IL-3),
interleukin-6 (IL-6), interleukin-11 (IL-11), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and basic fibroblast growth factor
(bFGF).4,5
Megakaryocytic differentiation is accompanied by changes in cell
morphology, notably an increase in size and the appearance of
demarcation membranes, and by the sequential expression of a number of
genes coding for specific cell surface markers, cytokines, and cytokine
receptors. Early genes, such as mpl coding for the TPO
receptor9 and the GPIIb gene10 are
first expressed in CD34+ cells, but are still expressed in
the late stages leading to platelet production. On the other hand,
platelet factor 4 (PF4), GPIIb-IIIa (integrin In contrast to GPIIb-IIIa, but also PF4 or vWF, the time course of
GPIb-V-IX expression during MK maturation has not been studied in
detail. The GPIb-V-IX complex, a member of the leucine-rich glycoprotein family, is a vWF receptor supporting the initial rolling
and adhesion of platelets on the subendothelium of damaged blood
vessels.14,31,32 Previous studies of megakaryoblasts and
MK cultured from bone marrow cells suggested a delayed time course of
GPIb Another unanswered question concerns the timing of the expression of
the 4 subunits forming the GPIb-V-IX complex. These subunits are
encoded by separate genes and despite their coexpression at the
platelet surface there is no clear indication of coordinated expression
during megakaryocytopoiesis. On the contrary, there is indirect
evidence in favor of the separate regulation of GPV expression in MK
because (1) studies in transfected cells show that the GPIb-IX complex
does not require GPV for its efficient expression33; (2)
among GPIb+ cells only the Dami cell line expressed
GPV34; and (3) GPV knockout mice have platelets displaying
normal surface expression of the GPIb-IX complex.35,36
The aims of this study were to use techniques of in vitro MK culture
using cord blood CD34+ cells to follow the temporal
expression of the 4 subunits of the GPIb-V-IX complex during MK
maturation, to compare the results to those for GPIIb-IIIa, and to
assess possible differences in the temporal expression of the 4 subunits of GPIb-V-IX.
Purification and culture of CD34+ cells
The CD34+ progenitor cells were isolated using an
immunomagnetic separation system (MACS: Miltenyi Biotec, Bergisch
Gladbach, Germany), as previously described by Miltenyi.37
Mononuclear cells were labeled with an anti-CD34 monoclonal antibody
(QBEND/10) recognizing a denaturation-resistant epitope on the CD34
antigen, washed in PBS, and incubated with microbeads, which bind
QBEND/10. The labeled cells were then resuspended in PBS containing 1%
bovine serum albumin (BSA) and 5 mmol/L EDTA to prevent platelet
contamination and purified on the MACS system according to the
manufacturer's instructions. To improve the CD34+ purity,
the cells were passed twice over the column, and the percentage of
CD34+ cells as determined by flow cytometry generally
exceeded 98% to 99%.
