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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4108-4118
GATA-1 Regulates Growth and Differentiation of Definitive Erythroid
Lineage Cells During In Vitro ES Cell Differentiation
By
Naruyoshi Suwabe,
Satoru Takahashi,
Toru Nakano, and
Masayuki Yamamoto
From the Center for Tsukuba Advanced Research Alliance and Institute
of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan; and
Institute of Microbial Diseases, Osaka University, Osaka, Japan.
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ABSTRACT |
Although the importance of GATA-1 in both primitive and definitive
hematopoietic lineages has been shown in vivo, the precise roles played
by GATA-1 during definitive hematopoiesis have not yet been clarified.
In vitro differentiation of embryonic stem (ES) cells using OP9 stroma
cells can generate primitive and definitive hematopoietic cells
separately, and we have introduced a method that separates
hematopoietic progenitors and differentiated cells produced in this
system. Closer examination showed that the expression of erythroid
transcription factors in this system is regulated in a differentiation
stage-specific manner. Therefore, we examined differentiation of GATA-1
promoter-disrupted (GATA-1.05) ES cells using this system.
Because the GATA-1.05 mice die by 12.5 embryonic days due to
the lack of primitive hematopoiesis, the in vitro analysis is an
important approach to elucidate the roles of GATA-1 in definitive
hematopoiesis. Consistent with the in vivo observation, differentiation
of GATA-1.05 mutant ES cells along both primitive and
definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of  y-globin mRNA, the
blastlike cells accumulated in the definitive stage showed  -globin
mRNA expression at approximately 70% of the wild type. Importantly,
the TER119 antigen was expressed and porphyrin was accumulated in the
definitive cells, although the levels of both were reduced to
approximately 10%, indicating that maturation of definitive erythroid
cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more
colony-forming activity, termed CFU-OP9. These results suggest that the
GATA-1.05 mutation resulted in proliferation of
proerythroblasts in the definitive lineage.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIETIC CELLS serve as an important
model system to analyze how lineage-specific transcription factors
actively contribute to the regulation of the multi-lineage cell
differentiation process. A number of transcription factors that
regulate hematopoietic cell differentiation processes have been
identified and characterized.1-3 We are interested in the
regulation of erythroid differentiation and have been analyzing the
functional roles of several erythroid transcription factors. The
expression of erythroid transcription factors that have been
characterized are not restricted to the erythroid lineage, but rather
each factor shows a specific expression profile in several tissues or
cell types including the erythroid lineage. Transcription factors
expressed in erythroid cells include GATA-1, GATA-2, NF-E2, AML1, EKLF,
Myb, PU1, rbtn2, and SCL/tal-1.4-12 These transcription
factors have been suggested to work through the formation of a protein
network and have been shown to be necessary for the development
and/or function of erythroid lineage cells.13-22
Recently developed mouse technologies, such as transgenic
overexpression or disruption of specific genes in vivo, have been shown
to be very powerful for the functional analysis of transcription factors. However, these manipulations often result in embryonic lethality of the genetically engineered mice and thus make it impossible to analyze potential functions at later stages. Furthermore, to introduce a transgene to mice, selection of the vector is always technically intricate, so that even upon successful introduction of a
transgene, expression of endogenous functions is artificially changed
due to the ectopic overexpression of the transgene. An alternative and
supplemental approach is to use cultured cell lines that reflect
characteristics of hematopoietic lineages, because cultured cells are
easy to manipulate. However, the disadvantage of the use of
immortalized cell lines is that they always reflect only restricted
aspects of in vivo differentiation processes and sometimes behave
differently from their physiological counterparts.
In vitro differentiation of embryonic stem (ES) cells may overcome the
detrimental aspects of the above-mentioned methods. Particularly, an
advantage of the system is the ability to assess directly properties of
manipulated ES cells that have been used to generate gene-disrupted
mice. For instance, both knockout and knock-down analyses of the
erythroid transcription factor GATA-1 resulted in embryonic lethality
due to failure of yolk sac hematopoiesis,5,6 indicating
that GATA-1 is necessary for the differentiation of primitive
hematopoietic cells. However, because the GATA-1-deficient mice die by
12.5 embryonic days, which is before the full commencement of
definitive hematopoiesis, alternative approaches are necessary to
investigate definitive hematopoiesis.
Because the GATA-1 gene localizes to the X-chromosome23 and
ES cells usually have a male karyotype, homologous recombination needs
to be executed in only once to generate ES cells with a homozygous
genotype for GATA-1 disruption. Taking advantage of this, several
reports have already been published in which the GATA-1-disrupted ES cells were used.24-27 In one report an
embryoid body formation method was used, which allowed the
investigators to analyze both primitive and definitive hematopoiesis
from ES cells in vitro.25 Importantly, GATA-1-null ES
cells failed to generate primitive progenitors in that
analysis.25 Definitive progenitors, in contrast, were
normal in number but underwent developmental arrest and apoptosis at
the proerythroblast stage. Arrested GATA-1( )-definitive
proerythroblasts expressed GATA target genes, such as -major globin
mRNA, at approximately normal levels.25 An erythroid cell
line (G1E) was also generated from in vitro differentiated
GATA-1() ES cells.27 G1E cells proliferate as
immature erythroblasts yet complete erythroid maturation upon restoration of GATA-1 function, so that rescue of terminal erythroid maturation in G1E cells was used as a cellular assay system in which to
evaluate the functional relevance of domains of GATA-1.
Among various ES cell differentiation systems,28-30 the
method using OP9 cells has several remarkable advantages, among which is their potential to differentiate along erythroid lineage to finally
give rise to enucleated red blood cells. ES cell-derived hematopoietic
cells start floating during differentiation and can be obtained with
culture media without any protease treatment.31 Therefore,
cell-surface markers of the ES cell-derived hematopoietic cells could
be analyzed by fluorescence-activated cell sorter (FACS). We also have
introduced a method that separates hematopoietic progenitors and
differentiated cells produced in this system. These important progress
enabled us to assess precisely the function and target genes of GATA-1.
