Tissue-derived mesenchymal stromal cells used as vehicles for anti-tumor therapy exert different in vivoeffects on migration capacity and tumor growth
© Belmar-Lopez et al.; licensee BioMed Central Ltd. 2013
Received: 24 July 2012
Accepted: 19 April 2013
Published: 28 May 2013
Mesenchymal stem cells (MSCs) have been promoted as an attractive option to use as cellular delivery vehicles to carry anti-tumor agents, owing to their ability to home into tumor sites and secrete cytokines. Multiple isolated populations have been described as MSCs, but despite extensive in vitro characterization, little is known about their in vivo behavior.
The aim of this study was to investigate the efficacy and efficiency of different MSC lineages derived from five different sources (bone marrow, adipose tissue, epithelial endometrium, stroma endometrium, and amniotic membrane), in order to assess their adequacy for cell-based anti-tumor therapies. Our study shows the crucial importance of understanding the interaction between MSCs and tumor cells, and provides both information and a methodological approach, which could be used to develop safer and more accurate targeted therapeutic applications.
We first measured the in vivo migration capacity and effect on tumor growth of the different MSCs using two imaging techniques: (i) single-photon emission computed tomography combined with computed tomography (SPECT-CT), using the human sodium iodine symporter gene (hNIS) and (ii) magnetic resonance imaging using superparamagnetic iron oxide. We then sought correlations between these parameters and expression of pluripotency-related or migration-related genes.
Our results show that migration of human bone marrow-derived MSCs was significantly reduced and slower than that obtained with the other MSCs assayed and also with human induced pluripotent stem cells (hiPSCs). The qPCR data clearly show that MSCs and hiPSCs exert a very different pluripotency pattern, which correlates with the differences observed in their engraftment capacity and with their effects on tumor growth.
This study reveals differences in MSC recruitment/migration toward the tumor site and the corresponding effects on tumor growth. Three observations stand out: 1) tracking of the stem cell is essential to check the safety and efficacy of cell therapies; 2) the MSC lineage to be used in the cell therapy needs to be carefully chosen to balance efficacy and safety for a particular tumor type; and 3) different pluripotency and mobility patterns can be linked to the engraftment capacity of the MSCs, and should be checked as part of the clinical characterization of the lineage.
KeywordsMesenchymal stromal cells Migration In vivo imaging Tumor growth Pluripotency
Human mesenchymal stem cells or mesenchymal stromal cells (MSCs) are multipotent progenitor cells or adult stem cells that exhibit the ability to migrate and engraft into tumor sites when delivered systemically . However, determining the most appropriate clinical application of MSCs is hampered by the current lack of knowledge about how these cells behave in vivo. The precise mechanisms behind the recruitment of MSCs to tumor sites and their migration across the endothelium are not yet fully understood. It is probable that damaged tissue expresses specific receptors or ligands to make possible trafficking, adhesion, and extravasation of MSCs to the site of damage and recruitment to inflammation sites, using a mechanism similar to leukocyte migration [2–4].
The most likely cause of specific migration is the release of chemotactic gradients from the tumors, which may enable MSCs to home to, and modulate, the tumor microenvironment [5, 6]. Owing to these properties and their ability to modulate the activity of immune cells, MSCs could function as cellular delivery vehicles for anti-tumor agents [7–9].
MSCs were first identified in the 1960s in the stromal compartment of bone marrow [10, 11], and since then, they have been isolated from a wide variety of adult [12–20] and fetal (both first and second trimester) tissues, including blood, liver, bone marrow, placenta, and umbilical cord [21–25], using similar techniques . The best-characterized source for adult human stem cells is bone marrow, and both bone marrow-derived human MSCs (BM-hMSCs) and adipose-derived human MSCs (hASCs) have become attractive candidates because these tissues are rich sources of MSCs and are easy to collect. The other tissue-derived MSCs share a number of important characteristics with BM-hMSCs, including expression of cell surface marker, ability to adhere to plastic, and capacity to differentiate into cells of mesenchymal lineage under appropriate conditions . Despite extensive investigations, the effect of unmodified MSCs on tumor progression remains unclear. Many studies have shown that MSCs promote tumor progression and metastasis, whereas others have reported that MSCs suppress tumor growth . The contradictions in these findings may be attributable to the variability and heterogeneity in adult stem cells from different sources, or to differences in isolation methods and in vitro culture conditions. Further development of an efficient and safe cell-based therapy will require the in vivo tracking of engrafted MSCs to ensure that they reach their destination. In vivo imaging techniques provide a continuum observation rather than a single snapshot of conventional post-mortem histological analyses.
The aim of our work was to investigate the efficacy and efficiency of five different MSC lineages, in order to assess their adequacy for use as cell-based anti-tumor therapies. Our study shows the crucial importance of understanding the interaction between MSCs and tumor cells, and provides both information and a methodological approach, which could be used to develop safer and more accurately targeted therapeutic applications. The pluripotency expression pattern of MSCs was studied and compared with that obtained in human induced pluripotent stem cells (hiPSCs). Furthermore, the effects exerted on migration-related gene expression in tumors obtained from animals after 24 days of systemic MSC injection were also analyzed.
A human cervical cancer cell line (HeLa; Cancer Research UK Cell Services, London Research Institute, Clare Hall Laboratories, Herts, UK) and human PN3 fibroblasts (kindly supplied by Dr Liu (Imperial College, London, UK)) were used. Cells were cultured in DMEM containing 10% FBS and antibiotics (Lonza, Verviers, Belgium), at 37°C in 5% CO2.