These cells were cultured in serum-free STEM Monoclonal antibodies
Flow cytometry The MoAbs used in flow cytometry were ALMA.12, ALMA.16, V.1, ALMA.12-Alexa 488, V.1-Cy3, FITC-GAM, FITC-anti-CD15, FITC-anti-CD38, PE-anti-CD41, and PerCP-anti-CD34.Indirect labeling. Cells (2 × 104) were incubated with 2 µg ALMA.12, ALMA.16, or V.1 in PBS containing 1% BSA (PBS/BSA) for 15 to 20 minutes at 4°C. After 2 washes in PBS/BSA, the samples were resuspended in 100 µL PBS/BSA and further labeled by addition of 2 µL FITC-GAM for 15 to 20 minutes at 4°C in the dark. The cells were then washed twice, incubated with 2 µL each of PerCP-anti-CD34 and PE-anti-CD41 for 15 to 20 minutes at 4°C in the dark and finally washed and resuspended in 200 µL PBS/BSA. Direct labeling. Cells (2 × 104) in PBS/BSA were triple labeled by incubation for 15 to 20 minutes at 4°C in the dark with different combinations of PerCP-anti-CD34, PE-anti-CD41, FITC-anti-CD15, FITC-anti-CD38, V.1-Cy3, and ALMA.12-Alexa 488. The cells were then washed and resuspended in 200 µL PBS/BSA. Samples were analyzed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson) and PerCP-IgG1, PE-IgG1, FITC-IgM, and FITC-IgG1 as nonspecific controls.Cell sorting The CD34+ cells purified as described above were double labeled with PE-anti-CD41 and ALMA.12-Alexa 488, or with PE-anti-CD41 and an anti-GPV revealed by an Alexa 488-GAM for 30 minutes at 4°C, and washed twice. Cells were sorted using a Coulter Elite flow cytometer (Coulter Electronics, Margency, France). CD41+GPIb  and
CD41+GPIb+ populations were selected at day
4, and CD41+GPV and
CD41+GPV+ populations were selected at day 7. The purity of the sorted populations was confirmed by analysis on a
FACSCalibur cytometer (Becton Dickinson).
Clonogenic cell assay Methylcellulose assays for burst-forming units-erythrocytes.
Freshly sorted CD41+GPIb Megakaryocytic progenitor assays.
Freshly sorted CD41+GPIb Ploidy values Cells labeled with anti-GPV and anti-CD41 were fixed overnight in 70% ethanol at 4°C. The fixed cells were washed and resuspended in 300 µL PBS containing propidium iodide (50 µg/mL; Sigma, Saint Quentin Fallavier, France) and RNase A (0.2 µg/mL; Sigma) for 30 minutes at 4°C. Samples were analyzed on a FACSCalibur flow cytometer using CellQuest software. The ploidy distribution was determined by setting markers at the nadirs between peaks.Immunocytochemistry Cells in RPMI 1640 were seeded on glass coverslips coated with poly-L-lysine, at a density of 5 × 104/mL, and incubated for 30 to 40 minutes at 37°C. The medium was removed and the cells were fixed in PBS-3.7% paraformaldehyde for 10 minutes at room temperature, rinsed twice in PBS, and quenched by incubation in PBS-0.1 mol/L glycine for 10 minutes at room temperature. After 2 washes in PBS, the cells were permeabilized in PBS buffer containing 0.02% BSA and 0.005% saponin (SBP buffer) for 20 minutes, at room temperature. The cells were subsequently incubated with the primary antibodies (V.1, anti-CD41, or ALMA.12-Alexa 488) diluted in SBP for 30 minutes at room temperature, washed 3 times for 5 minutes in SBP, incubated for 30 minutes with the secondary antibodies (Cy3-GAM, Alexa 488-GAM, or FITC-GAM), washed 3 times for 5 minutes in SBP, and then incubated for 30 minutes with the tertiary antibodies (ALMA.12, ALMA.16, and V.1 directly conjugated with Cy3). Finally, the coverslips were washed twice for 5 minutes in SBP and twice for 5 minutes in PBS and mounted upside down on a slide in Mowiol.The following dilutions were used: ALMA.16, 1:200; V.1, 1:200; anti-CD41, 1:200; FITC-GAM, 