Comparative Proteomic Analysis of Human CD34+ Stem/Progenitor Cells and Mature CD15+ Myeloid Cells
http://www.100md.com
《干细胞学杂志》
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@iupui.edu
ABSTRACT
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 . Human umbilical cord blood is a valuable source of hematopoietic stem and progenitor cells used for stem cell transplantation . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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
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. 1). The isolated CD34+ cell population only contained 1.86% CD15+CD34– cells, whereas purified CD15+ cell population only contained 0.28% CD15–CD34+ cells.
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 2. 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 3 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 4, 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.
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.
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 1. Quantitative changes in average protein expression levels between different cell types are shown in parts per million (PPM), as defined by the software.
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 1 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.
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 . 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 . 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 . 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 . 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
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
Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet 2000;1:57–64.
Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105–111.
Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U SA 1989;86:3828–3832.
Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992;89:4109–4113.
Gluckman E. Hematopoietic stem-cell transplants using umbilical-cord blood. N Engl J Med 2001;344:1860–1861.
Broxmeyer HE, Smith FO. Cord blood hematopoietic cell transplantation. In: Blume KG, Forman SJ, Appelbaum FR eds. Thomas’Hematopoietic Cell Transplantation. Malden, MA: Blackwell Science Ltd, 2004;550–564.
Visser JW, Bauman JG, Mulder AH et al. Isolation of murine pluripotent hemopoietic stem cells. J Exp Med 1984;159: 1576–1590.
Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells, I: the separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence. Exp Hematol 1985;13:999–1006.
Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells, II: stem cells of long-term bone marrow-reconstituted recipients. Exp Hematol 1988;16:245–249.
Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;240:58–62.
Muller-Sieburg C, Torok-Storb B, Visser JW et al. Hematopoietic stem cells: animal models and human transplantation. In: Compans RW, Cooper M, Koprowski H et al., eds. Current Topics in Microbiology and Immunology, Vol. 177. New York: Springer-Verlag, 1992;1–251.
Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1:661–673.
Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 2000;287: 1442–1446.
Tao W, Broxmeyer HE. Towards a molecular understanding of hematopoietic stem and progenitor cells. In: Broxmeyer HE, ed. Cord Blood: Biology, Immunology, Banking, and Clinical Transplantation. Bethesda, MD: AABB Press, 2004:69–105.
de Wynter EA, Buck D, Hart C et al. CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors. STEM CELLS 1998;16:387–396.
Blystad AK, Holte H, Kvaloy S et al. High-dose therapy in patients with Hodgkin’s disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells. Bone Marrow Transplant 2001;28:849–857.
Lanza F, Campioni D, Moretti S et al. CD34(+) cell subsets and long-term culture colony-forming cells evaluated on both autologous and normal bone marrow stroma predict long-term hematopoietic engraftment in patients undergoing autologous peripheral blood stem cell transplantation. Exp Hematol 2001;29:1484–1493.
Bettelheim P. Cluster report: CD15. In: Knapp W, ed. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press, 1989:798–799.
Kannagi R. CD15 workshop panel report. In: Kishimoto T, ed. Leucocyte Typing VI: White Cell Differentiation Antigens. Proceedings of the Sixth International Workshop and Conference Held in Kobe, Japan, 10–14 November, 1996. New York: Garland Publishing, 1998:348–352.
Zahler S, Kowalski C, Brosig A et al. The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method. J Immunol Methods 1997;200:173–179.
Rappsilber J, Mann M. What does it mean to identify a protein in proteomics? Trends Biochem Sci 2002;27:74–78.
Zhu H, Bilgin M, Snyder M. Proteomics. Annu Rev Biochem 2003;72:783–812.
Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature 2003;422:198–207.
Patterson SD, Aebersold RH. Proteomics: the first decade and beyond. Nat Genet 2003;33(suppl):311–323.
Ferguson PL, Smith RD. Proteome analysis by mass spectrometry. Annu Rev Biophys Biomol Struct 2003;32:399–424.
Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601–604.
Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597–600.
Terskikh AV, Miyamoto T, Chang C et al. Gene expression analysis of purified hematopoietic stem cells and committed progenitors. Blood 2003;102:94–101.
Akashi K, He X, Chen J et al. Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood 2003;101:383–389.
Lian Z, Kluger Y, Greenbaum DS et al. Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line. Blood 2002;100:3209–3220.
Lian Z, Wang L, Yamaga S et al. Genomic and proteomic analysis of the myeloid differentiation program. Blood 2001;98:513–524.
Lewis ID, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001;97:3441–3449.
Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736–3749.
Takatoku M, Sellers S, Agricola BA et al. Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates. J Clin Invest 2001;108:447–455.
