Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities
- Reinhold J Medina†1,
- Christina L O'Neill†1,
- Mark Sweeney1,
- Jasenka Guduric-Fuchs1,
- Tom A Gardiner1,
- David A Simpson1 and
- Alan W Stitt1Email author
© Medina et al; licensee BioMed Central Ltd. 2010
Received: 3 November 2009
Accepted: 13 May 2010
Published: 13 May 2010
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).
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.
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.
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.
Endothelial Progenitor Cells (EPCs) are a minor population of mononuclear cells circulating in peripheral blood . 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 microvasculopathies, 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 . 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 .
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) . OECs are also known as endothelial colony-forming cells (ECFCs) 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 , and debate about whether these putative EPCs represent the 'bona fide' EPC . 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 . 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 . 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.
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, R3-IGF-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 . Monocytes were isolated from peripheral blood by positive selection using CD14 MicroBeads and an autoMACs separator (Miltenyi Biotec, Bergisch Gladbach, Germany). Human dermal microvascular endothelial cells (DMECs) were purchased from PromoCell GmbH (Heidelberg, Germany) and cultured in endothelial cell basal medium MV (Promocell).
RNA extraction and microarray analysis
Total RNA was extracted using an RNAqueous kit (Ambion, Cambridgeshire, UK). 1 μg of RNA from each cell sample was labelled and hybridised to an Illumina WG-6 v3.0 Expression Beadchip. Samples included in the array analysis were: 3 biological replicates for PB-derived eEPCs and OECs, 3 technical replicates for CB-derived eEPCs and OECs, and single samples for controls DMECs and monocytes. Gene expression data obtained from Illumina Beadstudio was normalised using 'R' bioconductor with 'lumi' package . Data was processed and analysed using the National Institute on Aging (NIA) array, The Database for Annotation, Visualisation and Integrated Discovery (DAVID), GenePattern, and visual anaysis tool (visANT) software.
RT was performed with 500 ng of RNA using random hexamers and Superscript II (Invitrogen, Paisley, UK). Primers were designed using Primer BLAST and tested with AmplifX software. Primer sequences are shown in Additional File 1 Table S1. Conventional RT-PCR was performed in a 30 μl reaction volume containing 1 μl of cDNA, 0.2 μM sense and anti-sense primers designed for the particular gene of interest (Invitrogen), 1× PCR buffer (Invitrogen), 10 mM dNTP mix (Roche, Mannheim, Germany), and 1 μl DNA polymerase (Invitrogen). PCR was performed for 30 cycles using a thermocycler (ABI 2720, Applied Biosystems, Foster City, CA). PCR products were resolved by 2% agarose gel electrophoresis.
Real time RT-PCR
Quantitative real time RT-PCR reactions were performed in a 10 μl volume containing 2 μl of 1:15 diluted cDNA template, 0.5 μM of sense and anti-sense primers (Invitrogen), and 5 μl of SYBR green mastermix (Qiagen, Crawley, UK). PCR was performed for 45 cycles with denaturation at 94°C for 15 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds using a LightCycler 480 (Roche). Standard curves for quantification of PCR products were constructed using serial dilutions of pooled cDNA.
Protein Extraction and 2D gel electrophoresis
Protein was extracted by lysing cells in 9 M urea, 2 M thiourea, 4% CHAPS buffer. 7 cm immobilized 4-7 pH gradient (IPG) strips (GE Healthcare Life Sciences, Buckinghamshire, UK) were rehydrated overnight at room temperature in 125 μl of sample containing 100 μg protein. Isoelectric focusing (IEF) was performed on a Pharmacia Biotech Multiphor II tray in a stepwise fashion (Step 1: 500 volts, 1 mA, 5 watts(W) and 5 volt hours (Vhrs); step 2: 3500 volts, 1 mA, 5 W and 5200 Vhrs; and step 3: 3000 volts, 1 mA, 5 W and 3500 Vhrs). After IEF, reduction and alkylation of thiol groups was performed by immersing strips in equilibration buffer containing 10 mg/ml dithiothreitol (DTT) for 15 minutes, and then 27 mg/ml of iodoacetamide (Sigma-Aldrich, UK) for 15 minutes, respectively. Equilibrated strips were placed horizontally on top of NuPAGE 4-12% Bis-Tris gels (Invitrogen) and secured in place by covering it with 0.5% agarose gel (w/v) made up in running buffer and containing trace amounts of bromophenol blue. Electrophoresis was performed starting at 50 V for 30 minutes, then 100 V for up to 2 hours. Gels were fixed for 1 hour in 40% methanol (v/v), 7% acetic acid (v/v) and immersed in Colloidal coomassie solution (Sigma) overnight at room temperature with gentle agitation. Gels were de-stained with 10% acetic acid (v/v), 1% glycerol (v/v) de-stain solution, and scanned using an Odyssey imaging system (Licor Biosciences, UK). Image analysis was performed with Progenesis PG220 software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).
