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TRANSLATIONAL AND CLINICAL RESEARCH

Constitutive Expression of the ATP-Binding Cassette Transporter ABCG2 Enhances the Growth Potential of Early Human Hematopoietic Progenitors

^aDepartment of Medicine III, University of Munich-Grosshadern, Munich, Germany;
^bClinical Cooperative Group Leukemia, Helmholtz Zentrum Muenchen-German Research Center for Environmental Health, Munich, Germany;
^cDepartment of Translational Oncology, National Center for Tumor Diseases and German Cancer Research Center, Heidelberg, Germany

Key Words. ABCG2 • Human progenitor cells • Hematopoiesis

Correspondence: Correspondence: Michaela Feuring-Buske, M.D., Department of Medicine III, Ludwig-Maximilans-University Munich-Grosshadern, Clinical Cooperative Group, "Leukemia," Helmholtz Zentrum Muenchen-German Research Center for Environmental Health, Marchioninistrasse 25, 81377 Munich, Germany. Telephone: 49-89-7099-402; Fax: 49-89-7099-400; e-mail: feuring{at}gsf.de

Received on July 4, 2007; accepted for publication on November 17, 2007.

First published online in STEM CELLS EXPRESS  November 29, 2007.

	ABSTRACT

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The ATP-binding cassette transporter, ABCG2, is a molecular determinant of the side population phenotype, which is enriched for stem and progenitor cells in various nonhematopoietic and hematopoietic tissues. ABCG2 is highly expressed in hematopoietic progenitors and silenced in differentiated hematopoietic cells, suggesting a role of ABCG2 in early hematopoiesis. To test whether ABCG2 is involved in human hematopoietic development, we retrovirally transduced umbilical cord blood-derived early hematopoietic cells and analyzed hematopoiesis in vitro and in vivo. ABCG2 increased the number of clonogenic progenitors in vitro, including the most primitive colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte, by twofold (n = 14; p < .0005). Furthermore, ABCG2 induced a threefold increase in the replating capacity of primary colonies (n = 9; p < .01). In addition, ABCG2 impaired the development of CD19⁺ lymphoid cells in vitro. In transplanted NOD/SCID mice, the ATP-binding cassette transporter decreased the number of human B-lymphoid cells, resulting in an inversion of the lymphoid/myeloid ratio. ABCG2 enhanced the proportion of CD34⁺ progenitor cells in vivo (n = 4; p < .05) and enhanced the most primitive human progenitor pool, as determined by limiting dilution competitive repopulating unit assay (p < .034). Our data characterize ABCG2 as a regulatory protein of early human hematopoietic development.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Hematopoietic stem cells (HSC), residing at the top of the hematopoietic hierarchy, possess the unique abilities to self-renew and produce progenitors that give rise to differentiated blood cells throughout the life of an individual. This ordered process of self-renewal and differentiation depends on a complex regulatory system, involving various transcription factors, cytokines, and interaction with the hematopoietic niche [1]. One of the key features of HSC and early hematopoietic progenitors, as well as other tissue stem cells, is the expression of high levels of ATP-binding cassette (ABC) transporter proteins, such as MDR1 and ABCG2 [2]. ABCG2 or Breast Cancer resistance protein, a member of the G subfamily of ABC proteins, is known to transport a wide variety of drugs and dyes out of the cells, including the fluorescent Hoechst 33342. This Hoechst efflux property has been increasingly exploited to isolate Hoechst-low cells that are termed the side population (SP), using fluorescence-activated cell sorting [3]. SP cells from several different tissues have been demonstrated to have stem cell and early progenitor characteristics [3–6]. During hematopoiesis, ABCG2 is highly expressed in the CD34⁺CD38^– stem cell and progenitor-enriched compartment, with downregulation of its expression in differentiated cells [7, 8]. This expression pattern was shown to be typical for genes involved in the regulation of early hematopoiesis, suggesting an important role of ABCG2 in earlier stages of hematopoiesis.

