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Intramyocardial Injections of Human Mesenchymal Stem Cells following Acute Myocardial Infarction Modulate Scar Formation and Improve Left Ventricular Function

Intramyocardial Injections of Human Mesenchymal Stem Cells following Acute Myocardial Infarction... Cell Transplantation, Vol. 21, pp. 1697–1709, 2012 0963-6897/12 $90.00 + .00 Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368911X627462 Copyright  2012 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com Intramyocardial Injections of Human Mesenchymal Stem Cells Following Acute Myocardial Infarction Modulate Scar Formation and Improve Left Ventricular Function Jan Otto Beitnes,* Erik Øie,† Aboulghassem Shahdadfar,‡ Tommy Karlsen,‡# Regine M. B. Müller,‡ Svend Aakhus,* Finn P. Reinholt,§¶ and Jan E. Brinchmann‡# *Department of Cardiology, Oslo University Hospital, Oslo, Norway †Research Institute for Internal Medicine, Oslo University Hospital, Oslo, Norway ‡Institute of Immunology, Oslo University Hospital, Oslo, Norway §Department of Pathology, Oslo University Hospital, Oslo, Norway ¶Institute of Pathology, University of Oslo, Oslo, Norway #Norwegian Center for Stem Cell Research, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Cell therapy is a promising treatment modality to improve heart function in acute myocardial infarction. However, the mechanisms of action and the most suitable cell type have not been finally determined. We per- formed a study to compare the effects of mesenchymal stem cells (MSCs) harvested from different tissues on LV function and explore their effects on tissue structure by morphometry and histological staining for species and lineage relationship. MSCs from skeletal muscle (SM-MSCs) and adipose tissue (ADSCs) were injected in the myocardium of nude rats 1 week after myocardial infarction. After 4 weeks of observation, LVEF was significantly improved in the SM-MSCs group (39.1%) and in the ADSC group (39.6%), compared to the placebo group (31.0%, p < 0.001 for difference in change between groups). Infarct size was smaller after cell therapy (16.3% for SM-MSCs, 15.8% for ADSCs vs. 26.0% for placebo, p < 0.001), and the amount of highly vascularized granulation tissue in the border zone was significantly increased in both groups receiving MSCs (18.3% for SM-MSCs, 22.6% for ADSCs vs. 13.1% for placebo, p = 0.001). By in situ hybridization, moderate engraftment of transplanted cells was found, but no transdifferentiation to cardiomyocytes, endothelial cells, or smooth muscle cells was observed. We conclude that MSC injections lead to improved LVEF after AMI in rats predominantly by reduction of infarct size. After 4 weeks, we observed modulation of scar formation with significant increase in granulation tissue. Transdifferentiation of MSCs to cardiomyocytes or vascular cells did not contribute significantly in this process. MSCs from skeletal muscle and adipose tissue had similar effects. Key words: Cell therapy; Mesenchymal stem cells (MSCs); In situ hybridization; Immunofluorescence; Echocardiography; Animal model INTRODUCTION In the clinical setting, skeletal myoblasts and bone marrow cells have been used in different applications, but clinical Despite advances in revascularization and medical trials have so far not provided the evidence needed for rou- therapy in the last decade, acute myocardial infarction tine implementation of this approach. Mesenchymal stem (AMI) and heart failure are still important causes of cells (MSCs) are present in many tissues, can be expanded morbidity and mortality in industrialized countries (18). rapidly when cultured, and have been shown to differenti- Although recent evidence suggest the presence of car- ate also to muscular tissues, including smooth muscle cells diac stem cells and a slow turnover of cells in the heart and cardiomyocytes (2,4,28,38). MSCs tolerate hypoxia, throughout life (5,6), AMI leads to a permanent loss of secrete angiogenic factors, and have been shown to improve contractile elements and the subsequent formation of a vascularization and perfusion (3,30). MSCs also lack con- fibrous scar. stitutive surface expression of major histocompatibility Regeneration of contractile myocardium has been a tar- complex (MHC) class II, making the cells less immuno- get for cell therapy research for more than a decade (24). genic in allotransplantation. Thus, MSCs have properties Received January 13, 2011; final acceptance June 25, 2011. Online prepub date: March 8, 2012. Address correspondence to Jan E. Brinchmann, M.D., Ph.D., Norwegian Center for Stem Cell Research, Oslo University Hospital Rikshospitalet and University of Oslo, Domus Medica, Sognsvannsveien 9, PO Box 1121, Blindern, 0317 Oslo, Norway. Tel: +47 22 84 04 89; Fax: +47 22 85 10 58; E-mail: [email protected] 1697 1698 BEITNES ET AL. that suggest that they may be beneficial in AMI, chronic antibiotics. On day 5, the medium containing nonadherent heart failure, and angina pectoris, perhaps also as allogenic cells was discarded, and fresh culture medium was added. off-the-shelf therapy in acute settings (1,9,37). When the cells reached 50% confluence, plastic adherence Previous studies have investigated the therapeutic was interrupted with trypsin-EDTA, and the cells were effects of MSCs isolated predominantly from bone mar- plated into new flasks at 5,000 cells/cm . After the first row and adipose tissue and have shown that both may passage (P1), amphotericin B was removed and 10% FBS stimulate myocardial regeneration and recovery of left was used instead of 20% for the duration of the cultures. ventricular (LV) function (20,21,25,27,31,35). However, Muscle cells were expanded up to P5 for two donors and the mechanisms responsible for the beneficial effects P7 for one donor. At P2 and immediately before transplan- remain incompletely understood. Also, MSCs obtained tation, CD56 cells were removed using magnetic beads from different tissues may have different regenerative directly coupled to mouse anti-human CD56 monoclonal capacity (16), but this has not been broadly investigated antibody (MAb) (Miltenyi Biotech, Bergisch Gladbach, in the setting of intramyocardial injection after AMI. Germany) and LS columns as described by the manu- Consequently, we designed the present study to com- facturer (Miltenyi Biotech), leaving a CD56- population, pare the effect of MSCs derived from human adipose tis- which has previously been described as MSCs (8). Cell sue (ADSCs) with MSCs isolated from human skeletal viability was always >90% (data not shown). Flow cytom- muscle (SM-MSCs) following injection into the border etry showed that no more than 3% of CD56 cells were left zone and infarct area of immunodeficient rats 1 week in the suspension at P2 and last passage. after the induction of large myocardial infarctions. We Isolation and Culture of Human Adipose Tissue-Derived used echocardiography to measure myocardial function- MSCs (ADSCs) ality and histological analyses combined with tissue mor- phometry to determine the size of the postinfarct scar, the Adipose tissue (AT) was obtained as part of routine lipo- density of blood vessels in the scar, the border zone, and suction procedures from healthy donors. The donors provided the healthy myocardial tissues, and, finally, the fraction of written informed consent, and the collection and storage of the border zone identifiable as granulation tissue. In addi- AT and ADSCs was approved by the regional committee for tion, we used fluorescence in situ hybridization (FISH) ethics in medical research (approval No. 2.2007.132). The and immunohistochemistry to identify human cells in the stromal vascular fraction (SVF) was separated from AT as rat heart and to determine if they had transdifferentiated described previously (7). Briefly, lipoaspirate (300–1,000 ml) into cells normally resident in the heart. was washed repeatedly with Hanks’ balanced salt solution (HBSS) (Life Technologies-BRL, Paisley, UK) containing MATe RIALS AND Me THODS 100 IU/ml penicillin, 100 IU/ml streptomycin, and 2.5 μg/ml Isolation and Culture of Human Skeletal amphotericin B and digested for 45 min on a shaker at Muscle-Derived MSCs 37°C using 0.1% collagenase A type 1 (Sigma). After cen- Skeletal muscle tissue was obtained from the gracilis trifugation at 400 × g for 10 min, floating adipocytes were and semitendinosus muscles removed from patients under- removed. The remaining SVF cells were resuspended in going surgery of the anterior cruciate ligament. The donors HBSS containing 2% FBS. Tissue clumps were allowed provided written informed consent, and the collection and to settle for 1 min. Suspended cells were filtered through storage of muscle tissue and SM-MSCs was approved 100-μm and then 40-μm cell sieves (Becton Dickinson, San by the regional committee for ethics in medical research Jose, CA). Cell suspensions (15 ml) were layered onto 15 ml (approval no. 1.2006.740). Ten to 20 g of skeletal muscle Lymphoprep gradient separation medium (Axis Shield, was washed three times in PBS containing 100 IU/ml peni- Oslo, Norway) in 50-ml tubes. After centrifugation (400 × g, cillin, 100 IU/ml streptomycin, and 2.5 g μ /ml amphotericin 30 min), cells at the gradient interface were collected, washed, B (Sigma Aldrich, St. Louis, MO), minced with scissors, and resuspended in culture medium containing 10% FBS rewashed, and centrifuged at 100 × g for 5 min. The muscle and antibiotics. Immediately after separation, ADSCs were fragments were digested for 60 min at 37°C in 1.5 mg/ml separated from the remaining cells using magnetic cell sort- + + collagenase IA (Sigma) in 20–40 ml DMEM/F12 (Gibco, ing. Endothelial cells (CD31 ) and leukocytes (CD45 ) were Paisley, UK). The digested fragments were centrifuged at removed using magnetic beads directly coupled to mouse 300 × g for 10 min and resuspended in 5–10 ml trypsin- anti-human CD31 and CD45 Mab (Miltenyi Biotech) and LS EDTA (Sigma) in a new culture flask for 20 min at 37ºC. columns as described by the manufacturer. Flow cytometry + + The enzymatic reaction was stopped by adding 1 ml fetal showed that no more than 5% of CD31 and CD45 cells bovine serum (FBS, Cambrex, Verviers, Belgium), and the were left in the suspension. Cells were washed, resuspended cell solution was centrifuged at 300 × g for 10 min. The and seeded in DMEM/F12 containing 20% FBS and antibiot- isolated cells were washed once and subsequently cultured ics. On day 7, culture medium containing nonadherent cells in DMEM/F12 containing 20% FBS, amphotericin B, and was