 |
Introduction |
Hematopoiesis is regulated by the orchestration of several processes, including cell proliferation, cell differentiation, and cell death. Apoptosis, also referred to as programmed cell death, regulates the survival of progenitor
cells and the turnover of mature elements, which are important for maintaining the homeostasis of hematopoietic
cells (1). Growth factors are thought to play important roles
in the apoptosis of hematopoietic progenitors. They act as
survival factors for hematopoietic precursors, and the hematopoietic progenitors succumb to apoptosis in their absence (2). On the other hand, the survival of hematopoietic cells is also controlled by the Bcl-2 protein family (5). Family members such as Bcl-2, Mcl-1, A1, and Bcl-XL are expressed
in hematopoietic cells and are considered to function as repressors of apoptotic cell death (9).
In mouse embryogenesis, erythropoiesis originates in the
yolk sac blood islands, beginning at approximately embryonic day 7.5, then migrates to the fetal liver, spleen, and
eventually to the bone marrow (10). Embryonic primitive
erythropoiesis in the yolk sac and definitive erythropoiesis
in the fetal liver, spleen, and bone marrow produce primitive erythrocytes (EryP)1 and definitive erythrocytes (EryD),
respectively. Although the glycoprotein hormone erythropoietin (EPO) was initially characterized as stimulating
both the proliferation and differentiation of EryD progenitors, it has been shown that EPO maintains the viability of
primarily EryD progenitor cells (11). The dependence of
the survival of EryD on EPO is reported in various experimental systems, including Friend virus anemia strain-infected
murine splenic erythroid progenitors, the erythroleukemia
cell line, and murine fetal liver erythroid progenitors (2, 14-
16). These studies showed that survival of the late erythroid
progenitors (CFU-E) of EryD was dependent on EPO and
that the EPO-dependent period lasted from the CFU-E stage to the beginning of hemoglobin synthesis. Gene targeting experiments with EPO and EPO receptor revealed that
EPO has important roles in primitive erythropoiesis (12, 13).
We found that the survival of immature EryP was also dependent on EPO, as was that of EryD progenitors (our unpublished data). Thus, EPO prevents apoptosis of both EryP
and EryD during their immature state.
The protein Bcl-2 prevents apoptosis triggered by various stimuli, including chemotherapeutic drugs, 
irradiation, viral infections, oxidant stress, and notably growth
factor deprivation (6). Bcl-2 and Bcl-XL act as cell death
repressors, whereas Bax and the alternatively spliced bcl-x
gene product, Bcl-XS, act as cell death promoters (6, 17,
18). A family of bcl-2 genes participates in the regulation of
cell survival in multiple cell lineages, including the hematopoietic lineage. Constitutive overexpression of Bcl-2 suppresses apoptosis in hematopoietic precursors by growth
factor withdrawal, and overexpression of Bcl-XL also suppresses apoptosis (19, 20). Both Bcl-2 and Bcl-XL have recently been reported to be involved in regulating erythroid
progenitors and survival (21). However, all of the evidence is circumstantial, and it is uncertain how bcl-x functions during erythroid differentiation under physiological
conditions. In this study, we analyzed the function of bcl-x
in erythropoiesis using mouse embryonic stem (ES) cells in
which both alleles of bcl-x were disrupted (26). The
production of immature EryP and EryD by bcl-x
/ ES cells
was normal. Unexpectedly, however, prominent apoptotic cell death of both EryP and EryD occurred at the very end
of erythroid maturation. These data clearly show that Bcl-X
is essential in the late erythroid maturation stage.
| |
Materials and Methods |
Target Disruption of the bcl-x Gene.
E14 ES cells derived from
strain 129/Ola were used throughout the experiment. ES bcl-x+/+,
bcl-x+/, and bcl-x/ cell lines were produced as described previously (21). Genomic DNA containing the bcl-x locus was isolated
from a library of mouse strain 129/Sv DNA. A 1.8-kb XhoI-BamHI fragment containing most of the bcl-x coding region was
replaced with either a PGK-neo polyadenylated (poly A) cassette
or a PGK-hyg poly A cassette. Both targeting vectors contain
6.0-kb 5' and 1.0-kb 3' regions of homology with the drug-resistance markers and a PGK-tk poly A cassette. Transfection and selection were performed as described (31). DNA prepared from
ES cells was digested with EcoRV, transferred to a nylon membrane, and then hybridized with the 0.4-kb KpnI-PstI probe that
flanked the 3' homology region. The expected sizes of wild-type
bcl-x, mutant bcl-x with the neo targeting vector, and mutant bcl-x
with the hyg targeting vector were 9.8, 7.0, and 5.5 kb, and were
detected in wild-type, bcl-x+/, and bcl-x/ ES clones, respectively.
Production of Chimeric Mice and Analysis of the Contribution of ES
Cells.
ES bcl-x+/+, bcl-x+/, and bcl-x/ cells were injected into
the 3.5-d post-coitum blastocysts from C57BL/6 mice to generate chimeric mice. GPI (glucose phosphoisomerase) isozymes
were used to analyze the contribution of ES cells in various organs of chimeric mice. The GPI isozyme of B6 mice and of 129/
Ola mice from which E14 ES cells were established are GPI-1A
and GPI-1B, respectively. Separation and detection of GPI isoenzymes were performed essentially as described (32). Chimeric
mice were perfuged with PBS to eliminate blood cell contamination, and dissected tissues were kept at -70°C. Frozen tissue samples were thawed and gently homogenized in water, and cells
were then lysed by three rounds of freezing and thawing. After
centrifugation of the homogenates, the supernatants were diluted
with water and then electrophoresed on Titan III Zip Zone cellulose acetate plates (Helena Laboratories) in Tris-glycine buffer
(25 mM Tris, 200 mM glycine, pH 8.5) for 4 h at 150 V at 4°C.