In this study, the GATA-1 knock-down mutant ES cells, which
were also referred to as GATA-1.05 mutant ES
cells,5 were examined in the OP9/ES cell in vitro
differentiation system. The results clearly show that GATA-1 and other
erythroid transcription factors are expressed in wild-type ES
cell-derived hematopoietic cells, following the in vivo expression
profiles. However, maturation of both primitive and definitive
erythroid cells is blocked during differentiation of GATA-1.05
mutant ES cells, but the timing of the arrest appears to be different.
On the other hand, growth of the erythroid progenitors (named as
CFU-OP9) was markedly induced by the GATA-1.05 mutation,
demonstrating that GATA-1 regulates both proliferation and
differentiation of erythroid cells.
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MATERIALS AND METHODS |
Cell culture.
E14 ES cells were used in this study.32 ES cells were
maintained in the undifferentiated condition by using embryonic
fibroblasts as feeder cells.33 OP9 cells were cultured as
previously described.31,34 ES cells were differentiated in
vitro following the method as reported previously,31 with
minor modifications. We added 10 µg/mL stem cell factor (SCF; R&D
Systems, Minneapolis, MN) and 2 IU/mL erythropoietin (EPO;
Chyugai Pharmaceutical, Tokyo, Japan).
Colony assays.
ES-derived hematopoietic cells were cultured in semisolid
Iscove's modified Dulbecco's medium (IMDM) supplemented
with 0.8% methylcellulose, 20% fetal bovine serum (FBS), 1% bovine
serum albumin (BSA), and cytokines. Cytokines supplemented for
colony-forming unit-erythroid (CFU-E) assay were 2 IU/mL
human EPO and for CFU-granulocyte and monocyte (CFU-GM)
assay were 100 U/mL murine GM-colony-stimulating factor (GM-CSF;
PeproTech, St James' Square, London, UK), 100 U/mL murine
interleukin-3 (IL-3; Genzyme, Cambridge, MA), and 10 ng/mL
human IL-6 (PeproTech). Numbers of CFU-E were counted 2 days and those
of CFU-GM were counted 7 days after the start of the culture.
Quantitative reverse transcriptase-polymerase chain reaction
(RT-PCR) assays.
Total RNA was isolated by single-step RNA extraction system (RNA-zol,
Tel-Test, Friendswood, TX). cDNA was synthesized with Superscript reverse transcriptase (Life Technologies, Rockville, MD) and amounts of the cDNAs were adjusted by dilution
to produce equal amounts of hypoxanthine guanine
phosphoribosyl transferase (HPRT) amplicon. PCR was performed
using GeneAmp PCR system 9600 (Perkin-Elmer, Norwalk,
CT) on a regimen of 94°C for 20 seconds, ramp time
for 1 minute, 55°C for 1 second, and 72°C for 1 minute for
cycle number as described in Table 1.
The sequence of the primers used in this study is listed in Table
1. Each primer set is designed to locate at least one intron between
the two primer annealing sites to distinguish RT-PCR amplicon from that originated from genomic DNA template. Table 1 also lists cycle number for each set of primers. To assure linear amplification of
test samples, control samples were always monitored, and ranges were
determined in which dilution of the control sample results in linear
reduction of the signal intensity. The cycle number was set to give
rise to the intensity of each test sample always within this range.
Flow cytometry analysis.
Harvested ES cells in floating fraction were washed twice with
phosphate-buffered saline (PBS) and concentrated in 50 µL of 4%
BSA/PBS solution. First antibody was added to this solution and
incubated for 20 minutes on ice. Anti-TER119, -Mac-1, and -B220
monoclonal antibodies (PharMingen, San Diego, CA) were
used as the first antibodies. After washing with PBS, fluorescein
isothiocyanate-conjugated anti-rat Ig (PharMingen) was added to the
suspension of cells. These cells were also stained with 4 µg/mL
propidium iodide/PBS and subjected for FACS analysis with FACS Calibur
(Becton Dickinson, Franklin Lakes, NJ). Cells lost their
viability were counted as propidium iodide-positive cells. These cells
were gated out when measuring each first antibody-positive cells.
Measurement of porphyrin content.
A total of 5 × 104 cells in the floating fraction
were prepared for porphyrin analysis as described.35
Porphyrin fluorescence was measured by a Hitachi F-2000 fluorescence
spectrophotometer (Hitachi Ltd, Hitachi, Japan) in
comparison with hemin standard solution.
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RESULTS |
A method to facilitate fractionation of hematopoietic progenitors from
differentiated cells.
To examine the contribution of transcription factors to
hematopoietic cell differentiation processes, we cultured ES cells on
OP9 cells.31 During culture in differentiation
medium, we found that the ES cells were clustered adhering to
and submerging beneath the OP9 cell layer
(Fig 1A). The ES cells show a typical cobblestone formation, which is known to be characteristic
for early hematopoietic progenitors,36,37
indicating that this ES cell differentiation system can represent the
early hematopoietic cell differentiation process in addition to the
previously identified terminal maturation process of several
hematopoietic lineages.
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| Fig 1.
Fractionation of hematopoietic progenitors from
differentiated cells in OP9/ES cell differentiation system. (A)
Formation of cobblestone is observed with a phase-contrast
microscopy at day 14 of ES cell culture. Dark round cells indicated by
arrows are hematopoietic progenitors lying beneath OP9
layer. Large irregular-shaped cells indicated by arrowheads are OP9
cells. (B) Schematic presentation of the method to prepare floating and
adherent fractions. First, floating fractions are harvested with
culture media. Adherent cells are then disaggregated transiently by
trypsin and transferred to a new culture dish. After a 1-hour
incubation, which makes OP9 cells adhere to the dish, the floating
cells are obtained from media (adherent fraction).