All MSC media were supplemented with 10% FBS and antibiotics. BM-hMSCs were obtained from Lonza and maintained in DMEM low glucose (1.0 g/l) and hypoxic conditions (3% O2). hASCs were obtained from Invitrogen (UK) and cultured in MesenPro RS Basal Medium and MesenPro RS Growth Supplement (Gibco, Paisley, UK). Human epithelial endometrium-derived stem cells or hEESCs (also known as endometrial epithelial stem cell lines; ICEp) and human stroma endometrium-derived stem cells or hESSCs (also known as endometrial stromal stem cell lines; ICEs) were supplied by Dr Carlos Simon from IVI (Valencia, Spain) [12, 13]. Cells were maintained in DMEM F-12 under hypoxic conditions (3% O2) and dishes were pre-treated with 0.1% gelatin solution (Sigma-Aldrich Chemie GmBh, Munich, Germany). Human amniotic membrane mesenchymal stem cells or hAMCs were obtained from Cellular Engineering Technologies (CET), (Coralville, IA, USA) and were maintained in DMEM high glucose (4.5 g/l) and 10 ng/ml basic human fibroblast growth factor (hFGFb;Gibco). Cells were used between passages 5 to 8.
The hiPSCs (human IPSC line 2 F8) were kindly supplied by Dr Austin Smith (University of Cambrige, UK) and cultured in knockout DMEM (Gibco), 15% knockout serum (Gibco), 1× NEAA (Lonza), 0.1 mmol/l β-mercaptoethanol (Sigma-Aldrich), 10 ng/ml hFGFb, and antibiotics at 37°C in 5% CO2. Cells were seeded on a PN3 feeder cell monolayer inactivated with mitomycin C (Sigma-Aldrich).
Characterization of MSCs was verified by flow cytometry. The negative surface markers used were CD45, CD34, and HLA-DR, and the positive ones were CD90, CD73, and CD105, plus CD9 and CD13 (the last two markers refers only to hEESCs and hESSCs, respectively). The antibodies used were CD13 FITC-conjugated (Immunostep, Salamanca, Spain), CD34 Percp/Cy5.5-conjugated (Becton Dickinson Co., Madrid, Spain), CD9 PE-conjugated (Millipore Corp., Billerica, MA, USA), CD45 PerCP/Cy5.5-conjugated (Becton Dickinson), CD73 PE-conjugated (BD), CD90 PE-conjugated (Becton Dickinson), CD105 FITC-conjugated (R&D Systems Inc., Minneapolis, MN, USA), and HLA-DR APC-conjugated (Immunostep). Briefly, cells were incubated in PBS supplemented with 2% FBS and specific antibodies at 4°C for 30 minutes. Then, cells were washed and fixed in 1% paraformaldehyde (Sigma-Aldrich) before FACS analysis (FACSAria system; Becton Dickinson).
In vitroMSC differentiation
For adipogenesis, MSCs were kept for 21 days in 1× basal medium (STEMPRO® Adipocyte Differentiation Basal Medium; Invitrogen Corp., Carlsbad, CA, USA), 1× supplement (STEMPRO® Adipogenesis Supplement; Invitrogen) and antibiotics. When differentiation was finished, cells were stained with Oil Red O solution (Sigma-Aldrich). For osteogenesis, MSCs were maintained for two weeks in DMEM medium containing 10% FBS, 50 μg/ml ascorbic acid, 100 nmol/l dexamethasone, and 10 mmol/l β-glycerophosphate disodium salt hydrate (Sigma-Aldrich) and antibiotics. Osteocyte formation was evaluated by staining with Alizarin Red S (Sigma-Aldrich). Images were visualized under a microscope (AE31; Motic Group Co. Ltd, Causeway Bay, Hong Kong) equipped with a camera (2500 Moticam; Motic Group) and Motic Imaging Plus 2 software (version 0.23).
Adenoviral vectors and infections
The hNIS gene is endogenously expressed mainly in the thyroid and stomach, and is responsible for iodide concentration. In cells expressing hNIS, gamma ray-emitting radioisotopes such as 99mTc are accumulated, and can be imaged by SPECT-CT, and thus the hNIS gene can be used as a reporter gene . The adenoviral vector AdhNIS (also known as Ad10) used in this work was based on adenovirus serotype 5, and the hNIS gene is driven by the immediate-early cytomegalovirus promoter. AdhNIS was constructed and amplified as previously described . The amount of infective adenoviral vector per cell (pFUs/cells) in culture media was expressed as multiplicity of infection (MOI). Previously, adenoviral infection efficiency was determined using adenoviral vector AdGFP testing at 100, 250, 500, and 1000 MOI, as in our previous study  (data not shown). For the adenoviral infection with AdhNIS, viruses were diluted in serum-free culture media to 500 MOI, added to cells, and incubated at 37°C for 1 h. The complete medium was then added and cells were maintained for 24 h until used in the in vivo experiments.
All procedures were carried out under a project license approved by the Ethics Committee for Animal Experiments from the University of Zaragoza (Spain). The care and use of animals was performed in accordance with the Spanish Policy for Animal Protection RD1201/05.
Experimental in vivodesign
Female BALB/c nu/nu mice 6–8 weeks old (Harlan UK Ltd (Bicester, Oxfordshire, UK) and Harlan Interfauna Iberica (Barcelona, Spain)) received subcutaneous (SC) injections of 2 × 106 HeLa cells suspended in 200 μl PBS for the generation of subcutaneous xenograft tumors. When these tumors reached 50 mm3 in size, mice were randomly divided into different groups, and intravenous injections of MSC were performed. For MRI experiments, animals were separated into six groups (n = 4/group). Group 1 (BM-hMSCs injected); group 2 (hASCs injected); group 3 (hAMCs injected); group 4 (hESSCs injected); group 5 (hEESCs injected); and group 6 (control; PBS injected). For SPECT-CT experiments, animals were separated into seven groups (n = 4/group), with groups 1 to 5 as above, and groups 6 and 7 being injected with hiPSCs or PBS (control), respectively.