The stainings were performed by overlaying the mixture consisting of 2 ml of 0.2 M Tris-HCl (pH 8.0), 0.1 ml each of 0.25 M
magnesium acetate, 10 mg/ml NADP, and 100 mg/ml fructose
6-phosphate, and 0.2 ml of MTT, 0.05 ml of 2.5 mg/ml phenazine methosulfate, 5 µl glucose 6-phosphate dehydrogenase (140 U/ml; Sigma Chemical Co.), and 5 ml of 2% agarose. The GPI
isozyme bands appeared after a few minutes in the dark. Density
of the bands was analyzed by densitometer.
The hemoglobin type of B6 mice and 129/Ola mice are single
(Hbbs/Hbbs) and diffuse (Hbbd/Hbbd), respectively, and were used
to evaluate the contribution of ES cells in circulating erythrocytes
of chimeric mice. These two types of hemoglobin can be distinguished by electrophoresis. Cellulose acetate electrophoresis
of cystamine-modified hemoglobins was performed essentially as
described (33). Whole blood in PBS containing 50 mM EDTA
was layered onto 2 vol of Histopaque-1077 (Sigma Chemical
Co.) and centrifuged at 3,500 g for 20 min at room temperature.
The pellet, enriched for RBCs, was collected. 10 µl purified
RBCs was added to 300 µl cystamine lysis buffer (12.5 mg/ml
cystamine dihydrochloride, 1 mM dithiothreitol, 0.55% ammonium hydroxide) and agitated to lyse the RBCs. The samples were applied to Titan III cellulose acetate plates and run in TBE
buffer (0.18 M Tris, 0.10 M boric acid, 0.002 M EDTA) for 40 min at 300 V. The plates were placed in staining solution (1% Ponceau S, 5% TCA) for 10 min and rinsed in three changes of
5% acetic acid for 10 min each. The percentage contributions of
ES cells in adult chimera were examined using the allotype of GPI from various nonhematopoietic organs, such as the liver and kidney. The hemoglobin type analysis data were obtained from the
chimera in which the contribution of ES cells to nonhematopoietic
organs was >50%.
ES Cells and Their Differentiation Induction.
ES bcl-x+/+, bcl-x+/,
and bcl-x/ cells were cultured on embryonic fibroblasts as
feeder cells in the presence of a saturated dose of leukemia inhibitory factor using the standard procedure (31). The culture of
OP9 stromal cells and the differentiation induction method were
carried out as described (27). OP9 stromal cells were maintained
in 
-MEM (Life Technologies, Inc.) supplemented with 20%
FCS (Summit) and standard antibiotics (27, 34). 105 ES cells were
transferred onto confluent OP9 stromal cells in 10-cm culture
dishes (Nunc). After day 3 of the induction, human recombinant
EPO (provided by Kirin Brewery Co. Ltd.) was added at a final
concentration of 2 U/ml during the differentiation induction.
The induced cells were trypsinized at day 5, and 106 cells were
transferred onto fresh OP9 cells on a 10-cm plate. Nonadherent
cells were harvested on day 6, 7, or 8 to obtain EryP. On day 10, all of the cells on individual 10-cm plates were harvested by vigorous pipetting and transferred to individual 10-cm plates with a
fresh OP9 cell layer, and then both adherent and nonadherent cells were harvested to obtain EryD after day 11.
Hematopoietic Colony Formation from Day 8 Hematopoietic Clusters.
To determine the differentiation capacity of bcl-x+/+ and
bcl-x/ ES cells, on day 8 of the differentiation induction 30 hematopoietic clusters were picked and transferred to semisolid culture medium containing IL-3 (50 U/ml) and EPO (2 U/ml),
which promote erythroid and myeloid cell growth. As previously
reported, the day 8 hematopoietic clusters have a clonal origin
and can differentiate into erythroid and various myeloid lineages
under these conditions (27). 5 d after transfer into this myeloid
permissive semisolid media, individual colonies were picked,
cytospin specimens were stained with May-Grunwald Giemsa,
and the emerged blood cells were typed.
Purification and Counting Viability of the Induced Cells.
More than
75% of the differentiation-induced cells between days 6 and 8 were EryP, and the same proportion of cells between days 11 and
13 were EryD. In some experiments, the purification of EryP and
EryD was carried out with metrizamide step gradient centrifugation. The cells were washed once with Tyrode's buffer containing 0.1% gelatin. 1-5 × 106 cells in 1 ml of the Tyrode's buffer
were layered on a step gradient of 2.0 ml of 30% wt/vol metrizamide (Nacalai Tesque) and 2.0 ml of 15% wt/vol metrizamide.
The cells were centrifuged at room temperature for 20 min at
400 g at the interface between the 15% metrizamide and the 30%
metrizamide. The cells remaining at this interface were collected
and washed three times with -MEM with 20% FCS. After the
purification, >98% of the cells were dianisidine-positive erythroid
cells, with a viability of 95-98%.
