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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-01-0220.
HEMATOPOIESIS
From the Terry Fox Laboratory, British Columbia Cancer
Agency, and the Department of Medical Genetics and Department of
Medicine, University of British Columbia, Vancouver, British Columbia,
Canada; the GSF-National Research Center for Environment and Health,
and Department of Medicine III, Grosshadern, Ludwigs-Maximilian
University Munich, Munich, Germany; INSERM U421, Faculté de
Médecine, Créteil, France; and Laboratory of Molecular
Genetics of Hemopoietic Stem Cells, Clinical Research Institute of
Montreal, Montreal, Quebec, Canada.
Identification of the molecular mechanisms that can promote human
hematopoietic stem cell amplification is a major goal in experimental
and clinical hematology. Recent data indicate that a variety of
regulatory molecules active in early development may also play a role
in the maintenance of hematopoietic stem cells with repopulating
activity. One important class of early developmental genes determining
hematopoietic development are homeobox transcription factors. Here, we
report that retrovirally mediated expression of the homeobox gene
HOXB4 rapidly triggers an increase in the number of
human hematopoietic cord blood cells with stem cell and progenitor cell
properties detected both by in vitro and in vivo assays. This growth
enhancement extended across primitive myeloid-erythroid and B-lymphoid
progenitors but did not lead to alterations in the balance of
lymphomyeloid reconstitution in vivo, suggesting that HOXB4
does not affect control of end-cell output. These findings reveal
HOXB4 as a novel, positive regulator of the primitive
growth activity of human hematopoietic progenitor cells and underline
the relevance of early developmental factors for stem cell fate decisions.
(Blood. 2002;100:862-868) The remarkable proliferative capacities of
primitive undifferentiated cells within the hematopoietic system
provide for the lifelong maintenance of blood cell production and an
extensive regenerative capacity. Identification of extrinsic and
intrinsic regulators that control the self-renewal, lineage commitment
and initial differentiation processes within these cells remains a key
goal of experimental and clinical hematology.1-3 Molecular regulators influencing hematopoietic stem cell expansion have attracted
particular attention because of the importance of these cells for a
variety of clinical applications, including hematopoietic stem
cell-based gene therapy and rescue of the hematopoietic system after
myeloablative therapeutic strategies. Indeed, considerable progress has
been made in delineating and exploiting the properties of multiple
hematopoietic cytokines to achieve expansion of primitive hematopoietic
cells in vitro.4-8 Expansion of both murine and human
hematopoietic stem cells (HSCs) with long-term repopulating ability,
however, is still limited. Recent findings intriguingly point to the
possibility that regulatory molecules known for their growth-promoting
roles in early developmental processes may also affect HSC activity.
These include growth factors such as bone morphogenetic protein (BMP),
which plays a pivotal role in the patterning of the embryonic ventral
mesoderm and in inducing hematopoiesis from mesodermal tissue, and
sonic hedgehog (SHH), a key segment polarity and patterning gene in the
embryo.9-12
Homeobox transcription factors, first recognized as an evolutionarily
conserved gene family critical to the control of embryonic development,
have emerged as another important class of developmental genes
determining early hematopoietic development.13,14 Multiple HOX family members are expressed in the most primitive hematopoietic stem cell-enriched populations, whereas their expression is
consistently down-regulated to undetectable levels in terminally
differentiating CD34 Retroviral constructs
Isolation and transduction of human cells
In vitro progenitor assays
