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HEMATOPOIESIS
From the Institut National de la Santé et de la
Recherche Médicale (INSERM) U506, Hôpital Paul Brousse,
Villejuif, France; INSERM U238, Centre d'Etudes Atomique,
Grenoble, France; INSERM U363, Hôpital Cochin and INSERM U344,
Hôpital Necker, Paris, France.
The Mpl receptor plays an important role at the level of adult
hematopoietic stem cells, but little is known of its function in
embryonic and fetal hematopoiesis. We investigated the signals sent by
the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and
during embryonic stem (ES) cell hematopoietic commitment. Mpl was found
to be expressed only from day 6 of ES cell differentiation into
embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES
cells or in fetal progenitors and studied the effects on
hematopoietic differentiation. To avoid the inadvertent effect
of thrombopoietin, we used a chimeric receptor, PM-R,
composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in
hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver
hematopoietic progenitors as compared with that of EPO-R and that it
promotes hematopoietic commitment of ES cells. Our findings demonstrate
for the first time the specific role of Mpl in early embryonic
or fetal hematopoietic progenitors and stem cells.
(Blood. 2002;100:2063-2070) Mpl is the receptor for thrombopoietin (TPO), the
primary regulator of megakaryocytopoiesis and platelet
production.1,2 TPO controls the number and ploidy
of megakaryocytes and supports their full maturation into
platelet-producing cells.1-3 TPO also stimulates the
development of megakaryocyte colonies from marrow progenitors in
vitro,4 but several reports indicate that TPO, like other
hematopoietic growth factors, has a broader range of activities. TPO
has been shown to enhance erythroid cell proliferation5-7 and to accelerate red blood cell recovery if administered after myelosuppressive therapy.6,8,9 Furthermore, TPO
administration led to an expansion of colony-forming units
granulocyte-macrophage (CFU-GMs) and burst-forming units-erythroid
(BFU-Es) in normal mice8 and to an increase of CFU-mix in
rhesus monkeys.10 More recently, several studies have
shown that TPO alone or in combination with early-acting growth factors
can stimulate ex vivo expansion of early hematopoietic progenitors,
which maintain their capacity for multilineage
differentiation.11-15 The phenotype of c-mpl- and
TPO-deficient mice was also consistent with a role of TPO and its
receptor on early hematopoietic progenitors. Indeed, in addition to the
expected thrombocytopenia and defect in megakaryocytes and
megakaryocytic progenitors, these mice had a reduced number of myeloid
progenitors16,17 and a dramatic shortage in hematopoietic stem cells.18 Clues as to the role of Mpl and TPO in
primitive hematopoietic progenitors may have come from the initial
biologic properties of myeloproliferative leukemia
virus (MPLV): in this virus, the truncated form of the Mpl receptor,
v-mpl, which contains the transmembrane and cytoplasmic domains and
only 43 amino acids of the extracellular domain, was able to promote
the proliferation and terminal differentiation of both committed and
multipotential stem cells.19,20
If the role of Mpl within adult hematopoietic stem cells is now well
established, nothing is known about their embryonic or fetal
counterparts. Therefore, we decided to investigate signals transduced
by the Mpl cytoplasmic domain first in fetal liver hematopoietic
progenitors and then during embryonic stem (ES) cells hematopoietic
commitment. In the in vitro model of hematopoietic differentiation of
ES cells, Mpl was found to be expressed only from day 6 of
differentiation in embryoid bodies (EBs).21
Accordingly, we expressed Mpl in undifferentiated ES cells and studied
the consequences of this early expression on hematopoietic
differentiation. To prevent the unwanted effect of TPO during cell
culture, in both models we used a chimeric receptor, PRL-R/MPL
(thereafter named PM-R), composed of the extracellular domain of PRL
receptor and the transmembrane and cytoplasmic domain of Mpl. By using the cytoplasmic and transmembrane domains of c-mpl in the chimeric PM-R
receptor, it allowed us to study a kind of activatable v-mpl receptor,
which could be triggered by a hormone that does not play a role in
hematopoietic differentiation (PRL). We assessed the specificity of
fetal liver hematopoietic progenitor and ES cell responses to Mpl by
comparing PM-R with another PRL-R chimeric receptor that includes the
cytoplasmic domain of erythropoietin-R (EPO-R) (PE-R, for PRL-R/EPO-R)
and that has been already used to investigate the signaling specificity
of cytokine receptors in fetal liver hematopoietic progenitors. From
these former studies, it was concluded that the cytoplasmic domains of
cytokine receptors are interchangeable for their ability to support the
terminal differentiation of hematopoietic progenitor
cells.22
Data presented herein show that in fetal liver, the cytoplasmic domain
of the MPL receptor is indeed active only in immature hematopoietic
progenitors and transduces unique signals compared to the cytoplasmic
domain of EPO-R. In addition, this region of MPL promotes hematopoietic
commitment of ES cells.