1:150; ALMA.12-Cy3, 1:500; ALMA.16-Cy3, 1:66; V.1-Cy3, 1:500; ALMA.12-Alexa 488, 1:250; Cy3-GAM, 1:800; and Alexa 488-GAM, 1:400. Samples were examined and photographed under a DMR HC fluorescence microscope (Leica, Vienna, Austria) using an oil immersion objective (63 × or 100 ×). Confocal laser scanning microscopy and image analysis Confocal microscopy was performed using a Zeiss laser scanning microscope (LSM 410 invert, Göttingen, Germany) equipped with an oil immersion lens (63 ×, numerical aperture = 1.4). Cy2, Alexa 488, and FITC emissions were excited with the argon 488 nm line, Cy3 with the He/Ne 543 nm line, and Cy5 with the He/Ne 633 nm line. The emission signals were filtered with a Zeiss 515-565 nm filter (Cy2, Alexa 488, and FITC), with a long pass 595 nm filter (Cy3) or with a long pass 650 nm filter (Cy5). Nonspecific fluorescence was assessed by incubating the cells with the secondary fluorescent-labeled antibodies and measuring the average emission intensity for each fluorochrome, which was then subtracted from all specific images.Analysis of transcripts Total cellular RNA was extracted by the thiocyanate-guanidium method of Chomczynski and Sacchi.39 Briefly, 107 cells were lysed in 1 mL TriPure (Boehringer), extracted with 0.2 mL chloroform and centrifuged at 11 000 rpm for 15 minutes at 4°C. The aqueous phase containing RNA was precipitated with isopropanol and the pellet washed in 70% (v/v) cold ethanol.Complementary DNA (cDNA) was synthesized from 50 ng total RNA from
CD34+ cells using a Ready To Go: T-Primed First-Strand Kit
(Pharmacia Biotech, Uppsala, Sweden), in a reaction volume of 33 µL.
Polymerase chain reaction (PCR) amplifications were carried out with an
Expand High Fidelity PCR System (Boehringer) in a volume of 100 µL,
using 200 µmol/L each dNTP, 0.4 µmol/L of the reverse and forward
primers, 1.5 mmol/L MgCl2, 2 µL cDNA, and 2.6 U of a mix
containing Taq DNA polymerase and Pwo DNA polymerase. Amplifications
were performed in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk,
CT) for 2 minutes at 94°C, followed by 30 cycles of 1 minute at
94°C, 2 minutes at 55°C (for glyceraldehyde-3-phosphate
dehydrogenase [GAPDH], GPIb The direct and reverse primers were respectively P1 and P2 for GAPDH
(EMBL/Genbank: HSGAPDR-X01677),40 P3 and P4 for GPIIb (EMBL/Genbank: HUMGPIIBA-M34480),41 P5 and P6 for GPV
(EMBL/Genbank: HSGPV-Z23091),42 and P7 and P8 for GPIb
Maturation of megakaryocytes during the course of the liquid culture During the first days of culture, the proliferation was maximal: the number of cells was amplified 3 times during the first 4 days (Figure 1A). Then, the proliferation decreased, reaching a plateau between days 10 to 14. Figure 1B shows that during the high proliferative stage of the culture, the cells had the aspect of undifferentiated progenitors. These cells were about 8 µm in diameter with thin chromatin. They grew and differentiated from day 6 to day 9 into 15-µm diameter megakaryoblasts with denser chromatin. At day 12, most of the cells were about 25 to 30 µm in diameter with polylobular nuclei. By day 13, they measured approximately 30 to 40 µm in diameter and cells with the appearance of mature MK were frequent in the culture, with filopodia and polylobular nuclei.
Early expression of GPIIb-IIIa and delayed expression of GPIb-V-IX during megakaryocyte maturation Surface expression of GPIb, GPIX, and GPV was determined by
flow cytofluorimetry and compared to that of GPIIb in double-labeling experiments (Figure 2). Typical
expression profiles of these markers are shown on Figure 2A and 2B.
