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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 550-559
The Glucocorticoid Receptor Cooperates With the Erythropoietin Receptor
and c-Kit to Enhance and Sustain Proliferation of Erythroid Progenitors
In Vitro
By
Marieke von Lindern,
Wolfgang Zauner,
Georg Mellitzer,
Peter Steinlein,
Gerhard Fritsch,
Klaus Huber,
Bob Löwenberg, and
Hartmut Beug
From the Institute of Hematology, Erasmus University, Rotterdam, The
Netherlands; the Institute of Molecular Pathology, Vienna, Austria; the
Children's Cancer Research Institute, St. Anna Kinderspital, Vienna,
Austria; and the Department of Laboratory Medicine, Donauspital
SMZ-Ost, Vienna, Austria.
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ABSTRACT |
Although erythropoietin (Epo) is essential for the production of
mature red blood cells, the cooperation with other factors is required
for a proper balance between progenitor proliferation and
differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid
progenitors. We examined the role of corticosteroids in the in vitro
expansion of primary human erythroid cells in liquid cultures and
colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone,
cooperated with Epo and stem cell factor to induce erythroid
progenitors to undergo 15 to 22 cell divisions, corresponding to a
105- to 106-fold amplification of erythroid
cells. Dex acted directly on erythroid progenitors and maintained the
colony-forming capacity of the progenitor cells expanded in liquid
cultures. The hormone delayed terminal differentiation into
erythrocytes, which was assayed by morphology, hemoglobin accumulation,
and the expression of genes characteristic for immature cells.
Sustained proliferation of erythroid progenitors could be induced
equally well from purified erythroid burst-forming units (BFU-E), from
CD34+ blast cells, and from bone marrow depleted from
CD34+ cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE DEVELOPMENT OF mature, peripheral
blood cells from pluripotent stem cells in the bone marrow is a complex
process in which lineage commitment, proliferation, differentiation,
and cell survival have to be perfectly balanced. Pluripotent stem cells
are maintained via self renewal (ie, proliferation without detectable
entry of a differentiation pathway), while, in addition, they are able
to give rise to multipotent and committed progenitors. The latter
progenitors differentiate along a specific cell lineage(s) in a process
thought to occur according to a fixed program, dictating both the
remaining number of cell divisions and the time of entry into terminal
differentiation.1,2 Because of genetic changes, the
regulation of commitment and differentiation is disregulated in leukemia.
Leukemic transformation of avian erythroid progenitors by the
oncoproteins v-ErbA and v-ErbB, present in avian erythroblastosis virus, appeared to be based on a mechanism that is latently present in
normal cells.3 Normal avian erythroid progenitors express tyrosine kinase receptors related to v-ErbB (ie, c-ErbB and c-Kit, using transforming growth factor  [TGF] and avian
stem cell factor [SCF] as ligands) and nuclear hormone
receptors with a function similar to v-ErbA (estrogen receptor and
glucocorticoid receptor).3 Simultaneous stimulation of
these two receptor types in normal bone marrow cells results in the
outgrowth of committed erythroid progenitors that resemble leukemic
cells transformed by v-ErbA and v-ErbB as regards their abilities to
proliferate without differentiation for 25 to 30 generations.4-8
This progress in identifying the players that regulate the balance
between proliferation and differentiation in normal avian erythropoiesis prompted us to study whether similar mechanisms operate
in human erythropoiesis. If a similar synergism between tyrosine kinase
receptors and steroid hormones would apply to human erythropoiesis, it
may be possible to establish mass cultures of human erythroid
progenitors through the addition of recombinant factors and hormones
that would selectively provoke proliferation and yield cell numbers
large enough to perform biochemical and molecular studies.
In mammals, erythropoietin (Epo) and SCF were shown to be crucial
factors in erythropoiesis. Mice with a homozygous disruption of the
gene encoding Epo or the Epo-receptor (EpoR) die at day 13.5 of
gestation due to a lack of erythrocytes.9 Erythroid burst-forming units (BFU-E) and erythroid colony-forming units (CFU-E) can be isolated from the fetal liver of these
mice, but cells lacking the EpoR cannot mature to
erythrocytes.9,10 Mice that lack SCF or c-Kit, its
receptor, suffer from severe anemia.11,12 c-Kit is
expressed on the cell surface of the earliest progenitor up to the
stage of the basophilic erythroblast.13 In the presence of
SCF, erythroid progenitors undergo an increased number of cell
divisions before they develop into mature
erythrocytes.14,15
The role of steroids in mammalian erythropoiesis has remained elusive.
In macrophages and lymphocytes, glucocorticoids induce apoptosis due to
inhibition of AP1 and NF B transcription complexes.16 However, glucocorticoids were shown to increase the number of erythroid
colonies grown in vitro from normal bone marrow,17-19 and
treatment of nonanemic patients with the synthetic glucocorticoid receptor (GR) ligand prednisone has been suggested to enhance erythropoiesis.20 Thus, the GR may have a stimulatory role
in mammalian erythropoiesis similar to what was found in avian
erythroid progenitors.