ABCG2 expression is also found in acute myeloid leukemia (AML), which originates from transformed hematopoietic stem and progenitor cells. Several studies linked the expression of ABCG2 to therapeutic outcome in patients with AML; two independent studies in adult AML patients showed no correlation between ABCG2 expression and complete remission (CR) rate, but with overall survival being inferior in patients with high ABCG2 expression [9, 10]. In 59 children with de novo AML, a higher ABCG2 expression was observed in patients who did not reach CR [11]. It was reported that ABCG2 is frequently upregulated in patients with AML at relapse and that expression of ABCG2 is associated with secondary AML in elderly patients [12, 13]. In another study, ABCG2-positive AML cases showed an increased risk of relapse [14]. Aberrant overexpression of ABCG2 has also been demonstrated in CD34⁺ CML cells [15]. Furthermore, the SP phenotype, in combination with expression of CD34 and absence of CD38, was associated with normal stem cells in patients with AML, in contrast to leukemic stem cells, which were SP-negative CD34⁺CD38^– or SP-positive CD34^–CD38^– [16]. Taken together, these studies suggest that upregulated ABCG2 expression might be associated with malignant stem cell behavior in myeloid leukemias.

So far, there are no data analyzing the impact of elevated ABCG2 expression on early human hematopoiesis. In a previous study, expression of human ABCG2 in mouse bone marrow (BM) cells expanded the SP and caused a reduction in the mature cell numbers [2]. Of note, Abcg2-knockout mice did not display any defect in steady state hematopoiesis [8], suggesting that in the overexpression studies, ABCG2 expression in differentiating cells, in which expression of the transporter protein is normally silenced, probably had a deleterious effect. Studies on Mdr1, a related ABC transporter, have shown that it can confer drug resistance to hematopoietic cells in Mdr1-transgenic mice [17]. It was further shown that retrovirally driven expression of the ABC transporter induced myeloproliferative disorder or leukemia in a murine bone marrow transplantation [18–20]. However, it could be demonstrated that retroviral insertional mutagenesis played a key role in the development of leukemias in this model [20]. In addition, other murine and primate models did not show development of any hematopoietic disturbances after retroviral or transgenic expression of the ABC transporter, indicating that, most likely, aberrant expression of Mdr1 on its own had no leukemogenic potential [17, 21, 22].

In this study, we investigated the role of ABCG2 overexpression on human hematopoietic development. For this, we retrovirally engineered stem and progenitor cells derived from human umbilical cord blood (CB) to constitutively express ABCG2 and analyzed the impact on hematopoiesis in vitro and in vivo. Our data demonstrate that ABCG2 affects hematopoiesis in these model systems at the level of early human progenitor cells.

	MATERIALS AND METHODS

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Collection and Enrichment of Human CD133⁺ Cells
Umbilical CB was collected in heparinized syringes according to institutional guidelines following normal full-term deliveries. Informed consent was obtained in all cases. Low-density CB mononuclear cells were collected by separation on Pancoll (PAN Biotech, Aidenbach, Germany, http://www.pan-biotech.com). CD133⁺ cells were enriched from CB mononuclear fraction using immunoselection with the miniMACS system (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). Enriched samples were cryopreserved in fetal bovine serum (FBS) and 10% dimethyl sulfoxide and stored in liquid nitrogen until use.

Retroviral Vectors
A 2.2-kilobase AfeI-XhoI fragment coding for the full-length ABCG2 gene (Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, http://www.dkfz.de/en/forschung/index.html) was subcloned into the HpaI-XhoI sites of murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-enhanced yellow fluorescent protein (EYFP) retroviral expression vector (kindly provided by R.K. Humphries, Terry Fox Laboratory, University of British Columbia, Vancouver, BC, Canada). The construct generated (ABCG2-YFP) was used for the preparation of stable packaging cell lines. As a control, the empty MSCV-IRES-EYFP (YFP) was used (Fig. 1A).