Six-week long-term culture-initiating cell (LTC-IC) assays were carried out using pre-established irradiated murine fibroblasts genetically engineered to produce human IL-3, G-CSF, and SF as feeder layers.25 Both bulk assays and limiting dilution assays, initiated 2 days and 2 to 7 days after termination of transduction, respectively, were performed. In the latter case, cell numbers ranging from 1 to 12 800 GFP+ control or HOXB4-GFP+ transduced cells were sorted into 96-well plates (Nunc, Naperville, IL) and LTC-IC frequencies were calculated using Poisson statistics and the method of maximum likelihood with the assistance of the L-calc software (Stem Cell Technologies). To set up liquid suspension cultures, transduced GFP+ cells were placed in the same cytokine-supplemented serum-free medium described above and then aliquots removed at the times indicated for CFC assays. B-cell progenitor activity was assessed by plating 2 × 104 GFP+CD34+ cells on murine MS-5 cells in RPMI 1640 with 10% fetal calf serum (FCS) and 5% human AB serum with either 50 ng/mL SF, 10 ng/mL IL-2, and 10 ng/mL IL-15 (R & D Systems, Minneapolis, MN) or with these same factors plus 10 ng/mL IL-7 (R & D Systems), 100 ng/mL FL, and 50 ng/mL thrombopoietin (TPO; Genentech, San Francisco, CA), conditions permissive for B-lymphoid development in addition to varying degrees of myeloid cell development. After 3 or 6 weeks, both adherent and nonadherent cells were collected and analyzed by FACS for the expression of CD34, CD38, myeloid (CD15, CD33), lymphoid (CD3, CD19, CD20), erythroid (glycophorin A [GlyA], CD71), and megakaryocytic (CD41) and natural killer (NK) cell (CD56) markers (see below). In vivo assays The NOD/LtSz-scid/scid (NOD/SCID) mice were bred and maintained in the animal facility of the British Columbia Cancer Research Centre (Vancouver, BC, Canada) in microisolator cages containing autoclaved food and water. Test cells were injected intravenously into sublethally irradiated mice (350 cGy from a 137Cs source given at 6-12 weeks of age) and marrow cell aspirates performed 3, 6, and 10 weeks later.19 Mice were killed 6 to 18 weeks after transplantation, and the cells from both tibiae and femurs of each mouse collected for additional analyses. The absolute number of cells in the marrow of each mouse was calculated assuming that the contents of both femurs and both tibiae represent 25% of the total marrow.Flow cytometry Cells were suspended in cold Hanks balanced salt solution, supplemented with 5% pooled normal human serum (HBSS, Stem Cell Technologies) and were then incubated with an antimouse Fc receptor antibody 2.4G2 to block nonspecific antibody binding. The percent positive cells was determined after excluding nonviable (propidium iodide [PI]+) cells and at least 99.9% of cells labeled with isotype control antibodies. Separate aliquots of cells were stained for 30 minutes at 4°C, with the antihuman CD45-phycoerythrin (PE; Becton Dickinson) and antihuman CD71-PE antibodies (OKT9) to quantitate the total number of human cells present (CD45+/71+), with antihuman CD34 8G12-Cy5, antihuman CD19-PE, and antihuman CD20-PE (Becton Dickinson) to quantitate the number of human B cells (CD34 CD19+) present, and with antihuman
CD15-PE (Becton Dickinson) to quantitate the number of human myeloid
cells present. A detection limit of more than 20 CD45+
human cells per 2 × 104 cells analyzed and at least 5 human B cells plus at least 5 human myeloid cells per
2 × 104 cells analyzed was used to identify positively
engrafted and lymphomyeloid-engrafted mice,
respectively.19 Additional antibodies used for certain
analyses included antihuman GlyA-PE, 10F7 antihuman CD33-PE (Becton
Dickinson), antihuman CD41a-PE (Pharmacia Biotech, QC, Canada), and
antihuman CD38-PE (Becton Dickinson).
Frequencies of lymphomyeloid stem repopulating (referred to as competitive repopulating units or CRUs)6,7 were calculated from the proportions of mice in a given experiment, or set of identical experiments, that were negative for lymphomyeloid engraftment using Poisson statistics and the method of maximum likelihood with the assistance of the L-calc software (Stem Cell Technologies). Statistical analysis Statistical tests were performed using the Student t test (software STATISTICA 5.1, StatSoft, Tulsa, OK).
Retroviral transduction of HOXB4 in human
Lin CB cells were transduced with
HOXB4 virus-conditioned medium using a 5-day transduction
protocol previously optimized for transduction of the control GFP
vector used here.24 In the present experiments (n = 14),
a mean transduction efficiency of 48% (20%-80%) and 23% (8%-56%)
was achieved with the GFP and B4-GFP virus, respectively, with an
equivalent frequency of CD34+ cells in the GFP+
fraction in both cases (mean, 12%).