discarded, and fresh culture medium was added. When MESENCHy MAL STEM CELLS IN AMI 1699 the cells reached 50% confluence, plastic adherence was (GAPDH; Taqman assay no Hs99999905_m1), which was interrupted with trypsin-EDTA, and the cells were plated into used as endogenous control. For staining procedures, cells new flasks at 5,000 cells/cm . After P1, amphotericin B was were fixed with 4% formalin and subsequently incubated for removed and 10% FBS was used instead of 20% for the dura- 10 min with Oil-Red O and Alizarin Red S to visualize lipid tion of the cultures. ADSCs from three donors were expan- droplets and calcium deposition, respectively. ded to P5. Prior to injection, cell viability was always >90% Animal Model and Surgical Procedures (data not shown). NiH rnu/rnu (Charles River, France) nude, athymic rats Verification of the Cells as MSCs by Differentiation and were bred at the Oslo University Hospital, Rikshospitalet Flow Cytometry Studies animal facility. Animals were maintained in a specific pathogen-free environment in positive pressure rooms with To verify that the cultured cells were actually MSCs, a standard 12-h day/12-h night cycle. Eighty-two animals SM-MSCs and ADSCs were differentiated along adipogenic were included in the study, as 18 surviving animals in each and osteogenic lineages and analyzed by flow cytometry for group were required to achieve sufficient power. MSC markers. For flow cytometric analysis, cells were incu- Rats aged 8–11 weeks were anesthetized with 1% iso- bated with directly conjugated antigen-specific or -irrelevant flurane, intubated, and ventilated by a rodent ventilator with monoclonal antibodies (Mabs) at room temperature for 20 min, 1% isoflurane in a mixture of 1/3 O and 2/3 N O. A left washed with PBS, and fixed in 1% paraformaldehyde. Mabs 2 2 thoracotomy was made in the fifth intercostal space, and used were CD56/PE (phycoerythrin), CD105/APC (allophy- the proximal portion of the left anterior descending (LAD) cocyanin), CD44/PE, human leukocyte antigen (HLA) ABC/ coronary artery was rapidly ligated by an intramural suture Cy-Chrome, CD34/FITC (fluorescein isothiocyanate), CD45/ to induce anterior wall myocardial infarction (26,29). The FITC (all BD Biosciences, CA), CD105/APC and HLA DR/ chest was closed, 0.05 mg/kg buprenorphine was adminis- APC (Diatec, Oslo, Norway), 144/PE (eBioscience, http:// tered subcutaneously for analgesia, and animals were moni- www.ebioscience.com/), and CD146/FITC (AbD Serotec, tored for 1 h. On day 6 post-MI, rats were sedated with 1.2 % Kidlington, UK). Cells were analyzed using a FACSCalibur isoflurane inhalation, and transthoracic echocardiography flow cytometer (Becton Dickinson). For adipogenic differ- was performed. Animals with LV fractional shortening entiation, confluent cultures were incubated in DMEM/F12 (FS) < 25% were selected for further studies. The fol- containing 10% FBS, 0.5 μ M 1- methyl-3 isobutylxanthine, lowing day (day 7), animals were randomized to receive 1 μ M dexamethasone, 10 μ g/ml insulin (Novo Nordisk, ADSCs, SM-MSCs, or placebo (cell growth medium Copenhagen, Denmark), and 100 μ M indomethacin (Dumex- without cells). Sedation, intubation, and anesthesia were Alpharma, Copenhagen, Denmark). For osteogenic differen- performed as described. The chest was reopened by ster- tiation, cells were incubated at 3,000 cells/cm in DMEM/ notomy, the infarct zone localized, and a total of 3 × 10 F12 containing 10% AS or FBS, 100 nM dexamethasone, ADSCs or 3 ´ 10 SM-MSCs suspended in 150 μl medium 10 mM ß-glycerophosphate, and 0.05 mM l -ascorbic acid-2- or placebo (150 μ l medium only) was injected with a 30-G phosphate. Fresh differentiation medium was replaced every needle as three equal aliquots in the border zone and one in third day. After 4 weeks, differentiated cells were examined the infarct zone. The chest was then closed and desufflated by real-time (RT)-PCR and staining procedures. For real-time through an 18-G drain. Postoperative analgesia and moni- RT-PCR, total RNA was extracted from cells using Trizol rea- toring were performed as described above. Four weeks gent (Invitrogen, Carlsbad, CA). Following DNase treatment after cell injection (day 35), repeated echocardiography (Ambion, Austin, TX), RNA was quantified by spectropho- was performed. Animals were then euthanized, and the tometry (Nanodrop, Wilmington, DE). Reverse transcrip- hearts were removed for histological analysis. tion (RT) was performed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Abingdon, UK) with 200 Echocardiography ng total RNA per 20 μl RT reaction. Relative quantification (RQ) was performed using the 7300 Real-Time RT PCR sys- Transthoracic echocardiography was performed on tem (Applied Biosystems) and Taqman Gene Expression sedated animals in supine position with a Vivid 7 scanner assays following protocols from the manufacturer (Applied and a 14-MHz Linear Array Probe (GE Vingmed Ultrasound, Biosystems). Taqman assays used were for osteomodulin Horten, Norway). Parasternal long-axis and two short-axis (OMD; Hs00192325_m1) and peroxisome proliferator- cine loops (midpapillary and basal) were acquired. M-mode activated receptor gamma (PPARG; Hs01115513_m1). All registration including LV wall thickness, endocavitary samples were run in triplicates (each reaction: 1.0 μl cDNA, diameter, and fractional shortening (FS) was obtained at total volume: 25 μl). The thermo cycling parameters were the midventricular level. LV ejection fraction (LVEF) was 95°C for 10 min followed by 40 cycles of 95°C for 15 s and calculated by the area–length method from the long-axis 60°C for 1 min. All samples were scaled relative to the expres- cine loop. We also calculated a wall motion score index sion level of glyceraldehyde 3-phosphate dehydrogenase (WMSI) in a 13-segment model using the long-axis and two 1700 BEITNES ET AL. short-axis cine loops (22). All analyses were performed analysis was performed on a randomly superimposed blinded to treatment allocation. Measurements were per- 150 × 150 m μ grid, dividing intersections with granulation formed on three consecutive heart cycles and averaged. tissue (hits) by the total number of myocardial intersec- tions (hits + nonhits) on the grid. Cardiomyocytes and col- Histological Analysis lagenized scar tissue were excluded from the granulation Hearts were fixed with 4% phosphate-buffered for- tissue. The pericardial zone and the right ventricle were malin for 24 h, dehydrated, and embedded in paraffin. not included in the analysis. Infarct size was assessed in 12 random hearts from each To assess vascular density, sections were stained group, while assessment of vascular density and border with goat anti-mouse vascular endothelial (VE) cad- zone quantification were performed in 10 random hearts herin primary antibodies (R&D Systems, Minneapolis, from each group. The sample sizes were based on power MN), biotinylated rabbit anti-goat secondary antibodies calculations using the results from a preliminary analysis (Dako, Glostrup, Denmark), and streptavidin peroxi- of four animals in each group. dase (Dako). Hematoxylin was used for contrast stain- Hearts were cut on a Leica microtome to obtain a stack ing. Morphometry was performed on images collected of 3-m μ short-axis sections covering the entire LV with at 400× magnification on a 15 × 15 μm randomly super - 1-mm gap between sections for estimation of both infarct imposed grid. Two representative fields in the infarct size and vascular density. For estimation of infarct size, zone (scar tissue), border zone, and remote myocardial we used Masson–Goldner staining to differentiate scar segments were counted on six representative sections tissue (green-colored collagen) from cardiomyoctes (red/ from 10 random animals in each group. The number of pink). Morphometry was performed by point counting intersections on vessel lumen and wall was divided by images collected at 12.5× magnification on a randomly the total number of intersections on the relevant tissue superimposed 250 × 250- m μ grid, and intersections were on the section to obtain the density of VE cadherin- recorded as hits on infarct zone or not (Fig. 1). The per- positive vessels. As larger vessels (diameter > 50 μm) centage infarct area on each section was determined by the were rare events within the myocardium and the scar number of infarct zone intersections on the grid divided tissue, these were not included in the analysis. by the total number of intersections. The sum of scores For identification of the transplanted cells, we per- for all slides covering the entire LV was used to estimate formed FISH with a FITC-conjugated probe specific for the total infarct volume (13). To quantify the granulation the human Alu sequence (Alu-positive control probe, tissue in the border zone, we obtained one 100× magni- Ventana, Tucson, AZ) on sections incubated in a Ventana fication image from the border zone on both sides of the Discovery machine according to the manufacturer’s infarction (Fig. 2). Granulation tissue was defined as loose instructions. The sections were subsequently qualitatively connective tissue, rich in blood vessels. A morphometric evaluated by fluorescence microscopy. Figure 1. Tissue sections stained with Masson Goldener without (A) and with (B) a superimposed grid for calculation of infarct size (magnification: ´12.5). Viable myocardium is stained red, and the hits are marked with green small circles. Scar tissue is stained green, and the hits are marked with blue small circles. MESENCHy MAL STEM CELLS IN AMI 1701 Figure 2. Representative images (magnification: ×100) from the border zone in the mesenchymal stem cell (MSC) group (A) and placebo group (B). Hematoxylin staining (blue) and anti-vascular endothelial (VE)-cadherin with peroxidase (brown). To investigate the possibility of in situ differentiation Statistics of transplanted cells, tissue sections with Alu-positive cells Data are presented as mean ± SD. Values are presented were counterstained with myocardium-, smooth muscle-, for all animals included at baseline and all animals sur- and endothelium-specific antibodies. Primary mouse anti- viving to follow-up, respectively. All continuous data were human Mabs specific for anti-smooth muscle actin (SMA, analyzed by one-way ANOVA with Bonferroni correction Dako), anti-desmin (Dako), anti-CD31 (Dako), and anti- for multiple comparisons. Categorical variables were ana- Nkx 2.5 (R&D Systems) and Alexa 555-conjugated anti- lyzed by chi-square tests. SPSS version 16.0 software was mouse secondary antibodies (Molecular Probes, Eugene, used. Statistical significance was assigned if p < 0.05. OR) were used. We also applied rabbit anti-Troponin I pri- mary antibody (Abcam, Cambridge, UK) with biotinylated Re SULTS goat anti-rabbit (Vector Labs, Burlingame, CA) and Alexa Animals and Cells 594-conjugated streptavidin (Molecular Probes). Nuclei were stained with DAPI. Sections from human hearts and The three groups of rats were well matched for physi- hearts from rats in the placebo group were used as positive ological characteristics except for weight at entry into the and negative controls, respectively. study, where the animals in the placebo group had mar- An Olympus microscope with AnalySIS software ginally higher body weight (Table 1). No significant dif- (OlympusSIS, Münster, Germany) was used for mor- ference in weight change or mortality was found during phometry. Multiplane fluorescence microscopy was per- the study. The verification of the CD56- phenotype of the formed on a Zeiss Axioplan 2 microscope (Göttingen, SM-MSCs is shown in Figure 3. Both ADSCs (7) and Germany) with ISIS software (Metasystems, Altlussheim, SM-MSCs were verified as bona fide MSCs by expres- Germany). All image analyses were performed blinded sion of CD105, CD73, CD90, and HLA class I and failure regarding treatment allocation. to express CD34, HLA DR, and hematopoietic markers Table 1. Animal Characteristics SM-MSCs ADSCs Placebo (Baseline n = 26, (Baseline n = 26, (Baseline n = 30, Follow-up n = 20) Follow-up n = 20) Follow-up n = 18) p Age (days) 63±6 67±10 67±9 0.17 Weight, baseline (g) 174±34 180+43 199±42 0.05 Weight at end of 208±45 220±46 231±47 0.44 study (g) Weight change (g) 35±21 34±18 40±18 0.63 Tibia length (mm) 36.1±1.6 36.4±1.6 36.7±1.5 0.53 Mortality 6 (23%) 6 (23%) 12 (40%) 0.27 Values are mean ± SD or number (percentage). p Values by one-way ANOVA, except for mortality where the chi-square method was used. SM-MSCs, skeletal muscle-derived mesenchymal stem cells; ADSCs, adipose tissue-derived stem cells. 1702 BEITNES ET AL. Figure 3. Characterization of the skeletal muscle (SM)-MSCs by flow cytometry. Histograms in one column represent results from each one of the three donors. Tracings represented by thick lines; no fill are from cells incubated with irrelevant control antibodies. Tracings represented by thin lines; gray fill are from cells incubated with antigen-specific antibodies. HLA, human leukocyte antigen. MESENCHy MAL STEM CELLS IN AMI 1703 Figure 4. Assays for osteogenic and adipogenic differentiation of SM-MSCs and adipose tissue-derived MSCs (ADSCs) performed after 4 weeks in differentiation medium. Quantitative RT-PCR for osteomodulin (OMD) (A) and peroxisome proliferator-activated receptor g (PPARG) (B) were performed on undifferentiated and differentiated cells from three donors (D1–D3) each for SM-MSC and ADSC (different donors for the two cell populations). All results presented are scaled relative to the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Staining assays (C) were for calcium deposition by Alizarin Red S to show osteogenic differ- entiation (top) and lipid droplets by Oil-Red O to show adipogenic differentiation (bottom) on cells from the same donors. The 200-μ m scale bar in the lower right image is representative for all images in the panel. 1704 BEITNES ET AL. Table 2. Left Ventricular Function Measured by Echocardiography SM-MSCs ADSCs Placebo (Baseline n = 26, (Baseline n = 26, (Baseline n = 30, Follow-up n = 20) Follow-up n = 20) Follow-up n = 18) p LVEDd (mm), baseline 7.58 ± 0.68 7.37 ± 0.52 7.29 ± 0.73 0.25 LVEDd (mm), follow-up 8.80 ± 0.80 8.53 ± 0.94 8.46 ± 0.79 0.43 LVEDd (mm), change 1.24 ± 0.88 1.22 ± 0.74 1.30 ± 0.89 0.96 LVESd (mm), baseline 6.41 ± 0.63 6.11 ± 0.65 6.08 ± 0.87 0.19 LVESd (mm), follow-up 7.23 ± 0.88 6.68 ± 1.24 7.06 ± 0.88 0.23 LVESd (mm), change 0.82 ± 0.80 0.68 ± 1.06 1.17 ± 0.96 0.27 FS (%), baseline 15.4 ± 3.2 17.4 ± 4.04 16.9 ± 4.9 0.22 FS (%), follow-up 18.1 ± 4.1 19.9 ± 6.4 16.7 ± 4.0 0.15 FS (%), change 2.8 ± 3.7 1.9 ± 4.1 -1.2 ± 4.9 0.01* LVEF (%), baseline 33.1 ± 7.3 35.6 ± 8.0 31.6 ± 10.4 0.24 LVEF (%), follow-up 39.1 ± 9.7 39.6 ± 6.8 31.0 ± 8.3 0.003 LVEF (%), change 4.9 ± 6.8 2.5 ± 7.7 -3.9 ± 7.3 0.002† WMSI, baseline 1.96 ± 0.17 1.90 ± 0.20 1.94 ± 0.22 0.47 WMSI, follow-up 1.82 ± 0.21 1.80 ± 0.20 1.89 ± 0.19 0.37 WMSI, change -0.12 ± 0.14 -0.06 ± 0.15 0.02 ± 0.27 0.09 Values are mean ± SD. p Value by ANOVA. LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; FS, fractional shortening; LVEF, left ventricular ejection fraction; WMSI, wall motion score index. *p = 0.01 between SM-MSCs and placebo, p = 0.08 between ADSCs and placebo. †p = 0.002 between SM-MSCs and placebo, p = 0.03 between ADSCs and placebo. Echocardiography CD45, CD14, CD3, and CD19 prior to injection (Fig. 3) (7). Also, both cell populations from all donors showed LV dimensions and function were similar between robust differentiation toward adipogenic and osteogenic groups at baseline, with mean LVEF 33 ± 10% 1 week lineages (Fig. 4). For unknown reasons, the staining for after ligation of LAD indicating substantial myocar- mineralization following osteogenic differentiation of dial infarctions (Table 2). During 4 weeks of follow-up, SM-MSCs from donor 1 was only patchy, but the upregu- we observed significant remodeling with 1.3 ± 0.8 mm lation of osteomodulin following differentiation of these (p < 0.001) increase in left ventricular end-diastolic diam- cells was robust and similar to that observed for the other eter (LVEDd). LV systolic function as measured by FS SM-MSC donors (Fig. 4A). improved by 1.9 percentage points (%) in the ADSC- Figure 5. Left ventricle (LV) function by echocardiography. FS, fractional shortening; LVEF, left ventricular ejection fraction. *See Table 2 for details. MESENCHy MAL STEM CELLS IN AMI 1705 Table 3. Tissue Morphometrics Table 4. Vascular Density SM-MSCs ADSCs Placebo p Vascular SM-MSC ADSC Placebo Density (%) (n = 10) (n = 10) (n = 10) p Infarct size 16.3 ± 4.0 15.8 ± 4.9 26.0 ± 6.8 <0.001* (% of (n = 13) (n = 13) (n = 12) Scar tissue 2.3 ± 0.8 2.2 ± 0.8 1.4 ± 0.6 0.009* myocardium) Remote 9.3 ± 1.9 9.0 ± 2.1 9.3 ± 2.1 0.95 Granulation 18.3 ± 3.7 22.6 ± 6.0 13.1 ± 5.3 0.001† myocardium tissue Border zone 22.1 ± 3.1 20.8 ± 5.2 20.2 ± 2.3 0.50 (% of border (n = 12) (n = 12) (n = 10) Values are mean ± SD. zone) *p = 0.01 for difference between SM-MSCs and placebo, p = 0.03 for difference between ADSCs and placebo. For SM-MSCs versus ADSCs, Values are mean ± SD. p = n.s. *p < 0.001 between SM-MSCs and placebo, p < 0.001 between ADSCs and placebo, and p = 1.00 between SM-MSCs and ADSCs. †p = 0.02 between SM-MSCs and placebo, p < 0.001 between ADSCs and placebo, and p = 0.05 between SM-MSCs and ADSCs. the cell-treated groups was classified as granulation tis- sue. Vascular density (Table 4) was low in the collagen- ized scar tissue (overall 2.0 ± 0.8%), higher in the remote treated group ( p = 0.08 vs. placebo) and by 2.8% in the myocardium (9.2 ± 2.0%), and highest in the granulation SM-MSC group ( p = 0.01 vs. placebo), compared to a 1.2% tissue (21.0 ± 3.7%), with p < 0.001 for difference in vas- decrease in the placebo group (Table 2, Fig. 5). LVEF cular density between tissues. Vascular density in the scar increased 2.5% in the ADSC group ( p = 0.03 vs. placebo) tissue was significantly higher in the groups receiving and 4.9% in the SM-MSC group ( p = 0.002 vs. placebo), SM-MSCs or ADSCs as compared to the control group. compared to a 3.9% decrease in the placebo group. There There was no significant difference between the three was also a trend ( p = 0.09) suggesting improved WMSI in groups for vascular density in the granulation tissue or the groups receiving cell therapy. The relative improve- within the remote myocardium. ment in LV function tended to be better in the SM-MSC In Situ Hybridization and Immunohistochemistry group compared to the ADSC group, but the difference In the transplanted hearts, a small number of Alu- did not reach statistical significance. positive human cells were retrieved in clusters, mainly in Morphometry the peripheral region of the infarct zone and in the border Infarct size at the end of study (Table 3) was sig- zone (Fig. 6). No donor-derived cells were found within nificantly lower in the SM-MSC group (16.3%) and the the preserved myocardium. The Alu-positive cells did not ADSC group (15.8%) compared to the placebo group stain positive for SMA, desmin, CD31, TnI, and Nkx (26.0%), indicating significant effect of cell therapy 2.5, indicating that differentiation to cardiomyocyte, ( p < 0.001 vs. placebo for both groups). Within the bor- smooth muscle cell, or endothelial cell phenotypes had der zone, a significantly higher proportion of the tissue in not occurred. Figure 6. Alu sequence-positive cells by fluorescence in situ hybridization (FISH). (A) Cluster of Alu-positive cells in the infarct zone. Blue represents nuclei stained with DAPI. Green and blue merged (bright green) represents transplanted cells stained with DAPI and Alu probe. Magnification: ×200. (B) Same as in (A). Magnification: ×600. 