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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 4-10
Role of c-mpl in Early Hematopoiesis
By
Gregg P. Solar,
William G. Kerr,
Francis C. Zeigler,
Darren Hess,
Christopher Donahue,
Frederic J. de Sauvage, and
Dan L. Eaton
From the Departments of Cardiovascular Research, Molecular Oncology
and Flow Cytometry, Genentech Inc, South San Francisco, CA; and the
Department of Molecular and Cellular Engineering, Institute for Human
Gene Therapy, University of Pennsylvania, Philadelphia.
 |
ABSTRACT |
Recently, several lines of evidence have indicated an expanded role
for thrombopoietin (TPO) and its receptor, c-mpl, in
hematopoiesis. In addition to being the primary physiological regulator
of platelet production, it is now apparent that TPO also acts during
early hematopoiesis. To futher define the role of TPO in early
hematopoiesis we have identified discrete murine and human stem cell
populations with respect to c-mpl expression and evaluated their
potential for hematopoietic engraftment. Fluorescence-activated cell
sorter analysis of enriched stem cell populations showed the presence of c-mpl expressing subpopulations. Approximately 50% of the murine fetal liver stem cell-enriched population,
AA4+Sca+c-kit+,
expressed c-mpl. Analysis of the murine marrow stem cell population LinloSca+c-kit+ showed that
70% of this population expressed c-mpl. Expression of c-mpl was also
detected within the human bone marrow
CD34+CD38 stem cell progenitor pool and
approximately 70% of that population expressed c-mpl. To rigorously
evaluate the role of TPO/c-mpl in early hematopoiesis we compared the
repopulation capacity of murine stem cell populations with respect to
c-mpl expression in a competitive repopulation assay. When
comparing the fetal liver progenitor populations,
AA4+Sca+c-kit+c-mpl+
and
AA4+Sca+c-kit+c-mpl,
we found that stem cell activity segregates with c-mpl expression. This
result is complemented by the observation that the
LinloSca+ population of c-mpl
gene-deficient mice was sevenfold less potent than
LinloSca+ cells from wild-type mice in
repopulating activity. The engraftment potential of the human
CD34+CD38c-mpl+ population
was evaluated in a severe combined immunodeficient-human bone model. In comparison to the CD34+
CD38c-mpl population, the
CD34+CD38c-mpl+ cells showed
significantly better engraftment. These results demonstrate a
physiological role for TPO and its receptor, c-mpl, in regulating early
hematopoiesis.
| |
INTRODUCTION |
THROMBOPOIETIN (TPO) is the primary
physiological regulator of platelet production and was initially
thought to be a lineage dominant factor primarily affecting
megakaryocytopoiesis.1,2 Like other hematopoietic growth
factors, however, several lines of evidence indicate that TPO has a
more pleiotropic range of activities. This was first realized when TPO
was found to accelerate red blood cell recovery in myelosuppressed
mice,3-5 as well as to synergize with erythropoietin (EPO)
to stimulate erythroid proliferation in
vitro.5,6 In addition, in vivo administration of TPO caused an expansion of colony-forming unit
granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid
(BFU-E) in normal mice3 and expanded CFU-mixed in rhesus
monkeys.7 It was also shown that megakaryocytopoietic
activities of TPO are initiated at the early progenitor cell
level.8 Recently, several in vitro studies have shown that
TPO alone or in combination with the early acting growth factors, c-kit
ligand (KL), interleukin-3 (IL-3), or Flt-3 ligand (FL), stimulates the
proliferation of early hematopoietic progenitors.9-19
Significantly, progenitors expanded by TPO in combination with KL or FL
retain a primitive phenotype and maintain the capacity for multilineage
differentiation, even in the presence of TPO.9,12-14,16-18
TPO also enhances the expansion and viability of
CD34+CD38 progenitors in
culture.19
The phenotypic alteration of c-mpl- and TPO-deficient
mice also indicate a role for TPO in regulating hematopoietic
progenitors. As expected, the primary phenotype of these mice is severe
thrombocytopenia. However, detailed analysis of their marrow shows
deficiencies in their progenitor pool as well.20-23 In
addition to reduced megakaryocyte progenitors, both TPO- and
c-mpl-deficient mice show significant reductions in
neutrophil, GM, erythroid, and multilineage progenitors in the bone
marrow (BM), spleen, and peripheral blood (PB).20,21 A
reduction in blast cell-colony-forming cell (CFC) was also observed in
c-mpl-deficient mice.21 TPO administration to
TPO-deficient mice restores their progenitors to normal or
above-normal levels.22 These latter observations further
indicate that TPO may act to maintain and/or expand early
progenitors.