HOXB4 expression increases the production of secondary CFCs both in semisolid and liquid suspension cultures As an initial test of possible HOXB4 effects on human hematopoietic cell proliferative potential, we examined the ability of transduced (GFP+) CD34+ CB cells to generate colonies of erythroid and myeloid colonies in standard methylcellulose assays. Primary assays did not show any significant differences in either the total number or type of colonies detected after 14 days (48 ± 6 versus 37 ± 6 CFC/200 cells plated from the HOXB4 and control cells, respectively). However, replating of the cells harvested from these primary cultures into secondary CFC assays revealed 5-fold more CFC for HOXB4 cells compared with controls (mean of 4600 ± 1600 secondary CFC/200 initially plated cells versus 983 ± 580 in the controls; n = 6; P = .03; Figure 2A). Analysis of the types of cells present in the secondary colonies derived from the HOXB4-transduced cells further showed a selective increase in erythroid colonies (P = .03) compared with the control (n = 6; Figure 2B). Notably, in 3 of 6 experiments, there were no detectable secondary erythroid CFC colonies in the control cultures, whereas high numbers were observed in the cultures of HOXB4-transduced cells (> 103) per 200 initially plated cells. Interestingly, both erythroid and myeloid secondary colonies produced showed a normal morphology indistinguishable from those obtained from the cells transduced with the control GFP vector based on microscopic analysis of single plucked Wright-Giemsa-stained colonies and immunophenotyping. Furthermore, enforced HOXB4 expression did not induce formation of secondary blast colonies in any of the experiments in contrast to previous findings with HOXA1019 (data not shown). Thus, constitutive expression of HOXB4 significantly increased the proliferative capacity of transduced human CB cells without apparently blocking terminal differentiation, once it was initiated.
Similar results were obtained when CFC assays were performed on
transduced cells maintained in serum-free liquid suspension cultures.
These showed no significant difference after 1 week in the number of
total nucleated cells present in cultures of HOXB4 or
control GFP-transduced cells
(3.1 × 105 ± 0.8 and
3.4 × 105 ± 0.3 at week 1, respectively). However,
the cultures of HOXB4-transduced cells were found to contain
significantly greater numbers of CFC (compared with cultures of
GFP+ control cells, P < .01, with a
difference ranging from 2-fold after 1 week to 14-fold at 6 weeks
Figure 3, n = 3). This was associated
with a net increase in the number of CFCs present after 6 weeks in the
cultures of HOXB4-transduced cells, whereas in the GFP
control arm, the total number of CFCs present declined about 3-fold
during the same time period (Figure 3). This increase in CFC numbers
compared with the control included myeloid as well as erythroid
progenitors throughout the 6-week duration of the experiments (2.2- versus 5.7-fold increases after 1 week, respectively; 14-fold increase
in myeloid CFCs and 4.2 × 103 erythroid CFCs for
HOXB4 versus none for the control after 6 weeks; Figure 3).
HOXB4 expression amplifies the number of cells with LTC-IC activity The GFP+ CD34+ cells were also assayed for LTC-IC activity to evaluate potential effects of HOXB4 expression on more primitive hematopoietic cells. Bulk LTC-ICs for the detection of clonogenic progenitor cell output were initiated 2 days after termination of transduction (n = 6; Figure 4). HOXB4 LTCs contained nearly 10-fold more CFCs after 6 weeks than the control cultures and more than 90% of the CFCs obtained under LTC conditions were granulopoietic in both experimental arms (data not shown). To determine whether the increased CFC output by HOXB4-transduced cells was due to the number of cells detectable as LTC-IC or to an enhanced output of CFCs per LTC-IC, LTC-IC frequencies were determined by limiting dilution analysis after 4 to 7 days in vitro culture of CD34+/GFP+ or CD34+/HOXB4-GFP+ cells. As shown in Figure 4, the initial LTC-IC frequency of HOXB4-transduced CB cells was significantly increased (~5-fold; P < .001) compared with the control, whereas the progenitor yield per LTC-IC was essentially unchanged. Thus, the major impact of HOXB4 overexpression was a rapid increase in the number of cells with the primitive functional capacity of LTC-ICs.