Hemoglobin-containing cells were confirmed with dianisidine
staining as reported previously (35). To examine EPO responsiveness (the experiment shown in Fig. 3), 3.0 × 105/ml dianisidine-positive differentiation-induced cells were cultured in 6-well
plates containing 20% FCS supplemented with -MEM in the
absence or presence of 2 U/ml EPO without the OP9 cell layer. The viability of the cells was examined using the trypan blue dye
exclusion method and calculated by counting >200 cells. May-Grunwald Giemsa staining of cytospin specimens was also carried
out to examine the morphological changes of apoptotic EryP.
The number of hemoglobin-containing cells and the percentage
of viable cells are reported as mean ± SD. The t test was used for
statistical analysis, using StatView software.
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 3.
Percentage of viable erythroid lineage
cells in the presence or absence of EPO. Purified
day 6.5 EryP (A and B) and purified day 11.5 EryP
(C and D) derived from bcl-x+/+ and bcl-x/
(clone 18) ES cells were cultured in the presence or
absence of EPO until the indicated day. The percentage of viable cells is shown as the mean ± SD
of six dishes. The data shown are representative of
three independent experiments. bcl-x/ (clone 3a)
ES cells gave results similar to bcl-x/ (clone 18)
ES cells.
|
|
Electrophoretic Analysis of DNA Fragmentation.
After culture for
18 h in the presence or absence of 2 U/ml EPO, 106 cells were
harvested by centrifugation at 200 g for 10 min. Low molecular
weight DNA was extracted following the method of Sellins and
Cohen (36). One quarter of the extracted DNA was electrophoresed in a 2.0% agarose gel and stained with ethidium bromide.
| |
Results |
No Contribution of bcl-x Null ES Cells to Circulating Adult
Definitive Erythrocytes.
ES cells of bcl-x+/+, bcl-x+/, and
bcl-x/ genotypes were injected into the blastocysts of
C57BL/6 mice to assess their ability to differentiate into
various organs in vivo. There were no differences in the
growth of parental bcl-x+/+, bcl-x+/, and bcl-x/ ES cells
(data not shown). Chimeric mice of >80% chimerism by coat color were analyzed for the contribution of the injected ES cells in various organs based on the activity of
GPI-1 isozymes. E14 ES cell-derived cells express the
GPI-1A isozyme, which is easily distinguishable from
the GPI-1B isozyme of the C57BL/6-derived cells (37). As
for heart, kidney, and muscle, there were no differences in
the contribution of parental bcl-x+/+, bcl-x+/, and bcl-x/
ES cells (Table I). On the other hand, the contribution of bcl-x/ ES cells to lymphoid organs such as spleen and thymus
was significantly lower than that of bcl-x+/+ or bcl-x+/ ES
cells. This result is compatible with a previous report on the
shortened life span of bcl-x/ immature lymphocytes (21).
Two bcl-x/ ES cell lines (clones 18 and 3a) were analyzed for the function of the bcl-x gene in hematopoiesis.
Host blastocysts from the strain C57BL/6 are homozygous
for the Hbbs 
-globin haplotype (corresponding to the
"single" band in Fig. 1, lane 1). In contrast, 129/Ola mice,
from which the ES cell line of this study was established,
are homozygous for the Hbbd haplotype (corresponding to
the "major" and "minor" bands in Fig. 1, lane 2). The proportion of major and minor hemoglobin shows the contribution of the injected ES cells to mature circulating EryD
in the chimeric mice. When bcl-x+/+ or bcl-x+/ ES cells
were used for chimera production, the contribution of the
ES cells to the circulating EryD was proportional to the
contribution of ES cells to the other organs. However,
when bcl-x/ ES cells were used, no contribution of the
ES cells to circulating EryD was detected, despite their significant contribution to the other nonlymphohematopoietic organs (Fig. 1). These data clearly show that bcl-x has an
essential role for the in vivo production of EryD. In addition, the results from the chimeric mice demonstrate that
the contribution of bcl-x to EryD production is cell autonomous, since the hematopoietic microenvironment in the
chimeric animal could not complement the defective EryD
production from bcl-x/ ES cells.
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Contribution of ES-derived cells to mature adult definitive
erythrocytes of chimeras. Hemoglobin was analyzed in the peripheral
blood of C57BL/6 mice (lane 1) and 129/Ola mice (lane 2). The Hbbs
haplotype (single) is specific for host blastocysts of strain C57BL/6, and
the Hbbd haplotype (diffuse; major and minor) is specific for strain 129/
Ola from which the ES cell line used in this study was established. Peripheral blood samples of chimeras made with bcl-x+/+ ES cells (lanes 3 and 4), bcl-x+/ ES cells (lanes 5 and 6), bcl-x/ ES cells, clone 18 (lanes
7-9), and bcl-x/ ES cells, clone 3a (lanes 10-12) were analyzed to examine the contribution of ES cells.
|
|
The process of definitive erythroid lineage cell production can be divided into two stages. The earlier stage is
commitment and involves differentiation from multipotential progenitor cells to committed erythroid lineage cells.
The later stage is proliferation and maturation of the committed EryD progenitors. Two possibilities might account
for the failure of EryD production by bcl-x/ ES cells.
