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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-04-1050.
NEOPLASIA
From the Biomedical Research Centre, Department
of Medical Genetics, University of British Columbia, Vancouver,
Canada and the Department of Developmental and Molecular
Biology, Albert Einstein College of Medicine, Bronx,
NY.
Acute chicken leukemia retroviruses, because of their capacity to
readily transform hematopoietic cells in vitro, are ideal models to
study the mechanisms governing the cell-type specificity of
oncoproteins. Here we analyzed the transformation specificity of 2 acute chicken leukemia retroviruses, the Myb-Ets- encoding E26 virus
and the ErbA/ErbB-encoding avian erythroblastosis virus (AEV). While
cells transformed by E26 are multipotent (designated "MEP" cells),
those transformed by AEV resemble erythroblasts. Using antibodies to
separate subpopulations of precirculation yolk sac cells, both viruses
were found to induce the proliferation of primitive erythroid
progenitors within 2 days of infection. However, while AEV induced a
block in differentiation of the cells, E26 induced a gradual shift in
their phenotype and the acquisition of the potential for multilineage
differentiation. These results suggest that the Myb-Ets oncoprotein of
the E26 leukemia virus converts primitive erythroid cells into
proliferating definitive-type multipotent hematopoietic progenitors.
(Blood. 2003;101:1103-1110) The first leukemia-inducing oncogenes discovered
are those transduced by acute avian leukemia viruses. These genes
encode truncated or mutated forms of cellular proteins. Acute leukemia viruses are relatively common in chickens, probably reflecting this
species' unusual susceptibility to the transforming effects of a
variety of oncoproteins. More than 10 different isolates of acute avian
leukemia viruses have been identified, each inducing a distinct type of
leukemia within a matter of weeks in vivo and transforming cultured
hematopoietic cells within a few days of infection.1 Cells
transformed in vivo and in vitro closely resemble each
other.2,3 Thus, for example, hematopoietic cells
transformed by the Myb-encoding avian myeloblastosis virus (AMV)
resemble monoblasts while cells transformed by the ErbB and ErbA
proteins of the avian erythroblastosis virus (AEV) resemble erythroblasts both in the animal and in tissue culture.4-6
Crude fractionation experiments using adherence and cytotoxic
antibodies as selection methods suggested that these viruses block the
differentiation of their respective target cells.7
Additional experiments have shown that transformation specificity is a
property mediated by the individual viral oncogene(s) and not by the
specificity of the viral envelope.8
An exception to the rule is the avian acute leukemia E26 virus, which
encodes a fused oncoprotein between the truncated cellular transcriptional activators c-Myb and c-Ets-1. This virus was originally described as an erythroleukemia virus, based on the expression in
leukemic cells of the erythroid-specific histone H5 and the observation
that E26-transformed hematopoietic colonies contained a small number of
mature erythroid cells.9 In addition, myelomonocytic precursors ("myeloblasts") were seen within the leukemic cell population of infected animals, and this cell type could be transformed in culture as well.2,3 The virus therefore transforms 2 different cell types and induces a mixed "erythroid"/myeloid
leukemia. Subsequent studies have shown that the transformed
"erythroid" cells actually resemble thromboblasts (the avian
equivalents of megakaryocytes, which give rise to thrombocytes) and
that they are, in fact, multipotent. Thus, they can be induced to
differentiate into erythrocytes and thrombocytes by inactivation of the
viral Ets and Myb proteins, respectively,10,11 and into
eosinophils and myeloblasts (granulocyte/macrophage precursors12) by activation of the Ras and protein kinase
C (PKC) pathways.12,13 In addition, E26-transformed cells
(called MEPs for Myb-Ets-transformed progenitors) express cell surface antigens characteristic of both normal, definitive hematopoietic progenitors and thrombocytes.12,14-16 In summary,
E26-transformed MEP cells can differentiate not only into myeloblasts
but also into 3 additional hematopoietic lineages and thus resemble
adult multipotent hematopoietic progenitors.
