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Stem Cells Vol. 24 No. 11 November 2006, pp. 2592 -2602
doi:10.1634/stemcells.2005-0434; www.StemCells.com
© 2006 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

HOX Decoy Peptide Enhances the Ex Vivo Expansion of Human Umbilical Cord Blood CD34+ Hematopoietic Stem Cells/Hematopoietic Progenitor Cells

Hirokazu Tanakaa,b, Itaru Matsumurab, Kiminari Itoha, Asako Hatsuyamaa, Masayuki Shikamuraa, Yusuke Satohb, Toshio Heikec, Tatsutoshi Nakahatac, Yuzuru Kanakurab

aDepartment of Regenerative Medicine, Institute of Biomedical Research and Innovation, Kobe, Japan;
bDepartment of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka, Japan;
cDepartment of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Key Words. Ex vivo expansion • Hematopoietic stem/progenitor cells • HOX • Peptide mimetics

Correspondence: Hirokazu Tanaka, M.D., Ph.D., Department of Hematology and Oncology, Osaka University Graduate School of Medicine, C9, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. Telephone: 81-6-6879-3871; Fax: 81-6-6879-3879; e-mail: htanaka{at}fbri.org

Received November 6, 2005; accepted for publication July 14, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
HOX transcription factors play important roles in the self-renewal of hematopoietic cells. HOX proteins interact with the non-HOX homeobox protein PBX1 to regulate, both positively and negatively, the expression of target genes. In this study, we synthesized a decoy peptide containing the YPWM motif from HOX proteins (decoy HOX [decHOX]), which was predicted to act as a HOX mimetic, and analyzed its effects on self-renewal of human cord blood CD34+ cells. We were able to deliver decHOX into approximately 70% of CD34+ cells. By examining the expression of HOX target genes c-myc and p21waf1/cip1, we confirmed that decHOX enhanced HOX functions. After 7 days of culture in serum-free medium containing a cytokine cocktail, cultures treated with decHOX had approximately twofold-increased numbers of CD34+ cells and primitive multipotent progenitor cells compared with control cells. Furthermore, decHOX-treated cells reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficiency mice more rapidly and more effectively (more than twofold greater efficiency, as determined by a limiting dilution method) than control cells. decHOX-treated cells were also able to repopulate secondary recipients. Together, these results indicate that in combination with growth factors and/or other approaches, decHOX might be a useful new tool for the ex vivo expansion of hematopoietic stem/progenitor cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Human umbilical cord blood (CB) is a useful source of hematopoietic stem cells (HSCs) for transplantation. In fact, during the last few years, an increasing number of patients have received CB transplants [1]. However, clinical applications of CB are inevitably limited by the fact that the number of HSCs in each CB sample is insufficient for many adult patients. Also, compared with transplantation of HSCs from the bone marrow or HSCs mobilized into peripheral blood, the recovery of hematopoiesis is rather delayed in patients receiving CB transplants, partly because of the insufficient number of transplanted HSCs and progenitor cells and the persistent quiescence of CB HSCs that sometimes accompanies lethal complications [1]. Therefore, it is of particular interest to expand CB HSCs ex vivo and to develop strategies for hastening hematopoietic recovery after CB transplantation in vivo [2]. Regarding strategies for ex vivo expansion, the most important problem is to preserve the functions and properties of HSCs, that is, self-renewal and multipotency, during culturing. At present, the use of cytokines is the most promising and practical strategy for this purpose. To establish the culture conditions most suitable for expansion of HSCs, a number of investigators have used various cytokine combinations [2, 3]. When their effects were compared by long-term reconstitution assays in transplanted mice, the combination of stem cell factor (SCF), FLT3 ligand (FL), thrombopoietin (TPO), and IL-6/soluble IL-6 receptor (sIL-6R) was found to expand HSCs most efficiently, with a 4.2-fold increase in severe combined immunodeficient (SCID)-repopulating cells (SRC) [4]. Several patients have received the transplantation with cytokine-expanded CB HSCs, and these cells were transplanted without serious toxicities [5, 6]. However, although increased numbers of infused CB HSCs were shown to correlate with good outcomes, cytokine-expanded CB HSCs did not shorten the nadir period after transplantation, indicating the limited usefulness of cytokines for ex vivo expansion of CB HSCs. Thus, further improvement in ex vivo expansion procedures is necessary to prepare more efficient HSCs.

During the last few years, several molecules that can contribute to HSC self-renewal have been identified and characterized. These include external signaling molecules such as Wnt [7–10], bone morphogenic protein (BMP) [11], Sonic hedgehog (SHH) [12], and Notch ligands [13–15]. Furthermore, endogenous transcriptional modulators such as HOXB4 and Bmi-1 have been shown to be important for HSC self-renewal [16–18]. Among these, HOXB4 is of particular interest because it promotes prominent expansion of HSCs without causing leukemia. When HOXB4 was introduced into murine or human HSCs by gene transfer or protein delivery, these HSCs could be expanded without losing their normal potentials for differentiation into all lineages and for long-term repopulation, with a few exceptions [16, 19–22]. In addition to HOXB4, other HOX homeobox transcription factors play important roles in the proliferation and differentiation of hematopoietic cells [23, 24]. For example, HOXA9 regulates HSCs by mediating the expression of a variety of gene families [25, 26]. HOXA5/A10 and HOXB6 induce differentiation toward the myelomonocytic or erythroid lineage, respectively [27–30]. Furthermore, other HOX transcription factors, especially paralogous groups from A, B, and C, are expressed in normal hematopoietic cells; however, their physiological functions have not been elucidated.