Tao W, Hangoc G, Hawes JW et al. Profiling of differentially expressed apoptosis-related genes by cDNA arrays in human cord blood CD34(+) cells treated with etoposide. Exp Hematol 2003;31:251–260.
Decker ED, Zhang Y, Cocklin RR et al. Proteomic analysis of differential protein expression induced by ultraviolet light radiation in HeLa cells. Proteomics 2003;3:2019–2027.
Delves PJ, Roitt IM. The immune system: second of two parts. N Engl J Med 2000;343:108–117.
Tarlinton D. Germinal centers: form and function. Curr Opin Immunol 1998;10:245–251.
Wong MW, Henry RW, Ma B et al. The large subunit of basal transcription factor SNAPc is a Myb domain protein that interacts with Oct-1. Mol Cell Biol 1998;18:368–377.
Henry RW, Sadowski CL, Kobayashi R et al. A TBP-TAF complex required for transcription of human snRNA genes by RNA polymerase II and III. Nature 1995;374:653–656.
Sifers RN. Insights into checkpoint capacity. Nat Struct Mol Biol 2004;11:108–109.
Wickner S, Maurizi MR, Gottesman S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 1999;286:1888–1893.
Ellgaard L, Molinari M, Helenius A. Setting the standards: quality control in the secretory pathway. Science 1999;286: 1882–1888.
Mootha VK, Bunkenborg J, Olsen JV et al. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 2003;115:629–640.
Yeung K, Seitz T, Li S et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 1999; 401:173–177.
Lorenz K, Lohse MJ, Quitterer U. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 2003; 426:574–579.
Hengst U, Albrecht H, Hess D et al. The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors. J Biol Chem 2001;276:535–540.
Moore C, Perry AC, Love S et al. Sequence analysis and immunolocalisation of phosphatidylethanolamine binding protein (PBP) in human brain tissue. Brain Res Mol Brain Res 1996;37:74–78.
Bevilacqua G, Sobel ME, Liotta LA et al. Association of low nm23 RNA levels in human primary infiltrating ductal breast carcinomas with lymph node involvement and other histopathological indicators of high metastatic potential. Cancer Res 1989;49:5185–5190.
Chang CL, Zhu XX, Thoraval DH, et al. Nm23-H1 mutation in neuroblastoma. Nature 1994;370:335–336.
Gilles AM, Presecan E, Vonica A et al. Nucleoside diphosphate kinase from human erythrocytes: structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme. J Biol Chem 1991;266: 8784–8789.
Rosengard AM, Krutzsch HC, Shearn A et al. Reduced Nm23/Awd protein in tumour metastasis and aberrant Drosophila development. Nature 1989;342:177–180.
Postel EH, Berberich SJ, Flint SJ et al. Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science 1993;261:478–480.
Okabe-Kado J, Kasukabe T, Honma Y et al. Identity of a differentiation inhibiting factor for mouse myeloid leukemia cells with NM23/nucleoside diphosphate kinase. Biochem Biophys Res Commun 1992;182:987–994.
Lombardi D, Lacombe ML, Paggi MG. nm23: unraveling its biological function in cell differentiation. J Cell Physiol 2000;182:144–149.
Duncan R, Bazar L, Michelotti G et al. A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev 1994;8:465–480.
Blackwood EM, Eisenman RN. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 1991;251:1211–1217.
Grandori C, Mac J, Siebelt F et al. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo. EMBO J 1996;15:4344–4357.
Blaschke RJ, Monaghan AP, Schiller S et al. SHOT, a SHOX-related homeobox gene, is implicated in craniofacial, brain, heart, and limb development. Proc Natl Acad Sci U S A 1998;95:2406–2411.
Clement-Jones M, Schiller S, Rao E et al. The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome. Hum Mol Genet 2000;9:695–702.(Wen Taoa,b,c, Mu Wangb,d,)
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@iupui.edu
ABSTRACT
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 . Human umbilical cord blood is a valuable source of hematopoietic stem and progenitor cells used for stem cell transplantation . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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
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. 1). The isolated CD34+ cell population only contained 1.86% CD15+CD34– cells, whereas purified CD15+ cell population only contained 0.28% CD15–CD34+ cells.
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 2. 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 3 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 4, 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.
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.
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 1. Quantitative changes in average protein expression levels between different cell types are shown in parts per million (PPM), as defined by the software.
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 1 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.
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 . 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 . 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 . 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 . 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
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
Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet 2000;1:57–64.
Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105–111.
Broxmeyer HE, Douglas GW, Hangoc G et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U SA 1989;86:3828–3832.
Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci U S A 1992;89:4109–4113.
Gluckman E. Hematopoietic stem-cell transplants using umbilical-cord blood. N Engl J Med 2001;344:1860–1861.
Broxmeyer HE, Smith FO. Cord blood hematopoietic cell transplantation. In: Blume KG, Forman SJ, Appelbaum FR eds. Thomas’Hematopoietic Cell Transplantation. Malden, MA: Blackwell Science Ltd, 2004;550–564.
Visser JW, Bauman JG, Mulder AH et al. Isolation of murine pluripotent hemopoietic stem cells. J Exp Med 1984;159: 1576–1590.
Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells, I: the separation and enrichment of stem cells homing to marrow and spleen on the basis of rhodamine-123 fluorescence. Exp Hematol 1985;13:999–1006.
Bertoncello I, Hodgson GS, Bradley TR. Multiparameter analysis of transplantable hemopoietic stem cells, II: stem cells of long-term bone marrow-reconstituted recipients. Exp Hematol 1988;16:245–249.
Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;240:58–62.
Muller-Sieburg C, Torok-Storb B, Visser JW et al. Hematopoietic stem cells: animal models and human transplantation. In: Compans RW, Cooper M, Koprowski H et al., eds. Current Topics in Microbiology and Immunology, Vol. 177. New York: Springer-Verlag, 1992;1–251.
Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1994;1:661–673.
Weissman IL. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 2000;287: 1442–1446.
Tao W, Broxmeyer HE. Towards a molecular understanding of hematopoietic stem and progenitor cells. In: Broxmeyer HE, ed. Cord Blood: Biology, Immunology, Banking, and Clinical Transplantation. Bethesda, MD: AABB Press, 2004:69–105.
de Wynter EA, Buck D, Hart C et al. CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors. STEM CELLS 1998;16:387–396.
Blystad AK, Holte H, Kvaloy S et al. High-dose therapy in patients with Hodgkin’s disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells. Bone Marrow Transplant 2001;28:849–857.
Lanza F, Campioni D, Moretti S et al. CD34(+) cell subsets and long-term culture colony-forming cells evaluated on both autologous and normal bone marrow stroma predict long-term hematopoietic engraftment in patients undergoing autologous peripheral blood stem cell transplantation. Exp Hematol 2001;29:1484–1493.
Bettelheim P. Cluster report: CD15. In: Knapp W, ed. Leucocyte Typing IV: White Cell Differentiation Antigens. Oxford: Oxford University Press, 1989:798–799.
Kannagi R. CD15 workshop panel report. In: Kishimoto T, ed. Leucocyte Typing VI: White Cell Differentiation Antigens. Proceedings of the Sixth International Workshop and Conference Held in Kobe, Japan, 10–14 November, 1996. New York: Garland Publishing, 1998:348–352.
Zahler S, Kowalski C, Brosig A et al. The function of neutrophils isolated by a magnetic antibody cell separation technique is not altered in comparison to a density gradient centrifugation method. J Immunol Methods 1997;200:173–179.
Rappsilber J, Mann M. What does it mean to identify a protein in proteomics? Trends Biochem Sci 2002;27:74–78.
Zhu H, Bilgin M, Snyder M. Proteomics. Annu Rev Biochem 2003;72:783–812.
Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature 2003;422:198–207.
Patterson SD, Aebersold RH. Proteomics: the first decade and beyond. Nat Genet 2003;33(suppl):311–323.
Ferguson PL, Smith RD. Proteome analysis by mass spectrometry. Annu Rev Biophys Biomol Struct 2003;32:399–424.
Ivanova NB, Dimos JT, Schaniel C et al. A stem cell molecular signature. Science 2002;298:601–604.
Ramalho-Santos M, Yoon S, Matsuzaki Y et al. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science 2002;298:597–600.
Terskikh AV, Miyamoto T, Chang C et al. Gene expression analysis of purified hematopoietic stem cells and committed progenitors. Blood 2003;102:94–101.
Akashi K, He X, Chen J et al. Transcriptional accessibility for genes of multiple tissues and hematopoietic lineages is hierarchically controlled during early hematopoiesis. Blood 2003;101:383–389.
Lian Z, Kluger Y, Greenbaum DS et al. Genomic and proteomic analysis of the myeloid differentiation program: global analysis of gene expression during induced differentiation in the MPRO cell line. Blood 2002;100:3209–3220.
Lian Z, Wang L, Yamaga S et al. Genomic and proteomic analysis of the myeloid differentiation program. Blood 2001;98:513–524.
Lewis ID, Almeida-Porada G, Du J et al. Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system. Blood 2001;97:3441–3449.