View larger version (19K): [in this window] [in a new window]   Figure 1. Retroviral constructs and gene transfer. (A): Schematic representation of the retroviral constructs used for transduction of human lin– cells. (B): K562 cells were transduced with ABCG2-YFP virus (K562-ABCG2) or YFP control virus (K562-YFP). After 48 hours, transduced cells were fixed, permeabilized, and stained using anti-ABCG2 antibody. A PE-labeled secondary antibody (goat anti-mouse antibody) was used to detect ABCG2-positive cells by fluorescence-activated cell sorting (FACS). (C): Reverse transcription-polymerase chain reaction analysis of retrovirally transduced K562 and PG13 packaging cell lines. (D): Gene transfer efficiencies of ABCG2-YFP-transduced and YFP (control)-transduced CD133+ cells were analyzed 48 hours after final transduction for the expression of YFP, using FACS to determine the gene transfer efficiency. (E): Representative dot plots, showing the CD34+ YFP+ cell populations that were sorted for experiments in both experimental arms (indicated are the sort gates, R2). Abbreviations: EYFP, enhanced yellow fluorescent protein; IRES, internal ribosome entry site; LTR, long-terminal repeats; PE, phycoerythrin; SCF, stem cell factor; YFP, yellow fluorescent protein.

Prestimulation and Transduction of Human Cells
Frozen CD133⁺ cells were thawed and prestimulated before transduction. Cells at 1 x 10⁵ cells per milliliter were cultured for 48 hours in serum-free medium (SFM) consisting of Iscove's modified Dulbecco's medium (IMDM; Gibco, Karlsruhe, Germany, http://www.invitrogen.com) supplemented with 20% bovine serum albumin, insulin, and transferrin (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com), 10^–4 M mercaptoethanol (Sigma-Aldrich), and 40 µg/ml low-density lipoproteins (Sigma-Aldrich). The following recombinant human cytokines were added to the SFM: 100 ng/ml Flt-3 ligand, 100 ng/ml steel factor, 20 ng/ml interleukin (IL)-3, 20 ng/ml IL-6, and 20 ng/ml granulocyte colony-stimulating factor (G-CSF) (ImmunoTools, Friesoythe, Germany, http://www.immunotools.de). After 48 hours, cells were resuspended in filtered virus-conditioned medium (VCM) from producer cells, supplemented with the same cytokine combination, and plated on tissue culture dishes (Corning Life Sciences, Corning, NY, http://www.corning.com) that were preloaded twice with VCM, each time for 30 minutes. Protamine sulfate (5 µg/ml) was added to enhance viral transduction. This procedure was repeated on the next two consecutive days for a total of three transductions. For in vitro studies, aliquots of these cells were transferred to fresh SFM with cytokines and then incubated for an additional 48 hours prior to staining with phycoerythrin (PE)-labeled anti-CD34 antibody and isolation of the YFP⁺/CD34⁺ cells on a FACSVantage (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) (Fig. 1E).

Hoechst Stainings for Side Population Analysis
CB CD133⁺ cells transduced with ABCG2-YFP or YFP were cultured for 2 days in SFM with the same cytokine combination described above. The cells were then harvested and resuspended in Dulbecco's modified Eagle's medium containing 2% FBS and 10 mM HEPES buffer (Sigma-Aldrich). Cells (2 x 10⁶) (comprising YFP-positive and YFP-negative cells after transduction) were stained with 5 µg/ml Hoechst 33342 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) for 120 minutes at 37°C under swirling motion. An aliquot of cells was simultaneously treated with 50 µM Verapamil (Sigma-Aldrich) for the entire Hoechst staining period to block ABCG2-mediated efflux. Cells were spun down and resuspended in cold Hanks' balanced saline solution containing 2% FBS, 10 mM HEPES buffer, and 2 µg/ml propidium iodide. Side population analyses were performed on a BD FACStar instrument (BD Biosciences, San Diego, http://www.bdbiosciences.com) as described previously [3].

