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HEMATOPOIESIS
From the Department of Clinical Chemistry, Microbiology
and Immunology, Ghent University, Ghent University Hospital, Ghent,
Belgium; and the Flanders Interuniversity Institute for Biotechnology,
Belgium.
Homeobox genes are well known for their crucial role during
embryogenesis but have also been found to be critically involved in
normal and leukemic hematopoiesis. Because most previous studies focused on the role of aberrant HOX gene expression in
leukemogenesis and because HOX-A10 is expressed in human
CD34+ precursor cells, this study investigated whether
HOX-A10 also plays a pivotal role in normal
hematopoietic-lineage determination. The effect of enforced expression
of this transcription factor on hematopoietic differentiation of highly
purified human cord-blood progenitors was examined by using in vitro
assays. In fetal thymic organ cultures, a dramatic reduction in cells
expressing high levels of HOX-A10 was observed, along with
absence of thymocytes positive for CD3+ T-cell receptor
Hematopoietic stem cells can differentiate into all
types of blood cells, although how this lineage determination occurs is poorly understood. Several transcription factors that control this
process at the molecular level have been identified.1-3 These factors enhance or direct expression of lineage-specific genes.
Most of these identified regulators of transcription are critical for
one specific hematopoietic lineage. Thus, Pax-5,4 early
B-cell factor,5 and Sox-46 are essential for
B-cell development; GATA-37 and T-cell factor
18 for T-cell differentiation; c-myb for early myeloid
differentiation9; and GATA-1 for red blood cell
formation.10 Other factors control more than one blood
cell lineage. Low levels of expression of PU.1 drive hematopoietic
progenitor cells toward the B-cell lineage, whereas a high
concentration of PU.1 results in myeloid
differentiation.11 On the other hand, Ikaros plays a
crucial role in lymphoid but not myeloid
development.12
Several studies have suggested that HOX genes,
originally identified as key regulators of embryonic
development,13,14 also play a crucial role in both normal
and leukemic hematopoiesis.15,16 Of the 39 members of the
HOX family identified in humans, most HOX-A and
HOX-B cluster genes were preferentially expressed in CD34+ bone marrow precursor cells in gene-expression
studies, and these genes were activated from 3' to 5' during
hematopoiesis,17 similar to the situation during embryonic
development.18 This spatial and temporal colinear
expression pattern was also observed during activation of natural
killer (NK) cells19 and T cells.20
Functional studies of HOX genes in hematopoiesis done by
generating knockout mice are lacking, except for investigations of HOX-A9. Mice deficient in HOX-A9 have serious
hematopoietic defects in the lymphoid, erythroid, and myeloid
compartments.21 Most information on the role of
HOX genes in hematopoiesis is derived from studies in which
one HOX gene was constitutively expressed in normal murine
bone marrow. Overexpression of HOX-B4 was found to produce
an increased number of transplantable hematopoietic stem cells without
changing their differentiation potential; thus, no malignant disease
resulted.22 On the other hand, enforced expression of
HOX-B3 increased the number of mature granulocyte-macrophage colony-forming cells, perturbed B-cell differentiation, and skewed T-cell progenitors toward the Expression of HOX-A10 was shown to be restricted to
CD34+ precursor cells and early stages of myeloid
differentiation.26 In one study, overexpression of this
gene in human progenitor cells resulted in severely perturbed
hematopoiesis, a dramatic reduction in B-cell differentiation, and a
myeloproliferative effect.27 No data on the effect of
continuous expression of HOX-A10 on the development of T and
NK cells, monocytes, and dendritic cells were reported.
In this study, we analyzed the role of HOX-A10 in
hematopoietic-lineage determination by producing overexpression of this gene in CD34+ cord-blood (CB) progenitor cells with use of
retrovirus-mediated gene transfer. Such overexpression resulted in a
severe disruption of the T-, B-, and NK-cell differentiation pathways
and granulocytic precursor cells were unable to mature fully. In
contrast, the monocytic differentiation pathway was enhanced. These
findings suggest that HOX-A10 is an important regulator of
lineage determination in human stem cells and provides a novel marker
for monocytic differentiation.