Hormones, antibodies, and cytokines
Plasmids and DNA constructs
Full-length SalI murine MPL cDNA26 was introduced in an antisense orientation at the XhoI site of the MSCV vector. Orientation of the insert was checked by BamHI digestion. ES cell culture R1 ES cells (kindly provided by Andras Nagy)27 and CJ7 ES cells28 were maintained in an undifferentiated state on gelatinized tissue culture dishes in Dulbecco modified essential medium (DMEM, Gibco, Life Technologies, Cergy-Pontoise, France), supplemented with 15% selected fetal calf serum (FCS) (Biological Industries, Kibbutz Beit Haemek, Israel), 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin (Gibco, Life Technologies), 150 µM monothioglycerol (Sigma, France), and 1000 U/mL of recombinant leukemia inhibitory factor (LIF) (Gibco, Life Technologies).ES cell electroporation ES cell culture medium was replaced with fresh medium 3 hours before electroporation. ES cells were then trypsinized, and 7.106 cells were resuspended in 800 µL of electroporation buffer29 in the presence of 40 µg of linearized plasmid MSCV, MSCV P-R, MSCV PE-R, or MSCV PM-R. Electroporation was performed at 250 V and 500 µF (Gene Pulser, Biorad, Brussels, Belgium). After a 10-minute incubation at 37°C, cells were resuspended in ES medium and plated in 6 gelatinized Petri dishes (10 cm in diameter) in the presence of 200 µg/mL geneticin (Gibco, Life Technologies). ES medium plus geneticin was then replaced at day (D)1, D2, D3, D5, and D7. Individual colonies were picked around D8 to D10 and plated in 96-well plates. When cells became confluent, they were trypsinized and split in half in two 96-well plates. One plate was used for freezing the clones, and the other for expression analysis by whole cell blot of the transgene.30Production of retroviruses and retroviral infections Retrovirus production. MSCV vectors encoding the different receptors were transfected transiently into the ecotropic packaging cell line BOSC 23,31 and viruses were prepared as described previously.32 All the viruses used in this study had a titer around 1.106 neoR cfu/mL. Retroviral infection of ES cells. Freshly passaged ES cells were resuspended at 3 × 105 cells in 4 mL viral supernatant + 1000 U/mL LIF with 8 µg/mL polybrene, plated in 60-mm gelatinized dishes, and incubated as usual with one change of the viral supernatant over 24 hours. After infection, adherent cells were rinsed with phosphate-buffered saline (PBS) (Gibco, Life Technologies), and fresh medium was added for a further 24-hour culture period. At 48 hours after infection, ES cells were transferred to fresh dishes with 200 µg/mL geneticin (Gibco, Life Technologies). Geneticin-resistant polyclonal populations were then expanded prior to further studies. Infection of fetal liver hematopoietic progenitor cells Single-cell suspensions of fetal livers were prepared from day-12.5 C57/Bl6 or PRL-R KO fetuses, respectively.33 5.106 nucleated cells were resuspended in 10 mL of viral supernatant in the presence of 8 µg/mL polybrene (Sigma, France) and incubated for 3 to 4 hours at 37°C with retroviral supernatant. Following infection, cells were washed in Iscove modified Dulbecco medium (IMDM) (Gibco, Life Technologies) and plated in methylcellulose medium for clonogenic cell assays. A fraction of cells from each infection (4.106 cells) was cultured for 48 hours in IMDM with 20% FCS, in the presence of 500 ng/mL O-PRL and 50 ng/mL mSCF for fluorescence-activated cell-sorter (FACS) analysis of receptor expression.Receptor expression analysis Reverse transcription-polymerase chain reaction (RT-PCR).
Undifferentiated ES cells, EBs, or individual hematopoietic
colonies were lysed with Trizol (Gibco, Life Technologies) (1 mL per
106 cells or 100 µL per hematopoietic colony), and total
RNA was extracted as recommended by the manufacturer. Then 1 µg or less of RNA was incubated for 30 minutes at 37°C with 0.2 U
RQ1 DNAse (Promega, Madison, WI) in reverse transcriptase (RT) buffer
(Gibco, Life Technologies) in a final volume of 20 µL. MoMulV RT
(Gibco, Life Technologies) was added, and the mixture was further
incubated for 1 hour at 37°C. The volume of cDNA was then adjusted up
to 200 µL, depending of the initial amount of RNA used in the
reaction. Samples were submitted to 35 cycles of amplification with Taq Polymerase (Gibco, Life Technologies) under conditions recommended by
the manufacturer. Then 5 µL of sample was used with primers specific for RNase mapping.