Transplantation of Human Cells in NOD/SCID Mice
Eight-week-old NOD-LtSz-scid/scid (NOD/SCID) mice (Taconic, Lille Skensved, Denmark, http://www.taconic.com) were sublethally irradiated with 2.75 Gy 24 hours before injection using a ¹³⁷Cs -source. For transplantation, transduced cells were washed, counted, resuspended in phosphate-buffered saline (PBS), and injected into the lateral tail vein of irradiated mice (300–400 µl/mouse), along with 1 x 10⁶ irradiated (15 Gy) CB mononuclear cells as carrier cells. The mice were sacrificed 8 weeks post-transplantation according to institutional guidelines. BM from the femurs and tibiae were flushed using IMDM containing 10% fetal calf serum (FCS) and 0.2 mM EDTA and collected for analysis of human engraftment.

Flow Cytometry Analyses
Cells harvested from the mouse BM were resuspended in cell-wash buffer (IMDM with 3% FCS, 0.2 mM EDTA) and treated with 1% ammonium chloride solution (20 minutes on ice) for selective removal of red cells. The cells were washed again using cell-wash buffer, passed through a cell strainer (60-µm pore size; BD Biosciences), and analyzed for cell viability by trypan blue exclusion. Approximately 10⁶ cells were resuspended in PBS (with 3% FBS) and incubated on ice for 10 minutes with an anti-mouse IgG Fc-receptor antibody, 2.4G2 (Miltenyi Biotec), to block nonspecific antibody binding. Separate aliquots were then incubated for 30 minutes on ice with a mouse isotype-matched control antibody or antibodies against the following human antigens: CD45-PE, CD34-PE, CD38-APC, CD33-PE and CD15-PE, CD19-APC, CD3-APC, CD41a-PE, and glycophorin A-PE (Gly A). All antibodies were purchased from BD Pharmingen (Heidelberg, Germany, http://www.bdbiosciences.com/index_us.shtml).

In Vitro Progenitor Assays
Hematopoietic colony-forming cells (CFC) were assayed using methylcellulose-based medium (MethoCult H4434; StemCell Technologies). YFP⁺ CD34⁺ cells were diluted with IMDM + 2% FBS to 10 x the final concentration(s) required for plating. For a duplicate assay, 0.3 ml of diluted cells was added to a 3-ml MethoCult tube, which was mixed vigorously. After 5 minutes, 1.1 ml of cell-methylcellulose mixture was dispensed into 35-mm culture dishes using a sterile 3-ml syringe and 16-gauge blunt-end needle. The 35-mm culture dishes were placed in a CO₂ incubator at 37°C and >95% humidity. CFC numbers were evaluated after an incubation period of 12–14 days and distinguished into following classes: blast-forming unit-erythroid/colony-forming unit-erythroid (BFU-E/CFU-E), colony-forming unit-granulocyte, macrophage (CFU-GM), colony-forming unit-macrophage (CFU-M), and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). Secondary CFC assays were performed by replating aliquots of cells obtained by harvesting 14-day-old primary CFC cultures as previously described [23]. Lymphoid cell and natural killer (NK) cell development was assessed by plating 5 x 10⁴ YFP⁺CD34⁺ cells on murine MS-5 cells in RPMI 1640 with 10% FCS and 5% human AB serum with 50 ng/ml stem cell factor (SCF), 10 ng/ml IL-2, and 10 ng/ml IL-15 as previously described [24]. After 3 weeks, both adherent and nonadherent cells were collected and analyzed by fluorescence-activated cell sorting (FACS) for the expression of CD34, myeloid (CD33), lymphoid (CD19, CD10), and NK cell (CD56) markers.

Six-week long-term culture-initiating cell (LTC-IC) limiting dilution 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]. Cell numbers ranging from 1 to 12,800 YFP⁺ control or ABCG2-YFP⁺-transduced cells were sorted out into 96-well plates (Sarstedt, Nuremberg, Germany, http://www.sarstedt.com). LTC-IC frequencies were calculated using Poisson statistics and the method of maximum likelihood with the assistance of the L-calc software (StemCell Technologies).