Monoclonal antibodies
Rat anti-mouse MoAb CD45-Cychrome (30F1 1.1; PharMingen) was used to
gate out mouse cells during flow cytometry, and anti-Fc Mice
Purification of CD34+ CB stem cells Cord blood was obtained and used in accordance with the guidelines of the Medical Ethical Commission of Ghent University Hospital. Umbilical CB was obtained from full-term, healthy newborns. Within 24 hours after CB collection, mononuclear cells were isolated by using a Lymphoprep density gradient (Nyegaard, Oslo, Norway), resuspended in 9 vol fetal-calf serum (FCS; Invitrogen, Carlsbad, CA) and 1 vol dimethyl sulfoxide (Serva, Heidelberg, Germany), and cryopreserved in liquid nitrogen until use. After the CB cells were thawed and washed, they were labeled with glycophorin A, CD19, CD41, and FITC CD7. For immunomagnetic depletion, the cells were resuspended in 1 mL cold phosphate-buffered saline (PBS) and 2% FCS and mixed with 1 mL prewashed (to remove the preservative) sheep anti-mouse immunoglobulin-coated Dynabeads (Dynal AS, Oslo, Norway) to obtain a 1:5 ratio of cells to Dynabeads. After 30 minutes at 4°C, the suspension was subjected to a magnetic field in a magnetic particle concentrator (Dynal AS). The supernatant containing the unlabeled cells was recovered. The remaining cells were resuspended in 0.2 mL PBS and 2% FCS and labeled with CD34-PE and FITC-labeled CD1, CD3, CD4, and CD8. Finally, cells that were positive for CD34-PE and negative for FITC (called CD34+Lin cells) were sorted on a
fluorescence-activated cell-sorter scanner (FACS; Vantage; BDIS).
Sorted cells were checked for purity, which was always at least
99.0%.
Cell cultures All cultures were maintained at 37°C in a humidified atmosphere containing 7.5% (vol/vol) carbon dioxide in air. Cells were cultured in Iscoves modified Dulbecco medium (IMDM) supplemented with penicillin (100 IU/mL), streptomycin (100 µg/mL), and 10% heat-inactivated FCS (complete IMDM; Invitrogen). The CD34+Lin CB stem cell coculture on MS-5
cells28 was done in complete IMDM containing 10%
heat-inactivated human AB serum (BioWhittaker, Walkersville, MD) and
5% FCS instead of 10% FCS. Fetal thymic organ cultures (FTOCs) were
done in complete IMDM containing 10% heat-inactivated human AB serum
(BioWhittaker) instead of 10% FCS.
Cloning of human HOX-A10 cDNA We previously used bicistronic vectors with a gene of interest linked to a downstream internal ribosome entry site (IRES) and a marker gene that allow independent translation of the products of both genes in the transduced target cells.29 The HOX-A10 cDNA was isolated from the Bluescript SK-positive-HA10 plasmid30 (Dr C. Largman, University of California VA Medical Center, San Francisco, CA) by using the restriction enzymes BbsI (New England Biolabs, Beverly, MA) and EcoRI (Roche Diagnostics, Mannheim, Germany). The BbsI site was made to have a blunt end by using T4 DNA polymerase (Roche Diagnostics). This DNA fragment was then cloned into the LZRS-IRES-enhanced green fluorescent protein (EGFP) retroviral vector,29 cut with SnaBI (Roche Diagnostics) and EcoRI, to generate the LZRS-HOX-A10-IRES-EGFP vector. The LZRS-IRES-EGFP retroviral vector was used as a control. Direct sequencing (ABI; Perkin Elmer, Foster City, CA) confirmed the integrity of the construct and showed that the clone used contained the published coding region of the human HOX-A10 gene (GenBank accession number AF040714).Generation of HOX-A10 encoding retrovirus The Phoenix-A-based amphotropic packaging cell line (Dr P. Achacoso and Dr G.P. Nolan, Stanford University School of Medicine, Stanford, CA) was transfected with the LZRS-IRES-EGFP and LZRS-HOX-A10-IRES-EGFP plasmids by using calcium phosphate precipitation (Invitrogen) to generate both retroviruses. The viral supernatant was stored in aliquots at 70°C until use. The IRES-EGFP
and HOX-A10-IRES-EGFP batches used in this study contained
approximately 9 × 105 and 1 × 106
transducing units/mL, respectively, titrated on Jurkat cells (ATCC).