1706 BEITNES ET AL. DISCUSSION also seems to have been inhibited. Such a scenario would lead to the survival of more contractile myocardial tissue, In this study, intramyocardial injection of both ADSCs to a larger amount of persisting granulation tissue, and and SM-MSCs 1 week after AMI led to a substantial to smaller areas of scar tissue in the cell-treated hearts. decrease in infarct size and a significant improvement in This line of events would explain our observations. LVEF compared with injections of cell culture medium Some of the molecules known to be secreted by MSCs only. The proportion of granulation tissue within the bor- that might contribute in this model are prostaglandin E2, der zone was significantly increased in both treatment known to inhibit fibrosis in other systems (32), and the groups. There was a trend for better functional improve- anti-inflammatory factor tumor necrosis factor (TNF)-a- ments in the SM-MSC-treated group compared to the induced protein 6, which is thought to mediate the effect ADSC group, but this did not reach significance. A small on myocardial infarction induced by intravenous injection number of transplanted cells were still present after 4 of MSCs (17). One observation that might speak against weeks, but they did not express markers of differentiation our hypothesis is the small number of Alu-positive cells toward cardiomyocyte, endothelial, or smooth muscle noted in the rat hearts after 4 weeks. Functional and mor- lineages. phometric differences on the scale observed in the current Our study confirms the favorable effect of MSC injec- study would intuitively seem to require a large number of tions after AMI reported by other groups (9,19,20,27). surviving cells. However, similar effects on LV function We also show that the reason why the cell-treated animals was reported in the study by Imanishi et al., although vir- maintained greater myocardial contractile power was tually all the transplanted cells were lost within 4 weeks that, following induction of the same-size anterior wall (15). One possible explanation to this observation could myocardial infarctions, the MSC-treated rats developed a be that a much larger number had survived initially and much smaller scar area at 4 weeks than the placebo-treated that these cells gradually succumbed during the following animals. In fact, the difference in infarct size between the period, as indicated in cell-tracking studies (15,34,36). If cell-treated and placebo-treated rats was even higher than so, enough cells may have been present to secrete bio- expected from the differences in LV function. Our data active compounds for a sufficient period of time to affect suggest that this may in part be explained by a larger the surrounding tissues. This hypothesis is supported by amount of granulation tissue in the border zone in groups the observation that, when MSC survival was enhanced receiving MSCs. The granulation tissue was immature, as by the transduction of the anti-apoptotic akt gene, the the content of fibrous elements was low. Cardiomyocytes effects of intramyocardial injection of MSCs on cardiac were not observed in the granulation tissue. The granu- function and infarct size were also enhanced (11). Several lation tissue was not included in the parameter “infarct factors may influence the number of cells observed. size,” as it stained Masson Goldener negative, but we The cells must be handled properly before injection, as believe that this tissue would eventually have turned into detachment from the plastic surface rapidly changes the fibrous scar tissue. Even so, the injection of human MSCs MSC morphology and may trigger anoikis. As the heart is must have led to a considerable reduction in the size of small and contracting at 3–400 beats/min, some cells are the infarct. probably also lost by myocardial perforation and endo- As could be predicted, the density of small blood ves- ventricular injections. Also, the intramural pressure during sels in the granulation tissue was much higher than that systole is high, which may facilitate extrusion of injected observed for the healthy myocardium, with no difference cells (34). Although MSCs are immunoprivileged, allo- observed between the granulation tissue in the three groups. immune responses may influence cell survival. Grinnemo However, the amount of granulation tissue was signifi- et al. reported lymphocyte infiltration and rapid loss of cantly higher in the borderzone in cell-treated hearts. The cells after MSC therapy in nude rats, suggesting allo- MSCs were injected 1 week after induction of the myo- immune rejection (12). Aggregation of lymphocytes cardial infarction, at a time when highly vascular granu- was not observed in proximity to Alu-positive cells in lation tissue had presumably already been established in our study, and cell survival may have been favored by parts of the infarct zone. Within and also bordering this the use of young animals, as nude rats improve cellular granulation tissue, there may have been cardiomyocytes immune competence with age. However, we believe that affected by ischemia that were not yet dead and endog- our observation of surviving cells at 4 weeks is reliable. enous cardiac progenitor cells that might have promoted Membrane markers used to identify human cells, like DiI cardiac regeneration. MSCs are known to secrete a large or PKH 26, may persist in the tissue after the cells have number of bioactive compounds. Some of these may have died because membrane fragments may be phagocytosed acted on ischemic cardiomyocytes to promote their sur- by cells in the vicinity or fragments of the stained mem- vival and on progenitor cells to stimulate cell division and brane may fuse with the membrane of neighboring cells. differentiation. In the process, the formation of scar tissue Similarly, following transplantation of cells transfected MESENCHy MAL STEM CELLS IN AMI 1707 with green fluorescence protein, the fluorescent proteins Re Fe Re NCe S may leak from the transfected cells to be taken up by 1. Amado, L. C.; Saliaris, A. P.; Schuleri, K. H.; St John, M.; nearby host cells, which may then give a false impression Xie, J. S.; Cattaneo, S.; Durand, D. J.; Fitton, T.; Kuang, J. Q.; Stewart, G.; Lehrke, S.; Baumgartner, W. W.; Martin, of surviving cells long after the transplanted cells have B. J.; Heldman, A. W.; Hare, J. M. Cardiac repair with actually died. In contrast, no study has to date shown a intramyocardial injection of allogeneic mesenchymal stem false-positive signal for surviving cells using the human- cells after myocardial infarction. Proc. Natl. Acad. Sci. specific Alu probe employed in this study. USA 102:11474–11479; 2005. Based on very early studies of transplantation of cells 2. Arminan, A.; Gandia, C.; Bartual, C.; Garcia-Verdugo, J. M.; Lledo, E.; Mirabet, V.; Llop, M.; Barea, J.; Montero, between organs, the hypothesis has been advocated that J. A.; Sepulveda, P. Cardiac differentiation is driven by committed cells from one organ might transdifferentiate NKX2.5 and GATA4 nuclear translocation in tissue spe- to a phenotype typical of another, when placed into the cific mesenchymal stem cells. Stem Cells Dev. 18:907– microenvironment of that organ, particularly in the con- 918; 2008. text of organ damage (10,14). We did not see evidence 3. Au, P.; Tam, J.; Fukumura, D.; Jain, R. K. Bone marrow derived mesenchymal stem cells facilitate engineering of long-lasting that the transplanted MSCs transdifferentiated to become functional vasculature. Blood 111:4551–4558; 2008. human-derived cardiomyocytes or other cardiac cells 4. Bartunek, J.; Croissant, J. D.; Wijns, W.; Gofflot, S.; de that might have contributed to the functional and tissue Lavareille, A.; Vanderheyden, M.; Kaluzhny, y .; Mazouz, structural effects observed here. Only a limited number N.; Willemsen, P.; Penicka, M.; Mathieu, M.; Homsy, of studies have reported post-transplant expression of C.; De Bruyne, B.; McEntee, K.; Lee, I. W.; Heyndrickx, G. R. Pretreatment of adult bone marrow mesenchymal cardiomyocyte markers, CD 31 or SMA in vivo. The ex - stem cells with cardiomyogenic growth factors and repair tent of cell engraftment and survival has been low, and of the chronically infarcted myocardium. Am. J. Physiol. only a small fraction of engrafted cells have differenti- Heart Circ. Physiol. 292:H1095–H1104; 2007. ated toward other lineages (23,30,33). Thus, although we 5. Beltrami, A. P.; Barlucchi, L.; Torella, D.; Baker, M.; cannot exclude that transdifferentiation may take place in Limana, F.; Chimenti, S.; Kasahara, H.; Rota, M.; Musso, E.; Urbanek, K.; Leri, A.; Kajstura, J.; Nadal-Ginard, B.; rare individual cells or after longer observation periods, Anversa, P. Adult cardiac stem cells are multipotent and this is clearly not the mechanism behind the functional support myocardial regeneration. Cell 114:763–776; 2003. and structural improvements following MSC injection 6. Bergmann, O.; Bhardwaj, R. D.; Bernard, S.; Zdunek, S.; observed in the present study. Barnabe-Heider, F.; Walsh, S.; Zupicich, J.; Alkass, K.; Finally, this study was also undertaken to determine Buchholz, B. A.; Druid, H.; Jovinge, S.; Frisen, J. Evidence for cardiomyocyte renewal in humans. Science 324:98– if MSCs isolated from different organs would result 102; 2009. in different functional outcomes. We and others have 7. Boquest, A. C.; Shahdadfar, A.; Fronsdal, K.; shown that this is the case when MSCs are used to gen- Sigurjonsson, O.; Tunheim, S. H.; Collas, P.; Brinchmann, erate cartilage in vitro (16). Unfortunately, this question J. E. Isolation and transcription profiling of purified remains unanswered after the current study. Although uncultured human stromal stem cells: Alteration of gene expression after in vitro cell culture. Mol. Biol. Cell there was a trend toward better results in the functional 16:1131–1141; 2005. tests for the animals treated with SM-MSC, this did 8. Crisan, M.; y ap, S.; Casteilla, L.; Chen, C. W.; Corselli, M.; not reach statistical significance upon direct compari- Park, T. S.; Andriolo, G.; Sun, B.; Zheng, B.; Zhang, L.; son and was not reflected in the morphometric data. Norotte, C.; Teng, P. N.; Traas, J.; Schugar, R.; Deasy, B. M.; Other questions left unanswered are the optimal time Badylak, S.; Buhring, H. J.; Giacobino, J. P.; Lazzari, L.; Huard, J.; Peault, B. A perivascular origin for mesenchy- delay between the onset of acute myocardial infarction mal stem cells in multiple human organs. Cell Stem Cell and the injection of the MSCs and whether there is a 3:301–313; 2008. functional difference between using MSCs from iso- 9. Dai, W.; Hale, S. L.; Martin, B. J.; Kuang, J. Q.; Dow, J. S.; genic or genetically different individuals. Still, at this Wold, L. E.; Kloner, R. A. Allogeneic mesenchymal stem time, MSCs appear as a potentially interesting adjuvant cell transplantation in postinfarcted rat myocardium: Short- and long-term effects. Circulation 112:214–223; 2005. treatment modality for selected patients following acute 10. Gehring, W. Clonal analysis of determination dynamics myocardial infarction. in cultures of imaginal disks in Drosophila melanogaster. Acknowledgments: The authors would like to thank Linda Dorg Dev. Biol. 16:438–456; 1967. for valuable assistance in histological staining procedures, 11. Gnecchi, M.; He, H.; Melo, L. G.; Noiseaux, N.; Morello, and Klaus Beiske, M.D. for providing and supervising the use F.; De Boer, R. A.; Zhang, L.; Pratt, R. E.; Dzau, V. J.; of the Zeiss microscope and ISIS software. J. O. Beitnes was Ingwall, J. S. 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Cell 13:4279– Seleznik, G.; Larson, S.; Wentworth, B.; O’Callaghan, M.; 4295; 2002. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Transplantation SAGE

Intramyocardial Injections of Human Mesenchymal Stem Cells following Acute Myocardial Infarction Modulate Scar Formation and Improve Left Ventricular Function

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© 2012 Cognizant Comm. Corp.