In this report, we demonstrate that the addition of the GR ligand
dexamethasone (Dex) to Epo and SCF allowed the establishment of mass
cultures of normal erythroid progenitors from mononuclear cells of
human umbilical cord blood, bone marrow, and peripheral blood.
Erythroid cells in these mass cultures underwent 15 to 20 cell
divisions. Cultures of purified BFU-E, as well as CD34+ and
even CD34 cells could be expanded up to
106-fold. Morphologically, 95% of the cells in the culture
appeared as proerythroblasts, which could be induced to terminal
erythroid differentiation upon removal of SCF and Dex. Single-cell
cloning experiments suggested that Dex acts directly on human erythroid progenitors, maintaining their colony-forming capacity and delaying their differentiation.
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MATERIALS AND METHODS |
Cells and cell culture.
Neonatal cord blood, bone marrow, or buffy coat from peripheral blood
was obtained from healthy volunteers after informed consent was
received. Mononuclear cells were purified by centrifugation over a
Ficoll gradient and seeded in CFU-E medium as described,5,6 with minor modifications: conalbumin was replaced by human transferrin (iron-loaded; Sigma, St Louis, MO), chicken serum was
omitted and only fetal calf serum (FCS; 12%; GIBCO, Grand Island,
NY) was used. Unless mentioned otherwise, we used 0.5 U/mL
recombinant human Epo (a kind gift from Janssen-Cilag, Tilburg, The
Netherlands), 100 ng/mL recombinant human SCF (a kind gift
from Amgen [Thousand Oaks, CA] or purchased from R&D
[Minneapolis, MN]), 40 ng/mL recombinant human
insulin-like growth factor-I (IGF-I; R&D), 1 U/mL human insulin
(Actrapid; 40 IU/mL; Bayer-Leverkusen, Leverkusen,
Germany), 5 × 107 mol/L Dex (Sigma), and 5 × 107 mol/L Dex-antagonist ZK112,993 (a kind
gift from Schering, Madison, NJ); obtained through Dr E. Müllner, Biocenter, Vienna, Austria). In initial experiments,
CD34+ cells were purified from cord blood on CellPro
columns. For the separation of CD34+ cells from bone
marrow, Minimacs columns (Miltenyi Biotec, Bergisch Gladbach, Germany)
were used. Total mononuclear cells were initially plated at a density
of 10 × 106 cells/mL. During the first 5 days of
culture, cell density was reduced to 1 to 2 × 106/mL.
Small mature cells and apoptotic cells were removed once or twice
during the first week of the culture by centrifugation through Percoll
step-gradients (density, 1.075 g/mL; Pharmacia, Uppsala,
Sweden). Such a purification was always performed when cultures were started from mononuclear cells derived from buffy coat or
cord blood; they could be omitted for bone marrow cultures. Cells were
kept at 1 to 2 × 106 through daily dilutions or
medium changes with fresh medium containing factors. Cells were counted
on an electronic cell counter (CASY-1; Schärfe-System,
Reutlingen, Germany).
Fluorescence-activated cell sorting (FACS) analysis.
Cells were stained using directly labeled antibodies against human cell
surface markers (DAKO [Vienna, Austria] and Serotec [Vienna,
Austria]) according to the manufacturer's directions. Analysis was
performed using a FACScan (Becton Dickinson, San Jose, CA). Propidium
iodide-positive cells were excluded from analysis. To sort BFU-E and
blast cells, 100 × 106 frozen mononuclear bone marrow
cells were thawed and CD34+ cells were purified using a
MACS CD34 isolation kit (Miltenyi Biotec) according to the guidelines
of the supplier. The CD34 fraction was labeled with
CD34-Texas Red (TR), and the CD34+ fraction
was labeled with CD34-TR, CD38-phycoerythrin (PE), and CD71-fluorescein isothiocyanate (FITC) (Becton Dickinson)
in one incubation. Cells were washed and resuspended in
phosphate-buffered saline (PBS) plus 1% bovine serum albumin (BSA).
Cells were sorted using a FACS-Vantage (Becton Dickinson). After
sorting, cell samples were counted, centrifuged on slides, and seeded
in human CFU-E medium with factors as described. The initial cell
numbers in Fig 6 are recalculated to the total number of cells obtained
after sorting.
Determination of hemoglobin accumulation and cell morphology.
