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HEMATOPOIESIS
From the Departments of Clinical Oncology, Molecular
and Developmental Biology, and Molecular Medicine, Division of DNA
Biology and Embryo Engineering, Center for Experimental Medicine, The
Institute of Medical Science, The University of Tokyo, Japan;
Department of Pediatrics, Kochi Medical School, Nankoku, Japan; the
Department of Pediatrics, Graduate School of Medicine, Kyoto
University, Japan; and the Howard Hughes Medical Institute and
Department of Molecular and Medical Pharmacology, University of
California at Los Angeles School of Medicine.
Although erythropoietin (EPO) and its receptor (EPOR) are crucial
for the proliferation, survival, and terminal differentiation of
erythroid progenitors, it remains to be elucidated whether EPOR-unique
signaling is required for erythropoiesis. To address this issue, human
granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor
(hGMR)-transgenic mice and heterozygous EPOR mutant mice were crossed
by in vitro fertilization. In methylcellulose clonal culture of fetal
liver (FL) cells of generated hGMR-expressing EPOR Erythropoietin (EPO) is a lineage-restricted
cytokine and plays a central role in the proliferation,1,2
differentiation,3,4 and survival5 of
erythroid progenitors. It exerts its function through EPO receptor
(EPOR), a member of the class I cytokine receptor family.6
Disruption of EPO or the EPOR gene in mice leads to embryonic death at
embryonic day (E) 11 to 13.5 of gestation owing to a defect of fetal
liver (FL) definitive erythropoiesis.7-9 Even in the
absence of EPO or EPOR, FL contains normal numbers of erythroid
burst-forming units (BFU-Es) and erythroid colony-forming units
(CFU-Es), indicating that EPO and EPOR are not required for the
commitment of hematopoietic stem cells to erythroid lineage, but
support progenitor cell survival and expansion beyond the CFU-E stage
in definitive erythropoiesis.7,9 On the other hand,
primitive erythropoiesis, which is the first erythropoiesis to occur in
the developing mouse embryo, takes place in blood islands of E7 yolk
sac (YS).10 Primitive erythropoiesis yields unique
erythrocytes, distinguishable from those in definitive erythropoiesis
by their morphology and the hemoglobin types they express. Primitive
erythrocytes are nucleated cells containing embryonic as well as adult
hemoglobins, whereas definitive erythrocytes are smaller nonnucleated
red cells committed only to adult hemoglobin synthesis. In
EPO We previously generated transgenic (Tg) mice expressing human
granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptors (hGMRs) consisting of ligand-binding Generation of hGMR-expressing
EPOR Cell preparation
Analysis of expression of hGMR and EPOR in embryos Genomic DNA was prepared from embryos removed from YS or FL by standard methods, to distinguish expression of hGMR , hGMR , and
mouse EPOR. The PCR primer sets used are shown in Table
1. PCR was performed in PCR buffer, 200 µM each deoxynucleotide, 300 nM each primer and 2 U/mL Taq polymerase in a final volume of 15 µL. The reaction profiles
were 28 cycles at 94°C for 30 seconds, 58°C for 40 seconds, and
72°C for 50 seconds for hGMR and hGMR; and 25 cycles at 94°C
for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes for EPOR. A
Gene Amp PCR System 2400 (Perkin-Elmer, Wellesley, MA) was
used. PCR products were run on a 1.2% agarose gel and made visible by
ethidium bromide (0.5 µg/mL) staining.
In vitro culture Methylcellulose clonal culture was prepared by means of a modification of a technique reported previously.14 Briefly, 1 mL culture medium containing 1 × 104 E12.5 FL cells,MEM, 1.2% methlycellulose (Shinetsu Chemical, Tokyo, Japan),
30% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT), 1%
deionized fraction V bovine serum albumin (BSA) (Sigma Chemical, St
Louis, MO), 100 µM mercaptoethanol (Eastman Organic Chemicals,
Rochester, NY), and cytokines was plated in 35-mm suspension culture
dishes (#1008, Becton Dickinson Labware, Franklin Lakes, NJ).