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Cell Transplantation, Vol. 21, pp. 1697–1709, 2012 0963-6897/12 $90.00 + .00 Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368911X627462 Copyright  2012 Cognizant Comm. Corp. E-ISSN 1555-3892 www.cognizantcommunication.com Intramyocardial Injections of Human Mesenchymal Stem Cells Following Acute Myocardial Infarction Modulate Scar Formation and Improve Left Ventricular Function Jan Otto Beitnes,* Erik Øie,† Aboulghassem Shahdadfar,‡ Tommy Karlsen,‡# Regine M. B. Müller,‡ Svend Aakhus,* Finn P. Reinholt,§¶ and Jan E. Brinchmann‡# *Department of Cardiology, Oslo University Hospital, Oslo, Norway †Research Institute for Internal Medicine, Oslo University Hospital, Oslo, Norway ‡Institute of Immunology, Oslo University Hospital, Oslo, Norway §Department of Pathology, Oslo University Hospital, Oslo, Norway ¶Institute of Pathology, University of Oslo, Oslo, Norway #Norwegian Center for Stem Cell Research, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway Cell therapy is a promising treatment modality to improve heart function in acute myocardial infarction. However, the mechanisms of action and the most suitable cell type have not been finally determined. We per- formed a study to compare the effects of mesenchymal stem cells (MSCs) harvested from different tissues on LV function and explore their effects on tissue structure by morphometry and histological staining for species and lineage relationship. MSCs from skeletal muscle (SM-MSCs) and adipose tissue (ADSCs) were injected in the myocardium of nude rats 1 week after myocardial infarction. After 4 weeks of observation, LVEF was significantly improved in the SM-MSCs group (39.1%) and in the ADSC group (39.6%), compared to the placebo group (31.0%, p < 0.001 for difference in change between groups). Infarct size was smaller after cell therapy (16.3% for SM-MSCs, 15.8% for ADSCs vs. 26.0% for placebo, p < 0.001), and the amount of highly vascularized granulation tissue in the border zone was significantly increased in both groups receiving MSCs (18.3% for SM-MSCs, 22.6% for ADSCs vs. 13.1% for placebo, p = 0.001). By in situ hybridization, moderate engraftment of transplanted cells was found, but no transdifferentiation to cardiomyocytes, endothelial cells, or smooth muscle cells was observed. We conclude that MSC injections lead to improved LVEF after AMI in rats predominantly by reduction of infarct size. After 4 weeks, we observed modulation of scar formation with significant increase in granulation tissue. Transdifferentiation of MSCs to cardiomyocytes or vascular cells did not contribute significantly in this process. MSCs from skeletal muscle and adipose tissue had similar effects. Key words: Cell therapy; Mesenchymal stem cells (MSCs); In situ hybridization; Immunofluorescence; Echocardiography; Animal model INTRODUCTION In the clinical setting, skeletal myoblasts and bone marrow cells have been used in different applications, but clinical Despite advances in revascularization and medical trials have so far not provided the evidence needed for rou- therapy in the last decade, acute myocardial infarction tine implementation of this approach. Mesenchymal stem (AMI) and heart failure are still important causes of cells (MSCs) are present in many tissues, can be expanded morbidity and mortality in industrialized countries (18). rapidly when cultured, and have been shown to differenti- Although recent evidence suggest the presence of car- ate also to muscular tissues, including smooth muscle cells diac stem cells and a slow turnover of cells in the heart and cardiomyocytes (2,4,28,38). MSCs tolerate hypoxia, throughout life (5,6), AMI leads to a permanent loss of secrete angiogenic factors, and have been shown to improve contractile elements and the subsequent formation of a vascularization and perfusion (3,30). MSCs also lack con- fibrous scar. stitutive surface expression of major histocompatibility Regeneration of contractile myocardium has been a tar- complex (MHC) class II, making the cells less immuno- get for cell therapy research for more than a decade (24). genic in allotransplantation. Thus, MSCs have properties Received January 13, 2011; final acceptance June 25, 2011. Online prepub date: March 8, 2012. Address correspondence to Jan E. Brinchmann, M.D., Ph.D., Norwegian Center for Stem Cell Research, Oslo University Hospital Rikshospitalet and University of Oslo, Domus Medica, Sognsvannsveien 9, PO Box 1121, Blindern, 0317 Oslo, Norway. Tel: +47 22 84 04 89; Fax: +47 22 85 10 58; E-mail: [email protected] 1697 1698 BEITNES ET AL. that suggest that they may be beneficial in AMI, chronic antibiotics. On day 5, the medium containing nonadherent heart failure, and angina pectoris, perhaps also as allogenic cells was discarded, and fresh culture medium was added. off-the-shelf therapy in acute settings (1,9,37). When the cells reached 50% confluence, plastic adherence Previous studies have investigated the therapeutic was interrupted with trypsin-EDTA, and the cells were effects of MSCs isolated predominantly from bone mar- plated into new flasks at 5,000 cells/cm . After the first row and adipose tissue and have shown that both may passage (P1), amphotericin B was removed and 10% FBS stimulate myocardial regeneration and recovery of left was used instead of 20% for the duration of the cultures. ventricular (LV) function (20,21,25,27,31,35). However, Muscle cells were expanded up to P5 for two donors and the mechanisms responsible for the beneficial effects P7 for one donor. At P2 and immediately before transplan- remain incompletely understood. Also, MSCs obtained tation, CD56 cells were removed using magnetic beads from different tissues may have different regenerative directly coupled to mouse anti-human CD56 monoclonal capacity (16), but this has not been broadly investigated antibody (MAb) (Miltenyi Biotech, Bergisch Gladbach, in the setting of intramyocardial injection after AMI. Germany) and LS columns as described by the manu- Consequently, we designed the present study to com- facturer (Miltenyi Biotech), leaving a CD56- population, pare the effect of MSCs derived from human adipose tis- which has previously been described as MSCs (8). Cell sue (ADSCs) with MSCs isolated from human skeletal viability was always >90% (data not shown). Flow cytom- muscle (SM-MSCs) following injection into the border etry showed that no more than 3% of CD56 cells were left zone and infarct area of immunodeficient rats 1 week in the suspension at P2 and last passage. after the induction of large myocardial infarctions. We Isolation and Culture of Human Adipose Tissue-Derived used echocardiography to measure myocardial function- MSCs (ADSCs) ality and histological analyses combined with tissue mor- phometry to determine the size of the postinfarct scar, the Adipose tissue (AT) was obtained as part of routine lipo- density of blood vessels in the scar, the border zone, and suction procedures from healthy donors. The donors provided the healthy myocardial tissues, and, finally, the fraction of written informed consent, and the collection and storage of the border zone identifiable as granulation tissue. In addi- AT and ADSCs was approved by the regional committee for tion, we used fluorescence in situ hybridization (FISH) ethics in medical research (approval No. 2.2007.132). The and immunohistochemistry to identify human cells in the stromal vascular fraction (SVF) was separated from AT as rat heart and to determine if they had transdifferentiated described previously (7). Briefly, lipoaspirate (300–1,000 ml) into cells normally resident in the heart. was washed repeatedly with Hanks’ balanced salt solution (HBSS) (Life Technologies-BRL, Paisley, UK) containing MATe RIALS AND Me THODS 100 IU/ml penicillin, 100 IU/ml streptomycin, and 2.5 μg/ml Isolation and Culture of Human Skeletal amphotericin B and digested for 45 min on a shaker at Muscle-Derived MSCs 37°C using 0.1% collagenase A type 1 (Sigma). After cen- Skeletal muscle tissue was obtained from the gracilis trifugation at 400 × g for 10 min, floating adipocytes were and semitendinosus muscles removed from patients under- removed. The remaining SVF cells were resuspended in going surgery of the anterior cruciate ligament. The donors HBSS containing 2% FBS. Tissue clumps were allowed provided written informed consent, and the collection and to settle for 1 min. Suspended cells were filtered through storage of muscle tissue and SM-MSCs was approved 100-μm and then 40-μm cell sieves (Becton Dickinson, San by the regional committee for ethics in medical research Jose, CA). Cell suspensions (15 ml) were layered onto 15 ml (approval no. 1.2006.740). Ten to 20 g of skeletal muscle Lymphoprep gradient separation medium (Axis Shield, was washed three times in PBS containing 100 IU/ml peni- Oslo, Norway) in 50-ml tubes. After centrifugation (400 × g, cillin, 100 IU/ml streptomycin, and 2.5 g μ /ml amphotericin 30 min), cells at the gradient interface were collected, washed, B (Sigma Aldrich, St. Louis, MO), minced with scissors, and resuspended in culture medium containing 10% FBS rewashed, and centrifuged at 100 × g for 5 min. The muscle and antibiotics. Immediately after separation, ADSCs were fragments were digested for 60 min at 37°C in 1.5 mg/ml separated from the remaining cells using magnetic cell sort- + + collagenase IA (Sigma) in 20–40 ml DMEM/F12 (Gibco, ing. Endothelial cells (CD31 ) and leukocytes (CD45 ) were Paisley, UK). The digested fragments were centrifuged at removed using magnetic beads directly coupled to mouse 300 × g for 10 min and resuspended in 5–10 ml trypsin- anti-human CD31 and CD45 Mab (Miltenyi Biotech) and LS EDTA (Sigma) in a new culture flask for 20 min at 37ºC. columns as