The mechanism of E26-mediated transformation has been studied in some
detail. Earlier studies showed that the fusion between Myb and Ets
proteins is essential for leukemogenesis because animals infected with
a viral construct expressing Myb and Ets as separate proteins only
rarely developed the disease. In the few animals that did
develop leukemia, rearrangements in the viral genome were found
to have taken place that led to an in-frame refusion of the
oncoproteins. Similarly, cells transformed in vitro with the
"split" Myb/Ets virus exhibited an erythroid phenotype and lacked
the capacity for multilineage differentiation.17 To assess the relative contributions of the Ets and Myb moieties of the oncoprotein to the biology of the virus, mutants were produced that
rendered either of these moieties temperature-sensitive for DNA
binding.10,11,18,19 In aggregate, these studies suggested that a functional DNA-binding domain of the Myb portion of the fusion
protein is necessary to block thrombocytic (and macrophage) differentiation of E26-transformed MEP cells,11,18 while
an active Ets domain is required to block erythroid (and to a lesser extent eosinophil and myleolomonocytic)
differentiation.10,19
These observations raised the question about the target cells of E26
virus: Does the virus transform multipotent cells, or does it transform
a committed progenitor, imposing a new fate? In the present study we
have investigated the mechanism of transformation specificity of E26
and AEV viruses by infecting sorted cells from day 2 precirculation
chicken blastoderms. Surprisingly, our results indicate that E26 target
cells correspond to committed primitive erythroid progenitors and that
these are shared with those of AEV. E26 virus, but not AEV, thus
appears to endow primitive erythroid cells with the capacity for
multilineage differentiation, a property that is normally associated
with definitive-type hematopoietic cells.
Cells and tissue culture
Antibodies, FACS analysis, and cell sorting
PCR analyses of hemoglobins RNA was isolated using the RNeasy kit (Qiagen) and reverse transcriptase-polymerase chain reaction (RT-PCR) performed on 1 µg total RNA per sample. Primer pairs 332-351 and 1575-1556 were used for chicken -globin transcripts26 and primer pairs
425-442 and 1432-1415 for chicken  -globin.27 The
-globin was amplified at 60°C for 20 cycles and -globin at
57°C for 23 cycles. Probes used for hybridization were obtained by
cloning the respective PCR products. Exposure of the gels was for
10 minutes.
E26 and AEV target cells reside in the yolk sac of precirculation chicken blastoderms The first blood cells in chickens arise after about 20 hours of incubation within the yolk sac, where they are located in the lateral and posterior parts of the area vasculosa surrounding the embryo (stages 10 to 1228; Figure 1A). After about another 5 hours of incubation, the blood islands fuse to form the vascular system and establish the blood circulation. Because earlier work showed that cell suspensions from 2-day-old chick blastoderms can readily be transformed by both E26 and AEV viruses,9 we wanted to determine whether the target cells for transformation by these viruses are located in the embryo proper or in the yolk sac. For this purpose, 2-day-old blastoderms (stages 9 to 11) were dissected into yolk sac and embryo, and the yolk sac was further dissected into anterior and posterior regions as shown in Figure 1A. Suspensions of these tissues were then infected with either E26 or AEV or mock-infected and seeded in methylcellulose-containing medium at 39°C. As illustrated in Figure 1B, colonies of mock-infected cells consisted of primitive erythrocytes at day 4, while colonies transformed by E26 and AEV consisted of a mixture of blastlike cells and some mature primitive erythrocytes. As shown in Figure 1C, mock-infected cells from posterior and anterior yolk sac yielded 17 000 and 11 000 erythroid colonies per 105 cells seeded, respectively, while cells from the embryo proper yielded 170 colonies, most of which were probably derived from yolk sac contaminants. In comparison, posterior and anterior yolk sac cells infected with E26 virus yielded 350 and 150 transformed colonies, respectively, while embryos yielded no transformed colonies. Similar data were obtained with AEV, except that for all fractions the yield of transformed colonies was about 15-fold higher than with E26 virus. Thus, relative to the total number of erythroid colony-forming cells obtained in the mock infections, about 2% of the total colonies were transformed by E26 virus while about 30% were transformed by AEV virus. We conclude that in stage 9 to 11 chick blastoderms most of the target cells for E26 and AEV viruses are contained within the yolk sac and not in the embryo and are mostly located in the posterior part of the yolk sac. This correlates with the sites where the highest numbers of blood islands can be observed and that generate the highest numbers of erythroid colony-forming cells.