HOX proteins have been demonstrated to interact with non-HOX homeobox family proteins (i.e., PBX and MEIS) at the DNA sequence 5'-TGATNNAT(G/A)(G/T)-3' in the regulatory region of target genes [31, 32]. These protein complexes regulate target gene expression both positively and negatively, dependent on binding to coactivators or corepressors such as CBP/p300, histone deacetylases, or NcoR/SMRT [33–36]. For a subset of HOX proteins, the formation of a HOX-PBX-DNA ternary complex is mediated through both the HOX homeodomain and a short, conserved YPWM motif located just upstream of the HOX homeodomain [37, 38]. The interaction between the YPWM motif of HOX and the third {alpha}-helix in the homeodomain of PBX1 is thought to modify HOX-PBX1 DNA-binding affinity and transcriptional activity [39–41]. In addition, it was reported that PBX1 expressed in HSCs is a negative regulator of HOXB4-mediated self-renewal of HSCs [42]. Consistent with this report, a very recent study demonstrated that although DNA-binding activities are necessary for HOXB4 to expand HSCs ex vivo, the interaction with PBX1 is dispensable for this function [43].

In an attempt to expand potent CB HSCs with high efficiency, we synthesized a peptide containing the YPWM motif from HOX, which was predicted to modify HOX function by inhibiting binding between the YPWM motif in endogenous HOX and the PBX1 homeodomain. Here we show that this decoy HOX (decHOX) peptide augments the cytokine-dependent ex vivo expansion of CD34-positive hematopoietic stem/progenitor cells (CD34+ hHSCs/HPCs), and these cells have the ability to reconstitute hematopoiesis more effectively and rapidly in mice that received transplants.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Peptide Synthesis
Peptides were synthesized at Greiner Bio-One (Tokyo, Japan, http://www.gbo.com/en) with purities of more than 95%. Synthetic peptides were lyophilized and stored at –20°C until use.

Reagents and Antibodies
Recombinant human SCF, TPO, IL-6, and sIL-6R were provided by Kirin Brewery (Tokyo, Japan, http://www.kirin.co.jp/english/). Recombinant human FL was purchased from R&D Systems Inc. (Minneapolis, http://www.rndsystems.com). Anti-asialo-GM1 antibody (Ab) was purchased from Wako Chemical (Osaka, Japan, http://www.wako-chem.co.jp/english). Antibodies (Abs) against HOXB4 (N-18) and PBX1 (P-20) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com).

Plasmids
The expression vectors for HOXB4 and PBX1 were kindly provided by Dr. R. K. Humphries (British Columbia Cancer Agency, Vancouver, BC, Canada) and Dr. M. Featherstone (McGill University, Montreal, QC, Canada), respectively.

Preparation of Glutathione S-Transferase Fusion Proteins
Mutants of PBX1 were generated by polymerase chain reaction (PCR) and subcloned into pGEX-5X-1 (GE Healthcare Bio-science Corp., Piscataway, NJ, http://www.gehealthcare.com). Glutathione S-transferase (GST)-PBX1 fusion proteins were produced in Escherichia coli and purified as described previously [44].

In Vitro Binding Assays Using the BIAcore System
To assess in vitro binding between decHOX and PBX1, we used the BIAcore system (Biacore AB, Uppsala, Sweden, http://www.biacore.com/lifesciences/index.html). The details of this system are described elsewhere [45]. Briefly, we immobilized decHOX on the surface of CM5 sensor chips. Solution containing each GST-PBX1 fusion protein was injected over the sensor chips. Binding kinetics were monitored by changes in the weight of sensor chips and evaluated as arbitrary resonance units (RUs).

Mice
Nonobese diabetic/Shi-severe combined immunodeficient (NOD/SCID) mice, which lack mature lymphocytes and circulating complement proteins and have defective macrophages, were obtained from Jackson Laboratory (Bar Harbor, ME, http://www.jax.org). The mice were kept in microisolator cages on laminar flow racks in a clean experiment room and fed an irradiated, sterile diet and autoclaved, acidified water. Animal care was in accordance with institutional guidelines.

Cell Preparation
Human umbilical CB was obtained from normal, full-term deliveries upon obtaining informed consent. After sedimentation of the red blood cells with 6% hydroxyethyl starch (HES), mononuclear cells (MNCs) were separated by Ficoll-Hypaque density gradient centrifugation. CD34+ cells were purified from MNCs using a MACS Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). After purification, over 95% of the separated cells were confirmed to be CD34+ by flow cytometric analysis (data not shown). Each experiment was performed with cord blood CD34+ cells derived from the same sample.

Suspension Cultures
Purified CD34+ cells were seeded at a cell density of 1–2 x 104 cells per milliliter in 24-well tissue plates (Falcon, Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) with QBSF-60 serum-free medium (Quality Biological, Inc., Gaithersburg, MD, http://www.qualitybiological.com) containing SCF (100 ng/ml), FL (100 ng/ml), TPO (10 ng/ml), IL-6 (100 ng/ml), and sIL-6R (100 ng/ml). Cells were cultured in humidified air with 5% CO2 at 37°C.

Protein Delivery
Synthetic peptides were delivered into 293T and CB CD34+ cells using the Profect Protein Delivery System (Targeting Systems, Santee, CA, http://www.targetingsystems.com) according to the manufacturer's instructions.

Colony Assays
Cells were seeded into methylcellulose medium (MethoCult GF H4434V; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) at a density of 2.5 x 102 cells per 35-mm dish and were cultured with 5% CO2 at 37°C. All cultures were performed in triplicate, and the numbers of colonies were counted after 10 days.

Reconstitution Assays Using NOD/SCID Mice
Transplantation assays using NOD/SCID mice were performed according to procedures described previously [4] with some modifications. Briefly, 6–8-week-old NOD/SCID mice were total-body irradiated (TBI) with a dose of 2.4 Gy (60 Co) and then transplanted with the whole of peptide-treated cells or 2 x 104 freshly isolated CD34+ cells through the tail vein. Because natural killer cell activity is retained in NOD/Shi-scid mice, the recipients were injected i.p. with 400 µl of phosphate-buffered saline containing 20 µl of anti-asialo-GM1 Ab immediately before cell transplantation. Identical treatments were performed on days 7 and 14. The proportion of reconstituted human cells in peripheral blood (PB) or bone marrow (BM) was assessed by flow cytometry with the anti-human CD45 Ab. For secondary transplantations, bone marrow cells were obtained from tibiae and femurs of the first mice that received transplants 12 weeks after transplantation, and 0.5 x 107 total bone marrow chimeric cells were injected into secondary NOD/SCID recipients subjected to immunosuppressive treatment before and after transplantation as described above (n = 5). Six weeks after transplantation, the presence of transplanted human cells in recipient BM was confirmed by flow cytometry as described above.