One is the commitment failure of the multipotential hematopoietic progenitor cells into erythroid lineage cells, and the other is the proliferation and maturation failure of the
committed erythroid cells. To analyze these possibilities, in
vitro differentiation induction from bcl-x/ and bcl-x+/+
ES cells was carried out using OP9 stromal cells. The differentiation capacity of day 8 in vitro differentiation-induced
hematopoietic progenitor cells was examined. The day 8 hematopoietic clusters were of clonal origin, and most of
them could differentiate into multiple hematopoietic lineages, including the definitive erythroid lineage (27). There
were no differences in the number of day 8 hematopoietic
clusters induced from bcl-x+/+ or bcl-x/ ES cells (data not
shown). As shown in Table II, there were also no significant differences in the types of colonies that developed from the day 8 hematopoietic clusters in methylcellulose
semisolid media containing IL-3 and EPO as growth factors. These data show that bcl-x is not necessary for the differentiation of the definitive erythroid lineage from multipotential hematopoietic progenitors. Since Bcl-X is essential
for the production of fully mature EryD, bcl-x seemed to
play important roles during the maturation of EryD after
commitment to the erythroid lineage.
Number and Viability of the In Vitro Differentiation-induced
Erythroid Lineage Cells.
To further analyze the function of
bcl-x in the production of erythroid lineage cells, in vitro
differentiation induction into erythroid lineage cells from
bcl-x/ and bcl-x+/+ ES cells was carried out using OP9
stromal cells in the presence of EPO. The number of hemoglobin-containing dianisidine-positive cells was counted
between days 6 and 8 and between days 12 and 14. As previously reported, EryP and EryD appear in the former and
latter periods, respectively (29). On day 6, the number of
bcl-x/ EryP was the same as bcl-x+/+ EryP. However, on
day 8, the difference between the number of bcl-x/ EryP
and bcl-x+/+ EryP became pronounced. As shown in Table
III, on day 8 the number of bcl-x/ EryP was only ~10%
that of bcl-x+/+ EryP. Moreover, the number of bcl-x/
EryP on day 8 was ~10% that of the day 7 bcl-x/ EryP,
suggesting that cell death occurred between these days. Similar results were obtained with EryD. There was no difference in the number of bcl-x/ EryD and bcl-x+/+ EryD
on day 12. But, the difference became significant with maturation, and the number of EryD originating from bcl-x/
ES cells was about one quarter that from bcl-x+/+ ES cells
on day 14.
The percentage of viable cells was next examined, because apoptotic cell death of bcl-x/ erythroid cells was
suspected (Table IV). Here again, there were no significant
differences between the day 7 EryP and the day 12 EryD,
but the differences became significant thereafter. The percentages of viable cells mainly reflect viable erythroid cells, because the vast majority of the cells during differentiation induction belong to the erythroid lineage. More than 80%
of the cells harvested between days 7 and 8, and >90% of
the cells harvested between days 12 and 14 were EryP and
EryD, respectively, when the bcl-x+/+ ES cells were induced for differentiation. The percentage of viable bcl-x/
EryP seems relatively high for the very low number of bcl-x/ EryP (Table IV). This apparent discrepancy was probably due to the removal of dead bcl-x/ EryP by adherent
macrophages. Electron microscopic features at about day 8 of the differentiation induction showed macrophages with
prominently phagocytosed dead EryP (data not shown).
Cell Death of In Vitro Differentiation-induced Erythroid Lineage Cells.
The morphological features and DNA fragmentation of the induced cells were analyzed to confirm
that the decreased number and viability of bcl-x/ cells
were due to apoptosis. Immature EryP and EryD were purified by metrizamide density gradient separation on days 6 and 12 of the differentiation induction, respectively. At
these times, no differences in number and viability were
detectable between the bcl-x+/+ and the bcl-x/ erythroid
cells as shown above. Using this purification method, >98% of the purified cells were erythroid lineage cells and
their viability was 95-98%. These purified EryP and EryD
were cultured on OP9 cells for 2 d in the presence of EPO,
and the cells were harvested. Their morphological and molecular features were then examined. The bcl-x+/+ EryP
were viable and had mature morphology on day 8. In
contrast, the bcl-x/ EryP had fragmented nuclei with
clumped chromatin, suggestive of apoptosis. On day 14, the vast majority of the cells were enucleated mature EryD
when the bcl-x+/+ ES cells were induced for differentiation,
whereas enucleated EryD were rarely observed when the
bcl-x/ ES cells were induced. The hemoglobinized bcl-x/ EryD were mainly nucleated erythroblasts. Thus, it
was difficult to find viable, fully mature EryP and EryD on
days 8 and 14 of the differentiation induction of bcl-x/
ES cells, respectively, although immature EryP and EryD
were equally viable on days 6 and 12, respectively. Low
molecular weight DNA was extracted from the cells, and
agarose gel electrophoresis was carried out (Fig. 2). The
nucleosomal DNA ladder, which is characteristic of apoptotic cells, was observed to be significantly more abundant
in the bcl-x/ erythroid lineage cells than in the bcl-x+/+
erythroid lineage cells. These data clearly demonstrate that the bcl-x/ erythroid lineage cells underwent apoptosis
during the end stage of maturation.
View larger version (74K):
[in this window]
[in a new window]
|
Fig. 2.
