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Stem Cells 2004;22:1003-1014 www.StemCells.com
© 2004 AlphaMed Press

Comparative Proteomic Analysis of Human CD34+ Stem/Progenitor Cells and Mature CD15+ Myeloid Cells

Wen Taoa,b,c, Mu Wangb,d, Emily D. Vossb, Ross R. Cocklinb, Jaime A. Smithb, Scott H. Coopera,c,e, Hal E. Broxmeyera,c,d,e

a Department of Microbiology and Immunology,
b Department of Biochemistry and Molecular Biology,
c Walther Oncology Center, and
d Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana, USA;
e Walther Cancer Institute, Indianapolis, Indiana, USA

Key Words. Hematopoietic stem and progenitor cells • Proteomics • CD15+ myeloid cells • Cord blood

Correspondence: Wen Tao, Ph.D., Walther Oncology Center, Indiana University School of Medicine, 950 West Walnut Street, Room 302, Indianapolis, Indiana 46202, USA. Telephone: 317-274-7568; Fax: 317-274-7592; e-mail: wetao{at}iupui.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human CD34+ cells, highly enriched for hematopoietic stem and progenitors, and CD15+ cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34+ and CD15+ cells. This suggests that CD34+ cells have a relatively larger proteome than mature CD15+ myeloid cells and production of many stem/progenitor cell–associated proteins ceases or is dramatically down-regulated as the CD34+ cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34+ cells. Most of the identified proteins in CD34+ cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34+ and CD15+ cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hematopoietic stem cells are rare cells found in bone marrow, fetal liver, and umbilical cord blood that are capable of self-renewal and differentiation to all types of mature blood cells [1,2]. Human umbilical cord blood is a valuable source of hematopoietic stem and progenitor cells used for stem cell transplantation [3–6]. Phenotypic and functional properties of murine and, to a lesser extent, human hematopoietic stem and progenitor cells have been extensively characterized both in vitro and in vivo. Populations greatly enriched for murine or human hematopoietic stem cells can be prospectively isolated based on cell-surface markers and their ability to cause an efflux in supravital fluorescent dyes [7–14]. Human CD34+ cells are highly enriched for hematopoietic stem and progenitor cells and are capable of maintaining a life-long supply of all hematopoietic lineages. These cells have also been used clinically to support high-dose chemotherapy or radiation therapy in patients with blood cancers [15–17]. In contrast, human CD15 (Lewis X or X-hapten) is a carbohydrate antigen whose expression is restricted to the more terminally differentiated myeloid lineage within the hematopoietic system. Human CD15 is expressed mainly on mature granulocytes (neutrophils and eosinophils) and to a varying degree on monocytes, but not on lymphocytes or basophils in peripheral blood [18–20]. Therefore, human CD34+ and CD15+ cells represent two distinct maturation/differentiation stages within the hematopoietic hierarchy and can be used to study functions of hematopoietic stem and progenitor cells as well as normal hematopoietic differentiation.

The proteome is the cell-specific protein complement of the genome and encompasses all proteins that are expressed in a cell at a given time [21]. The unique identity and functionality of a given cell are largely determined by the spectrum of proteins expressed in that cell. Therefore, characterizing the proteome for a cell type and determining how the proteome changes during development, in response to extrinsic stimuli, and in disease states are pivotal for elucidating the molecular mechanisms underlying many fundamental biological processes [22]. The availability of complete sequences of several genomes, including the human genome, coupled with the recent advances in high-resolution two-dimensional (2D) gel electrophoresis, image analysis algorithms, and mass spectrometry have made it possible to systematically identify and quantify large sets of proteins expressed in a given cell or tissue. The combination of high-resolution 2D gel electrophoresis or liquid chromatography with mass spectrometry has become a standard approach to proteomics [23–25]. Over the past several years, different populations of murine and human hematopoietic stem/progenitor cells under normal as well as neoplastic conditions have been characterized using gene expression profiling [26–29]. These studies have defined a molecular signature for stem cells (stemness) and have uncovered numerous genes specifically expressed in various populations of hematopoietic stem and progenitor cells. Recently, genomic and proteomic approaches have been used to quantitatively study the temporal patterns of protein and mRNA expression during retinoic acid–induced differentiation in a mouse promyelocytic cell line [30,31]. However, little is known about the large-scale protein components of primary human hematopoietic stem/progenitor cells or differentiated mature cells of a particular lineage.