To determine hemoglobin accumulation, 3 × 50 µL aliquots of the
cultures were removed and processed for photometric determination of
hemoglobin.21 To analyze cell morphology, cells were
cytocentrifuged onto slides and stained with histological dyes and
neutral benzidine for hemoglobin.22 Images were taken using
a CCD camera (Photometrics, Tucson, AZ) and a blue filter
(480 nm), so that mature cells (stained yellow to brownish) appear
darkly stained. Images were processed with Adobe Photoshop (Adobe
Systems Inc, San Jose, CA).
Colony assays.
To determine the number of colony-forming cells and their expansion
potential, Ficoll-purified bone marrow cells were cultured in CFU-E
medium and subsequently seeded at the indicated cell numbers per well
in 96-well plates, using CFU-E medium complemented with 1 mg/mL
fibrinogen and 0.1 U/mL thrombin immediately before aliquoting 50 µL
of cell suspension per well. After coagulation, another 50 µL of
medium was added containing combinations of factors at double the
concentrations indicated above. Every second day, the medium above the
clot was replaced by fresh medium with factors. To determine
hemoglobinization of the colonies, 10 µL acid benzidine staining
solution (a freshly prepared mix of 150 µL 10% acetic acid, 20 µL
3% free benzidine base in acetic acid, and 10 µL 30% H2O2) was added to the medium above the clots.
Northern blot.
RNA was isolated from erythroid progenitor cells using the method of
Chomczynski and Sacchi23 with minor modifications. Cells
were lysed in GITC buffer and NaAc, pH4.0, was added to 25 mmol/L. The
solution was extracted with H2O-saturated phenol plus
chloroform and isoamylalcohol. RNA was precipitated with isopropanol
and dissolved in 10 mmol/L Tris, pH 7, 1 mmol/L EDTA, 0.2% sodium
dodecyl sulfate (SDS). Proteinase K was added to 200 µg/mL for 30 minutes at 37°C. RNA was extracted with
phenol:chloroform:isoamylalcohol (25:24:1) pH 7 and precipitated with
ethanol. Ten to 20 µg of RNA was run on a formaldehyde-containing
agarose gel and transferred to nylon filters (Gene Screen; Gene Vest
Inc, Toronto, Canada) using conventional
procedures.24 Probes used were the entire ORF of human
GATA-1, c-myb, c-Kit, or RBTN2/TTG-2.
Phosphotyrosine blot.
Cultured cells were washed once with PBS and incubated in medium
without growth factors for 12 hours. Cells were washed once with PBS,
suspended in medium, and treated 10 minutes at 37°C with either 10 U/mL rhEpo, 1 µg/mL SCF, 400 ng/mL IGF-I, 100 ng/mL interleukin-3
(IL-3), 100 ng/mL granulocyte-macrophage colony-stimulating factor
(GM-CSF), or 100 ng/mL insulin. Cells (1 × 106) were
lysed in 20 µL lysis buffer (1% Triton X-100, 50 mmol/L Tris-HCl, pH
8.0, 100 mmol/L NaCl, 1 mmol/L sodium orthovanadate, 10 µg/mL
aprotinin, and 2 µg/mL leupeptin). Cell lysates were cleared by
centrifugation for 15 minutes at 15,000 rpm before use. Lysates derived
from 5 × 105 cells were run on a 8%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
nitrocellulose membranes (DuPont, Wilmington, DE). The
Western blot was probed with the antiphosphotyrosine antibody 4G10
(Upstate Biotechnology, Lake Placid, NY).
| |
RESULTS |
Sustained proliferation of immature human erythroid progenitors.
CD34-selected cells or the total fraction of mononuclear cells derived
from human cord blood were seeded in CFU-E medium supplemented with
Epo, SCF, TGF, estradiol, and Dex. After a lag-phase of 6 days, cell
numbers started to increase, with a doubling time of 20 to 24 hours,
resulting in a 105-fold increase over a 14-day period
(Fig 1A). Cell samples were taken from the
cultures at subsequent days, and cytological analysis showed typical
erythroblasts at various stages of maturation (Fig 1B). After 18 days
of culture, both the growth rate and the fraction of cells with a
proerythroblast morphology decreased, whereas the number of partially
mature erythroid cells increased (Fig 1B, panels 2 and 3). When cells
were harvested from the culture, separated by Percoll, and reseeded in
secondary cultures, the growth rate of the cultures was reestablished
using the low-density cell fraction containing immature erythroblasts.
Rapid growth could be repetitively restored by successive rounds of
purification (Fig 1A, top panel). This indicated that immature
progenitors recoverable from the culture had retained the capacity to
proliferate (a 106-fold expansion equivalent to ~20
population doublings).
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| Fig 1.