Retroviral gene transfer Sorted CD34+ Lin CB cells were
cultured in complete IMDM supplemented with stem cell factor (SCF; 100 ng/mL), ftl3/flk-2 ligand (FL; 100 ng/mL), and thrombopoietin (TPO, 20 ng/mL) for 2 days (all cytokines from R&D Systems Europe, Abingdon,
United Kingdom). Afterward, the cells were transduced once in 24 hours.
For transduction, 2 to 15 × 104 cells were seeded on
96-well culture plates coated with RetroNectin (Takara Biomedicals,
Otsu Shiga, Japan) in a final volume of 150 µL containing 75 µL
retroviral supernatant and 75 µL complete IMDM supplemented with 200 ng/mL SCF, 200 ng/mL FL, and 40 ng/mL TPO to keep the final cytokine
concentration constant. After transduction, cells were harvested and
washed to remove the virus particles, transduction efficiency was
determined, and the cells were used in subsequent assays.
FTOCs Thymic lobes were isolated from fetal NOD-SCID mice on day 14 or 15 of pregnancy.31 Hanging drops were prepared in Terasaki plates by adding to each well 25 µL complete medium containing the progeny of 10 × 103 CD34+Lin
CB cells transduced as described above. One fetal thymic lobe was added
to each well, and the plates were inverted to form hanging drops and
incubated for 48 to 72 hours. After incubation, on day 0 of FTOC, the
lobes were removed, washed in complete medium, put on a Nuclepore
filter (Costar, Cambridge, MA) resting on a Gelfoam sponge (Upjohn,
Kalamazoo, MI) soaked in complete medium in a 6-well tissue-culture
plate (BDIS), and cultured for 10 to 25 days. Subsequently, the lobes
were mechanically disrupted with a tissue grinder to obtain a
single-cell suspension that was used in flow cytometry.
MS-5 stromal cell cultures Transduced CD34+ Lin CB stem cells
were incubated in the presence of various human cytokines in 24-well
plates precoated with confluent MS-5 cells derived from murine
marrow28 (Dr Coulombel, Institut Gustave Roussy, Villejuif,
France, with the permission of Kirin Brewery, Tokyo, Japan). For NK- or
B-cell development, 2 × 103 CD34+
Lin CB cells were cultured for 3 weeks in the presence of
SCF (50 ng/mL), interleukin-2 (IL-2) (5 ng/mL), and IL-15 (10 ng/mL)
(for NK cells) or SCF (50 ng/mL) and IL-7 (20 ng/mL) (for B cells). Monocytic and granulocytic differentiation was assessed by culturing 1 × 103 CD34+ Lin CB cells for
2 weeks in the presence of SCF (50 ng/mL), FL (50 ng/mL), TPO (20 ng/mL), and granulocyte-macrophage colony-stimulating factor (GM-CSF;
10 ng/mL) (monocytic) or SCF (50 ng/mL), FL (50 ng/mL), TPO (20 ng/mL),
and granulocyte colony-stimulating factor (G-CSF; 10 ng/mL) (granulocytic).
Generation of dendritic cells EGFP-positive (EGFP+) CD14+ monocytes generated from IRES-EGFP- and HOX-A10-IRES-EGFP-transduced CD34+ Lin CB stem cells after 9 days of MS-5
stromal cell culture were sorted to a purity of at least 98.5%.
Between 10 × 103 and 25 × 103 of these
monocytes were cultured for 4 days in the presence of GM-CSF (10 ng/mL), tumor necrosis factor  (TNF-; 10 ng/mL) and IL-4 (20 ng/mL) in a flat-bottomed, 96-well plate to generate mature dendritic
cells.32
Calculation of absolute cell numbers and statistical analysis Because of the difference in virus concentration between the IRES-EGFP and HOX-A10-IRES-EGFP retroviral batches used and the differences in transduction efficiency among experiments, the number of transduced cells at the beginning of culture was not the same in the 2 cultures and in all experiments. To compare the yields of cells obtained from EGFP+-transduced CD34+Lin CB progenitors and
EGFP+-transduced CD34+Lin CB
progenitors positive for HOX-A10, we multiplied the absolute number of EGFP+ cells (obtained by multiplying the total
number of cells counted under a microscope by the fraction of
EGFP+ human cells determined by FACS analysis) by a factor
so that the number of EGFP+ cells at initiation of the
cultures was 1 × 103 for both EGFP+- and
HOX-A10-positive EGFP+-transduced
CD34+Lin CB cells. Statistical analysis was
done with the nonparametric paired Wilcoxon test (SPSS, version 9.0;
SPSS, Chicago, IL).