15 µg total RNA was precipitated in ethanol with 105 cpm
of a 320-bp riboprobe fragment of the extracellular domain of PRL-R and
5.104 cpm of actin riboprobe (250 bp), in the presence of
0.5 M ammonium acetate. After centrifugation, pellets were resuspended
in the hybridization buffer (RPAII kit, Ambion, Austin, TX), heated 3 to 4 minutes at 90°C, and incubated overnight at 45°C
(hybridization). Then 200 µL of digestion buffer containing
3 U RNase A and 125 U RNase T1 was added. After a 30-minute incubation
at 37°C, the stop/precipitation solution provided with the kit was
added; double-stranded RNAs were precipitated 15 minutes at FACS analysis. 105 fetal liver or ES infected cells were incubated with 50 µg/mL monoclonal antibody M110 for 30 minutes at 4°C. Cells were then washed 2 times in PBS-0.5% BSA and incubated 30 minutes with a phycoerythrin (PE)-conjugated goat anti-mouse F(ab')2. After 2 more washes in PBS-0.5% BSA, cells were analyzed by 2-color flow cytometry on a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). ES cell differentiation ES cells were induced to differentiate toward hematopoiesis following the classical 2-step model.21 ES cells were washed twice and incubated in ES medium without LIF 1 to 2 hours prior to differentiation. After trypsinization, 3000 cells were suspended in 1.5 mL complete medium for hematopoietic differentiation and plated in duplicate 35-mm bacterial Petri dishes (Greiner, Frickenhausen, Germany). This differentiation medium consisted of 1.1% methylcellulose (Methocel high viscosity, Fluka AG, Buchs, Switzerland) in IMDM-Glutamax (Gibco, Life Technologies) supplemented with 15% FCS (Biological Industries), 10 µg/mL insulin (Sigma, France), 300 µg/mL transferrin (Roche-Boerhinger), 10 µM hemin, 450 µM monothioglycerol, 50 U penicillin, and 500 µg/mL streptomycin (Gibco, Life Technologies). ES cells were allowed to form EBs for 10 to 12 days at 37°C, in modular incubator chambers (Billups-Rothenberg, Del Mar, CA), in a gas mixture composed of 5% CO2 and 6% O2 (Air Liquide, Mitry-Mory, France) in 100% humidity. To optimize hematopoietic differentiation, a mixture of 7 cytokines (7 GF), composed of 50 ng/mL SCF, 25 ng/mL IL-3, 10 ng/mL IL-6, 10 ng/mL IL-11, 10 ng/mL G-CSF, 2 U/mL EPO, and 50 ng/mL bFGF was added to the medium. In some experiments, a more restricted combination composed of 2 U/mL EPO, 50 ng/mL SCF, 25 ng/mL IL-3, and 500 ng/mL O-PRL (EIPS) has been used. EBs obtained after 10 to 12 days of differentiation were collected and resuspended in 500 µL of a dissociation medium containing IMDM with 10% FCS, 0.2% collagenase B (Roche-Boehringer), and 200 U/mL deoxyribonuclease 1 (Roche-Boehringer) and kept at 37°C in a water bath for 30 minutes. Dissociation of EBs was achieved by vigorous pipetting, and cells were then washed twice in IMDM. After counting, cells were submitted to hematopoietic progenitor cell assays in methylcellulose.Hematopoietic progenitor cell assays Clonogenic cultures were performed as previously described.26 Briefly, cells were plated in 1 mL 1% methylcellulose in IMDM supplemented with 30% FCS, 1% crystallized bovine serum albumin (Sigma), and 100 µM 2-mercaptoethanol (Sigma, France) in the presence of appropriate cytokines. For fetal liver cells, assays were performed with either 2 U/mL EPO or 500 ng/mL O-PRL or with 2 U/mL EPO, 10 ng/mL IL-3, and 20 ng/mL TPO (EIT). For ES cells, assays were performed with either the 7 GF or EIPS cocktail. Samples were plated in duplicate 35-mm bacterial Petri dishes for each experimental point and incubated at 37°C in a humidified incubator containing 5% CO2.
PM-R infection of fetal liver hematopoietic cells does not efficiently support mature erythroid colony development, but allows generation of all types of myeloid hematopoietic colonies in the presence of PRL To study the signals sent by the MPL cytoplasmic domain into hematopoietic progenitor cells from fetal origin, we expressed a chimeric PM-R receptor in fetal liver progenitors after retroviral infection. This receptor is composed of the extracellular domain of rabbit prolactin receptor (P-R) and the transmembrane and cytoplasmic domains of MPL (Figure 1A). The use of such a chimeric receptor exhibits 2 advantages: first, it allows activation of MPL-dependent pathways in the absence of TPO with a hormone that is not involved in hematopoiesis; and second, a chimeric receptor composed of the extracellular domain of PRL-R and of the cytoplasmic domain of EPO-R (PE-R) has already been used successfully in fetal liver cells.22,34,35 cDNAs encoding PM-R, PE-R, and P-R (used as control) (Figure 1A) were subcloned into the MSCV retroviral vector, and high-titer retroviral supernatants of MSCV P-R, MSCV PE-R, and MSCV PM-R were produced. We first checked the expression and function of these receptors in the IL-3-dependent Ba/F3 cell line. Infected cells proliferated when culture medium was supplemented with 10 ng/mL O-PRL instead of IL-3, while parental Ba/F3 or Ba/F3 infected with MSCV did not (data not shown).