To rigorously characterize the role of c-mpl and TPO in hematopoiesis
we have identified discrete murine hemopoietic progenitor populations
with respect to c-mpl, and evaluated their capacity to repopulate
lethally irradiated mice. Similarly, the repopulation capacity of
hematopoietic progenitors from c-mpl gene-deficient mice was
also determined. To further expand our analysis we have characterized
the expression of c-mpl on human stem cell populations and their
functional capabilities using the severe combined immunodeficient-human (SCID-hu) bone model. Collectively, these data show that the
expression of c-mpl is important in early hematopoiesis.
| |
MATERIALS AND METHODS |
Flow cytometric analysis of murine fetal liver and BM.
Murine fetal liver stem cell populations were isolated from
AA4.1+ (called AA4+) cells derived
by immune panning from mid-gestation fetal liver essentially as
previously described.8 The AA4+ cells were
subsequently stained using directly conjugated antibodies against
murine c-kit (fluorescein isothiocyanate [FITC]), Sca-1 (R-PE)
(Pharmingen, San Diego, CA), and biotinylated hamster anti-murine c-mpl.24 For the biotinylated antibodies, streptavidin
Red-670 (GIBCO-BRL, Grand Island, NY) was used for detection.
BM populations were derived using femurs from C57/BL/6 mice. Briefly,
the femurs were obtained and flushed with PBS/2% fetal calf serum
(FCS) and a single cell suspension was made. Any remaining red blood
cells were removed by lysis with 10 mmol/L NH4Cl. The mononuclear fraction was then incubated with monoclonal antibodies (MoAbs) specific for lineage markers consisting of rat anti-mouse CD4/L3T4, CD8/Ly 2,3, B-220/CD45R (Caltag, South San Francisco, CA),
Gr-1, Mac-1, and Ter-119 (Pharmigen, San Diego, CA). Lineage marker
positive cells were then removed by magnetic bead depletion with a
sheep anti-rat antibody conjugated to magnetic beads (Dynal, Great
Neck, NY). The lineage depleted cells were then incubated with MoAbs
specific for: c-kit (FITC), Sca-1 (R-PE) (purchased from Pharmigen),
goat anti-rat Lin (Cascade Blue; Molecular Probes, Eugene, OR), and
biotinylated hamster anti-murine c-mpl.24 A second step
using Streptavidin Red-670 (GIBCO-BRL) was included for the
biotinylated samples. All fetal liver or BM samples were analyzed or
sorted on a Coulter EPICS Elite (Hialeah, FL) dual-laser flow cytometer
configured with a Coherent model 302 Krypton laser (407 nm) and argon
laser (488 nm). The appropriate isotype controls were included for each
experiment. All analysis excluded propidium iodide positive cells and
all cells with high forward or right angle light scatter.
Competitive repopulation of fetal liver progenitors and
c-mpl-deficient BM.
