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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3658-3668
Growth and Differentiation of Human Stem Cell
Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro
By
Birgit Panzenböck,
Petr Bartunek,
Markus Y. Mapara, and
Martin Zenke
From the Max-Delbrück-Centre for Molecular Medicine, MDC,
Berlin, Germany; and the Humboldt University Berlin, Virchow Klinikum,
Robert-Rössle-Klinik, Berlin, Germany.
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ABSTRACT |
Stem cell factor (SCF) and erythropoietin (Epo) effectively support
erythroid cell development in vivo and in vitro. We have studied here
an SCF/Epo-dependent erythroid progenitor cell from cord blood that can
be efficiently amplified in liquid culture to large cell numbers in the
presence of SCF, Epo, insulin-like growth factor-1
(IGF-1), dexamethasone, and estrogen. Additionally, by
changing the culture conditions and by administration of Epo plus
insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral
blood stem cells and human bone marrow and can be isolated, amplified,
and differentiated in vitro under the same conditions. Thus, highly
homogenous populations of SCF/Epo-dependent erythroid progenitors can
be obtained in large cell numbers that are most suitable for further
biochemical and molecular studies. We demonstrate that such cells
express the recently identified adapter protein p62dok that
is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly
induced when cells differentiate. Thus, the in vitro system described
allows the study of molecules and signaling pathways involved in
proliferation or differentiation of human erythroid cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE DEVELOPMENT OF mature red blood cells
from hematopoietic stem/progenitor cells is tightly controlled by
multiple protein factors that are required for both limited growth and terminal differentiation. The two most prominent cytokines that regulate erythropoiesis are erythropoietin (Epo) and stem cell factor
(SCF; also referred to as mast cell growth factor [MGF], Steel factor
[SLF], or Kit ligand [KL]).1,2 Epo is a 34-kD glycoprotein that promotes survival, proliferation, and differentiation of erythroid progenitor cells in vivo and in vitro.1 SCF,
although initially identified by its ability to stimulate proliferation of multipotent hematopoietic progenitor cells, is effective also in
supporting growth of already committed progenitors, such as, erythroid
and myeloid progenitors, thereby acting synergistically with
lineage-restricted cytokines, such as Epo, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF).3-6 Additionally, in more recent studies,
the nuclear hormone receptors for dexamethasone and estrogen,
glucocorticoid receptor (GR) and estrogen receptor (ER), respectively,
have been implicated in causing sustained proliferation of erythroid
progenitor cells of chicken in vitro,7-10 whereas the
nuclear hormone receptors for thyroid hormone (T3), all-trans retinoic
acid (all-trans RA), and 9-cis RA (c-erbA/thyroid hormone receptor
[TR], retinoic acid receptor [RAR], and RXR, respectively) were
found to promote erythroid differentiation.11-14
Hematopoietic growth factors have been successfully used for selective
amplification of a particular subset of hematopoietic progenitor cells
in culture. Most of these in vitro experiments use colony assays in
semisolid culture media, in some instances followed by short-term
liquid culture.3-6,15 In these systems, hematopoietic
progenitors will, depending on the specific growth factors added,
develop into discrete colonies. However, the number of cells obtained
from such colonies that can be further propagated and studied are, in
most instances, too low for a more extensive biochemical and functional
analysis. Additionally, colony assays are one-step continuous cultures
with limited possibilities to manipulate the culture conditions, eg, by
addition or withdrawal of specific factors. Furthermore, colony
formation is, in most instances, the outcome of a limited number of
cell divisions followed by commitment and terminal differentiation.
Thus, colony assays normally do not allow study of cell proliferation
and differentiation separately as individual genetic programs that
determine cell fate. Most of these limitations can be overcome in
liquid culture systems that selectively support growth of the cell type
wanted and where cells can be induced to differentiate by changing
culture conditions.
Previous studies with human erythroid colony-forming cells (ECFC) from
peripheral blood established that SCF and Epo, if applied simultaneously, are the most prominent factors required for in vitro
growth of erythroid progenitor cells, whereas Epo alone shifts the
propensity of progenitor cells towards differentiation.5,6 However, so far, the signaling mechanisms of c-kit/SCF receptor and Epo
receptor have been studied mainly in established and/or engineered cell lines,16-19 and their analysis in primary
human progenitor cells has just commenced.15 One reason is
that primary human erythroid progenitors are difficult to obtain as
homogenous cell populations in sufficiently high cell numbers to allow
more detailed biochemical and molecular studies. In the chicken,
homogenous populations of erythroid progenitors can readily be
generated in vitro from bone marrow samples that are cultured in the
presence of SCF and/or transforming growth factor type  (TGF) and steroid hormones.7-10,20,21 Furthermore,
retroviral vectors have been used to introduce and express in chicken
erythroid cells various receptor tyrosine kinases, nuclear hormone
receptors, and transcription factors that modulate erythroid cell
growth and differentiation.7,21,22
In this study, we describe conditions that allow the selective
amplification in liquid culture of SCF/Epo-dependent erythroid progenitor cells from human cord blood, CD34+ peripheral
blood stem cells, and bone marrow to large cell numbers that are most
suitable for further functional, biochemical, and molecular studies.
Under growth conditions, cells express the recently identified adapter
protein p62dok that is involved in signaling downstream of
c-kit/SCF receptor. We also demonstrate that SCF/Epo-dependent
erythroid progenitors can be induced to terminally differentiate in
vitro and thereby faithfully recapitulate erythroid differentiation in
culture. After induction of differentiation, cells effectively
upregulate expression of the cyclin-dependent kinase (CDK) inhibitors
p21cip1 and p27kip1. Thus, in this experimental
system, molecules involved in growth control and differentiation of
erythroid cells can now be identified and studied, both during normal
erythropoiesis and in the pathological state.
