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Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4425-4435
By
From the Department of Biochemistry, School of Medical Sciences,
University of Bristol, Bristol, UK; and the Bristol Institute for
Transfusion Sciences, Bristol, UK.
Phenotypic analysis of hematopoietic stem and progenitor cells has
been an invaluable tool in defining the biology of stem cell
populations. We use here flow cytometry to examine the expression of
human erythroid-specific surface markers during the maturation of early
committed erythroid cells derived from cord blood in vitro. The
temporal order of the expression of erythroid specific markers was as
follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp,
glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The
time at which some of these markers appeared suggests possible roles
for some of these erythroid-specific polypeptides during the
differentiation of these committed progenitors. The early appearance of
Kell gp raises the possibility that it may have an important role in
the early stages of hematopoiesis or cell lineage determination. Kell
gp may also be a useful marker for the diagnosis of erythroleukemia.
The late expression of Lu gp suggests it may be involved in the
migration of erythroid precursors from the marrow. Fy gp is also
expressed late consistent with a role as a scavenger receptor for
cytokines in the bone marrow and circulation. Rh c antigen appeared
before Rh D antigen, and it is suggested that this may reflect a
reorganization of the developing erythroid cell membrane involving the
Rh polypeptides and other components, including GPA and Band 3.
ERYTHROPOIESIS IS A highly regulated
process by which pluripotent stem cells are recruited from bone marrow
or fetal liver and subsequently differentiate into erythrocytes to be
released into blood.1 Erythropoiesis was originally
characterized by the use of model animal cell systems, such as the
mouse erythroleukemia (MEL) cell line, and avian-nucleated erythroid
cells.2,3 Some of the results obtained from these model
systems have since been replicated in human systems. The process of
erythropoiesis can be divided into a number of discrete parts:
recruitment of primitive committed progenitor cells (burst-forming unit
erythroid [BFU-E] and colony-forming unit-erythroid [CFU-E]),
commitment to the erythroid lineage in erythropoietin-dependent and
independent stages, and enucleation of mature erythroblasts and their
release into the blood stream. Investigators have been able to describe the assimilation of the cytoskeletal network through ankryin, band 4.1, and band 3. However, even this description was found to differ between
the mouse and human models (for review, see Hanspal et
al4).
Previous studies in the human system have used nonerythroid surface
antigens to classify the earliest progenitors. BFU-E have been
shown5 to predominantly have the phenotype
CD34high CD45RAlow CD71high.
Another study showed that selection for CD33 could be used in conjunction with selection for CD34 to enrich erythroid
progenitors.6 Recently, placental cord blood (CB) has been
used to study human haematopoiesis.7-10 CB is enriched for
CD34+ cells, which include all of the stem cell progenitor
populations so far defined. These CB progenitors have been found to be
slightly more mature (have less self-renewal activity) than their adult marrow counterparts, but have a higher short-term proliferative capacity. Using different cytokine combinations, it is possible to
minimize the proliferation of nonerythroid cell lineages. When used in
combination with FK506, which effects an increase in numbers of
BFU-E11 in culture, this is a useful model system to study human erythropoiesis.
Although previous studies have looked at the expression of some human
blood group-active proteins in human bone marrow,12-17 a
comprehensive study of the temporal expression of erythroid-specific markers has not been reported. In recent years, there has been a rapid
increase in our understanding of the structure and function of the
proteins that express blood group antigens. The concomitant development
of monoclonal antibodies (MoAbs) against these proteins has made
possible a systematic investigation of the surface expression of blood
group-active polypeptides during erythropoiesis. In this report, we
provide new information on the order of appearance of
erythroid-specific markers during erythropoiesis from CB progenitors and also identify early surface markers that might be useful in the
diagnosis of erythroleukemia.18,19
Hematopoietic Growth Factors
Cell Preparation