In this study, we used a proteomic approach, which combines 2D gel electrophoresis with mass spectrometry, to systematically identify and quantify a spectrum of cytosolic proteins differentially present in human CD34+ stem/progenitor cells and in mature CD15+ myeloid cells from cord blood. Our study presents, for the first time, global cellular protein constituents in human CD34+ and CD34– depleted CD15+ cells and has identified changes in cellular protein composition during myeloid differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Purification and Cell Culture
Umbilical cord blood was collected into sterile tubes containing the anticoagulant heparin and used within 12 hours after collection. All procedures were done according to guidelines established at Indiana University School of Medicine. Low-density mononuclear cells were isolated by centrifugation on Ficoll-Paque PLUS (Amersham Biosciences, Piscataway, NJ). After washing, CD34+ cord blood cells were isolated from the low-density mononuclear cells using Direct CD34 Progenitor Isolation Kit (Miltenyi Biotec, Auburn, CA). CD34+ cell-depleted populations were then cultured overnight in Iscove’s Modified Dulbecco’s Medium (IMDM; Biowhittaker, Walkersville, MD) containing 10% fetal bovine serum (FBS; Hy Clone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Biowhittaker) with the following cytokines: 50 ng/ml recombinant human stem cell factor (SCF; UCB Research, Cambridge, MA), 100 U/ml human interleukin 3 (Bio Vision Research Products, Mountain View, CA), and 10 ng/ml human granulocyte-colony stimulating factor (Amgen, Thousand Oaks, CA). Subsequently, mature CD15+ myeloid cells were isolated from CD34+ cell-depleted populations using CD15 magnetic microbeads (Miltenyi Biotec). It has been shown that the function of normal mature granulocytes isolated by CD15 magnetic microbeads is not altered [20]. The purity of isolated CD34+ and CD15+ cells was determined by flow cytometry analysis of the isolated cells stained with a phycoerythrin-conjugated anti-CD34 antibody (Becton Dickinson, Mountain View, CA) and a fluorescent isothiocyanate–conjugated anti-CD15 antibody (BD Phar Mingen, San Diego, CA). To expand numbers of CD34+ cells, immunomagnetically isolated CD34+ cells were cultured for 5 days in IMDM containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and the following cytokines: 50 ng/ml SCF, 100 ng/ml recombinant human FLT3-ligand (Flt3-L; Immunex Corp., Seattle), and 100 ng/ml recombinant human thrombopoietin (Tpo; Genentech, Inc., South San Francisco, CA). Medium was changed once at day 3 [32–35].

Two-Dimensional Gel Electrophoresis
Isolated mature CD15+ myeloid cells and day-5 cultured CD34+ stem and progenitor cells were washed twice with phosphate-buffered saline and pelleted by brief centrifugation. The CD15+ or CD34+ cells were then lysed in a buffer containing 1.5% Triton X-100 at 4°C for 1 hour. Insoluble materials were removed by centrifugation at x16,000g. Lysis buffer containing 9 M urea, 4% Igepal CA-630 (NP-40, Sigma, St. Louis), 1% dithiothreitol (DTT), and 2% ampholytes (pH 8–10.5) was added to Triton X-100–soluble protein extracts. Two-dimensional gel electrophoresis was performed as described previously [36]. Briefly, samples were normalized by Bradford protein assay to 1 mg/ml using rehydration buffer containing 6 M urea, 375 mM Tris-HCl (pH 8.8), 2% SDS, and 20% glycerol. A 200-µl aliquot of the normalized protein sample solution was loaded on a linear gradient Immobiline Dry Strip (IPG strip, Amersham Biosciences, pH 3–10, 110 x3 x0.5 mm). Duplicate IPG strips were used for each sample. After the IPG strips were rehydrated, first-dimension isoelectric focusing (IEF) was performed at 21°C using the Bio-Rad PROTEAN IEF Cell for 120,000 Vh. After IEF, the IPG strips were equilibrated in the rehydration buffer plus 2% DTT for 20 minutes at room temperature. The IPG strips were then alkylated with 2.5% iodoacetamide in the rehydration buffer for 20 minutes. Subsequently, the IPG strips were washed twice in MOPS buffer (1 M 3-N-morphilino-propane sulfonic acid, 1M Tris base, 69.3 mM SDS, and 20.5 mM EDTA) and loaded onto precast NuPage Bis-Tris Gels (Invitrogen, Carlsbad, CA) with ReadyPrep overlay agarose containing trace bromophenol blue (Bio-Rad, Hercules, CA). The second-dimension electrophoresis was conducted in MOPS buffer at 200 V for 1 hour. Polyacrylamide gels were then fixed (50% ethanol, 1.7% H3PO4), stained with Coomassie blue (0.1% Coomassie Brilliant Blue R250, 40% methanol, and 10% acetic acid), and destained (40% methanol and 10% acetic acid) to visualize the separated proteins.