Outgrowth of erythroid progenitors from neonatal cord
blood, human bone marrow, or peripheral blood. (A) CD34+
cells isolated from neonatal cord blood were cultured in CFU-E medium
complemented with Epo, SCF, IGF-I, and Dex (lower part). Cell density
was kept at 1.5 to 3 × 106 cells/mL and total cell
numbers were enumerated at the times indicated. At day 14 (arrowhead),
immature low-density cells were purified from the mass culture using
Percoll 1.072 g/mL and reseeded (top part). This procedure was repeated
several times. (B) At day 8 (1), day 12 (2), and day 17 (3) after
initiating the culture, cells were cytocentrifuged onto slides and
stained with histochemical dyes plus a specific stain for hemoglobin
that makes hemoglobinized cells appear dark in this
figure.22 Numbers of panels correspond to numbered arrows
in (A), indicating the time of aliquot removal. (C and D)
Ficoll-purified cells from bone marrow (3 healthy donors) or from
peripheral blood (5 untreated, healthy donors) were cultivated as
described in the legend to (A) and cumulative cell numbers were
calculated. In case of the peripheral blood-derived cells, apoptotic
cells and lymphocytes were removed by centrifugation through Percoll
1.072 g/mL at days 4 and 6 of culture.
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Cultures of human bone marrow or mononuclear cells derived from
peripheral blood showed very similar expansion kinetics under the same
culture conditions (Fig 1C and D). To determine more accurately the in
vitro life span of human erythroid progenitors proliferating in these
cultures, mononuclear cells from cord blood were seeded into the above
medium under limiting dilution conditions.7,25 Cells from
wells containing one single colony only were expanded until extinction.
During propagation, cell numbers were counted and total cell numbers
were enumerated. From more than 80% of these single colonies between 1 × 105 and 4 × 106 cells were
generated, which is equivalent to an average amplification of 16 to 22 generations.
Characterization of the cells grown in cultures supplemented with
Epo, SCF, and Dex.
FACS analysis of the cells grown in cultures supplemented with Epo,
SCF, and Dex showed that most of the cells expressed an immature
phenotype (80% to 95% CD71+ and 50% to 80%
CD117+ cells). Approximately 20% of the cells were
maturing erythroid cells (CD36+ and GPA+) and
5% to 7% of the cells were of the myeloid or lymphoid lineage (Table 1). Cells lacked CD34 expression,
even when stained at only 5 days of culture (data not shown). In
addition, cells from several independent cultures were tested in colony
assays. Cells taken from 13- or 15-day-old cultures of cord blood
formed predominantly erythroid bursts and only a very small number of
myeloid colonies (Table 2). These data
indicate that the expansion of cultures obtained from mononuclear cells
of neonatal cord blood or bone marrow in medium supplemented with Epo,
SCF, and Dex is mainly the result of proliferation of erythroid cells.
Finally, we examined whether the cultured cells were responsive to
distinct factors in short-term proliferation assays (incorporation of
tritiated thymidine) and in a biochemical assay (induction of tyrosine
phosphorylation). The cells incorporated tritiated thymidine in
response to Epo. SCF and IGF-I did not induce DNA synthesis if added
alone, but they enhanced the incorporation of tritiated thymidine when
added together with a suboptimal concentration of Epo. The cells did
not respond to TGF or other epidermal growth factor
(EGF)-like ligands, IL-3 or GM-CSF (data not shown). The erythroid proliferation factors Epo, SCF, and IGF-I induced tyrosine phosphorylation of multiple substrate proteins, as detected with antiphosphotyrosine antibodies in a Western blot
(Fig 2). No phosphorylation of substrates
was detected when cells were stimulated with IL-3, GM-CSF, or EGF-like
substrates (Fig 2 and data not shown).
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| Fig 2.
Tyrosine phosphorylation induced by activation of
receptors for growth factors and cytokines. Erythroid progenitors were
withdrawn from growth factors overnight and subsequently stimulated for
10 minutes with the cytokines indicated, using 10-fold higher
concentrations than used in liquid cultures. Cell lysates were
separated on acrylamide gels and subjected to Western blot analysis
using the antiphosphotyrosine antibody 4G10. Arrows indicate the
position of the size markers used.
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In conclusion, cultures established from bone marrow or cord blood in
the presence of Epo, SCF, and Dex consisted of 90% to 95% erythroid
progenitors. In the following, we used these cultures to determine how
corticosteroids contribute to the in vitro expansion of erythroid progenitors.
Human erythroid progenitors require glucocorticosteroids for
sustained proliferation.
In all avian erythroid progenitors, the GR ligand Dex was indispensable
for progenitor self-renewal.7,26 However, the major role of
Dex in the outgrowth of erythroid progenitors from human bone marrow
mononuclear cells could still be mediated by nonerythroid accessory
cells. Human bone marrow cells were grown in Epo, SCF, and either Dex
or the GR-antagonist ZK. Amplification of erythroid progenitors from
bone marrow cells of several independent donors was at least 10-fold
reduced after culture for 10 days in the presence of ZK as compared
with cultures supplemented with Dex (Fig 3A
and data not shown). Addition of the GR-antagonist ZK was required to
unveil the effect of Dex, due to the presence of corticosteroids in
serum. In the absence of Dex or ZK, the proliferation of erythroid
progenitors depends very much on the serum batch used. Previously, we
showed that corticosteroids, as a major factor required for the
proliferation of avian erythroid progenitors, are removed from serum by
freon/charcoal treatment. The addition of ZK112,993 to serum had the
same effect.7
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| Fig 3.