Timed pregnant C57/BL/6 mice were killed and the murine day 14 fetal
liver cell populations were isolated as described above. The following
enriched populations were then sorted:
AA4+Sca+c-mpl+ and
AA4+Sca+c-mpl. For the
competitive repopulation analysis of c-mpl-deficient mice, the
BM population LinloSca+ was isolated from
either homozygote mutant mice (c-mpl/) or
their wild-type (c-mpl+/+) littermate controls. Young adult
male C57/BL/6 Ly 5.2 mice were obtained from NCI and used as
recipients. A minimum of five animals was used per experimental group.
Whole-body irradiation (1,100 rads, 190 cGy/min) was administered as a
single dose from a 137Cs source. One million nucleated BM
cells from the Ly 5.2 mice were used as a source competitor, and mixed
with 1,000 or 2,000 fetal liver donor cells or 10,000 BM-derived donor
cells. Cells were administered via tail-vein injection and PB samples
(50 to 100 µL) were obtained via the retro-orbital sinus 4, 12, and
24 weeks postreconstitution. The percentage of Ly 5.1 (A20.1) donor cells was determined indirectly by staining with biotin-conjugated Ly
5.2 MoAb (A20.1.7). Briefly, PB was collected in 10 U/mL heparin, 1 mmol/L EDTA in phosphate-buffered saline (PBS) and immediately placed
on ice. Erythrocytes were removed by the addition of 5 vol of 4%
Dextran T500 (Pharmacia) in PBS followed by incubation at room
temperature for 20 minutes. The red blood cell-depleted fraction was
then centrifuged for 5 minutes at approximately 200g. The
remaining red blood cells were then lysed using 10 mmol/L NH4Cl. The mononuclear fraction was then resuspended in
PBS/2% FCS. This procedure removed nearly 100% of the erythrocytes
(the remaining red blood cells were excluded by size gating) while leaving the leukocytes 95% viable by propidium iodide exclusion. Fluorescence-activated cell sorter (FACS) analysis of Ly 5.2 PB cells
was nearly identical to cells prepared by lysing. Controls for the
specificity of Ly 5.2 staining included cells from Ly 5.1 or Ly 5.2 animals and cells from animals repopulated with Ly 5.2 competitor cells
only. To confirm repopulation by the donor Ly 5.1 cells in all
lineages, PB cells and the BM mononuclear fraction were stained in
conjunction with Ly 5.1 for B220, CD4, CD8 (lymphoid lineages), and
Gr-1/Mac-1 (myeloid lineages).
The relative repopulating ability of each putative stem cell population
was compared by calculating repopulation units (RU) as described by
Harrison et al.25 Each RU represents the same repopulating
ability as 1 × 105 fresh marrow cells. It
has been determined that the long-term repopulating stem cell content
of fresh marrow is one stem cell per 105 total marrow
cells.25 Therefore, the number of RU from each donor
population is defined by the following equation:
Isolation of CD34+ cells from human BM.
Fresh human BM aspirates were obtained from healthy donors (Poietic
Technologies, Gaithersburg, MD). The mononuclear cells were then
enriched for CD34 by immunomagnetic positive selection (Miltenyi
Biotech, Inc, Auburn, CA) according to the manufacturer's instruction.
CD34 content was assessed by FACS and purity was routinely greater than
90%. The CD34+ fraction was then stained with murine MoAbs
against human CD34 (FITC), CD38 (R-PE), (Becton Dickinson, San Jose,
CA), and c-mpl (biotin).26 The biotinylated samples were
then subsequently stained with Streptavidin Red-670 (GIBCO-BRL).
SCID-hu bone mice.
CB-17 scid/scid mice were implanted with fetal bone as
previously described.27 Briefly, human fetal bone fragments
from femurs, tibias, and humeri of 17- to 22-week gestational fetuses were implanted subcutaneously in 8-week-old CB-17 scid/scid
mice. The BM fragments were allowed to engraft for 8 weeks. Mice were then bled via the retro-orbital sinus and engraftment of the bone fragments was determined by flow cytometric analysis of the blood samples with MoAbs specific for human HLA class 1 epitopes (One Lambda,
Canoga Park, CA).