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We designed a method to separate cells submerged beneath or adhering
tightly to the OP9 feeder from those loosely adhering to or floating
from the OP9 cell layer; we used the quickly adhering nature of the OP9
cells to culture dishes. As summarized in Fig 1B, cells spontaneously
floating into the culture supernatant were first collected
("floating fraction"). Then adherent cells including OP9 cells
were trypsinized, transferred to a new culture dish, and incubated for
1 hour to let OP9 cells adhere to the culture dish. Floating cells
after the incubation were obtained from the resulting culture
supernatant. Approximately 90% of OP9 cells were removed through this
procedure, with only 0.5% loss of the ES-derived cells ("adherent
fraction").
The adherent fraction contains more progenitors than the floating
fraction.
When the number of progenitors in each fraction was measured by means
of a colony assay, approximately 10 times more CFU-E and CFU-GM were
detected in the adherent fraction than in the floating fraction
(Fig 2). Thus, this result shows the
successful fractionation of hematopoietic progenitors from
differentiated hematopoietic cells derived from the ES cells. The
OP9/ES cell differentiation system has been shown to be augmented by
the addition of EPO and inhibited by blocking the signaling through
c-Kit.38 Therefore, we added EPO and
SCF to the culture medium to improve induction efficiency. Whereas the
addition of the cytokines did not influence the number of total
floating cells and that of dianisidine-positive cells during the first
7 days of culture, by day 14 the numbers of total floating cells and
dianisidine-positive cells were both increased to approximately 40 times more than those of the ES cells cultured without the addition of
the cytokines (data not shown). Therefore, based on this observation,
we used the two cytokines in this study to force the progenitor cells
in the adherent fraction to differentiate.
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| Fig 2.
Adherent fraction contains more progenitors than does the
floating fraction. Numbers of CFU-E and CFU-GM in the day 11 culture
are shown. Gray bar and hatched bar show numbers of hematopoietic
colonies in the adherent and floating fractions, respectively.
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Expression profiles of erythroid transcription factors in the
differentiation system.
To examine how erythroid differentiation proceeds in the ES cell
culture system, adherent and floating fractions were collected at
several time points from the in vitro ES cell culture system. Expression levels of mRNA encoding  y- and -major globins were first measured by RT-PCR. In this and following analyses, amounts of
the cDNAs were adjusted by dilution to produce equal amount of HPRT
amplicon and, after the adjustment, the relative percentage was
calculated based on the maximal gene expression. Both y- and
-major globin mRNAs were found to be more abundant in the floating
fraction than in the adherent fraction (Fig
3A). It should be noted that expression of the two globin genes peaked
at different time points. In the case of y-globin, floating cells at
day 8 showed peak-level expression and then the expression decreased, whereas the expression of -major globin increased gradually and reached peak levels at days 11 and 14 (Fig 3A). These data are consistent with previous observations for the OP9 system, in that primitive hematopoiesis appears early at approximately day 8, whereas
definitive hematopoiesis appears late at around day 14.38
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| Fig 3.
Expression profiles of erythroid transcription factors in
the OP9/ES cell differentiation system. Adherent and floating fractions
were collected at time points indicated from the in vitro ES cell
culture system. Concentrations of all the prepared cDNAs were adjusted
to produce equal amounts of HPRT amplicon. Expression levels of mRNA
encoding y- and -major globins were first measured by RT-PCR (A).
The expression profiles of erythroid transcription factors during the
differentiation of ES cells were then examined. The expression profiles
are categorized into three groups. The first group includes GATA-1,
p45, EKLF, and SCL (B); the second group consists of GATA-2 and
c-myb (C); and the third group includes Nrf2 and GATA-3 (D).
Each group is shown as a pair of boxes; the left box represents the
adherent fraction and the right box represents the floating fraction.
Highest expression for each factors is set to 100%.
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We next investigated the expression profiles of erythroid transcription
factors during the differentiation of ES cells into hematopoietic
cells. Each sample was obtained at the same timing as described above.
We found that the expression profiles are categorized into three
groups. The first group includes GATA-1, p45, EKLF, and SCL (Fig 3B).
mRNAs for these transcription factors were undetectable at day 0, but
the expression was gradually increased during cell culture. These mRNA
expression levels were more abundant in the floating fraction than in
the adherent fraction. Thus, the expression of this group of
transcription factors was suggested to increase along with the progress
of erythroid differentiation. The results also showed that these
factors are expressed in primitive as well as definitive hematopoietic
cells.
The second group consists of GATA-2 and c-myb
(Fig 3C). The mRNAs for these factors were expressed more abundantly in
the adherent fraction than in the floating fraction, and this profile was similar to that of c-kit. In particular, the expression of GATA-2 mRNA is so low in the floating fraction that we could not detect
expression in the RT-PCR analysis. Because progenitors are enriched in
the adherent fraction, this result shows that both transcription
factors are mainly expressed in hematopoietic progenitors.
The third group includes Nrf2 and GATA-3 (Fig 3D). Nrf2 mRNA expression
was uniformly observed in both the adherent and floating fractions
throughout the differentiation period, so that no correlation of Nrf2
to hematopoietic cell differentiation was found in this ES cell
differentiation system. In contrast to Nrf2, GATA-3 mRNA was
undetectable in all samples. This is quite intriguing if we consider
the fact that GATA-3 is an important transcription factor for T-cell
development39,40 and that T-cell differentiation has never
been observed in this OP9/ES differentiation system.31 The
failure to induce expression of GATA-3 gene may explain why it has,
until now, been impossible to differentiate ES cells into T
cells.