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MATERIALS AND METHODS |
Cells and cell culture.
Cord blood cells, scheduled for discard and collected according to
institutional guidelines, were obtained after normal full-term pregnancies. After placental delivery, the umbilical veins were cannulated and aspirated. Approximately 30 to 40 mL cord blood was
routinely recovered and collected in syringes containing 100 U sodium
heparin (Novo Nordisk Pharma, Mainz, Germany) per
milliliter of cord blood. Residual blood clots were removed by passage
through a 70-µm cell strainer (Becton Dickinson, Mountain View,
CA) and light-density, mononuclear cells were isolated
using Ficoll-Hypaque centrifugation (density 1.077 g/mL; Eurobio,
Paris, France). Cells were plated at 4 × 106 cells/mL
(days 1 through 3) and later at 2 × 106 cells/mL and
cultured at 37°C in 5% CO2 atmosphere and high
humidity (95%). Partial medium changes were performed daily.
Mobilized peripheral blood mononuclear cells were collected by
apheresis from patients with breast cancer after obtaining informed
consent followed by CD34+ selection using a CEPRATE LC34
(CellPro Inc, Bothell, WA) or Isolex 300 device (Baxter Inc, Santa Ana,
CA) to enrich CD34+ peripheral blood stem cells, as
published.23,24 CD34+ cells (2 to 10 × 106) with 85% to 99% purity were used per experiment and
cultured as described above at 2.5 × 106 cells/mL
cell density.
The culture medium used was a modification of the growth medium
established previously for growth of erythroid progenitors of
chicken.9-11,29,25 In brief, culture medium consisted of Dulbecco's modified Eagle's medium (DMEM; GIBCO-BRL, Paisley, United
Kingdom) containing 15% fetal calf serum (FCS; Boehringer Mannheim,
Mannheim, Germany), 1% deionized, delipidated, dialyzed bovine serum albumin (fraction V; Sigma, St Louis, MO),
15% distilled water, 1.9 mmol/L sodium bicarbonate, 0.1 mmol/L
 -mercaptoethanol, 0.128 mg/mL iron-saturated human transferrin
(Sigma), and 100 U/mL penicillin and streptomycin (GIBCO-BRL). Culture
medium was supplemented with 1 U/mL recombinant human Epo (rhuEpo;
Recormon 1000; 1.2 × 105 U/mg; Boehringer Mannheim,
Mannheim, Germany), 100 ng/mL recombinant human SCF (rhuSCF; Amgen Inc,
Thousand Oaks, CA), 40 ng/mL long R3 insulin-like growth
factor-1 (IGF-1; Sigma), 10 6 mol/L
dexamethasone (Sigma), and 106 mol/L -estradiol
(Sigma). To monitor cell proliferation, cells were counted daily with
an electronic cell counter device (CASY1; Schärfe Systems,
Reutlingen, Germany) and cumulative cell numbers were determined.
During the initial phase of establishing the culture, cells were
subjected to Ficoll-Hypaque centrifugation to remove debris and dead
cells, if required. Similarly, Ficoll-Hypaque centrifugation was used
to remove mature and partially mature erythrocytes and dead cells that
accumulated during late stages of culture.
To induce differentiation, human erythroid progenitor cells were
recovered at day 9 of culture (see above), washed twice with serum-free
medium, and seeded at 4 × 106 cells/mL in culture
medium containing 1 U/mL rhuEpo and 1 µg/mL recombinant human insulin
(rhuIns; Actrapid HM40; Novo Nordisk Pharma). Medium was partially
replaced daily by fresh culture medium plus factors. Erythroid
differentiation was monitored by measuring cell size (CASY1;
Schärfe Systems) and by staining cytospin preparation for
hemoglobin (see below). If required, cells of different differentiation
stages were purified by Percoll density centrifugation.26
Proliferation assay.
Cell proliferation was assessed quantitatively by measuring the rate of
3H-thymidine incorporation. Cells (2 × 104 per well) were incubated in microtiter plates for 48 hours at 37°C in 100 µL culture medium containing various growth
factors or combinations thereof or without factor.
3H-thymidine (0.75 µCi per well; specific activity, 29 Ci/mmol; Amersham, Buchler, Braunschweig, Germany) was
added and cells were incubated for 2 hours. Cells were then lysed by
one cycle of freeze/thawing, harvested onto filter plates (Packard
Instruments, Meriden, CT), and subjected to liquid scintillation
counting. Average values of triplicate samples (counts per minute
[cpm]) were normalized to 1 × 105 cells seeded.
Colony assay.
Cord blood cells (5 × 104) before culture and 1 × 103 cells at day 6 of culture were plated in 1-mL
aliquots in methylcellulose medium on 35-mm plastic culture dishes.
Methylcellulose medium contained 0.9% methylcellulose in Iscove's
modified Dulbecco's medium (IMDM; MethoCult H4100; Stemcell
Technologies Inc, Vancouver, British Columbia, Canada), supplemented
with 10% heat-inactivated FCS, 1% detoxified bovine serum albumin
(BSA), 2 mmol/L L-glutamine, 0.1 mmol/L -mercaptoethanol, 0.128 mg/mL iron-saturated human transferrin (Sigma), 2 U/mL rhuEpo, 200 ng/mL rhuSCF, 2 × 106 mol/L -estradiol, and
2 × 106 mol/L dexamethasone. Cultures were
incubated for 14 days in 5% CO2 and high humidity at
37°C. Duplicate plates were analyzed for colonies that contained 30 or more cells using a stereo microscope. Burst-forming units-erythroid
(BFU-E) and colony-forming units erythroid (CFU-E) type colonies were
evaluated at days 12 through 14. Similarly, colony-forming units
granulocyte, erythrocyte, monocyte, macrophage (CFU-GEMM) colonies and
colony-forming units macrophage (CFU-M) colonies were identified
morphologically and evaluated.