described by the manufacturer. Flow cytometry + + The enzymatic reaction was stopped by adding 1 ml fetal showed that no more than 5% of CD31 and CD45 cells bovine serum (FBS, Cambrex, Verviers, Belgium), and the were left in the suspension. Cells were washed, resuspended cell solution was centrifuged at 300 × g for 10 min. The and seeded in DMEM/F12 containing 20% FBS and antibiot- isolated cells were washed once and subsequently cultured ics. On day 7, culture medium containing nonadherent cells in DMEM/F12 containing 20% FBS, amphotericin B, and was discarded, and fresh culture medium was added. When MESENCHy MAL STEM CELLS IN AMI 1699 the cells reached 50% confluence, plastic adherence was (GAPDH; Taqman assay no Hs99999905_m1), which was interrupted with trypsin-EDTA, and the cells were plated into used as endogenous control. For staining procedures, cells new flasks at 5,000 cells/cm . After P1, amphotericin B was were fixed with 4% formalin and subsequently incubated for removed and 10% FBS was used instead of 20% for the dura- 10 min with Oil-Red O and Alizarin Red S to visualize lipid tion of the cultures. ADSCs from three donors were expan- droplets and calcium deposition, respectively. ded to P5. Prior to injection, cell viability was always >90% Animal Model and Surgical Procedures (data not shown). NiH rnu/rnu (Charles River, France) nude, athymic rats Verification of the Cells as MSCs by Differentiation and were bred at the Oslo University Hospital, Rikshospitalet Flow Cytometry Studies animal facility. Animals were maintained in a specific pathogen-free environment in positive pressure rooms with To verify that the cultured cells were actually MSCs, a standard 12-h day/12-h night cycle. Eighty-two animals SM-MSCs and ADSCs were differentiated along adipogenic were included in the study, as 18 surviving animals in each and osteogenic lineages and analyzed by flow cytometry for group were required to achieve sufficient power. MSC markers. For flow cytometric analysis, cells were incu- Rats aged 8–11 weeks were anesthetized with 1% iso- bated with directly conjugated antigen-specific or -irrelevant flurane, intubated, and ventilated by a rodent ventilator with monoclonal antibodies (Mabs) at room temperature for 20 min, 1% isoflurane in a mixture of 1/3 O and 2/3 N O. A left washed with PBS, and fixed in 1% paraformaldehyde. Mabs 2 2 thoracotomy was made in the fifth intercostal space, and used were CD56/PE (phycoerythrin), CD105/APC (allophy- the proximal portion of the left anterior descending (LAD) cocyanin), CD44/PE, human leukocyte antigen (HLA) ABC/ coronary artery was rapidly ligated by an intramural suture Cy-Chrome, CD34/FITC (fluorescein isothiocyanate), CD45/ to induce anterior wall myocardial infarction (26,29). The FITC (all BD Biosciences, CA), CD105/APC and HLA DR/ chest was closed, 0.05 mg/kg buprenorphine was adminis- APC (Diatec, Oslo, Norway), 144/PE (eBioscience, http:// tered subcutaneously for analgesia, and animals were moni- www.ebioscience.com/), and CD146/FITC (AbD Serotec, tored for 1 h. On day 6 post-MI, rats were sedated with 1.2 % Kidlington, UK). Cells were analyzed using a FACSCalibur isoflurane inhalation, and transthoracic echocardiography flow cytometer (Becton Dickinson). For adipogenic differ- was performed. Animals with LV fractional shortening entiation, confluent cultures were incubated in DMEM/F12 (FS) < 25% were selected for further studies. The fol- containing 10% FBS, 0.5 μ M 1- methyl-3 isobutylxanthine, lowing day (day 7), animals were randomized to receive 1 μ M dexamethasone, 10 μ g/ml insulin (Novo Nordisk, ADSCs, SM-MSCs, or placebo (cell growth medium Copenhagen, Denmark), and 100 μ M indomethacin (Dumex- without cells). Sedation, intubation, and anesthesia were Alpharma, Copenhagen, Denmark). For osteogenic differen- performed as described. The chest was reopened by ster- tiation, cells were incubated at 3,000 cells/cm in DMEM/ notomy, the infarct zone localized, and a total of 3 × 10 F12 containing 10% AS or FBS, 100 nM dexamethasone, ADSCs or 3 ´ 10 SM-MSCs suspended in 150 μl medium 10 mM ß-glycerophosphate, and 0.05 mM l -ascorbic acid-2- or placebo (150 μ l medium only) was injected with a 30-G phosphate. Fresh differentiation medium was replaced every needle as three equal aliquots in the border zone and one in third day. After 4 weeks, differentiated cells were examined the infarct zone. The chest was then closed and desufflated by real-time (RT)-PCR and staining procedures. For real-time through an 18-G drain. Postoperative analgesia and moni- RT-PCR, total RNA was extracted from cells using Trizol rea- toring were performed as described above. Four weeks gent (Invitrogen, Carlsbad, CA). Following DNase treatment after cell injection (day 35), repeated echocardiography (Ambion, Austin, TX), RNA was quantified by spectropho- was performed. Animals were then euthanized, and the tometry (Nanodrop, Wilmington, DE). Reverse transcrip- hearts were removed for histological analysis. tion (RT) was performed using the High-Capacity cDNA Archive Kit (Applied Biosystems, Abingdon, UK) with 200 Echocardiography ng total RNA per 20 μl RT reaction. Relative quantification (RQ) was performed using the 7300 Real-Time RT PCR sys- Transthoracic echocardiography was performed on tem (Applied Biosystems) and Taqman Gene Expression sedated animals in supine position with a Vivid 7 scanner assays following protocols from the manufacturer (Applied and a 14-MHz Linear Array Probe (GE Vingmed Ultrasound, Biosystems). Taqman assays used were for osteomodulin Horten, Norway). Parasternal long-axis and two short-axis (OMD; Hs00192325_m1) and peroxisome proliferator- cine loops (midpapillary and basal) were acquired. M-mode activated receptor gamma (PPARG; Hs01115513_m1). All registration including LV wall thickness, endocavitary samples were run in triplicates (each reaction: 1.0 μl cDNA, diameter, and fractional shortening (FS) was obtained at total volume: 25 μl). The thermo cycling parameters were the midventricular level. LV ejection fraction (LVEF) was 95°C for 10 min followed by 40 cycles of 95°C for 15 s and calculated by the area–length method from the long-axis 60°C for 1 min. All samples were scaled relative to the expres- cine loop. We also calculated a wall motion score index sion level of glyceraldehyde 3-phosphate dehydrogenase (WMSI) in a 13-segment model using the long-axis and two 1700 BEITNES ET AL. short-axis cine loops (22). All analyses were performed analysis was performed on a randomly superimposed blinded to treatment allocation. Measurements were per- 150 × 150 m μ grid, dividing intersections with granulation formed on three consecutive heart cycles and averaged. tissue (hits) by the total number of myocardial intersec- tions (hits + nonhits) on the grid. Cardiomyocytes and col- Histological Analysis lagenized scar tissue were excluded from the granulation Hearts were fixed with 4% phosphate-buffered for- tissue. The pericardial zone and the right ventricle were malin for 24 h, dehydrated, and embedded in paraffin. not included in the analysis. Infarct size was assessed in 12 random hearts from each To assess vascular density, sections were stained group, while assessment of vascular density and border with goat anti-mouse vascular endothelial (VE) cad- zone quantification were performed in 10 random hearts herin primary antibodies (R&D Systems, Minneapolis, from each group. The sample sizes were based on power MN), biotinylated rabbit anti-goat secondary antibodies calculations using the results from a preliminary analysis (Dako, Glostrup, Denmark), and streptavidin peroxi- of four animals in each group. dase (Dako). Hematoxylin was used for contrast stain- Hearts were cut on a Leica microtome to obtain a stack ing. Morphometry was performed on images collected of 3-m μ short-axis sections covering the entire LV with at 400× magnification on a 15 × 15 μm randomly super - 1-mm gap between sections for estimation of both infarct imposed grid. Two representative fields in the infarct size and vascular density. For estimation of infarct size, zone (scar tissue), border zone, and remote myocardial we used Masson–Goldner staining to differentiate scar segments were counted on six representative sections tissue (green-colored collagen) from cardiomyoctes (red/ from 10 random animals in each group. The number of pink). Morphometry was performed by point counting intersections on vessel lumen and wall was divided by images collected at 12.5× magnification on a randomly the total number of intersections on the relevant tissue superimposed 250 × 250- m μ grid, and intersections were on the section to obtain the density of VE cadherin- recorded as hits on infarct zone or not (Fig. 1). The per- positive vessels. As larger vessels (diameter > 50 μm) centage infarct area on each section was determined by the were rare events within the myocardium and the scar number of infarct zone intersections on the grid divided tissue, these were not included in the analysis. by the total number of intersections. The sum of scores For identification of the transplanted cells, we per- for all slides covering the entire LV was used to estimate formed FISH with a FITC-conjugated probe specific for the total infarct volume (13). To quantify the granulation the human Alu sequence (Alu-positive control probe, tissue in the border zone, we obtained one 100× magni- Ventana, Tucson, AZ) on sections incubated in a Ventana fication image from the border zone on both sides of the Discovery machine according to the manufacturer’s infarction (Fig. 2). Granulation tissue was defined as loose instructions. The sections were subsequently qualitatively connective tissue, rich in blood vessels. A morphometric evaluated by fluorescence