Biomaterials 35 (2014) 8528e8539

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Magnetic targeting of cardiosphere-derived stem cells with ferumoxytol nanoparticles for treating rats with myocardial infarction Adam C. Vandergriff a, b, 1, Taylor M. Hensley a, 1, Eric T. Henry a, b, 1, Deliang Shen c, Shirena Anthony d, Jinying Zhang c, Ke Cheng a, b, e, * a

Department of Molecular Biomedical Sciences and Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, NC, USA c Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China d Department of Biology, North Carolina State University, NC, USA e The Cyrus Tang Hematology Center, Soochow University, Suzhou, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 May 2014 Accepted 15 June 2014 Available online 16 July 2014

Stem cell transplantation is a promising therapeutic strategy for acute or chronic ischemic cardiomyopathy. A major limitation to efficacy in cell transplantation is the low efficiency of retention and engraftment, due at least in part to significant early “wash-out” of cells from coronary blood flow and heart contraction. We sought to enhance cell retention and engraftment by magnetic targeting. Human cardiosphere-derived stem cells (hCDCs) were labeled with FDA-approved ferumoxytol nanoparticles Feraheme® (F) in the presence of heparin (H) and protamine (P). FHP labeling is nontoxic to hCDCs. FHPlabeled rat CDCs (FHP-rCDCs) were intracoronarily infused into syngeneic rats, with and without magnetic targeting. Magnetic resonance imaging, fluorescence imaging, and quantitative PCR revealed magnetic targeting increased cardiac retention of transplanted FHP-rCDCs. Neither infusion of FHP-rCDCs nor magnetic targeting exacerbated cardiac inflammation or caused iron overload. The augmentation of acute cell retention translated into more attenuated left ventricular remodeling and greater therapeutic benefit (ejection fraction) 3 weeks after treatment. Histology revealed enhanced cell engraftment and angiogenesis in hearts from the magnetic targeting group. FHP labeling is safe to cardiac stem cells and facilitates magnetically-targeted stem cell delivery into the heart which leads to augmented cell engraftment and therapeutic benefit. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Cardiac stem cells Magnetic targeting Myocardial infarction Ferumoxytol MRI Superparamagnetic iron oxide nanoparticles

1. Introduction Most families in the United States are impacted by cardiovascular disease. Cardiovascular disease remains the leading cause of death and disability in Americans. On average, cardiovascular disease kills one American every 37 s. Death rates have improved, but new treatments are urgently needed. Stem cell transplantation is a promising therapeutic strategy for acute or chronic ischemic cardiomyopathy [1]. A major limitation in cell transplantation is the low efficiency of retention and engraftment [2]. Acute (75% confluence. FHP nanocomplexes dissolved in serum-free media were added to the cells in culture and incubated for 12 h. Following incubation in serum free media, an equal amount of complete media containing 20% Fetal Bovine Serum (FBS) was then added and the cells were incubated overnight for each cell type. Cells were washed with Phosphate-Buffered Saline (PBS) (Invitrogen) to remove the residual FHP particles. For brevity, the FHP nanocomplex-labeled human CDCs and rat CDCs were hereafter referred as FHPhCDCs and FHP-rCDCs, respectively. 2.3. Prussian blue staining For Prussian blue staining [4], cells or tissue sections were fixed in 2% glutaraldehyde for 10 min at 4  C, and then immersed in 1% potassium ferrocyanide and 3% HCl solution (Sigma) for 30 min. After several washes with D.I. water, cell nuclei were counter-stained with nuclear fast red (Sigma) for 5 min. Then the cells were washed again with D.I. water twice. After dehydration with methanol (3 washes, 70% once and 100% twice) and xylene, the slides were finally mounted in DPX mounting media (Sigma) before observation. 2.4. Transmission electron microscopy In preparation for TEM, cells were spun down, media removed, and resuspended in 4% formaldehyde and 1% gluteraldehyde fixative (4F:1G) and stored at 4  C. At a later date, the samples were stained following the procedure in Ref. [13]. Some samples were not post-stained to allow for easier detection of FHP nanoparticles. Sections of cell samples were imaged using a FEI/Philips EM 208S/Morgagni transmission electron microscope at the NCSU Laboratory for Advanced Electron and Light Optical Methods (LAELOM). 2.5. In vitro cytotoxicity assay In vitro cytotoxicity assays were performed using FHP-hCDCs and control (nonlabeled) hCDCs. Cell viability was assessed by Trypan blue exclusion. To evaluate apoptosis, cells were fixed, and apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL assay using the In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. The proliferation rates of FHP-CDCs and control CDCs were assessed with by Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc., Rockville, MD), and cell cycling was evaluated by immunocytochemistry

staining of Ki67 (cell proliferation marker). Cell migration was evaluated with a trans-well cell migration assay plate (BD Biosciences) [14]. Reactive oxygen species (ROS) generation was measured with an Image-IT® LIVE Green Reactive Oxygen Species Detection Kit (Life Technologies) [4]. As positive controls for the TUNEL and ROS assay, human CDCs were incubated with 100 mM H2O2 in the medium for 24 h to induce cell apoptosis and ROS generation. 2.6. Microarray gene expression analysis

