Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

Medina et al. BMC Medical Genomics 2010, 3:18 http://www.biomedcentral.com/1755-8794/3/18 Open Access RESEARCH ARTICLE Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities Research article Reinhold J Medina†, Christina L O'Neill†, Mark Sweeney, Jasenka Guduric-Fuchs, Tom A Gardiner, David A Simpson and Alan W Stitt* Abstract Background: The term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs). Methods: Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. Results: Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. Conclusions: This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature. Background Endothelial Progenitor Cells (EPCs) are a minor population of mononuclear cells circulating in peripheral blood [1]. Although rare in comparison to other blood cells, EPCs are capable of facilitating vascular repair in different ischaemic tissues, therefore they have been regarded as promising candidates for inducing therapeutic angiogenesis in multiple diseases such as acute myocardial infarction, unstable angina, stroke, diabetic microvascul* Correspondence: 1 Centre for Vision & Vascular Science, School of Medicine, Dentistry & BioMedical Science, Queen's University Belfast, Belfast, BT12 6BA, UK † Contributed equally Full list of author information is available at the end of the article opathies, pulmonary arterial hypertension, atherosclerosis, and ischaemic retinopathies [2-6]. EPCs are classically described as cells expressing a combination of an endothelial marker (VEGFR2) and a progenitor marker (CD34/CD133), however there is considerable debate surrounding this definition, because none of these markers are fully specific [7,8]. It has been reported that CD34+ CD133+ VEGFR2+ cells are haematopoietic and may not actually be true EPCs [9]. Indeed, a methodological comparison of six flow cytometric approaches for EPC quantification using CD34 and VEGFR2 markers has demonstrated only poor to moderate agreement between methods [10]. © 2010 Medina et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons BioMed Central Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medina et al. BMC Medical Genomics 2010, 3:18 http://www.biomedcentral.com/1755-8794/3/18 An alternative approach to isolate EPCs from peripheral or umbilical cord blood utilizes in vitro culture and this consistently produces two distinct EPC subtypes which have been named as early EPCs (eEPCs) and Outgrowth endothelial cells (OECs) [11]. OECs are also known as endothelial colony-forming cells (ECFCs)[12] or late EPCs because of their late appearance in culture. Although clear differences have been shown between these two endothelial progenitors, there is still concern surrounding their nature [8], and debate about whether these putative EPCs represent the 'bona fide' EPC [7]. It has been previously demonstrated that both subsets contribute to angiogenesis, but through different mechanisms. eEPCs act in a paracrine manner, while OECs directly incorporate into resident vasculature [13,14]. A range of pre-clinical and clinical studies using EPCs have yielded inconsistent outcomes in terms of therapeutic benefit, implying that a precise EPC definition needs to be elucidated [15]. Moreover, many clinical trials still use very heterogeneous populations of cells such as unfractionated bone marrow or freshly isolated CD34 + cells [2,4]. Clearly, an accurate EPC definition based on a broad range of molecular characteristics is needed. If sub-populations could be precisely characterized, this would facilitate the use of the most appropriate cell therapy in future clinical trials [16]. The aim of this study was to provide a thorough, unbiased analysis of the phenotype of the two EPC subsets isolated in vitro; eEPCs and OECs. This was achieved using combined transcriptomic and proteomic analysis to establish a highly detailed molecular fingerprint of these important endothelial progenitors. Methods Cell Isolation and culture This study was approved by the Office for Research Ethics Committees Northern Ireland (ORECNI 08/NIR02/ 20). EPCs were isolated from human peripheral blood (PB) and umbilical cord blood (CB). Fresh human PB (40 mls) was obtained under full ethical approval from three female volunteer subjects aged 25-35, non-smokers, not receiving any medication and without any clinical diagnosis; while CB-derived mononuclear cells (MNCs) were purchased from AllCells LLC (California, USA). MNCs were isolated from PB by density gradient centrifugation. To obtain eEPCs, MNCs were seeded at a density of 2 × 106 cells/ml onto fibronectin coated petri dishes and cultured in complete EBM-2 MV medium (Lonza Ltd., Slough, UK) that contained hEGF, VEGF, hFGF-B, R3IGF-1 and was supplemented with 10% FBS. OECs were obtained by seeding MNCs onto collagen coated wells at a density of 1 × 107 cells/ml using complete EBM-2 medium with the same supplements as described above [17]. Monocytes were isolated from peripheral blood by Page 2 of 13 positive selection using CD14 MicroBeads and an autoMACs separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Human der (...truncated)