Progenitor Cell Purification Cells bearing the CD34 antigen were isolated from the mononuclear population by positive selection using the MiniMACS magnetic beads system (Miltenyi Biotec Ltd, Bisley, Surrey, UK), according to the manufacturer's instructions, which are briefly summarized here. Mononuclear cells (1 × 108) were resuspended in sterile, degassed phosphate-buffered saline (PBS) containing 0.5% BSA, 0.5 mmol/L EDTA, pH 7.2, and washed once. Fifty microliters to 100 µL of mouse antihuman CD34 biotinylated MoAb (QBEND/10) was added to the pellet and the mixture was then incubated for 20 minutes at 4°C in an end-over-end rotator. Cells were then washed once in PBS and resuspended in PBS (300 µL) containing streptavidin-coated paramagnetic MiniMACS microbeads (50 to 100 µL). They were then incubated for a further 20 minutes. The cells were washed, resuspended in PBS (400 µL), passed through a 30-µm cell strainer, and applied to a primed MiniMACS column. The column was washed 4 times with PBS (0.5 µL) while attached to the magnet. The magnet was removed and the column was then washed with 1 mL of PBS to elute the CD34-selected cells. Purity of the CD34 enriched population of cells was assessed using mouse antihuman CD34-fluorescein isothiocyanate (FITC) MoAb (Birma-K; DAKO, Ely, Cambridge, UK) on FACstar Plus and FACSCalibur flow cytometers (Becton Dickinson, Cowley, Oxford, UK), as described below. Birma-K and QBEND/10 bind to a different epitopes on CD34.Clonogenic Assays To measure progenitor content, CD34+ cells were removed every 2 days and cultured at 1,000 cells/mL in semisolid media (0.9% methylcellulose with 10% agar) in leukocyte-conditioned medium (Stem Cell Technologies Inc, Northampton, UK) and 3 U/mL erythropoietin. Colonies were scored after 14 days in secondary culture for BFU-E, colony-forming unit-granulocyte-myeloid (CFU-GM), and colony-forming unit-mixture (CFU-MIX) using an inverted microscope, according to established criteria. CFU-MIX were defined as colonies containing erythroid progenitors as well as cells of any other lineage.Cell Culture in Suspension Suspension cultures were performed using a serum-free culture medium, as detailed in Sposi et al20 and Lebowski et al.21 The culture contained Iscove's modified Eagle's medium (IMEM) supplemented with BSA fraction V (10 mg/mL), iron-saturated human transferrin (1 mg/mL), human low-density lipoprotein suspension (40 mg/mL), human insulin (10 mg/mL), sodium pyruvate (0.1 mmol/L), ferrous sulphate (40 mmol/L), and nucleosides (10 ng/mL each). All products were purchased from Sigma Ltd, unless otherwise stated. Cells were seeded at 1 to 2 × 105 cells/mL and were maintained at 37°C in sealed T25 flasks (Becton Dickinson, Oxford, UK) flushed with a mixture of 6% O2/7% CO2/87% N2, for up to 21 days. Passaging was performed as necessary. These cultures were supplemented with recombinant cytokines at the following concentrations: IL-3 (10 ng/mL), SCF (100 ng/mL), and erythropoietin (3 U/mL). All cytokines were obtained from R&D Systems Europe Ltd Abingdon, Oxon, UK. FK506 (Prograf; 0.1 ng/mL)11 immunosuppressant was also included.Cytospin Preparation and Staining Aliquots of 5 × 104 cells were removed from cultures on various days, washed in PBS containing 1% BSA, and resuspended to a final volume of 300 mL. They were then centrifuged on to frosted glass slides at 2,000 rpm for 3 minutes in a cytofuge (Shandon Southern, Sewickley, PA). Slides were air-dried and then fixed in 100% methanol for 5 minutes. The slides were stained using a stock solution of 1 g Eosin yellowish (BDH; Poole, Dorset, UK) in 600 mL methanol together with 3 g Azur B (BDH) in 400 mL dimethyl sulfoxide (DMSO) for 5 minutes. Slides were washed in H2O and stained for a further 25 minutes in stock stain diluted 1:16 with a diluent buffer consisting of 11.5 g HEPES in 5 L H2O adjusted to pH 6.8. Slides were quick dried in an acetone bath and blotted.Analysis of Cell Surface Antigen Expression by Flow Cytometric Analysis Cells were removed from culture on various days and analyzed for cell surface antigen expression using CellQuest software on FACstar Plus or FACSCalibur flow cytometers (Becton Dickinson) gated for low side light scatter (SSC). Mean fluorescent intensity (FLI) was used as a measure of Ab binding. Cultured cells (1 × 105) were removed in PBS supplemented with 1% BSA (PBS-A) and incubated in human AB serum for 15 minutes at room temperature (20°C to 25°C) before the addition of an isotype-matched mouse IgG (as a negative control) or with one of the panel of MoAb detailed in Table 1. After incubation with the primary mouse Ab for 30 minutes at 4°C, the cells were washed once in PBS-A. The cells were then incubated for 30 minutes with an appropriate volume of F(ab')2 goat antimouse IgG-R-phycoerythrin (RPE; diluted 1:15), and the cells were washed once in PBS-A. The cells were subsequently incubated with the second mouse MoAb or human MoAb (1 hour at 37°C), and the cells were washed once in PBS-A. Finally, either F(ab')2 rabbit antimouse IgG-FITC (diluted 1:25) or F(ab')2 rabbit antihuman IgG-FITC (diluted 1:20), as appropriate, was added for 30 minutes on ice or 30 minutes at room temperature for mouse and rabbit Ab, respectively. (Fluorescently labeled Ab were supplied by DAKO). In triple-labeling studies, the RPE-Cy5 directly conjugated Ab was added last. All the reactions were performed under conditions of antibody saturation. Electronic compensation between the three fluorescent signals (FITC, RPE, and RPE-Cy5) was set using cell samples stained with single fluorochromes.
