REGENERATIVE MEDICINE

REGENERATIVE MEDICINE Remote Transplantation of Mesenchymal Stem Cells Protects the Heart Against Ischemia-Reperfusion Injury MIHAI BOGDAN PREDA, a,b,c TORUNN RïNNINGEN, b ALEXANDRINA BURLACU, a,d MAYA SIMIONESCU, a JAN ØIVIND MOSKAUG, b GURO VALEN c Key Words. Mesenchymal stem cells Remote transplantation Cardioprotection In vivo imaging Heme oxygenase-1 Pentraxin 3 a Institute of Cellular Biology and Pathology Nicolae Simionescu of the Romanian Academy, Bucharest, Romania; b Department of Biochemistry and c Department of Physiology, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway; d Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania Correspondence: Mihai Bogdan Preda, Ph.D., Laboratory of Stem Cell Biology, Institute of Cellular Biology and Pathology Nicolae Simionescu, 8 B.P.Hasdeu, Sector 5, Bucharest, Romania. Telephone: 40-213194518; Fax: 40-213194519; e-mail: bogdan.preda@icbp.ro ABSTRACT Cardioprotection can be evoked through extracardiac approaches. This prompted us to investigate whether remote transplantation of stem cells confers protection of the heart against ischemic injury. The cardioprotective effect of subcutaneous transplantation of na ıve versus heme oxygenase-1 (HMOX-1)-overexpressing mouse mesenchymal stem cells (MSC) to mice was investigated in hearts subjected to ischemia-reperfusion in a Langendorff perfusion system. Mice were transplanted into the interscapular region with na ıve or HMOX-1 transfected MSC isolated from transgenic luciferase reporter mice and compared to sham-treated animals. The fate of transplanted cells was followed by in vivo bioluminescence imaging, revealing that MSC proliferated, but did not migrate detectably from the injection site. Ex vivo analysis of the hearts showed that remote transplantation of mouse adipose-derived MSC (masc) resulted in smaller infarcts and improved cardiac function after ischemia-reperfusion compared to sham-treated mice. Although HMOX-1 overexpression conferred cytoprotective effects on masc against oxidative stress in vitro, no additive beneficial effect of HMOX-1 transfection was noted on the ischemic heart. Subcutaneous transplantation of MSC also improved left ventricular function when transplanted in vivo after myocardial infarction. Plasma analysis and gene expression profile of na ıve- and HMOX-1-mASC after transplantation pointed toward pentraxin 3 as a possible factor involved in the remote cardioprotective effect of masc. These results have significant implications for understanding the behavior of stem cells after transplantation and development of safe and noninvasive cellular therapies with clinical applications. Remote transplantation of MSC can be considered as an alternative procedure to induce cardioprotection. STEM CELLS 2014;32:2123 2134 Received August 16, 2013; accepted for publication February 1, 2014; first published online in STEM CELLS EXPRESS February 27, 2014. VC AlphaMed Press 1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.1687 INTRODUCTION After several decades of encouraging preclinical studies, stem cell therapy for myocardial regeneration moved into clinical trials early in 2000 [1, 2]. Disappointingly, the overall effect of regenerative stem cell therapy to improve human heart function is not considerable, showing at most 2% 4% improvement of left ventricular ejection fraction [3, 4]. Many issues are yet to be resolved, that is, which cells should be delivered, how many, where to, and how to increase the integration of transplanted cells into the host tissue. Although several reports indicated that stem cells delivered to the heart differentiated into cardiomyocytes [5 9], other studies indicated that they did not [10, 11] and suggested that their beneficial effects were of paracrine nature [12]. Among various stem cell populations proposed as exogenous sources of cells for cardiac repair therapy [13], mesenchymal stem cells (MSC) have several advantages. These include their immune privilege, trophic activity [14], potential to differentiate into specific cell types [15], and to promote vascularization [16]. MSC are multipotent cells [17] residing in virtually all postnatal organs and tissues [18]. The mechanisms underlying their therapeutic effects are not clearly defined. However, it is known that they produce and secrete a broad variety of cytokines, chemokines, and growth factors, acting through paracrine mechanisms to ensure cytoprotection [19]. Thus, it is likely that the improvement of cardiac function afforded by MSC is due to secreted factors which attenuate inflammation and promote cell survival [12]. Such secreted factors may include tumor necrosis factor a (TNF-a), interleukin-1b (IL-1b), interleukin-6 (IL-6), interleukin 1 receptor antagonist (IL-1Rn), interleukin-10 (IL-10), nitric oxide synthase 2 STEM CELLS 2014;32:2123 2134 www.stemcells.com VC AlphaMed Press 2014