Comparison of cell surface antigens between E26 and AEV leukemic cells and 2-day yolk sac cells Before attempting to identify the specific target cells for the 2 virus strains, we compared the phenotypes of leukemic cells transformed by E26 and AEV viruses with those of normal 2-day blastoderm cells using antibodies that detect lineage-restricted cell surface antigens. Figure 2 shows fluorescence-activated cell sorter (FACS) profiles of leukemic cells isolated from the blood of diseased chicks infected 1 month earlier with E26 or AEV, respectively, using antibodies that detect lineage-restricted cell surface antigens. In the E26-infected animal, about two thirds of the leukemic cells displayed an "MEP"/thromboblast phenotype, expressing high levels of MEP17 ( 21
integrin20), MEP21 (also called PCLP-1, podocalyxin, thrombomucin14,15,21), and MEP2616 antigens.
These cells were negative for the erythroid cell antigens JS3
(HEMCAM23) and JS4 as well as for the myeloid antigen
MYL51/2 and the eosinophil antigen EOS47
(melanotransferrin24). The other third of the cells
exhibit a "myeloblast" phenotype, being MYL51/2-positive but
negative for all other antigens (with the exception of MEP17, which is
weakly expressed in myeloblasts). In contrast, AEV leukemia cells
exhibited an erythroid phenotype, expressing JS3, JS4, and weakly MEP26
antigen but not MEP21 and EOS47 antigens. These data confirm earlier
observations showing that E26 leukemia cells resemble normal
multipotent progenitor/thrombocytic cells while AEV cells resemble
erythroid cells.10,11,16
To determine the cell surface phenotype of 2-day yolk sac cells, suspensions from dissected stage 9 to 11 blastoderms were stained with the above antibodies and analyzed by FACS. As shown in Figure 2, these cells were weakly positive for MEP17, MEP21, and MEP26 antigens, strongly positive for JS3 and JS4 antigens, and negative for EOS47 and MYL51/2 antigens. These results demonstrate that precirculation chick yolk sac cells strongly express erythroid markers but are essentially negative for the multipotent progenitor/thrombocytic marker MEP21. Thus, these primitive hematopoietic precursors more closely resemble AEV- rather than E26-transformed cells. Yolk sac target cells of both E26 and AEV leukemia viruses correspond to primitive erythrocyte progenitors To identify the target cells of E26 and AEV in the precirculation chick yolk sac, suspensions of 2-day blastoderm pools were stained with the erythroid-specific JS4 antibody and sorted into antigen-positive and -negative fractions. Reanalysis of the sorted fractions by FACS showed that the JS4+ cells were 99% and the JS4 cells were 98% pure. Benzidine staining revealed
that 75% to 85% of the JS4+ cells expressed hemoglobin,
while the JS4 cells contained less than 1%
hemoglobin-positive cells. Next, cells from each fraction were
mock-infected or infected with either E26 or AEV virus and seeded in
35-mm dishes containing methylcellulose. Colony formation was scored
between days 3 and 10 of culture. In the uninfected samples, of 50 000
cells seeded, about 20 000 primitive erythrocyte colonies were
obtained with the JS4+ cell fraction while only around 100 colonies were obtained with the JS4 fraction (primitive
erythrocyte colonies could be recognized by their red color and
consisted of 4 to 20 cells that grew in tight clusters that
disintegrated within 4 to 5 days). In addition to the erythrocyte
colonies, occasional single macrophages or macrophage clusters
consisting of 2 to 10 cells could be detected in both fractions, with
2- to 3-fold higher proportions in the JS4 fraction
(about 20 vs 45). Finally, thrombocyte colonies consisting of 4 to 16 small dispersed hemoglobin-negative cells could be seen in the
JS4+ but not in the JS4 fraction (up to about
30 colonies). Because these colonies disintegrated within 2 to
3 days, their number could not be assessed accurately.