SCID-hu bone mice were administered whole-body irradiation of 200 rads
from a 137Cs source and sorted human stem cells were then
injected directly into a bone graft using a Hamilton syringe. Mice were
then killed 8 weeks postinjection, HLA disparate grafts were removed,
and marrow mononuclear cells were analyzed by FACS for donor HLA
contribution as well as CD19, CD33, and CD34 (Becton Dickinson).
| |
RESULTS |
Expression of c-mpl on murine stem cell populations.
To determine if a murine primitive hematopoietic cell population
enriched for stem cell activity expressed c-mpl, we isolated AA4.1+ (refered to as AA4+ here) cells from
midgestational murine fetal liver by immunopanning and quantified the
expression of c-mpl on the AA4+ fraction that is
double-positive for the Sca-1 marker (called Sca here) and c-kit. The
AA4+Sca+c-kit+ fraction has been
shown to be highly enriched for stem cell activity.28,29 Approximately 50% of the AA4+ cells that were positive for
both Sca and c-kit (AA4+Sca+c-kit+)
were c-mpl+. This c-mpl positive fraction
(AA4+Sca+c-kit+ c-mpl+)
constituted less than 0.1% of the total fetal liver
(Fig 1A).
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| Fig 1.
(A) Expression of c-mpl of murine fetal liver stem cell
populations. Fetal liver stem cells were enriched for AA4 and stained with MoAbs to murine Sca, c-kit, and c-mpl as described in Materials and Methods. Fetal liver cells positive for both Sca and c-kit were
gated and the resultant c-mpl positive cells are shown. Analysis was
done a minimum of four times. (B) Expression of c-kit, Sca, and c-mpl
on murine BM hematopoietic progenitors. The mononuclear fraction from
BM was isolated by a density gradient and stained with a Lin cocktail
of antibodies (see Materials and Methods). Lin stained cells were then
removed by magnetic bead depletion. The lineage-depleted cells were
then stained using directly conjugated MoAbs against Sca (R-PE), c-kit
(FITC), c-mpl (biotin), and the Lin cocktail (Cascade Blue). The
biotinylated c-mpl was detected using Streptavidin Red-670. Analysis
was repeated three times. (C) Expression of CD34, CD38, and c-mpl on
enriched human BM progenitor cells. CD34 cells were isolated as
described. The enriched cells were then stained with MoAbs to CD34
(FITC), CD38 (R-PE), and c-mpl (Red -670). Cells were then analyzed on
a Coulter Epics Elite flow cytometer as described. BM cells were
enriched for CD34 using an immunomagnetic column. Analysis was repeated
six times.
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In adult murine marrow, we analyzed the expression of c-mpl on the
LinloSca+c-kit+ population which is
enriched for stem cell activity.30 In these experiments the
mononuclear fraction of BM was depleted of lineage-committed progenitors with immunomagnetic beads (see Materials and Methods) and
the Linlo mononuclear cells were then further fractionated
into the LinloSca+c-kit+
population. FACS analysis of the
LinloSca+c-kit+ population showed
that 70% of this population was c-mpl+ (Fig 1B). This
population comprised less than 0.1% of total adult BM. As expected, we
detected c-mpl on the Linhi cells (data not shown) that are
presumably the lineage committed megakaryocyte progenitors.
Expression of c-mpl on human stem cell progenitor populations.
To evaluate the presence of c-mpl on human progenitors we analyzed the
expression of c-mpl on the CD34+CD38
stem cell progenitor population derived from human marrow. The CD34+CD38 population comprises 10% of
the total CD34+ population and is highly enriched for
multiprogenitor and stem cell activity.31-33
CD34+ cells were isolated from adult BM by immunomagnetic
positive selection (>90% purity), then stained with MoAbs against
human CD34, CD38, and c-mpl for FACS analysis. Similar to the
expression pattern on murine progenitors, approximately 70% of the
CD34+CD38 population expressed c-mpl
(Fig 1C).