Analysis of hemoglobin content and surface antigen expression.
For analysis of cell morphology and hemoglobin content, cells were
cytocentrifuged onto glass slides (700 rpm for 7 minutes; Cytospin 2;
Shandon Inc, Pittsburgh, PA) and stained with neutral benzidine and
histological dyes, as previously described.27 Photographs
were taken with Axiophot II microscope and Kontron ProgRes 3012 CCD
camera (Zeiss, Jena, Germany) and processed with Adobe Photoshop
software (Adobe Systems Inc, San Jose, CA).
Surface antigen expression of erythroid cells was analyzed by flow
cytometry. Therefore, cells were preincubated with 1% BSA (fraction V;
Sigma) and 1% human IgG (Beriglobin; Behringwerke, Marburg, Germany)
in phosphate-buffered saline (PBS) for 1 hour and then reacted with
specific antibodies (1 hour). Immunophenotyping used monoclonal
antibodies to CD3 (anti-LEU-4, clone SK7; Becton Dickinson), CD14
(IOM2, clone RM052; Immunotech, Marseille, France), CD19 (HD37; DAKO,
Glostrup, Denmark), CD29 (MAR4; Pharmingen, San Diego,
CA), CD34 (anti-HPCA-1, clone My10; Becton Dickinson), CD44 (IM7; Pharmingen), CD49d (9F10; Pharmingen), CD71 (Ber-T9; DAKO),
CD117 (YB5.B8; Pharmingen), band 3 (BIII-136; Sigma), and glycophorin
A/B (E3; Sigma), followed by reaction with fluorescein isothiocyanate
(FITC)-conjugated antimouse IgG (Fc specific; 45 minutes; Sigma). Cells
were washed twice and resuspended in PBS containing 1% BSA and
propidium iodide (2 µg/mL; Sigma) for gating on viable cells. For
flow cytometry, a FACScalibur device with CELLQuest software (Becton
Dickinson) was used.
Western blotting.
To induce tyrosine phosphorylation of receptors in response to ligand,
cells were washed twice with serum-free medium, incubated for 6 hours
in culture medium without growth factors, and treated at 37°C for 5 minutes with the following factors: 10 U/mL rhuEpo, 1 µg/mL rhuSCF,
400 ng/mL IGF-I, 200 ng/mL recombinant human interleukin-3 (rhuIL-3;
Novartis, Vienna, Austria), 200 ng/mL recombinant human epidermal growth factor (rhuEGF; Boehringer Mannheim),
200 ng/mL rhuGM-CSF (Novartis), or 10 µg/mL rhuIns. Cells were
harvested and then lysed in 20 µL lysis buffer per 1 × 106 cells (50 mmol/L Tris HCl, pH 8.0, 100 mmol/L NaCl, 1%
Triton X-100, 1 mmol/L Na3VO4). Protein lysates
were clarified by centrifugation (14,000 rpm for 1 minute), and
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE; 10% acrylamide). Proteins were transferred onto
nitrocellulose membranes (BA85; Schleicher and Schüll,
Dässel, Germany) by using a semidry blotting
apparatus (Pharmacia LKB, Uppsala, Sweden).
Blocking of membranes and reaction of specific antibodies was performed
essentially as described before.28 Briefly, nitrocellulose filters were blocked for 2 hours with blocking buffer (3% BSA, 1 mmol/L EDTA, 0.05% Tween-20 in Tris-buffered saline [TBS]), rinsed
with wash buffer (50 mmol/L Tris HCl, pH 8.0, 0.1 mol/L NaCl, 0.1%
Tween-20; 10 minutes), and incubated for 1 hour with monoclonal
antiphosphotyrosine antibody (4G10; Upstate Biotechnology Inc, Lake
Placid, NY). Monoclonal antihuman p21cip1 and
antimouse p27kip1 antibodies (Transduction
Laboratories, Lexington, KY), monoclonal antihuman band 3 antibody (Sigma), or polyclonal rabbit antihuman p62dok
antibody29 (kindly provided by N. Carpino, Cold Spring
Harbor, NY) were used accordingly. Filters were then washed (50 minutes, wash buffer) and reacted with a horseradish peroxidase-labeled antimouse or antirabbit IgG conjugate (diluted 1:3000; Amersham) in 5%
nonfat milk powder in TBS (45 minutes). Filters were washed again (50 minutes, see above), and immunocomplexes were detected using enhanced
chemiluminescence (ECL reagents; Amersham) and Amersham Hyperfilm ECL
films.
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RESULTS |
In vitro growth of erythroid progenitors from cord blood.
Human erythroid progenitor cells from cord blood were grown in liquid
culture in the presence of SCF, Epo, IGF-1, dexamethasone, and estrogen
using culture conditions modified from growth of chicken red blood cell
progenitors.9,10,25 These conditions and the specific
factor combination used were also found to be optimal for sustained
growth of human erythroid progenitor cells (data not shown). Cells were
counted daily and cumulative cell numbers were determined.