HOXB4 promotes the development of B cells in vitro To evaluate whether the growth-promoting effects of HOXB4 might extend to the lymphoid pathway, we examined its effect on the generation of B-lineage cells in vitro. Transduced-positive CD34+ CB cells were cultured for 6 weeks on MS-5 fibroblast feeders in media with 2 different growth factor cocktails supportive of B-lymphoid development (SF, IL-2, and IL-15 with or without IL-7, TPO, and FL, respectively) and then assessed by FACS for the number of CD19+ cells present in addition to cells of myeloid and NK lineages using a range of markers. Both conditions also supported a significant level of myeloid cell growth and these constituted the dominant cell type in cultures examined at 3 or 6 weeks with no significant difference observed in either total cell number or CD15+ myeloid cells or CD56+ NK cells. However, as shown in Table 1, under both conditions, the proportion and absolute number of CD19+ B cells present was significantly higher in the cultures of HOXB4-transduced cells (~2- to 8-fold after 3 weeks and 3- to 20-fold after 6 weeks; P < .04). When we analyzed the effect of constitutive HOXB4 expression on generation of CD34+/CD19+ B-cell precursors, the difference was even more pronounced: at week 3, HOXB4 induced a 31-fold (P < .02) and 16-fold (P < .0005) increase with 3 and 6 cytokines, respectively. Thus, in culture conditions permissive for B lymphopoiesis, constitutive expression of HOXB4 promoted production of B cells in sharp distinction to effects observed with HOXA10.19
HOXB4 expression increases the number of cells with repopulating activity detectable in NOD/SCID mice Because we had seen that constitutive expression of HOXB4 significantly amplified the number of cells with LTC-IC activity, we assessed whether HOXB4 would also expand long-term repopulating stem cells using the CRU assay and the xenograft NOD/SCID mouse model. The CRU frequency was determined by limit dilution analysis in 3 cohorts of mice (n = 5) transplanted with CB infected either with the HOXB4 or the GFP virus. Mice were injected with the CB progeny of an original input of lin cells containing 2.5 × 105
CD34+ cells, one fourth or one eighth of this cell number,
respectively, after 24 hours or less in vitro culture after infection.
There was no significant difference in the proportion of
CD34+GFP+ cells after transduction between the
control and B4-GFP transduced CB cells and cells were
injected without any preselection of CD34+ GFP+
cells. The CRU frequency was calculated by Poisson statistics for the
same starting number of GFP+ cells for both experimental
arms at the time point of transplantation taking into account the
number of lymphomyeloid-engrafted mice per dilution. Figure
5A shows the FACS analysis of a
representative mouse from the HOXB4 and GFP cohort after
injection of 2.5 × 105 CD34+ cells 6 weeks
after transplantation with positive lymphomyeloid engraftment.
Strikingly, HOXB4-GFP-transduced CB had a 3-fold increase
and a 4-fold increase in the CRU frequency as determined at weeks 3 and
6 after transplantation, respectively (P = .02; Figure 5B).
In contrast to its effect on the number of primitive repopulating hematopoietic cells, constitutive expression of HOXB4 (as documented by the continued expression of the linked GFP gene at readily detectable levels through the observation period [Figure 5A]) did not alter the normal differentiation program of human progenitor cells in vivo; proportions of engrafted lymphoid (CD19+/CD45+), myeloid (CD15+/CD45+), megakaryocytic (CD41+/CD45+), and erythroid (glycophorin+/CD45+) human cells were determined at weeks 3, 6, and 10 after transplantation by femoral bone marrow aspiration and again at 18 weeks when the mice were killed (n = 3). Constitutive expression of HOXB4 did not alter the proportions of the different lineages at any time point compared to the nontransduced compartment of the HOXB4 mice and the transduced and nontransduced compartment of the control mice. This was confirmed when absolute cell numbers were calculated after sacrificing the animals (data not shown). Thus, enhanced and extended expression of HOXB4 significantly augmented the number of primitive cells with repopulating activity, but did not alter differentiation in vivo.