2124 Remote Transplantation of MSC is Cardioprotective (NOS2), chemokine (C-X-C motif) ligand 5 (CXCL5), transforming growth factor-b1 (TGF-b1), pentraxin 3 (PTX3), and TNF-ainduced protein 6 (TSG-6) due to their involvement in inflammation, cell migration, engraftment, chemotaxis, or cardioprotection [20 23]. Some experimental studies have shown enhanced therapeutic effects of MSC through their genetic engineering prior to transplantation [24 26]. Ischemic conditioning is an endogenous way to protect the heart from ischemia-reperfusion injury using repetitive brief episodes of ischemia, evoking myocardial protection when applied either locally or distantly [27 29]. Expanding upon the principle of remote therapy to protect the heart, gene therapy through delivery of DNA to peripheral organs has been performed successfully. Remote gene therapy induced protection of the heart from an ischemic episode when DNA encoding for insulin-like growth factor I [30] or vascular endothelial growth factor [31] was injected into the skeletal muscle. Similar, we showed that DNA encoding for hypoxia-inducible factor 1-alpha (HIF-1a) [32] or heme oxygenase-1 (HMOX-1) [33], injected into the skeletal muscle, was beneficial for heart cells ex vivo, in vitro, and in vivo [32, 33]. These findings highlighted the possibility of protecting the injured heart through extracardiac approaches. Our previous data showed that the cardioprotection provided by HIF-1a was similar to that of its direct target HMOX- 1. Nevertheless, HIF-1a overexpression in skeletal muscle caused a general angiogenesis, which might be procarcinogenic in vivo [32]. Therefore, HMOX-1 was concluded as a better choice to obtain cardioprotective effects without causing angiogenesis and was selected for further studies. HMOX-1 degrades heme into three end-metabolic products with individual cardioprotective effects: free iron, carbon monoxide, and biliverdin, which is rapidly converted to bilirubin [33]. Besides the cardioprotective effect of HMOX-1 in remote gene therapy, several other studies have shown that transfection of MSC with HMOX-1 before local transplantation also protected the infarcted heart [25, 34, 35]. This study hypothesized that remote transplantation of MSC could protect the heart against ischemia-reperfusion injury, and that overexpression of HMOX-1 in MSC prior to remote transplantation might enhance the cardioprotection. We report here that subcutaneous transplantation of MSC induced cardioprotection against ischemia. However, there was no additive beneficial effect of HMOX-1 overexpression compared to na ıve MSC. Moreover, remote transplantation of cells improved left ventricular function even when performed after myocardial infarction, an observation that may have clinical implications. MATERIALS AND METHODS Animals The mice were housed and used in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86 23. revised 1996) and all experiments were approved by the local ethics committee for animal research. C57BL/6 male mice 2 3 months old with conventional microbiological status (NOVA-SCB, Nittedal, Norway) were acclimatized for 14 days before experiments in a controlled environment of 12/12 hours light/dark cycle, 23 C, 55% 60% VC AlphaMed Press 2014 humidity, with chow (RM3 from NOVA-SCB) and water ad libitum. Male luciferase reporter mice (Luc 1 mice) on C57BL/6 background [36] expressing Luciferase driven by the promoter of c-glutamylcysteine synthetase heavy subunit [37] were used for isolation of MSC from the adipose tissue. Reporter mice transgenic for luciferase driven by NF-jB-binding sites [38] were used for assessment of inflammatory response after cell transplantation. Isolation and Transfection of Mouse MSC Mouse adipose-derived MSC (masc) were isolated from Luc 1 mice as previously described [39]. For details, please refer to Supporting Information Methods. The masc were used between 8th and 13th passages. Mouse bone marrow-derived MSC, used in experiments of cell transplant after in vivo myocardial infarction, were previously obtained and characterized [40]. masc grown in six-well plates were transiently transfected with 1.6 mg of vector pcdna3.1 carrying either HMOX-1 [32] or enhanced green fluoresecent protein (EGFP) as control to estimate the transfection efficiency using Lipofectamine 2000 reagent (Life Technologies, Oslo, Norway) according to the manufacturer s instructions. Expression of HMOX-1 was evaluated by real-time PCR and Western blot 24 hours after transfection, corresponding to the time of transplantation. The protection conferred by HMOX-1 overexpression in masc was evaluated 24 hours after transfection by exposing the cells to 200 mm H 2 O 2 for 1 hour. These conditions were chosen after performing dose-response studies (results not shown). Cell viability was determined 24 hours after H 2 O 2 - induced oxidative stress both by trypan blue exclusion assay with an automated cell counter (Life Technologies, Oslo, Norway) and the lactate dehydrogenase (LDH) assay, according to the manufacturer s instructions (Roche, Oslo, Norway). The experiments were performed three times in triplicates. Adipogenic and Osteogenic Differentiation of masc Multipotency of masc was assessed by their ability to generate adipocytes and osteoblasts in vitro, when cultured in specific differentiation media. For details, please refer to the Supporting Information Methods. Flow Cytometry Assay masc were phenotypically evaluated by flow cytometry for the expression of Sca-1, CD90, CD105, CD106, CD44, CD29, CD73, CD11b, and CD45. For experimental details, please refer to the Supporting Information Methods. In Vivo Bioluminescence Imaging To assess the correlation between cell number and bioluminescent signal, increasing numbers of masc (1 3 10 5,23 10 5, 3 3 10 5, and 4 3 10 5 cells) were seeded in six-well plates. Images were recorded and the correlation coefficient R 2 between number of cells and luminescence signal was calculated. To monitor the engraftment and possible migration of masc after subcutaneous injection into the interscapular region, four mice were imaged at 1 hour, 3 days, 5 days, and 7 days after cell transplantation using the IVIS Spectrum CT system (PerkinElmer, Oslo, Norway). This was performed by intraperitoneal injection of D-luciferin (130 mg/kg b.wt.) 7 minutes before imaging. Surface images were then analyzed using Living Image 4.3.1 software (PerkinElmer, Oslo, Norway) STEM CELLS