Flow cytometry Before labeling, cells were suspended in PBS, 1% bovine serum albumin, and 0.1% sodium azide. In all experiments in which human cells were stained in the presence of mouse cells, the mixture of cells was preincubated for 15 minutes with saturating amounts of anti-Fc RII/III MoAb to avoid nonspecific binding of MoAbs by the
murine cells. Subsequently, the cells were stained with a panel of
MoAbs. Dead cells were gated out by propidium iodide; murine thymocytes
in FTOCs were gated out by using mouse CD45-Cychrome. For each
analysis of MS-5 stromal cell cultures, at least one staining was done
with the human CD45 antibody to show that gates were set on human
cells. Negative controls included isotype MoAbs conjugated with the
corresponding fluorochrome. The cells were analyzed on a FACScalibur
instrument (BDIS) with an argon-ion laser tuned at 488 nm and a
red-diode laser at 635 nm. Forward light scattering, orthogonal
scattering, and 4 fluorescence signals were determined and stored in
list-mode data files. Data acquisition and analysis were done with use
of CellQuest software (BDIS).
Continuous expression of HOX-A10 in CB progenitors severely hampers their lymphoid differentiation potential T-cell development.
To analyze the effect of HOX-A10 overexpression on human
T-cell development, CD34+ Lin
Cell-surface staining with T-cell-specific markers after 25 days of FTOC showed that within the few remaining HOX-A10-transduced thymocytes, the fraction of CD4+ CD8  double-positive cells was reduced in comparison
with that in control transduced cells (Figure 1B) or untransduced cells (data not shown). Moreover, virtually no TCR--positive
CD3+ thymocytes were detected among the
HOX-A10-EGFP-transduced cells, and despite a slight increase in the
fraction of TCR- -positive CD3+ T cells, there was no
obvious skewing of T-cell progenitors toward the T-cell lineage
(Figure 1B).
NK-cell and B-cell development.
To study the effect of HOX-A10 overexpression on development
of NK and B cells, the progeny cells of 2000 transduced
CD34+ Lin
After 3 weeks of culture, a significant decline in the percentage of HOX-A10-IRES-EGFP-transduced cells compared with that of control IRES-EGFP-transduced cells had occurred in the NK-cell cultures (Figure 2). In addition, the percentage of human CD45+ CD56+ cells was dramatically reduced in the EGFP+ gated cells of the HOX-A10-transduced cultures compared with control transduced cultures (Figure 2) or untransduced cells (data not shown). As a result, the absolute number of NK cells in cultures with HOX-A10 overexpression had decreased an average of 7-fold compared with those in control cultures (Table 1).
Overexpression of HOX-A10 has different effects on the myeloid differentiation potential of CB progenitors Monocyte differentiation.
The effect of HOX-A10 overexpression on monocyte
differentiation was analyzed by coculturing transduced CB progenitor
cells on the MS-5 stromal cell line in the presence of the human
cytokines SCF, FL, TPO, and GM-CSF. Analysis was done 2 weeks after
initiation of the cultures. There was a slightly higher increase in the
percentage of EGFP+ cells in HOX-A10-IRES-EGFP-transduced
cultures compared with control transduced cultures, thereby showing
that continuous expression of HOX-A10 slightly favors
expansion or survival under these culture conditions (Figure
3A). Monocytic cells were characterized
by expression of CD14, CD4, and HLA-DR. There was always a higher percentage of CD14+ cells in HOX-A10-IRES-EGFP-transduced
cells than in control IRES-EGFP-transduced cells (Figure 3A) or
untransduced cells (data not shown). Most of these cells expressed low
levels of HLA-DR and CD4, which is characteristic of monocytes. The
generation of monocytes was confirmed by Wright-Giemsa staining on
sorted EGFP+ CD14+ cells; this assessment
showed that 95% of IRES-EGFP-transduced CD14+ cells
(Figure 3B) and 99% of HOX-A10-IRES-EGFP-transduced CD14+
cells (Figure 3C) contained mature monocytes and some macrophages. As a
result, the absolute number of CD14+ monocytic cells
generated from HOX-A10-IRES-EGFP-transduced CB progenitor cells was,
on average, 6-fold higher than that generated from
IRES-EGFP-transduced precursor cells (Table 1).
