Cell Transplantation, Vol. 23, pp. 195–206, 2014 Printed in the USA. All rights reserved. Copyright 2014 Cognizant Comm. Corp.
0963-6897/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X659871 E-ISSN 1555-3892 www.cognizantcommunication.com
comparison of Human Adipose-Derived Stem Cells and Bone Marrow-Derived Stem Cells in a Myocardial Infarction Model Jeppe Grøndahl Rasmussen,*† Ole Frøbert,‡ Claus Holst-Hansen,§ Jens Kastrup,¶ Ulrik Baandrup,#** Vladimir Zachar,† Trine Fink,† and Ulf Simonsen* *Department of Biomedicine, Pulmonary and Cardiovascular Pharmacology, Aarhus University, Aarhus, Denmark †Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark ‡Department of Cardiology, Örebro University Hospital, Örebro, Sweden §Department of Cardiology, Head and Heart Centre, Aalborg Hospital, Århus University Hospital, Aalborg, Denmark ¶Cardiac Stem Cell Laboratory, The Heart Centre, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark #Department of Pathology, Vendsyssel Hospital, Hjørring, Denmark **Center for Clinical Research, Aalborg University, Aalborg, Denmark
Treatment of myocardial infarction (MI) with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal MI models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of MI using a fully grown non-immune-compromised rat model. Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague–Dawley rats were randomized to receive intramyocardial injections of adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, or phosphate-buffered saline 1 week following induction of MI. After 4 weeks, left ventricular ejection fraction (LVEF) was improved in the adipose-derived stem cell group, and scar wall thickness was greater compared with the saline group. Adipose-derived as well as bone marrow-derived mesenchymal stem cells prevented left ventricular end diastolic dilation. Neither of the cell groups displayed increased angiogenesis in the myocardium compared with the saline group. Adipose-derived stem cells from a human ischemic patient preserved cardiac function following MI, whereas this could not be demonstrated for bone marrow-derived mesenchymal stem cells, with only adipose-derived stem cells leading to an improvement in LVEF. Neither of the stem cell types induced myocardial angiogenesis, raising the question whether donor age and health have an effect on the efficacy of stem cells used in the treatment of MI. Key words: Myocardial infarction; Cell therapy; Left ventricular function; Remodeling; Angiogenesis
introduction Transplantation of mesenchymal stem cells (MSCs) in the treatment of myocardial infarction (MI) has been shown to improve left ventricular function in animal models (21,23,24,30,33). Paracrine effects of the transplanted cells plays a major role in preserving cardiac function (11,28,44), and induction of angiogenesis as well as a strengthened scar area are often reported (5,37). Preconditioning MSCs in a hypoxic environment prior to their use in the treatment of MI improves the effect of stem cell transplantation (20,40), possibly because of increased production of proangiogenic and antiapoptotic cytokines and improved stem cell engraftment (16,17,26,27,40).