ES cells with the GATA-1.05/Y genotype differentiate into blastlike
cells.
We recently succeeded in making ES cells with a GATA-1 knock-down or
GATA-1.05 allele.5 To further dissect the function of GATA-1, we analyzed the GATA-1.05/Y ES cells using the
OP9/ES cell in vitro differentiation system. The GATA-1.05
mutant ES clones produced slightly decreased numbers of floating cells
at both days 8 and 14 (Fig 4A [see
page 4113]). In the day 8 floating cells, representing primitive
hematopoiesis, GATA-1 mRNA expression was below detection levels even
with the sensitive RT-PCR method (Fig 4B). In contrast, GATA-1 mRNA
expression was detectable in the definitive lineage (ie, see the day 14 floating cells), although the expression level was significantly lower
than that of the wild type. Through quantitative PCR analysis, we found
that, compared with wild-type ES cells, only 7.8% of GATA-1 was
expressed in the day 14 GATA-1.05 cells (data not shown).
Wright-Giemsa staining of the wild-type day 14 floating cells showed
erythroid cells at various differentiation stages, including the
enucleated mature red blood cell (Fig 4C). Importantly, we found an
increased number of blastlike cells. Approximately 80% of cells in the
floating fraction from the mutant ES clones are the blastlike cells and the cells appeared morphologically to be proerythroblasts (Fig 4D).
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| Fig 4.
ES cells with GATA-1.05/Y genotype differentiate
into blastlike cells. Differentiation of GATA-1.05/Y ES
cells were examined using the OP9/ES cell in vitro differentiation
system. (A) Numbers of floating cells produced by GATA-1.05
mutant ES clones at both day 8 and 14. (B) Quantitative RT-PCR analysis
of GATA-1 mRNA expression in the wild-type and GATA-1.05
ES cells. (C and D) Wright-Giemsa staining of the wild-type and
mutant day 14 floating cells.
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Previous analysis of GATA-1 knockout ES cells showed that
EPO-responsive cells differentiated along the erythroid lineage and
died at the proerythroblast stage.25 However,
identification of these stages relied solely on morphological
observations and no surface marker studies were conducted in that
analysis. Because only a small number of ES cell-derived hematopoietic
cells could be recovered in the previous method, FACS analysis was
technically not feasible. In contrast, the OP9/ES cell differentiation
system produces a large quantity of floating cells derived from the ES cells and is, therefore, suitable for FACS analysis.31
We exploited this improvement and examined the expression of
lineage-specific surface antigens on the cells of the day 14 floating
fraction from both wild-type and GATA-1.05 mutant ES cells.
FACS analysis using three surface antigens showed that cells with
TER119 antigen, a late erythroid marker, were detected in both the
wild-type and mutant floating fractions. To our surprise, TER119-positive cells occupied 82% of the total GATA1.05
mutant floating fraction (Fig 5). This
number was similar to that of the blastlike cells observed in the
floating fraction from the mutant ES cells (see above). However, the
important finding here is that, whereas the wild-type floating cells
showed an intensity range of 102 to 103 for the
TER119 antigen, the mutant floating cells did not show such strong
intensity, indicating that GATA-1.05 mutant definitive cells
express a relatively low level of the TER119 antigen. Mac-1 and B220
antigen-positive cells also existed in both the wild-type and mutant
floating fractions. Numbers and intensities of the latter two
antigen-positive cells were almost similar to those from the wild-type
ES clones (Fig 5). Thus, the blastlike cells in the mutant floating
fraction are erythroid lineage cells. The differentiation of
GATA-1.05 mutant ES cells along the definitive erythroid
lineage reaches the stage at which the TER119 antigen is expressed,
albeit at a low level, but the maturation of the cells is arrested at
this stage.
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| Fig 5.
FACS analysis of the lineage-specific surface antigens.
Cells of day 14 floating fraction from both wild-type and
GATA-1.05 mutant ES cells were analyzed with FACS using three
surface antigens as indicated in the figure. Note that, whereas the
wild-type floating cells showed 102 to 103
fluorescence intensity range for TER119 antigen, the mutant floating
cells did not show such strong intensity. Upper three histograms show
results of wild-type cells, while lower three show results of
GATA-1.05 mutant cells.
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Decrease of porphyrin content and mRNAs for heme biosynthesis enzymes
in GATA-1.05 mutant hematopoietic cells.
The stage of erythroid cells in the OP9/ES cell differentiation system
was monitored not only by the morphological appearance but also by
hemoglobin staining using dianisidine. Although approximately 43% of
the wild-type cells in the day 8 floating fraction stained positive for
dianisidine (Fig 6A), we could not find any dianisidine-positive cells
in the GATA-1.05 mutant cells (Fig 6B). This indicates that the
mutant ES cells cannot differentiate into mature primitive erythroid
cells, being in very good agreement with our previous in vivo analysis
showing that primitive erythroid cells were arrested at the immature
stage.5
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| Fig 6.
Decrease of porphyrin content in GATA-1.05 mutant
hematopoietic cells. (A and B) Dianisidine staining of the wild-type
and GATA-1.05 mutant cells in the day 8 floating fraction,
respectively. (C) Color comparison of floating cell pellet at day 14. (D) Measurement of the porphyrin content in the cells. The fluorescence
spectra in lysate of both wild-type and GATA-1.05 floating
fraction showed the specific profile of porphyrins. The spectrum of the
higher peak shows wild type and that with the lower peak shows the
mutant results.