To preserve apico-basal polarity and architecture of cell monolayers, cells were grown on glass coverslips and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C overnight. After post-fixation in 1% osmium tetroxide, cells were dehydrated with increasing alcohol concentrations and embedded in Spurr resin. Ultrathin sections for both horizontal and vertical planes were prepared using an ultramicrotome, placed on copper grids (Agar Scientific Ltd, UK), stained with uranyl acetate and lead citrate, and examined using a JEOL 100 CX transmission electron microscope.
Human peripheral blood-derived eEPCs and OECs have distinct genomic profiles
Identification of differentially expressed transcripts between PB.OECs and eEPCs: Top 20 transcripts higher in eEPCs.
Complement component 1, q subcomponent, C chain
TYRO protein tyrosine kinase binding protein
Secreted phosphoprotein 1
Fc fragment of IgE
Lymphocyte cytosolic protein 1
Glycoprotein nmb, transcript variant B
ADAM-like, decysin 1
Major histocompatibility complex, class II, DR alpha
Membrane-spanning 4-domains, subfamily A, member 6A
Major histocompatibility complex, class II, DM alpha
Selenoprotein P, plasma 1
Matrix metallopeptidase 9
Transmembrane 4L six family member 19
Colony stimulating factor 1 receptor
S100 calcium binding protein A9
Matrix metallopeptidase 7
Integrin beta 2, lymphocyte antigen 1
Identification of differentially expressed transcripts between PB.OECs and eEPCs: Top 20 transcripts higher in OECs.
EGF-containing fibulin-like extracellular matrix protein 1
Procollagen-lysine, transcript variant 2
Four and a half LIM domains 2
Connective tissue growth factor
Guanine nucleotide binding protein
Transmembrane 4L six family member 1
Matrix metallopeptidase 1
S100 calcium binding protein A16
Cysteine-rich, angiogenic inducer, 61
C-type lectin domain family 14, member A
Homo sapiens dickkopf homolog 1
Homo sapiens PDZ and LIM domain 1
Endothelial cell-specific molecule 1
SRY (sex determining region Y)-box 18
Laminin, alpha 5
Principal component analysis (PCA) further demonstrated the differences between the mRNA expression profiles of eEPCs and OECs. Using a single component (PC1) it was possible to separate samples into two groups; one containing the three eEPC biological replicates with a high PC1 value, and other group with the three OEC biological replicates showing a low PC1 value (Figure 1D). The PCA biplot analysis highlighted some differentially expressed genes for each group; eEPC genes included HLA-DRA, CD36, CD14, and complement 1QC, while OEC genes comprised caveolin1, VE-cadherin, CD34, and endothelial specific molecule 1, among others (Figure 1E).
OECs mRNA fingerprint closely resembles endothelial cells, eEPCs resemble monocytes
Discrepancies exist in the published literature on whether EPCs have monocytic features  or not. There is evidence to demonstrate CD14+ cells can generate EPCs that exhibit revascularising properties [20, 21]. On the other hand, it has also been suggested that blood monocytes only mimic endothelial progenitors and do not directly contribute to vascular network formation . Furthermore, differentially expressed genes described above (Figures 1 and 2, Tables 1 and 2) suggest monocytic and endothelial characteristics for eEPCs and OECs respectively. Therefore, to elucidate and differentiate the expressed phenotypes of endothelial progenitors, eEPC and OEC transcriptomes were directly compared with monocytes and endothelial cells. Monocytes were isolated from fresh peripheral blood by CD14 positive selection using a magnetic cell sorter and human dermal microvascular endothelial cells (DMECs) were chosen as mature endothelial cells. Total RNA from these two cell types was extracted and used as comparator transcriptomes. CB-derived as opposed to PB-derived eEPCs and OECs were also included in transcriptomic analysis as it has been suggested that foetal blood may represent a richer EPC source than adult peripheral blood [23, 24].
Comparative proteomic analysis of EPCs, monocytes and mature endothelial cells
OECs have characteristic endothelial ultrastructural features
This study has revealed highly distinguishing transcript signatures for eEPCs and OECs and clearly demonstrates that these cells represent distinct EPC populations. We show novel evidence based on transcriptomic-, proteomic-, and ultrastructural- analysis to indicate that eEPCs are haematopoietic cells with a monocytic-like molecular profile, while the OEC molecular fingerprint suggests close association to the endothelial lineage. These findings suggest OECs as candidates to establish cell therapies for ischaemic diseases because of their unmistakable endothelial nature. On the other hand, eEPCs should be used with caution because of their monocytic nature and possible role enhancing tissue inflammation.
In 1997, eEPCs were the first putative endothelial progenitors isolated in vitro by culturing CD34+ VEGFR2+ mononuclear blood cells on fibronectin  and confirming a subsequent increase in expression of endothelial cell-associated markers such as CD34, CD31, VEGFR2, Tie2, and E-selectin. Later, this protocol was slightly modified by eliminating the cell sorting step and the so-called CFU-Hill colony assay was developed as a commercial kit to quantify EPCs . In recent years, the assertion that CD34+ VEGFR2+ cells are bona fide EPCs has been challenged , and the cells growing as CFU-Hill colonies have been genetically linked to primitive haematopoietic cells  and suggested to be mainly composed of monocytes and T cells . Furthermore, it has been recently demonstrated that monocytes can acquire endothelial markers in vitro by uptake of platelet microparticles, as a simple transfer of antigens CD31 and vWF .