Gel electrophoresis of low molecular weight DNA extracted from the cells after
culture of purified day 6 differentiation-
induced EryP and day 12 differentiation-induced
EryD in the presence of EPO for 48 h. DNA
extracted from the cells from the day 6 induced bcl-x+/+ EryP (lane 1), the day 6 induced
bcl-x/ EryP (lane 2), the day 12 induced bcl-x+/+ EryD (lane 3), and the day 12 induced
bcl-x/ EryD (lane 4).
|
|
Effects of Bcl-X and EPO on Apoptotic Cell Death.
EPO is
required by immature erythroid lineage cells to prevent apoptosis. To analyze the roles of EPO and Bcl-X during the maturation of erythroid cells, an EPO deprivation experiment was performed. On days 6.5 and 11.5 of the differentiation induction, immature EryP and EryD were purified.
These erythroid cells were not considered to be late-stage
erythroid progenitors, but rather immature erythroid cells,
because hemoglobinization had already begun but the cells
still showed an immature morphology. After purification
on days 6.5 and 11.5, the bcl-x+/+ and bcl-x/ erythroid
lineage cells were cultured without OP9 stromal cells in
the presence or absence of EPO. 1 and 1.5 d after the culture, the viability of the cells was examined (Fig. 3). Deprivation of EPO at this stage affected the viability of EryP
much more severely than EryD; however, the results of the
examination of EryP and EryD were essentially the same.
In the presence of EPO, there were significant differences
in the viability of bcl-x+/+ and bcl-x/ erythroid cells (P < 0.0005 by t test). Even in the absence of EPO, the differences in the viability were significant (P < 0.005 by t test).
Furthermore, EPO deprivation decreased the viability of
both EryP and EryD even in the context of bcl-x null (P < 0.0001 by t test). Taken together, EPO deprivation and the
bcl-x null mutation affected cell death additively.
| |
Discussion |
bcl-x, a member of the bcl-2 family of apoptosis regulatory genes, can be alternatively spliced to produce two protein isoforms, Bcl-XL and Bcl-XS (6, 17, 18). Bcl-XL exhibits remarkable structural homology with Bcl-2 and
inhibits apoptotic cell death. Evidence from studies of cell
lines and transgenic mice suggests that the bcl-2 gene family
plays a role in the survival of erythroid lineage (22, 23, 25).
The expression pattern of bcl-x obtained from primary human erythroid cells and mouse erythroblasts infected with
the anemia-inducing strain of Friend virus (FVA) suggests that bcl-x among bcl-2 gene family members is the principal
antiapoptotic regulator during late erythroid differentiation
(24). Bcl-X is strongly increased during the terminal differentiation stages of human and mouse erythroblasts in the
presence of EPO, reaching maximum transcript and protein levels at the time of maximum hemoglobin synthesis.
This increase in Bcl-X expression leads to an apparent level
~50 times greater than the level in proerythroblasts before EPO stimulation. In contrast, neither mouse nor human
erythroblasts express Bcl-2 transcript or protein. The levels
of other Bcl-2 family members, Bax and Bad proteins, remain relatively constant throughout differentiation, but diminish at the end of terminal differentiation near the time
of enucleation. These data on the expression pattern of the
bcl-2 gene family products imply that bcl-x is the critical
member of the bcl-2 family during erythroid differentiation.
Furthermore, the increased apoptotic cell death of hematopoietic cells in bcl-x/ fetal liver and the absence of defects
in the fetal liver of bcl-2/ mice support the hypothesis
that Bcl-X, not Bcl-2, is the important factor in erythropoiesis (21, 38). However, there is no direct evidence
for the role of bcl-x in erythropoiesis, despite this circumstantial evidence.
To examine the critical physiological roles of the bcl-x
gene on hematopoiesis, chimeric mice production and
OP9 in vitro differentiation induction were carried out using bcl-x/ ES cells. There was no contribution by bcl-x/
ES cells to the circulating EryD in the chimeric mice, demonstrating that bcl-x is indispensable for the full maturation
of EryD. Defects in erythropoiesis were analyzed in detail
using in vitro differentiation induction from ES cells by
coculturing the cells on the macrophage colony-stimulating factor-deficient OP9 stromal cell line (the OP9 system
[28]). Two waves of erythroid cell production were observed when ES cells were cocultured with OP9 stromal
cells. The development of hematopoietic cells in this OP9
system is very similar to that observed in developing mouse
embryos (27, 29, 41). The first wave of erythropoiesis, appearing between days 6 and 8 of the induction, and the
second wave, appearing after day 10 of the induction, correspond to primitive and definitive erythropoiesis, respectively, by morphological and biochemical criteria (29). Our
data clearly show that apoptotic cell death of bcl-x/ erythroid lineage cells was observed only at the end of maturation in both primitive and definitive erythropoiesis.
bcl-x-deficient mice die at about embryonic day 13 (21).
Extensive apoptotic cell death is evident in hematopoietic
cells in fetal liver. There is a threefold increase in TUNEL
(for terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling)-labeled apoptotic nuclei in histologically
identifiable hematopoietic cells in embryonic day 12.5 bcl-x/ liver compared with wild-type tissue. These data suggest that erythropoiesis in the fetal liver of bcl-x/ mice is
impaired because the vast majority of fetal liver hematopoietic cells at this gestational stage are erythroid lineage cells.
