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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 27, 2001 as doi:10.1096/fj.00-0705fje. |
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* Institute of Medical Biochemistry, Division of Molecular Biology,
Institute of Molecular Pathology, Vienna Biocenter, A-1030 Vienna, Austria; and
Institute of Cancer Research, A-1090 Vienna, Austria
2Correspondence: Institute of Medical Biochemistry, Division of Molecular Biology, Dr. Bohr-Gasse 9, A-1030 Vienna. E-mail: em{at}mol.univie.ac.at
SPECIFIC AIMS
We tried to establish serum-free culture conditions for primary as well as immortal p53-deficient murine erythroblasts, which should allow to study sustained proliferation as well as maturation of immature cells into enucleated erythrocytes in precisely defined combinations of proliferation or differentiation factors. If it could be verified that such a cell system faithfully executes essential steps of normal erythropoiesis, this would permit characterization of molecular mechanisms involved in red cell maturation by expression profiling.
PRINCIPAL FINDINGS
In vitro culture, phenotypical characterization, and
differentiation of primary mouse erythroblasts
Detailed molecular characterization of terminal
erythropoiesis in the mouse has so far been hampered by the lack of
suitable in vitro culture models. The available primary cell systems
suffer from insufficient immature cell numbers and from the
heterogeneity of these cells with respect to stage of maturity. In
contrast, immortal erythroleukemic cell lines show aberrant
factor/hormone dependence and fail to execute a normal in vivo
erythroid differentiation program (see below).
Primary avian and human erythroblasts can undergo long-term
proliferation in factors crucial for erythroid progenitor expansion in
stress erythropoiesis (erythropoietin, stem cell factor, and
dexamethasone; Epo, SCF, Dex). Using this factor combination, we
established long-term erythroblast cultures from the mouse, using a
serum free culture medium (StemPro, Life Technologies) and determining
optimal concentrations of the above factors. These primary erythroblast
cultures showed sustained proliferation for 15–20 days, allowing an
overall expansion of >10,000-fold before undergoing senescence.
Synchronous red cell differentiation was induced by replacing the
proliferation factors with high concentrations of Epo plus insulin
(Ins) (Fig. 1A
). The cells underwent 3–4 accelerated ‘differentiation
divisions’ within 48 h, characterized by a massively shortened
G1 phase and a drastic size decrease. After
72 h, the cells had arrested in G1 and had enucleated in
their majority, showing hemoglobin levels similar to peripheral blood
erythrocytes in vivo (Fig. 1B
).
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Immortal erythroblasts from p53-deficient mice are
genetically stable and capable of normal terminal differentiation
To overcome the problem of a finite life span of our
primary erythroblasts, the above culture conditions were applied to
erythroblasts from p53-/- mice, fibroblasts of
which proliferate indefinitely without a Hayflick crisis. Indeed, both
bulk- as well as clonal cultures (clone I/11, picked at random from a
set of 37 well differentiating clones) could be cultivated for >16
months while retaining full factor dependence, in vivo-like terminal
differentiation capacity, and genetic stability if kept under optimal
medium conditions. Data on the synchronous size decrease and
enucleation as well as changes of cell surface expression of
characteristic markers indicated that clone I/11 represents a committed
erythroid precursor with properties of a pro-erythroblast (Fig. 1C
, D
), highly similar in all parameters tested to the
respective wild-type cells (also compare Fig. 1A
, B
).
To further substantiate that the cells were differentiating according to the normal program, expression patterns of several well known erythroid-specific transcription factors were determined by Western blotting in synchronously maturing I/11 cells. Gata-1 and SCL/Tal-1 were transiently induced between 16 and 36 h, followed by a late up-regulation of NF-E2, which shortly preceded globin accumulation. In contrast, the inhibitors of differentiation Id1 and Id2 were both expressed in proliferating cells, but not detectable anymore after 36 h Our results perfectly agree with the presumed roles of these transcription factors during the normal differentiation program, deducted mainly from genetically modified mice.
Molecular Characterization of erythroid differentiation by gene
expression profiling
The unlimited life span of I/11 erythroblasts, combined with their
ability to faithfully reproduce the in vivo erythroid differentiation
program in culture, prompted us to employ this novel system for mRNA
expression profiling, using filter based arrays containing 588 cDNAs
selected for their key regulatory functions. Kinetics of mRNA
expression were determined by analyzing samples from proliferating I/11
cells and cultures induced to differentiate for 6, 20, and 48 h,
respectively, to properly assess gradual as well as transient changes.
For several target mRNAs, the results perfectly agreed with predictions
from earlier work on the expression kinetics during differentiation.