Image Analysis
The Coomassie blue–stained 2D gels were scanned and digitized under visible light at 200 µm/pixel resolution using the Flour-S MAX Multi Imager system (Bio-Rad). The same scanning conditions were used for each 2D gel in a matched set. Image data were analyzed using PDQuest 2D Analysis software (Bio-Rad). The background values for the gels were subtracted, and the spot peaks were located and quantified. To minimize variation due to experimental factors, the intensity of each spot on a gel was normalized on the basis of the total integrated optical density for that gel. The quantity of protein in a given spot was defined as parts-per-million of the total integrated optical density. The image analysis software was used to create a Match Set consisting of all the gels to be analyzed followed by generating a reference template that contains the spot data from all of the gels in the Match Set. Subsequently, gel matching and quantitative and qualitative spot comparisons across gels were performed. Each gel in the Match Set was matched to the reference template, with landmark proteins (those uniformly expressed in all of the gels) used to quickly and accurately match all gels. The positively identified spots were manually inspected and evaluated. The abundance of individual proteins was calculated using a quantitative analysis set within the PDQuest software, which allows for the construction of a fold change comparison chart for any given expressed proteins [36].

Identification of Proteins by Mass Spectrometry
After quantitative analysis, protein spots of interest were cut out from replicate gels using an automated spot excision robot, PROTEAN 2D Spot Cutter (Bio-Rad). Then the excised protein spots were robotically processed for mass spectrometry using the Mass PREP Workstation (Perkin-Elmer) as described [36], and for a limited number of proteins, the samples were also prepared manually. Briefly, the excised protein gel pieces placed in individual wells of a 96-well plate were destained with 50% acetonitrile and 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide. After alkylation, proteins in gel pieces were digested with 6 ng/µl trypsin overnight at 37°C. The resulting peptides were desalted using ZipTips containing C18 resin, mixed with the matrix solution containing {alpha}-cyano-4-hydroxycinnamic acid, and analyzed using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Micro-mass, Manchester, UK). Peptide mass fingerprinting spectra were recorded in positive reflection mode of the MALDI-TOF mass spectrometer. The time of flight was measured using the following parameters: 3,400-V pulse voltage, 15,000 source voltage, 500-V reflection voltage, 1,950-V microchannel plate voltage, and low mass gate of 500 d. All peptide samples were measured as monoisotopic masses, and internal calibration was performed using autolytic peaks of bovine trypsin (M + H+, m/z 842.5099, and m/z 2211.1045) in the same series as the samples to be measured. For protein identification, the measured peptide mass profiles were compared with the theoretical peptide masses derived from tryptic digests of known human proteins in the protein database of the National Center for Biotechnology Information using ProFound® search engine.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cells and Purity
We used primary umbilical cord blood CD34+ and the corresponding CD34-depleted CD15+ cells to define a broad range of cytosolic proteins differentially present in human hematopoietic stem/progenitor cells and in mature myeloid cells. Cord blood CD34+ cells were immunomagnetically isolated and expanded as described in Materials and Methods. CD15+ cells were then purified from the same cord bloods that were depleted of CD34+ cells. The purity of 2-day cultured CD34+ cord blood cells and the corresponding isolated CD15+ cells was respectively 89% and 85%, as determined by flow cytometry (Fig. 1Go). The isolated CD34+ cell population only contained 1.86% CD15+CD34– cells, whereas purified CD15+ cell population only contained 0.28% CD15–CD34+ cells.