Limiting Dilution Analysis
The frequencies of human HSCs that were capable of repopulating in NOD/SCID mice in freshly isolated CB CD34+ cells and peptide-treated cells were quantified by a limiting dilution analysis as described previously [46–48]. In this analysis, to avoid graft rejection, the recipients were treated with TBI in combination with anti-asialo-GM1 Ab immediately before and after transplantation (days 7, 14, 21, and 28). Data from several limiting dilution experiments were pooled and analyzed by applying Poisson statistics to the single-hit model. Frequencies were calculated using the maximum likelihood estimator.

Luciferase Assays
The details of the –1,137-c-myc-Luc vector, containing a 1,653-base pair (bp) fragment of the c-myc promoter (–1,137 to +516), were described previously [49]. To construct 3 x HB4(–316)-Luc and 3 x HB4(–72)-Luc, three tandem repeats of HOXB4-responsive elements at the indicated positions in the insulin-like growth factor-binding protein (IGFBP)-1 promoter were subcloned into pGL3 basic-TK-Luc. The sequences of the HOXB4-responsive elements were as follows: HB4(–316), 5'-CTTGTGTCAATTAAAGA and HB4(–72), 5'-GCGCTGCCCAATCATTAA. Luciferase assays were performed with a Dual-Luciferase Reporter System (Promega, Madison, WI, http://www.promega.com) as previously described [50]. Briefly, 293T cells (2 x 105 cells) were seeded in a 60-mm dish and cultured for 24 hours. Using the calcium phosphate coprecipitation method, cells were transfected with 6 µg of pcDNA3-HOXB4 alone or in combination with 6 µg of pCS2-PBX1a, along with 2 µg of reporter gene and 10 ng of pRL-CMV, a Renilla luciferase expression vector. After 12 hours, cells were washed, serum-starved for 24 hours, and subjected to luciferase assays. In some experiments, various doses of synthetic peptides were delivered into 293T cells 24 hours prior to luciferase assays.

Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay (EMSA) was performed as previously described [51]. The double-stranded oligonucleotide HB4(–316) (described above) was used as a probe or competitor.

Semiquantitative Reverse Transcription PCR Analysis
Total RNA was isolated from 5 x 104 cells using a Concert Micro-to-Midi Total RNA Purification System (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Reverse transcription PCR (RT-PCR) was performed using a SuperScript One-Step RT-PCR system (Invitrogen) according to the manufacturer's instructions with forward/reverse primer sets as follows: c-myc, 5'-CTT CTG CTG GAG GCC ACA GCA AAC CTC CTC and 5'-CCA ACT CCG GGA TCT GGT CAC GCA GGG; p21waf1/cip1, 5'-ACA GCA GAG GAA GAC CAT GT and 5'-GGT ATG TAC ATG AGG AGC TG; and ß-actin, 5'-GGC GGC AAC ACC ATG TAC CCT and 5'-AGG GGC CGG ACT CGT CAT ACT.

Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were performed with a ChIP Assay Kit (Upstate, Charlottesville, VA, http://www.upstate.com). Briefly, after transfection with various expression vectors, 293T cells were fixed with 1% formaldehyde. After isolation of nuclear extract, the chromatin was sonicated. Then, protein-DNA complexes were immunoprecipitated with 2 µg of anti-PBX1, anti-HOXB4, or anti-actin Ab. Immunoprecipitated DNA was eluted and subjected to PCR analysis with the following primer pair to amplify 420 bp of the human IGFBP-1 promoter (M59316 [GenBank] ): forward primer, 5'-GGC ATT GTT TTC TGC GTT TGA GAA CTG CTG; reverse primer, 5'-CTG GAC ACA GCG CGC ACC TTA TAA AGG GCA. After electrophoresis, PCR products were visualized with ethidium bromide staining.

Statistical Analysis
Data are presented as mean ± SEM or mean ± SD. The statistical significance of the data was determined by the Mann-Whitney U test or Student's t test. The significance level was set at .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
The Synthetic Peptide decHOX Binds Directly to the Homeodomain of PBX1
In this study, we attempted to expand CB CD34+ hHSC/HPCs by modifying the function of HOX family proteins. For this purpose, we designed and synthesized a peptide designated decHOX, which was expected to inhibit the interaction between HOX and PBX1. decHOX contains the YPWM motif of HOX, used for its cooperative interaction with PBX1 [36, 37], and the nuclear localization signal (NLS) of the SV40 large T antigen (Fig. 1A) [52]. The negative control (NC) peptide contains the unrelated amino acid sequence CINEVA. To evaluate the efficiency of peptide delivery into CB CD34+ hHSC/HPCs and the subsequent kinetics, FL protein was conjugated to the N termini of both peptides.


Figure 1
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  		Figure 1. Binding of decHOX to GST-PBX1. (A): The structures of the 5'-FL synthetic peptides 5'-FL-decHOX and 5'-FL-NC are indicated. (B): GST-PBX1 fusion proteins expressed in E. coli were purified by glutathione-Sepharose 4B beads, and their qualities and quantities were confirmed by Coomassie staining. (C): In vitro binding of GST-PBX1 to decHOX evaluated by the BIAcore system, with decHOX attached to the sensor chip. To examine the kinetics of the binding and dissociation, various GST-PBX1 proteins were injected onto the sensor chip for 180 seconds and then washed with HEPES-buffered saline for 180 seconds. Abbreviations: decHOX, decoy HOX; delC, C-terminal deletion; FL, fluorescein; GST, glutathione S-transferase; HD, homeobox domain; M.W., molecular weight; NC, negative control; NH NLS, nuclear localization signal; RU, resonance unit.