Iron-oxide labeling and cell-viability assay
MSCs were magnetically labeled with superparamagnetic iron oxide (SPIO; Endorem, Guerbet, France), as previously described . SPIO is an oxide nanoparticle solution with a total iron content of 11.2 mg Fe/ml. Labeling with SPIO acts by reducing the transverse relaxation time on T2-weighted MRI scans. Cells were incubated with the labeling medium containing 100 μg/ml iron for 24 h. After labeling, cells were washed to remove residual contrast agent.
Viability of the iron oxide-labeled MSCs was evaluated by performing a long-term (10 days) in vitro exclusion test with Trypan blue (Sigma-Aldrich).
Animals bearing the tumor xenograft were separated into six groups (n = 4/group) when the tumors reached 50 mm3. MSCs were labeled with SPIO as described above. Groups 1 to 5 received an intravenous injection of 106 SPIO-labeled MSCs, while the control group (group 6) received intravenous injection of PBS. Scans were performed at 3, 10, 17, and 24 days after injection. The MRI experiments were performed (Pharmascan system; Bruker Medical GmBH, Germany, http://www.bruker-biospin.com/pharmascan.html) using a 7.0-T horizontal-bore superconducting magnet, equipped with a 1H selective surface coil and a Bruker gradient insert with 90 mm inner diameter (maximum intensity 360 mT/m). All data were acquired using Paravision software (Bruker). Anesthesia was initiated using oxygen (1 l/min) containing 4% isofluorane, and maintained during the experiment with 1 to 1.5% isofluorane in O2. T2-weighted spin-echo anatomical images were acquired by rapid acquisition with relaxation enhancement (RARE) sequence in axial (12 slices) and sagittal (8 slices) orientations and the following parameters: TR 3000 ms, TE 60 ms, RARE factor 8, average 3, FOV 30 × 30 mm, acquisition matrix 256 × 256, corresponding to an in-plane resolution of 117 × 117 μm2 and slice thickness of 1.00 mm. Tumors were measured every 2 days and tumor volume was calculated using the formula:
tumor volume = 1/2 L × S2, where L is long side and S is short side.
Prussian blue staining and histological analysis
At day 24, the animals in the in vivo MRI experiments were euthanized. Tumors and tissues were obtained, fixed in formalin, and embedded in paraffin wax. Sections of 4 μm were obtained from the blocks for staining with haematoxylin and eosin (Sigma-Aldrich). Staining with Prussian blue (Sigma-Aldrich) was used to detect labeled iron particles in the cells, in accordance with the manufacturer’s instructions. The number of blue-stained (positive) cells per high-power field (HPF) was calculated by counting the cells in at least five HPFs per section, with a minimum of five sections per sample examined.
Animals bearing tumor xenograft were separated into seven groups (n = 4/group) when the tumors reached 50 mm3. MSCs were infected with AdhNIS as described above. Groups 1 to 6 received intravenous injection of 106 hNIS-labeled MSCs and the control group (group 7) received intravenous injection of PBS. At 3, 10, 17, and 24 days post-injection, all groups received an intravenous dose of 18.5 MBq of 99mTc. Anesthesia was initiated by inhaled oxygen (1 l/min) containing 4% isofluorane, and maintained during the experiment with 1 to 1.5% isofluorane in O2. The mice were scanned using a nano-SPECT-CT scanner for small animals (Bioscan, Paris, France). A tomogram was taken, and the limits of the scan were determined. A CT and a SPECT whole-body scan were performed, with a time of 100 seconds per acquisition. The images were reconstructed with the MEDISO software (Medical Imaging Systems, Budapest, Hungary); fusion of SPECT and CT images was carried out using PMOD software (Biomedical Imagen Quantification, Basel, Switzerland). Tumors were measured every two days and tumor volume was calculated using the formula for tumor volume given above.
Quantification of radioisotope (99mTc) accumulation was carried out using InVivoScope software (Medical Imaging Systems). Fused SPECT/CT images were used to draw the voxel-guided specific volume of interest (VOI) to accurately quantify the total activity associated with the whole tumor volume. Quantification of 99mTc accumulation was expressed as ratio of tumor uptake to muscle uptake.
Primers used for PCR
Real-time quantitative PCR
TaqMan® gene expression assays used to amplify the pluripotency-related genes
Amplicon size, nucleotides
Primers used to amplify the migration-related genes
GenBank accession number
Sequence (5′ → 3′)
Results are reported as mean ± SEM. Statistical evaluation of data was carried out using the SPSS Statistics software package (version 17.0; IBM SPSS, Chicago, IL, USA). Normal distribution of the variables was analyzed by means of the Kolmogorov-Smirnov test followed by the Tukey HSD test, except for KLF4 and REX1 expression in the pluripotency qPCR assays, whose distribution was non-parametric and so analysis was performed using the Kruskal-Wallis test. P<0.05 was considered significant.
To confirm whether labeled MSCs injected systemically into animals are able to migrate, proliferate, and engraft into the microenvironment of tumors, two non-invasive imaging techniques were performed in the present study: 1) SPECT-CT using hNIS as reporter gene, and 2) MRI labeled with iron nanoparticles (SPIO).
Cell-viability assay of SPIO-labeled MSCs
Before performing MRI scans, viability of SPIO-labeled MSCs was determined by Trypan blue exclusion assay. Cell-viability values, compared the control group (100%), with 99.23 ± 3.18% in the BM-hMSC group, 101.53 ± 5.6% in the hASC group, 96.4 ± 1.75% in the hEESC group, 100.28 ± 0.97% in the hESSC group, and 98.05 ± 6.15% in the hAMC group, thus revealing that labeling with SPIO did not significantly affect cell viability and proliferation.