Preda, Rïnningen, Burlacu et al. 2125 and quantification of bioluminescence was performed by manually defining regions of interest and reported as photons/second/square centimeter/steradian. The heart, spleen, regional lymphatic ganglions, and the surrounding adipose tissue were harvested for PCR analysis to detect any low-graded migration of luciferase-expressing masc. Cell Transplantation Two experimental series of male C57BL/6 mice (10 12 weeks old) were prepared for evaluation of the cardioprotective effect of MSC in cell transplantation studies: in the first series the infarct was induced in Langendorff system 3 days after cell transplantation, and in the second the infarct was induced in vivo, before cell transplantation. In the first series, the effect of na ıve versus HMOX-1 transfected masc was evaluated in mice randomly divided into three groups: (a) sham group (n 5 7), which received 50 ml Dulbecco s modified Eagle s medium (DMEM)/F-12 culture medium by subcutaneous injection; (b) na ıve masc group (n 5 7), which were subcutaneously injected with 10 6 masc in 50 ml DMEM/F-12 medium; and (c) HMOX-1-mASC group (n 5 6), which were subcutaneously injected with the same number of HMOX-1-transfected cells in 50 ml DMEM/F-12 medium (24 hours after transfection). Cells or culture medium were injected into the interscapular region and after 3 days, hearts were removed for Langendorff perfusion, blood samples were collected from the thoracic cavity, and the cell pellets were excised for further analysis. In the second series, the mice were subjected to myocardial infarction through ligating the left coronary artery (LCA) and randomly divided into two groups (n 5 5 in each), which received either vehicle (50 ml DMEM, control group) or 10 6 bone marrowderived MSC (transplanted group). Injections were given into the interscapular region within the first hour after infarction. Histology and Immunohistochemistry The implanted cell pellets were dissected out from the mice and used for histochemical analysis and Sca-1 detection. For experimental details, please refer to Supporting Information Methods. Gene Analysis The presence of the luciferase gene in different organs after subcutaneous transplantation of masc was assessed by PCR analysis. The expression of several genes involved in migration, engraftment, chemotaxis, and/or cardioprotection was assessed in cultured masc and implanted cell pellets 3 days after transplantation. For details about gene expression analysis, please refer to Supporting Information Methods. Western Blot HMOX-1 expression was determined 24 hours after transfection in cell lysates of na ıve and HMOX-1-mASC. The plasma levels of PTX3 and TSG-6 were also quantified by Western blot 3 days after cell transplantation, corresponding to the time of heart isolation for Langendorff-perfusion. For experimental details, please refer to Supporting Information Methods. Plasma Bilirubin Measurement Bilirubin, an end-product of HMOX-1 activity, was measured in mouse plasma using Bilirubin Assay Kit (Abnova, Heidelberg, Germany), according to the manufacturer s recommendations. Isolated Heart Perfusion Hearts were isolated 3 days after cell transplantation and Langendorff-perfused as previously described [32]. Briefly, the hearts were isolated from mice anesthetized with pentobarbital (60 mg/kg). The aorta was cannulated and the hearts were retrogradely perfused with oxygenated Krebs-Henseleit buffer at a constant pressure of 60 mmhg. A balloon was inserted into the left ventricle for measurement of heart rate (HR) and left ventricular systolic and end-diastolic pressures (LVSP and LVEDP). Left ventricular developed pressure (LVDP) was calculated as the difference between LVSP and LVEDP. The coronary flow (CF) was measured with a flow meter (Transonic Systems, Elsloo, The Netherlands). Data were continuously collected (Chart software, ADInstruments, Oxford, United Kingdom) and after 20 minutes of stabilization, 25 minutes global ischemia was induced by clamping the inflow tubing. This was followed by 60 minutes of reperfusion. LVEDP was set to 5 10 mmhg preischemically in all groups. Only hearts having LVSP more than 60 mmhg, CF 1 4 ml/minute, and HR more than 300 beats per minute at the end of the stabilization period were included in the experiments. To assess the extent of the infarction at the end of reperfusion, hearts were sectioned in 1 mm slices and stained with 1% 2,3,5- triphenyltetrazolium chloride (TTC) solution at 37 C for 15 minutes. Both sides of each section were imaged with a Nikon Coolpix camera. Infarct size was determined by image analyses by a researcher blinded to the protocol. Myocardial Infarction Myocardial infarction was induced by permanent LCA ligation, as previously described [41]. Briefly, mice were anesthetized with a mixture of ketamine/xylasine (100:10 mg/kg b.wt.) and orotracheally intubated by cannulating the trachea with a 20G blunt needle attached to a mouse ventilator (UGO BASILE, Comerio VA, Italy) via a plastic Y-connector. The chest was shaved and left thoracotomy was made in the fourth intercostal space. The ligation was made 1 mm distal from the tip of the left auricle with a 7-0 polypropylene suture (BBraun, Bucharest, Romania). After ligation, the chest cavity was closed and the muscles and skin were separately sutured with 6-0 polypropylene sutures (BBraun, Bucharest, Romania). Voluntary respiration of the animals was restored by gently removing the intubation needle. After the surgery, mice were evaluated for specific ECG modifications and those which did not show ST high elevation were removed from the study. For analgesia, all animals were subcutaneously injected with buprenorphine hydrochloride (0.1 mg/kg Temgesic) prior to surgery and on the first postoperative day. Echocardiography Echocardiography under isoflurane anesthesia was used for cardiac function analysis before and after myocardial infarction. The measurements were obtained using a Vevo 2100 ultrasound system (Visualsonics, Amsterdam, The Netherlands) equipped with a 30 MHz MicroScan transducer. B-mode images of the parasternal long- and short-axis view as well as M-mode images at the midpapillary level in the parasternal short axis view were obtained and archived to be used in the determination of left ventricular (LV) function. Calculations of LV function were made using the Simpsons method and included ejection fraction, fractional shortening, and cardiac output. www.stemcells.com VC AlphaMed Press 2014