 
First, we examined in vitro binding between decHOX and several GST-PBX1 fusion proteins using the BIAcore system. In this system, the analyte protein is injected onto the sensor chip, the surface of which is covered by the immobilized partner ligand. Binding of the ligand to the analyte is monitored by an increase in arbitrary RUs. Prior to this analysis, we purified several GST-PBX1 fusion proteins (Fig. 1B, left panel) and confirmed their qualities and quantities by Coomassie Brilliant Blue staining (Fig. 1B, right panel). Injection of either GST-full-length PBX1 protein (GST-PBX1 FL) or GST-PBX1 homeobox domain (HD) protein (GST-PBX1 HD) over the decHOX surface resulted in a significant increase in RUs with a lapse of 3 minutes for the binding reaction (Fig. 1C), and these signals increased in a dose-dependent manner (data not shown). In contrast, GST alone and GST-PBX1 delC (lacking the HD) did not bind appreciably to decHOX. After the binding reaction, we injected HEPES-buffered saline for 180 seconds. During this dissociation reaction, GST-PBX1 HD bound to decHOX more stably than GST-PBX1 FL (Fig. 1C). These results suggest that GST-PBX1 FL and GST-PBX1 HD bind to decHOX, probably through HD.

decHOX Can Modulate the Transcriptional Activity of HOX-PBX
To assess the effects of decHOX on HOX-PBX-mediated gene expression, we performed luciferase assays with three types of reporter genes for HOXB4, one containing the c-myc promoter (–1,137-c-myc-Luc) and the other two containing IGFBP-1 promoters (3 x HB4[–316]-Luc and 3 x HB4[–72]-Luc) as responsive elements, as described previously [49, 53]. Although HOXB4 activated –1,137-c-myc-Luc 5.2-fold in 293T cells, PBX1 suppressed this induction (Fig. 2B). However, this inhibitory effect of PBX1 was decreased in a dose-dependent manner by pretreatment with decHOX. Similar responses were observed in assays using 3 x HB4(–316)-Luc and 3 x HB4(–72)-Luc (Fig. 2B). These results indicate that decHOX can enhance the activity of HOXB4 that is suppressed by PBX1.


Figure 2
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  		Figure 2. Effects of decHOX on DNA-binding and transcriptional activities of the HOX/PBX complex. (A): To construct –1,137-c-myc-Luc, a 1,653-base pair fragment of the c-myc promoter (–1137 to +516) was subcloned into the plasmid pSP72-Luciferase [47]. To generate 3 x HB4(–316)-Luc and 3 x HB4(–72)-Luc, three tandem repeats of HOXB4-responsive elements in the IGFBP-1 promoter at the indicated locations were subcloned into TK-pGL3 basic-Luc at just upstream of the murine minimal TK promoter linked to the firefly luciferase gene, and their sequences were as indicated. (B): 293T cells (2 x 105 cells) seeded in a 60-mm dish were transfected with 6 µg of pcDNA3-HOXB4 alone or in combination with 6 µg of pCS2-PBX1a along with 2 µg of reporter gene and 10 ng of pRL-CMV. After 12 hours, cells were washed, serum-starved for 24 hours, and subjected to luciferase assays using a Dual Luciferase Reporter Assay System. In some experiments, various doses of synthetic peptides were delivered into 293T cells 24 hours prior to luciferase assays. Results are shown as mean ± SD of triplicate cultures. (C): 293T cells were transfected with PBX1 together with HOXB4 WT or HOXB4 AA. After 36 hours, nuclear extract was isolated and subjected to electrophoretic mobility shift assay (EMSA) with probes of 3 x HB4(–316). In competition assays, a 200-fold excess of unlabeled wt or mt competitor oligonucleotide was added to the binding mixture. In some experiments, various doses of synthetic peptides were delivered into 293T cells 24 hours prior to EMSA. (D): 293T cells transfected with the indicated expression vectors were fixed with 1% formaldehyde. After the isolation of the nuclear extract, chromatin was sonicated. Then, protein-DNA-binding complexes were immunoprecipitated with the 2 µg of the indicated antibodies. Immunoprecipitated DNA was subjected to polymerase chain reaction (PCR) analysis with a primer pair that amplifies 420 base pairs of the human IGFBP-1 promoter. PCR products were electrophoresed onto the agarose gel and visualized with ethidium bromide staining. Abbreviations: decHOX, decoy HOX; IGFBP-1, insulin-like growth factor-binding protein; IP, immunoprecipitation; mt, mutant; WT, wild type.

 
In a previous study using EMSA, mutant HOX proteins that cannot bind to PBX1 were shown to have defects in DNA-binding activities [39–41]. In contrast, it was reported that the interaction with PBX1 is not necessary for HOXB4 to induce HSC self-renewal [43]. Because decHOX was designed to inhibit the interaction between HOX and PBX1, we evaluated the effect of decHOX on the DNA-binding activity of HOXB4-PBX1 using EMSA. For this purpose, we transfected 293T cells with wild-type (WT) HOXB4 or mutant HOXB4 harboring a WM->AA mutation in the YPWM motif (designated HOXB4 AA; Fig. 2C). Protein(s) in nuclear extracts from 293T cells transfected with PBX1 and HOXB4 WT bound to the HB4(–316) probe (Fig. 2C, lane 2). This band was abolished by WT DNA competitor (Fig. 2C, lane 3) but not by mutant (mt) competitor (Fig. 2C, lane 4), implying that it contains the HOXB4 WT-PBX1 complex. In contrast, proteins in the nuclear extract from HOXB4 AA-transfected cells scarcely bound to the probe, indicating the importance of the YPWM motif for the DNA-binding activity of HOXB4-PBX1 (Fig. 2C, lane 5). Also, pretreatment with decHOX inhibited DNA binding of the HOXB4 WT-PBX1 complex in a dose-dependent manner (Fig. 2C, Lanes 6 and 7). These results suggest that decHOX inhibits the interaction between HOXB4 and PBX1 in the ex vivo EMSA binding experiment, thereby suppressing the DNA-binding activity of the HOXB4 WT-PBX1 complex. However, in ChIP assays, which reflect the in vivo DNA-binding state of transcription factors more precisely than EMSA, the HOXB4 AA-PBX1 complex bound to the endogenous IGFBP-1 promoter as efficiently as the HOXB4 WT-PBX1 complex (Fig. 2D, top and second panels, lane 4 vs. lane 5). Also, decHOX barely influenced the DNA-binding activity of the HOXB4 WT-PBX1 complex (Fig. 2D, top and second panels, lane 4 vs. lane 6). Furthermore, we obtained similar results from ChIP assays using two additional primer sets that amplify different sites in the IGFBP-1 promoter (data not shown). From these results, we speculated that HOXB4 is capable of binding to DNA regardless of its interaction with PBX1. However, since we found that PBX1 bound to target DNA in the presence of HOXB4 AA, it is also possible that the YPWM is not required for the interaction between HOXB4 and PBX1. Because the latter speculation is inconsistent with several previous reports indicating the essential role of the YPWM motif in the interaction between HOXB4 and PBX1 [37, 38], further studies using several endogenous promoter sequences will be required to draw a definite conclusion.