In vivotropism of MSCs to tumors, detected by non-invasive imaging techniques
hMSCs did not exert any signal at day 3, and their highest signal was found at day 10 (1.92 ± 0.72), after which it decreased until day 24 (1.07 ± 0.12). In hiPSCs and in the other hMSCs assayed, various levels of signal were detected from day 3, with hASCs presenting the lowest signal (1.44 ± 0.69) and hESSCs the highest (2.01 ± 0.18). Figure 4B shows the differences at day 3. The detected signals increased for the following determinations, with MSCs reaching their maximum level at day 17, when hESSCs also had their highest values (3.84 ± 0.41), whereas the highest signal for hiPSCs (6.73 ± 1.23) occurred at day 24. For MSCs, the detected signals decreased after 24 days, with the highest values being for hAMCs (3.07 ± 0.29) and hESSCs (2.89 ± 0.69), and the lowest for hASCs (2.46 ± 0.68) and hEESCs (2.52 ± 0.72). The tumor:muscle uptake ratio showed the differences between the migration ability of MSCs and hiPSCs (Figure 4C).
Effects on xenograft tumor growth
In general, expression levels of migration-related genes in tumors obtained from HeLa cells and MSC or hiPSC injection were significantly lower than those of control samples. CXCL12 expression was significantly decreased in all groups, with the smallest decrease seen in BM-hMSCs (−4.4 ± 0.04-times), and the largest in hASCs, hEESCs, and hESSCs (between −9-fold and −11-fold). For CCL5, expression levels were significantly decreased in tumors obtained from BM-hMSCs (−1.69 ± 0.25-fold), hEESCs (−2.31 ± 0.08-fold), and hESSCs (−2.58 ± 0.08-fold), but were significantly increased in hAMCs (1.73 ± 0.07-fold) and hiPSCs (0.85 ± 0.1-fold). In the hASC group, the expression level was similar to that of the control samples (−0.25 ± 0.12-fold). CCL2 and CXCR4 expression levels were significantly decreased in tumors resulting from MSC injection, with the hAMC group showing the lowest decrease (−1.05 ± 0.07-fold and −0.98 ± 0.12-fold, respectively), whereas the hiPSC group had significantly increased expression levels (5.53 ± 0.18-fold and 0.60 ± 0.33-fold, respectively). Finally, MMP-2 expression was significantly decreased in tumors composed of HeLa cells and BM-hMSCs (−6.28 ± 0.22-fold), hASCs (−6.99 ± 0.12-fold), hEESCs (−5.62 ± 0.13-fold), and hAMCs (−3.09 ± 0.06-fold) but was significantly increased in hESSCs (2.28 ± 0.07-fold) and hiPSCs (6.48 ± 0.14-fold). These findings highlight, once again, the differences between MSCs and hiPSCs. These differences may be responsible for the different migration patterns and probably play a role in the effects on the microenvironment in tumors.
MSCs have been promoted as an attractive option for cellular delivery vehicles to carry anti-tumor agents, owing to their ability to home into tumor sites and secrete cytokines [28, 33, 34]. Previous studies have shown that systemic delivery of MSCs does not result in engraftment into healthy organs, but that they do migrate in various in vivo tumor models, although this mechanism has not yet been fully elucidated [35–42]. Additionally, MSCs can also counteract inflammation by suppressing host immune responses  and by secreting anti-inflammatory cytokines .
The ability of MSCs and hiPSCs to migrate into tumor sites after systemic injection into mice is confirmed in the present study. HeLa-based subcutaneous xenograft tumors showed extensive MSC engraftment, and BM-hMSC migration was significantly lower and slower than that obtained by the other MSCs and hiPSCs. All cell types were detected by MRI in a similar way to recent results obtained with MSCs by other groups . Signals were obtained from the first (day 3) until the final (day 24) imaging determination, except in BM-hMSCs, which highlights the capacity of these cells to persist in tumor sites. These results and the differences between BM-hMSCs and the other cell types were confirmed by iron-particle staining and the detection of hNIS in tumor samples. Using SPECT-CT images and hNIS expression, another study has shown BM-hMSC migration towards a breast-cancer model . In contrast to our observations, hNIS expression in that study was detected in tumors from day 3 after MSC injection, which may be attributed to the differences between donors, tumor models, and the size of the pre-established tumor.
To clarify whether pluripotency plays a role in MSC migration to tumors, a pluripotency marker panel was analyzed. These studies revealed diminished expression of SOX2, NANOG, OCT4, KLF4, and REX1 compared with the control sample (hiPSCs). hEESCs and hESSCs, which displayed the highest signals in the imaging techniques, also had significant expression of KLF4, NANOG, and REX1 with respect to the other MSCs, although it was much lower than that shown by hiPSCs. These differences in pluripotency patterns may influence the differences in migration and engraftment of MSCs and hiPSCs.
Tumor growth confirmed the ability of MSCs to migrate into tumors, and the significant differences between BM-hMSCs, the other MSCs, and hiPSCs. In a previous PC3 prostate xenograft model , intra-tumoral injection of hASCs also induced larger tumors compared with the control group, although the differences between control and hASC-induced tumors were smaller at the final time point (<2-fold vs. 3-fold in ours). These differences may be attributed to the different tumor model used and the different procedures for hASC injection. However, there are also studies reporting inhibitory effects on tumor growth in an in vivo tumor model following MSC injection, using different approaches from ours [46, 47]. The dose of MSCs delivered and the timing of injection have been highlighted as determining factors in the promotion or inhibition of tumor growth .
Although the mechanisms behind homing are not yet fully understood, the most likely cause of preferential migration is the release of chemotactic gradients from tumors. MSCs have a wide range of chemokine and cytokine receptors on their cell surface, which respond functionally to their ligands in vitro, whereas in vivo, their modification implies changes in migration behavior [48–52].