MSCs from both bone marrow and adipose tissue have been used in animal infarction models (5,24). Although promising effects have been reported using adipose-derived stem cells (ASCs) compared to bone marrow mononuclear cells from mice (22), the potential of human ASCs needs yet to be determined in the treatment of MI. The MSCs used in many animal trials have been from young animals or of human origin derived from young healthy donors (22,32,36). Given the fact that the conducted human clinical trials have been using autologous transplantation of bone marrow-derived cells (10,19,29,43), the effect of using cells from elderly patients with cardiac ischemia needs to be elucidated. Age and health status of the donor
Received May 30, 2012; final acceptance November 16, 2012. Online prepub date: December 4, 2012. Address correspondence to Trine Fink, Laboratory for Stem Cell Research, Department of Health Science and Technology, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg, Denmark. Tel: +45-9940-7550; E-mail:
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are particularly important, as previous studies have demonstrated a reduced effect on left ventricular ejection fraction (LVEF) and angiogenesis when bone marrow-derived cells were derived from old donors and donors with cardiomyopathy (7,13). In view of the very recent findings that mesenchymal stem cells from adipose tissue have a significantly later onset of senescence than those from bone marrow (41), a head-to-head comparison between human ASCs and bone marrow-derived mesenchymal stem cells (BMSCs) from the same patient needs to be conducted. Furthermore, when xenotransplantations are used in these transplantation models, young immunosuppressed or athymic animals are most often used (5,15,18,21). The effect of immunosuppression on MSCs in the ischemic heart is unknown. However, an often used immunosuppressant, cyclosporine A, is known to have beneficial effects on the myocardial infarct (MI) size in an ischemia–reperfusion model due to its blocking effect on the mitochondrial transition pore (2,12). The use of immunosuppression might not be necessary as MSCs themselves have been shown to have immunosuppressive effects upon transplantation (3,6,38), and human MSCs have previously been used in an immune-competent xenotransplantation model (14,31,39). Hypoxic preconditioning of MSCs before their use in transplantation has previously been shown to enhance MSC survival and efficacy (16,35). In this study, we directly compared hypoxically preconditioned human ASCs and BMSCs from an elderly patient suffering from coronary atherosclerosis, in the treatment of MI, using a rat model. The cell therapy was given to an MI model in immunecompetent, fully grown animals, without administration of immunosuppressant drugs. MATERIALS AND Methods The present study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Animal care was provided in accordance with the University of Aarhus guidelines and policies for the use of laboratory animals. Mesenchymal Stem Cells This study conforms to the Declaration of Helsinki. All protocols were approved by the local ethics committee in Copenhagen (H-D-2007-0057). Human MSCs were obtained from an 84-year-old male patient with verified coronary artery disease undergoing coronary artery bypass surgery at the Heart Center at Copenhagen University Hospital (Rigshospitalet, Denmark). The patient had a preserved LVEF, normal lung function, hyperlipidemia, and benign prostatic hyperplasia. Medication included a loop diuretic, potassium supplement, acetylic salicylic acid, a statin, a calcium antagonist (dihydropyridine), an angiotensin II receptor blocker in combination with hydrochlorothiazide, a long-acting nitrate, and finasteride.
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Aspiration of 200 ml subcutaneous adipose tissue from the upper abdomen and 40 ml bone marrow from the sternum was performed during bypass surgery. MSCs were isolated from adipose tissue and bone marrow as previously described (8,9,25). Briefly, adipose tissue was washed in phosphate-buffered saline (PBS, Life Techno logies, Frederick, MD, USA) and incubated with collagenase buffer [0.6 PZ U/ml Collagenase NB6 (Serva Electrophoresis, Heidelberg, Germany) dissolved in Hanks’ balanced salt solution (HBSS, Life Technologies)] at 37°C for 60 min. The digested tissue was filtered through a 60-µm Steriflip filter (Millipore, Copenhagen, Denmark) and centrifuged at 400 × g for 10 min, and the pelleted cells were resuspended in Ortho-Mune lysing solution (Hospital Pharmacy, Copenhagen, Denmark) in order to lyse erythrocytes. The cells were seeded, and