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In contrast, 67% of wild-type cells and 32% of the GATA-1.05
mutant cells in the day 14 floating fraction were positive for dianisidine staining. We suspect that cells containing small amounts of
hemoglobin might all be counted as dianisidine-positive, because dianisidine staining is a very sensitive method. Therefore, the considerably high number of dianisidine positive cells in the mutant
floating fraction might not reflect the actual difference in expression
level of hemoglobin. Indeed, when the colors of floating cell pellets
were compared at day 14, the pellet of the mutant cells was apparently
white and that of the wild-type cells was red (Fig 6C).
This observation suggested that either globin protein or porphyrin
content in the floating fraction of GATA-1.05 might be diminished considerably. Therefore, we measured the porphyrin content
in the cells. To this end, an excitation beam of 400-nm wave length was
used to emit fluorescence from the cell extracts, and the
characteristic pattern of fluorescence spectrum with a peak emission at
600 nm was monitored using a fluorescence spectrophotometer. The
fluorescence spectra from lysates of both wild-type and
GATA-1.05 floating fractions showed the specific profile of
porphyrins (Fig 6D). Importantly, porphyrin content in the
GATA-1.05 mutant floating cells was reduced to 13% of that of
the wild-type cells. This observation thus shows that heme synthesis is
severely affected in the GATA-1.05 mutant definitive erythroid
cells and that differentiation of the GATA-1.05 mutant ES
clones along the definitive erythroid lineage is arrested before
heme/porphyrin synthesis is fully commenced.
Impaired expression of heme biosynthetic enzymes in GATA-1.05 mutant.
To ask how porphyrin synthesis proceeds in this ES cell differentiation
system or how it is affected in the GATA-1.05 mutant cells, we
examined expression of mRNAs encoding four heme biosynthetic pathway
enzymes in the wild-type and GATA-1.05 mutant hematopoietic cells using RT-PCR. mRNAs for erythroid-specific 5-aminolevulinate synthase (ALAS-E), 5-aminolevulinate dehydratase (ALAD),
porphobilinogen deaminase (PBGD), and ferrochelatase (FC), the first
three and the last enzymes of this pathway, could not be detected in
the uninduced ES cells (Fig 7; day 0 in the
adherent fraction). The mRNAs for these enzymes first became detectable
in the floating fraction cells by day 8, indicating that these mRNAs
are expressed in the primitive hematopoietic cells (Fig 7).
Expression of mRNAs for these enzymes was significantly induced in the
floating fraction by day 11 and adherent fractions by day 14. These
results indicate that the expression of the heme pathway enzymes was
induced when hematopoietic progenitor cells committed to and started
differentiation along the erythroid lineage.
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| Fig 7.
Impaired expression of heme biosynthetic enzymes in
GATA-1.05 mutant. The expression of mRNAs for four heme
biosynthetic pathway enzymes were examined in the wild-type and
GATA-1.05 mutant hematopoietic cells. Amounts of the cDNAs used
for the RT-PCR analyses were adjusted by dilution to produce equal
amount of HPRT amplicon. The expression of two -type globin mRNAs
was also examined. y-globin mRNA is expressed only in primitive
erythroid cells, whereas -major globin mRNA is expressed in
definitive erythroid cells.
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In contrast to the expression profiles in the wild-type ES cells,
expression of these mRNAs was significantly decreased in the
GATA-1.05 mutant floating fraction cells. In day 8 floating fraction of the GATA-1.05 mutant cells, mRNAs for the four heme biosynthetic enzymes were all below detection level. In day 14 mutant
floating fraction, the relative expression levels of ALAS-E, ALAD,
PBGD, and FC mRNAs were 2.2%, 9.4%, 25.9%, and 10%, respectively, of the wild-type ES-derived cells (Fig 7). The decrease of heme pathway
enzymes resulted in the reduction in porphyrin content (see Fig 6D).
These observations unequivocally show that the heme pathway enzyme
genes are regulated, either directly or indirectly, by GATA-1, and that
one of the major contributions of GATA-1 to the erythroid
differentiation process is to activate the expression of heme
biosynthetic pathway enzymes.
Expression of two -type globin mRNAs was examined in parallel. While
y-globin mRNA is expressed only in primitive hematopoiesis and
behaves as a marker gene for primitive hematopoiesis, -major globin
mRNA is expressed in definitive hematopoietic cells. In GATA-1.05 mutant cell differentiation, the expression
of y-globin mRNA was not detectable at any time. Expression of
-major globin mRNA also was not observed at day 8. This is consistent with the observation that GATA-1 expression was not
detectable during primitive hematopoiesis. On the other hand, -major
globin mRNA expression was not so severely affected in the mutant
floating fraction at day 14 (69% of wild type). This was an
unexpectedly high level of expression because GATA-1 was expressed at
only 7.8% of the normal level and porphyrin was accumulated at 13% in
the GATA-1.05 mutant cells. These data indicate that the
expression of heme biosynthetic enzyme genes depends more profoundly on
GATA-1 than does the -major globin gene.
GATA-1.05 mutant erythroid cells cease differentiation without losing
viability.
Data obtained thus far indicate that the GATA-1.05 mutant cells
in the day 14 floating fraction have already committed to the
definitive erythroid lineage, but further maturation was arrested. In
this regard, there existed a possibility that erythroid cells all died
after certain stages of differentiation, so that mature erythroid cells
were not detected in this experiment. Indeed, erythroid cells derived
from GATA-1 knockout ES cells were reported to undergo apoptosis after
the proerythroblast stage in a different ES cell differentiation
system.37 Therefore, we examined viability of the cells in
the floating fraction by two independent methods, ie, trypan blue dye
exclusion assay and propidium iodide staining. In the former method,
more than 90% of both the wild-type and mutant cells were counted as
viable cells. In the latter method, 79% of the wild-type and 82% of
the mutant cells were also found to be viable
(Table 2). These results indicate that the
prominent increase of immature erythroid cells in the GATA-1.05
mutant is not caused by death or disappearance of differentiated cells, but rather is caused by maturation arrest of erythroid cells.