The data on defective EryD production by the OP9 system
are consistent with these in vivo data. During in vitro
differentiation, although significant numbers of bcl-x/
erythroblasts survived, almost no enucleated erythrocytes
could be detected. This result shows that the pivotal function of bcl-x is expressed at the late stage of erythroid maturation. The critical role of bcl-x seems to be brought about
by the remarkable increase of Bcl-X protein at the end of
erythroid maturation. In vitro differentiation induction
shows that apoptotic cell death of EryP also occurred at the
late stage of maturation, which is consistent with primitive
erythropoiesis in the bcl-x/ mice. The effect of bcl-x on
primitive erythropoiesis was not examined extensively because of the difficulty counting EryP numbers correctly in
tiny mouse embryos. However, the following two lines of
evidence strongly suggest that EryP production in bcl-x/
mice was impaired to some extent. First, bcl-x/ mice
were paler than the control mice at day 12.5 of gestation (21; our unpublished data). At this gestational stage, >95%
of the erythrocytes are still EryP, although the relative percentage of EryP begins to decrease (42). Second, bcl-x/
mice died at day 13 of gestation, which is earlier than the
mutant mice lacking only definitive hematopoiesis by gene
targeting of c-myb (43). The c-myb targeted mice were severely anemic by day 15; however, the mutant mice appeared normal at day 13 of gestation. On the other hand,
EPO signal-deficient mice, which have a partial defect in
primitive erythropoiesis and a complete defect in definitive
erythropoiesis, die at day 13 of gestation, as early as bcl-x/
mice (12, 13). Meanwhile, it is reasonable to consider that a
similar time course of cell death of bcl-x/ EryP and bcl-x/ EryD would reflect a similar underlying molecular
mechanism of cell death caused by the null mutation of
bcl-x. The cause of cell death might be explained by the relationship between massive heme synthesis at the end of
maturation of erythroid lineage cells and the antioxidant
function of Bcl-XL (24).
Of the various methods of in vitro hematopoietic differentiation from ES cells, the OP9 system has several remarkable advantages, among which are their potential to differentiate into fully mature blood cells and the feasibility of
analyzing the cells quantitatively (26, 27). To analyze the
defective erythropoiesis from bcl-x/ ES cells, quantitative
analysis of the fully mature erythroid cells was necessary.
However, such analysis is almost impossible by the conventional in vitro differentiation induction method with embryoid body formation. The other substantial advantage of
the OP9 system is that hematopoietic microenvironment
and hematopoietic cells can be analyzed separately by this
method. It is well known that hematopoiesis is maintained
by the hematopoietic microenvironment, such as stromal
cells. By the conventional embryoid body formation method, both hematopoietic microenvironment and hematopoietic cells are induced from ES cells and are unseparable.
But with the OP9 system, hematopoietic cells are induced
from ES cells while the hematopoietic microenvironment
is provided by OP9 stromal cells. It is concluded from the
defective EryD production in the chimeric mice that this
defect is cell autonomous. In addition, the defective erythropoiesis of the bcl-x/ genotype with the OP9 system strongly
supports this conclusion.
The production of definitive erythroid lineage cells is controlled by EPO (11). EPO induces the proliferation and prevents the apoptotic cell death of EryD. The antiapoptotic
effect of EPO on EryD was observed from late erythroid
progenitors (CFU-E) until the onset of hemoglobinization
(2, 14). In other words, EPO-deprived apoptotic cell
death is hardly at all observed at the end of maturation
when maximal hemoglobin synthesis occurs. On the other
hand, massive apoptotic cell death of bcl-x/ EryD was
observed after day 13 of differentiation induction. It is reasonable to consider that the accumulation of Bcl-X (probably Bcl-XL) resulting from EPO stimulation prevents the
apoptotic cell death of terminally differentiated erythroid
cells. However, the accumulation of Bcl-X cannot be the
only way to explain the antiapoptotic effect of EPO, because EPO prevents apoptotic cell death to some extent
even in the absence of Bcl-X (Fig. 3). Taken together, it is
likely that EPO has dual roles to prevent apoptotic cell
death at different differentiation stages.
Address correspondence to Toru Nakano, Department of Molecular Cell Biology, Research Institute for
Microbial Diseases, Osaka University, Yamada-Oka 3-1, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-8361; Fax: 81-6-6879-8362; E-mail: tnakano{at}biken.osaka-u.ac.jp
The authors thank Dr. Yoshihide Tsujimoto for discussions, and Kirin Brewery Co. Ltd. for their kind gift
of human recombinant EPO.
This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, the
Research for the Future Program of Japanese Society for Promotion of Sciences (JSPS-RFTF98L01101), the Naito Memorial Foundation, and the Novartis Foundation (Japan) for the Promotion of Science.
| 1.
|
Koury, M.J..
1992.
Programmed cell death (apoptosis) in hematopoiesis.
Exp. Hematol
20:
391-394
[Medline].
|
| 2.
|
Koury, M.J., and
M.C. Bondurant.
1990.
Erythropoietin retards DNA breakdown and prevents programmed death in
erythroid progenitor cells.
Science.
248:
378-381
[Abstract/Free Full Text].
|
| 3.
|
Williams, G.T.,
C.A. Smith,
E. Spooncer,
T.M. Dexter, and
D.R. Taylor.
1990.
Haemopoietic colony stimulating factors
promote cell survival by suppressing apoptosis.
Nature.