after 24 h the medium was changed, disposing of all nonadherent cells. The bone marrow aspirate was anticoagulated with heparin (Hospital Pharmacy, Copenhagen, Denmark), and the mononuclear cells were separated with Lymphoprep (Medinor, Copenhagen, Denmark) using density gradient centrifugation. Contaminating erythrocytes were lysed in Ortho-Mune lysing solution. The cells were seeded, and after 24 h the medium was changed, disposing of all non adherent cells. Growth media contained a-modified Eagle’s medium (a-MEM), 1% penicillin, 1% streptomycin, 0.5% gentamicin (all from Invitrogen, Taastrup, Denmark), and 10% fetal calf serum (FCS; Europa Bioproducts Ltd., Cambridge, UK). To evaluate whether an endothelial phenotype had been achieved, the ability to take up acetylated lowdensity lipoprotein (LDL) cholesterol labeled with DiI (1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate) (DiI-Ac-LDL) (Biomedical Technologies, Stoughton, MA, USA) was examined. The cells were plated in 48-well plates (Costar, Acton, MA, USA), and after 24 h the medium was changed from standard growth medium to Endothelial Cell Growth Medium 2 (ECGM2) (PromoCell, Heidelberg, Germany). After 10 days of growth, the cells were incubated with DiI-Ac-LDL diluted 1:20 in the ECGM2 for 4 h, washed in media and PBS before fixation with 4% formalin (Bie & Berntsen, Herlev, Denmark), and then observed in a fluorescence microscope (Zeiss Axio Observer Z1; Zeiss, Göttingen, Germany). Fluorescence-Activated Cell Sorting Cells were harvested using 5–10-min incubation with acutase (PAA, Laboratories, Pasching, Austria) and incubated with antibodies at 4°C for 30 min. Antibodies were conjugated with allophycocyanin–Alexa 750 (APC-Alexa 750), fluorescein isothiocyanate (FITC), phycoerythrin (PE), or phycoerythrin–Texas Red (ECD). The MSCs were characterized based on a panel of antibodies including cluster of differentiation 13 (CD13)–ECD, CD31–FITC,
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CD34–APC, CD45–APC–Alexa 750, CD146–PE, CD90– FITC, CD73–PE, and CD166–PE (all from BD Bioscience, Albertslund, Denmark) and CD105–PE (R&D Systems Europe, Abingdon, UK). Analyses were done using 10,000 cells per sample on a Navios flow cytometer (Beckman Coulter, Copenhagen, Denmark), and data analysis was performed using FlowJo 7.2 software (TreeStar, Ashland, OR, USA). Proliferation Assay Proliferation capability was measured essentially as previously described (45). Briefly, ASCs and BMSCs were seeded at a density of 300/cm2 in 48-well plates and cultured at both 1% and 20% oxygen. On days 0, 2, 4, 6, 8, and 10, the cells were lysed in 200 µl 0.02% sodium dodecyl sulfate (SDS; Sigma-Aldrich, Brøndby, Denmark) in DNase-free water (Life Technologies), after which the cell number in the samples was determined using the PicoGreen dsDNA Quantitation Kit (Invitrogen). Aliquots of 100 µl cell lysate were mixed with 100 µl PicoGreen diluted 1:200 in 1× TE buffer, placed in a black microtiter plate (Thermo Fisher Scientific, Rockford, IL, USA), and incubated on a rocking shaker for 10 min. Fluorescence was measured using a Wallac 1420 Victor Multilabel Counter (PerkinElmer, Hvidovre, Denmark) with excitation at 485 nm and emission at 535 nm. A standard curve based on known Lambda DNA (CyQuant; Molecular Probes, OR, USA) concentrations was used to calculate sample DNA concentrations. The number of cells was calculated using a theoretical calculated value of 6.6 pg DNA per cell. Based on the exponential phase of the growth curve, the cell doubling time was calculated. The experiment was performed twice as two independent experiments, each in triplicate. Apoptosis Assay To determine if hypoxic culture affected the cells with respect to apoptosis, ASCs and BMSCs were seeded at a density of 104 cells/cm2 in 24-well culture plates (Corning, Amsterdam, The Netherlands) and cultured at 20% oxygen for 24 h, after which half the plates were transferred to 1% oxygen. After 24 h of additional culture, the medium was removed, and cells were washed twice with PBS. Then the cells were incubated with 2.5 mm/ml Hoechst 33342 (Invitrogen) to visualize nuclei and 1 ng/ml YO-PRO-1 (Invitrogen) to identify apoptotic cells. The stains were dissolved in PBS, and cells were incubated with the dyes at 37°C for 1 h. At the end of the incubation, the cells were washed twice with PBS, fixed in 4% formalin, and analyzed with fluorescence microscopy (Zeiss Axio Observer Z1). The level of apoptosis in each well was determined by evaluating four image fields at 20× magnification. The analysis was carried out through two independent experiments, each performed in triplicate.