Figure 1A shows that the specific culture conditions used effectively supported logarithmic growth of erythroid progenitor cells from cord blood until day 15 to 18. This yields a
cumulative cell number of 3 to 5 × 108 cells per 10 mL cord blood, corresponding to an overall amplification in cell number
of about 500- to 1,000-fold. However, because the starting cell
population contains only a minority of erythroid progenitors (<1%,
see below), this represents a 105-fold net increase in the
number of erythroid progenitor cells within 15 to 18 days of culture.
Thereafter, cells ceased proliferation and cell numbers decreased due
to an increased rate of spontaneous differentiation and cell death (see
below). Thus, the experimental conditions used allow sustained growth
of erythroid progenitors to large cell numbers, although their
expansion is eventually limited due to a restricted self-renewal
capacity and/or lifespan.
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| Fig 1.
Growth kinetics of erythroid progenitor cells from cord
blood. (A) Human erythroid progenitor cells from cord blood were grown
in liquid culture in the presence of SCF, Epo, IGF-1, dexamethasone,
and estrogen. Cumulative cell numbers (calculated per 10 mL cord blood)
of three representative experiments determined in regular time
intervals are shown. (B) Aliquots of the cultures shown in (A) were
subjected to cytocentrifugation and staining with neutral benzidine and
histological dyes and analyzed for the proportion of proerythroblasts
(proerbl), polychromatic and orthochromatic erythroblasts (pchrom and
ochrom erbl, respectively), erythrocytes (ery), granulocytes (gran),
macrophages (mac), and lymphocytes (lymph). The starting cell
population (day 0) consisted mainly of granulocytes,
monocyte/macrophages, lymphocytes, and some blast-like progenitor
cells. Only nucleated cells are evaluated at day 0. (C) Photographs of
the cells shown in (A) and (B) after cytocentrifugation and
histological staining.
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The growth potential of erythroid progenitors from cord blood was also
assessed in colony assays. Therefore, cells were seeded either at the
day of isolation or after 6 days of liquid culture (see above) in
semisolid methylcellulose medium supplemented with SCF, Epo, and
steroid hormones. Two weeks later, the number of colonies representing
different stages of differentiation and lineage commitment were
determined. In this assay, the starting cell population yielded 3 × 102 BFU-E and CFU-E type colonies per
105 cells seeded and about 50 CFU-M and CFU-GEMM type
colonies. Most importantly, when cells were grown in liquid culture for
6 days and then seeded in colony assays, the number of erythroid
colonies that developed had increased by 100-fold (3 × 102 BFU-E and CFU-E type colonies per 103 cells
seeded) and only very few nonerythroid colonies were observed (<3%).
This finding demonstrates that the specific conditions used for liquid
culture effectively supported the outgrowth of cells with a
differentiation capacity confined to the erythroid lineage.
To further extend this conclusion, cells were analyzed by morphological
criteria using cytospin preparations stained with neutral benzidine and
histological dyes. Cord blood samples (which were depleted from
erythrocytes by Ficoll purification) consisted mainly of granulocytes,
monocyte/macrophages, lymphocytes, and some blast-like cells (Fig 1B
and C). Mature erythrocytes, which were not efficiently removed during
Ficoll purification, and a low number of polychromatic erythroblasts
(2% to 3%) were also present. This picture changed dramatically after
5 to 8 days of culture in the presence of SCF, Epo, IGF-1, and steroid
hormones. Proerythroblast-like progenitors clearly represented the
major cell population with some residual macrophages and granulocytes (Fig 1B and C). At days 9 to 13 of culture, the cell population was
completely erythroid, comprising 86% to 92% of proerythroblasts and
some polychromatic and orthochromatic erythroblasts (3% to 7% and 5%
to 7%, respectively; Fig 1B). With prolonged time in culture, the rate
of spontaneous differentiation further increased, and at day 20 the
cell population consisted of approximately 45% polychromatic and
orthochromatic erythroblasts; at day 25, 1% to 2% mature enucleated
erythrocytes were found.
The outgrowth of erythroid progenitors was also monitored by assessing
expression of specific cell surface proteins by flow cytometry. At day
9 of culture, cells effectively expressed, as expected, c-kit/SCF
receptor (CD117), high levels of transferrin receptor (CD71), and low
levels of glycophorin A/B and band 3 (Fig
2). Cells also expressed high levels of the adhesion molecules 41
integrin VLA-4 (CD29, CD49d) and CD44; no expression of T- and
B-cell-specific and myeloid markers (CD3, CD19, and CD14, respectively) was detected (data not shown). Thus, at this point in
time, the culture represented a homogenous population of erythroid cells. This conclusion is further supported by the observation that all
cells can be effectively induced to undergo erythroid differentiation
(see below).
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| Fig 2.
Cell surface expression profile of erythroid progenitor
cells. Erythroid progenitor cells were analyzed by flow cytometry for
expression of c-kit/SCF receptor (CD117), transferrin receptor (CD71),
glycophorin A/B, and band 3 as indicated (grey). Control cells were
incubated with FITC-labeled secondary antibody only (white). Cells at
day 9 of culture are shown.
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Growth factor dependence of erythroid progenitor cells.