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  		Figure 1. Purity of isolated human CD34+ and CD15+ umbilical cord blood cells. Isolated CD34+ cells after 2 days in culture and purified CD15+ cells were stained with a phycoerythrin-conjugated anti-CD34 antibody and a fluorescent isothiocyanate–conjugated anti-CD15 antibody. The samples were then analyzed by flow cytometry. Quadrant gates were set based on staining of isotype control antibodies. Percentages of cells are indicated within the defined quadrants.

 
Proteomic Analyses of Human CD34+ and CD15+ Cord Blood Cells
To survey cytosolic protein compositions of hematopoietic stem/progenitor cells and of mature myeloid cells, isolated human CD34+ and CD15+ cord blood cells were extracted with Triton X-100 detergent, and the Triton X-100–soluble proteins were then separated on duplicate 2D gels with wide-range (pH 3–10), linear IPG strips in the first dimension. Representative Coomassie blue–stained 2D maps of human CD34+ and CD15+ cells are shown in Figure 2Go. The patterns of resolved protein spots on duplicate gels for each sample were very consistent. Using PDQuest 2D Analysis software, we analyzed and compared the 2D gels from the different cell types, and the abundance of individual protein spots on each gel was quantified. On average, approximately 460 protein spots on each gel were detected. To avoid inaccuracy resulting from imperfect alignment of 2D gel images by the software, approximately 140 differentially expressed (> twofold) protein spots were manually and individually inspected, evaluated, and chosen for further analyses. Figure 3Go displays images and histograms, representing detected levels of protein expression, of nine representative protein spots that showed significant changes between CD34+ and CD15+ cells. In general, at least a twofold difference in protein expression levels between different cell types could be reliably detected. As shown in Figure 4Go, quantification of all of the selected protein spots revealed that 112 and 15 proteins were differentially expressed or post-translationally modified in human CD34+ and CD15+ cord blood cells, respectively. These results demonstrated that CD34+ stem/progenitor cells contain much larger numbers of their specific proteins compared with CD15+ mature myeloid cells, which suggests that the CD34+ stem/progenitor cells have a relatively larger proteome than mature CD15+ myeloid cells; production of many stem cell–associated proteins completely ceased or was dramatically downregulated as the CD34+ cells differentiated toward mature blood cells. In addition to specific functions that individual proteins can perform, the larger proteome of hematopoietic stem cells may not only afford these cells to be multipotent but may also provide a basis for lineage choice upon differentiation. Differentiation of hematopoietic stem cells into a particular lineage could be controlled by shutting down expression of a specific set of proteins and activating a limited number of lineage-related proteins.



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  		Figure 2. Representative two-dimensional (2D) gel images of cytosolic proteins extracted from human CD34+ (A) and CD15+ (B) cord blood cells. Samples containing 200-µg total proteins were subjected to isoelectric focusing (IEF) in linear IPG strips with pH of approximately 3 to 10, followed by SDS-PAGE. The 2D gels were then stained with Coomassie blue, scanned, and digitized. Image data were analyzed, and the abundance of individual proteins was calculated using PDQuest software. Subsequently, approximately 140 differentially expressed protein spots from different sets of gels were excised, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

 


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  		Figure 3. Images of nine representative Coomassie blue–stained two-dimensional gel protein spots that showed significant changes between human CD34+ and CD15+ cells and their corresponding histograms depicting quantified expression levels. Each bar in a histogram for a protein spot represents the mean protein quantity value (the horizontal black line between white portions of the bar) and the standard deviation (white portions of the bar). Bars on the left and right in each histogram correspond to the samples from human cord blood CD34+ and CD15+ cells, respectively. Standard spot (SSP) numbers are numbers assigned to each protein spot by PDQuest software, and each SSP number uniquely identifies that protein. Please also see footnote a in Table 1Go. Quantitative changes in average protein expression levels between different cell types are shown in parts per million (PPM), as defined by the software.

 


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  		Figure 4. Number of proteins found to be predominantly present or post-translationally modified in human cord blood CD34+ or CD15+ cells. Individual protein spots identified from CD34+ and CD15+ cells were matched, quantified, and compared. Fold changes (> twofold) were calculated for each protein between the CD34+ and CD15+ samples.