microscopy. Figure 1. Tissue sections stained with Masson Goldener without (A) and with (B) a superimposed grid for calculation of infarct size (magnification: ´12.5). Viable myocardium is stained red, and the hits are marked with green small circles. Scar tissue is stained green, and the hits are marked with blue small circles. MESENCHy MAL STEM CELLS IN AMI 1701 Figure 2. Representative images (magnification: ×100) from the border zone in the mesenchymal stem cell (MSC) group (A) and placebo group (B). Hematoxylin staining (blue) and anti-vascular endothelial (VE)-cadherin with peroxidase (brown). To investigate the possibility of in situ differentiation Statistics of transplanted cells, tissue sections with Alu-positive cells Data are presented as mean ± SD. Values are presented were counterstained with myocardium-, smooth muscle-, for all animals included at baseline and all animals sur- and endothelium-specific antibodies. Primary mouse anti- viving to follow-up, respectively. All continuous data were human Mabs specific for anti-smooth muscle actin (SMA, analyzed by one-way ANOVA with Bonferroni correction Dako), anti-desmin (Dako), anti-CD31 (Dako), and anti- for multiple comparisons. Categorical variables were ana- Nkx 2.5 (R&D Systems) and Alexa 555-conjugated anti- lyzed by chi-square tests. SPSS version 16.0 software was mouse secondary antibodies (Molecular Probes, Eugene, used. Statistical significance was assigned if p < 0.05. OR) were used. We also applied rabbit anti-Troponin I pri- mary antibody (Abcam, Cambridge, UK) with biotinylated Re SULTS goat anti-rabbit (Vector Labs, Burlingame, CA) and Alexa Animals and Cells 594-conjugated streptavidin (Molecular Probes). Nuclei were stained with DAPI. Sections from human hearts and The three groups of rats were well matched for physi- hearts from rats in the placebo group were used as positive ological characteristics except for weight at entry into the and negative controls, respectively. study, where the animals in the placebo group had mar- An Olympus microscope with AnalySIS software ginally higher body weight (Table 1). No significant dif- (OlympusSIS, Münster, Germany) was used for mor- ference in weight change or mortality was found during phometry. Multiplane fluorescence microscopy was per- the study. The verification of the CD56- phenotype of the formed on a Zeiss Axioplan 2 microscope (Göttingen, SM-MSCs is shown in Figure 3. Both ADSCs (7) and Germany) with ISIS software (Metasystems, Altlussheim, SM-MSCs were verified as bona fide MSCs by expres- Germany). All image analyses were performed blinded sion of CD105, CD73, CD90, and HLA class I and failure regarding treatment allocation. to express CD34, HLA DR, and hematopoietic markers Table 1. Animal Characteristics SM-MSCs ADSCs Placebo (Baseline n = 26, (Baseline n = 26, (Baseline n = 30, Follow-up n = 20) Follow-up n = 20) Follow-up n = 18) p Age (days) 63±6 67±10 67±9 0.17 Weight, baseline (g) 174±34 180+43 199±42 0.05 Weight at end of 208±45 220±46 231±47 0.44 study (g) Weight change (g) 35±21 34±18 40±18 0.63 Tibia length (mm) 36.1±1.6 36.4±1.6 36.7±1.5 0.53 Mortality 6 (23%) 6 (23%) 12 (40%) 0.27 Values are mean ± SD or number (percentage). p Values by one-way ANOVA, except for mortality where the chi-square method was used. SM-MSCs, skeletal muscle-derived mesenchymal stem cells; ADSCs, adipose tissue-derived stem cells. 1702 BEITNES ET AL. Figure 3. Characterization of the skeletal muscle (SM)-MSCs by flow cytometry. Histograms in one column represent results from each one of the three donors. Tracings represented by thick lines; no fill are from cells incubated with irrelevant control antibodies. Tracings represented by thin lines; gray fill are from cells incubated with antigen-specific antibodies. HLA, human leukocyte antigen. MESENCHy MAL STEM CELLS IN AMI 1703 Figure 4. Assays for osteogenic and adipogenic differentiation of SM-MSCs and adipose tissue-derived MSCs (ADSCs) performed after 4 weeks in differentiation medium. Quantitative RT-PCR for osteomodulin (OMD) (A) and peroxisome proliferator-activated receptor g (PPARG) (B) were performed on undifferentiated and differentiated cells from three donors (D1–D3) each for SM-MSC and ADSC (different donors for the two cell populations). All results presented are scaled relative to the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Staining assays (C) were for calcium deposition by Alizarin Red S to show osteogenic differ- entiation (top) and lipid droplets by Oil-Red O to show adipogenic differentiation (bottom) on cells from the same donors. The 200-μ m scale bar in the lower right image is representative for all images in the panel. 1704 BEITNES ET AL. Table 2. Left Ventricular Function Measured by Echocardiography SM-MSCs ADSCs Placebo (Baseline n = 26, (Baseline n = 26, (Baseline n = 30, Follow-up n = 20) Follow-up n = 20) Follow-up n = 18) p LVEDd (mm), baseline 7.58 ± 0.68 7.37 ± 0.52 7.29 ± 0.73 0.25 LVEDd (mm), follow-up 8.80 ± 0.80 8.53 ± 0.94 8.46 ± 0.79 0.43 LVEDd (mm), change 1.24 ± 0.88 1.22 ± 0.74 1.30 ± 0.89 0.96 LVESd (mm), baseline 6.41 ± 0.63 6.11 ± 0.65 6.08 ± 0.87 0.19 LVESd (mm), follow-up 7.23 ± 0.88 6.68 ± 1.24 7.06 ± 0.88 0.23 LVESd (mm), change 0.82 ± 0.80 0.68 ± 1.06 1.17 ± 0.96 0.27 FS (%), baseline 15.4 ± 3.2 17.4 ± 4.04 16.9 ± 4.9 0.22 FS (%), follow-up 18.1 ± 4.1 19.9 ± 6.4 16.7 ± 4.0 0.15 FS (%), change 2.8 ± 3.7 1.9 ± 4.1 -1.2 ± 4.9 0.01* LVEF (%), baseline 33.1 ± 7.3 35.6 ± 8.0 31.6 ± 10.4 0.24 LVEF (%), follow-up 39.1 ± 9.7 39.6 ± 6.8 31.0 ± 8.3 0.003 LVEF (%), change 4.9 ± 6.8 2.5 ± 7.7 -3.9 ± 7.3 0.002† WMSI, baseline 1.96 ± 0.17 1.90 ± 0.20 1.94 ± 0.22 0.47 WMSI, follow-up 1.82 ± 0.21 1.80 ± 0.20 1.89 ± 0.19 0.37 WMSI, change -0.12 ± 0.14 -0.06 ± 0.15 0.02 ± 0.27 0.09 Values are mean ± SD. p Value by ANOVA. LVEDd, left ventricular end-diastolic diameter; LVESd, left ventricular end-systolic diameter; FS, fractional shortening; LVEF, left ventricular ejection fraction; WMSI, wall motion score index. *p = 0.01 between SM-MSCs and placebo, p = 0.08 between ADSCs and placebo. †p = 0.002 between SM-MSCs and placebo, p = 0.03 between ADSCs and placebo. Echocardiography CD45, CD14, CD3, and CD19 prior to injection (Fig. 3) (7). Also, both cell populations from all donors showed LV dimensions and function were similar between robust differentiation toward adipogenic and osteogenic groups at baseline, with mean LVEF 33 ± 10% 1 week lineages (Fig. 4). For unknown reasons, the staining for after ligation of LAD indicating substantial myocar- mineralization following osteogenic differentiation of dial infarctions (Table 2). During 4 weeks of follow-up, SM-MSCs from donor 1 was only patchy, but the upregu- we observed significant remodeling with 1.3 ± 0.8 mm lation of osteomodulin following differentiation of these (p < 0.001) increase in left ventricular end-diastolic diam- cells was robust and similar to that observed for the other eter (LVEDd). LV systolic function as measured by FS SM-MSC donors (Fig. 4A). improved by 1.9 percentage points (%) in the ADSC- Figure 5. Left ventricle (LV) function by echocardiography. FS, fractional shortening; LVEF, left ventricular ejection fraction. *See Table 2 for details. MESENCHy MAL STEM CELLS IN AMI 1705 Table 3. Tissue Morphometrics Table 4. Vascular Density SM-MSCs ADSCs Placebo p Vascular SM-MSC ADSC Placebo Density (%) (n = 10) (n = 10) (n = 10) p Infarct size 16.3 ± 4.0 15.8 ± 4.9 26.0 ± 6.8 <0.001* (% of (n = 13) (n = 13) (n = 12) Scar tissue 2.3 ± 0.8 2.2 ± 0.8 1.4 ± 0.6 0.009* myocardium) Remote 9.3 ± 1.9 9.0 ± 2.1 9.3 ± 2.1 0.95 Granulation 18.3 ± 3.7 22.6 ± 6.0 13.1 ± 5.3 0.001† myocardium tissue Border zone 22.1 ± 3.1 20.8 ± 5.2 20.2 ± 2.3 0.50 (% of border (n = 12) (n = 12) (n = 10) Values are mean ± SD. zone) *p = 0.01 for difference between SM-MSCs and placebo, p = 0.03 for difference between ADSCs and placebo. For SM-MSCs versus ADSCs, Values are mean ± SD. p = n.s. *p < 0.001 between SM-MSCs and placebo, p < 0.001 between ADSCs and placebo, and p = 1.00 between SM-MSCs and ADSCs. †p = 0.02 between SM-MSCs and placebo, p < 0.001 between ADSCs and placebo, and p = 0.05 between SM-MSCs and ADSCs. the cell-treated groups was classified as granulation tis- sue. Vascular density (Table 4) was low in the collagen- ized scar tissue (overall 2.0 ± 0.8%), higher in the remote treated group ( p = 0.08 vs. placebo) and by 2.8% in the myocardium (9.2 ± 2.0%), and highest in the granulation SM-MSC group ( p = 0.01 vs. placebo), compared to a 1.2% tissue (21.0 ± 3.7%), with p < 0.001 for difference in vas- decrease in the placebo group (Table 2, Fig. 5). LVEF cular density between tissues. Vascular density in the scar increased 2.5% in the ADSC group ( p = 0.03 vs. placebo) tissue was significantly higher in the groups receiving and 4.9% in the SM-MSC group ( p = 0.002 vs. placebo), SM-MSCs or ADSCs as compared to the control group. compared to a 3.9% decrease in the placebo group. There There was no significant difference between the three was also a trend ( p = 0.09) suggesting improved WMSI in groups for vascular density in the granulation tissue or the groups receiving cell therapy. The relative improve- within the remote myocardium. ment in LV function tended to be better in the SM-MSC In Situ Hybridization and Immunohistochemistry group compared to the ADSC group, but the difference In the transplanted hearts, a small number of Alu- did not reach statistical significance. positive human cells were retrieved in clusters, mainly in Morphometry the peripheral region of the infarct zone and in the border Infarct size at the end of study (Table 3) was sig- zone (Fig. 6). No donor-derived cells were found within nificantly lower in the SM-MSC group (16.3%) and the the preserved myocardium. The Alu-positive cells did not ADSC group (15.8%) compared to the placebo group stain positive for SMA, desmin, CD31, TnI, and Nkx (26.0%), indicating significant effect of cell therapy 2.5, indicating that differentiation to cardiomyocyte, ( p < 0.001 vs. placebo for both groups). Within the bor- smooth muscle cell, or endothelial cell phenotypes had der zone, a significantly higher proportion of the tissue in not occurred. Figure 6. Alu sequence-positive cells by fluorescence in situ hybridization (FISH). (A) Cluster of Alu-positive cells in the infarct zone. Blue represents nuclei stained with DAPI. Green and blue merged (bright green) represents transplanted cells stained with DAPI and Alu probe. Magnification: ×200. (B) Same as in (A). Magnification: ×600. 