Long-term self-renewing stem cell activity segregates with c-mpl
expression.
In this study, we used a competitive repopulation assay to compare the
long-term functional abilities of murine hematopoietic stem cell
populations with respect to c-mpl expression. Briefly, progenitor-enriched donor populations derived from either
mid-gestational fetal liver or adult BM were mixed with 106
competitor cells from BM of C57/BL6 Ly 5.2 animals and then injected into lethally irradiated C57/BL6 Ly 5.2 recipients. At the 4-, 12-, and
24-week time points, the relative contribution of donor cells to
hematopoietic lineages was determined by FACS analysis of the Ly 5.1 and Ly 5.2 allotypes.
Using this model we directly compared the repopulating activity of
AA4+Sca+c-mpl+ and
AA4+Sca+c-mpl fetal liver
cells derived from wild-type mice (Table
1). This direct comparison shows that all of the long-term repopulating activity of the AA4+Sca+ population segregated
with c-mpl expression. At the 24-week time point more than 50% of the
myeloid and lymphoid cells were derived from the
AA4+Sca+c-mpl+ donor cells with
equal contribution to each of these hematopoietic compartments. At 24 weeks the AA4+Sca+c-mpl+ population
exhibited a reconstituting activity of 220 RU. In contrast, the
AA4+Sca+c-mpl cells
exhibited no repopulating activity at the 24-week time point. The
AA4+Sca+c-mpl population did
exhibit short-term reconstituting activity, indicating that this
population consists primarily of committed progenitors. These data
indicate that the enriched fetal liver AA4+Sca+
population can be fractionated into functionally and phenotypically different populations based on differential expression of c-mpl, and
that c-mpl expression in this subset is associated with pluripotent repopulating activity.
To further expand this analysis, we isolated
LinloSca+ cells from the BM of
c-mpl-deficient mice and their wild-type littermate controls.
The LinloSca+ cells from wild-type animals
contained significantly more RU when compared with
LinloSca+ cells from the
c-mpl-deficient mice (37 v 5). Preferential engraftment of
the lymphoid or myeloid compartments was not observed with the
LinloSca+ donor populations isolated from
either wild-type or c-mpl-deficient mice. In addition, for the
mice injected with wild-type LinloSca+ cells,
the majority of lymphoid and myeloid cells were derived from this donor
population (Table 1).
To evaluate the in vivo functional characteristics of the human
CD34+CD38c-mpl+ population
in comparison to the
CD34+CD38c-mpl
fraction, the multilineage engraftment potential of these populations was compared in the SCID-hu bone model.27 Thirty thousand
cells of each phenotype were injected directly into the bone fragments of the SCID-hu animals and at 8 weeks postinjection, the mice were
killed, and mononuclear cells from the grafts were analyzed by FACS.
Cell suspensions were made of each graft and analyzed for overall donor
contribution by FACS using HLA haplotype-specific antibodies. In
addition, the contribution of donor cells to myeloid (CD33), lymphoid
(CD19), and hematopoietic progenitors (CD34) was determined. As shown
in Table 2, the
CD34+CD38c-mpl+ population
engrafted better (donor HLA) than the other populations. The combined
results of two separate experiments show that the CD34+CD38c-mpl+ population
engrafted in 90%(9 of 10) of the mice injected with this cell
population. In contrast, of the mice receiving the
CD34+CD38c-mpl cell
population only 22% (2 of 9) showed donor derived reconstitution. In
addition, the contribution of donor derived cells to the progenitor (CD34+), myeloid (CD33+), or lymphoid
(CD19+) compartments was higher with the
CD34+CD38c-mpl+ than the
CD34+CD38c-mpl
population 8 weeks postreconstitution. This was especially true for the
CD34+ progenitor population. In both experiments, with mice
injected with the
CD34+CD38c-mpl+ cells,
greater than 22% of CD34+ cells were donor
derived. This compares to 2% to 4% donor-derived CD34+ cells for the two mice that engrafted with the
CD34+CD38c-mpl cells.