Expression of Major Erythroid Specific Molecules: Rh Glycoprotein (gp), Glycophorin A (GPA), and Band 3 Cultures were established from CD34+-selected CB progenitors and sampled at different times for expression of erythroid-specific surface markers. Initially, the morphology of the cells was similar in size and shape to that of a small lymphocyte, with visible nucleoli and a small rim of cytoplasm. By days 4 to 5, the cells were generally larger, with the nuclear-to-cytoplasm ratio decreasing and the cells resembling classical pronormoblasts. The quantity of primitive BFU-E (>50,000 cells) depleted over the time course of the experiment until they were almost entirely absent from day 8 onwards. There was a concurrent increase in smaller mature BFU-E (~1,000 cells) from day 6 onwards. This increase peaked at day 8 and slowly decreased over the rest of the culture period. By day 13, erythropoiesis had progressed to the pyknotic erythroblast, and the overall percentage of these cells increased progressively over the next 7 days. However, this was coupled with a notable decrease in cellular viability, with increased cellular apoptosis (as assayed by annexin-V-FITC; data not shown) and number of endocytic vacuoles. Erythroid enucleation was not observed at any time during the culture period. Flow cytometric analysis was performed using double-labeling to determine precisely the temporal relationship between the expression of different antigens. The conditions used were such that the results obtained were independent of the order in which primary antibodies were added.Rh gp.
A substantial proportion of the cells isolated initially displayed a
phenotype of CD34high Rh gpmed
(Fig 1, day 0). These are mature precursors
present in the initial sample22 that are not the main focus
of this study. The cells of interest are CD34high Rh
gpneg, which represent 3% of cells in the experiment
displayed in Fig 1 (day 2). By day 4, 34% of cells in this culture had
this phenotype. The percentage of cells with this phenotype decreased
by day 13, and there was a concomitant increase in the proportion of
cells with the phenotype CD34neg Rh gphigh.
This population of cells appears to be the major proliferating cohort
of early progenitors in the culture, but other minor cohorts are also
present. We interpret these data as being consistent with the
progressive maturation of CD34high Rh gplow
cells to CD34neg Rh gphigh erythroblasts.
GPA.
Double-labeling with MoAb against CD34 and GPA gives results similar to
those obtained using labeling for CD34 and Rh gp. The major cohort of
cells on day 6 had the phenotype CD34high
GPAneg. By day 13, these cells were no longer detectable
and the predominant phenotype in the culture was CD34low
GPAhigh (data not shown). To examine when GPA was expressed
relative to Rh gp, double-labeling experiments were performed. The data presented in Fig 2A show that, by day 6, a
substantial population of the cells (19%) were Rh gphigh
GPAneg. The relative levels of expression of both Rh gp and
GPA appear to increase over time in culture (Fig 2A). Similar results
were obtained when two different anti-GPA MoAbs, R10 and R18 (which do
not recognize sialic acid-dependent epitopes),23 were used, indicating that the extent of sialylation of GPA is not a factor in the
recognition of expression of this polypeptide (data not shown). These
results indicate that expression of GPA follows that of Rh gp.
Band 3. Double-labeling experiments comparing the expression of Rh gp and band 3 (band 3) showed that 72% of cells had the phenotype Rh gpmed band 3low by day 6 (Fig 2B). This population was reduced to 24% by day 9 with a concomitant appearance of, and increase in, cells with a phenotype of Rh gpmed Band 3high. Double-labeling using anti-GPA and Band 3 showed that cells of the phenotype GPAneg Band 3high could not be detected, whereas GPAmed Band 3neg cells were present (Fig 2C). These results indicate that Band 3 is expressed after GPA in the culture system. Expression of Less Abundant Erythroid Proteins: Kell, Landsteiner Wiener (LW), Lutheran (Lu), and Duffy (Fy) The expression of the Kell, LW, Lu, and Fy proteins in these cultures was examined in relation to the expression of the major proteins described in the previous section.Kell gp.