In a culture of E8.0 YS cells, 1 mL culture medium contained one half
of whole YS cells obtained from one embryo because of a small quantity
of available YS cells. Serum-free culture was prepared as
described elsewhere.15 The culture medium contained 1%
deionized crystallized globulin-free BSA (Calbiochem-Behring, La Jolla,
CA), 300 µg/mL fully iron-saturated human transferrin (Sigma
Chemical, St Louis, MO), 96 µg/mL cholesterol (Nacalai Tesque, Kyoto, Japan), and 100 nM sodium selenite (Sigma Chemical), instead of FBS and fraction V BSA. The dishes were incubated at 37°C
in a humidified atmosphere flushed with 5% O2 and 5%
CO2. Colony formation was monitored at appropriate times:
day 3 for erythroid colonies and day 7 for erythroid bursts,
erythrocyte-containing mixed hematopoietic colonies, and nonerythroid
colonies such as granulocyte-macrophage and mast cell colonies. To
assess the accuracy of in situ identification of the colonies,
individual colonies were lifted by means of an Eppendorf micropipette
under direct microscopic visualization, spread on glass slides with the
use of a cytocentrifuge (Cytospin 2; Shandon, Pittsburgh, PA), and then
stained with May-Grunwald-Giemsa (Muto Chemica, Tokyo, Japan). E8.0 YS
cells or E12.5 FL cells were also incubated in suspension culture as
reported previously.14 One milliliter of culture mixture
containing 1 × 106 cells per milliliter of E12.5 FL
cells, one third of whole E8.0 YS cells obtained from one embryo or
their progenies in the culture, MEM, 10% FBS, 1% deionized
fraction V BSA, 100 µM mercaptoethanol, and designated cytokines was
incubated in 12-well tissue plates (Nunc, Roskilde, Denmark) for 3 days
at 37°C in a humidified atmosphere flushed with 5% O2
and 5% CO2.
Cytokines used in the present study were human EPO, hGM-CSF, mouse stem cell factor (SCF), and mouse IL-3, which were kindly provided by Kirin Brewery (Tokyo, Japan). All the cytokines were pure recombinant molecules and were used at concentrations that induced an optimal response in methylcellulose clonal culture of murine adult bone marrow cells. These concentrations are 2 U/mL for human EPO, 100 ng/mL for mouse SCF, and 20 ng/mL for mouse IL-3.15-17 To determine the optimal concentration of hGM-CSF, methylcellulose culture was prepared with various concentrations (80, 10, 10/8, 10/82, 10/83, and 0 ng/mL). Analysis of adult and embryonic globin chains and transcription factors Poly(adenylic acid) (poly[A]) messenger RNA (mRNA) was prepared from single colonies harvested on day 7 of methylcellulose culture or E8.0 YS and E12.5 FL mouse embryo cells stimulated by hGM-CSF or EPO for 3 days, by means of a commercial kit (QuickPrep Micro mRNA Purification Kit, Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). For RT-PCR, first-strand complementary DNA (cDNA) synthesis with the use of random hexamer pd(N)6 (Pharmacia Biotech, Cambridge, United Kingdom) and Molony murine leukemia virus reverse transcriptase first-strand cDNA synthesis kit (Toyobo Biochemicals, Tokyo, Japan) was carried out as described elsewhere,18 with the use of poly(A) mRNA isolated from single colonies or cultured cells. The PCR reaction mixtures contained quantities of cDNA equivalent to 1 × 10 6, 105,
104, 103, 102, and
101 of those derived from 1 × 105
hGMR-expressing EPOR/ or EPOR+/+ FL cells,
one third of whole YS cells obtained from one hGMR-expressing EPOR+/+ mouse embryos or their progenies in the culture.
The PCR reaction was carried out with the use of the primer sets shown
in Table 1. Reactions were controlled by including amplification
primers for mouse -actin, and were incubated at 94°C for 30 seconds, 55-65°C for 1 minute, and 72°C for 2 minutes. PCR products
were run on a 1.5% agarose gel and made visible by ethidium bromide (0.5 µg/mL) staining.