Values for GPIIb and GPIb obtained for one CB sample, representative
of 6 individual experiments, are shown in Figure 2C. GPIb, GPIX, and
GPV were not detected at day 0, whereas about 15% of the cells were
positive for GPIIb. The GPIIb labeling did not correspond to background
generated by residual platelets because EDTA treatment of the cells
during labeling did not change the percentage of CD41+
cells (data not shown). From days 4 to 14, there was increased expression of the GPIb, GPV, and GPIX subunits at the cell surface. At day 14, when most cells had differentiated morphologically into MK,
GPIb+ cells represented about 77% of the total
population, whereas about 95% of the cells expressed GPIIb. Dot-plot
analysis indicated that all GPIb+ cells were also
GPIIb+ (Figure 2B). Similar results were obtained for GPV
and GPIX (data not shown). Interestingly, although GPIIb+
cells evolved as a single population from mildly to strongly labeled
cells from day 7 to day 14, GPIb+, GPV+, and
GPIX+ cells evolved from a single to 2 distinct
populations, with mild and strong expression, respectively. This
suggests that at different maturation stages, 2 megakaryocytic
populations could coexist, both expressing CD41, but displaying
different levels of GPIb-V-IX complexes.
The delay in GPIb
We also tested the expression of CD34, CD15, and CD38 (Figure 3B). CD34 expression decreased progressively during differentiation, as previously described for bone marrow cell cultures11 and was totally absent by days 9 to 10. At day 0, 95% of CD34+ cells were also CD38+, but this later marker disappeared more rapidly than CD34 during the MK differentiation. To check for the presence of myeloid precursors, we analyzed the expression of CD15, the Lewis x (Lex) antigen, which is a granulomonocytic marker, absent from the MK surface. This marker remained at low levels (10%-15%) throughout culture, confirming that cells committed to the MK pathway were selectively amplified. GPV appears later than GPIb and GPIX (Figure 3). To confirm this observation, we performed additional FACS analysis of GPV and GPIb expression during the first 5 days of culture. To avoid potential bias generated by the secondary antibodies, we used MoAbs directly coupled to fluorophores (anti-GPIb-Alexa 488 and anti-GPV-Cy3). At day 2, GPV
expression was low (approximately 3%) as compared to 10% of GPIb
(Figure 4A). At day 3, the difference
between both expressions decreased (8% and 11%, respectively). The
delay of GPV expression was also observed at the mRNA level, because
GPV transcript was detected, in parallel reverse transcription
(RT)-PCR analysis, a day later than GPIb. Futhermore, FACS analysis
and RT-PCR experiments confirmed the presence of GPIIb protein and
transcript at day 0 (Figure 4). In conclusion, GPIIb,
GPIb, and GPV appear sequentially during the early stages of
megakaryocytopoiesis.
CD41+GPIb-V-IX and
CD41+GPIb-V-IX+ cell populations had different
clonogenic properties, we sorted CD41+GPIb
and CD41+GPIb+ day 4 and
CD41+GPV and
CD41+GPV+ cells at day 7. Figure
5A shows that at day 4 double-positive sorted cells produce fewer BFU-E than
CD41+GPIb cells (65% ± 10% BFU-E
versus 90% ± 9%), in methylcellulose assay. This observation was
confirmed at day 7 for the CD41+GPV+ and the
CD41+GPV populations. Moreover, cells sorted
at day 7 produced fewer BFU-E than cells sorted at day 4, indicating
that as the cells differentiate, they progressively lose their
clonogenic capacities. Using collagen medium, we observed only MK
colonies. The number of MK colonies obtained with the double-positive
cells at day 4 was significantly lower than that obtained with the
CD41+GPIb population (233 ± 37 versus
321 ± 42, respectively). The number of MK colonies decreased
significantly at day 7 (4- to 5-fold), whereas the ratio between the 2 populations was the same as at day 4 (Figure 5B).
Cell ploidy distribution of the CD41+GPV+
cells at day 7 is different from that of
CD41+GPV cells were 2N, whereas 13% and 2%
of the CD41+GPV+ cells were 4N and 8N,
respectively.