Next we introduced PM-R, P-R, or PE-R receptors into day-12.5 fetal
liver hematopoietic precursor cells by retroviral infection and
examined the ability of infected cells to generate colonies when
cultured in semisolid methylcellulose medium in the presence of EPO or
PRL. An aliquot was cultured in parallel in liquid medium with PRL and
SCF for 36 to 48 hours and used to measure the percentage of infected
cells by quantifying the fraction of M110-positive cells by FACS
analysis. As shown in Figure 1B, the same proportion of fetal liver
cells (11% to 14%) were found to express P-R, PE-R, or PM-R,
indicating that the transduction efficiency was roughly the same for
the 3 viruses. Colonies were observed 2 and 7 to 8 days later. As
described previously,22 both P-R and PE-R gave rise to
similar numbers of CFU-e-derived colonies, equally hemoglobinized, in
the presence of PRL. Interestingly enough, PM-R supported only a
restricted number of less hemoglobinized CFU-e-derived colonies (Figure 2A). No significant difference
was observed in CFU-e numbers between the different receptors in the
presence of EPO (Figure 2B). Cultures grown in the presence of PRL were
incubated further and observed 5 to 6 days later for the formation of
colonies derived from more primitive progenitors. As shown in Figure
3A, only PM-R-infected cells were able
to generate all types of myeloid colonies (GM, E, E/MK, G/MK, MK, and
GEMM), in significant numbers. The same results were obtained with
12.5-day fetal liver hematopoietic precursor cells from PRL-R KO mice
(Figure 3A), which do not exhibit any hematopoietic defect (M.S. and
N.B., unpublished data, May 1997). In the presence of a mixed
myeloid stimulus (EPO + IL-3 + TPO), however, no significant
difference was observed between the different receptors (Figure 3B). In
addition, when the colonies grown under these conditions were analyzed
by RT-PCR for the presence of the chimeric receptors, 8% were found
positive for P-R, 26% for PE-R, and 13% for PM-R. Therefore, the
ability of PM-R-infected cells to generate all types of myeloid
colonies in the presence of O-PRL cannot be explained by a better
infection of fetal liver hematopoietic progenitors by the retroviral
vector carrying PM-R. Taken together, these results indicate that in
12.5-day fetal liver, the MPL cytoplasmic domain signals in rather
primitive hematopoietic progenitors.
Introduction of chimeric receptors into ES cells We next compared the signals sent by the cytoplasmic domains of MPL and EPO-R at the earliest stages of development and differentiation of the hematopoietic system by introducing the same chimeric receptors into ES cell lines. Undifferentiated CJ7 ES cells were transduced with MSCV (control), and MSCV P-R, MSCV PE-R, or MSCV PM-R by electroporation or by retroviral infection. After neomycin selection, 3 PM-R- and 2 PE-R-electroporated clones were selected by whole cell blot for high expression of chimeric receptors and amplified. Comparison by RNase mapping of PM-R expression in these clones and in the bulk PM-R-infected population (PMI) demonstrated that retroviral infection was the most rapid and efficient way to induce prominent expression of the chimeric receptor in undifferentiated ES cells, as shown for PM-R in Figure 4A. All receptors were also introduced by retroviral infection into undifferentiated R1 ES cells, and all of the experiments described thereafter were performed in parallel in CJ7 and R1 ES cells. Expression of the receptors at the surface of undifferentiated ES cells was checked by flow cytometry using M110 monoclonal antibody 10 days after infection (at the end of geneticin selection). As illustrated in Figure 4B, 30% to 40% of the R1 ES cell population was found to express the chimeric receptors at their surface.
Expression of PM-R chimeric receptor enhances hematopoietic differentiation of ES cells after PRL stimulation Transduced ES cells were then induced to differentiate toward hematopoiesis following the classical 2-step model in the presence of the 7-growth factor cocktail (7GF cocktail) (no O-PRL added). By RT-PCR analysis, chimeric receptor expression was at least identical or higher 12 days after the initiation of differentiation (D12) than in the starting population of ES cells (D0) (Figure 5A), indicating that the expression of chimeric receptors was maintained during the first steps of hematopoietic differentiation. Hematopoietic progenitors included within D12 embryoid bodies (EBs) were analyzed in parallel in semisolid medium with the 7GF cocktail. EBs derived from P-R and PE-R ES cells contained an identical number of CFU-GM, BFU-E, and CFU-Mix progenitors as MSCV EBs. In contrast, an influence of the PRL-R/MPL receptor already could be detected both on the number of E and early Mix colonies (90 ± 16.2 BFU-E in PM-R EBs vs 54 ± 23.2 in MSCV EBs, and 28.6 ± 10.9 CFU-Mix in PM-R EBs vs 15.8 ± 13.4 in MSCV EBs) and on the size and appearance of all colonies (Figure 5B). This has been observed in all subsequent experiments. When transduced CJ7 ES cells were induced to differentiate under the same conditions, an increased number of CFU-Mix colonies was also observed (data not shown). The weak amount of prolactin contained within fetal bovine serum (FBS), although much lower than 500 ng/mL (between 6 ng/mL and 50 ng/mL in the lots of FBS used in these experiments), seemed sufficient to trigger the chimeric receptors expressed at the surface of ES cells under those conditions.