In the experiment shown in Fig 3A, 22% of
cells had the phenotype CD34high Kelllow by day
2. However, by day 6, most of the cells in the culture (67%) had the
phenotype CD34neg Kellhigh. It is apparent that
only 10% of cells were Kellneg by day 6 of culturing,
compared with some 30% when stained by anti-Rh gp MoAb (Fig 1, day 6),
and 59% were reactive with anti-GPA MoAb (data not shown). There was a
progressive decrease in HLA-DR and CD34 reactivity during the early
stages of the culture, whereas Kell reactivity increased (Fig 3B and
C). These data also show that CD34 reactivity was lost before HLA-DR
activity. Double-staining with anti-Kell and anti-Rh gp MoAbs on day 6 showed that 52% of cells had the phenotype Kellmed Rh
gpneg (Fig 3D). These results suggest that Kell gp is a
lineage-specific marker that appears earlier than Rh gp.
LW gp.
Double-labeling of cells with anti-LW and anti-Kell MoAbs demonstrated
that LW immunoreactivity appeared after the Kell gp and at the same
time as Rh gp, on day 2 (Fig 4A). By day 7, it is apparent that some 10% of cells display the phenotype
LWlow GPAneg, with the majority being
LWlow GPA+ (55%; Fig 4A). These data clearly
demonstrate that expression of LW appears after Kell gp and at
approximately the same time as Rh gp, but before GPA.
Lu gp. Double-labeling with anti-Lu and either anti-GPA or anti-Band 3 MoAbs demonstrated that the Lu gp is expressed after GPA and Band 3 in this in vitro culture system (Fig 4B and C). On day 10, 36% of cells were GPAmed Luneg, whereas few, if any, GPAneg Lulow were found (Fig 4B). On day 12, 21% of cells were Band 3med Luneg and only 6% were Band 3neg Lulow (Fig 4B). Fy gp. Similar experiments performed using anti-Fy3 showed that expression of Fy gp paralleled that of Lu gp in the culture system (Fig 4C). Expression of Rh polypeptides.
Two mouse MoAbs (R6A and BRIC 6954,55) were used to detect
the expression of Rh polypeptides in the cultures. Double-labeling experiments with R6A and anti-GPA MoAb are shown in
Fig 5A. In the experiment shown on day 8, 65% of the cells were GPAmed R6Aneg, but by
day 10 the proportion of GPAmed R6Aneg was
reduced to 41%, and 60% of the cells were GPAmed
R6Alow. Double-staining with BRIC 69 and anti-Band 3 (Fig
5B) showed that there was a wide range of expression of BRIC 69 on the
cells in the cultures, but a substantial number of the cells (27%)
clearly expressed Band 3 but showed little or no expression of BRIC 69. Very similar results were obtained with R6A (Fig 5B). When the expression of R6A was compared with that of the Lu gp in the experiment shown (Fig 5C), a substantial proportion of cells were
R6Amed Luneg on day 16, whereas very few cells
were found in the R6Aneg Lulow gate. These
results demonstrate that the Rh polypeptides detected by BRIC 69 and
R6A are expressed after GPA, but before Lu gp. The expression of the
Rh-related epitopes detected by R6A/BRIC 69 appears to develop after
the expression of Band 3. These data clearly show that the epitopes on
the Rh polypeptides recognized by BRIC 69 and R6A do not appear at the
same time as Rh gp.
A systematic study of the expression of erythoid-specific antigens
during erythropoiesis has not been reported previously. Recent
developments in cell culture and the availability of MoAbs to
erythrocyte antigens have made such a study possible. Our results are
summarized in Fig 7. In the in vitro system
that we have studied, the most abundant membrane proteins in the mature
red blood cell are expressed in the following order: Rh gp, GPA, and
Band 3. The Kell gp was found to be expressed before the Rh gp and the Lu and Fy gps after Band 3. The epitopes of the Rh antigenic system show a complex pattern of expression that is discussed below.
The authors greatly thank Dr P.A. Judson and J.S. Smythe for their
advice and help during this study. We are grateful for the CB samples
made available by Prof J. Hows (University of Bristol, Bristol, UK) and
the staff at Southmead Hospital Maternity Unit, Bristol,
UK. MoAb LA18.18 was a gift from Prof A.E.K. von dem Borne, BS56 and BS58 were gifts from Dr H. Sonneborn, MS47 was a gift
from Dr K. Thompson, and monoclonal anti-FY3 was from Dr M. Uchikawa.
Submitted August 10, 1998; accepted February 17, 1999.
Supported in part by an MRC Collaborative Studentship.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part as an abstract at the 39th American Society of
Hematology Meeting, December 5-9, 1997 (Blood 90:175b, 1997 [abstr,
suppl 1, part 2]). Address reprint requests to David J. Anstee, PhD, Bristol
Institute for Transfusion Sciences, Southmead, Bristol BS10 5ND, UK;
e-mail: david.anstee{at}nbs.nhs.uk.
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TOP
ABSTRACT
INTRODUCTION
/Cl