decHOX Is Efficiently Delivered into CB CD34+ and Colocalizes with PBX1
Next, we introduced 5'-FL-decHOX into CB CD34+ hHSC/HPCs and analyzed the efficiency of delivery by examining fluorescein intensity with flow cytometry. CD34+ hHSC/HPCs were isolated from CB using AutoMACS and cultured in QBSF-60 serum-free medium containing SCF, FL, TPO, IL-6, and sIL-6R. After culturing for 24 hours, FL-decHOX or FL-NC was delivered into CD34+ cells using the Profect Protein Delivery System. Immediately after delivery (day 0; total culture day 2), 76.2% of CB CD34+ cells were fluorescein-positive (Fig. 3A). Fluorescein intensity decreased with time in culture and was scarcely detectable at day 7. This result suggested that the direct influence of decHOX on CD34+ hHSC/HPCs is limited to 7 days.


Figure 3
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  		Figure 3. Expression and intracellular localization of decHOX in hHSC/HPCs. (A): FL-decHOX was transferred into CD34+ cells by the Profect Protein Delivery System, and fluorescence intensity was assessed by flow cytometry at the indicated times. (B): Cytospin preparations of the FL-NC (upper panel)- or FL-decHOX (lower panel)-delivered CD34+ cells were fixed, permeabilized, and incubated with a rabbit anti-human PBX1 antibody (Ab) for 1 hour and then with the anti-rabbit IgG Ab AlexaFluor 546. Cells were rinsed with phosphate-buffered saline containing Hoechst 33342. The stained cells were observed under a confocal laser microscope. Abbreviations: decHOX, decoy HOX; MFI, mean fluorescence intensity; NC, negative control.

 
Next, we examined the subcellular localizations of PBX1, decHOX, and the NC peptide in hHSC/HPCs using fluorescent microscopy. Forty-eight hours after peptide delivery, both peptides were predominantly detected in the nucleus because of the respective nuclear localization signals. In NC-delivered cells, PBX1 was mainly localized in the cytosol (Fig. 3B, upper panel). On the other hand, in decHOX-delivered cells, PBX1 colocalized with decHOX in the nucleus (Fig. 3B, lower panel). These results suggested that decHOX could interact with PBX1 and colocalized with PBX1 in the nucleus.

decHOX Can Modulate HOX/PBX-Mediated Gene Expression in CB hHSC/HPCs
PBX1 is known to modulate the function of HOX proteins both positively and negatively [30, 32, 33]. For example, HOXB4 induces the expression of c-myc in HSCs, and this effect is suppressed by PBX1. On the other hand, HOXA10-mediated expression of p21waf1/cip1 is enhanced by PBX1 in myelomonocytic progenitors [28]. To assess the effects of decHOX on the function of HOX proteins in CB cells, we examined the expression of these two target genes. First, to characterize CD34+CD38+ and CD34+CD38– cells after the ex vivo expansion, we sorted these cells after 9 days in culture and performed methylcellulose colony assays (Fig. 4A). It was reported that CD34+CD38– cells can develop from CD34+CD38+ cells through the loss of CD38 expression during culturing with cytokines [54]. We found that the primitive colony, mixed hematopoietic colony-forming unit (CFU-Mix) was formed from CD34+CD38– cells but not from CD34+CD38+ cells. Therefore, we supposed that CD34+CD38– cells were more primitive than the CD34+CD38+ cells that developed after ex vivo culturing. Next, we treated CB CD34+ cells with 5'-FL-decHOX, cultured for 48 hours, and subjected them to flow cytometric analysis. At that point, 43.1% of the cultured cells were fluorescein+CD38+, 30.5% were fluorescein+CD38–, 7.82% were fluorescein–CD38+, and 18.2% were fluorescein–CD38– (Fig. 4C). Then, we sorted the cells from each fraction and subjected them to semiquantitative RT-PCR analysis. In the CD34+CD38– immature cell fraction, c-myc expression was increased in the decHOX-delivered fluorescein+ fraction compared with the fluorescein– control fraction (Fig. 4D, top panel, lane 1 vs. lane 3). Conversely, in the CD34+CD38+ mature fraction, the expression of p21waf1/cip1 was decreased in the fluorescein+ fraction compared with the fluorescein– fraction (Fig. 4D, middle panel, lane 2 vs. lane 4). Together, these results suggest that decHOX binds to PBX1 as a HOX decoy and cancels both the positive and negative effects of PBX1 on HOX proteins in CD34+ hHSC/HPCs.