The characterization of molecular mechanisms to expand human stem cells has major medical implications with respect to therapeutic strategies based on stem cell transplantation. Earlier studies have shown that cytokine-induced proliferation of progenitor-enriched populations is characterized by induction of lineage commitment and terminal differentiation accompanied by a rapid loss of stem cell activity.2,8 An intriguing alternative to use of extrinsic growth factors is to harness the function of intrinsic regulators that may be upstream of cytokine receptor-mediated signal transduction pathways. We now show that constitutive expression of the clustered homeobox gene HOXB4 can rapidly lead to increased numbers in vitro of cells detectable at the level of NOD/SCID repopulating cells, LTC-ICs, and committed clonogenic progenitors. Furthermore, enforced expression did not block terminal differentiation or changed lineage distribution in vivo. These data thus reveal HOXB4 as a novel growth stimulatory regulator of primitive human hematopoietic cells. Intriguingly, constitutive expression of HOXB4 induced a rapid and significant increase in the number of cells with LTC-IC activity because cells were plated into the LTC-IC detection assay as soon as 48 hours as well as 7 days after transduction. This rapid onset of a HOXB4 effect on the number of human primitive hematopoietic cells was confirmed in the CRU assay in which HOXB4-expressing cells were injected into NOD/SCID mice within 24 hours after transduction and by this time CRU numbers were increased by some 4-fold over control transduced cells, as determined by limit dilution assay. Constitutive expression of HOXB4 might influence the growth kinetics of primitive progenitor cells, for example, by shortening the doubling time, or enhance self-renewal of HSCs. However, these data also suggest the interesting possibility of recruitment of cells, which have already left the primitive progenitor compartment, thus enhancing the number of cells detectable as LTC-ICs or CRUs. Both explanations are consistent with previous findings in the murine system, in which constitutive expression of HOXB4 shortened the doubling time of hematopoietic progenitor cells and induced an accelerated regeneration of CRUs in lethally irradiated recipient mice (eg, 25% of normal CRU level versus 0.2% in the control 2 weeks after transplantation).22 The striking effect on CRU numbers observed after only 24 hours after infection culture will also make it of interest to further assess in future experiments, the potential to achieve significant in vitro expansion of human CRUs with more extended culture periods as has recently been demonstrated in the murine model.26 The impact of the 3'-located Antennapedia-like HOXB4 on early human hematopoietic development contrasts with effects so far described for other HOX genes on human hematopoietic progenitor cells. Thus, constitutive expression of the 5'-located Abdominal-B-like Hox gene HOXA10 resulted in competitive growth advantage of myeloid cells and blockage of differentiation with formation of blast colonies in vitro and ex vivo.19 The differential impact of HOXB4 and HOXA10 is furthermore highlighted in their opposite effects on B-cell differentiation and erythropoiesis with HOXA10 overexpression impairing B-cell development, whereas HOXB4 induced a marked enhancement of B lymphopoiesis.19 The mechanisms that lead to the differential gene effect are not known, but data point to a pivotal role of the TALE homeobox genes and Hox cofactors Meis1 and Pbx1 for the specification of Hox gene effects in the hematopoietic system. Importantly, HOXB4 cannot interact directly with Meis1 but only with Pbx1 in contrast to HOXA10, which can interact directly with both proteins.13,18 HOXA5 overexpression, also in contrast to HOXB4, has been shown to impair erythroid differentiation and enhance formation of colonies with undifferentiated blasts.27 Furthermore, HOXB7 overexpression has been associated with the induction of persistent proliferation of a blast population in vitro, but without modifying the total number of hematopoietic progenitor cells.28 The effect of HOXB4 is somewhat reminiscent of effects recently reported for BMP-4 or SHH, which were originally described for their essential role in early developmental cell fate decisions9,11; both factors are able to maintain or increase human stem cells with repopulating capacity in vitro without altering lineage differentiation.10-12 Interestingly, both genes are linked to homeobox genes: SHH induces expression of Bmp-4 and of the 5'-located Hox genes Hoxd-11 as well as Hoxd-13, while Hoxd-12 regulates SHH expression in a positive feedback loop.29,30 BMP-4 regulates Hoxc-8 expression31 and acts together with the homeobox gene Mix.1 in inducing embryonic hematopoiesis.9 Our data characterize HOXB4 as a potentially powerful positive mediator of the maintenance and expansion of human stem cells and provide a new avenue to manipulate and further elucidate the basis for human hematopoietic stem cell fate decisions. These in vitro and in vivo models will facilitate the dissection of the molecular mechanisms underlying the HOXB4-induced stem cell proliferation in the human cellular milieu. Furthermore, they will allow tests of whether stem cell amplification by HOXB4 can be further augmented by mutating distinct motifs of the gene such as the PBX YPWM interacting motif as reported previously in the murine system.32
The expert assistance of Ms Patty Rosten for technical support and Ms Colleen MacKinnon in the preparation of the manuscript is gratefully acknowledged.