Some authors have shwon that MSCs secrete a large panel of chemokines such as CXCL12, CCL2, and CCL5, which implies activation of the MAPK, FAK, and STAT signaling pathways, and the induction of biological responses [48, 49]. Moreover, tumors produce a wide range of chemokines and cytokines, which may act as ligands for MSC receptors [35, 53]. Of the different pairs of receptor/ligand described as responsible for migration, the CXCL12/CXCR4 pair should be highlighted. This has been studied in numerous works, both in vitro and in vivo, and using different tumor models or MSCs sources [35, 49–51, 54–59]. Loss of expression in MSC´s receptors  implies a loss in cell-migration ability, indicating the great importance of these axes in MSC migration.
The relationship between homing and the inflammatory state has been assessed, and studies have been conducted in which BM-hMSCs were pre-treated with factors involved on inflammation (such as tumor necrosis factor, MMP2, CXCL12 and CCL5) . But some studies were contradictory [35, 50], which it could be due to the variation in donors or cell-culture conditions (confluence, hypoxia, and passages) .
Our results show that, in general, when MSCs engraft into tumors, migration-related gene expression is decreased (whereas hiPSCs exert an increase) except for the CXCL12/CXCR4 axis, causing a significant depletion in the expression of both markers when MSCs are present. The exceptions were hiPSCs, in which CXCR4 expression increased. The reason for this may be that CXCL12 expression is also linked to more immature cell fractions, with higher expression in less committed stages of differentiation, that is, in cells closer to the embryonic state . With regard to hiPSCs, this study and another previous work  are the first to highlight their in vivo migration ability to and long-term engraftment into tissue damage areas.
Besides the release of chemotactic gradients from the tumors, other explanations for MSCs migration could be the hypoxic conditions produced by tumor cells, which may cause MSCs to increase the expression of migratory signals , and thus confer the ability to cross the biological barriers . However, in our study, a direct correlation between the migration patterns and the absence of oxygen does not seem to exist (data not shown).
Finally, a recent dialogue mediated by exosomes between MSCs from the bone marrow and tumoral cells in patients with melanoma  has been shown. The degree of exosome release by the various MSCs could determine the migratory differences between those cells, depending both on their area of origin and also on their exosome targeting.
This study clarifies the in vivo capacity of different types of MSCs and hiPSCs to migrate and engraft into a tumor model. The results reveal that the adult stem cells assayed were able to enter tumor sites and remain there, at least until the end of the experiments. Their different pluripotency pattern may also play a role in the differences between these cell types, in which higher imaging signals but also higher tumor growth were obtained in those with highest expression of pluripotency genes. Moreover, MSCs and hiPSCs also exerted different migration-related gene profiles in tumors, which could be attributed to the anti-inflammatory effect exerted by MSCs, thereby diminishing expression of the migration marker. Our promising data support the importance of MSCs as therapeutic gene carriers in anti-tumor treatments, and highlight the importance of choosing the most suitable lineage for specific tumor lesion. Further understanding of the role of MSCs in tumors and the promising possibility to use them as therapeutic gene vehicles may lead to potentially fruitful treatment approaches.
CB-L is a pre-doctoral staff member at the Instituto Aragones de Ciencias de la Salud-IIS Aragón, Zaragoza, Spain. GM is a post-doctoral fellow at the Instituto Aragones de Ciencias de la Salud-IIS Aragón, Zaragoza, Spain. DO is a visiting teacher at the Department of Medical Biochemistry and Microbiology, Uppsala University, Sweden. JB is a laboratory technician at the Queen Mary University of London, UK. CS is Full Professor of Obstetrics and Gynecology at the University of Valencia (Spain) and Scientific Head of the IVI. IC is Principal Investigator at IVI Foundation, Valencia, Spain. MI is Professor of Biochemistry and Dean of Faculty of Health Sciences at the University Francisco de Vitoria, Spain. JCR is Head of the Viral Vectors Unit at the Centro Nacional de Investigaciones Cardiovasculares, Spain. PL-L and MQ are Principal Investigators at the Instituto de Investigaciones Biomédicas Alberto Sols, Spain. PM-D is ARAID Foundation Principal Investigator, professor at University Francisco de Vitoria, Spain, and Head of the Gene and Therapy Group at the Instituto Aragones de Ciencias de la Salud-IIS Aragon, Zaragoza, Spain.
Ad10 Adenoviral vector containing human sodium iodine symporter gene
Activated protein C
Bone marrow-derived human mesenchymal stem cell
Chemokine (C-C motif) ligand 2
Chemokine (C-C motif) ligand 5
Chemokine (C-X-C motif) ligand 12
C-X-C chemokine receptor type 4
Fluorescence-activated cell sorting
Fetal bovine serum
Field of view
Human amniotic fluid mesenchymal stem cell
Human amniotic membrane mesenchymal stem cell
Human adipose-derived stem cell
Human epithelial endometrium-derived stem cell
Human stromal endometrium-derived stem cell
Human induced pluripotent stem cell
Human sodium iodine symporter
Honestly significant difference
Endometrial epithelial stem cell lines
Endometrial stromal stem cell lines
Monocyte chemotactic protein-1
Multiplicity of infection
Magnetic resonance imaging
Mesenchymal stem cell
Peridinin chlorophyll protein complex
Quantitative polymerase chain reaction
(Regulated on Activation, Normal T cell Expressed and Secreted)
Rapid acquisition with relaxation enhancement
Stromal cell-derived factor-1
Single-photon emission computed tomography/X-ray computed tomography
Super-paramagnetic iron oxide
Volumes of interest.