2.8. Finite element analysis of the NdFeB magnet The magnetic field simulation was performed using the open-source software Finite Element Method Magnetics (http://www.femm.info/wiki/HomePage). Finite element analysis was performed on the 1.3 T NbFeB magnet (Edmund Scientifics, Tonawanda, NY; diameter ¼ 9.5 mm; height ¼ 5 mm) to be used in subsequent rat studies. A density plot of the field lines was produced to show the shape of the field. The field magnitude along the z-axis directly above the magnet was calculated as well to show the decay of the field with distance. 2.9. Rat model of ischemia/reperfusion Animal care was in accordance to the Institutional Animal Care and Use Committee (IACUC) guidelines. A rat ischemia/reperfusion model was used [5]. Female WKY rats (Charles River Laboratories) (n ¼ 58 total) underwent left thoracotomy in the 4th intercostal space under general anesthesia. The heart was exposed and myocardial infarction was produced by 45 min ligation of the left anterior descending coronary artery, using a 7-0 silk suture. The suture was then released to allow coronary reperfusion. Intracoronary injection was achieved by injection into the left ventricle cavity during a 25 s temporary aorta occlusion with a looped suture. Animals were randomized into three treatment groups: 1) Control, intracoronary injection of 200 mL PBS; 2) FHP-CDC, intracoronary injection of 500,000 FHP-labeled syngeneic CDCs in 200 mL PBS; 3) FHP-CDC þ Magnet, intracoronary injection of 500,000 FHP-labeled syngeneic CDCs in 200 mL PBS with a superimposed 1.3 T circular NdFeB magnet during and after the cell injection for 10 min [4]. The chest was closed and the animal was allowed to recover after all procedures. 2.10. Magnetic resonance imaging (MRI) 24 h after injection, magnetic resonance imaging (MRI) of the heart was accomplished by a gradient echo sequence to produce a T2* weighting with a Bruker Biospec 9.4T small animal MRI system. A 60 mm diameter linear transmit/receive coil was utilized for excitation and a 4 channel array coil designed for rat heart imaging was used for improved signal-to-noise data acquisition. Retrospective gating was accomplished with Bruker IntraGate® software to produce 10 cine images for each slice. In addition, one coronal cine image was acquired which dissected the heart to verify the presence of iron particles. 2.11. Fluorescence imaging (FLI) 24 h after cell injection, the hearts and lungs were excised, washed with PBS, and placed in a Xenogen IVIS imaging system (Caliper Life Sciences, Mountain View, CA) to detect RFP fluorescence. Excitation was set at 550 nm and emission was set at 580 nm. Exposure time was set at 5 s and kept the same during the entire imaging session.

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2.12. Quantification of engraftment by real-time PCR Quantitative PCR was performed 24 h after cell injection in 5 animals from each cell-injected group to quantify cell retention. We injected CDCs from male donor WK rats into the myocardium of female recipients to utilize the detection of SRY gene located on the Y chromosome. The whole heart was harvested, weighed, and homogenized. Genomic DNA was isolated from aliquots of the homogenate corresponding to 12.5 mg of myocardial tissue, using commercial kits (DNA Easy Minikit, Qiagen). The TaqMan® assay (Applied Biosystems, Carlsbad, CA) was used to quantify the number of transplanted cells with the rat SRY gene as template (forward primer: 5'-GGA GAG AGG CAC AAG TTG GC-3', reverse primer: 5'-TCC CAG CTG CTT GCT GAT C-3', TaqMan probe: 6FAM CAA CAG AAT CCC AGC ATG CAG AAT TCA G TAMRA; Applied Biosystems). A standard curve was generated with multiple dilutions of genomic DNA isolated from male hearts. All samples were spiked with equal amounts of female genomic DNA as control. The copy number of the SRY gene at each point of the standard curve was calculated with the amount of DNA in each sample and the mass of the rat genome per cell. For each PCR reaction, 50 ng of template DNA was used. Real-time PCR was performed with a real-time PCR System (Applied Biosystems). Cell numbers per mg of heart tissue and percentages of retained cells of the total injected cells were calculated. 2.13. ELISA for cardiac troponin-I, transferrin and ferritin All assays were run according to vendor's protocol (Rat Cardiac Troponin-I ELISA, Life Diagnostics, cat no 2010-2-HS; Rat Transferrin and Rat Ferritin, Immunology Consultants Laboratory Inc, cat# E-25TX and E-25F). Serum samples were assayed undiluted for the cTnI ELISA. Serum was diluted 1:40,000 for Transferrin and 1:40 for Ferritin analysis using the provided sample buffer. The absorbance was measured at 450 nm at the assay endpoint and the values of all analytes were expressed in nanograms per milliliter. 2.14. Morphometric heart analysis For morphometric analysis, animals were euthanized at 3 weeks (after cardiac function assessment) and the hearts were harvested and frozen in OCT compound.