To demonstrate the impact of Mpl signaling on ES cell
hematopoietic commitment, control MSCV, P-R, PE-R, and PM-R ES cells were also induced to differentiate in the presence of 500 ng/mL of PRL
in a minimal cocktail (PEIS). This allowed activating the chimeric receptors from the beginning of differentiation. No difference in the number and morphology of D12 EBs was observed under those conditions. EBs were then dissociated and analyzed in semisolid medium
in the presence of either the 7GF or PEIS cocktail. In the presence of
the 7GF cocktail, EBs derived from PM-R ES cells gave rise to higher
numbers of CFU-GM, BFU-E, and CFU-Mix than MSCV- and P-R-derived
EBs. They also generated higher numbers of BFU-E and CFU-Mix
than PE-R (Figure 6, 1.PEIS/2.7GF).
Interestingly, no such increase was observed for CFU-megakaryocytes
(CFU-MKs). This difference was even greater when the content
of EBs was analyzed in PEIS, since under these conditions, only PM-R ES
cells were found to give rise to all kinds of hematopoietic
progenitors, with a substantial number of CFU-Mix (44 ± 27 CFU-Mix
per 105 EB cells plated) (Figure 6, 1.PEIS/2.PEIS). The
results presented in Figure 6 are representative of 2 independent
experiments conducted with ES R1 cells. However, it is important to
note that this increase in the content of hematopoietic progenitors in
PM-R EBs has been consistently observed, regardless of which ES cell
line (R1 or CJ7) or conditions for hematopoietic differentiation were
used. These data indicate that early Mpl signaling favored the
hematopoietic differentiation of ES cells by stimulating the production
of progenitors.
Introduction of antisense MPL into CJ7 ES cells inhibits their hematopoietic differentiation in vitro To check for the functional relevance of Mpl signaling in ES cell differentiation, we used an antisense strategy. Full-length murine Mpl cDNA was subcloned in MSCV, in an antisense orientation. MSCV MPL antisense (MPLAS) DNA was introduced into ES CJ7 cells by electroporation, and 3 clones expressing high levels of MPLAS RNA were isolated after neomycin selection, as performed for the PRL-R/MPL receptor.We first checked whether MPLAS expression influenced the formation of
EBs. As shown in Table 1, except
in one clone (MPLAS-3), MPLAS did not affect the total number of D12
EBs. However, a decrease in red (hematopoietic) EBs was observed in all
clones. This decrease was dramatic in MPLAS-1 and MPLAS-3 (87% and
79%, respectively, compared to MSCV) and reached 32% for MPLAS-2.
This was consistently observed, even when we optimized the conditions
for hematopoietic differentiation by introducing MS5 stromal cells
during the first step of differentiation.21 Upon analysis
of hematopoietic progenitors present in these EBs, the number of
hematopoietic colonies derived from MPLAS ES clones was sharply
decreased (Table 2). MPLAS-2 was the
clone that produced the highest number of colonies, although this was
still reduced by 84%. As shown in Figure
7, all types of colonies decreased in all
clones (89% to 96% decrease for CFU-GM, 79% to 97% for BFU-E, 84%
to 100% for CFU-Mix, and 78% to 98% for mixed colonies with MKs).
Production of megakaryocytes was also severely impaired in these
cultures (97% to 99.7%) (Table 2).
During the past few years, an accumulation of data has
demonstrated that the Mpl receptor and its ligand, TPO, which were first described to be involved in the primary regulation of
megakaryopoiesis and thrombopoiesis, also played an important role at
the level of adult hematopoietic stem cells.11,12,36-41
This concept has been supported by the effects of inactivation of the
Mpl/TPO pathway.16,17 c-mpl We introduced chimeric receptors composed of the extracellular domain
of PRL-R and the cytoplasmic domain of Mpl or EPO-R (PM-R and PE-R), as
well as the parental rabbit PRL-R, into hematopoietic progenitors
prepared from C57Bl6 or PRL-R Introduction of PM-R into ES cells allowed us to study the impact of Mpl signaling on hematopoietic commitment of a totipotent embryonic stem cell. ES cells are maintained in an undifferentiated state in the presence of LIF. When allowed to form 3-dimensional structures known as embryoid bodies (EBs), ES cells are able to differentiate into various cell types, including hematopoietic cells, in the presence of growth factors.45,46 The expression of genes coding for hematopoietic growth factors, their receptors, and other early markers characteristic of hematopoietic cells has been studied by several groups, and EPO-R is one of the earliest receptors expressed during ES cell differentiation (PCR signal detected between D0 and D1).47-49 Mpl expression is detected later, starting on D6 of differentiation.21 The introduction of chimeric receptors into ES cells allowed us to stimulate Mpl, EPO-R, and PRL-R signaling as soon as D0 of differentiation, by addition of large amounts of PRL (500 ng/mL). Under those conditions, D12 EBs generated from PM-R ES cells contained more hematopoietic progenitors of all types compared to D12 P-R or PE-R EBs. This was even more dramatic when EBs were analyzed in the presence of a more restricted growth factor combination that contained PRL (PEIS) (Figure 6, lower panel). Our results indicate that only PM-R expression was able to promote the earliest stages of hematopoiesis. Interestingly, stimulation of Mpl signaling both in fetal liver hematopoietic progenitors and ES cells did not lead to a notable increase in the megakaryocytic compartment. This is in agreement with the report of Goncalves et al, who showed that introduction of Mpl into murine bone marrow stem cells does not result in preferential commitment toward the megakaryocytic lineage.50 However, our results indicated that early Mpl signaling enhanced the