Intracellular Staining with ABCG2 Antibody
K562 cells (1 x 10⁶) transduced with ABCG2-YFP and YFP viruses were resuspended in 200 µl of Cytofix (BD Biosciences) to fix the cells, incubated for 20 minutes at 4°C, and washed twice with 1 x Permwash buffer (BD Biosciences). The cells were centrifuged at 280g for 5 minutes. The pellet was resuspended in 50 µl of PBS, and 0.2 µg of primary anti-BCRP antibody (Millipore GmbH, Schwalbach, Germany, http://www.millipore.com) was added and incubated for 15 minutes at 4°C. After incubation, the cells were washed twice with 1 x Permwash buffer, resuspended again in 50 µl of PBS, and stained with the fluorochrome (PE)-conjugated goat anti-mouse secondary antibody (BIOMOL International LP, Hamburg, Germany, http://www.biomol.com) for 15 minutes at 4°C in dark. After staining, the cells were washed twice with 1 x Permwash buffer, resuspended in 500 µl of PBS, and analyzed using the FACSCalibur.

Polymerase Chain Reaction
PG13 cells stably transfected with ABCG2-YFP (PG13-ABCG2) and empty vector (PG13-YFP), as well as K562 cells transduced using VCM from the PG13-ABCG2 and PG13-YFP (K562-ABCG2 and K562-YFP respectively), were assessed for ABCG2 mRNA expression. RNA was isolated using TRIzol reagent, and cDNA was prepared using ThermoScript (both reagents from Gibco), according to the manufacturer's instructions. Expression of ABCG2 (forward, 5'-ATTGAAGGCAAAGGCAGATG-3'; reverse, 5'-TGAGTCCTGGGCAGAAGTTT-3') and human β-Actin (forward, 5'-CTTCAACACCCCAGCCAT-3'; reverse, 5'-TAATGTCACGCACGATTTCC-3') was assessed using polymerase chain reaction (PCR) under the following conditions: an initial 4-minute denaturation at 96°C; followed by 30 cycles of 45 seconds of denaturation at 94°C, 45 seconds of annealing at 56°C, and 45 seconds of extension at 72°C; followed by a final extension at 72°C for 10 minutes.

Statistical Analysis
Differences between two groups were assessed by a two-tailed Student's t test (Excel; Microsoft, Redmond, WA, http://www.microsoft.com). Competitive repopulating unit (CRU) frequencies were determined using Poisson statistics and the method of maximum likelihood with the assistance of the L-calc software (StemCell Technologies).

	RESULTS

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Efficient Retroviral Transduction of ABCG2 in Human Hematopoietic Cells
The complete cDNA of ABCG2 was cloned into the bicistronic vector with IRES-EYFP cassette based on the MSCV viral backbone (ABCG2-YFP). The MSCV-IRES-EYFP vector was used as a control (YFP) (Fig. 1A). High-titer clonal viral producer cells were generated from the GALV pseudotyped PG13 packaging cell line for both constructs. Gene expression in transduced K562 cells was demonstrated by reverse transcription-PCR and intracellular staining of ABCG2 protein (Fig. 1B, 1C). Transduction efficiency of human CB-derived cells was determined by measuring the YFP fluorescence 48 hours after the end of transduction (n = 19). Of the CD133⁺ cord blood cells exposed to ABCG2-YFP VCM or the YFP VCM, 23% (± 5%) and 54% (± 4%), respectively, expressed YFP when examined by FACS, demonstrating high-efficiency gene transfer for both vectors (Fig. 1D, 1E). In the experiment shown in Figure 2, 16% of the cells in the ABCG2 arm were successfully transduced (84% stayed YFP-negative after transduction). When the SP was quantified in this mixed population, we observed a 29-fold increase in the SP compared with cells transduced with the YFP control cells despite the fact that only a minority of the cells expressed the ABC transporter (Fig. 2A). However, when we determined the size of the SP only in the successfully transduced compartment in the ABCG2 experimental arm by gating on the ABCG-EYFP cells and compared it with the size of the SP in the YFP-negative fraction, there was a substantial difference with 6.25% SP cells versus 0.02% SP cells, respectively (Fig. 2B). Induction of the SP phenotype by ABCG2 expression was confirmed in the human K562 cell line (supplemental online Fig. 1).