This included early down-regulation of the SCF receptor c-kit, the
glucocorticoid receptor (GR), and c-myb, a potential target of ligand
activated GR. Several other mRNAs for genes with a known function late
in erythropoiesis, like glucose transporter 1, CHOP-10 (also known as
Gadd153), and the erythroid-specific transcription factor NF-E2 were
up-regulated as predicted. Together with results on many other relevant
genes (see electronic full length version) the observed expression
changes indicated that application of this cell system to expression
profiling should allow to draw reliable conclusions on the gene network
regulating erythropoiesis in vivo.
As a first step, ‘cluster’ analysis was performed to group all
mRNAs according to similarities in their expression pattern. A group of
19 genes (out of 588 examined; see also Fig. 2
) was down-regulated early, preceding the general decline in metabolic
activity late in maturation. Some of these genes, therefore, may
function in sustained proliferation. Forty-five transcripts were
transiently induced, pointing toward a function in the actual
differentiation process, while—contrary to the general trend of late
decrease—11 genes were massively up-regulated at the latest time point
(Fig. 2)
. In consequence, the latter group could contribute to the
mature phenotype. Several of the clustered genes were expected to play
roles in erythroblast self renewal, signaling via erythropoietin
receptor (EpoR) and/or via c-Kit, although their expression profile
during terminal differentiation had not been previously described. For
example, the mRNAs for PKB, PLC-beta, and fli-1 were decreasing early,
transcript abundance for the erythroid-specific transcription factor
EKLF was transiently increasing, whereas vav and PKC-theta mRNAs,
although highly expressed, did not significantly change over the entire
period. There also was a group of genes, whose expression so far has
not been linked with the definition of an erythroid phenotype. A
concise evaluation of possible functions of several target genes
is contained in the electronic full length version, the entire set of
raw data is available at
‘http://emb1.bcc.univie.ac.at/molb/expression-profiling/I11dif.htm’.
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For various, randomly picked candidate genes (EKLF, NF-E2, c-myb, junB, pim-1, smad1) the mRNA expression profiling data were successfully verified by Northern blot analysis and found to behave exactly as deduced from the array screen. For some genes encoding transcription factors, signal transducers, or cell cycle regulators (c-Kit, Cdk4, Stat5ab, Bcl-XL, Pim-1, IL3-R-beta), we also analyzed differential expression at the protein level. In every case, differentiation-specific changes in protein abundance closely matched alterations in mRNA expression, both in p53-deficient I/11- as well as in wild-type fetal liver-derived erythroblasts.
CONCLUSIONS AND SIGNIFICANCE
Until recently, extensive biochemical and molecular studies of
erythropoiesis were mainly restricted to established cell lines, which
are in their majority of limited physiological significance. Therefore,
important results in this area frequently had to come from analysis of
phenotypes of genetically modified mice or studies of ES cell
differentiation. Erythroblasts from p53-deficient mice, closely
resembling the properties of wild-type cells analyzed in parallel,
offered to combine these two approaches as detailed in the following.
First, the cells fulfilled the following necessary criteria:
1) unlimited life span in vitro to allow analysis of clonal
strains and repetition of experiments without variations in cell
batches, 2) complete dependence on growth factors and
hormones regulating erythroid progenitor expansion in vivo (i.e., in
stress erythropoiesis), and 3) terminal red cell maturation
comparable to that in vivo, depending on physiological differentiation
factors (see Fig. 2
). The potential problem of genetic instability of
p53-/- cells could be kept under control by
permanently maintaining optimal culture conditions and repeated
comparison to cultures from wild-type erythroblasts. Second, the
extended life span of wild-type cells in serum free media also allows
phenotypical and medium-scale biochemical characterization of cells
from genetically modified mice. This approach has already been used on
erythroblasts from mice deficient in GR, Stat5, c-Raf, etc. If
necessary, unlimited cell numbers will be accessible by breeding
respective mice on a p53-/- background.
Furthermore, both wild-type- and p53-/- cells
allow genetic modification by retroviral expression (already done for
c- and v-ErbB, bcl-XL, Fli-1, dominant-negative
Stat5, etc.).
Third, another major advantage of the described cell system is its
usefulness for expression profiling. By trying to cover the entire
genome, these experiments are extremely demanding with respect to time
and cost. Thus, it is particularly important to use cell systems that
allow conclusions of physiological significance. We have shown that our
cell system fulfills this requirement, permitting the identification of
meaningful gene clusters and interesting candidates for further study
(Fig. 2)
. Expression profiling using 13 k mouse oligonucleotide
chips (Affymetrix) is currently set up to repeat the screen described
here at a larger scale, including a comparison between
p53-/- and wt-erythroblasts, which differed
only in expression of very few genes. Our cell systems are now being
used in other screens to identify genes involved in EpoR/c-Kit
signaling and in GR function, i.e., to unravel how these diverse
receptors cooperate to stimulate proliferation induction and inhibit
differentiation at the same time.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0705fje ; to cite this
article, use FASEB J. (April 27, 2001) 10.1096/fj.00-0705fje 
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