Sections every 100 mm (8 mm thickness) were prepared. Masson's trichrome staining was performed as described by the manufacturer's instructions (HT15 Trichrome Staining Kit; Sigma). Images were acquired with a PathScan Enabler IV slide scanner (Advanced Imaging Concepts, Princeton, NJ). From the Masson's trichrome-stained images, morphometric parameters including viable myocardium and scar size were measured in each section with NIH ImageJ software. The percentage of viable myocardium as a fraction of the scar area (risk region) was quantified as described [4]. 2.15. Cardiac function assessment Rats underwent echocardiography 24 h (baseline) and 3 weeks post-MI using Vevo™ Imaging System (VisualSonics, Toronto, Canada). Hearts were imaged twodimensionally in long-axis views at the level of the greatest left ventricular (LV) diameter. LV ejection fraction (LVEF) was measured with VisualSonics V1.3.8 software from views taken through the infarcted area. 2.16. Histology The animals were euthanized and the hearts were harvested and frozen in OCT compound. Sections every 100 mm of the infarct and infarct border zone area (8 mm thickness) were prepared and immunocytochemistry was performed using the following primary antibodies: rabbit anti-RFP (Abcam, Cambridge, MA, USA), mouse anti-CD68 (Abcam), and rabbit anti-von Willebrand factor (vWF; Abcam). Secondary antibodies were also purchased from Abcam. Images were taken by a Zeiss confocal microscopy system. 2.17. Statistical analysis Results are presented as mean ± SD unless specified otherwise. All the comparisons between any 2 groups were performed using 2-tailed unpaired Student's t test. Comparison among more than 2 groups was analyzed by One-Way ANOVA followed by Bonferroni post-hoc test. Differences were considered statistically significant when p < 0.05.

Fig. 1. Ferumoxytol labeling of CDCs. A: Representative Prussian blue staining images showing human CDCs incubated with Feraheme® alone, Feraheme þ Protamine, and Feraheme þ Heparin þ Protamine. Cells were fixed, stained for Prussian blue (iron) and counter-stained with nuclear red. Non-labeled human CDCs were used as Control. Scale Bars ¼ 100 mm. B: Quantitation of Prussian blue-positive cells (n ¼ 3). The Feraheme þ Heparin þ Protamine method was employed for all labeling studies afterward and the labeled CDCs were referred as FHPeCDC for brevity. C: Appearance of FHPeCDCs after centrifugation and magnet attraction. D: TEM images showing the intracellular uptake of Feraheme in CDCs. * indicates p < 0.05 when compared to the Fh group.