| |
DISCUSSION |
Recent in vitro data clearly indicate that TPO may function not only to
expand the megakaryocyte compartment, but also to play a role in the
expansion and maintenance of early progenitors. TPO alone is capable of
activating quiescent progenitors into cycle13,18 and acts
in synergy with either IL-3, KL, or FL to expand the early progenitor
pool.9-19 In support of these observations, we have
recently shown that hematopoietic progenitors expanded in a stroma-free
liquid culture using a cytokine cocktail consisting of FL, KL, and TPO
maintain their long-term lymphohematopoietic repopulating activity in
vivo.34 The hematopoietic deficiencies that occur in
c-mpl and TPO-deficient mice, which exhibit a
significant reduction of multipotent progenitors, further indicate a
role for TPO/c-mpl in early hematopoiesis.20,21
At the molecular level, expression of c-mpl mRNA has been detected in
human CD34+cells35 and noncycling
progenitors.36 FACS analysis shows that c-mpl is expressed
on the stem cell enriched murine fetal liver population
AA4+Sca+.8 To further characterize
stem cell populations that express c-mpl, we have identified discrete
progenitor populations derived from murine and human sources that
express c-mpl. FACS analysis of murine BM
(LinloSca+c-kit+) or fetal liver
(AA4+Sca+c-kit+) stem cell
populations shows that c-mpl is expressed on at least 50% of each of
these stem cell-enriched populations.
The differential expression of c-mpl within the
AA4+Sca+ fetal liver progenitor population
allowed evaluation of its functional characteristics with respect to
c-mpl expression. Using a murine competitive repopulation
model,25 we directly compared the in vivo hematopoietic
reconstituting activity of
AA4+Sca+c-mpl+ and
AA4+Sca+c-mpl murine fetal
liver cell populations. These results show that all of the long-term
repopulating activity of the fetal liver progenitor population
(AA4+Sca+) segregates with c-mpl expression.
Repopulating activity showed equal contribution to the myeloid and
lymphoid compartments by the donor cell population
(AA4+Sca+c-mpl+). The
AA4+Sca+c-mpl population
exhibited a low level of short-term reconstituting activity, indicating
that this population is devoid of true stem cell activity. Furthermore,
marrow progenitors (LinloSca+) derived from
c-mpl gene-deficient mice showed a significant (sevenfold)
reduction in repopulating activity when compared with the
LinloSca+ progenitor population derived from
wild-type littermates. The segregation of stem cell activity with c-mpl
expression on the fetal liver AA4+Sca+
population indicates that c-mpl is expressed on a pluripotent stem cell
and that this subpopulation may further define a long-term self-renewing stem cell.
It is interesting to note that although the level of hematopoietic
progenitors is diminished by 50% in adult
c-mpl/mice, day 14 fetal liver
progenitors are not reduced in
c-mpl/ mice,21
suggesting that TPO does not play a critical role in early fetal
hematopoiesis. However, the inability of the
AA4+Sca+c-mpl fetal liver
cells to repopulate in the adult mouse indicates that c-mpl expression
may be essential for fetal liver progenitors to exhibit repopulating
activity in the adult marrow microenviroment. Because the
LinloSca+ progenitor population from the
c-mpl/mice had a low level of stem cell
activity and that only platelets, and not other hematopoietic blood
cells, are affected in these mice, suggests that for adult
hematopoiesis, c-mpl may play a crucial role in the function of
primitive stem/progenitors cells only when the homeostasis of the
hematopoietic compartment is perturbed. Thus, expression of c-mpl on a
subset of stem/progenitor cells may allow them to respond to feedback
mechanisms that maintain homeostasis in the hematolymphoid compartment.
As was observed for the murine progenitor populations, c-mpl is also
expressed on the stem cell-enriched progenitor population, CD34+CD38, derived from human marrow.