View larger version (25K): [in this window] [in a new window]   Figure 2. ABCG2 expression induces side population in umbilical cord blood-derived CD133+ cells. (A) CD133+ cells were transduced with ABCG2-YFP and YFP alone. Gene transfer efficiency was 16% in the ABCG2 arm and 28% in the YFP arm. Both cell populations were stained with Hoechst 33342 dye after 2 days of culture without preselcting for successfully transduced cells. ABCG2-YFP-transduced cells were also incubated with 5 µM Verapamil to block ABCG2-mediated efflux. The percentage of the side population is indicated. (B): Percentage of SP cells in the transduced (R3) and nontransduced (R4) cell fraction of the ABCG2-YFP experimental arm. Abbreviations: FSC-H, forward scatter-height; YFP, yellow fluorescent protein.

View larger version (10K): [in this window] [in a new window]   Figure 3. Effect of ABCG2 on primary and secondary colony formation. (A): Colonies derived from ABCG2-YFP- and YFP-transduced progenitors. Human cord blood-derived CD34+ cells were transduced with ABCG2-YFP or YFP vector and plated in methylcellulose CFC assays. The frequency and primary colony types are indicated. Columns represent mean (± SEM) frequencies from 14 independent experiments. (B): For secondary colony formation, methylcellulose dishes of primary CFC cultures derived from ABCG2-YFP-and YFP-transduced progenitors were harvested on day 14, and the progenitor cells, resuspended in Iscove's modified Dulbecco's medium 2% fetal bovine serum, were replated in secondary methylcellulose cultures. Secondary colony formation was assessed after 14 days. Mean values of secondary colony formation of nine independent experiments are indicated by columns (± SEM). p values are indicated. Abbreviations: BFU-E/CFU-E, blast-forming unit-erythroid/colony-forming unit-erythroid; CFC, colony-forming cells; CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GM, colony-forming unit-granulocyte, macrophage; CFU-M, colony-forming unit-granulocyte, macrophage.

Overexpression of ABCG2 Impairs the Ability of CB-Derived Hematopoietic Cells to Differentiate Toward B Cells In Vitro
To test whether constitutive expression of ABCG2 affects B-cell differentiation in vitro, 5 x 10⁴ transduced CD34⁺ cells were sorted and cocultured on MS-5 cells along with a cocktail of three cytokines (SCF, IL-2, IL-15) for 3 weeks (n = 3) (Table 1). The majority of cells generated in these cultures were myeloid CD33-positive cells (data not shown). Expression of ABCG2 reduced the total output of CD19⁺ B cells by sixfold (ABCG2-YFP, 7.7 x 10² ± 533; YFP, 5.15 x 10³ ± 3,050 per 5 x 10⁴ CD34⁺ YFP⁺ plated initially; p < .05). ABCG2 did not show any CD34⁺ CD10⁺ cells compared with the control (ABCG2-YFP, 0; YFP, 312 ± 158 per 5 x 10⁴ CD34⁺ YFP⁺ cells plated initially). In contrast, CD56⁺ NK cells were not significantly different in ABCG2-transduced arm (ABCG2-YFP, 5.5 x 10³ ± 3,060; YFP, 8.1 x 10³ ± 3,720 per 5 x 10⁴ CD34⁺ YFP⁺ plated initially).