Fluorescence microscopy analysis of the GPIb ,
and GPV would account for the differences observed in cell surface
expression of these molecules. At day 0, labeling of GPIIb was
observed, but labeling of GPIb or GPV (Figure
7) was negative, consistent with the FACS
and RT-PCR studies. By day 7 a large number of permeabilized cells
contained GPV molecules within their cytoplasm, whereas at this stage,
the membrane-bound GPV was only detected on 25% of the cells (Figure
3A). Almost all the GPIIb+ cells were also stained
intracellularly for GPIb. All GPIb+ or
GPV+ cells were positive for GPIIb, consistent with the
FACS results. Results for GPIX were identical to those obtained for
GPIb (data not shown). These experiments clearly demonstrate the
sequential expression of GPIIb, GPIb, and GPV during MK maturation,
with stages including intracellular retention of the subunits of the GPIb-V-IX complex.
Cellular localization of GPIb , GPIX, and GPV from day 0 to day 14 was obtained by confocal laser scanning microscopy (Figure 8).
Different combinations of antibodies against the 3 subunits and against
GPIIb were used in double- and triple-labeling experiments. At day 0, GPIIb was present on the cell surface, whereas GPIb and GPV were
undetectable. At day 4, GPIb was found in the cytoplasm and on the
cell surface. At day 7, some GPV and GPIb surface colocalization
(yellow) was observed (GPIbGPV double labeling). However, most
GPIb was localized at the cell surface, whereas the GPV molecule was
principally detected in a cytoplasmic pool (green in GPIIbGPV double
labeling and in GPIbGPV). In contrast, by day 7 GPIX and GPIb
subunits were almost completely colocalized at the cell surface. At day 10, all 3 subunits colocalized on the cell surface, and the cells began
to produce filopodia. Finally, by day 14 mature MK were observed with
colocalization of all the markers tested on the cell body and
on filopodia.
This is the first report addressing in detail the relative
kinetics of expression of the platelet GPIb-V-IX subunits during MK
maturation. Although GPIb-V-IX is considered to be a very specific marker of platelets, unlike GPIIb, it has been little studied during MK
differentiation. This complex is essential for the adhesive functions
of platelets, but its role in MK maturation is unknown. That GPIb plays
an active role in late stage of MK differentiation is suggested by the
presence of abnormally large platelets in GPIb-deficient
Bernard-Soulier patients as well as abnormal demarcation membranes in
their bone marrow MK, which was recently confirmed on
GPIb The study of the sequential development of MK and temporal surface expression of the GPIb-V-IX complex was made possible due to improved culture conditions allowing in vitro differentiation of MK. These culture conditions included TPO, IL-6 and IL-11, a mixture of powerful MK differentiation inducing cytokines,47,48 and IL-3, that has been described for acting synergistically with TPO to support the formation of multiples types of hematopoietic colonies including multilineage colonies.5 It has also been reported that IL-3 alone could support CFU-MK colony growth in vitro.4 The combination of IL-3, IL-6, and TPO appears to be additive as measured by colony growth.4 Moreover, the combination of IL-11 plus TPO resulted in a synergistic enhancement of the number of CFU-MK colonies. Using this cocktail of cytokines, we observed that the cells underwent typical MK morphologic maturation with development of polylobular nuclei and formation of filopodia and proplatelets. The time course of cell surface markers was typical for MK differentiation with down-regulation of CD34 and up-regulation of GPIb-V-IX and GPIIb-IIIa. Our present results performed at early stages of MK differentiation
revealed that GPIb-V-IX appearance was markedly delayed as compared to
that of GPIIb-IIIa. The CD34+ population already contained
a significant fraction of GPIIb+ cells (15%), consistent
with previous studies using CD34+ cells derived from bone
marrow1,11,13,49 or cord blood.50,51 FACS
analysis and immunofluorescence studies of permeabilized cells detected
no expression of GPIb on the first day of culture, which was confirmed
at the mRNA level by RT-PCR studies. We then tested whether the cells
that already expressed GPIIb-IIIa, but not yet the GPIb-V-IX complex,