2126 Remote Transplantation of MSC is Cardioprotective Figure 1. Characterization of na ıve and HMOX-1 transfected mouse adipose-derived mesenchymal stem cell (masc). (A): Phasecontrast microscopy illustrating preconfluent na ıve masc at passage 5. (B): Oil red O staining demonstrating the adipogenic differentiation of masc after incubation with adipocyte-differentiation culture medium. Phase-contrast microscopy revealed cellular accumulation of lipid droplets after 2 weeks of incubation. (C): Alizarin red staining demonstrating osteogenic differentiation of masc. Light microscopy revealed large amounts of calcification nodules accumulated after 2 weeks of incubation. (D): Fluorescence-activated cell sorting analysis for phenotypic characterization of masc profiles. (E): mrna (normalized to GAPDH) and protein expression of HMOX-1 (normalized to b-actin) in nontransfected (na ıve) and HMOX-1-mASC (HMOX-1), 24 hours after transfection. (F): HMOX-1 mrna (normalized to GAPDH) in na ıve and HMOX-1-mASC before transplantation (before) and removed from the injection site 3 days after transplantation (after). Control denotes na ıve masc before transplantation. (G): masc viability evaluated by trypan blue exclusion assay at 24 hours after exposure to H 2 O 2 200 mm for 1 hour. H 2 O 2 treatment reduced the viability of na ıve cells, whereas HMOX-1-mASC were more resistant. Values in panels (D) (F) are mean 6 SEM of n 5 3 experiments. Abbreviation: HMOX-1, heme-oxygenase-1. Statistical Analyses Data were analyzed with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla CA, USA) and presented as mean 6 SEM. Comparisons between groups were done using paired t tests with two-tailed distribution and two-way ANOVA using a Bonferroni post hoc test. Statistical significance was defined as p <.05. RESULTS Characterization of masc VC AlphaMed Press 2014 masc isolated from Luc 1 mice were proliferative in culture and exhibited the characteristic flattened fibroblastic morphology in phase-contrast microscopy (Fig. 1A). Their multipotency was demonstrated by the ability to generate adipocytes and osteoblasts. Thus, when cultured in adipogenic differentiation medium for 2 weeks, masc generated adipocytes, as revealed by the intracellular accumulation of lipid droplets showed by oil red O staining (Fig. 1B). When cultured in osteogenic differentiation medium for 2 weeks, masc gave rise to cells that accumulated large amounts of calcification nodules stainable by Alizarin Red (Fig. 1C). Flow cytometry analysis showed that masc were highly positive for Sca-1 (stem cell antigen-1), CD105 (endoglin), CD90 (Thy-1 cell surface antigen), CD106 (vascular cell adhesion molecule-1), CD29 (integrin b1), and CD44 (receptor for hyaluronic acid) (Fig. 1D). These cells did not express the hematopoietic markers CD11b (integrin a M ) or CD45 (leukocyte common antigen), nor did they express CD73 (ecto-5 0 -nucleotidase) (Fig. 1D). Taken together, the phenotype of the masc in our culture conditions was Sca-1 pos /CD105 pos /CD90 pos /CD106 pos /CD29 pos /CD44 pos /CD11b neg / CD45 neg /CD73 neg. Transfection of masc with HMOX-1 Confers Protection Against the Oxidative Stress In Vitro The efficiency of masc transfection as evaluated by introducing EGFP plasmid in masc was around 30% (results not STEM CELLS

Preda, Rïnningen, Burlacu et al. 2127 shown). Twenty-four hours after transfection, a 23-fold increase in HMOX-1 mrna and a 8-fold increase in HMOX-1 protein expression were observed (Fig. 1E). Notably, 3 days after subcutaneous transplantations of these cells both na ıve and HMOX-1 transfected cells exhibited a downregulation of HMOX-1 compared to cells before transplantation. However, expression of HMOX-1 in transfected cells remained significantly higher after transplantation as compared to na ıve cells (Fig. 1F). The cytoprotection conferred by HMOX-1 was estimated by trypan blue exclusion assay after exposure of masc to H 2 O 2. Figure 1E illustrates a 14.5% 6 2.1% decrease in viability after 1-hour-exposure of na ıve masc to 200 mm H 2 O 2 followed by 24 hours of culture in normal conditions. In contrast to na ıve cells, the viability of HMOX-1-cells was significantly higher after H 2 O 2 treatment, demonstrating protection conferred by HMOX-1 to cells against oxidative stress. This result was validated by LDH assay, which revealed a lower cytotoxicity of the transfected versus na ıve cells when exposed to H 2 O 2 (Supporting Information Fig. S1). Subcutaneously Transplanted masc Proliferate But Do Not Migrate Detectably from the Injection Site The stability of masc integration in vivo was followed by bioluminescent imaging (BLI). The bioluminescent signal was first determined in vitro as a function of cell number. A linear correlation between the number of cells (1 3 10 5,23 10 5,33 10 5, and 4 3 10 5 ) and BLI signal was found (Fig. 2A). These data indicated that bioluminescence imaging is a reliable tool for quantifying the reporter gene-carrying cells after