This work was supported by FIS (PI080750), DGA (PI041/08, B84, PI086/09), MMA Fund (ICS/08/0050), PROMETEO/2008/163; CTQ-2010-20960-C02-02; S2010/BMD-2349, PIPAMER-0912, and PIPAMER-1214. CB-L was funded by fellowships ICS/08/0050 and DGA PI-086/09, GM by PIPAMER-0912, and PMD by the Araid Fund. We are grateful to Dr Hugo Cabedo and Dr Jose Antonio Gómez-Sánchez for help with the qPCR studies, and to Juan Miguel Sanchez, Camino Latorre, and Rebeca Guerrero for technical assistance. We also thank Professor James McCue and Dr Nancy d’Cruz for their assistance in language editing.
- Kansas GS: Selectins and their ligands: current concepts and controversies. Blood. 1996, 88: 3259-3287.PubMedGoogle Scholar
- Rüster B, Göttig S, Ludwig RJ, Bistrian R, Müller S, Seifried E, Gille J, Henschler R: Mesenchymal stem cells display coordinated rolling and adhesion behavior on endothelial cells. Blood. 2006, 108: 3938-3944.View ArticlePubMedGoogle Scholar
- Schweitzer KM, Dräger AM, van der Valk P, Thijsen SF, Zevenbergen A, Theijsmeijer AP, van der Schoot CE, Langenhuijsen MM: Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues. Am J Pathol. 1996, 148: 165-175.PubMedPubMed CentralGoogle Scholar
- De Miguel MP, Fuentes-Julian S, Blazquez-Martinez A, Pascual CY, Aller MA, Arias J, Arnalich-Montiel F: Immunosuppressive properties of mesenchymal stem cells: advances and applications. Curr Mol Med. 2012, 12: 574-591.View ArticlePubMedGoogle Scholar
- Salem HK, Thiemermann C: Mesenchymal stromal cells: current understanding and clinical status. Stem Cells. 2010, 28: 585-596.PubMedGoogle Scholar
- Kidd S, Caldwell L, Dietrich M, Samudio I, Spaeth EL, Watson K, Shi Y, Abbruzzese J, Konopleva M, Andreeff M, Marini FC: Mesenchymal stromal cells alone or expressing interferon-beta suppress pancreatic tumors in vivo, an effect countered by anti-inflammatory treatment. Cytotherapy. 2010, 12: 615-625.View ArticlePubMedGoogle Scholar
- Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M: Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Cancer Res. 2002, 62: 3603-3608.PubMedGoogle Scholar
- Yong RL, Shinojima N, Fueyo J, Gumin J, Vecil GG, Marini FC, Bogler O, Andreeff M, Lang FF: Human bone marrow-derived mesenchymal stem cells for intravascular delivery of oncolytic adenovirus Delta24-RGD to human gliomas. Cancer Res. 2009, 69: 8932-8940.View ArticlePubMedPubMed CentralGoogle Scholar
- Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP: Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 1968, 6: 230-247.View ArticlePubMedGoogle Scholar
- Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV: Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol. 1966, 16: 381-390.PubMedGoogle Scholar
- Baksh D, Song L, Tuan RS: Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med. 2004, 8: 301-316.View ArticlePubMedGoogle Scholar
- Cervelló I, Gil-Sanchis C, Mas A, Delgado-Rosas F, Martínez-Conejero JA, Galán A, Martínez-Romero A, Martínez S, Navarro I, Ferro J, Horcajadas JA, Esteban FJ, O’Connor JE, Pellicer A, Simón C: Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells. PLoS One. 2010, 5: e10964.View ArticlePubMedPubMed CentralGoogle Scholar
- Cervelló I, Mas A, Gil-Sanchis C, Peris L, Faus A, Saunders PT, Critchley HO, Simón C: Reconstruction of endometrium from human endometrial side population cell lines. PLoS One. 2011, 6: e21221.View ArticlePubMedPubMed CentralGoogle Scholar
- Chiu RC: Bone-marrow stem cells as a source for cell therapy. Heart Fail Rev. 2003, 8: 247-251.View ArticlePubMedGoogle Scholar
- Dubois SG, Floyd EZ, Zvonic S, Kilroy G, Wu X, Carling S, Halvorsen YD, Ravussin E, Gimble JM: Isolation of human adipose-derived stem cells from biopsies and liposuction specimens. Methods Mol Biol. 2008, 449: 69-79.PubMedGoogle Scholar
- Gargett CE, Schwab KE, Zillwood RM, Nguyen HP, Wu D: Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium. Biol Reprod. 2009, 80: 1136-1145.View ArticlePubMedPubMed CentralGoogle Scholar
- Shih YR, Kuo TK, Yang AH, Lee OK, Lee CH: Isolation and characterization of stem cells from the human parathyroid gland. Cell Prolif. 2009, 42: 461-470.View ArticlePubMedGoogle Scholar
- Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH: Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001, 7: 211-228.View ArticlePubMedGoogle Scholar
- Zvaifler NJ, Marinova-Mutafchieva L, Adams G, Edwards CJ, Moss J, Burger JA, Maini RN: Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res. 2000, 2: 477-488.View ArticlePubMedPubMed CentralGoogle Scholar
- Bieback K, Klüter H: Mesenchymal stromal cells from umbilical cord blood. Curr Stem Cell Res Ther. 2007, 2: 310-323.View ArticlePubMedGoogle Scholar
- Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM: Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood. 2001, 98: 2396-2402.View ArticlePubMedGoogle Scholar
- Gang EJ, Hong SH, Jeong JA, Hwang SH, Kim SW, Yang IH, Ahn C, Han H, Kim H: In vitro mesengenic potential of human umbilical cord blood-derived mesenchymal stem cells. Biochem Biophys Res Commun. 2004, 321: 102-108.View ArticlePubMedGoogle Scholar