A.C. Vandergriff et al. / Biomaterials 35 (2014) 8528e8539 Fig. 2. The effects of ferumoxytol labeling on cell viability, apoptosis, proliferation, cycling, migration, ROS generation, and gene expression. A: Cell viability assessed by Trypan blue staining. B: Confocal images showing cell apoptosis (red nuclei) evaluated by TUNEL assay. C: Cell proliferation measured with CCK-8 assay. D: Expression of cell cycling marker Ki67 in FHPeCDCs and control cells. E: Cell migration analyzed by trans-well migration assay. F: Confocal images showing ROS generation (green). G: Volcano plot of the microarray analysis. Red points indicate genes that exceeded both thresholds and were considered to have changed significantly. Blue points indicate genes that failed to exceed both thresholds. All experiments were run in triplicates. Bars ¼ 50 mm * indicates p < 0.05 when compared to the CDC group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3. Results 3.1. Labeling of human CDCs with FHP nanoparticles Prussian blue staining confirmed excellent particle uptake using the FHP method (Fig. 1A, “Fh þ Hep þ Pro” panel). Fh alone or Fh þ Pro combination did not label the cells well (Fig. 1A, “Fh” & “Fh þ Pro” panels). Non-labeled control cells did not exhibit Prussian blue staining (Fig. 1A, “Control” panel). Quantitation of Prussian blue-positive cells confirmed superior labeling efficiency using the Fh-Hep-Pro labeling strategy. FHPeCDCs exhibited brownish color after centrifugation as compared to Control (non-labeled) cells and formed a cell condensation when a magnet was placed on the tube wall (Fig. 1C). Transmission electron microscopy (TEM) revealed intracellular uptake of iron particles (Fig. 1D) in the lysosomal compartment of CDCs. 3.2. Cytotoxicity of FHP labeling Since our ultimate goal is to apply FHP-labeled cell therapy in human beings, we conducted our cytotoxicity study using hCDCs. FHP-hCDCs were indistinguishable with Control CDCs in cell viability (Fig. 2A), cell apoptosis (Fig. 2B), proliferation (Fig. 2C), expression of cell cycling marker Ki67 (Fig. 2D), cell migration (Fig. 2E) and ROS generation (Fig. 2F). Microarray gene analysis reviewed that out of the 27382 genes analyzed with the Human Affymetrix gene chip, only 35 genes showed significant changes in expression based on a criteria of fold change 2 (or  2) and pvalue  0.05 (Fig. 2G, Table 1). In addition, the cardiac (Fig. 3A) and endothelial (Fig. 3B) differentiation capacities of CDCs were not affected by FHP labeling. These compound results suggest FHP labeling is nontoxic to hCDCs. 3.3. Acute cell retention by magnetic targeting The distribution of magnetic flux density created by the NdFeB magnet was created by finite element analysis (Fig. 4A). The field magnitude along the z-axis directly above the magnet was calculated as well to show the decay of the field with distance (Fig. 4B). Syngeneic WKY male rat CDCs were labeled with FHP nanoparticles (Fig. 5A). We first tested the safety of a previously determined intracoronary cell dose in naïve animals (animals without myocardial infarction) [5]. Intracoronary infusion of 500,000 FHPrCDCs with or without magnetic targeting did not cause microembolic damage, as the cardiac troponin levels for all three groups are indistinguishable (Fig. 5B). We then went ahead to create a rat myocardial infarction model. Syngeneic WKY female rats underwent ischemia-reperfusion injury and received intracoronary infusion of 500,000 FHP-rCDCs, with or without magnetic targeting. We used both semi-quantitative (MRI and FLI) and quantitative (qPCR) methods to assess acute cell retention. Cardiac MRI at 24 h (Fig. 5C) revealed larger signal hypointensity area (indicating the presence of iron) in the FHP-rCDC þ mag group than that from the FHP-rCDC group, suggesting superior cell retention by magnetic targeting. Since MRI signal can produce false positive signal of cell engraftment [15], a subgroup of FHP-rCDCs were also transduced with RFP lentiviral particles to enable FLI and histological detention of engrafted cells. FLI at 24 h confirmed the MRI results: magnetic targeting increased cell retention in the heart and reduced off-target cell retention in the lungs (Fig. 5D). As a more precise mean of measuring cell retention, we transplanted male rat FHP-rCDCs into female recipient hearts to enable the use of Y chromosome-specific PCR to quantify the exact numbers of cells in the heart. Quantitative PCR revealed the FHP-rCDC þ magnet group exhibited >3-fold greater cell retention rates than the FHP-rCDC group (Fig. 5E).

Table 1 Differentially expressed genes in human CDCs labeled with FHP labeling. Gene name

Description

Linear fold change

t-Test p-value

ANXA10 ASNS

Annexin A10 Asparagine synthetase (glutaminehydrolyzing) UDP-Gal:betaGlcNAc beta 1,3galactosyltransferase, polypeptide 2 Chemokine (CeC motif) ligand 11 Cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) ChaC, cation transport regulator homolog 1 (Escherichia coli) Cytidine monophospho-Nacetylneuraminic acid hydroxylase, pseudogene Chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) Cytochrome P450, family 1, subfamily B, polypeptide 1 ets homologous factor Gap junction protein, alpha 5, 40 kDa Insulin-like growth factor binding protein 5 Integrin alpha 11 Kynureninase Uncharacterized LOC100505633 Uncharacterized LOC284561 Microfibrillarassociated protein 4 Membrane metalloendopeptidase MicroRNA 361 Nicotinamide