in vivo transplantation. To track the fate of masc after subcutaneous transplantation, four male C57BL/6 mice were subcutaneously injected with masc and photons were registered and imaged at different time intervals to follow the survival, proliferation, and migration of cells from the transplantation site. A volume of 50 ml DMEM/F-12 medium containing 10 6 masc was injected into the interscapular region, and the mice were imaged at 1 hour and 3, 5, and 7 days after transplantation. Compared to 1 hour, the images revealed a slow decrease of BLI signal 3 days after transplantation (Fig. 2B, 2C), without reaching a statistical significance. This decrease might be due to a slight mortality of cells immediately after transplantation. Nonetheless, this decline was followed by an increase of the signal during the following 5 and 7 days (Fig. 2B), suggesting a doubling of grafted cells between 3 and 7 days after transplantation, possibly after an initial period of adaptation to the new microenvironment. As illustrated by in vivo imaging, the cells did not migrate detectably from the injection site up to 7 days after transplantation (Fig. 2C). However, as in vivo imaging may not be sensitive enough for the detection of a small number of cells, organs were collected for luciferase gene analysis by PCR. No amplification was found in the heart and only trace amounts were detected in the spleen and local lymphatic ganglions. Also, a small positive signal was found in the surrounding adipose tissue dissected from transplantation site (results not shown). Together, these data strongly indicated that subcutaneously transplanted masc proliferated to some extent and did not migrate significantly from the injection site. To estimate whether stem cell multipotency was affected by the local environment, the presence of Sca-1 was evaluated in the cell implants 3 days after the implantation. As illustrated in Figure 2D, the cells still expressed Sca-1 after this interval. Furthermore, inflammatory cells did not significantly invade the transplanted area, as demonstrated by hematoxylin-eosin staining (Fig. 2E). This result was also confirmed by experiments in which the inflammation was measured in NF-jB reporter mice after subcutaneous transplantation of masc. In contrast to the high inflammation induced by local application of a small amount of phorbol myristate acetate, only a small and local induction of NF-jB activity was observed following masc transplantation (Supporting Information Fig. S2). Both results are in good agreement with the immune privileged status of MSC. Subcutaneous Transplantation of MSC Protects the Heart Against Ischemic Injury To assess whether masc remote transplantation has an effect on ischemia-reperfusion injury, Langendorff perfusion was used 3 days after cell transplantation or sham treatment. Following heart isolation, the aorta was cannulated and mounted in a constant pressure-langendorff system. After stabilization, each heart was subjected to global ischemia followed by reperfusion. As expected, hearts from sham-treated mice had a large increase of LVEDP during reperfusion (Fig. 3A). This increase was attenuated in hearts from mice transplanted with na ıve masc or HMOX-1 1 masc (Fig. 3A). However, no difference was found between the two masc groups (Fig. 3A). LVDP was depressed during reperfusion of hearts in shamtreated mice (Fig. 3B). The depression of LVDP was attenuated in hearts of both experimental groups preimplanted with masc, with no difference between the two groups (Fig. 3B). There were no differences in HR or CF between sham and masc groups (Fig. 3C, 3D). Infarct size as measured with TTC was 28.8% 6 2.2% of the left ventricle in sham-treated mice (Fig. 3E, 3F). The infarct size was reduced by preimplantation of mice with either na ıve (18.9% 6 2%) or HMOX-1-mASC (18.1% 6 2.2%) 3 days prior to isolated heart perfusion, with no difference between masc groups. These data demonstrated that remote transplantation of MSC protects the heart against subsequent ischemic injury. Next, we questioned whether MSC remote transplantation could functionally improve the heart performance following myocardial infarction. To this aim, mice were subjected to myocardial infarction by LCA ligation and subsequent subcutaneously transplanted either with vehicle or MSC. Short-axis M-mode echocardiographic analysis revealed that MSCtreated animals had a better preserved anterior wall motion at 7 days postinfarction, as compared to control group (Fig. 4A). MSC remote transplantation was associated with an improvement in left ventricular function postinfarction. Thus, statistically significant increases in fractional shortening (16.55 6 3.52 vs. 9.57 6 1.22) and cardiac output (15.08 6 1.28 vs. 10.59 6 1.95) were noted 7 days after LCA ligation in MSC-treated, compared to control group (Fig. 4B). Furthermore, the ejection fraction showed a trend toward an improvement in MSC-treated animals versus sham-treated animals. However, this trend did not reach statistical significance. The beneficial effects induced by MSC remote transplantation were only transient and were not maintained at later times after infarction, suggesting that the protection induced by transplanted cells was of paracrine nature. www.stemcells.com VC AlphaMed Press 2014