GATA-1.05 mutant hematopoietic cells grow rapidly after replating.
Because fetal liver cells from the GATA-1.05/Y mice did not
form erythroid colonies efficiently in a standard colony
assay,5 an alternative approach was necessary to assess the
colony-forming activity of GATA-1.05 hematopoietic progenitors.
In this regard, the adherent fraction cells of the OP9/ES cell
differentiation system are found to contain hematopoietic progenitors,
which form colonies on the OP9 feeder cell layer (see Fig 1). It has
already been shown that the hematopoietic progenitors in the OP9/ES
cell culture system have the ability to form colonies when replated onto the OP9 layer, and the number of these replating colonies tightly
correlates with those of hematopoietic cells finally
obtained.31
To investigate the nature of the GATA-1.05 hematopoietic
progenitors, the adherent fraction cells were isolated at day 14 of the
culture and seeded onto another OP9 layer after dilution. Colonies were
found to be formed after 3 days in culture, and we referred to the
progenitors as CFU-OP9 in this study. The CFU-OP9 number of
GATA-1.05 mutant and wild-type cells were counted at this
stage. To our surprise, whereas the wild-type adherent fraction cells
produced only 9 ± 1 CFU-OP9/105 cells, the
GATA-1.05 mutant cells produced more than 200 CFU-OP9/105 cells (Fig 8). This
result thus indicates that the cells in the day 14 adherent fraction of
the GATA-1.05 mutant contain a dramatic increase in CFU-OP9
progenitors compared with wild type, and it also suggests that GATA-1
deficiency promotes the growth of immature hematopoietic cells.
View larger version (19K):
[in this window]
[in a new window]
| Fig 8.
Replating efficiency of wild-type and GATA-1.05
mutant ES cell-derived hematopoietic progenitors. The adherent
fraction cells contain hematopoietic progenitors that can form colonies
on the OP9 feeder layer (CFU-OP9). The adherent fraction cells at day
14 were isolated and seeded on another OP9 layer. After 3 days in
culture, colonies formed on OP9 were counted from three independent
GATA-1.05 mutant ES cell lines and wild-type ES cells.
|
|
Erythroid transcription factor expression in GATA-1.05 mutant
hematopoietic cells.
The GATA-1.05 mutation probably also introduced perturbation
into the transcription factor regulatory network. Therefore, we
analyzed the consequence of the perturbation on expression of other
erythroid-specific transcription factors and the following observations
are important. First, the expression of EKLF was not observed in the
mutant day 8 floating fraction cells (Fig 9). This is in sharp contrast to the situation with wild-type ES cells
(Fig 9; see also Fig 3B). Similarly, the expression of p45 and SCL in
the same fraction decreased to 50% and 70%, respectively. As the
expression level of GATA-1 is practically undetectable in primitive
hematopoietic cells of the GATA-1.05 mutant, this observation
suggests that GATA-1 is essential for the expression of the EKLF gene
in the primitive erythroid cells.
View larger version (58K):
[in this window]
[in a new window]
| Fig 9.
Expression of erythroid transcription factors in the
wild-type and GATA-1.05 mutant hematopoietic cells. To
understand the consequence of the perturbation introduced by the
GATA-1.05 mutation, expression of mRNAs for five erythroid
transcription factors was monitored by RT-PCR analysis.
|
|
Secondly, the expression of GATA-2 was induced markedly in the day 11 and 14 floating fraction of GATA-1.05 mutant cells, whereas the
expression was undetectable in the wild-type floating fraction.
Increase of GATA-2 mRNA in GATA-1-deficient cells has also been
reported in a different ES cell differentiation system.25 c-myb expression also was increased in this fraction. It should be noted that the day 14 floating fraction of GATA-1.05 mutant contains approximately eightfold more blastlike cells than the wild-type fraction. Therefore, we normalized the increase of GATA-2 using the c-myb datum, assuming that the increase in
c-myb expression might reflect the enrichment of
proerythroblasts. Even after the normalization, the GATA-2 expression
was increased more than 100-fold in the GATA-1.05 mutant
floating fraction than in the same fraction of wild type. Thus, this
result suggests that GATA-2 gene expression is induced in each cell in
the absence of GATA-1.
| |
DISCUSSION |
In vitro ES cell differentiation is powerful in analyzing mechanisms of
hematopoiesis. Of the various ES cell differentiation systems available
to date, the OP9 system has a unique feature in that mature
hematopoietic cells can be obtained as floating cells in culture
media.31 Detachment of the cells from the OP9 feeder cell
layer is most likely caused by the maturation of the cells, because the
cells adherent to the OP9 feeder layer are immature progenitors.
Applying the OP9 system to the GATA-1.05 mutant ES cells, we
have examined the consequence of GATA-1 deficiency in the
differentiation of both primitive and definitive erythroid lineages and
of perturbation of the transcription factor regulatory network
generated by the mutation. The results unequivocally showed that the
GATA-1 deficiency results in maturation arrest in both the primitive
and definitive erythroid lineages, although the timing of the arrest is
distinct in each lineage. In contrast, the replating activity of the ES
cell-derived hematopoietic progenitor, which we refer to as CFU-OP9,
is stimulated more than 20-fold by the GATA-1.05 mutation,
suggesting that loss of GATA-1 favors growth of progenitor cells. Thus,
GATA-1 appears to regulate both growth and differentiation of erythroid
lineage cells during in vitro ES cell differentiation.