In the infected cultures a subset of colonies proliferated and became
macroscopically visible after 1 week. No such large colonies were seen
in the mock-infected cultures. A quantitative evaluation of the results
(experiment nos. 1 and 2, Table
1) shows that, as for the
erythrocyte colonies seen in the uninfected controls, most of the E26-
and the AEV-transformed colonies were obtained with cells from the
JS4+ fraction, with a transformation efficiency of 1.5% to
2.5% for E26 and 10% to 20% for AEV relative to the number of normal
erythroid colonies observed. In another experiment, cells were sorted
using the JS3 antibody. Although the separation of the erythroid
colony-forming cells was not as clean as with the JS4 antibodies (there
was still a significant number in the JS3
E26 target cells express hemoglobin The finding that most JS4 antigen-positive cells express hemoglobin and that early E26-transformed colonies contain mature erythroid cells (Figure 1C) raises the possibility that the virus is capable of reprogramming committed primitive erythroid cells, conferring upon them an MEP phenotype. Indeed, in numerous assays we observed emergence of MEP-type cells from small primitive erythroid colonies. To study this possibility and to determine the time at which transformants can first be detected with E26 virus, cultures infected by the virus were photographed at different times after infection. For this purpose, JS4+ cells were sorted from 2-day blastoderms, infected with E26 virus, and seeded in methylcellulose cultures. The plates were pretreated with collagen, a ligand for the integrin21, to facilitate the identification of the transformed cells (MEP cells, through expression of the MEP17 antigen, 21 integrin, become
adherent to extracellular matrix, whereas normal erythroid cells do
not.20 Thus, transformed cells can be identified
by their ability to spread on collagen). At 18 hours after seeding of
E26-infected cells, 2 fields were randomly selected, marked on the
bottom of the plate, and photographs of the fields taken at various
intervals for up to 120 hours. Selected images collected from field no.
1 are shown in Figure 3. Normal erythroid
cells and colonies could be identified by their reddish coloration,
indicative of hemoglobin expression. Most of the colonies stopped
dividing after about 3 days and began to disintegrate (blue arrowheads
in Figure 3). In contrast, one colony (black asterisk in Figure 3)
continued to proliferate and contained well over 100 cells at 120 hours
and too many to count at 150 hours. This E26-transformed colony
consisted of a mixture of transformed cells, recognizable by their
plastic adherence and grayish coloration, and of normal erythroid
cells, recognizable by their reddish coloration (best seen in the
images taken at 66, 74, and 120 hours). Tracing the origin of this
colony to the 18-hour time point showed that it derived from a 2-cell
cluster whose cells appeared to be hemoglobin-low or -negative (more
obvious by direct microscopic inspection). This colony progressed into 3 weakly hemoglobin-positive cells at 24 hours and 5 partially hemoglobin-positive cells at 42 hours, one of which probably represents the first transformed cell (red arrowhead, Figure 3). From this point
on the colony proliferated rapidly, containing 20 cells at 66 hours.
These observations suggest that the target cells of E26 express
hemoglobin and that they can become reprogrammed to acquire a
hemoglobin-negative "MEP" phenotype. At the same time, some of the
E26-infected cells continue to differentiate into erythrocytes, as had
already been indicated by the appearance of benzidine-stained
E26-transformed colonies isolated 4 days after infection (Figure 1B,
middle panel).
E26-transformed clones undergo a gradual transition from an erythroid to an MEP phenotype The presence of erythroid cells in the E26-transformed colonies could also be shown by FACS analysis. Thus, of 13 transformed clones derived from JS4+ yolk sac cells, analyzed at day 17 after infection, the average percentage of cells expressing MEP21 was 61.1% (SD ± 23.7%), JS4 antigen 29.5% (SD ± 18.0%), JS3 antigen 15.8% (SD ± 11.3%), and MYL51/2 3.0% (SD ± 2.1%). Two AEV-transformed clones analyzed in comparison revealed that they were essentially negative for MEP21 (7.5%) but highly positive for JS4 and JS3 antigens (67% and 95%, respectively). These results are shown in Figure 4A for 2 E26-transformed clones (chosen to illustrate the variations observed) and for 1 AEV-transformed clone. When examined for the type of hemoglobin produced, using semiquantitative PCR, E26-transformed colonies were found to express both-globin (an embryonic -globin) and adult
-globin (Figure 4B). The fact that JS4+ erythroid cells
were still seen in E26 clones that had grown to more than a
million cells within 2 weeks after infection suggested that they
retained their erythroid differentiation potential for more than 20 cell divisions. To determine whether at this stage the clones continued
to drift toward an MEP phenotype, 4 of the E26-transformed colonies
were monitored for MEP21 and JS4 expression after an additional week in
culture. In addition, the clones were stained with benzidine at both
time points to determine hemoglobin expression. As shown in Figure 4B,
the proportion of MEP21+ cells increased in all clones,
with a corresponding decrease in JS4-expressing cells. Correlating with
this, the percentage of benzidine-positive cells in these colonies
(which ranged from less than 1% to 10% at day 17 after infection)
decreased during the same time period. These observations indicate that
E26-transformed colonies consist of variable proportions of erythroid
and MEP type cells and that during culture the colonies progressively lose their erythroid differentiation potential while acquiring MEP
properties. Such a selection of cells with an MEP phenotype might also
occur during leukemogenesis in vivo because E26 leukemia cells are
typically MEP21+ and JS4 (Figure
1A).