2128 Remote Transplantation of MSC is Cardioprotective Figure 2. Behavior of luciferase expressing mouse adipose-derived mesenchymal stem cell (masc) after in vivo transplantation. (A): Representative images of bioluminescent signal of increasing numbers of masc (as indicated on the top of each well) seeded on sixwell plates. Note the linear correlation between the cell number and the BLI signal expressed as radiance in photons per second per square centimeter per steradian (p/s/cm 2 /sr). The experiments were done in triplicates. (B): Quantification of BLI signal at the site of transplantation in mice subcutaneously injected with masc. The quantified BLI signal is shown as mean 6 SEM of four animals. (C): BLI images of a representative mouse at various time intervals after transplantation with masc. (D): Fluorescence microscopy illustrating the expression of Sca-1 antigen on cells, 3 days after transplantation. (scale bar 5 50 mm). (E): Histological evaluation (hematoxylin-eosin staining) of masc; no infiltration of inflammatory cells is noted 3 days after transplantation (scale bar 5 50 mm). Abbreviation: BLI, bioluminescent imaging. Local Microenvironment Modifies Gene Expression in masc After Transplantation There were no differences in the plasma level of bilirubin among the three animal groups (Supporting Information Fig. S3). This suggested that the increased HMOX-1 mrna level in HMOX-1-mASC was either nonfunctional after transplantation or translated into an insufficient increase of enzyme to produce plasma accumulation of heme end-products. To identify other putative candidates responsible for the observed cardioprotective effects, expression of several genes VC AlphaMed Press 2014 involved in migration, engraftment, chemotaxis, and/or cardioprotection was evaluated in masc before and after transplantation. Transplanted cells were carefully dissected and separated from the skin at the time of heart isolation for Langendorff perfusion and analyzed by RT-PCR and real-time PCR. The RT-PCR data revealed that TNF-a, IL-1b, IL-1Rn, IL-10, NOS2, and CXCL5 were poorly or not expressed in na ıve and HMOX-1-mASC in culture conditions, but were greatly induced in both groups after in vivo transplantation, suggesting that the local microenvironment influenced the expression (Fig. STEM CELLS

Preda, Rïnningen, Burlacu et al. 2129 Figure 3. Cardioprotective effect conferred by subcutaneous transplantation of masc. (A): The increase of LVEDP during postischemic reperfusion is attenuated in both na ıve and HMOX-1-mASC transplanted groups. (B): The depression of LVDP is reduced by both masc treatments. (C, D): No significant differences in heart rate (C) and (D) coronary flow are observed. (E): Representative transverse sections of hearts stained with TTC at the end of reperfusion, (viable tissue red and necrotic tissue unstained). (F): Quantification of infarct size in the three experimental groups; note the reduction in infarct extent in the mice pretreated with masc with no difference between na ıve and HMOX-1-mASC. Abbreviations: HMOX-1, heme-oxygenase-1; HR, heart rate; LVEDP, left ventricular end-diastolic pressure; masc, mouse adipose-derived mesenchymal stem cell. 5A). Among the activated genes, several differences were noted between na ıve and transfected cells and therefore they were further quantified by real-time PCR. The analysis showed that the expression of the proinflammatory cytokines TNF-a and IL-1b, as well as the antiinflammatory cytokines IL-1Rn and IL-10, was significantly upregulated in vivo in HMOX-1- transfected cells as compared to na ıve cells. In contrast, the upregulated levels of the angiogenic molecules NOS2 and CXCL5 were not statistically different between the two groups (Fig. 5B). Other genes, namely TGF-b1, IL-6, Ptgs2, PTX3, and TSG-6, were expressed by masc not only after transplantation but also in normal culture conditions. Except for TGFb1, a gene involved in MSC growth and differentiation, these genes modified their expression levels after transplantation (Fig. 5C). Thus, the proinflammatory cytokine IL-6 was upregulated after HMOX-1 transfection, but its expression decreased after subcutaneous transplantation. In addition, Ptgs2 (prostaglandin-endoperoxide synthase 2), an immune modulatory gene activated by inflammation [21], was similarly expressed in na ıve and HMOX-1-mASC, yet was markedly downregulated after transplantation in both groups. In contrast, PTX3 and TSG-6, known for their cardioprotective functions [22, 23], were significantly upregulated after in vivo transplantation, in both na ıve- and HMOX-1-mASC groups (Fig. 5C). PTX3 Is a Candidate for the Cardioprotective Effect of masc Remote Transplantation In the light of the cardioprotective effects of PTX3 and TSG-6 and similar increases in mrna expression in both na ıve and HMOX-1-mASC groups after in vivo transplantation, we hypothesized that PTX3 and TSG-6 were synthesized in vivo by both na ıve and HMOX-1-mASC and exerted their protective effects at remote sites through secretion into the circulation. To test this hypothesis, protein levels of PTX3 and TSG-6 were quantified by Western blot in plasma collected at the time of www.stemcells.com VC AlphaMed Press 2014

2130 Remote Transplantation of MSC is Cardioprotective Figure 4. Effect of subcutaneous transplantation of MSC on left ventricular function after myocardial infarction (MI). (A): Representative images of echocardiographic analysis measured from short-axis M-mode-based echocardiography showing improved contractility in MSC-treated mice as compare to control group. (B): Echocardiographic analysis of fractional shortening, cardiac output, and ejection fraction using the Simpsons method. Results are expressed as mean 6 SEM. Abbreviation: MSC, mesenchymal stem cells. heart isolation for Langendorff perfusion. We found an increase of PTX3 protein in the plasma of mice transplanted with na ıve and HMOX-1-mASC as compared to plasma from sham group, with no significant difference between the two groups (Fig. 5D). Concomitantly, no significant difference was noted in the TSG-6 plasma levels between