hematopoietic differentiation of ES cells. The physiological significance of this effect was strengthened when Mpl antisense RNA was
expressed in control CJ7 ES cells. When induced to differentiate toward
hematopoiesis in the presence of a rich combination of cytokines (7GF),
3 independent MPLAS CJ7 clones gave rise to nonhemoglobinized embryoid
bodies with a marked decrease in the total number of hematopoietic
colonies (80% to 100%). These results are somewhat contradictory to
the observation that in vitro, c-mpl This contradiction between the effects of an inactivated gene and the antisense approach on the in vitro hematopoietic differentiation of ES cells has been described already for the vav gene.53-55 In a knockout strategy, a gene important but not essential for hematopoietic development can be functionally complemented at the beginning of ES cell differentiation in the embryo, while in an antisense approach, the persistent presence of the antisense might act upon various pathways involved in hematopoietic differentiation. Accordingly, it is possible that persistent presence of MPLAS blocks other pathways involved in hematopoietic differentiation or apoptosis, while inactivation of Mpl can be functionally substituted for other signals. Nevertheless, the 2 approaches may provide complementary insights into the potential functions and roles of a given gene. In short, our results strongly suggest that Mpl plays a role during the hematopoietic commitment of embryonic cells and is important in the establishment of definitive hematopoiesis in the embryo.
We would like to thank Bruno Peault for his support and critical comments on the manuscript, and Jean-Philippe Rosa for the helpful discussion. We are indebted to Prof Jack Levin for critical reading, useful comments, and constant support.
Submitted August 7, 2001; accepted May 14, 2002.
Supported by the Institut National de la Santé et de la Recherche Médicale and the Association pour la Recherche contre le Cancer (grant 1397). C.C. is supported by the Ministère de l'Education de la recherche et de la Technologie (MERT) and by the Association pour la Recherche contre le Cancer.
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: Michèle Souyri, Institut National de la Santé et de la Recherche Médicale, U506, Hôpital Paul Brousse, 14, avenue Paul-Vaillant Couturier, 94807 Villejuif Cedex, France; e-mail: msouyri{at}infobiogen.fr.
1. Eaton DL, de Sauvage FJ. Thrombopoietin: the primary regulator of megakaryocytopoiesis and thrombopoiesis. Exp Hematol. 1997;25:1-7[Medline] [Order article via Infotrieve].
2.
Kaushansky K.
Thrombopoietin: the primary regulator of platelet production.
Blood.
1995;86:419-431
3.
Cramer EM, Norol F, Guichard J, et al.
Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.
Blood.
1997;89:2336-2346
4.
Broudy VC, Lin NL, Kaushansky K.
Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro.
Blood.
1995;85:1719-1726
5.
Fibbe WE, Heemskerk DP, Laterveer L, et al.
Accelerated reconstitution of platelets and erythrocytes after syngeneic transplantation of bone marrow cells derived from thrombopoietin pretreated donor mice.
Blood.
1995;86:3308-3313 6. Kaushansky K, Broudy VC, Grossmann A, et al. Thrombopoietin expands erythroid progenitors, increases red cell production, and enhances erythroid recovery after myelosuppressive therapy. J Clin Invest. 1995;96:1683-1687[Medline] [Order article via Infotrieve].
7.
Kobayashi M, Laver JH, Kato T, Miyazaki H, Ogawa M.
Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors.
Blood.
1995;86:2494-2499 8. Kaushansky K, Lin N, Grossmann A, Humes J, Sprugel KH, Broudy VC. Thrombopoietin expands erythroid, granulocyte-macrophage, and megakaryocytic progenitor cells in normal and myelosuppressed mice. Exp Hematol. 1996;24:265-269[Medline] [Order article via Infotrieve].
9.
Ulich TR, del Castillo J, Yin S, et al.
Megakaryocyte growth and development factor ameliorates carboplatin-induced thrombocytopenia in mice.
Blood.
1995;86:971-976
10.
Farese AM, Hunt P, Boone T, MacVittie TJ.
Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates.
Blood.
1995;86:54-59
11.
Ku H, Yonemura Y, Kaushansky K, Ogawa M.
Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice.
Blood.
1996;87:4544-4551
12.
Sitnicka E, Lin N, Priestley GV, et al.
The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells.
Blood.