The OP9/ES cell differentiation system allows us to analyze the
consequences of artificial gene manipulation in hematopoiesis even if
the loss of expression of a particular gene results in embryonic
lethality. GATA-1.05/Y mutant mice die due to the lack of
primitive hematopoiesis,5 so that the precise assessment of
the contribution of GATA-1 to the definitive lineage requires an in
vitro system like that presented here. In addition, because of the
nature of the system, detailed expression of the GATA-1 gene can be
examined in primitive and definitive hematopoietic lineages. Results of
the latter analysis clearly indicate that the GATA-1.05
mutation completely abolished (or knocked out) GATA-1 gene expression
in the primitive lineage and enfeebled (or knocked down) the expression
in the definitive lineage. As the GATA-1.05 mutation was
generated in ES cells by insertion of the neomycin-resistance gene in
front of the erythroid-specific promoter/first exon (IE promoter/exon),
we suspect that interference by the strong MC-1 promoter perhaps caused
the disruption of the IE promoter activity. Although the reason for the
difference in severity of the disruption in the primitive and
definitive lineages remains to be clarified, we recently found that
GATA-1 gene expression in the two hematopoietic lineages is regulated
by two distinct regulatory regions41 and we suspect that
this might be related to the emergence of the two different phenotypes.
We found in this study that the GATA-1.05 mutant ES cells can
differentiate along the primitive and definitive lineages and finally
give rise to the floating fraction. However, maturation of the
ES-derived progenitors ceased at proerythroblast stage and no further
maturation of the blasts was observed. Because this observation is
quite consistent with the previous report using GATA-1 gene knockout ES
cells,25 we conclude that the GATA-1.05 mutation is
similar to the null mutation of the GATA-1 gene in this respect and
that the residual amount of GATA-1 in our GATA1.05 system
cannot induce further maturation of the ES cells. The important finding
here is that, while the expression of TER119 erythroid-specific surface
antigen and porphyrin accumulation are detected in the definitive
erythroblasts of the wild type, expression of these erythroid markers
was also detectable, albeit weakly, in the GATA-1.05 ES-derived
cells. These results suggest that commitment of hematopoietic stem
cells to the erythroid lineage occurs without GATA-1, but GATA-1 is
necessary for the progenitors to further differentiate along this
lineage.
Proerythroblasts from the GATA-1.05 ES cells showed viability
essentially similar to those from the wild-type ES cells. Thus, significant cell death including apoptosis was suggested not to occur
in GATA-1.05 cells during the differentiation culture. In contrast, GATA-1-null ES cells were previously reported to have decreased viability due to apoptosis after the proerythroblast stage.27 One plausible explanation for this difference is
that the residual 7.8% GATA-1 in our GATA-1.05 system may be
sufficient to prevent apoptosis of differentiation-arrested blasts, but
the amount of GATA-1 is not sufficient to drive erythroid
differentiation of the cells. An alternative explanation is that the
presence of the OP9 cell layer may affect the fate of arrested
progenitor cells.
Erythroid progeniors derived from the GATA-1-null ES cell are reported
to express globin mRNAs at an approximately similar level to that of
erythroid cells derived from wild-type ES cells.25 Consistent with the observation, our present analysis shows that the
expression of -major globin gene is not so significantly affected by
the GATA-1.05 mutation. These observations raise a question as
to why the differentiation of erythroid cells is arrested in the
GATA-1.05 mutant erythroid cells. In this analysis we found that the expression of four heme biosynthetic enzyme genes is severely
affected by the GATA-1.05 mutation. The impairment is so severe that
porphyrin content in GATA-1.05 mutant cells is decreased to
approximately 13% of that in wild-type ES cell-derived hematopoietic
cells (Fig 6D). These observations show that one of the major
contributions of GATA-1 to the erythroid differentiation process is to
activate the expression of heme biosynthetic pathway enzymes. There
also exists a possibility that the decrease of intracellular heme
concentration may in turn cause the arrest of erythroid
differentiation.
We also found that the GATA-1.05 mutation augmented the
proliferative capacity to the progenitors that are still adherent to
the OP9 feeder layer. The proliferative capacity (ie, CFU-OP9) has been
examined by a replating assay (Fig 9) in which the lack of GATA-1
appears to induce the proliferation of progenitors. Consistent with this observation, forced expression of GATA-1 in NIH3T3
cells was reported to slow down cell-cycle progression.42 If seen from a different standpoint, the proliferation of progenitors should decrease in parallel with the cell undergoing differentiation. The consequence of the lack of GATA-1 in the GATA-1.05/ES cell differentiation system seems to correlate well with these physiological phenomena (see also reference 43). Thus, the function of GATA-1 is
suggested to redirect erythroid progenitors from proliferation to
differentiation.
The expression of mRNAs encoding erythroid transcription factors were
examined in the OP9/ES cell differentiation system. The results
indicate that expression of mRNAs for GATA-1, SCL, EKLF, and p45 was
induced during erythroid differentiation of wild-type ES cells and the
highest expression levels of these mRNAs were observed in the floating
fraction. In contrast, mRNAs for GATA-2 and c-myb showed their
peak expression at the adherent stage and their expression level was
decreased when cells floated from the feeder layer. We also found that
the expression of EKLF mRNA heavily depends on the presence of GATA-1
in primitive hematopoiesis, whereas GATA-2 mRNA is strongly suppressed
by GATA-1 in definitive hematopoiesis. These findings strongly argue
for the notion that the expression of EKLF and GATA-2 genes are
regulated by GATA-1. Consistent with these results, EKLF gene
expression was shown to be regulated through a GATA sequence in the
promoter in a transient transfection assay.44 A hypothesis
that emerged from these results was that the erythroid transcription
factors form a regulatory network, in which multiple factors interact
with each other to form a protein complex on regulatory
regions45 and also that the same factors crossregulate each
other. Apparently these transcription factors seem to regulate the
proliferation and differentiation of the erythroid lineage cells with
various positive and negative feedback pathways and/or
cascades. To further analyze these transcription factor regulatory
networks, it is necessary to manipulate expression of the gene by
targeted mutagenesis to examine the expression level of various
transcription factors in the ES cell differentiation system
characterized here.
| |
ACKNOWLEDGMENT |
We are grateful to Drs Shigeru Sassa for advice on porphyrin
measurement, Hiroaki Kodama for advice on OP9 cells, and Jorg Bungard
and Roger Patient for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted April 2, 1998;
accepted July 16, 1998.