the three animal groups (Fig. 5E). These findings indicated PTX3 as a candidate endocrine factor evoking the cardioprotective effect of masc when transplanted at sites remote from the heart. DISCUSSION Reports indicating that myocardial protection can be evoked through extracardiac approaches, such as remote ischemic conditioning or remote gene therapy [29, 32, 33] prompted us to assess whether remote transplantation of stem cells confers protection of the heart against ischemia injury. We used mouse bone marrow-derived and adipose-derived MSC and evaluated their ability to generate remote cardioprotection. These cells had a phenotype similar to other reported MSC lines of various origins [42 44] expressing Sca-1, CD105, CD44, CD29, and being negative for hematopoietic antigens VC AlphaMed Press 2014 (i.e., CD45 and CD11b). They had the capacity to generate adipocytes and osteoblasts under appropriate culture conditions. Their ability to secrete protective biological active factors and ease of isolation designated MSC as suitable tools for achieving cardiac repair [45]. The novel findings reported here are summarized as follows: (a) subcutaneously transplanted MSC survive and proliferate at the site of injection, with no detectable migration within 7 days after transplantation; (b) experimentally induced global ischemia of hearts isolated from mice with subcutaneously transplanted masc results in a smaller infarct size and a better cardiac function as compared to sham-treated mice; (c) subcutaneous transplantation of MSC improves the cardiac function postinfarct; (d) subcutaneous transplantation of masc induces changes in their gene expression profile, which may contribute to their cardioprotective effects; (e) PTX3 is possibly an endocrine factor evoking cardioprotection after MSC remote transplantation. Using luciferase positive cells we demonstrated that subcutaneously transplanted masc proliferated, but did not migrate extensively from the injection site, at least not within 7 days after transplantation. Our data corroborate well with previous studies showing local engraftment and long-term STEM CELLS

Preda, Rïnningen, Burlacu et al. 2131 Figure 5. Gene expression changes induced by subcutaneous transplantation of masc. (A): RT-PCR analysis illustrates the expression of TNF-a, IL-1b, IL-1Rn, IL-10, NOS2, and CXCL5 in na ıve and HMOX-1-mASC before transplantation (before) and 3 days after transplantation (after). (B): Quantification by real-time RT-PCR of the relative expression of TNF-a, IL-1b, IL-1Rn, IL-10, NOS2, and CXCL5 illustrating the differences between na ıve and HMOX-1-mASC after in vivo transplantation. (C): Real-time PCR analysis of TGF-b1, IL-6, Ptgs2, PTX3, TSG- 6, illustrating the differences between na ıve masc and HMOX-1-mASC before transplantation (before) and 3 days after transplantation (after). Control denotes na ıve masc before transplantation. (D, E): Quantification of plasma PTX3 and TSG-6 protein in sham-treated (sham), na ıve, and HMOX-1-mASC transplanted mice. Abbreviations: HMOX-1, heme-oxygenase-1; masc, mouse adipose-derived mesenchymal stem cell. survival of MSC at the site of injection when subcutaneously administered into NOD-SCID mice [46]. In our study, cell transplantation did not induce severe local inflammation and cells retained the Sca-1 marker expression 3 days after transplantation, suggesting that they were not significantly differentiated or modified by local environment and their properties were maintained in vivo. However, this does not exclude the possibility that transplanted cells may generate other cell types (parenchymal or stromal cells) or migrate to other places, at later times after transplantation. Importantly, transplanted MSC do not form teratomas for up to 17 months after subcutaneous transplantation [46]. www.stemcells.com VC AlphaMed Press 2014

2132 Remote Transplantation of MSC is Cardioprotective To our knowledge, this study is one of the first describing a protective effect of stem cell remote transplantation on heart damage. A study by Shabbir et al. [47] showed that MSC administered into the hind limb skeletal muscle improved ventricular function in a hamster heart failure model. The authors suggested the crosstalk between MSC and bone marrow endogenous progenitor cells as being the main mechanism for subsequent increase in myocardial c-kit 1 cells involved in cardiac repair. However, the migration ability of transplanted cells onto the heart was not addressed. Here, we demonstrated that subcutaneously transplanted masc at distant sites from the heart expressed paracrine/endocrine factors with cardioprotective effects against a subsequent ischemic stimulus. Although we identified PTX3 as a possible endocrine agent, other factors which were not measured might also have contributed to the observed protection. Furthermore, we cannot be sure that the transplanted cells were the only source of increased PTX3 in the murine circulation. The efficacy of MSC remote transplantation on cardioprotection was evaluated on hearts subjected to global ischemia followed by reperfusion, using a Langendorff perfusion system, as well as in vivo, on the infarcted hearts. Ex vivo analysis of the hearts showed that pretreatment of the animals with masc 3 days before heart isolation resulted in smaller infarct areas and improved cardiac function after ischemiareperfusion injury when compared to sham-treated mice. Surprisingly, no difference was noted between the na ıve- and HMOX-1-mASC transplanted groups in our study. Others have previously reported additional cardioprotective effects of HMOX-1-overexpressing MSC after intramyocardial transplantation [34, 35]. Furthermore, cardioprotection