View larger version (10K): [in this window] [in a new window]   Figure 4. Impact of ABCG2 on CFC output and frequency of LTC-IC. Assessment of CFC output and frequency of LTC-IC from week 6 LTC-IC assays. (A): Total yield of CFC per 1 x 106 initially plated cells (n = 5). (B): Frequency of LTC-IC per 106 cells determined in two independent limiting dilution LTC-IC assays. Abbreviations: CFC, colony-forming cells; LTC-IC, long-term culture-initiating cells; YFP, yellow fluorescent protein.

Although constitutive expression of ABCG2 in human NOD/SCID repopulating cells was compatible with lymphomyeloid engraftment, the proportion and absolute yield of human lymphoid and myeloid cells in the recipients of ABCG2-transduced cells were significantly perturbed: in the recipients of ABCG2-YFP-transduced CB cells, the proportion of CD19⁺ cells was 1.8-fold reduced compared with the YFP control (ABCG2-YFP, 27.5% ± 7%; YFP, 50.15% ± 4%; p < .05). At the same time, the proportion of myeloid CD15⁺ cells was 1.9-fold increased in the ABCG2-YFP-transduced CB cells compared with the YFP-transduced cells (ABCG2-YFP, 43% ± 10%; YFP, 22% ± 10%) (Fig. 5A). Within the myeloid compartment, the proportion of Gly A⁺ erythroid cells was notably enhanced in the ABCG2-YFP-positive compartment, with a threefold increase compared with YFP mice (ABCG2-YFP, 15.3% ± 2%; YFP, 5.1% ± 1.9%; p < .006) (Fig. 5A). Furthermore, in the ABCG2-YFP-transduced compartment, there was a 1.6-fold increase in the proportion of CD33⁺ cells compared with the YFP control (ABCG2-YFP, 77.5% ± 9%; YFP, 50% ± 5%; p < .05). The proportion of CD11b⁺ cells was also found to be increased by 1.7-fold in the mice transplanted with ABCG2-YFP-positive CB cells compared with the control (ABCG2-YFP, 62.7% ± 10%; YFP, 37% ± 4%; p < .03). In contrast, the generation of megakaryopoietic (CD41⁺) cells was not significantly changed.

View larger version (20K): [in this window] [in a new window]   Figure 5. Lymphomyeloid engraftment of ABCG2-transduced cells in NOD/SCID mice. (A): Detailed analyses of the type of transduced human cells in the NOD/SCID bone marrow (BM) were performed 8 weeks post-transplantation. The data are represented as the mean (± SEM) percentage of total transduced human cells in the BM of mice (n = 4). (B): Inversion of lymphoid-myeloid ratio in the ABCG2-YFP transduced cord blood (CB) cells. The median ratios of the absolute number of lymphoid (YFP+ CD19+) and myeloid (YFP+ CD15+) cells generated are shown (n = 4 in each arm). (C): Competitive repopulating unit (CRU) frequency from ABCG2-YFP- and YFP-transduced CB cells in NOD/SCID mice. Analysis of lymphomyeloid engraftment was determined by staining BM for CD19 and CD15 human cells 6 weeks post-transplantation. The CRU frequency was determined using Poisson statistics. The p values are indicated. Abbreviation: CRU, competitive repopulating unit; Gly A, glycophorin A; YFP, yellow fluorescent protein.

ABCG2 Increases the CRU Frequency
It was further analyzed whether ABCG2 expression would affect the hematopoietic progenitor pool in engrafted mice. The proportion of CD34⁺ progenitor cells was significantly enhanced by 50% in the ABCG2 group compared with control mice (ABCG2-YFP, 21.6% ± 2%; YFP, 14.6% ± 1.7%; n = 4; p < .05). These observations indicated that expression of ABCG2 increases the proportion of immature CD34⁺ cells. To determine whether ABCG2 affects the frequency of NOD/SCID repopulating cells, a limiting dilution CRU assay was performed. CB-derived Lin^– cells were transduced with ABCG2-YFP and YFP vectors and transplanted into six cohorts of sublethally irradiated NOD/SCID mice (n = 3–6 animals per cohort) in limiting dilution without any presorting. Mice were injected with the progeny of an original input of lin^– cells containing 2.5 x 10⁵ CD34⁺ cells 24 hours after transduction. The exact numbers of transplanted CD34⁺YFP⁺ cells were determined by keeping an aliquot of the transduced cell population in vitro to assess YFP expression 48 hours after the end of the transduction. The CRU frequency was calculated by Poisson statistics for the starting number of CD34⁺/YFP⁺ cells at the time point of transplant based on the number of lymphomyeloid-engrafted mice per dilution after a period of 6 weeks (Table 2). In the CRU assay, constitutive expression of ABCG2 induced a 2.7-fold increase in the frequency of CRU (p < .034) (Fig. 5C).