were functionally different from those expressing both markers. We
observed that the cells only expressing GPIIb-IIIa reproducibly
generated more BFU-E than the double-positive cells, which confirms
that single-positive GPIIb-IIIa+ cells are more immature
cells. The observation that cells sorted for the expression of the 2 distinct megakaryocytic markers GPIIb-IIIa and GPIb Using megakaryocytic-inducing conditions,
CD41+GPIb We then tested whether the ploidy distribution of the cells expressing only GPIIb-IIIa was different from that of GPIIb-IIIa/GPIb-V-IX double-positive cells. We were unable to detect 4N population before day 7. At day 7, 4N and 8N cells were detected in the double-positive population, whereas only 2N cells were detected in the GPIIb-IIIa single-positive population. However, we did not detect any MKs displaying ploidy above 8N, even at late stages of the differentiation. These results are consistent with previous studies,50,51 and suggest that MKs from CB could undergo fewer endoreplications than those of adult bone marrow. The physiologic significance of this difference in ploidization is not clear. The observation that most cell lines expressing GPIIb-IIIa do not express GPIb-V-IX might be explained by the delayed appearance of GPIb-V-IX, because most cell lines display an erythromegakaryocytic phenotype fitting with a blockage at an immature stage. Hence, it is possible that the few cell lines that express GPIb-V-IX such as Dami25 or M-O7e22 have acquired a more mature phenotype, although this issue requires further clarification. There is to date no clear explanation for the different temporal
expression of GPIIb and GPIb-V-IX. Detailed analysis of the GPIIb gene
promoter has revealed the importance of Ets and GATA elements for
transcription in the megakaryocytic lineage52 and the
additional role of a repressor element active in nonmegakaryocytic cell
lines.53 Initial functional analyses of the promoters of GPIb Our detailed analysis on the early days of culture revealed a lag of a
day in GPV expression (at the protein and mRNA levels), possibly due to
different transcriptional regulation. The same delay was observed when
analyzing the intracellular and membrane compartments by fluorescence
or confocal microscopy. At day 0, cells were only positive for surface
GPIIb; GPIb In conclusion, this study confirms that GPIb-V-IX represents a later
marker of MK maturation than GPIIb and further demonstrates that GPV
has a delayed temporal pattern of expression. This distinct behavior,
as compared to GPIb
The authors wish to thank Sylvette Chasserot-Golaz (INSERM U.338, Strasbourg, France) for confocal laser scanning microscopy, Annie Falkenrodt (EFS-Alsace, Strasbourg, France) for help with morphologic analyses, and Denis Clay (INSERM U.268, Villejuif, France) for helpful discussions and support during this study. We are also grateful to Pierre Charbord (INSERM U.506, Villejuif, France) and Jean-Philippe Rosa (INSERM U.348, Paris, France) for critical reading of the manuscript.
Submitted December 20, 1999; accepted August 23, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Georges Uzan, Hôpital Paul Brousse, 12-14 avenue Paul Vaillant Couturier, F-94807 Villejuif Cedex, France; e-mail: guzan{at}infobiogen.fr.
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
IIb
3 integrin and GPIb-V-IX
complex identify distinct stages in the maturation of CD34+
cord blood cells to megakaryocytes
Top
Abstract
Introduction
-thromboglobulin, von Willebrand factor
(vWF),
30 cells) were counted on 5 individual
plates using an inverted microscope. BFU-E-type colonies were
evaluated at days 12 through 14. Similarly, BFU-E/MK, colony-forming
units of granulocytes (CFU-G), colony-forming units of macrophages
(CFU-M), colony-forming units of granulocytes and macrophages (CFU-GM), and colony-forming units of granulocytes, erythrocytes, monocytes, macrophages (CFU-GEMM) colonies were identified morphologically and counted.
), GPIb
), and GPIIb-IIIa
(CD41, 
) was followed by flow cytometry (Figure 
,
CD41+GPV
,
CD41+GPV+. (A) The percentage of positive cells
represents the proportion of BFU-E colonies compared to the total
number of colonies obtained in methylcellulose medium. (B) Number of
megakaryocytic colonies obtained in collagen medium.