Piacibello W, Sanavio F, Severino A et al. Engraftment in nonobese diabetic severe combined immunodeficient mice of human CD34(+) cord blood cells after ex vivo expansion: evidence for the amplification and self-renewal of repopulating stem cells. Blood 1999;93:3736–3749.
Takatoku M, Sellers S, Agricola BA et al. Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates. J Clin Invest 2001;108:447–455.
Tao W, Hangoc G, Hawes JW et al. Profiling of differentially expressed apoptosis-related genes by cDNA arrays in human cord blood CD34(+) cells treated with etoposide. Exp Hematol 2003;31:251–260.
Decker ED, Zhang Y, Cocklin RR et al. Proteomic analysis of differential protein expression induced by ultraviolet light radiation in HeLa cells. Proteomics 2003;3:2019–2027.
Delves PJ, Roitt IM. The immune system: second of two parts. N Engl J Med 2000;343:108–117.
Tarlinton D. Germinal centers: form and function. Curr Opin Immunol 1998;10:245–251.
Wong MW, Henry RW, Ma B et al. The large subunit of basal transcription factor SNAPc is a Myb domain protein that interacts with Oct-1. Mol Cell Biol 1998;18:368–377.
Henry RW, Sadowski CL, Kobayashi R et al. A TBP-TAF complex required for transcription of human snRNA genes by RNA polymerase II and III. Nature 1995;374:653–656.
Sifers RN. Insights into checkpoint capacity. Nat Struct Mol Biol 2004;11:108–109.
Wickner S, Maurizi MR, Gottesman S. Posttranslational quality control: folding, refolding, and degrading proteins. Science 1999;286:1888–1893.
Ellgaard L, Molinari M, Helenius A. Setting the standards: quality control in the secretory pathway. Science 1999;286: 1882–1888.
Mootha VK, Bunkenborg J, Olsen JV et al. Integrated analysis of protein composition, tissue diversity, and gene regulation in mouse mitochondria. Cell 2003;115:629–640.
Yeung K, Seitz T, Li S et al. Suppression of Raf-1 kinase activity and MAP kinase signalling by RKIP. Nature 1999; 401:173–177.
Lorenz K, Lohse MJ, Quitterer U. Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 2003; 426:574–579.
Hengst U, Albrecht H, Hess D et al. The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors. J Biol Chem 2001;276:535–540.
Moore C, Perry AC, Love S et al. Sequence analysis and immunolocalisation of phosphatidylethanolamine binding protein (PBP) in human brain tissue. Brain Res Mol Brain Res 1996;37:74–78.
Bevilacqua G, Sobel ME, Liotta LA et al. Association of low nm23 RNA levels in human primary infiltrating ductal breast carcinomas with lymph node involvement and other histopathological indicators of high metastatic potential. Cancer Res 1989;49:5185–5190.
Chang CL, Zhu XX, Thoraval DH, et al. Nm23-H1 mutation in neuroblastoma. Nature 1994;370:335–336.
Gilles AM, Presecan E, Vonica A et al. Nucleoside diphosphate kinase from human erythrocytes: structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme. J Biol Chem 1991;266: 8784–8789.
Rosengard AM, Krutzsch HC, Shearn A et al. Reduced Nm23/Awd protein in tumour metastasis and aberrant Drosophila development. Nature 1989;342:177–180.
Postel EH, Berberich SJ, Flint SJ et al. Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science 1993;261:478–480.
Okabe-Kado J, Kasukabe T, Honma Y et al. Identity of a differentiation inhibiting factor for mouse myeloid leukemia cells with NM23/nucleoside diphosphate kinase. Biochem Biophys Res Commun 1992;182:987–994.
Lombardi D, Lacombe ML, Paggi MG. nm23: unraveling its biological function in cell differentiation. J Cell Physiol 2000;182:144–149.
Duncan R, Bazar L, Michelotti G et al. A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif. Genes Dev 1994;8:465–480.
Blackwood EM, Eisenman RN. Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc. Science 1991;251:1211–1217.
Grandori C, Mac J, Siebelt F et al. Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo. EMBO J 1996;15:4344–4357.
Blaschke RJ, Monaghan AP, Schiller S et al. SHOT, a SHOX-related homeobox gene, is implicated in craniofacial, brain, heart, and limb development. Proc Natl Acad Sci U S A 1998;95:2406–2411.
Clement-Jones M, Schiller S, Rao E et al. The short stature homeobox gene SHOX is involved in skeletal abnormalities in Turner syndrome. Hum Mol Genet 2000;9:695–702.(Wen Taoa,b,c, Mu Wangb,d,)