	DISCUSSION

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In addition to its effect on clonogenic progenitors, constitutive expression of ABCG2 was able to expand the human CD34⁺ compartment in transplanted NOD/SCID mice. Importantly, the limiting dilution CRU assay demonstrated that the ABC transporter can affect most primitive human progenitor cells by enhancing the frequency of NOD/SCID repopulating cells. In contrast, ABCG2 did not significantly enhance the LTC-IC frequency or the number of clonogenic progenitors generated per LTC-IC in vitro. However, studies have shown that the LTC-IC assay and the CRU assay monitor two different classes of human progenitor cells [31].

So far, it is unknown how ABCG2 regulates early human hematopoiesis. Taking into account that ABCG2 acts as an efflux pump, it is conceivable that constitutive expression of ABCG2 in human progenitor cells critically induces efflux of substrates that are necessary for lineage differentiation, thereby causing accumulation of early hematopoietic progenitors or impairing differentiation into B-lymphoid lineages. Furthermore, it was shown that physiologic hypoxia is essential for the proliferation of hematopoietic precursors during development and that low oxygen levels play a fundamental role in the maintenance of normal stem cell function [32, 33]. It was also demonstrated that protection of hematopoietic stem and progenitor cells under conditions of low oxygen requires ABCG2 and that this protective function of the transporter protein is linked to its ability to remove porphyrin from cells. Interestingly, ABCG2 is activated in hypoxia through hypoxia response elements present on the ABCG2 promoter [34]. Therefore, overexpression of ABCG2 in early hematopoietic progenitors might facilitate survival and expansion of primitive human progenitor cells and affect lineage differentiation in vivo [34, 35].

Our results differ from those of the nonhuman primate models [28]. This might be due to the fact that the impact of the ABCG2 efflux pump might be context-dependent. For instance, differences in the murine and primate stem cell biology or differences with regard to the microenvironmental stem cell niche might be responsible for the observed discrepancies. Thus, in contrast to the findings in the autologous macaque model, which did not show any effect of ABCG2 on hematopoietic cell differentiation, we observed a skewed lymphoid/myeloid differentiation in the NOD/SCID xenotransplantation model. However, as in the macaque model, we did not observe any block in differentiation of hematopoietic cells. An insight into the natural substrates of ABCG2, which yet remain to be defined, would surely help in understanding the influence of ABCG2 on cell fate decisions in vivo.

Taken together, our data indicate that balanced expression of ABCG2 is crucial for normal human hematopoietic development and that its constitutive expression affects the behavior of early human progenitors and their development into more mature cell stages. This model system will help to further elucidate the role of ABC transporters in the regulation of early human progenitors and help to dissect the molecular mechanisms underlying the role of ABCG2 in hematopoiesis.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Nicole Behm and Andrea Focke for technical assistance, Bianka Ksienzyk for cell sorting, and the members of the GSF animal facility for the excellent breeding and maintenance of animals. We also thank the Department of Obstetrics and Gynaecology of the Klinikum Grosshadern, Munich, Germany, for supplying umbilical cord blood samples. We are also thankful to Dr. Wolfgang Beisker (Helmholtz Zentrum Muenchen-Institute of Toxicology, Neuherberg, Germany) for help in performing the SP analysis. This work was supported by Grant 70-2968-FE I from the German Cancer Foundation (to M.F.-B.).

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Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 

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