1996;87:4998-5005 13. Gilmore GL, DePasquale DK, Lister J, Shadduck RK. Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells. Exp Hematol. 2000;28:1297-1305[CrossRef][Medline] [Order article via Infotrieve]. 14. Murray LJ, Young JC, Osborne LJ, Luens KM, Scollay R, Hill BL. Thrombopoietin, flt3, and kit ligands together suppress apoptosis of human mobilized CD34+ cells and recruit primitive CD34+ Thy-1+ cells into rapid division. Exp Hematol. 1999;27:1019-1028[CrossRef][Medline] [Order article via Infotrieve].
15.
Yagi M, Ritchie KA, Sitnicka E, Storey C, Roth GJ, Bartelmez S.
Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin.
Proc Natl Acad Sci U S A.
1999;96:8126-8131
16.
Alexander WS, Roberts AW, Nicola NA, Li R, Metcalf D.
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl.
Blood.
1996;87:2162-2170
17.
Carver-Moore K, Broxmeyer HE, Luoh SM, et al.
Low levels of erythroid and myeloid progenitors in thrombopoietin- and c-mpl-deficient mice.
Blood.
1996;88:803-808
18.
Kimura S, Roberts AW, Metcalf D, Alexander WS.
Hematopoietic stem cell deficiencies in mice lacking c-Mpl, the receptor for thrombopoietin.
Proc Natl Acad Sci U S A.
1998;95:1195-1200 19. Souyri M, Vigon I, Penciolelli JF, Heard JM, Tambourin P, Wendling F. A putative truncated cytokine receptor gene transduced by the myeloproliferative leukemia virus immortalizes hematopoietic progenitors. Cell. 1990;63:1137-1147[CrossRef][Medline] [Order article via Infotrieve].
20.
Solar GP, Kerr WG, Zeigler FC, et al.
Role of c-mpl in early hematopoiesis.
Blood.
1998;92:4-10 21. Berthier R, Prandini MH, Schweitzer A, Thevenon D, Martin-Sisteron H, Uzan G. The MS-5 murine stromal cell line and hematopoietic growth factors synergize to support the megakaryocytic differentiation of embryonic stem cells. Exp Hematol. 1997;25:481-490[Medline] [Order article via Infotrieve].
22.
Socolovsky M, Dusanter-Fourt I, Lodish HF.
The prolactin receptor and severely truncated erythropoietin receptors support differentiation of erythroid progenitors.
J Biol Chem.
1997;272:14009-14012 23. Hawley RG, Lieu FH, Fong AZ, Hawley TS. Versatile retroviral vectors for potential use in gene therapy. Gene Ther. 1994;1:136-138[Medline] [Order article via Infotrieve]. 24. Lacombe C, Dusanter I, Gobert S, Muller O, Gisselbrecht S, Mayeux P. Intracellular pathways activated by erythropoietin. Ann NY Acad Sci. 1994;718:223-230[Medline] [Order article via Infotrieve]. 25. Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367-382[Medline] [Order article via Infotrieve].
26.
Cocault L, Bouscary D, Le Bousse KC, et al.
Ectopic expression of murine TPO receptor (c-mpl) in mice is pathogenic and induces erythroblastic proliferation.
Blood.
1996;88:1656-1665
27.
Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC.
Derivation of completely cell culture-derived mice from early-passage embryonic stem cells.
Proc Natl Acad Sci U S A.
1993;90:8424-8428
28.
Swiatek PJ, Gridley T.
Perinatal lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20.
Genes Dev.
1993;7:2071-2084
29.
van den Hoff MJ, Moorman AF, Lamers WH.
Electroporation in `intracellular' buffer increases cell survival.
Nucleic Acids Res.
1992;20:2902 30. Wendling F, Vigon I, Souyri M, Tambourin P. Myeloid progenitor cells transformed by the myeloproliferative leukemia virus proliferate and differentiate in vitro without the addition of growth factors. Leukemia. 1989;3:475-480[Medline] [Order article via Infotrieve].
31.
Pear WS, Nolan GP, Scott ML, Baltimore D.
Production of high-titer helper-free retroviruses by transient transfection.
Proc Natl Acad Sci U S A.
1993;90:8392-8396 32. Challier C, Cocault L, Flon M, et al. A new feature of Mpl receptor: ligand-induced transforming activity in FRE rat fibroblasts. Oncogene. 2000;19:2033-2042[CrossRef][Medline] [Order article via Infotrieve].
33.
Ormandy CJ, Camus A, Barra J, et al.
Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse.
Genes Dev.
1997;11:167-178
34.
Socolovsky M, Fallon AE, Lodish HF.
The prolactin receptor rescues EpoR-/- erythroid progenitors and replaces EpoR in a synergistic interaction with c-kit.
Blood.
1998;92:1491-1496 35. Fichelson S, Chretien S, Rokicka-Piotrowicz M, et al. Tyrosine residues of the erythropoietin receptor are dispensable for erythroid differentiation of human CD34+ progenitors. Biochem Biophys Res Commun. 1999;256:685-691[CrossRef][Medline] [Order article via Infotrieve].
36.