Monocyte-derived dendritic cell differentiation.
To test whether the generated monocytes were able to differentiate into
mature dendritic cells, we sorted both IRES-EGFP- and
HOX-A10-IRES-EGFP-transduced CD14+ EGFP+ cells
after 9 days of culture under monocyte culture conditions (Figure
4A) and then cultured the cells for 4 days in the presence of GM-CSF, TNF-
Granulocytic differentiation.
Differentiation of granulocytes from retrovirally transduced CB
progenitors was studied by culturing the cells under the same conditions used for monocytic differentiation, except that GM-CSF was
replaced with G-CSF. Cultures were initiated with the same progeny of
transduced cells used for monocytic differentiation (Figure 3A), and
analysis was done after 2 weeks of culture. In contrast to the results
under the monocyte culture conditions, the fraction of
EGFP+ cells was less increased in
HOX-A10-IRES-EGFP-transduced cultures compared with control transduced
cultures (Figure 5A). Moreover, the
fraction of CD15+ cells was reduced in these cells
overexpressing HOX-A10, resulting in a one-third reduction
in the absolute number of granulocytes generated from
HOX-A10-transduced CB progenitors compared with controls
(Table 1). Wright-Giemsa staining on sorted CD15+
EGFP+ cells from both cultures showed that virtually all
CD15+ cells were granulocytic cells at different stages of
development. However, in HOX-A10-IRES-EGFP-transduced
CD15+ cells (Figure 5C), aside from an increase in cells
with blastlike morphologic features, virtually no mature granulocytes
with segmented nuclei were detected. In contrast, in
IRES-EGFP-transduced CD15+ cells, mature granulocytes were
clearly present (Figure 5B). In addition, the fraction of
HLA-DR-positive cells was significantly higher in the
HOX-A10-IRES-EGFP-transduced cells than in control transduced cells
(Figure 5A). Most of these cells were CD14+ (data not
shown), thus showing that there was an enhanced differentiation of
HOX-A10-overexpressing CB progenitors into the monocytic
differentiation pathway in the granulocyte culture conditions.
Together, these results show that overexpression of HOX-A10
enhances differentiation of CB progenitors into monocytic cells and
allows their differentiation into granulocytic precursor cells but that
final maturation of the granulocytic cells is blocked.
Most studies of homeobox genes have focused on their role in embryogenesis and oncogenesis. In this study, we investigated the effect of overexpression of HOX-A10 on differentiation of human stem cells into the myeloid and lymphoid lineages. We found that development of T, B, and NK cells is severely hampered in cells expressing high levels of HOX-A10, thus showing that continuous expression of this gene is detrimental to lymphoid development. In contrast, overexpression of HOX-A10 had various effects on differentiation of stem cells toward the myeloid lineage. Generation of monocytic cells was greatly enhanced and the cells were able to differentiate into dendritic cells. On the other hand, development of immature granulocytes was reduced only slightly, but their final maturation was blocked completely. Differentiation of stem cells toward the lymphoid lineage was severely hampered by continuous expression of HOX-A10. This was indicated by 2 different findings. First, there was a reduction in the fraction of EGFP+ cells in HOX-A10-IRES-EGFP-transduced cultures compared with control transduced cultures. This was most pronounced during T-cell differentiation in FTOCs but also clearly occurred under the NK-cell and B-cell culture conditions. Second, of the remaining EGFP+ cells, fewer HOX-A10-IRES-EGFP-transduced cells than control transduced cells differentiated into lymphocytes. This shows that lymphoid development from CB stem cells that overexpress HOX-A10 is reduced by a defect in both proliferation/survival and differentiation. Different effects on myeloid development were observed. Stem cells transduced with HOX-A10-IRES-EGFP encoding retrovirus showed slightly less proliferation/survival in cultures containing G-CSF compared with control transduced progenitors. This was accompanied by a small reduction in differentiation toward CD15+ cells, suggesting a slight reduction in granulocyte differentiation from HOX-A10-IRES-EGFP-transduced CB precursors. However, granulocytic cells derived from HOX-A10-IRES-EGFP-transduced stem cells were clearly immature, since virtually no cells present had segmented nuclei. Thus, although development of granulocytic precursor cells decreased only slightly, these cells were blocked from undergoing final maturation toward polymorphonuclear leukocytes. Monocytic