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Immunoblotting ASCs were lysed in 50 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.5% NP40, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 0.04% b-mercaptoethanol, 1 mM Na3VO4, and 1 Complete Mini Protease Inhibitor Cocktail (Roche Diagnostics, Hvidovre, Denmark). Protein concentrations in lysates were measured using the BCA Protein Assay Kit (Thermo Fisher Scientific). Samples of 40–60 µg total protein were denatured by heat, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Life Technologies), and transferred to nitrocellulose membranes (Invitrogen, Taastrup, Denmark) using the iBlot transfer equipment (Invitrogen) according to standard protocols. Membranes were incubated overnight at 4°C with primary antibodies rabbit polyclonal to hypoxia-inducible factor 1 (HIF-1a; Abcam, Cambridge, UK) 1:500 and mouse monoclonal to b-actin (Sigma-Aldrich) 1:10,000. Membranes were then incubated 1 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies rabbit polyclonal anti-mouse (Dako, Glostrup, Denmark) and goat polyclonal anti-rabbit (Santa Cruz Biotechnology, Heidelberg, Germany). PBS containing 5% skimmed milk (Sigma-Aldrich) and 1% Tween 20 (Sigma-Aldrich) was used for all dilutions. Finally, target proteins were visualized using enhanced chemiluminescence (Amersham ECL Plus; GE Healthcare Europe, Brøndby, Denmark), and signal acquisition was accomplished using a Kodak Image Station 4000 mm Pro (Carestream Health Denmark, Skovlunde, Denmark). ELISA Analysis of Conditioned Media ASCs were seeded in 12-well plates (Costar, Acton, MA, USA) with 10,000 cells/cm2. After 3 days, half of the cells were exposed to 1% oxygen for 24 h, and the other half of the cells were left at 20% oxygen. Before the last 24 h of culture, the medium was changed to a-MEM with 0.2% FCS. After 24 h of culture in serum-reduced medium, the medium was harvested and the cells were lysed in 0.02% sodium dodecyl sulfate in DNase-free water, after which the cell number in the samples was determined using the PicoGreen dsDNA Quantitation Kit. Fluorescence was measured with excitation at 485 nm and emission at 535 nm. The collected medium was analyzed using a commercially available ELISA vascular endothelial growth factor (VEGF) kit (R&D Systems Europe). The experiments were conducted twice as two independent experiments, each in quadruplicate. Culture of Rat Aortic Rings Rat aortic rings were derived from thoracic aortas aseptically removed from four 8-month-old male Sprague– Dawley rats (Taconic Europe, Ry, Denmark) after euthanization using CO2. The aortas were trimmed, removing
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fibroadipose tissue. The vessels were cross-sectioned into rings of 2 mm in length, and the rings were washed in five consecutive baths of serum-free a-MEM to remove blood clots. The rings were placed in a 50-μl drop of growth factor-reduced Matrigel (BD Biosciences, Albertslund, Denmark) at the bottom of a 48-well plate (Costar) with the luminal axis parallel to the bottom of the culture well. The culture plate was incubated at 37°C for 30 min to allow cross-linking of the Matrigel, then 300 μl conditioned media from MSCs cultured at different oxygen concentrations were added to each well. After 4 days, the number of endothelial outgrowths from the aorta rings was counted using a phase-contrast microscope (Zeiss Axio Observer Z1). The experiments were conducted twice as two independent experiments. Animals and Cell Transplantation All protocols were performed in accordance with and approved by the Danish Animal Research Committee (2007/561-1337). Animals were anesthetized with a mixture of ketamine (50 mg/kg) (Intervet, Ballerup, Denmark), xylazine (5 mg/kg) (Bayer HealthCare, Animal Health, Copenhagen, Denmark), and acepromazine (0.75 mg/kg) (Pharmaxim, Helsingborg, Sweden), intubated and ventilated with a mixture of room air, oxygen, and