Borge OJ, Ramsfjell V, Veiby OP, Murphy MJ, Lok S, Jacobsen SE.
Thrombopoietin, but not erythropoietin, promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro.
Blood.
1996;88:2859-2870
37.
Ramsfjell V, Borge OJ, Veiby OP, et al.
Thrombopoietin, but not erythropoietin, directly stimulates multilineage growth of primitive murine bone marrow progenitor cells in synergy with early acting cytokines: distinct interactions with the ligands for c-kit and FLT3.
Blood.
1996;88:4481-4492
38.
Borge OJ, Ramsfjell V, Cui L, Jacobsen SE.
Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+.
Blood.
1997;90:2282-2292
39.
Piacibello W, Sanavio F, Garetto L, et al.
Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood.
Blood.
1997;89:2644-2653
40.
Piacibello W, Sanavio F, Severino A, et al.
Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells.
Blood.
1999;93:3736-3749 41. Ueda T, Tsuji K, Yoshino H, et al. Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor. J Clin Invest. 2000;105:1013-1021[Medline] [Order article via Infotrieve].
42.
Goldsmith MA, Mikami A, You Y, et al.
Absence of cytokine receptor-dependent specificity in red blood cell differentiation in vivo.
Proc Natl Acad Sci U S A.
1998;95:7006-7011
43.
Stoffel R, Ziegler S, Ghilardi N, Ledermann B, de Sauvage FJ, Skoda RC.
Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo.
Proc Natl Acad Sci U S A.
1999;96:698-702
44.
Zeng H, Masuko M, Jin L, Neff T, Otto KG, Blau CA.
Receptor specificity in the self-renewal and differentiation of primary multipotential hemopoietic cells.
Blood.
2001;98:328-334 45. Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol. 1985;87:27-45[Medline] [Order article via Infotrieve]. 46. Wiles MV, Keller G. Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. Development. 1991;111:259-267[Abstract].
47.
Schmitt RM, Bruyns E, Snodgrass HR.
Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression.
Genes Dev.
1991;5:728-740
48.
Keller G, Kennedy M, Papayannopoulou T, Wiles MV.
Hematopoietic commitment during embryonic stem cell differentiation in culture.
Mol Cell Biol.
1993;13:473-486
49.
McClanahan T, Dalrymple S, Barkett M, Lee F.
Hematopoietic growth factor receptor genes as markers of lineage commitment during in vitro development of hematopoietic cells.
Blood.
1993;81:2903-2915
50.
Goncalves F, Lacout C, Villeval JL, Wendling F, Vainchenker W, Dumenil D.
Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation.
Blood.
1997;89:3544-3553 51. Filippi M, Porteu F, Le Pesteur F, et al. Embryonic stem cell differentiation to hematopoietic cells: a model to study the function of various regions of the intracytoplasmic domain of cytokine receptors in vitro. Exp Hematol. 2000;28:1363-1372[CrossRef][Medline] [Order article via Infotrieve]. 52. Mitjavila MT, Filippi MD, Cohen-Solal K, Le Pesteur F, Vainchenker W, Sainteny F. The Mpl-ligand is involved in the growth-promoting activity of the murine stromal cell line MS-5 on ES cell-derived hematopoiesis. Exp Hematol. 1998;26:124-134[Medline] [Order article via Infotrieve]. 53. Wulf GM, Adra CN, Lim B. Inhibition of hematopoietic development from embryonic stem cells by antisense vav RNA. EMBO J. 1993;12:5065-5074[Medline] [Order article via Infotrieve].
54.
Zhang R, Tsai FY, Orkin SH.
Hematopoietic development of vav-/- mouse embryonic stem cells.
Proc Natl Acad Sci U S A.
1994;91:12755-12759 55. Zmuidzinas A, Fischer KD, Lira SA, et al. The vav proto-oncogene is required early in embryogenesis but not for hematopoietic development in vitro. EMBO J. 1995;14:1-11[Medline] [Order article via Infotrieve].
© 2002 by The American Society of Hematology.
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  L. Petit-Cocault, C. Volle-Challier, M. Fleury, B. Peault, and M. Souyri Dual role of Mpl receptor during the establishment of definitive hematopoiesis Development, August 15, 2007; 134(16): 3031 - 3040. [Abstract] [Full Text] [PDF]
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 J. Staerk, C. Lacout, T. Sato, S. O. Smith, W. Vainchenker, and S. N. Constantinescu An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor Blood, March 1, 2006; 107(5): 1864 - 1871. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-actin and 10 µL with primers specific for chimeric receptors and rabbit PRL receptor. Sequences for 
20°C
and loaded on a 6% acrylamide gel, 8 M urea. After a 3-hour migration
at 250 V in 1 × TBE buffer, the gel was dried and exposed on
Hyperfilm MP (Amersham, France).
,
granulocyte-macrophage (-GM); 
, burst-forming unit-erythroid
(BFU-E); 
, mixed (-GEMM); 
, erythro-megakaryocyte (-EMK); 
,
granulo-megakaryocyte (-GMK); and 
, megakaryocyte (-MK).
/