differentiation was the only hematopoietic pathway analyzed that was not reduced by continuous expression of HOX-A10; in fact, differentiation toward this lineage was enhanced. This resulted from augmentation of proliferation/survival, since the fraction of EGFP+ cells increased more in HOX-A10-IRES-EGFP-transduced cultures than in controls, but also from increases in differentiation, since the percentage of CD14+ cells was higher in cells that continuously expressed HOX-A10. Wright-Giemsa staining showed that these CD14+ monocytic cells contained mature monocytes and some macrophages. Under appropriate culture conditions, these cells were able to differentiate into mature dendritic cells. Therefore, all these results suggest that HOX-A10 is a transcription factor that can drive hematopoietic precursor cells toward the monocytic differentiation pathway but not toward other hematopoietic lineages. Using an in vivo model, Buske et al27 showed that HOX-A10-IRES-EGFP-transduced CB progenitors contribute more to the myeloid lineage and less to the lymphoid lineage. In regard to lymphoid development, however, they analyzed only B-cell differentiation. Here, we showed that total lymphoid development was hampered, since differentiation of both T cells and NK cells was also reduced. Buske et al27 used CD15 as a marker for total myeloid differentiation and found that the fraction of CD15+ cells in HOX-A10-IRES-EGFP-transduced cells was greater than that in IRES-EGFP-transduced cells. This finding is in agreement with our results, since we found that lymphoid development was much more affected than myeloid development. However, CD15 alone is not sufficient as a marker for development of all myeloid lineages. Although Buske et al27 showed that terminal myeloid differentiation is inhibited by enforced expression of HOX-A10, we found that this is indeed the case for granulocytic differentiation, but that monocytic differentiation was not affected; in contrast, it was enhanced by overexpression of HOX-A10. This finding of enhanced monocytic differentiation is in agreement with results of a study showing that enforced expression of HOX-A10 facilitates differentiation of myelomonocytic U937 cells into monocytes/macrophages.34 It would be interesting to see whether generation of CD14+ monocytic cells is also enhanced in vivo.27 Because HOX-A10 belongs to a large family of transcription
factors, an intriguing question is which genes are regulated by this
protein. However, data on target genes of HOX proteins in general are limited. We showed that HOX-A10-IRES-EGFP-transduced hematopoietic precursor cells respond differently to various
combinations of cytokines and that both proliferation/survival and
differentiation are affected. Given the high level of responsiveness of
HOX-A10-IRES-EGFP-transduced progenitors to GM-CSF, it might be that
HOX-A10 is involved in regulating expression of the
corresponding receptor and perhaps of downstream signal-transduction
proteins. Alternatively, HOX-A10 itself may be a downstream
effector of the GM-CSF receptor and excess HOX-A10 mimics
this signaling pathway. It is interesting in this context that on
interferon- A summary of the effects on hematopoietic differentiation of enforced
expression of HOX-A10 in CB precursor cells as observed by
Buske et al27 and described here is provided in Figure
6. In accordance with the most widely
accepted scheme for hematopoiesis,36 the first option for
differentiating hematopoietic stem cells is to become either a lymphoid
or a myeloid multipotent progenitor. Lymphoid differentiation is
severely hampered by enforced expression of HOX-A10. This
could be due to inhibition of differentiation of stem cells toward the
common lymphoid precursor (CLP) or an inability of the CLP to
differentiate into mature lymphocytes. This question is currently
difficult to address because the human CLP has not been well
characterized, despite several studies in this area.37,38
In all situations, however, HOX-A10 seems to direct
progenitor cells toward the myeloid lineage. Along this differentiation
pathway, the common myeloid precursor can differentiate into the
megakaryocyte/erythrocyte pathway or the granulocyte/monocyte pathway.
Buske et al27 showed that development of erythrocytes is
severely inhibited by overexpression of HOX-A10. The effect of continuous HOX-A10 expression on the development of
megakaryocytes in humans has not been analyzed, but studies in mice by
Thorsteinsdottir et al30 found that such expression results
in an expansion of megakaryocyte blast cells. Whether this also occurs
in humans is not known, since differences among species can exist.