Dex enhances proliferation and colony-forming ability of
erythroid progenitors. (A) Human bone marrow cells were cultured in
medium complemented with Epo, SCF, IGF-1, and either Dex or ZK ( and
 , respectively). (B) At days 0, 4, and 8, cell aliquots of both
cultures were seeded in fibrinogen clots with 2, 5, and 15 cells per
well and medium complemented with Epo, SCF, IGF-1, and either Dex or ZK
(indicated by D or Z at the X-axis) was added. Previous liquid culture
conditions are indicated below by Dex or ZK. After 10 days, colonies
were stained with benzidine and counted, and the cell number per colony
was estimated as small (0 to 30 cells), medium (30 to 100 cells), or
large (>100 cells). Colony number is given per 100 bone marrow cells
seeded in the culture at day 0. The number above the bars indicates the
percentage of only partially hemoglobinized colonies.
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Subsequently, the colony-forming capacity of aliquots from the same
cultures was determined at successive intervals, using a limiting
dilution approach. Bone marrow cells were seeded in fibrinogen clots at
2, 5, and 15 cells per well in 96-well plates. The cells, grown in
liquid cultures in the presence of either Dex or ZK, were exposed to
Epo, IGF-I, and SCF and from each culture again to Dex and ZK.
Benzidine-positive colonies were scored after 10 days as small (4 to
30), medium (30 to 100), or large (>100 cells). At the start of the
culture (day 0), a similar number of colonies was obtained (~1.5%)
in the presence of either Dex or ZK. However, in the presence of ZK,
the colonies were smaller and more hemoglobinized (Fig 3B).
In liquid cultures containing Dex, the number of erythroid
colony-forming units (ECFU) had increased 20-fold in 4 days and 100-fold in 8 days, with 50% of these ECFU able to undergo at least
another 5 cell divisions (colony size, >30 cells). In contrast, bone
marrow cultures in presence of the GR-antagonist ZK resulted only in a
10-fold increase of ECFC after 4 days and no further amplification at 8 days of culture (Fig 3B). Irrespective of the conditions of liquid
culture, fibrinogen clots containing Dex yielded colonies that were
larger and less hemoglobinized and that exceeded 4 to 5 times in colony
number compared with fibrinogen clots containing ZK (Fig 3B). The
number of colonies per 100 cells seeded was constant under all
conditions, regardless of whether 15, 5, or 2 cells had been seeded per
well (data not shown).
These data are consistent with the notion that Dex supports sustained
proliferation of erythroid progenitors and that it acts directly on
these progenitors. Dex increased both the number of colony-forming
cells in the mass cultures and the proportion of immature colonies
derived from these colony-forming cells. The ability to form
medium-sized to large colonies in Dex did not change largely in the
Dex-treated mass cultures, but rapidly decreased in the presence of ZK.
Cooperation of the GR with the EpoR and c-Kit arrests human
proerythroblast differentiation and maintains the expression of
immature erythroblast markers.
We next tested in more detail how Dex affects the balance of
proliferation and differentiation in an established culture of erythroid progenitors. Aliquots from a 6-day-old culture of erythroid progenitors were seeded in media containing Epo, IGF-I, SCF, and either
Dex or the GR-antagonist ZK. Cell numbers were counted daily and
aliquots were processed to determine hemoglobin content and morphology.
Three days after applying the different conditions (Fig 4A), cultures containing ZK became
stationary, whereas cultures containing Dex continued to proliferate.
Concurrently, hemoglobin levels increased considerably in cells grown
in ZK-containing medium (Fig 4B). Cytospins showed that cultures
exposed to ZK differentiated completely into small, partially
enucleated erythrocyte-like cells and then disintegrated (Fig 4C,
bottom panels). In contrast, cultures kept in Dex retained their low
hemoglobin content, large size, and basophilic erythroblast morphology
throughout the experiment (Fig 4C, top panels). In the absence of Epo,
the cells neither proliferated nor differentiated irrespective of the
presence of Dex, ZK, or SCF, whereas the presence of Epo but not SCF
resulted in rapid differentiation, as shown elsewhere for human
cells27 as well as for avian progenitors.26 The
presence of Dex or ZK did not affect Epo-induced differentiation (data
not shown), which is in agreement with observations in avian cells in
which Dex cooperates with SCF to retain erythroid cells in an immature
state.7
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| Fig 4.
Dex supports proliferation and inhibits differentiation.