1706 BEITNES ET AL. DISCUSSION also seems to have been inhibited. Such a scenario would lead to the survival of more contractile myocardial tissue, In this study, intramyocardial injection of both ADSCs to a larger amount of persisting granulation tissue, and and SM-MSCs 1 week after AMI led to a substantial to smaller areas of scar tissue in the cell-treated hearts. decrease in infarct size and a significant improvement in This line of events would explain our observations. LVEF compared with injections of cell culture medium Some of the molecules known to be secreted by MSCs only. The proportion of granulation tissue within the bor- that might contribute in this model are prostaglandin E2, der zone was significantly increased in both treatment known to inhibit fibrosis in other systems (32), and the groups. There was a trend for better functional improve- anti-inflammatory factor tumor necrosis factor (TNF)-a- ments in the SM-MSC-treated group compared to the induced protein 6, which is thought to mediate the effect ADSC group, but this did not reach significance. A small on myocardial infarction induced by intravenous injection number of transplanted cells were still present after 4 of MSCs (17). One observation that might speak against weeks, but they did not express markers of differentiation our hypothesis is the small number of Alu-positive cells toward cardiomyocyte, endothelial, or smooth muscle noted in the rat hearts after 4 weeks. Functional and mor- lineages. phometric differences on the scale observed in the current Our study confirms the favorable effect of MSC injec- study would intuitively seem to require a large number of tions after AMI reported by other groups (9,19,20,27). surviving cells. However, similar effects on LV function We also show that the reason why the cell-treated animals was reported in the study by Imanishi et al., although vir- maintained greater myocardial contractile power was tually all the transplanted cells were lost within 4 weeks that, following induction of the same-size anterior wall (15). One possible explanation to this observation could myocardial infarctions, the MSC-treated rats developed a be that a much larger number had survived initially and much smaller scar area at 4 weeks than the placebo-treated that these cells gradually succumbed during the following animals. In fact, the difference in infarct size between the period, as indicated in cell-tracking studies (15,34,36). If cell-treated and placebo-treated rats was even higher than so, enough cells may have been present to secrete bio- expected from the differences in LV function. Our data active compounds for a sufficient period of time to affect suggest that this may in part be explained by a larger the surrounding tissues. This hypothesis is supported by amount of granulation tissue in the border zone in groups the observation that, when MSC survival was enhanced receiving MSCs. The granulation tissue was immature, as by the transduction of the anti-apoptotic akt gene, the the content of fibrous elements was low. Cardiomyocytes effects of intramyocardial injection of MSCs on cardiac were not observed in the granulation tissue. The granu- function and infarct size were also enhanced (11). Several lation tissue was not included in the parameter “infarct factors may influence the number of cells observed. size,” as it stained Masson Goldener negative, but we The cells must be handled properly before injection, as believe that this tissue would eventually have turned into detachment from the plastic surface rapidly changes the fibrous scar tissue. Even so, the injection of human MSCs MSC morphology and may trigger anoikis. As the heart is must have led to a considerable reduction in the size of small and contracting at 3–400 beats/min, some cells are the infarct. probably also lost by myocardial perforation and endo- As could be predicted, the density of small blood ves- ventricular injections. Also, the intramural pressure during sels in the granulation tissue was much higher than that systole is high, which may facilitate extrusion of injected observed for the healthy myocardium, with no difference cells (34). Although MSCs are immunoprivileged, allo- observed between the granulation tissue in the three groups. immune responses may influence cell survival. Grinnemo However, the amount of granulation tissue was signifi- et al. reported lymphocyte infiltration and rapid loss of cantly higher in the borderzone in cell-treated hearts. The cells after MSC therapy in nude rats, suggesting allo- MSCs were injected 1 week after induction of the myo- immune rejection (12). Aggregation of lymphocytes cardial infarction, at a time when highly vascular granu- was not observed in proximity to Alu-positive cells in lation tissue had presumably already been established in our study, and cell survival may have been favored by parts of the infarct zone. Within and also bordering this the use of young animals, as nude rats improve cellular granulation tissue, there may have been cardiomyocytes immune competence with age. However, we believe that affected by ischemia that were not yet dead and endog- our observation of surviving cells at 4 weeks is reliable. enous cardiac progenitor cells that might have promoted Membrane markers used to identify human cells, like DiI cardiac regeneration. MSCs are known to secrete a large or PKH 26, may persist in the tissue after the cells have number of bioactive compounds. Some of these may have died because membrane fragments may be phagocytosed acted on ischemic cardiomyocytes to promote their sur- by cells in the vicinity or fragments of the stained mem- vival and on progenitor cells to stimulate cell division and brane may fuse with the membrane of neighboring cells. differentiation. In the process, the formation of scar tissue Similarly, following transplantation of cells transfected MESENCHy MAL STEM CELLS IN AMI 1707 with green fluorescence protein, the fluorescent proteins Re Fe Re NCe S may leak from the transfected cells to be taken up by 1. Amado, L. C.; Saliaris, A. P.; Schuleri, K. H.; St John, M.; nearby host cells, which may then give a false impression Xie, J. S.; Cattaneo, S.; Durand, D. J.; Fitton, T.; Kuang, J. Q.; Stewart, G.; Lehrke, S.; Baumgartner, W. W.; Martin, of surviving cells long after the transplanted cells have B. J.; Heldman, A. W.; Hare, J. M. Cardiac repair with actually died. In contrast, no study has to date shown a intramyocardial injection of allogeneic mesenchymal stem false-positive signal for surviving cells using the human- cells after myocardial infarction. Proc. Natl. Acad. Sci. specific Alu probe employed in this study. USA 102:11474–11479; 2005. Based on very early studies of transplantation of cells 2. Arminan, A.; Gandia, C.; Bartual, C.; Garcia-Verdugo, J. M.; Lledo, E.; Mirabet, V.; Llop, M.; Barea, J.; Montero, between organs, the hypothesis has been advocated that J. A.; Sepulveda, P. Cardiac differentiation is driven by committed cells from one organ might transdifferentiate NKX2.5 and GATA4 nuclear translocation in tissue spe- to a phenotype typical of another, when placed into the cific mesenchymal stem cells. Stem Cells Dev. 18:907– microenvironment of that organ, particularly in the con- 918; 2008. text of organ damage (10,14). We did not see evidence 3. Au, P.; Tam, J.; Fukumura, D.; Jain, R. K. Bone marrow derived mesenchymal stem cells facilitate engineering of long-lasting that the transplanted MSCs transdifferentiated to become functional vasculature. Blood 111:4551–4558; 2008. human-derived cardiomyocytes or other cardiac cells 4. Bartunek, J.; Croissant, J. D.; Wijns, W.; Gofflot, S.; de that might have contributed to the functional and tissue Lavareille, A.; Vanderheyden, M.; Kaluzhny, y .; Mazouz, structural effects observed here. Only a limited number N.; Willemsen, P.; Penicka, M.; Mathieu, M.; Homsy, of studies have reported post-transplant expression of C.; De Bruyne, B.; McEntee, K.; Lee, I. W.; Heyndrickx, G. R. Pretreatment of adult bone marrow mesenchymal cardiomyocyte markers, CD 31 or SMA in vivo. The ex - stem cells with cardiomyogenic growth factors and repair tent of cell engraftment and survival has been low, and of the chronically infarcted myocardium. Am. J. Physiol. only a small fraction of engrafted cells have differenti- Heart Circ. Physiol. 292:H1095–H1104; 2007. ated toward other lineages (23,30,33). Thus, although we 5. Beltrami, A. P.; Barlucchi, L.; Torella, D.; Baker, M.; cannot exclude that transdifferentiation may take place in Limana, F.; Chimenti, S.; Kasahara, H.; Rota, M.; Musso, E.; Urbanek, K.; Leri, A.; Kajstura, J.; Nadal-Ginard, B.; rare individual cells or after longer observation periods, Anversa, P. 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Journal

Cell Transplantation – SAGE

Published: Aug 1, 2012

Keywords: Cell therapy; Mesenchymal stem cells (MSCs); In situ hybridization; Immunofluorescence; Echocardiography; Animal model

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