Indeed, overexpression of HOX-A10 in human stem cells caused
an inhibition of erythrocyte differentiation, but this was not the case
in murine cells.27,30 Analyses of granulocyte/monocyte
differentiation showed that terminal granulocytic differentiation was
inhibited whereas monocytic differentiation was enhanced. Therefore, at this lineage-decision point, it appears that HOX-A10 drives
the precursor cells toward the monocytic lineage.
All these data and the fact that expression of HOX-A10 is restricted to multipotential precursor cells and early myeloid progenitors show that HOX-A10 can act as a key regulator of hematopoietic stem cell differentiation. In the light of the crucial role of HOX genes during embryonic development, it is clear that this gene family has a similar important function during normal hematopoiesis. However, many members of the family require further investigation and target genes of HOX proteins must be identified.
We thank José De Bosscher, Evelien Naessens, Katrien Goeman, Koen Hugelier, Caroline Van Geyt, Veronique De Backer, Greet De Smet, Caroline Collier, and Achiel Moerman for technical assistance; An De Creus and Tessa Kerre for expert scientific advice; Dr Jan Philippé for help with Wright-Giemsa staining and analysis; the Department of Respiratory Diseases, Ghent University Hospital, for microscopical analysis; the Department of Obstetrics, Ghent University Hospital, for the CB samples; Dr L. Coulembel (Institut Gustave Roussy, Villejuif, France) and Kirin Brewery (Tokyo, Japan) for the MS-5 stromal cell line; Dr H. Spits (The Netherlands Cancer Institute, Amsterdam) for the gift of the IRES-EGFP construct; and Dr G.P. Nolan (Stanford University School of Medicine, CA) for the gift of the Phoenix-NA packaging cell line and the LZRS vector.
Submitted July 23, 2001; accepted October 17, 2001.
Supported by grants from Ghent University; the Flanders Institute for Biotechnology; and the Fund for Scientific Research Flanders, Belgium.
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: Georges Leclercq, Ghent University Hospital, 4 Blok A, De Pintelaan 185, B-9000 Ghent, Belgium; e-mail: georges.leclercq{at}rug.ac.be.
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© 2002 by The American Society of Hematology.
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  T. Taghon, I. Van de Walle, G. De Smet, M. De Smedt, G. Leclercq, B. Vandekerckhove, and J. Plum Notch signaling is required for proliferation but not for differentiation at a well-defined {beta}-selection checkpoint during human T-cell development Blood, April 2, 2009; 113(14): 3254 - 3263. [Abstract] [Full Text] [PDF]
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P. Van Vlierberghe, M. van Grotel, J. Tchinda, C. Lee, H. B. Beverloo, P. J. van der Spek, A. Stubbs, J. Cools, K. Nagata, M. Fornerod, et al. The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia Blood, May 1, 2008; 111(9): 4668 - 4680. [Abstract] [Full Text] [PDF]
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M. Magnusson, A. C. M. Brun, N. Miyake, J. Larsson, M. Ehinger, J. M. Bjornsson, A. Wutz, M. Sigvardsson, and S. Karlsson HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development Blood, May 1, 2007; 109(9): 3687 - 3696. [Abstract] [Full Text] [PDF]
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M. De Smedt, I. Hoebeke, K. Reynvoet, G. Leclercq, and J. Plum Different thresholds of Notch signaling bias human precursor cells toward B-, NK-, monocytic/dendritic-, or T-cell lineage in thymus microenvironment Blood, November 15, 2005; 106(10): 3498 - 3506. [Abstract] [Full Text] [PDF]
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V. Stove, E. Naessens, C. Stove, T. Swigut, J. Plum, and B. Verhasselt Signaling but not trafficking function of HIV-1 protein Nef is essential for Nef-induced defects in human intrathymic T-cell development Blood, October 15, 2003; 102(8): 2925 - 2932. [Abstract] [Full Text] [PDF]
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A. Thompson, M. F. Quinn, D. Grimwade, C. M. O'Neill, M. R. Ahmed, S. Grimes, M. F. McMullin, F. Cotter, and T. R. J. Lappin Global down-regulation of HOX gene expression in PML-RARalpha + acute promyelocytic leukemia identified by small-array real-time PCR Blood, February 15, 2003; 101(4): 1558 - 1565. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
). Original magnification B-C,
× 200.
and T cell receptor 
5' cluster side and proliferative function.
J Immunol.
1996;157:2462-2469