We next wanted to assess the individual contribution of Epo, SCF, and
IGF-1 for growth of erythroid progenitor cells (1) by determining
receptor phosphorylation in response to specific growth factors and (2)
by measuring the rate of DNA synthesis induced by factor. Therefore,
cells were incubated without factor for 6 hours; Epo, SCF, or IGF-1
were added; and 5 minutes later, cell lysates were prepared and
subjected to Western blot analysis using a phosphotyrosine-specific
monoclonal antibody. After the addition of Epo, tyrosine
phosphorylation of the 78-kD Epo receptor was clearly evident
(Fig 3A). Epo also induced tyrosine
phosphorylation of several other proteins, probably Jak2, Stat5, Shc,
and syp, which is in line with previous
studies.15,17,18,30,31 Phosphorylation of these proteins
was clearly specific for Epo and not seen for control cells. As
expected, SCF effectively induced tyrosine phosphorylation of the p145
c-kit/SCF receptor15 together with phosphorylation of a
faster migrating protein. Stripping and reprobing of the Western blot
with receptor specific antisera demonstrated that the 78-kD and 145-kD
tyrosine phosphorylated bands correspond to Epo receptor and c-kit/SCF
receptor, respectively (data not shown). Additionally, IGF-1 and
insulin led to tyrosine phosphorylation of the IGF-1 and insulin
receptor -chains, demonstrating that these cells express IGF-1 and
insulin receptors. No change in tyrosine phosphorylation was obtained
with, eg GM-CSF, IL-3, and EGF. Thus, the erythroid progenitors
obtained expressed receptors for Epo, SCF, IGF-1, and insulin but
lacked detectable levels of receptors for, eg, GM-CSF, IL-3, and EGF.
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| Fig 3.
Erythroid progenitor cells respond to Epo, SCF, IGF-1,
and insulin. (A) Erythroid progenitor cells were analyzed for specific
responses to Epo, SCF, GM-CSF, IGF-1, insulin, IL-3, and EGF by Western
blotting and staining with phosphotyrosine-specific monoclonal antibody
(4G10). One hundred micrograms of protein lysate per lane. Control, no
factor added. Cells at day 9 of culture are shown. (B)
3H-thymidine incorporation in response to factor of the
same cell preparation shown in (A). Factors were applied individually
or in various combinations as indicated. 3H-thymidine
incorporation was determined 48 hours after the addition of factors;
for details see Materials and Methods.
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To assess the effect of Epo, SCF, IGF-1, and insulin on progenitor cell
growth quantitatively, the rate of DNA synthesis in response to factor
was measured. Factors were added either individually or in combinations
thereof; 48 hours later, cells were assayed for their proliferative
response in 3H-thymidine incorporation assays. When
individual factors were applied, Epo and SCF were found to be the most
effective (Fig 3B). Furthermore, if applied simultaneously, the effect
of Epo and SCF was more than additive, indicating that both factors
synergized in inducing DNA synthesis in these cells. Administration of
dexamethasone and estrogen did not significantly augment
3H-thymidine incorporation, at least under the experimental
conditions used (FCS was not depleted from endogenous steroids). The
activity of IGF-1 and insulin on 3H-thymidine incorporation
was also very low (Fig 3 and data not shown). Most likely, the effects
of steroids, IGF-1, and insulin are more pronounced under serum-free
conditions. However, both IGF-1 and steroid hormones (which were not
effective individually) enhanced the rate of DNA synthesis induced by
Epo plus SCF and there was no further increase by the addition of other
factors such as, EGF, IL-3, and GM-CSF, which is in line with the
Western blotting data.
In conclusion, SCF and Epo, if added simultaneously, clearly represent
the most potent factors required for inducing proliferation of this
type of erythroid progenitor cell. To ensure optimal growth rates, the
addition of IGF-1 was also important. However, we emphasize that an
efficient outgrowth of erythroid progenitor cells from cord blood
samples was critically dependent on the administration of dexamethasone
and, to a lesser extent, of estrogen, which significantly reduced
the rate of spontaneous differentiation (B.P., P.B. and M.Z., data
not shown).
Erythroid progenitor cells differentiate into mature erythrocytes in
vitro.
Next, we determined whether the erythroid progenitor cells obtained
were capable of differentiating in vitro into fully mature enucleated
erythrocytes. Cells were withdrawn from growth factors and incubated in
the presence of Epo and insulin using culture conditions modified from
in vitro differentiation of red blood cell progenitors of
chicken.9-11,25 After the induction of differentiation, cells began to accumulate hemoglobin and gradually acquired the morphology of normal erythrocytes while undergoing 2 to 3 cell divisions followed by cell cycle arrest
(Figs 4 and
5A and data not shown). Parallel to the
onset of differentiation, a decrease in cell size was routinely
observed (Fig 5B). At day 3 of differentiation, orthochromatic
erythroblasts represented the majority of the cell population (Figs 4
and 5A). The proportion of orthochromatic erythroblasts further
increased to 72% at day 4; at this time, fully mature enucleated
erythrocytes constituted about 9% of the culture. As expected, cells
kept as an experimental control under growth conditions (SCF, Epo,
IGF-1, and steroid hormones; see above) did not differentiate (data not
shown). This demonstrates that, with the specific culture conditions
used, the erythroid progenitor cells obtained were fully competent in
differentiating terminally.
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| Fig 4.
Erythroid progenitor cells differentiate in vitro.
Erythroid progenitor cells were induced to differentiate in the
presence of Epo and insulin (see Materials and Methods). After 1, 2, 4, and 5 days of differentiation, cells were subjected to
cytocentrifugation and staining with neutral benzidine and histological
dyes and photographed. Day 0, undifferentiated cells cultured under
standard growth conditions. Please note the enucleated cells obtained
at day 4 to 5 of differentiation.
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| Fig 5.
Kinetics of in vitro differentiation of erythroid
progenitor cells. (A) Aliquots of the cultures shown in Fig 4 were
evaluated for the proportion of proerythroblasts (proerbl),
polychromatic and orthochromatic erythroblasts (pchrom and ochrom erbl,
respectively), and erythrocytes (ery). (B) Cell size profile of the
same cells as shown in (A) demonstrate reduction in cell size during
differentiation: undifferentiated cells, white; differentiated cells at
24, 48, and 72 hours, grey, dark grey, and black, respectively.