343:
76-79
[Medline].
|
| 4.
|
Collins, M.K.,
G.R. Perkins,
G. Rodriguez-Tarduchy,
M.A. Nieto, and
A. Lopez-Rivas.
1994.
Growth factors as survival
factors: regulation of apoptosis.
Bioessays.
16:
133-138
[Medline].
|
| 5.
|
Thompson, C.B..
1995.
Apoptosis in the pathogenesis and
treatment of disease.
Science.
267:
1456-1462
[Abstract/Free Full Text].
|
| 6.
|
Yang, E., and
S.J. Korsmeyer.
1996.
Molecular thanatopsis: a
discourse on the BCL2 family and cell death.
Blood.
88:
386-401
[Free Full Text].
|
| 7.
|
Chao, D.T., and
S.J. Korsmeyer.
1998.
Bcl-2 family: regulators of cell death.
Annu. Rev. Immunol.
16:
395-419
[Medline].
|
| 8.
|
Adams, J.A., and
S. Cory.
1998.
The Bcl-2 protein family:
arbiters of cell survival.
Science.
281:
1322-1326
[Abstract/Free Full Text].
|
| 9.
|
Nunez, G., and
M.F. Clarke.
1994.
The Bcl-2 family of
proteins: regulators of cell death and survival.
Trends Cell
Biol.
4:
399-402
.
[Medline] |
| 10.
| Rugh, R. 1968. The Mouse. Oxford University Press, Oxford.
|
| 11.
|
Ihle, J.N.,
F.W. Quelle, and
O. Miura.
1993.
Signal transduction through the receptor for erythropoietin.
Semin. Immunol.
5:
375-389
[Medline].
|
| 12.
|
Wu, H.,
X. Liu,
R. Jaenisch, and
H.F. Lodish.
1995.
Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor.
Cell.
83:
59-67
[Medline].
|
| 13.
|
Lin, C.-S.,
S.-K. Lim,
V. D'Agati, and
F. Costantini.
1996.
Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.
Genes Dev.
10:
154-164
[Abstract/Free Full Text].
|
| 14.
|
Spivak, J.L.,
T. Pham,
M. Isaacs, and
W.D. Hankins.
1991.
Erythropoietin is both a mitogen and a survival factor.
Blood.
77:
1228-1233
[Abstract/Free Full Text].
|
| 15.
|
Yu, H.,
B. Bauer,
G.K. Lipke,
R.L. Phillips, and
G. Van
Zant.
1993.
Apoptosis and hematopoiesis in murine fetal
liver.
Blood.
81:
373-384
[Abstract/Free Full Text].
|
| 16.
|
Koury, M.J.,
L.L. Kelley, and
M.C. Bondurant.
1994.
The fate
of erythroid progenitor cells.
Ann. NY Acad. Sci
718:
259-267
[Medline].
|
| 17.
|
Boise, L.H.,
M. Gonzalez-Garcia,
C.E. Postema,
L. Ding,
T. Lindsten,
L.A. Turka,
X. Mao,
G. Nunez, and
C.B. Thompson.
1993.
bcl-x, a bcl-2-related gene that functions as a
dominant regulator of apoptotic cell death.
Cell.
74:
597-608
[Medline].
|
| 18.
|
Gonzalez-Garcia, M.,
R. Perez-Ballestero,
L. Ding,
L. Duan,
L.H. Boise,
C.B. Thompson, and
G. Nunez.
1994.
bcl-XL is
the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria.
Development.
120:
3033-3042
[Abstract].
|
| 19.
|
Vaux, D.L.,
S. Cory, and
J.M. Adams.
1988.
Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc
to immortalize pre-B cells.
Nature.
335:
440-442
[Medline].
|
| 20.
|
Nunez, G.,
L. London,
D. Hockenbery,
M. Alexander,
J.P. McKearn, and
S.J. Korsmeyer.
1990.
Deregulated Bcl-2 gene
expression selectively prolongs survival of growth factor-
deprived hemopoietic cell lines.
J. Immunol.
144:
3602-3610
[Abstract].
|
| 21.
|
Motoyama, N.,
F. Wang,
K.A. Roth,
H. Sawa,
K. Nakayama,
K. Nakayama,
I. Negishi,
S. Senju,
Q. Zhang,
S. Fujii, et al
.
1995.
Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice.
Science.
267:
1506-1510
[Abstract/Free Full Text].
|
| 22.
|
Silva, M.,
D. Grillot,
A. Benito,
C. Richard,
G. Nunez, and
J.L. Fernandez-Luna.
1996.
Erythropoietin can promote
erythroid progenitor survival by repressing apoptosis through
Bcl-XL and Bcl-2.
Blood.
88:
1576-1582
[Abstract/Free Full Text].
|
| 23.
|
Benito, A.,
M. Silva,
D. Grillot,
G. Nunez, and
J.L. Fernandez-Luna.
1996.
Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL.
Blood.
87:
3837-3843
[Abstract/Free Full Text].
|
| 24.
|
Gregoli, P.A., and
M.C. Bondurant.
1997.
The roles of Bcl-X(L) and apopain in the control of erythropoiesis by erythropoietin.
Blood.
90:
630-640
[Abstract/Free Full Text].
|
| 25.
|
Lacronique, V.,
P. Varlet,
P. Mayeux,
A. Porteu,
S. Gisselbrecht,
A. Kahn, and
C. Lacombe.
1997.