Cells from a culture of neonatal cord blood cells were washed 5 days
after seeding and aliquots exposed to medium plus Epo, SCF, and Dex
( ) or Epo, SCF, and the GR-antagonist ZK 112,993 (). (A)
Proliferation was monitored by calculating cumulative cell numbers. (B)
Hemoglobin accumulation was determined at the days indicated and
plotted as milligrams of hemoglobin per 106 cells. (C) At
the days indicated by arrows in (A), cell samples from cultures
containing Dex (upper panels) or ZK 112,993 (lower panels) were
cytocentrifuged onto slides and stained cytochemically with a specific
stain for hemoglobin. Well-hemoglobinized cells show a dark to black
cytoplasm.
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Earlier work in the chicken had shown that Dex maintained the
expression of c-myb, which is a requirement for long-term
proliferation.7 Self-renewing avian erythroblasts also
express c-Kit. Upon induction of differentiation, c-myb and
c-Kit are rapidly downregulated. RBTN2 is a transcription factor
selectively expressed in immature erythroid cells (M.v.L., unpublished
data), which is required for progression past the
proerythroblast stage according to observations in RBTN2
 / mice.28 In contrast, expression of GATA-1
is required during terminal differentiation.29,30 We
examined whether Dex affects transcription of c-myb, c-Kit,
RBTN2, and GATA-1 in human erythroid progenitors. Aliquots from a
10-day-old culture were kept for 48 hours in medium containing
charcoal-stripped serum and growth factors, but no steroid hormones.
Subsequently, the GR agonist Dex or antagonist ZK were added, and cell
samples were processed daily for Northern blot analysis. mRNA levels of
c-myb, c-Kit, and RBTN2 increased in the presence of Dex,
whereas they remained low or declined in ZK
(Fig 5). In contrast, expression levels of
GATA-1 were not affected by activation or inactivation of the GR. The
apparent upregulation of c-myb, c-Kit, and RBTN2 is consistent
with the finding that Dex stimulates sustained proliferation of
immature erythroid cells and delays differentiation.
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| Fig 5.
Dex maintains high expression levels of c-myb,
c-Kit, and RBTN2. A culture of erythroid progenitor cells was kept for
2 days in medium containing Epo, SCF, IGF-I, and freon/charcoal
stripped serum (depleted from Dex). Thereafter, aliquots of the culture
were cultivated in the same medium supplemented with either Dex or ZK
112,993 at 106 mol/L. Total RNA was extracted from
untreated cells (day 0) or from cells cultured for 1, 2, or 3 days in
GR agonist or antagonist. Total RNA was analyzed on Northern blots,
using full-length c-DNA probes of c-myb, c-Kit, RBTN2, and
GATA-1. The ethidium staining is shown in the bottom panel as a loading
control.
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Origin of erythroid progenitors capable of sustained cell
proliferation.
We then set out to characterize the progenitor cell phenotype that was
responsive to stimulation of expansion by Epo, SCF, and Dex. Human bone
marrow cells were separated into CD34+ and
CD34 fractions. The CD34 fraction
was subjected twice to depletion of CD34+ cells and finally
contained less than 0.1% CD34+ cells by FACS analysis
(Fig 6A). The CD34+ cells were
stained for CD34, CD71, and CD38 and were sorted by FACS into two
subfractions: a blast fraction (CD34+, low CD71, low CD38;
gate R3, Fig 6C) and a BFU-E-enriched fraction (CD34+,
high CD71; gate R4, Fig 6C).
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| Fig 6.
Erythroid progenitors can be grown from
CD34+ and CD34 cell fractions. Mononuclear
bone marrow cells were fractionated into CD34 and
CD34+ fractions using Minimacs columns. The
CD34 fraction was repurified using the same columns (A
and B). Cells were stained for CD34 to verify the purity of the
CD34 and CD34+ fraction by FACS. The
CD34+ cells were costained with CD71 (horizontal axis)
and CD38 (vertical axis; C). Only CD34+ cells were gated
(A and B; gate R2). Fractions highly enriched for BFU-E (C; R4, high
CD71, medium CD38) or immature blast cells (C; R3; medium-low CD71,
medium-low CD38) were purified by sorting. CD34 cells
( ), the sorted BFU-E progenitors ( ), and the sorted immature
blast cells () were cultured in CFU-E medium supplemented with Epo,
SCF, IGF-I, and Dex. Cumulative cell numbers were calculated (D).
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After purification, the three subpopulations (blast cells, BFU-E
fraction, and CD34 cells) were seeded in medium
containing Epo, SCF, and Dex, and proliferation was monitored by daily
counting (Fig 6D). Approximately equal numbers of erythroid progenitors
could be grown from all three cell populations, which corresponded with
an expansion of 105-fold for the purified BFU-E fraction
(Fig 6D).