Colony formation by hGM-CSF and EPO
from E12.5 FL cells of
hGMR-expressing EPOR (hGMR+/ hGMR+/)EPOR/;
12 hGMR+/EPOR+/; 6 hGMR+/EPOR+/+; 4 hGMR/(hGMR/hGMR/)EPOR/;
13 hGMR/EPOR+/; and 5 hGMR/EPOR+/+ embryos. We then isolated
E12.5 FL cells from EPOR+/+hGMR/,
EPOR+/+hGMR+/,
EPOR/hGMR/, and
EPOR/hGMR+/ embryos. FL cells of
EPOR+/+ embryos contained both nucleated primitive
erythrocytes and nonnucleated definitive erythrocytes, but most of the
erythrocytes in FL cells of EPOR/ embryos were
nucleated primitive erythrocytes.
We carried out methylcellulose culture of 1 × 104 E12.5
FL cells with EPO (2 U/mL) or hGM-CSF (10 ng/mL) (Table
2; Figure 1A). EPO, but not hGM-CSF, stimulated
erythroid colony formation in EPOR+/+hGMR
We further carried out serum-free methylcellulose clonal culture of
hGMR+/ Globin gene expression in erythrocyte-containing colonies formed
from hGMR-expressing EPOR+/+
or EPOR / FL
cells by hGM-CSF originate from primitive or definitive hematopoiesis. We then randomly chose 4 erythroid bursts/erythrocyte-containing mixed
colonies formed from hGMR-expressing EPOR+/+ E12.5 FL cells
by hGM-CSF and analyzed the expression of globins in each colony
(Figure 3A, lanes 1 to 4). While 1 colony
(lane 2) expressed both - and -major globins, adult globins, and
the embryonic globin H1, 3 colonies (lanes 1, 3, and 4) expressed - and -major globins, but no or little H1 globin. We also
analyzed 4 erythroid bursts/erythrocyte-containing mixed colonies
formed from hGMR-expressing EPOR/ E12.5 FL cells by
hGM-CSF (Figure 3A, lanes 5 to 8). Three colonies (lanes 5 to 7)
expressed both adult and embryonic globins, and one colony expressed
adult globins and a low level of H1 globin. This result indicates
that definitive erythroid progenitors are present even in
EPOR/ embryos, although mature erythrocytes were
undetectable, in accordance with previous reports,7-9 and
that hGM-CSF stimulates both definitive and primitive erythroid
progenitors lacking EPOR but expressing hGMR.
Colony formation by hGM-CSF and EPO from E8.0 YS cells of mouse embryos expressing hGMR To compare the effects of hGM-CSF and EPO on primitive erythropoiesis in hGMR-expressing mouse embryos, we carried out the methylcellulose clonal culture of E8.0 YS cells with hGM-CSF or EPO (Table 3). The hGM-CSF and EPO stimulated the formation of similar numbers of erythroid bursts and erythrocyte-containing mixed colonies. RT-PCR showed that all 4 erythroid bursts/erythrocyte-containing mixed colonies formed by hGM-CSF and chosen randomly contained both adult and embryonic globins, confirming that these colonies were derived from primitive erythropoiesis (Figure 3B).
Globin gene expression in E12.5 FL and E8.0 YS cells of hGMR-Tg mice To examine the effects of hGM-CSF and EPO on the differentiation of embryonic erythroid cells, we analyzed the adult globins- and
-major globins and the embryonic globins, H1-  - and  -globins by semiquantitative RT-PCR using E12.5 FL cells of
hGMR-expressing EPOR+/+ and EPOR/ embryos.