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Interestingly, the starting cell preparation (day 9 of culture)
apparently contained two populations of erythroid progenitor cells that
differentiated with different kinetics. The majority of cells were
proerythroblasts, whereas a subpopulation of cells had presumably
already further advanced in maturation and exhibited morphological
characteristics of polychromatic cells. These cells differentiated with
faster kinetics and gave rise to orthochromatic erythroblasts by day 2 of differentiation. Differentiation of two apparently different
progenitor cell populations is also seen in the specific changes
observed in cell size (Fig 5B). However, we emphasize that eventually
all SCF/Epo-dependent progenitor cells differentiate into
orthochromatic erythroblasts and enucleated erythrocytes, indicating
that the culture conditions used effectively support normal erythroid
cell maturation in vitro.
Growth and differentiation in vitro of erythroid progenitors from
CD34+ peripheral blood stem cells and bone marrow.
Next, we determined whether CD34+ peripheral blood stem
cell preparations contain erythroid progenitor cells that behave
similarly to the erythroid progenitors from cord blood and can be
amplified and differentiated in vitro accordingly. Therefore,
CD34+ peripheral blood stem cells were obtained by
immunomagnetic bead affinity purification of leukapheresis preparations
and cultured in the presence of SCF, Epo, IGF-1, and steroid hormones
using the growth conditions described above. Cells were counted daily and cumulative cell numbers were determined. Under such culture conditions, erythroid progenitors from CD34+ peripheral
blood stem cells exhibited similar growth kinetics as the erythroid
progenitors derived from cord blood (data not shown). The outgrowth of
progenitor cells started at day 4 of culture, accompanied by an
increase in cell size (from 8.5 to 11 µm; data not shown). Cumulative
cell numbers increased until day 17, whereas in the following days,
cells stopped proliferating and cell numbers decreased.
Cells were also analyzed by morphology after cytospin centrifugation
and staining with histological dyes. At day 6 of culture, the cell
population comprised 88% proerythroblasts expressing high levels of
CD71/transferrin receptor (Fig 6A and B and
data not shown); some persisting granulocytes and macrophages were also
present. The culture was almost exclusively erythroid at day 9 to 10. At later stages, cells started to spontaneously differentiate (day 13 to 15), and an increasing number of polychromatic and orthochromatic
erythroblasts was found. The number of orthochromatic erythroblasts
further increased with time, and at days 21 and 25 of culture,
represented 52% and 62%, respectively, of the cell population. At
this time, the culture also contained some terminally differentiated
fully mature enucleated erythrocytes.
View larger version (67K):
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| Fig 6.
In vitro growth and differentiation of erythroid
progenitors from CD34+ peripheral blood stem cells. (A)
Erythroid progenitors from CD34+ peripheral blood stem
cells (starting cell population, day 0) were grown in liquid culture in
the presence of SCF, Epo, IGF-1, dexamethasone, and estrogen. At days
4, 9, and 14 of culture, cells were subjected to cytocentrifugation and
staining with neutral benzidine plus histological dyes. (B) Cultures of
erythroid progenitors from CD34+ peripheral blood stem
cells shown in (A) were analyzed for proportion of proerythroblasts
(proerbl), polychromatic and orthochromatic erythroblasts (pchrom and
ochrom erbl, respectively), erythrocytes (ery), granulocytes (gran),
macrophages (mac), and lymphocytes (lymph). The starting cell
population (day 0) consisted mainly of small progenitor cells (striped
box) and some granulocytes and macrophages as indicated. At day 0, only
nucleated cells are evaluated. (C) Erythroid progenitor cells were
induced to differentiate in the presence of Epo and insulin (see
Materials and Methods). After 2, 3, 4, and 5 days of differentiation,
cells were subjected to cytocentrifugation and staining with neutral
benzidine and histological dyes and evaluated for the proportion of
proerythroblasts, polychromatic and orthochromatic erythroblasts, and
erythrocytes as in (B).
|
|
Western blot analysis with a phosphotyrosine-specific monoclonal
antibody demonstrated that erythroid progenitors from CD34+
stem cell preparations express c-Kit/SCF receptor, Epo receptor, IGF-1
receptor, and insulin receptor, which were effectively phosphorylated in response to ligand (data not shown). GM-CSF, IL-3, EGF, and TGF
were without effect. Thus, the erythroid progenitors obtained from
CD34+ peripheral blood stem cells behaved similarly to
those isolated from cord blood: they exhibit similar growth kinetics
and expressed receptors for SCF, Epo, IGF-1, and insulin.
Additionally, these progenitors were fully competent in undergoing
terminal differentiation in response to Epo and insulin (Fig 6C). At
day 3, the culture exhibited 55% and 10% polychromatic and
orthochromatic erythroblasts, respectively, and 2% mature erythrocytes. By day 5 of differentiation, the number of orthochromatic erythroblasts and erythrocytes had further increased. At this stage,
the number of fully mature enucleated erythrocytes was found
consistently to be higher than that observed for differentiation of
erythroid progenitors from cord blood, suggesting that terminal differentiation in vitro of red blood progenitors from
CD34+ stem cells is more efficient.
In summary, the SCF/Epo-dependent erythroid progenitors present in
CD34+ peripheral blood stem cell preparations behaved very
similarly to the respective progenitor cells obtained from cord blood.