In all cultures, the increase in cell numbers reached a plateau at
approximately 15 days when the cells started to mature to
hemoglobinized cells. All cultures contained greater than 90% erythroid progenitors, as shown by cytocentrifugation and benzidine staining (data not shown). These results demonstrate that cultures of
committed erythroid progenitors can be established from cells of
varying maturity (including CD34 erythroid
progenitors, generally considered as [post-]CFU-E) in the presence
of Epo, SCF, and glucocorticoids.
| |
DISCUSSION |
In this report, we demonstrate that sustained proliferation of human
erythroid progenitors can be induced upon activation of EpoR, c-Kit,
and GR by their respective ligands. Mass cultures yielding
109 to 1011 erythroid cells were established
from a few million mononuclear cells derived from umbilical cord blood,
bone marrow, or peripheral blood. The cultures differed from previously
described cultures of erythroid progenitors by their dependence on
GR-ligands. These ligands were shown to exert their effect directly on
the erythroid progenitors, and they maintained the colony-forming ability of the progenitors expanded in liquid culture while suppressing their differentiation. The cultures consisted of greater than 90% pure
erythroid progenitors, apparent mainly as proerythroblasts and
basophilic erythroblasts and able to undergo approximately 15 to 20 generations in culture. These cultures of proliferating human
erythroblasts could be obtained from both CD34+ and
CD34 cells as well as from a cell fraction enriched
for BFU-E. This suggests that the expansion capacity of human erythroid
progenitors, like their counterparts in chicken and mouse, may be
greater than hitherto anticipated and that glucocorticoids play an
important role in the regulation of the proliferation/differentiation program.
A recent report described a similar approach to grow and differentiate
primary human erythroblasts.27,31 In this report, the
differentiation characteristics of these cells were characterized in
more detail.
In avian erythroblastosis virus (AEV)-transformed cells, a mutated
epidermal growth factor receptor (v-ErbB) cooperates with a mutated
nuclear receptor for thyroid hormone (v-ErbA), which results in
self-renewal of erythroid progenitors for the entire in vitro life-span
of a chicken cell32,33 (25 to 40 generations). Subsequently, normal erythroid progenitors (SCF/TGF progenitors) could be identified that express nonmutated members of receptor tyrosine kinases and nuclear hormone receptors and can be induced to
self-renewal in the presence of human recombinant TGF as a ligand
for c-ErbB, avian SCF, and both Dex and estradiol as steroid ligands.
Ligand activation of both the steroid hormone receptors and the
tyrosine kinases was essential for self-renewal of these cells.4-7,14 These progenitors are relatively rare, being
present at frequencies of only 1 in 15,000.6 Recently, a
more abundant avian erythroid progenitor was identified
(EpoR-progenitors, occurring at a frequency of 1 in 40026),
which is dependent on the presence of Epo, SCF, and Dex for
self-renewal.26,34 The life span of these EpoR-progenitors
was shorter than that of SCF/TGF progenitors (15 to 20 generations).
These avian progenitors closely correspond to those obtained in the
cultures of human erythroblasts. They do not respond to EGF-like
ligands or estradiol and terminally differentiate upon culture
progression rather than entering senescence as undifferentiated cells
typical of the SCF/TGF progenitors.
Differentiation of avian erythroid progenitors can be induced at any
time by removal of the self-renewal factors (TGF, E2, and Dex or
Epo, SCF, and Dex, respectively) and addition of differentiation factors (Epo plus insulin). Upon differentiation induction, the cells
undergo a fixed number of 5 cell divisions, during which cell cycle and
cell size control is profoundly altered.35 The human
progenitors grown from bone marrow or cord blood could be similarly
induced to differentiate into enucleated red blood cells, when the
proliferation factors Epo, SCF, and Dex were replaced by the
differentiation factors Epo and insulin. The cells underwent a limited
number of divisions, became rapidly smaller (from 900 fL in the
progenitors to ~250 fL in the mature cells), and accumulated hemoglobin levels comparable to peripheral blood erythrocytes (mean
cellular hemoglobin content of 35 to 40 g/dL; M.v.L., unpublished data). Taken together, these data strongly suggest that
control of erythroid proliferation and differentiation is comparable in mammalian and avian progenitors. It is therefore likely that previously described mechanisms regulating the balance of self-renewal versus terminal differentiation in avian cells similarly control the balance
between proliferation and differentiation in mammalian cells.3,25,33,36
Cultures of human erythroid progenitors could be established both from
CD34 and CD34+ cells, predominantly
containing cells with the morphology of proerythroblasts. Cultures
initiated from CD34+ cells only contained
CD34 cells by day 5. Cells with such a phenotype are
generally considered to be (post-)CFU-E. However, a CFU-E is
characterized by a very limited proliferation capacity (3 to 5 cell
divisions). Even BFU-E are reported to undergo a maximum of 10 cell
divisions,37 less than the 20 cell divisions induced by the
cooperation of cytokines and steroid hormones that we observed in our
cultures. This indicates that committed erythroid progenitors have a
larger expansion capacity than hitherto anticipated.