- In’t Anker PS, Noort WA, Scherjon SA, Kleijburg-van der Keur C, Kruisselbrink AB, van Bezooijen RL, Beekhuizen W, Willemze R, Kanhai HH, Fibbe WE: Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential. Haematologica. 2003, 88: 845-852.Google Scholar
- Anker PS I’t, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, Fibbe WE, Kanhai HH: Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells. 2004, 22: 1338-1345.View ArticleGoogle Scholar
- Zhang X, Hirai M, Cantero S, Ciubotariu R, Dobrila L, Hirsh A, Igura K, Satoh H, Yokomi I, Nishimura T, Yamaguchi S, Yoshimura K, Rubinstein P, Takahashi TA: Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue. J Cell Biochem. 2011, 112: 1206-1218.View ArticlePubMedGoogle Scholar
- Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR: Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284: 143-147.View ArticlePubMedGoogle Scholar
- Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006, 8: 315-317.View ArticlePubMedGoogle Scholar
- Klopp AH, Gupta A, Spaeth E, Andreeff M, Marini F: Concise review: Dissecting a discrepancy in the literature: do mesenchymal stem cells support or suppress tumor growth?. Stem Cells. 2011, 29: 11-19.View ArticlePubMedGoogle Scholar
- Groot-Wassink T, Aboagye EO, Glaser M, Lemoine NR, Vassaux G: Adenovirus biodistribution and noninvasive imaging of gene expression in vivo by positron emission tomography using human sodium/iodide symporter as reporter gene. Hum Gene Ther. 2002, 13: 1723-1735.View ArticlePubMedGoogle Scholar
- Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G: Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004, 15: 995-1002.View ArticlePubMedGoogle Scholar
- Martín-Duque P, Quintanilla M, McNeish I, Lopes R, Romero J, Romero D, Lemoine NR, Ramón y Cajal S, Vassaux G: Caspase-1 as a radio- and chemo-sensitiser in vitro and in vivo. Int J Mol Med. 2006, 17: 841-847.PubMedGoogle Scholar
- Henning TD, Boddington S, Daldrup-Link HE: Labeling hESCs and MSCs with iron oxide nanoparticles for non-invasive in vivo tracking with MR imaging. J Vis Exp. 2008, 13: 685.PubMedGoogle Scholar
- Loebinger MR, Janes SM: Stem cells as vectors for antitumor therapy. Thorax. 2010, 65: 362-369.View ArticlePubMedPubMed CentralGoogle Scholar
- Pereboeva L, Komarova S, Mikheeva G, Krasnykh V, Curiel DT: Approaches to utilize mesenchymal progenitor cells as cellular vehicles. Stem Cells. 2003, 21: 389-404.View ArticlePubMedGoogle Scholar
- Dwyer RM, Potter-Beirne SM, Harrington KA, Lowery AJ, Hennessy E, Murphy JM, Barry FP, O’Brien T, Kerin MJ: Monocyte chemotactic protein-1 secreted by primary breast tumors stimulates migration of mesenchymal stem cells. Clin Cancer Res. 2007, 13: 5020-5027.View ArticlePubMedGoogle Scholar
- Khakoo AY, Pati S, Anderson SA, Reid W, Elshal MF, Rovira II, Nguyen AT, Malide D, Combs CA, Hall G, Zhang J, Raffeld M, Rogers TB, Stetler-Stevenson W, Frank JA, Reitz M, Finkel T: Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi’s sarcoma. J Exp Med. 2006, 203: 1235-1247.View ArticlePubMedPubMed CentralGoogle Scholar
- Komarova S, Kawakami Y, Stoff-Khalili MA, Curiel DT, Pereboeva L: Mesenchymal progenitor cells as cellular vehicles for delivery of oncolytic adenoviruses. Mol Cancer Ther. 2006, 5: 755-766.View ArticlePubMedGoogle Scholar
- Loebinger MR, Eddaoudi A, Davies D, Janes SM: Mesenchymal stem cell delivery of TRAIL can eliminate metastatic cancer. Cancer Res. 2009, 69: 4134-4142.View ArticlePubMedPubMed CentralGoogle Scholar
- Pereboeva L, Curiel DT: Cellular vehicles for cancer gene therapy: current status and future potential. Bio-Drugs. 2004, 18: 361-385.Google Scholar
- Shinagawa K, Kitadai Y, Tanaka M, Sumida T, Kodama M, Higashi Y, Tanaka S, Yasui W, Chayama K: Mesenchymal stem cells enhance growth and metastasis of colon cancer. Int J Cancer. 2010, 127: 2323-2333.View ArticlePubMedGoogle Scholar
- Stoff-Khalili MA, Rivera AA, Mathis JM, Banerjee NS, Moon AS, Hess A, Rocconi RP, Numnum TM, Everts M, Chow LT, Douglas JT, Siegal GP, Zhu ZB, Bender HG, Dall P, Stoff A, Pereboeva L, Curiel DT: Mesenchymal stem cells as a vehicle for targeted delivery of CRAds to lung metastases of breast carcinoma. Breast Cancer Res Treat. 2007, 105: 157-167.View ArticlePubMedGoogle Scholar
- Studeny M, Marini FC, Dembinski JL, Zompetta C, Cabreira-Hansen M, Bekele BN, Champlin RE, Andreeff M: Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents. J Natl Cancer Inst. 2004, 96: 1593-1603.View ArticlePubMedGoogle Scholar
- Eiró N, Vizoso FJ: Inflammation and cancer. World J Gastrointest Surg. 2012, 4: 62-72.View ArticlePubMedPubMed CentralGoogle Scholar
- Dwyer RM, Ryan J, Havelin RJ, Morris JC, Miller BW, Liu Z, Flavin R, O’Flatharta C, Foley MJ, Barrett HH, Murphy JM, Barry FP, O’Brien T, Kerin MJ: Mesenchymal stem cell-mediated delivery of the sodium iodide symporter supports radionuclide imaging and treatment of breast cancer. Stem Cells. 2011, 29: 1149-1157.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin G, Yang R, Banie L, Wang G, Ning H, Li LC, Lue TF, Lin CS: Effects of transplantation of adipose tissue-derived stem cells on prostate tumor. Prostate. 2010, 70: 1066-1073.View ArticlePubMedPubMed CentralGoogle Scholar