Additionally, SCF/Epo-dependent erythroid progenitor cells were also
isolated from human bone marrow preparations, amplified, and
differentiated in vitro accordingly (data not shown).
p62dok and CDK inhibitor p21cip1 and
p27kip1 expression in erythroid cells.
Because the experimental system described above allows selective
programming of erythroid progenitor cells to either self-renewal or
terminal differentiation, we thought to determine the expression pattern of signaling molecules in such cells that have been implicated in either process. To this end, we investigated expression of p62dok, a recently identified adapter protein involved in
signal transduction downstream of receptor and nonreceptor tyrosine
kinases such as, c-kit/SCF receptor, bcr-abl, and
v-abl.29,32 Cells of different differentiation stages were
prepared and analyzed for p62dok expression in Northern and
Western blots using p62dok-specific probe and antibody
(kindly provided by J. Grimm and W. Birchmeier [MDC, Berlin, Germany]
and N. Carpino [Cold Spring Harbor, NY], respectively). We
demonstrate that proliferating SCF/Epo-dependent erythroid progenitor
cells express p62dok mRNA and protein
(Fig 7 and data not shown). Most
importantly, p62dok expression declines when cells
differentiate. Additionally, c-kit/SCF receptor expression also
decreases upon differentiation, which is in line with previous studies
on SCF-dependent erythroid progenitors of chicken20 (data
not shown). As expected, expression of the anion transporter band 3, used as an experimental control, was effectively upregulated during
differentiation (Fig 7).
View larger version (44K):
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| Fig 7.
p62dok, p21cip1, and
p27kip1 expression in proliferating and differentiating
erythroid cells. SCF/Epo-dependent erythroid progenitor cells at
different stages of differentiation were analyzed for
p62dok, p21cip1, and p27kip1
expression by Western blotting. Band 3 expression is shown to
demonstrate efficient differentiation. Lane 1, undifferentiated
progenitor cells at day 9 of culture. Lanes 2 through 4, differentiated
cells at day 3 of differentiation following fractionation by Percoll
density centrifugation. Samples were normalized for equal protein
loading per lane (30 µg). The proportion of proerythroblasts,
polychromatic and orthochromatic erythroblasts, and erythrocytes is
indicated.
|
|
Preliminary experiments suggested that the CDK inhibitor
p27kip1 is regulated during differentiation of erythroid
progenitors from chicken (P.B. and M.Z., unpublished
data). Thereupon, we analyzed p27kip1
expression in proliferating SCF/Epo-dependent erythroid progenitors and
differentiated cells. Figure 7 shows that p27kip1 protein
level dramatically increases when cells differentiate. Additionally,
expression of p21cip1, another member of the same CDK
inhibitor family, was also induced.
In summary, we demonstrate that SCF/Epo-dependent erythroid progenitor
cells from cord blood express the adapter protein p62dok
and the CDK inhibitors p21cip1 and p27kip1;
these molecules have been implicated in self-renewal and cell cycle
arrest, respectively, and undergo specific changes in expression when
cells differentiate. Thus, the experimental system described in this
report readily allows the analysis of molecules involved in growth and
differentiation of primary human erythroid progenitor cells.
| |
DISCUSSION |
In this report, we describe an SCF/Epo-dependent erythroid progenitor
that is efficiently obtained from cord blood, CD34+
peripheral blood stem cells, and bone marrow by using specific culture
conditions and SCF, Epo, IGF-1, and steroid hormones (dexamethasone and
estrogen). This progenitor can be selectively amplified in liquid
culture to large cell numbers (3 to 5 × 108 cells/10
mL cord blood). Whereas the starting cell preparations contain only a
minor population of erythroid progenitors (<1%, as determined in
colony assays), there was a 105-fold net amplification in
cell number within 15 to 18 days of culture. Additionally, the
progenitor cells obtained were fully competent of differentiating
terminally into erythrocytes in response to differentiation factors
(Epo, insulin). Thus, the experimental system described allows the
selective amplification of an erythroid progenitor in liquid culture to
homogenous cell populations and high cell numbers that allow a more
detailed biochemical, molecular, and functional analysis. We
demonstrate that this progenitor is critically dependent on SCF and Epo
for efficient growth in vitro and, therefore, refer to this cell as
SCF/Epo-dependent progenitor. IGF-1 appears not to be a crucial growth
factor (at least in the presence of SCF, Epo, and serum) but, if added,
does further improve the growth conditions. Additionally, efficient
outgrowth of SCF/Epo-dependent progenitors critically required
dexamethasone presumably by inhibiting spontaneous
differentiation.10 Culture conditions that additionally contained estrogen further enhanced progenitor cell outgrowth (B.P.,
P.B., and M.Z., unpublished data). Notably, the
SCF/Epo-dependent progenitor described in this study exhibits
properties very similar to an SCF-dependent erythroid progenitor
obtained from the bone marrow of chicken.10,20 This
progenitor can be effectively amplified in the presence of chicken SCF,
chicken serum, and steroid hormones without exogenous Epo (Epo is so
far not cloned for chicken). Thus, although the human progenitor
studied in this report requires both SCF and Epo for efficient growth
in culture, the erythroid progenitor of chicken appears to be solely
dependent on SCF and factors in chicken serum. However, these
progenitors are different from a TGF-dependent erythroid progenitor
obtained from chicken bone marrow in the presence of TGF (and
steroid hormones) which exhibits a considerably longer lifespan in
vitro.8,9,20 As demonstrated above, the human
SCF/Epo-dependent progenitor studied here did not express detectable
levels of TGF/EGF receptor and did not respond to the addition of
TGF and/or EGF.