Representative results are presented in Figure
4. The - and -major globin mRNAs
were detected at higher levels in hGMR-expressing EPOR+/+
than in EPOR/ FL cells, and embryonic globin levels
were similar or slightly higher in EPOR+/+ FL cells. When
hGMR-expressing EPOR+/+ FL cells were stimulated by EPO or
hGM-CSF for 3 days in suspension culture, a similar change in the
globin expression was observed. The expression of - and -major
globins showed no remarkable changes or was slightly higher as compared
with unstimulated cells, but embryonic globins decreased. When
hGMR-expressing EPOR/ FL cells were stimulated by
hGM-CSF for 3 days, the increase of - and -major globin RNA
levels and the decrease of embryonic globin RNA levels were also
observed. This result indicated that hGM-CSF, as well as EPO, induces
adult globin expression, but suppresses embryonic globin expression in
FL erythropoiesis of hGMR-Tg mouse embryo.
As shown in Figure 3A, however, E12.5 FL cells of hGMR-expressing mouse
embryo contained both primitive and definitive erythroid progenitors
whether they expressed EPOR or not. Therefore, to examine the effect of
hGM-CSF on globin expression by primitive erythroid cells in more
detail, we analyzed the expression of globins in E8.0 YS cells of
hGMR-expressing EPOR+/+ embryos (Figure
5). YS cells expressed all of the globins
examined, including adult globin. When YS cells were cultured with
hGM-CSF or EPO for 3 days, they induced the expression of globins
in a different fashion. The expression of
Gene expression of transcription factors in FL cells of hGMR-Tg mice We also examined the effects of hGM-CSF and EPO on the expression of GATA-1, erythroid Kruppel-like factor (EKLF), and cMyb in E12.5 FL cells of hGMR-expressing EPOR+/+ and EPOR/
embryos by semiquantitative RT-PCR (Figure
6). The expression of GATA-1 in
hGMR-expressing EPOR/ and EPOR+/+ FL cells
was similar, but the expressions of EKLF and cMyb were higher in
EPOR+/+ embryos. EPO and hGM-CSF showed a similar effect on
the expression of transcription factors in hGMR-expressing
EPOR+/+ FL cells. Both induced an increase of GATA-1 and a
decrease of cMyb. The EKLF expression slightly decreased. When
hGMR-expressing EPOR/ FL cells were stimulated by
hGM-CSF for 3 days, the expression of GATA-1 and EKLF increased, but
that of cMyb decreased.
We previously showed that hGM-CSF can stimulate
colony formation from erythroid progenitors in adult bone marrow of
Tg mice expressing hGMR.11
However, it remains unclear whether the activity of hGM-CSF in this
process is mediated by signaling through EPOR. Stopka et
al21 demonstrated that erythroid bursts derived from human
peripheral blood or bone marrow cells were positive for EPO mRNA,
suggesting that erythroid cells proliferate and differentiate in an
autocrine manner. It was also reported that With regard to this issue, 2 experiments were recently conducted.
Socolovsky et al25 have reported that a retrovirally
transduced prolactin receptor efficiently supports the differentiation
of BFU-E and CFU-E progenitors in FL of EPOR When E8.0 YS and E12.5 FL cells of hGMR-expressing embryo were cultured
with hGM-CSF, various types of colonies were formed in accordance with
our previous report that hGM-CSF stimulates various lineages of
hematopoietic progenitors in adult bone marrow.11 These
results indicate that hGM-CSF functions as a proliferation-promoting factor of hematopoietic progenitors as long as they express hGMR in
adult and embryonic hematopoiesis. In particular, analysis of globin
types expressed in individual erythrocyte-containing colonies formed
from hGMR-expressing EPOR We evaluated the differentiation of erythroid cells by examining the
expression of globins. Kieran et al8 showed the absence of
The differentiation of erythroid cells was also evaluated on the basis
of the expression of transcription factors. It was shown that RNA
encoding GATA-1 and EKLF was expressed normally in
EPOR All together, the results of present study demonstrated that hGM-CSF
can stimulate the proliferation and differentiation of primitive and
definitive erythroid progenitors independent of EPOR signal if they
express hGMR. The prolactin receptor and MPL also have been
demonstrated to promote the growth of EPOR
Submitted October 26, 2000; accepted July 25, 2001.
Supported by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kohichiro Tsuji, Department of Clinical Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: tsujik{at}ims.u-tokyo.ac.jp.
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Abstract
Introduction
/