Bcl-2 targeted
overexpression into the erythroid lineage of transgenic mice
delays but does not prevent the apoptosis of erythropoietin-deprived erythroid progenitors.
Blood.
90:
3050-3056
[Abstract/Free Full Text].
|
| 26.
|
Wiles, M.V..
1993.
Embryonic stem cell differentiation in
vitro.
Methods Enzymol
225:
900-918
[Medline].
|
| 27.
|
Nakano, T.,
H. Kodama, and
T. Honjo.
1994.
Generation of
lymphohematopoietic cells from embryonic stem cells in culture.
Science.
265:
1098-1101
[Abstract/Free Full Text].
|
| 28.
|
Nakano, T..
1995.
Lymphohematopoietic development from
embryonic stem cells in vitro.
Semin. Immunol.
7:
197-203
[Medline].
|
| 29.
|
Nakano, T.,
H. Kodama, and
T. Honjo.
1996.
Development
of primitive and definitive erythrocytes from different precursors in culture.
Science.
272:
722-724
[Abstract].
|
| 30.
|
Weiss, M.J., and
S.H. Orkin.
1996.
In vitro differentiation of
murine embryonic stem cells. New approaches to old problems.
J. Clin. Invest
97:
591-595
[Medline].
|
| 31.
| Wurst, W., and A.L. Joyner. 1993. Production of targeted
embryonic stem cell clones. In Gene Targeting: A Practical
Approach. A.L. Joyner, editor. IRL Press at Oxford University Press, Oxford. 33-61.
|
| 32.
| Bradley, A. 1987. Production and analysis of chimaeric mice. In
Teratocarcinomas and Embryonic Stem Cells. E.J. Robertson,
editor. IRL Press at Oxford University Press, Oxford. 113-152.
|
| 33.
|
Williams, B.O.,
E.M. Schmitt,
L. Remington,
R.T. Bronson,
D.M. Albert,
R.A. Weinberg, and
T. Jacks.
1994.
Extensive contribution of Rb-deficient cells to adult chimeric
mice with limited histopathological consequences.
EMBO
(Eur. Mol. Biol. Organ.) J
13:
4251-4259
[Medline].
|
| 34.
|
Kodama, H.,
M. Nose,
S. Niida,
S. Nishikawa, and
S.-I. Nishikawa.
1994.
Involvement of the c-kit receptor in the
adhesion of hematopoietic stem cells to stromal cells.
Exp.
Hematol.
22:
979-984
[Medline].
|
| 35.
|
Cooper, M.C.,
J. Levy,
L.N. Cantor,
P.A. Marks, and
R.A. Rifkind.
1974.
The effect of erythropoietin on colonial
growth of erythroid precursor cells in vitro.
Proc. Natl. Acad.
Sci. USA.
71:
1677-1680
[Abstract/Free Full Text].
|
| 36.
|
Sellins, K.S., and
J.J. Cohen.
1987.
Gene induction by -irradiation leads to DNA fragmentation in lymphocytes.
J. Immunol.
139:
3199-3206
[Abstract].
|
| 37.
| Hogan, B., R. Beddington, F. Costantini, and E. Lacy. 1994. Manipulating the Mouse Embryo. 2nd ed. Cold Spring Harbor
Laboratory Press, Plainview, NY. 376-377.
|
| 38.
|
Veis, D.J.,
C.M. Sorenson,
J.R. Shutter, and
S.J. Korsmeyer.
1993.
Bcl-2-deficient mice demonstrate fulminant lymphoid
apoptosis, polycystic kidneys, and hypopigmented hair.
Cell.
75:
229-240
[Medline].
|
| 39.
|
Nakayama, K.,
I. Negishi,
K. Kuida,
H. Sawa, and
D.Y. Loh.
1994.
Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.
Proc. Natl. Acad. Sci. USA.
91:
3700-3704
[Abstract/Free Full Text].
|
| 40.
|
Kamada, S.,
A. Shimono,
Y. Shinto,
T. Tsujimura,
T. Takahashi,
T. Noda,
Y. Kitamura,
H. Kondoh, and
Y. Tsujimoto.
1995.
bcl-2 deficiency in mice leads to pleiotropic abnormalities: accelerated lymphoid cell death in thymus and spleen,
polycystic kidney, hair hypopigmentation, and distorted small
intestine.
Cancer Res.
55:
354-359
[Abstract/Free Full Text].
|
| 41.
|
Era, T.,
T. Takahashi,
K. Sakai,
K. Kawamura, and
T. Nakano.
1997.
Thrombopoietin enhances proliferation and differentiation of murine yolk sac erythroid progenitors.
Blood.
89:
1207-1213
[Abstract/Free Full Text].
|
| 42.
|
Brotherton, T.W.,
D.H. Chui,
J. Gauldie, and
M. Patterson.
1979.
Hemoglobin ontogeny during normal mouse fetal development.
Proc. Natl. Acad. Sci. USA.
76:
2853-2857
[Abstract/Free Full Text].
|
| 43.
|
Mucenski, M.L.,
K. McLain,
A.B. Kier,
S.H. Swerdlow,
C.M. Schreiner,
T.A. Miller,
D.W. Pietryga,
W.J. Scott Jr., and
S.S. Potter.
1991.
A functional c-myb gene is required
for normal murine fetal hepatic hematopoiesis.
Cell.
65:
677-689
[Medline].
|
Top
Abstract
Introduction
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