Figure 4
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  		Figure 4. Effects of decoy HOX (decHOX) on target gene expression in hHSC/HPCs. Cord blood (CB) CD34+ cells were cultured in QBSF-60 serum-free medium containing cytokines (stem cell factor, 100 ng/ml; fluorescein [FL], 100 ng/ml; TPO, 10 ng/ml; IL-6, 100 ng/ml; and sIL-6R, 100 ng/ml) for 9 days. (A): Before and after culturing, the expression of CD34 and CD38 were examined by flow cytometry. (B): After culturing, CD34+CD38– and CD34+CD38+ cells were sorted and subjected to colony assays. (C): CB CD34+ cells were treated with FL-decHOX for 48 hours, and then CD38 and fluorescein expression was examined by flow cytometry. (D): Cells from each fraction were sorted and subjected to RT-PCR analysis. Abbreviations: APC, allophycocyanin; CFU-GM, colony-forming unit-granulocyte/monocyte precursor; CFU-mix, mixed hematopoietic colony-forming unit.

 
decHOX Enhances Cytokine-Dependent Ex Vivo Expansion of CB hHSC/HPCs
Next, we examined effects of decHOX on the growth and differentiation of CB CD34+ hHSC/HPCs. As shown in Figure 5, purified CD34+ cells were exposed to 5'-FL-decHOX or 5'-FL-NC for 24 hours. Then, 1 x 104 fluorescein+ cells were sorted and cultured in QBSF-60 serum-free medium containing SCF, FL, TPO, IL-6, and sIL-6R for 7 days, during which, medium dilution was performed as indicated. After these cultures, no apparent difference was observed between the total number of viable decHOX-treated and NC-treated cells (Fig. 6A). However, the proportion of CD34+ cells was significantly higher in decHOX-treated cells than in NC-treated cells (decHOX, 33.2%; NC, 17.9%) (Fig. 6B). Similar results were obtained from five independent experiments (data not shown). Accordingly, the fold expansion of CD34+ cells was higher in decHOX-delivered cells than in NC-delivered cells (decHOX, 32.5 ± 8.71-fold; NC, 17.2 ± 6.25-fold [n = 6] [p < .05]) (Fig. 6A). Furthermore, cultures treated with decHOX retained immature cells with CD34+CD38– or CD45+HLA-DR– phenotype more effectively than NC-treated cells (percentage CD34+CD38– cells: decHOX, 24.2% ± 6.67%; NC, 14.8% ± 6.17% [n = 3] [p < .05]; percentage CD45+HLA-DR– cells: decHOX, 37.2% ± 6.98%; NC, 18.8% ± 7.44% [n = 3] [p < .05]) (representative results from one experiment are shown in Fig. 6B). To characterize the ex vivo expanded cells, we analyzed the expression of lineage markers on these cells (Fig. 6C). Fluorescence-activated cell sorting analyses after 7 days in culture indicated that both NC- and decHOX-treated cells contained not only immature cells but also mature cells expressing lineage markers such as CD33 (myeloid), CD14 (monocytic), CD19 (B lymphoid), GPA (erythroid), and CD41 (megakaryocytic). Except for GPA and CD41 expression, the expression of these markers was notably lower in decHOX-delivered cells than in NC-delivered cells. Next, we analyzed the effects of decHOX on colony-forming activities of CB CD34+ hHSC/HPCs. As shown in Figure 6D, both decHOX- and NC-delivered cells, which contain approximately 33% and 18% of CD34+ cells, respectively (Fig. 6B), generated all types of colonies, and the numbers of colony-forming unit-erythrocyte precursor/burst-forming unit-erythroid precursor and colony-forming unit-granulocyte/monocyte precursor colonies were nearly the same in both cultures. However, decHOX more effectively yielded CFU-Mix primitive colonies than NC (CFU-Mix colonies per 250 cultured cells: decHOX, 15.3 ± 2.1; NC, 7.5 ± 0.8 [n = 6] [p < .05]). Together, these results suggest that although both immature progenitors and mature cells were amplified during culturing, decHOX selectively expands immature progenitors.


Figure 5
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  		Figure 5. Experimental design. CD34+ hematopoietic stem/progenitor cells were isolated from cord blood and cultured in serum-free medium containing cytokines. FL-decHOX or FL-NC was then delivered into CD34+ cells. Twenty-four hours after peptide delivery, approximately 70% of cultured cells were fluorescein-positive. Fluorescein-positive cells were sorted and cultured for 7 days. Medium dilutions were performed as indicated. Cultured cells were then subjected to FACS analyses, colony assays, and reconstitution assays using NOD/SCID mice. Abbreviations: decHOX, decoy HOX; FACS, fluorescence-activated cell sorting; FL, fluorescein; NC, negative control.

 


Figure 6
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  		Figure 6. Effects of decHOX on biological properties and functions of hHSC/HPCs cultured with cytokines. After treatment with fluorescein (FL)-decHOX or FL-NC, fluorescein-positive cells were sorted and cultured for 7 days. (A): The total number of viable cells and their surface phenotypes were examined. Results are shown as mean ± SD (n = 6). (B, C): Representative fluorescence-activated cell sorting data from one experiment are shown. (D): Cultured cells were subjected to methylcellulose colony assays using freshly isolated cells as a control. All cultures were done in triplicate and scored after 10 days. Results are shown as mean ± SD (n = 4). Abbreviations: BFU-E, burst-forming unit-erythroid precursor; CFU-E, colony-forming unit-erythrocyte precursor; CFU-GM, colony-forming unit-granulocyte/monocyte precursor; CFU-Mix, mixed hematopoietic colony-forming unit; CTL, control; decHOX, decoy HOX; FITC, fluorescein isothiocyanate; HLA, human leukocyte antigen; NC, negative control; PE, phycoerythrin.

 
decHOX-Treated hHSC/HPCs Reconstitute Hematopoiesis Rapidly and Efficiently in NOD/SCID Mice
Next, we assessed the effects of decHOX on engrafting abilities of CB CD34+ hHSC/HPCs by xenotransplantation into NOD/SCID mice. For this purpose, 2 x 104 CB CD34+ cells were treated with decHOX or NC and cultured in QBSF-60 containing cytokines for 7 days. Then, total cultured cells were transplanted into NOD/SCID mice that were treated with 2.4 Gy of TBI and i.p. injection of anti-asialo-GM1 Ab immediately before and after transplantation (days 7 and 14) (each group, n = 9). Also, 2 x 104 freshly isolated CB CD34+ cells derived from the same sample as the expanded cells were transplanted as a control (CTL). CTL cells are expected to contribute to hematopoiesis in approximately 10% of BM cells after 4 weeks under our experimental conditions using NOD/SCID mice. When decHOX-treated cells were transplanted, human CD45+ (hCD45+) cells constituted 9.17% of the BM cells 4 weeks after transplantation, whereas NC-treated cells yielded only 4.28% hCD45+ cells (Fig. 7A). In addition, the proportion of hCD34+ cells in the BM was increased by decHOX (decHOX, 3.05%; NC, 1.22%) (Fig. 7A). We also analyzed the lineage distributions of hCD45+ cells in BM of mice that received transplants of decHOX-treated cells at 4 and 8 weeks after transplantation. As shown in Figure 7A, transplanted decHOX-treated cells not only retained CD34+ cells but also generated CD33+ myeloid cells and CD19+ B cells more effectively than CTL and NC-treated cells.


