Hindawi Publishing Corporation Stem Cells International Volume 2013, Article ID 245695, 9 pages http://dx.doi.org/10.1155/2013/245695 Research Article Proteomic Profiling of Ex Vivo Expanded CD34-Positive Haematopoetic Cells Derived from Umbilical Cord Blood Heiner Falkenberg,1 Teja Falk Radke,2 Gesine Kögler,2 and Kai Stühler1 1 Molecular Proteomics Laboratory (MPL), Center for Biomedical Research (BMFZ), Heinrich Heine University, Universitatsstrasse¨ 1, 40225 Dusseldorf,¨ Germany 2 Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, 40225 Dusseldorf,¨ Germany Correspondence should be addressed to Kai Stuhler;¨ [email protected] Received 12 October 2012; Revised 25 January 2013; Accepted 7 February 2013 Academic Editor: David Allan Copyright © 2013 Heiner Falkenberg et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF), thrombopoietin (THPO), interleukin 6 (IL-6), and fms-related tyrosine kinase 3 ligand (FLT3lg). At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression. 1. Introduction (2) Clinical grade growth factors are only available for a limited number of cytokines and are expensive. Although several groups in preclinical and clinical settings have attempted ex vivo expansion of the cord blood (CB) (3) Moreover, none of the clinical experiences could product in order to increase haematopoetic progenitor and unequivocally document a clear benefit of infusion of granulocytenumbersandtoreducethedurationofposttrans- such ex vivo cytokine expanded components. plant neutropenia (summarized in [1]), the implementation + of protocols applying for instance several cytokines has been Since cytokine-driven ex vivo expansion of CD34 cells complicated by the following facts. from CB is being discussed controversially, other ways to improve haematopoetic engraftment time and reconstitution (1) CB transplants are frozen in the majority of banks in after CB transplantation are being explored including double asinglebag.Clinicaltrialswereperformedwithonly CB transplants (summarized in [2]) and cotransplantation + a fraction of CB unit expanded ex vivo with the larger of a single CB unit together with highly purified CD34 remainder infused unmanipulated. Therefore, the mobilized peripheral blood stem cells from a haploidentical expanded product usually could be infused only 10– or related or completely unrelated donor as a very promising 14 days after transplantation. Alternative approaches clinical approach [3]. focus on the expansion of one CB unit together with Recently Csaszar et al. presented an interesting approach a second nonmanipulated unit. for rapid expansion of human HSC by automated control 2 Stem Cells International Day 0 (presorting) Day 3 Day 7 5 10 105 105 4 10 104 104 3 10 103 103 2 10 102 102 CD34 PE CD34 PE CD34 PE 1 10 101 101 100 100 100 100 101 102 103 104 105 100 101 102 103 104 105 100 101 102 103 104 105 CD45 FITC CD45 FITC CD45 FITC (a) Representative development of CD34-expression during expansion. On day 0, cells were enriched by MACS (one column) and sorted for CD34high/CD45low-expression (gate R4). Isolated cells were expanded on 6-well plates in cytokine-supplemented media with SCF, TPO, FLT3-lg, and IL-6. After 3 days, most cells still expressed significant amounts of CD34 on the surface while after 7 days, less than20% could be classified as CD34high/CD45low. Total cells (fold) Total CD34+ (fold) CD34+ (%) 40 6 100 30 80 (fold) 4 + 60 20 40 2 CD34 (%) 10 20 Total cells (fold) Total Total CD34 Total 0 0 0 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Day Day Day CB1 CB3 CB1 CB3 CB1 CB3 CB2 CB2 CB2 (b) Expansion rates of total and CD34-positive cells. Within the first 3 days, total cell count increased 2.07-fold± ( 0.32) while only a slight + change in CD34-expression could be observed, hence the amount of CD34 cells also expanded by factor 2.00 (±0.32). Within further 4 days of cytokine-driven expansion, the total cell count reached a value of 25.30-fold (±2.93) while expression of CD34 decreased dramatically, resulting in only a 4.17-fold (±0.32) expansion of cells classified as CD34 positive. Figure 1: Umbilical cord blood cells were enriched by MACS and sorted for CD34high/CD45low-expression. of inhibitory feedback mechanism [4], whereas the group of direct analysis of clinical specimen [13–15]. Therefore, a label- Delaney et al. is focusing on Notch-mediated expansion [5]. freeapproachwaschosentoprofileexpressionchangesofex + + Although the CD34 expanded cell populations were well vivo expanded CD34 haematopoetic stem/progenitor cells characterized, no clear protein data are available to define the derived from umbilical cord blood. + changes in the CD34 population in the expanded product. This is the first report applying label-free proteomics to + Mass spectrometry (MS)-based proteomics allows rapid reveal proteomic changes during ex vivo expansion of CD34 assessment of changes in protein expression [6]. However, haematopoetic stem/progenitor cells derived from umbilical in spite of its potential, only few studies applied proteomics cord blood. The results clearly document significant changes to obtain insights into biological processes within cord- towards a protein biosynthesis phenotype already 3 days after + blood-derived CD34 cells [7–12]. With a broad spectrum expansion not reflected by the immunophenotype. of different techniques, analysis of cell lysate led to the + identification of up to 370 proteins [10]innativeCD34 cord blood cells. Nevertheless, biological processes occurring 2. Material and Methods during ex vivo expansion need further elucidation. 2.1. Collection of Cord Blood. CB was collected with the While these studies were limited to a detection of quali- + informed consent of the mother according to established tative description of native CD34 cells, emerging techniques methods [16]. Briefly, after delivery of the baby, the cord are in the position to identify more than thousands of proteins was doubly clamped and transected, and the blood was col- within a cell and also to quantify these proteins in parallel. lected in special collection bags containing citrate-phosphate In contrast to labeling-based quantification techniques such dextrose. In the experiments performed, only CB units not as ICAT, iTRAQ, TMTs or SILAC, label-free quantification suitable for banking due to exclusion criteria, such as low cell avoid any additional sample preparation step and allows number or low volume, were used (ethics committee approval no. 2975). Stem Cells International 3 20 Table 1: Number of proteins with expression profiles from day 0 to day 7. Discrimination between early, late, and long-term changes 15 d7 cord blood 3 as well as between transient and permanent regulations shows 10 d7 cord blood 2 that most significant changes occur early and remain regulated d7 cord blood 1 permanently. 5 d0 cord blood 3 Expression profile Proteins 0 d0 cord blood 2 d0 cord blood 1 Up 17 −5 Early transient d3 cord blood 3 Down 25 Component (20.2%) Component d3 cord blood 2 −10 Up 167 d3 cord blood 1 Early permanent −15 Down 151 −15 −10 −5 0 5 10 15 20 25 Up 38 Component (46.1%) Late Down 6 + Figure 2: Principal component analysis (PCA) of CD34 cells, Up 21 Long term showing that the three time points are distinct. Based on protein Down 15 expression data, the largest variance was observed between day 0 and day 3; compared to this, the variation between day 3 and day No regulation 903 7 is smaller. Only little variances were observed between the three In total 1343 different cord blood donors. ∗ 4 aliquot of app. 5 10 cells was stained against CD34/CD45 + 2.2. Isolation of CD34 -Positive Haematopoetic Cells. CD34- for flow cytometric analysis (FACSCanto; BD Biosciences). positive cells were isolated using a two-step procedure of magnetic activated cell sorting (MACS) and fluorescence 2.4. Cell Lysis and Sample Preparation. For protein extrac- activated cell sorting (FACS). tion, cells were lysed and homogenised in lysis buffer (2 M Firstly, mononuclear cells were isolated using Ficoll- thiourea, 7 M urea, 30 mM Tris-HCl, pH 8.0). After determi- based density centrifugation (Biochrom; Berlin, Germany) as nation of protein concentration using Bradford assay 10 gof described in previous publications [17]. Remaining erythro- proteins were loaded on a SDS-PAGE. After complete entry cytes were removed by ammonium-chloride lysis (10 min.; ∘ into the gel, the SDS-PAGE was stopped after 5 min. Gels were 4 C), and cells were washed twice with phosphate buffered + silver stained according to [18] and one band per sample was saline. Subsequent enrichment of CD34 cells was per- cut out. Bands were destained and washed, and proteins were formed by antibody labeling with paramagnetic beads against digested with trypsin (Promega, Mannheim, Germany).