Submitted January 25, 2002; accepted March 29, 2002.
Prepublished online as Blood First Edition Paper, April 30, 2002; DOI 10.1182/blood-2002-01-0220.
Supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Terry Fox Run; and the National Institutes of Health (grants no. HL65430 and DK48642). C.B. was supported by a grant from the Deutsche Forschungsgesellschaft (DFG), Bonn, Germany, and M.F.-B. by a grant from the Deutsche Krebshilfe, Bonn, Germany.
C.B. and M.F.-B. contributed equally to this article.
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: R. Keith Humphries, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, BC, V5Z 1L3, Canada; e-mail: khumphri{at}bccancer.bc.ca.
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H. Abdel-Azim, Y. Zhu, R. Hollis, X. Wang, S. Ge, Q.-L. Hao, G. Smbatyan, D. B. Kohn, M. Rosol, and G. M. Crooks Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl Blood, April 15, 2008; 111(8): 4064 - 4074. [Abstract] [Full Text] [PDF]
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 F. Ahmed, N. Arseni, H. Glimm, W. Hiddemann, C. Buske, and M. Feuring-Buske Constitutive Expression of the ATP-Binding Cassette Transporter ABCG2 Enhances the Growth Potential of Early Human Hematopoietic Progenitors Stem Cells, March 1, 2008; 26(3): 810 - 818. [Abstract] [Full Text] [PDF]
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A. Rizo, B. Dontje, E. Vellenga, G. de Haan, and J. J. Schuringa Long-term maintenance of human hematopoietic stem/progenitor cells by expression of BMI1 Blood, March 1, 2008; 111(5): 2621 - 2630. [Abstract] [Full Text] [PDF]
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R. Haddad, F. Pflumio, I. Vigon, G. Visentin, C. Auvray, S. Fichelson, and S. Amsellem The HOXB4 Homeoprotein Differentially Promotes Ex Vivo Expansion of Early Human Lymphoid Progenitors Stem Cells, February 1, 2008; 26(2): 312 - 322. [Abstract] [Full Text] [PDF]
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N. Miyake, A. C.M. Brun, M. Magnusson, K. Miyake, D. T. Scadden, and S. Karlsson HOXB4-Induced Self-Renewal of Hematopoietic Stem Cells Is Significantly Enhanced by p21 Deficiency Stem Cells, March 1, 2006; 24(3): 653 - 661. [Abstract] [Full Text] [PDF]
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H. J. Lawrence, J. Christensen, S. Fong, Y.-L. Hu, I. Weissman, G. Sauvageau, R. K. Humphries, and C. Largman Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells Blood, December 1, 2005; 106(12): 3988 - 3994. [Abstract] [Full Text] [PDF]
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 J. M. Bjornsson, N. Larsson, A. C. M. Brun, M. Magnusson, E. Andersson, P. Lundstrom, J. Larsson, E. Repetowska, M. Ehinger, R. K. Humphries, et al. Reduced Proliferative Capacity of Hematopoietic Stem Cells Deficient in Hoxb3 and Hoxb4 Mol. Cell. Biol., June 1, 2003; 23(11): 3872 - 3883. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