Glucocorticoids have pleiotropic effects on hematopoietic cells:
activation of the GR induces apoptosis in lymphoid and myelomonocytic cells, while it protects granulocytes from apoptosis38 and
stimulates proliferation of erythroid progenitors7 (this
report). Repression of macrophages and lymphocytes is caused by the
ability of the GR to interact with the transcription factors AP-1 and
NFB, independent of DNA binding by the GR, which results in
repression of transcription by these complexes.16 Several
members of the NFB family are also expressed in erythroid
progenitors.39 However, repression appears not essential
for the function of the GR in erythroid progenitors. Instead,
proliferation induction of avian erythroid cells is dependent on intact
DNA binding and transcriptional activation domains of the
GR.7 We recently confirmed this notion in mice carrying a
mutated GR, which lacked the ability to transactivate upon hormone
binding, but retained transrepressing activity.40 Erythroblasts could be grown from fetal livers of wild-type mice using
the same conditions described here, but not from fetal livers of the
GRdim/dim mice, confirming the crucial role of the GR and its
transactivation ability in mammalian erythroblast proliferation.
Although we could not expand erythroblasts from fetal livers of mice
deficient for the GR41 or from GRdim/dim
mice,40 these mice do not show obvious defects in normal in
vivo erythropoiesis. Studies addressing stress erythropoiesis in mice
showed that upregulation of erythroid progenitors (BFU-E and CFU-E) on
anemia induction occurred exclusively in the spleen and required a
functional c-Kit receptor tyrosine kinase.42 Recently, we
have obtained evidence that this upregulation of spleen erythroid
progenitors in anemic mice does not occur in the GR dim/dim mice (A. Bauer and H.B., manuscript in preparation). Because
glucocorticoids are highly upregulated on blood loss or injury, the GR
may play a physiological role in stress erythropoiesis.
The GR has been shown to interact with STAT5, resulting in enhanced
transcription of  -casein.43 Although disputed, DNA binding by the GR appears important for its synergistic action with
STAT5.44-46 This synergy has so far only been described for the -casein promoter. We tested expression of the STAT5 target gene
CIS47 in primary erythroid progenitors stimulated with or
without Epo in the presence of either Dex or the GR-antagonist ZK. Cis
expression was fully dependent on Epo-induced STAT5-phosphorylation, but independent of GR activation. Thus, we could not demonstrate synergy between STAT5 and the GR in erythroid progenitors. However, we
cannot exclude that this mechanism plays a role in the activation of
other target genes.
Among several genes tested that are critical for erythroid
self-renewal, the GR induced expression of c-myb. Notably,
constitutive expression of activated Myb (v-Myb) abrogated the
requirement for corticosteroids.7 Mice deficient in Myb die
at day 15 of gestation when they are severely anemic.48 One
of the genes regulated by c-Myb is c-Kit.49,50 In the
cultured erythroid progenitors, activation of the GR enhanced
expression of c-myb, c-Kit, and the erythroid transcription
factor RBTN2. One mechanism by which corticosteroids cooperate with SCF
and Epo may involve maintenance of c-Kit expression. SCF is required to
delay differentiation of erythroid progenitors.15 The EpoR
appears to form a complex with c-Kit,51 and
cross-phosphorylation of the receptors has been
reported.52-54 Constitutive overexpression of c-Kit in the absence of corticosteroids delays differentiation, but it is not sufficient to induce self-renewal of avian erythroid
progenitors.14 Thus, maintaining c-Kit expression is an
important contribution of the GR to sustained proliferation, but
induction of renewal divisions most likely involves additional genes
controlled by the GR via c-Myb and could involve transcription factors
such as RBTN2.
| |
ACKNOWLEDGMENT |
The authors thank Dr B. Royer-Pokora for the gift of the TTG2/RBTN2
probe, Eva Deiner and Petra Buchinger for expert technical assistance,
Saskia Buchwald and Dr B. Wognum for help and advice in FACS-sorting of
cells, and Drs I.P Touw and T. van Dijk for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted December 2, 1998; accepted March 22, 1999.
Supported by grants from the Forschungsförderungsfund für
die Gewerbliche Wirtschaft (FFF3/10628), the Dutch Cancer Society (EUR
95-1021), and the European Community (Biomed 2-PL951355).
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.
Address reprint requests to Marieke von Lindern, PhD,
Institute of Hematology, Erasmus University, p/o Box 1738, 3000 DR
Rotterdam, The Netherlands; e-mail: vonlindern{at}hema.fgg.eur.nl.
| |
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TOP
ABSTRACT
INTRODUCTION