 
The approximately 140 chosen protein spots from different sets of 2D gels were then excised, digested with trypsin, and analyzed by MALDI-TOF mass spectrometry. The identities of individual proteins were determined by comparing the recorded masses of fingerprint peptides with the theoretical peptide masses derived from tryptic digests of known human proteins in the protein database of the National Center for Biotechnology Information (NCBI) using ProFound search engine. Table 1Go lists the 47 positively identified proteins. The identified known proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Moreover, we identified at least eight novel proteins whose functions are unknown.


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  		Table 1. List of proteins enriched in CD34+ stem/progenitor cells and in mature CD15+ myeloid cells
 
Of note, multiple heat shock proteins and chaperonins, including members of the 60-kDa and 70-kDa heat shock protein families, chaperonin-containing TCP1 complex proteins, stress-induced phosphoprotein 1 (Hsp70/Hsp90-organizing protein), and hypothetical protein DKFZ p761K0511, were expressed predominantly in CD34+ cells. This indicates that CD34+ stem/progenitor cells possess the attributes of cells under stress, which is consistent with findings from genomic studies of highly purified murine hematopoietic stem cells [27]. Moreover, proteins involved in intracellular membrane traffic, including RAB7, valosin-containing protein, synatosomal-associated protein, and protein kinase C and casein kinase substrate in neurons 1, were also found to be differentially expressed in CD34+ cells. Thus, active protein folding and assembly machinery seems to be part of the molecular circuitry of hematopoietic stem and progenitor cells. These processes may be required for many of their basic functions. The presence of many heat shock proteins and chaperones in the stem and progenitor cells also renders these cells highly resistant to various environmental stresses.

Surveying the expression profiles of the positively identified proteins from the NCBI databases revealed that 31 CD34+ stem/progenitor cell–associated proteins, including RAB7, KH-type splicing regulatory protein, peptidylprolyl isomerase A, pyruvate kinase 3, and guanine nucleotide-binding protein beta polypeptide 2-like 1, are also expressed in germ cell tumors. This suggests that hematopoietic stem cells and germ cells share some similar subsets of proteins; perhaps these proteins are directly or indirectly involved in the maintenance of pluripotency of these cells. It is possible that basic molecular mechanisms necessary for maintaining pluripotency are conserved among different types of stem cells. Nearly all of the identified CD34+ cell–enriched proteins were found to be expressed in many types of tumors, including embryonal carcinoma and neuroblastoma. These results demonstrate that there exists a certain degree of molecular similarity between hematopoietic stem/progenitor cells and cancer cells. This supports the notion that similar signaling pathways may regulate self-renewal in stem cells and cancer cells [2]. Moreover, many proteins differentially expressed in human CD34+ cells have also been found to be expressed in brain, embryonic stem cells, and germinal center B cells. It is also of interest to note that germinal centers within secondary lymphoid tissues represent the sites where memory B cells are generated and where somatic hypermutation of the variable region of immunoglobulin genes occurs within B cells at high frequency [37,38]. In addition, prostatic binding protein and enolase 1 have been previously found to be differentially expressed in purified CD34+CD38– normal bone marrow cells (http://www.ncbi.nlm.nih.gov/UniGene; Uni Gene Cluster Hs.433863 and Uni Gene Cluster Hs.433455 Homo sapiens).

Several cell motility proteins, such as Tropomyosin 4, Fascin homolog 1 (an actin-bundling protein), gamma actin, beta tubulin, and hypothetical protein XP_037953, were found to be differentially expressed in human CD34+ stem/progenitor cells. Hypothetical protein XP_037953 is a member of the ERM (ezrin, radixin, moesin) family of proteins that is thought to link cytoskeletal components with proteins in the cell membrane. Hypothetical protein XP_037953 is highly homologous to neurofibromin 2. This suggests that hematopoietic stem cells may possess a unique cytoskeletal architecture or mobility compared with their mature descendants.