isoflurane 0.5% (Baxter, Allerød, Denmark) at 70 breaths per minute (depth of anesthesia was tested applying a painful stimulus). The heart was exposed through a left-side thoracotomy, and MI was induced in 45 male Sprague–Dawley rats (5 months old, weight 450–550 g; Taconic Europe) by ligation of the left anterior descending artery. Throughout surgery, the animals were kept on a small animal temperature-controlled (rectal probe) heating plate (Föhr Medical Instruments GMBH, Seeheim, Germany). Mortality was 40% with all deaths occurring within the first 24 h after induction of the MI. After 1 week and prior to the first echocardiography, the 27 surviving rats were randomized to receive ASCs, BMSCs, or PBS. After echocardiography and before receiving cells, one animal in the ASC group and two animals in the PBS group were excluded due to an ejection fraction above 75%. Transplantation was done by intramyocardial injection using a 30-gauge needle (Hamilton, Reno, NV, USA). The cells were suspended in PBS and injected at three places in the peri-infarct zone with a total transplantation of 1 × 106 cells. All cells were cultured for 24 h in 1% oxygen prior to transplantation. After surgery, the animals received buprenorphine (0.1 mg/kg SC) (Schering-Plough, Ballerup, Denmark) twice daily for 3 days. Echocardiography Rats were anesthetized with a mixture of ketamine, xylazine, and acepromazine. Echocardiography was per formed 7 and 35 days after MI using Vivid i (GE Healthcare)
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equipment fitted with a 10-MHz probe. Parasternal shortand long-axis pictures were obtained; from these pictures, the ejection fraction was calculated using the Teichholz formula. Rats have an echocardiographic measured ejection fraction of approximately 85% (4,42); therefore, based on echocardiography performed before treatment, only animals with an ejection fraction below 75% were included in the analysis, leaving the following groups: ASCs (n = 8), BMSCs (n = 9), and PBS (n = 7). All echocardiographic ana lyses were performed by an experienced operator blinded to the treatment protocol using the EchoPAC software (GE Healthcare). Histology After the animals were sacrificed, the hearts were excised, fixed in formalin (10%) and after dehydration, embedded in paraffin. Infarction size was determined stereologically on five Masson trichrome (Bie & Berntsen)stained sections from each animal. The diameter of the thinnest part of the left ventricular wall was measured in four sections and included in the analysis of scar wall thickness. Vascularization was quantified in the periinfarct area using sections stained with a-smooth muscle actin antibody (Dako), stereologically determining the area covered by vessels in five 1 × 0.5-mm areas and one control area in the noninfarct area. In order to detect remaining human cells, the sections were incubated with human-specific anti-mitochondria antibody (Millipore, Copenhagen, Denmark). BNP Analysis Blood was drawn when the animals were sacrificed and, after separating serum, analysis for rat B-type (brain) natriuretic peptide-45 (BNP-45) was performed using a commercially available ELISA kit [AssayMax, Rat BNP-45 (rBNP-45) ELISA kit; Assaypro, St. Charles, MO, USA]. Statistics Data were analyzed using SigmaPlot 11.0 software (Systat Software, Erkrath, Germany). When comparing more than two groups, a one-way analysis of variance (ANOVA) test combined with a Bonferroni post hoc test was used, and when comparing two samples, a Student’s t test was used. As BNP and infarct wall thickness data were not normally distributed, a Kruskall–Wallis oneway analysis of variance on ranks test combined with a Dunn post hoc test was used. Normal distributed data are presented as means ± standard error of the mean (SEM); nonnormal distributed data are presented using boxplots denoting median and lower and upper quartiles. A two-sided value of p