Supported in part by Grants-in-Aid from the Ministry of Education,
Science, Sports and Culture, Japanese Society for Promotion of Sciences
(JSPS) and CREST.
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 Masayuki Yamamoto, MD, PhD, Institute of
Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba
305-8575, Japan; e-mail: masiya{at}md.tsukuba.ac.jp.
| |
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Transgenic expression of BACH1 transcription factor results in megakaryocytic impairment
Blood,
April 15, 2005;
105(8):
3100 - 3108.
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M. A. Vodyanik, J. A. Bork, J. A. Thomson, and I. I. Slukvin
Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential
Blood,
January 15, 2005;
105(2):
617 - 626.
[Abstract]
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M. Nakano, K. Ohneda, H. Yamamoto-Mukai, R. Shimizu, O. Ohneda, S. Ohmura, M. Suzuki, S. Tsukamoto, T. Yanagawa, H. Yoshida, et al.
Transgenic over-expression of GATA-1 mutant lacking N-finger domain causes hemolytic syndrome in mouse erythroid cells
Genes Cells,
January 1, 2005;
10(1):
47 - 62.
[Abstract]
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R. Shimizu, T. Kuroha, O. Ohneda, X. Pan, K. Ohneda, S. Takahashi, S. Philipsen, and M. Yamamoto
Leukemogenesis Caused by Incapacitated GATA-1 Function
Mol. Cell. Biol.,
December 15, 2004;
24(24):
10814 - 10825.
[Abstract]
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M. Tanaka, J. Zheng, K. Kitajima, K. Kita, H. Yoshikawa, and T. Nakano
Differentiation status dependent function of FOG-1
Genes Cells,
December 1, 2004;
9(12):
1213 - 1226.
[Abstract]
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M. Garriga-Canut and S. H. Orkin
Transforming Acidic Coiled-coil Protein 3 (TACC3) Controls Friend of GATA-1 (FOG-1) Subcellular Localization and Regulates the Association between GATA-1 and FOG-1 during Hematopoiesis
J. Biol. Chem.,
May 28, 2004;
279(22):
23597 - 23605.
[Abstract]
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K. Umeda, T. Heike, M. Yoshimoto, M. Shiota, H. Suemori, H. Y. Luo, D. H. K. Chui, R. Torii, M. Shibuya, N. Nakatsuji, et al.
Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro
Development,
April 15, 2004;
131(8):
1869 - 1879.
[Abstract]
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C. Cerdan, A. Rouleau, and M. Bhatia
VEGF-A165 augments erythropoietic development from human embryonic stem cells
Blood,
April 1, 2004;
103(7):
2504 - 2512.
[Abstract]
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R. Shimizu, K. Ohneda, J. D. Engel, C. D. Trainor, and M. Yamamoto
Transgenic rescue of GATA-1-deficient mice with GATA-1 lacking a FOG-1 association site phenocopies patients with X-linked thrombocytopenia
Blood,
April 1, 2004;
103(7):
2560 - 2567.
[Abstract]
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N. Suzuki, N. Suwabe, O. Ohneda, N. Obara, S. Imagawa, X. Pan, H. Motohashi, and M. Yamamoto
Identification and characterization of 2 types of erythroid progenitors that express GATA-1 at distinct levels
Blood,
November 15, 2003;
102(10):
3575 - 3583.
[Abstract]
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M. Jackson, J. W. Baird, N. Cambray, J. D. Ansell, L. M. Forrester, and G. J. Graham
Cloning and Characterization of Ehox, a Novel Homeobox Gene Essential for Embryonic Stem Cell Differentiation
J. Biol. Chem.,
October 4, 2002;
277(41):
38683 - 38692.
[Abstract]
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S. Chung, T. Andersson, K.-C. Sonntag, L. Bjorklund, O. Isacson, and K.-S. Kim
Analysis of Different Promoter Systems for Efficient Transgene Expression in Mouse Embryonic Stem Cell Lines
Stem Cells,
March 1, 2002;
20(2):
139 - 145.
[Abstract]
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S. Saleque, S. Cameron, and S. H. Orkin
The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages
Genes & Dev.,
February 1, 2002;
16(3):
301 - 306.
[Abstract]
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S. Takahashi, R. Shimizu, N. Suwabe, T. Kuroha, K. Yoh, J. Ohta, S. Nishimura, K.-C. Lim, J. D. Engel, and M. Yamamoto
GATA factor transgenes under GATA-1 locus control rescue germline GATA-1 mutant deficiencies
Blood,
August 1, 2000;
96(3):
910 - 916.
[Abstract]
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T. Era and O. N. Witte
Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate
PNAS,
February 15, 2000;
97(4):
1737 - 1742.
[Abstract]
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S. Nishimura, S. Takahashi, T. Kuroha, N. Suwabe, T. Nagasawa, C. Trainor, and M. Yamamoto
A GATA Box in the GATA-1 Gene Hematopoietic Enhancer Is a Critical Element in the Network of GATA Factors and Sites That Regulate This Gene
Mol. Cell. Biol.,
January 15, 2000;
20(2):
713 - 723.
[Abstract]
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C. Heyworth, K. Gale, M. Dexter, G. May, and T. Enver
A GATA-2/estrogen receptor chimera functions as a ligand-dependent negative regulator of self-renewal
Genes & Dev.,
July 15, 1999;
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1847 - 1860.
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Top
Abstract
Introduction