Small nuclear RNA-activating complex polypeptide 4 (SNAPC4) and KIAA0912 protein were enriched in mature CD15+ myeloid cells. SNAPC4 is a Myb DNA-binding domain containing protein that interacts with transcription factor Oct-1, and SNAPC is required for transcription of human snRNA genes by RNA polymerase II and III [39,40]. KIAA0912 protein contains a small-conductance mechano-sensitive channel domain and a membrane-bound metal-lopeptidase domain. The function of KIAA0912 protein is at present unknown.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes a proteomic approach to systematically analyze the cytosolic proteins in human umbilical cord blood CD34+ cells and their corresponding CD15+ cells. This provides a bird’s-eye view of cytosolic protein expression patterns in primary human hematopoietic stem/progenitor cells and in their offspring, the more terminally differentiated myeloid cells. Comparison of human CD34+ stem/progenitor cells with differentiated CD15+ myeloid cells identified changes in cellular protein composition during myeloid differentiation; this information should further our understanding of the intracellular processes of myeloid differentiation and add insight into the functional capabilities of these distinct cell types. Although we believe that the proteins identified in this study are important for hematopoietic stem and progenitor cell function, we should also point out that how these proteins actually function in these cells and during hematopoietic differentiation is at present unknown. Perhaps studying each protein individually using gain of function, loss of function, and dominant-negative mutants would reveal how, when, and if these various proteins contribute to the self-renewal and differentiation of hematopoietic stem cells. Among approximately 140 2D protein spots analyzed by MALDI-TOF mass spectrometry, 47 proteins were positively identified and classified into a variety of functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Our results suggest that CD34+ stem/progenitor cells have a relatively larger proteome than mature CD15+ myeloid cells and that production of many stem cell–associated proteins completely ceases or is dramatically downregulated as CD34+ cells undergo differentiation toward mature blood cells. Moreover, multiple heat shock proteins and chaperones as well as proteins important for vesicular transport were found to be predominantly present in CD34+ cells. Recent studies have shown that protein-folding and quality-control systems in the endoplastic reticulum and in the secretory pathway function in parallel as part of post-translational checkpoints to ensure the fidelity and regulation of eukaryotic protein expression. That these systems are able to operate with great precision is vital for maintaining many essential biological processes [41–43]. Therefore, it appears that protein folding and post-translational quality control systems are highly active in hematopoietic stem cells. They may be part of regulatory networks that control the proteome of these stem cells and may be essential to their full functionality.

Given that an accurate and specific mass spectrum of fingerprinting peptides is obtained, the protein spot signal derived from a 2D gel can quantitatively represent the expression of the identified protein or distribution among different posttranslationally modified species of that protein. In most cases, a 2D protein spot identified by mass spectrometry represents the expression of that protein. There exist several possibilities for not being able to identify all of the proteins analyzed. First, we did not include any proteins that have Z scores very close to but less than 1 to ensure the quality and accuracy of protein identification. Second, sometimes the same protein was identified from multiple spots, which are most likely the post-translationally modified species of that protein. Third, the finite sensitivity of the mass spectrometric methodology acts as a bias against proteins of low abundance, from which no good mass spectra were obtained [44]. Finally, high-quality mass spectra were obtained from several protein spots, but the database search did not produce good matches.

More than a dozen proteins were found almost exclusively in the human CD34+ cell population. These include DKFZP566K023 protein, apolipoprotein F, tropomyosin, nonmetastatic cell 1 protein, and prostatic binding protein (Table 1Go). Prostatic binding protein (PBP or RKIP) has been shown to inhibit the phosphorylation and activation of mitogen-activated protein (MAP) kinase kinase by RAF1 [45]. This MAP kinase signaling cascade is a well-established pathway for controlling proliferation and differentiation of different cell types. Recently, it has been demonstrated that PBP is a physiologic inhibitor of G protein–dependent receptor kinase 2 (GRK2). After stimulation of G protein–coupled receptor, PBP becomes phosphorylated on serine-153 by a protein kinase C–dependent mechanism. The phosphorylated PBP then dissociates from RAF1 to associate with GRK2 and block its activity [46]. Because GRK2 seems to mediate agonist-specific desensitization of G protein–coupled receptors, the incoming receptor signal is markedly amplified both by releasing inhibition of RAF1 and by blocking receptor desensitization [46]. Inhibition of GRK2 by PBP, which decreases receptor desensitization, may also regulate motility and migration of hematopoietic stem cells, because most chemo-kine receptors are G protein–coupled receptors. PBP is also a novel serine protease inhibitor and exerts inhibitory activity against several serine proteases, including thrombin, neuropsin, and chymotrypsin [47]. In addition, PBP is strongly expressed in the cytoplasm of oligodendrocytes in brain and Schwann cells in nerve roots [48].