Figure 7
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  		Figure 7. Xenotransplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. (A): A total of 2 x 104 decHOX- or NC-delivered cells were sorted and subjected to the culture for 7 days. Whole expanded cells or 2 x 104 freshly isolated CD34+ cells were transplanted into 5–6-week-old NOD/SCID mice subjected to immunosuppressive treatment before and after transplantation (each group, n = 9). Four weeks after transplantation, the proportion of engrafted human cells in BM was assessed by flow cytometry with anti-hCD45-PE antibody (Ab). Four weeks and 8 weeks after transplantation, short-term repopulation abilities of the ex vivo-expanded cells were analyzed using BM and PB cells with the indicated Abs. Representative flow cytometry data obtained from BM cells are shown. (B): Kinetics of engraftment in PB and BM of NOD/SCID mice are indicated. Results are shown as mean ± SD (each group, n = 9). Abbreviations: BM, bone marrow; CTL, control; decHOX, decoy HOX; NC, negative control; PB, peripheral blood; PE, phycoerythrin; W, weeks.

 
We also analyzed the kinetics of short-term repopulation in the PB and BM of recipient mice. Two weeks after transplantation, hCD45+ cells were detectable in both BM and PB without significant differences in their frequencies among decHOX, NC, and CTL groups (Fig. 7B). However, at 4 weeks, the proportion of hCD45+ cells in PB was significantly higher in the decHOX group than in the NC and CTL groups (decHOX, 2.70% ± 0.36%; NC, 1.73% ± 0.14%; CTL, 1.72% ± 0.16%) (p < .05). This result suggests that decHOX reduces the delay in engraftment associated with CB transplantation (Fig. 7B). Also, the percentage of hCD45+ cells in BM was significantly higher in the decHOX group than in the CTL and NC groups at 8 weeks (decHOX, 29.0% ± 10.9%; CTL, 11.9% ± 6.64%; NC, 14.9% ± 7.91% [p < .05]).

Given the possibility that decHOX supports the expansion of long-term repopulating (LTR)-HSCs, we next calculated the expansion rate using a limiting dilution method. To obtain the highest possible levels of human cell engraftment, recipient mice were treated with TBI in combination with anti-asialo GM1 Ab immediately before and after transplantation (days 7, 14, 21, and 28). As shown in Figure 8A, the frequency of LTR-HSCs was calculated to be 1 in 6,017 freshly isolated CB CD34+ cells and 1 in 7,143 NC-treated cells. In contrast, the frequency of LTR-HSCs in decHOX-treated cells was calculated to be 1 in 3,573. Accordingly, the expansion of LTR-HSCs by decHOX was estimated to be 2.0-fold. Furthermore, in BM highly reconstituted with decHOX-treated human cells, we detected considerable proportions of hCD45+CD33+ cells (22.5%) and hCD45+hCD19+ cells (51.8%) 12 weeks after transplantation. In addition, hCD45+CD34+ cells were an estimated 8.81% of total BM cells (Fig. 8B).


Figure 8
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  		Figure 8. Long-term repopulating abilities in decHOX-delivered cells. (A): Frequencies of human HSCs capable of repopulating in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice in freshly isolated CB CD34+ cells (n = 26), NC-treated cells (n = 29), and decHOX-treated cells (n = 23) were quantified by limiting dilution analyses. (B): Twelve weeks after transplantation, the long-term repopulating ability of the decHOX-treated cells was analyzed by flow cytometry using BM from NOD/SCID mouse highly reconstituted with human cells. (C): Twelve weeks after transplantation, BM cells were isolated from NOD/SCID mice that received transplants of decHOX-treated cells and injected into secondary recipients (n = 5). Six weeks after transplantation, the proportions of engrafted human cells in BM were assessed by flow cytometry. Representative data are shown. Abbreviations: BM, bone marrow; CB, cord blood; decHOX, decoy HOX; FITC, fluorescein isothiocyanate; hCD, human cluster of differentiation; NC, negative control; PE, phycoerythrin; W, weeks.

 
To further examine the long-term reconstituting activity of decHOX-treated cells, we performed a secondary transplantation. Twelve weeks after the first transplantation, we isolated BM cells from two mice that received transplants of decHOX-treated cells. A mixture of these cells (58.2% and 8.0% of which were hCD45+ and hCD34+, respectively) was transplanted into five recipients at 4.0 x 105 hCD34+ cells per mouse. An apparent second engraftment was detected in the BM of 2 of 5 mice 6 weeks after the transplantation (representative data are shown in Fig. 8C). At this point, hCD45+ cells were an estimated 14.8% of the total BM cells in the recipient mouse. Furthermore, approximately 20% of hCD45+ cells expressed CD34 (3.17% of total BM cells). Taken together, these results indicate that CB CD34+ hHSCs/HPCs expanded by decHOX reconstitute hematopoiesis more rapidly and efficiently than control cells, and these cells have long-term reconstituting abilities in NOD/SCID mice.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
HOXB4-deficient mice do not exhibit obvious abnormalities in hematopoiesis except for a minor proliferative defect of HSCs detected by reconstitution assays, suggesting that HOXB4 is dispensable for normal hematopoiesis [55]. It was speculated that HOXB4 functional roles can be replaced by other HOX family members because mice lacking both HOXB4 and HOXB3 had more apparent defects in hematopoiesis than those lacking HOXB4 alone [56]. However, HOXB4 has attracted particular interest during the last few years because its gene transfer induced ~40-fold murine and ~30-fold human HSC expansion ex vivo, suggesting possible clinical applications [16, 21]. Regarding clinical use, there was an initial concern that constitutive expression of HOXB4 in HSCs might cause leukemia. This is because deregulated expression of HOXB8 was found in myeloid leukemia, and HOX family genes are sometimes involved in leukemogenic chromosomal translocations, such as t(7,11)(p15;p15), yielding NUP98-HOXA9 [57, 58]. However, HSCs expanded by HOXB4 treatment reconstituted all hematopoietic lineages in mice that received transplants mice without causing leukemia, indicating that HSCs expressing HOXB4 were regulated by the hematopoietic system [16]. To eliminate any deleterious effects caused by stable HOXB4 gene transfer, Krosl et al. tried to expand murine HSCs by delivering HOXB4 protein [20]. In that study [20], cell membrane-permeable, recombinant TAT-HOXB4 protein was added to the culture medium, inducing a fivefold net expansion of HSCs. Although TAT-HOXB4 was supposed to be delivered with high efficiency, its half-life was estimated to be only 1 hour. In addition, Amsellem et al. tried to expand human CB HSCs using HOXB4 protein [19]. They used HOXB4 protein secreted into the culture supernatant from cocultured MS-5 murine stromal cells, and this approach increased NOD/SCID mouse repopulating cells (SRCs) 2.5-fold. However, the efficiency of protein delivery was not very high, and the coculture system may not be practical for clinical applications. In contrast, our decHOX could be delivered into more than 70% of CB CD34+ hHSC/HPCs and was detected in these cells even after 4 days.