The expression pattern of c-mpl expression within this population is
similar to that observed for the murine progenitors with greater than
50% of CD34+CD38 expressing c-mpl.
These results are in contrast to those of Debili et al,37
who suggest that the c-mpl+ cells present in the
CD34+ fraction are primarily caused by late megakaryocyte
progenitors and transitional cells. Expression of c-mpl on
CD34+CD38 cells and the ability of TPO
to activate noncycling progenitors13 confirm that c-mpl is
indeed expressed on an early progenitor.
Until recently the study of long-term human hematopoiesis in vivo was
difficult because of the lack of an appropriate in vivo model. However,
using immunodeficient, nonobese diabetic (NOD)/SCID and
scid/scid mice, models have been developed in recent years to assay for
human hematopoietic stem cells and allow evaluation of their
multilineage repopulating capacity.27, 32,38
In this study we used the SCID-hu bone model27 to determine the effect of c-mpl expression on the in vivo reconstituting activity of the human progenitor CD34+CD38 cell
population. A direct comparison of the
CD34+CD38c-mpl+ and
CD34+CD38c-mpl cell
populations shows that c-mpl expression correlates with significantly
better donor-derived engraftment. This was apparent in both overall
engraftment and donor contribution to various hematopoietic lineages.
With equal numbers of cells injected, 90% of the mice receiving the
CD34+CD38c-mpl+ cells
engrafted compared with 22% engraftment for the mice receiving the
c-mpl fraction. In addition, a higher level of donor
contribution to the progenitor, myeloid, and lymphoid compartments was
observed with the
CD34+CD38c-mpl+ donor cells.
This was especially true for the CD34+ fraction in which
greater than 20% were donor derived using
CD34+CD38c-mpl+ cells
compared with only 4% for the
CD34+CD38c-mpl donor
cells or 8% for the c-mpl unenriched
CD34+CD38 fraction. Taken together,
these results suggest that the multilineage long-term reconstituting
activity of the human CD34+CD38 cell
population correlates with c-mpl expression.
The expanded role of TPO in hematopoiesis suggest that TPO may be
useful clinically for more than just alleviating thrombocytopenia. Recent in vitro studies have shown that TPO in combination with other
early growth factors can cause multilineage expansion of CD34+ cells in a long-term culture.17,33 Ex
vivo expansion would be a great benefit for the banking of umbilical
cord progenitors for future engraftment procedures. Additionally,
recent clinical trials have reported that TPO treatment results in an
increase in marrow hematopoietic progenitors and peripheral
CD34+ cells.39 These preliminary clinical
results again suggest that TPO may be useful not only as a
thrombopoietic agent but also in peripheral stem cell harvest
procedures.
| |
FOOTNOTES |
Submitted February 11, 1998;
accepted March 25, 1998.
Address reprint request to Dan L. Eaton, PhD, Genentech, 1 DNA Way,
South San Francisco, CA 94080.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Jim Chin for excellent technical assistance and Daniel Tumas
for critically reviewing the manusript.
| |
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[Abstract]
[Full Text]
[PDF]
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G. A. Millot, W. Vainchenker, D. Dumenil, and F. Svinarchuk
Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells
J. Cell Sci.,
January 6, 2002;
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J. W. Snow, N. Abraham, M. C. Ma, N. W. Abbey, B. Herndier, and M. A. Goldsmith
STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells
Blood,
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T. I. Pestina, J. L. Cleveland, C. Yang, G. P. Zambetti, and C. W. Jackson
Mpl ligand prevents lethal myelosuppression by inhibiting p53-dependent apoptosis
Blood,
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[Abstract]
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J. Levin, L. Cocault, C. Demerens, C. Challier, M. Pauchard, J. Caen, and M. Souyri
Thrombocytopenic c-mpl{-}/{-} mice can produce a normal level of platelets after administration of 5-fluorouracil: the effect of age on the response
Blood,
August 15, 2001;
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[Abstract]
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H. Zeng, M. Masuko, L. Jin, T. Neff, K. G. Otto, and C. A. Blau
Receptor specificity in the self-renewal and differentiation of primary multipotential hemopoietic cells
Blood,
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[Abstract]
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M. Majka, A. Janowska-Wieczorek, J. Ratajczak, K. Ehrenman, Z. Pietrzkowski, M. A. Kowalska, A. M. Gewirtz, S. G. Emerson, and M. Z. Ratajczak
Numerous growth factors, cytokines, and chemokines are secreted by human CD34+ cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner
Blood,
May 15, 2001;
97(10):
3075 - 3085.