The schematic in Figure 5 summarizes
how E26 transforms its target cells and how this differs from AEV
transformation. Both viruses induce the proliferation of a subset of
primitive erythroid progenitors and in the initial stages after
infection colonies transformed by the 2 viruses are morphologically
very similar, consisting of a mixture of blastlike cells and mature
erythrocytes (Figure 1B). However, while AEV-transformed cells maintain
an erythroid phenotype, cells infected by E26 virus gradually
down-regulate erythroid cell surface antigens while up-regulating
antigens characteristic of multipotent progenitors/thrombocytes,
resulting in an MEP phenotype. In parallel to the antigenic changes the
cells acquire multipotentiality.10-13 Thus, the mechanism
of cell transformation by E26 appears to involve a Myb-Ets-induced
cell fate change and thus differs from that seen with AEV. Whether
these changes represent an induced "differentiation" or
"dedifferentiation" of a committed precursor is not clear. Adopting
the classical view that a monopotent progenitor is more differentiated
than a multipotent progenitor, our observations would be compatible
with a dedifferentiation. Alternatively, they could also represent
forward differentiation because monopotent progenitors appear to arise
before multilineage progenitors and hematopoietic stem cells
during ontogeny.29 Ultimately, however, a distinction
between the 2 processes might be semantic, because there is increasing
evidence for the reversibility of differentiation among hematopoietic
cells (reviewed by Graf30). For example, committed
B-lineage cells have been shown to be reprogrammable to become
macrophages by overexpression of the Raf
oncogene.31
Another interpretation of our data is that E26 target cells represent
rare definitive multilineage progenitors present in precirculation yolk
sac, especially in view of the low transformation efficiency observed.
Although studies in the mouse have indicated the presence of small
numbers of definitive progenitors in the precirculation yolk
sac,32 several arguments speak against this possibility:
(1) Definitive multilineage, hematopoietic progenitors in the chick
emerge only after establishment of circulation, in day 2.5 to 3.0 yolk
sac,14 and have been shown to be recent immigrants from
intraembryonic sites (see also below). Thus, the use of precirculation
yolk sac precludes the possibility of such target cells in the assay.
(2) Most of the E26 target cells are contained within the erythroid
cell fraction of the precirculation yolk sac (as judged by their marker
expression and differentiation potential). In contrast, multilineage
progenitors in the postcirculation yolk sac are MEP21+,
JS4 The low transforming efficiency observed with E26 relative to AEV might be explained by an inefficient reprogramming of committed erythroid cells by the Myb-Ets oncoprotein. Consistent with this interpretation is the finding that superinfection of AEV-transformed erythroblasts with E26 virus induces cell death and that E26 infection of yolk sac cells can induce an accelerated disintegration of normal erythroid colonies. In addition, we have observed that the ts1.1 mutant of E26 (encoding a protein with a point mutation in Ets, Myb-Etsts) transforms yolk sac cells at similar efficiencies as AEV in cells infected at the permissive temperature (data not shown). This might be due to the fact that Myb-Etsts-transformed cells exhibit an immature erythroid phenotype, expressing hemoglobin and JS4 but no MEP21 antigen.10 Studies with chick-chick and chick-quail chimeras have suggested that during development the first hematopoietic cells arise in the yolk sac and that these are subsequently replaced by a definitive population of stem cells from intraembryonic sites (splanchnopleura and paraaortic foci).34-40 In addition to their separate sites of origin, primitive yolk sac progenitors differ from adult hematopoietic progenitors in that they are largely restricted to erythroid differentiation and undergo complete erythrocyte differentiation in only 1 to 2 days versus 1 to 2 weeks. Thus, the fact that E26 transforms primitive erythroid progenitors, giving rise to multilineage progenitors, raises the possibility that the virus induces a transition from primitive to definitive-type hematopoiesis. Whether this transition requires the induction of cell cycling is not known. Even in the absence of oncogenic transformation, there is evidence to suggest that primitive hematopoietic progenitors can undergo a shift toward definitive progenitors if normal patterns of transcription factor expression are perturbed. This has been shown most strikingly in mice lacking a single allele of the transcription factor AML-1, resulting in the precocious emergence of long-term repopulating (definitive) hematopoietic stem cells in the yolk sac and aorta-gonad-mesonephros (AGM) region.41 Studies with AEV have shown that the v-ErbA and v-ErbB oncoproteins each contribute to the induction of cell proliferation and differentiation arrest of erythroid cells. Thus, while v-ErbA (a mutated form of the thyroid receptor) cooperates with stem cell factor (SCF) under normal growth conditions,42 v-ErbB, a mutated form of the EGF receptor, abrogates the cell's need for erythropoietin and SCF under conditions of hypoxia.43 The mechanism of transformation by the Myb-Ets oncoprotein of E26 leukemia virus is less well understood at the molecular level. Each domain of the oncoprotein, if expressed on its own, is sufficient to induce the proliferation of erythroid cells in culture.44,45 When expressed together, they cooperate in inducing erythroid cell proliferation44 but they need to be fused to induce an MEP phenotype.17 An interesting possibility is that Myb-Ets forces a transition from primitive-to-definitive hematopoietic cells by mimicking a process that is exerted by c-Myb during normal development. Thus, the primitive to definitive cell transition is accompanied by an up-regulation of c-Myb,32 and mice with an inactivated c-Myb develop primitive but not definitive hematopoietic cells.46 Little is known about the relevant target genes of Myb-Ets. A strong candidate is bcl-2, which is up-regulated by Myb-Ets in myeloid cells and by c-Myb in T cells, probably by direct interaction with its promoter.47,48 Hox genes are another group of candidate target genes because these are often deregulated in human leukemias.49,50 In particular, Hox11, which is associated with oncogenesis in human beings and mice,51 transforms murine fetal liver cells with a phenotype similar to E26 transformants (K.M.M., unpublished observations 2001). In addition, it has recently been shown that overexpression of HoxB4 induces the expansion of definitive hematopoietic stem cells and confers long-term, multilineage reconstitution potential to primitive hematopoietic cells derived from the yolk sac.52 It will thus be interesting to determine whether E26-transformed cells, but not AEV-transformed cells, express HoxB4. The early embryonic origin of the E26 virus target cells described here
raises the question as to whether they represent a valid model for
leukemogenesis by the virus. This is likely to be the case because in
vitro-transformed yolk sac and bone marrow cells resemble leukemic
cells obtained within 3 to 4 weeks after infection of newly hatched
chicks.2,3,12 If the model described is relevant for
leukemia, can our finding of an E26-induced reprogramming of target
cells be extrapolated to human acute myeloid leukemia (AML)? It is
intriguing that cases of myeloid leukemia have been reported where the
leukemic cells exhibit specific immunoglobulin gene rearrangements also
seen in normal B cells from the same patient.53,54 These
observations have been explained by the transformation of a
multilineage precursor in which immunoglobulin rearrangements were
subsequently aberrantly initiated; however, it is also possible that a
B-lineage cell becomes reprogrammed by a transforming event into a
myeloid cell, perhaps as a consequence of a secondary mutation. In
addition, studies by Grignani et al55 have shown that
infection of CD34+, lin
We thank Inger Pettersson and Lidia Perez for excellent technical assistance and Ari Melnick for comments on the manuscript. K.M.M. is a Canadian Institutes of Health Research Scholar. Some of the work for this article was performed at the European Molecular Biology Laboratory, Heidelburg, Germany.
Submitted April 10, 2002; accepted September 2, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-04-1050.
Partially supported by a Canadian Institutes of Health Research Scholar grant (MT-15477) and a Grant-In-Aid from the Heart and Stroke Foundation of B.C. & Yukon.
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: Thomas Graf, Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, New York, NY 10461; E-mail: graf{at}aecom.yu.edu.
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  K. Fukushima, I. Matsumura, S. Ezoe, M. Tokunaga, M. Yasumi, Y. Satoh, H. Shibayama, H. Tanaka, A. Iwama, and Y. Kanakura FIP1L1-PDGFR{alpha} Imposes Eosinophil Lineage Commitment on Hematopoietic Stem/Progenitor Cells J. Biol. Chem., March 20, 2009; 284(12): 7719 - 7732. [Abstract] [Full Text] [PDF]
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 D. N. Levasseur, T. M. Ryan, K. M. Pawlik, and T. M. Townes Correction of a mouse model of sickle cell disease: lentiviral/antisickling {beta}-globin gene transduction of unmobilized, purified hematopoietic stem cells Blood, December 15, 2003; 102(13): 4312 - 4319. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