The growth conditions employed for amplification of the
SCF/Epo-dependent erythroid progenitor described in this paper are a
modification of those used for propagation of primary nontransformed or
oncogene-transformed erythroid cells from chicken bone
marrow9,10,25; various chicken specific components were
substituted by the respective mammalian factors. Cells are first
amplified under particular medium conditions in the presence of a
specific combination of factors that support cell proliferation (SCF,
Epo, IGF-1, and steroid hormones); changing the culture conditions by
replacing the growth promoting agents with appropriate differentiation
factors (Epo, insulin) induces their maturation and differentiated
orthochromatic erythroblasts and enucleated erythrocytes are obtained.
Differentiation was most effective when cells showed optimal growth
rates (day 8 to 10 of culture). Thus, the specific culture conditions
used direct the cells to either proliferation or differentiation.
The culture conditions used are different from the two-step culture
system by Fibach et al.33,34 This system involves a first,
Epo-independent phase in which early erythroid progenitors (eg, BFU-E
cells) proliferate and differentiate into CFU-E type progenitors. In a
second phase, cells are cultured in the presence of Epo and continue to
proliferate and to mature into orthochromatic erythroblasts and
enucleated erythrocytes. Although in such cultures high numbers of
largely pure erythroid cells can be obtained, the system does not
separate cell proliferation and terminal differentiation and thus does
not allow study of self-renewal and differentiation of erythroid cells
individually. This is clearly the advantage of the system described in
this report, because, by choosing the appropriate media conditions plus
factors, proliferating cells can be reprogrammed and directed to
differentiation. The culture conditions used in this report are also
different from the ones used by Krystal et al35 and
Taniguchi et al,36 which have, eg, insulin in growth
medium, whereas here insulin is used to induce differentiation. The
presence of insulin in the growth medium used in our study led to an
increased rate of differentiation, delayed growth kinetics, and a lower
overall cell number than control (data not shown). However, we
emphasize that the effect of insulin might be different, for example,
under serum free conditions such as those used by Krystal et
al.35
It will now be interesting to identify on a molecular level the
determinants that are important in controlling growth and differentiation of the erythroid progenitor cell described in this
report. As a first step, we have measured receptor phosphorylation for
SCF receptor, Epo receptor, IGF-1 receptor, and insulin receptor in
response to ligand. Additionally, we provide first evidence that such
progenitor cells express p62dok mRNA and protein.
p62dok represents a recently identified adapter molecule
that associates with p120 ras GTPase-activating protein (GAP) and that
is rapidly tyrosine phosphorylated upon activation of c-kit/SCF
receptor.29,32 Association of p62dok with GAP
correlates with tyrosine phosphorylation, indicating that
p62dok is a component of a signal transduction pathway
downstream of the c-kit/SCF receptor. Furthermore, both SCF-induced
tyrosine phosphorylation of p62dok and its constitutive
tyrosine phosphorylation in chronic myelogenous leukemia (CML) cells
correlate with cell proliferation, strongly supporting the view that
p62dok is an important molecule in mitogenic signaling.
Interestingly, as shown in this report, SCF/Epo-dependent erythroid
progenitor cells express p62dok and its expression declines
when cells cease proliferation and undergo terminal differentiation
into mature red blood cells. Thus, in these progenitor cells,
p62dok might be involved in signaling that induces cell
proliferation.
After induction of differentiation, SCF/Epo-dependent progenitor cells,
as demonstrated above, effectively upregulate the CDK inhibitors
p21cip1 and p27kip1 and undergo cell cycle
arrest. This finding is reminiscent of the increase of
p21cip1 and p27kip1, eg, during oligodendrocyte
differentiation.37,38 Cellular differentiation is
associated with a reduction in overall G1 CDK activity, which is, eg,
regulated through specific interactions of CDKs with CDK
inhibitors.39 p21cip1 and p27kip1
are members of the same family of CDK inhibitors and their upregulation during erythroid cell differentiation might affect G1 CDK activity and
therefore influence the propensity of erythroid progenitor cells to
undergo either cell cycle reentry or growth arrest. The specific
signals that induce p21cip1 and p27kip1 in
differentiating erythroid cells still remain elusive. Furthermore, in
erythroid progenitor cells of chicken differentiation induction causes
downregulation of D-type cyclins and CDK4,40 indicating that reprogramming of erythroid cell gene expression during
differentiation involves multiple components of the cell cycle
machinery. All of these findings provide further support for the
existence of two distinct genetic programs that determine erythroid
cell proliferation and differentiation, respectively. Information about
the molecular determinants and how they function in controlling cell
growth and differentiation cannot be reliably obtained from studies in established cell lines that exhibit aberrantly altered properties due
to their transformed and/or immortalized phenotype. The primary culture system described in this report is particularly well suited for
studying such signaling molecules and how they operate, both during
normal erythropoiesis and in the pathological state.
| |
ACKNOWLEDGMENT |
The authors are most grateful to Amgen Inc for providing recombinant
human SCF and to Novartis Ltd for recombinant human IL-3 and GM-CSF.
Additionally, we thank J. Grimm and W. Birchmeier for
p62dok-specific probe, N. Carpino for
anti-p62dok antibody, I. Körner for CD34+
peripheral blood stem cell purification, G. Blendinger and R. Franke
for tissue culture work, and I. Gallagher for typing the manuscript. We
are also most grateful to B. Dörken for continuous support.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted July 15, 1998.
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 Martin Zenke, PhD,
Max-Delbrück-Center for Molecular Medicine, MDC,
Robert-Rössle-Str. 10, D-13122 Berlin, Germany; e-mail:
zenke{at}mdc-berlin.de.
| |
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Top
Abstract
Introduction