The comprehensive transcriptomic data obtained in the current study strengthens the idea that eEPCs are indeed haematopoietic cells with a typical monocytic phenotype. Despite the fact that many studies have shown that eEPCs can promote therapeutic neovascularisation in ischaemic tissues , our data suggests that these cells should be utilised for therapy with caution and consideration of the underlying pathology they will treat. This is because the eEPC transcriptome profile indicates a sub-population of cells that are enriched for genes involved in inflammation and immune responses. Therefore, if injected into a pro-inflammatory microenvironment such as in ischaemic diabetic tissues, it is possible they could serve to exacerbate the preexisting pathology .
The second putative EPC isolated in vitro was firstly named endothelial outgrowth , also known as OECs, ECFCs, and late outgrowth endothelial cells. These progenitors were uniformly positive for endothelial markers CD146, thrombomodulin, VEGFR2, VE-cadherin, CD31, CD34, and typically showed greater proliferative potential than circulating endothelial cells. Our group alongside others have established that OECs immunophenotype consists of multiple endothelial markers and complete absence of haematopoietic markers such as CD45 and CD14 [23, 32]. The present study adds strong new evidence to prove that OECs possess intrinsic endothelial nature, as revealed in their transcriptome, proteome, and ultrastructure. OECs have been described to be directly involved in vascular repair by forming well-perfused human neo-vessels when injected subcutaneously as matrigel plugs in immune-deficient mice [12, 32] and it is important to highlight that the OEC interactome analysis identified essential vasculogenesis/angiogenesis components for cell signalling (Tie2, eNOS, Ephrins, TGFβ) and cell adhesion (adherens, tight and gap junctions, cell adhesion molecules, and collagens). This strongly indicates OECs possess intrinsic angiogenic properties, making them an attractive EPC sub-type to be tested for therapeutic angiogenesis.
Despite exponential growth of EPC literature, the primary origin of OECs is still uncertain. The first report confronting this issue affirms that while circulating endothelial cells are derived from the vessel wall, OECs arise from bone marrow-derived circulating angioblasts . Later, the existence of a 'vasculogenic zone' in the wall of adult human blood vessels that contains EPCs has also been suggested , and a complete hierarchy of endothelial progenitor cells including OEC-like cells were isolated from vessel wall-derived cultures . There is even a hypothesis that OECs are 'culture artifacts' arising from in vitro conditions . Finding the in vivo counterpart for the OECs will resolve this argument, however such a discovery remains challenging due to the rarity of OECs in peripheral blood. Moreover, the lack of a marker combination that uniquely identifies OECs further underscores the timeliness of the current study. The molecular profile for OECs presented in this investigation could ultimately help improve approaches for OECs isolation efficiency from blood or bone marrow.
An important question has emerged around whether adult PB, foetal CB or bone marrow offer the best source for EPC isolation. Many reports recognize the advantages of using CB for greater OEC isolation efficiency in comparison to PB , better ex-vivo cell number expansion, and even longer stability of vascular networks formed in vivo [35, 36]. The current study has found that gene expression profile of PB-derived OECs more closely resembles that of mature endothelial cells than those cells derived from CB. Our transcriptomic evaluation also revealed that expression levels of progenitor markers such as CD133, CD34, and C-Kit were higher in CB-derived OECs than in their PB counterparts indicating an apparent cell immaturity which is consistent with their more primitive developmental stage and higher proliferative capacity . By contrast, PB-derived OECs demonstrate higher expression of claudins -11 and -5; which may be indicative of a relative advantage when forming tight junctions with resident endothelium.
Using genome-wide transcriptional profiling, we have precisely characterized the molecular fingerprint of two distinct EPCs. Furthermore, these progenitor transcriptomes were rigorously compared to mature endothelial cells and monocytes to elucidate on their phenotypic nature, results from this transcriptome assessment were strengthened and confirmed by proteomics and ultrastructure studies. The finding that the molecular fingerprint of OECs corresponds to an endothelial phenotype, while eEPCs are similar to monocytes should inform future studies using these cells to promote revascularization of ischaemic tissues.
Many clinical trials using EPCs have yielded inconsistent outcomes in terms of therapeutic benefit, most of them using very heterogeneous populations of cells. Our results indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, as their transcript fingerprint, protein profile and ultrastructure clearly indicates they are fully committed to an endothelial phenotype. Our findings also suggest that eEPCs should be used with caution and consideration of the pathology they will treat. If injected into a pro-inflammatory microenvironment, it is possible they could exacerbate pre-existing pathology because of their monocytic nature.
We thank Dr. Lisa Colhoun for her expert technical assistance in electron microscopy, and Dr. Richard Pringle for help with 2D gel electrophoresis.
This work was supported by grants from Juvenile Diabetes Research Foundation (JDRF), Medical Research Council (MRC), TBF Thompson Trust and Guide Dogs for the Blind Association (GDBA).
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