- Cousin B, Ravet E, Poglio S, De Toni F, Bertuzzi M, Lulka H, Touil I, André M, Grolleau JL, Péron JM, Chavoin JP, Bourin P, Pénicaud L, Casteilla L, Buscail L, Cordelier P: Adult stromal cells derived from human adipose tissue provoke pancreatic cancer cell death both in vitro and in vivo. PLoS One. 2009, 4: e6278.View ArticlePubMedPubMed CentralGoogle Scholar
- Secchiero P, Zorzet S, Tripodo C, Corallini F, Melloni E, Caruso L, Bosco R, Ingrao S, Zavan B, Zauli G: Human bone marrow mesenchymal stem cells display anti-cancer activity in SCID mice bearing disseminated non-Hodgkin’s lymphoma xenografts. PLoS One. 2010, 5: e11140.View ArticlePubMedPubMed CentralGoogle Scholar
- Honczarenko M, Le Y, Swierkowski M, Ghiran I, Glodek AM, Silberstein LE: Human bone marrow stromal cells express a distinct set of biologically functional chemokine receptors. Stem Cells. 2006, 24: 1030-1041.View ArticlePubMedGoogle Scholar
- Ponte AL, Marais E, Gallay N, Langonné A, Delorme B, Hérault O, Charbord P, Domenech J: The in vitro migration capacity of human bone marrow mesenchymal stem cells: comparison of chemokine and growth factor chemotactic activities. Stem Cells. 2007, 25: 1737-1745.View ArticlePubMedGoogle Scholar
- Ringe J, Strassburg S, Neumann K, Endres M, Notter M, Burmester GR, Kaps C, Sittinger M: Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2. J Cell Biochem. 2007, 101: 135-146.View ArticlePubMedGoogle Scholar
- Sordi V, Malosio ML, Marchesi F, Mercalli A, Melzi R, Giordano T, Belmonte N, Ferrari G, Leone BE, Bertuzzi F, Zerbini G, Allavena P, Bonifacio E, Piemonti L: Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood. 2005, 106: 419-427.View ArticlePubMedGoogle Scholar
- Von Lüttichau I, Notohamiprodjo M, Wechselberger A, Peters C, Henger A, Seliger C, Djafarzadeh R, Huss R, Nelson PJ: Human adult CD34- progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev. 2005, 14: 329-336.View ArticlePubMedGoogle Scholar
- Dai LJ, Moniri MR, Zeng ZR, Zhou JX, Rayat J, Warnock GL: Potential implications of mesenchymal stem cells in cancer therapy. Cancer Lett. 2011, 305: 8-20.View ArticlePubMedGoogle Scholar
- Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA: Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell. 2005, 121: 335-348.View ArticlePubMedGoogle Scholar
- Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S: Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002, 109: 625-637.View ArticlePubMedPubMed CentralGoogle Scholar
- Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS, Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T: G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol. 2002, 3: 687-694.View ArticlePubMedGoogle Scholar
- Kang NH, Hwang KA, Kim SU, Kim YB, Hyun SH, Jeung EB, Choi KC: Human amniotic fluid-derived stem cells expressing cytosine deaminase and thymidine kinase inhibits the growth of breast cancer cells in cellular and xenograft mouse models. Cancer Gene Ther. 2012, 19: 412-419.View ArticlePubMedGoogle Scholar
- Yang DY, Sheu ML, Su HL, Cheng FC, Chen YJ, Chen CJ, Chiu WT, Yiin JJ, Sheehan J, Pan HC: Dual regeneration of muscle and nerve by intravenous administration of human amniotic fluid-derived mesenchymal stem cells regulated by stromal cell-derived factor-1α in a sciatic nerve injury model. J Neurosurg. 2012, 116: 1357-1367.View ArticlePubMedGoogle Scholar
- Bhoopathi P, Chetty C, Gogineni VR, Gujrati M, Dinh DH, Rao JS, Lakka SS: MMP-2 mediates mesenchymal stem cell tropism towards medulloblastoma tumors. Gene Ther. 2011, 18: 692-701.View ArticlePubMedPubMed CentralGoogle Scholar
- Dawson MR, Chae SS, Jain RK, Duda DG: Direct evidence for lineage-dependent effects of bone marrow stromal cells on tumor progression. Am J Cancer Res. 2011, 1: 144-154.PubMedGoogle Scholar
- Kortesidis A, Zannettino A, Isenmann S, Shi S, Lapidot T, Gronthos S: Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood. 2005, 105: 3793-3801.View ArticlePubMedGoogle Scholar
- Templin C, Zweigerdt R, Schwanke K, Olmer R, Ghadri JR, Emmert MY, Müller E, Küest SM, Cohrs S, Schibli R, Kronen P, Hilbe M, Reinisch A, Strunk D, Haverich A, Hoerstrup S, Lüscher TF, Kaufmann PA, Landmesser U, Martin U: Transplantation and tracking of human induced pluripotent stem cells in a pig model of myocardial infarction: assessment of cell survival, engraftment and distribution by hybrid SPECT-CT imaging of sodium iodide symporter trangene expression. Circulation. in press
- Vertelov G, Kharazi L, Muralidhar MG, Sanati G, Tankovich T, Kharazi A: High targeted migration of human mesenchymal stem cells grown in hypoxia is associated with enhanced activation of RhoA. Stem Cell Res Ther. 2013, 4: 5.View ArticlePubMedPubMed CentralGoogle Scholar
- Peinado H, Alečković M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, García-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat Med. 2012, 18: 883-891.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/11/139/prepub