Nonmetastatic cells 1 protein (NME1) was identified because of its reduced expression in highly metastatic tumor cells. Accordingly, NME1 mRNA levels are highest in cell lines with low metastatic potential [49]. Mutations in NME1 have been found in aggressive neuroblastomas [50]. Thus, NME1 seems to function as a negative regulator of cancer metastasis. NME1 encodes the A isoform of nucleoside diphosphate kinase. Nucleoside diphosphate kinase exists as a hexameric enzyme composed of the A (encoded by NME1) and B (encoded by NME2) isoforms [51]. It has been shown that human NME1 protein is highly homologous to a developmentally regulated protein in Drosophila encoded by the abnormal wing disc (awd) gene. Mutations in awd cause abnormal tissue morphology and necrosis as well as widespread aberrant differentiation in Drosophila, analogous to changes in malignant progression [52]. Moreover, it has been demonstrated that the NME1 protein can also function as a transcription factor for the c-MYC oncogene. MYC is an oncoprotein implicated in cell proliferation, differentiation, and apoptosis. NME1 binds to the MYC P1 promoter and is required for efficient transcription of MYC oncogene in vitro [53]. Therefore, NME1 can be both a suppressor of tumor metastasis and an activator of MYC. It is well established that there is an inverse relationship between MYC expression and cell differentiation. A differentiation-inhibiting factor in mouse myeloid leukemia cells has been identified as a murine homolog of NME1 [54]. Recent evidence also indicates that the NME genes are involved in control of normal development and cell differentiation [55]. Because NME1 is an important factor for inhibiting metastasis and differentiation of tumor cells, it is possible that NME1 may play a role in retention of hematopoietic stem cells in bone marrow niches as well as in control of their differentiation, although we do not yet know how NME1 functions in these stem cells. It is of great interest that far upstream element binding protein 1 (FUBP1 or FBP) and MAX protein isoform a (MAX) were found to be almost exclusively associated with human CD34+ cells. It has been shown that expression of 2.6-kb FUBP1 mRNA declines upon differentiation, and FUBP1 is present in undifferentiated but not differentiated cells. FUBP1 activates the far upstream element of c-MYC and stimulates expression of c-MYC in undifferentiated cells [56]. MAX protein is a member of the basic helix-loop-helix leucine zipper family of transcription factors. MAX is able to form homodimers and heterodimers with three other evolutionarily related transcription factors, MYC, MAD, and MXI1. MAX protein specifically associates with MYC to form a heterodimer that binds, with high affinity, to a specific set of DNA sequences called the enhancer box (the E box). The homodimers and heterodimers compete for the common E box–related DNA sites, and rearrangement among these dimers provides a complex system of transcriptional regulation [57,58]. Coupled with the fact that c-MYC is expressed at relatively high levels in human CD34+ cells, these results strongly support our previous proposition that c-MYC and signaling pathways regulated by c-MYC play pivotal roles in the maintenance, proliferation, and differentiation of hematopoietic stem/progenitor cells [35].

It is intriguing to note that short stature homeo box 2 (SHOX2 or SHOT) protein was found to be differentially associated with human CD34+ cells. SHOX2 belongs to the homeo box superfamily of proteins containing a 60-aminoacid motif that represents a DNA binding domain. Home-obox genes have been characterized extensively as transcriptional regulators involved in pattern formation during embryonic development in both invertebrate and vertebrate species. SHOX2 gene is only present in vertebrates, and human SHOX2 is implicated in craniofacial, brain, heart, and limb development [59]. SHOX2 expression during human embryonic development detected by in situ hybridization was reported to be in the pharyngeal arches, nasal process, cardiac inflow tract, limb, and genital tubercle [60].

Currently, the functions of DKFZP566K023 protein, KIAA0819 protein, CTCL tumor antigen se2-2, and KIAA0063 protein remain to be elucidated, and it would be of interest to study how these proteins may function in hematopoietic stem and progenitor cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
These studies were supported by United States Public Health Service grants RO1 HL 56416, RO1 DK 53674, and RO1 HL 67384 from the NIH to H.E.B. We would like to thank the Indiana Genomic Initiative for purchasing the mass spectrometers used in this study.

Wen Tao and Mu Wang contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet 2000;1:57–64.[Medline]

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Received April 14, 2004; accepted for publication June 4, 2004.



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