Because similar decoy peptides, such as NFAT and JNK-interacting-protein-1 (JIP-1) decoy peptides were shown to be harmless at the genomic level [59, 60], decHOX may be useful for clinical applications. Furthermore, it is possible to use decHOX in combination with HOX proteins [19, 20] to further augment the activity of HOXB4. However, it should be noted that Brun et al. reported that an excessive amount of HOXB4 introduced by an adenoviral vector system inhibits self-renewal of human CB HSCs and induces myeloid differentiation [22]. Therefore, it is necessary to determine the optimal amount of HOXB4 for enforcing HSC self-renewal.

Recently, DiMartino et al. generated Pbx1-null mice, which died at embryonic day 15 or 16 due to anemia, and reported that PBX1 is required for the maintenance of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets [61]. However, Krosl et al. demonstrated that antisense DNA against PBX1 apparently augmented self-renewal of HSCs overexpressing HOXB4 but was not effective on normal HSCs, suggesting that PBX1 may be a negative regulator of HOXB4-mediated self-renewal of HSCs [42]. In accordance with this result, we found that decHOX could enhance cytokine-mediated self-renewal of HSCs by modifying the function of HOXB4. Furthermore, we found that decHOX restored HOXB4 activity suppressed by PBX1 without affecting DNA-binding activities. The interaction between the YPWM motif in HOX and the homeodomain in PBX1 modifies the DNA-binding affinities of the proteins and their coactivator/corepressor binding-dependent transcriptional activities, and decHOX presumably affects the latter interactions. At present, we know that decHOX can augment HOXB4-dependent transcription of c-myc, which we recently identified as a key regulator of HOXB4- and Notch1-mediated HSC self-renewal [49]. However, HOX-PBX complexes regulate a number of genes in HSCs both positively and negatively. Also, PBX1 shows dual (positive and negative) effects on HOX-mediated transcription according to the target genes and/or the cellular context [31, 33, 34]. Thus, further studies to identify the target genes of HOX/PBX1 in HSCs would provide useful information to facilitate decHOX-mediated ex vivo amplification of HSCs.

To date, two groups of investigators have used ex vivo-amplified CB HSCs for transplantation. Shpall et al. [5] isolated CD34+ cells from CB. Forty percent of the isolated cells were expanded in medium containing SCF, G-CSF, and TPO for 10 days, and the remaining 60% were immediately transplanted or stored frozen until transplantation. After high-dose chemotherapy, 37 patients (25 adults, 12 children) received transplants of expanded CD34+ cells and nonexpanded cells with a median dose of 0.99 x 107 nucleated cells per kilogram. The median time to engraftment of neutrophils (neutrophil count > 500/µl) was 28 days (range, 15–49 days) and that of platelets (platelet > 20,000/µl) was 106 days (range, 38–345 days). The authors of that study concluded that although the ex vivo expansion of CB cells was feasible and safe, expanded HSCs did not improve the time to engraftment in recipients [5]. In a phase I trial, Jaroscak et al. [6] transplanted CB HSCs expanded by PIXY321, FL, and EPO into 28 patients with a median dose of 2.4 x 107 nucleated cells per kilogram. They also concluded that the ex vivo expanded CB HSCs were not effective in shortening the recovery period [6]. In contrast, the present study showed that decHOX can expand short-term as well as long-term SRC, thereby shortening the delay in hematopoietic recovery, suggesting that decHOX may be an effective tool to resolve this problem. Since HSC quiescence is regulated by p21waf1/cip1, p16INK4A, and p18INK4C and progenitor quiescence by p27kip2 [62–66], a basic study focusing on the expression of cell cycle control molecules in decHOX-transduced cells may clarify the mechanism of decHOX-mediated rapid recovery of hematopoiesis.

In conclusion, in the present study we demonstrated that decHOX can further augment cytokine-mediated ex vivo expansion of CB HSCs and that these expanded HSCs can restore hematopoiesis more rapidly and effectively than freshly prepared CB HSCs. However, it is necessary to further optimize treatment conditions, such as the method and timing of peptide delivery. Also, to enhance the effects of decHOX, it will be useful to explore the combined effects of other signals that can support HSC self-renewal, such as SHH and Wnt. We hope that our decHOX will eventually benefit patients with hematopoietic malignancies.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
We are grateful to N. Takada, K. Maruyama, and M. Hirose for technical support and animal care and Y. Ikegami for laboratory management. This work was supported by grants from the Ministry of Health, Labour and Welfare of Japan (number 18790672, to N.T.), Uehara Foundation (to K.Y.), and Hovansha Foundation (to K.Y.).


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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