was noticed by us after transfecting skeletal muscle cells with DNA encoding for HMOX-1 [32, 33]. In this study, it is possible that although HMOX-1-transfected masc had increased HMOX-1 expression at the time of transplantation and conferred ex vivo protection against oxidative injury, they might have been altered at the time of heart isolation 3 days after transplantation. This could be due to changes induced by the local microenvironment. In support of this hypothesis, animals transplanted with HMOX-1-mASC had no increased plasma bilirubin as compared to sham- or na ıve masc-treated animals, and in vivo downregulation of HMOX-1 gene expression occurred in both na ıve or HMOX-1-mASC 3 days after transplantation. Furthermore, gene expression patterns of pro- and anti-inflammatory factors in HMOX-1-mASC were altered both before transplantation and afterward. To mimic a more clinical scenario, we determined the therapeutic efficacy of transplanting MSC subcutaneously in mice after myocardial infarction. The results revealed that remote transplantation of MSC was associated with a mild improvement in left ventricular function after 7 days from infarct induction. The beneficial effects were however only short-lived. Further studies are required to optimize the number of cells in relation to the size and duration of the infarct. Intuitively, long-term secretion of beneficial factors from an adequate number of transplanted cells or multiple doses of cells given regularly would possibly provide a sustained effect. To uncover the putative candidates responsible for the cardioprotection induced by subcutaneous transplantation of masc, several genes involved in migration, engraftment, chemotaxis, and/or cardioprotection were analyzed in na ıve- and VC AlphaMed Press 2014 HMOX-1-transfected cells before and 3 days after transplantation. Gene transcripts enriched in cells after transplantation included cytokines and chemokines generally involved in immune regulatory effects. In addition, PTX3 and TSG-6 were identified as factors possibly involved in the cardioprotective effects. PTX3 is a protein that has both atheroprotective and cardioprotective functions [23, 48], while TSG-6 (a molecule induced by TNF-a) has an important role in remodelling of the ischemic heart by reducing the inflammatory response and infarct area [22]. Systemic administration of human MSC in a mouse model of chemically injured cornea reduced corneal inflammation without engraftment at the site of lesions, by producing and secreting antiinflammatory protein TSG-6 [49]. In our experiments, although both genes were upregulated in masc in vivo, only PTX3 (but not TSG-6) was significantly increased in the plasma of masc-transplanted mice, suggesting that this factor might be involved in the cardioprotection induced by remotely transplanted masc. However, it is not ruled out that an ischemic stimulus, when applied before cell transplantation, would stimulate the increase of plasmatic level of TSG-6, which could also contribute to the cardioprotection conferred by MSC remote transplantation. Stem cell delivery methods used in clinical studies are generally associated with poor engraftment, possibly caused by a local inflammatory environment [50, 51]. Furthermore, most clinical studies have delivered stem cells directly into the myocardium, which is a rather invasive strategy [52, 53]. The inefficiency of the existing approaches for cell transplant demands the development of other strategies for the treatment of acute and chronic myocardial ischemia. Our study suggests that stem cell transplantation at distant sites from the heart allows the engraftment and proliferation of cells at the site of implantation, and thus may become a more effective and less invasive strategy as compared to direct intracardiac delivery of stem cells for myocardial protection. However, experiments in larger animals must be performed before remote stem cell transplantation can be considered as a therapeutic approach. CONCLUSIONS This study provides evidence that subcutaneous transplantation of masc can be considered as a safe and less invasive procedure to induce protection and repair of the ischemic heart. Transplanted masc secrete molecules that are possibly involved in an endocrine/paracrine fashion. These results unravel new facets regarding the behavior of MSC after in vivo transplantation and may contribute significantly to the development of cell remote transplantation as a viable option for ischemia-reperfusion injury and other clinical applications. ACKNOWLEDGMENTS This work was supported by the National Health Association, University of Oslo, Norwegian Center for Stem Cell Research, Romanian Ministry of Education and Research (RU-TE88/2010 and PCCA-1-THERION 79/2012) and CARDIOPRO Project ID 143-ERDF cofinanced investment in RTDI for Competitiveness. M.B.P. acknowledges the financial support from the Research Council of Norway in the YGGDRASIL program and A.B. STEM CELLS

Preda, Rïnningen, Burlacu et al. 2133 acknowledges the financial support of POSDRU/89/1.5/S/ 55216, 2007 2013. AUTHOR CONTRIBUTIONS M.B.P.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; A.B.: collection and assembly of data and manuscript writing; T.R.: collection and assembly of data; M.S.: manuscript writing; J.Ø.M. and G.V.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript. J.Ø.M. and G.V. have a shared last authorship. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest. REFERENCES 1 Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: The BOOST randomised controlled clinical trial. Lancet 2004;364:141 148. 2 Sch achinger V, Assmus B, Britten MB, et al. 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