[Abstract]
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B. D. Car and V. M. Eng
Special Considerations in the Evaluation of the Hematology and Hemostasis of Mutant Mice
Vet. Pathol.,
January 1, 2001;
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[Abstract]
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M. Ballmaier, M. Germeshausen, H. Schulze, K. Cherkaoui, S. Lang, A. Gaudig, S. Krukemeier, M. Eilers, G. Strau{beta}, and K. Welte
c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia
Blood,
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[Abstract]
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H. Ema, H. Takano, K. Sudo, and H. Nakauchi
In Vitro Self-Renewal Division of Hematopoietic Stem Cells
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S.-M. Luoh, E. Stefanich, G. Solar, H. Steinmetz, T. Lipari, T. I. Pestina, C. W. Jackson, and F. J. de Sauvage
Role of the Distal Half of the c-Mpl Intracellular Domain in Control of Platelet Production by Thrombopoietin In Vivo
Mol. Cell. Biol.,
January 15, 2000;
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[Abstract]
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A. W. Greenberg, W. G. Kerr, and D. A. Hammer
Relationship between selectin-mediated rolling of hematopoietic stem and progenitor cells and progression in hematopoietic development
Blood,
January 15, 2000;
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T. Gainsford, H. Nandurkar, D. Metcalf, L. Robb, C. G. Begley, and W. S. Alexander
The residual megakaryocyte and platelet production in c-Mpl-deficient mice is not dependent on the actions of interleukin-6, interleukin-11, or leukemia inhibitory factor
Blood,
January 15, 2000;
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[Abstract]
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R. Tordjman, N. Ortega, L. Coulombel, J. Plouet, P.-H. Romeo, and V. Lemarchandel
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Blood,
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S. Fichelson, J.-M. Freyssinier, F. Picard, M. Fontenay-Roupie, M. Guesnu, M. Cherai, S. Gisselbrecht, and F. Porteu
Megakaryocyte Growth and Development Factor-Induced Proliferation and Differentiation Are Regulated by the Mitogen-Activated Protein Kinase Pathway in Primitive Cord Blood Hematopoietic Progenitors
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M. Yagi, K. A. Ritchie, E. Sitnicka, C. Storey, G. J. Roth, and S. Bartelmez
Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin
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R. Stoffel, S. Ziegler, N. Ghilardi, B. Ledermann, F. J. de Sauvage, and R. C. Skoda
Permissive role of thrombopoietin and granulocyte colony-stimulating factor receptors in hematopoietic cell fate decisions in vivo
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K. Kaushansky
Thrombopoietin and the Hematopoietic Stem Cell
Blood,
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Y. Miyakawa, P. Rojnuckarin, T. Habib, and K. Kaushansky
Thrombopoietin Induces Phosphoinositol 3-Kinase Activation through SHP2, Gab, and Insulin Receptor Substrate Proteins in BAF3 Cells and Primary Murine Megakaryocytes
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Y. Miyakawa, J. G. Drachman, B. Gallis, A. Kaushansky, and K. Kaushansky
A Structure-Function Analysis of Serine/Threonine Phosphorylation of the Thrombopoietin Receptor, c-Mpl
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Abstract
Introduction
Abstract