3.09 2.18

9.30E-05 0.000213

B3GALT2

CCL11 CDKN2B

CHAC1

CMAHP

CXCL6

CYP1B1

EHF GJA5 IGFBP5 ITGA11 KYNU LOC100505633 LOC284561 MFAP4 MME MIR361 NAMPT

2.07

0.00391

2.56

0.002098

2.3

1.90E-05

3.54

2.20E-05

2.06

1.21E-07

2.08

0.00165

2.02

0.00019

2.02 2.77

0.000928 0.001156

2.53

5.10E-05

2.7 2.95 2.02

5.00E-05 0.002661 6.00E-04

2.66

0.000375

2.69

2.00E-06

2.02

0.000106

2.15

0.004454

phosphoribosyltransferase, nicotinamide phosphoribosyltransferase-like 2.030.000438NEFMNeurofilament, medium polypeptide 2.030.000518NPR3Natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C) 2.470.002248PCK2Phosphoenolpyruvate carboxykinase 2 (mitochondrial) 2.460.00081PDGFRBPlatelet-derived growth factor receptor, beta polypeptide 2.062.50E-05PHGDHPhosphoglycerate dehydrogenase 2.120.000458PSAT1Phosphoserine aminotransferase 1 2.991.80E-05RAB27BRAB27B, member RAS oncogene family 3.57.00E-06RGCCRegulator of cell cycle 2.080.004473SCUBE3Signal peptide, CUB domain, EGF-like 3 2.180.000468SERPINB2Serpin peptidase inhibitor, clade B (ovalbumin), member 2, serpin peptidase inhibitor, clade B (ovalbumin), member 10 3.570.000575SOD2Superoxide dismutase 2, mitochondrial, uncharacterized LOC100129518 2.10.000512SULT1E1Sulfotransferase family 1E, estrogen-preferring, member 1 2.270.000181TNFSF4Tumor necrosis factor (ligand) superfamily, member 4 2.610.000765UNC5Bunc-5 homolog B (Caenorhabditis elegans) 2.030.000746VCAM1Vascular cell adhesion molecule 1 2.950.000376

3.4. Cardiac inflammation and iron overload 3 days after treatment, a subpopulation of rats in each group was sacrificed for histological analyses of cell engraftment and cardiac tissue density of macrophages (as an inflammatory indicator). In two neighboring tissue sections, Prussian blue staining

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Fig. 3. The effects of ferumoxytol labeling on cardiovascular differentiation. A: Representative confocal images showing differentiation of CD105POS CDCs (green) into alphaSAPOS cardiomyocytes (red), and a bar graph showing the percentage of alpha-SAPOS CDCs. B: : Representative confocal images showing differentiation of CD105POS CDCs (green) into vWRPOS endothelial cells (red), and a bar graph showing the percentage of vWRPOS CDCs. All experiments were run in triplicates. Scale bars ¼ 10 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Fig. 6A, blue spots circled with black) matched well with RFP signal (Fig. 6B, red spots circled with white), indicating iron staining may predict cell engraftment at least 72 h post-transplantation into the heart. Moreover, CD68POS macrophages (green) did not cluster in or sounded the injected FHP-CDCs (Fig. 6B), suggesting FHP particles did not provoke severe inflammatory response. Consistent with cell retention data at 24 h (Fig. 5), more RFPPOS cells were engrafted in the magnetic targeting group at 72 h post-transplantation (Fig. 6C). The overall tissue density of CD68POS macrophages was similar in all three groups including controls (Fig. 6D), further verifying that

FHP labeling and/or magnetic targeting did not induce or worsen inflammation in the injured heart. To exclude systematic iron overload caused by FHP-CDCs, serum ferritin and transferrin levels were measured at day 3; these were comparable to controls in both FHPeCDC-treated groups (Fig. 6E and F). 3.5. Structural and functional benefits from magnetic targeting Heart morphometry at 3 weeks showed severe LV chamber dilatation and infarct wall thinning in the Control group (PBS-

Fig. 4. Finite element analysis of magnetic field. A: A density plot showing the shape of the magnetic field generated by the 1.3 T NbFeB magnet. B: The field magnitude decay of with distance along the z-axis directly above the magnet.

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Fig. 5. Magnetic targeting boosts the retention of ferumoxytol-labeled CDCs in rats with ischemia/reperfusion. A: FHP-labeled rat CDCs. B: serum TnI values measured by ELISA 24 h after intracoronary injection of 500,000 cells into naïve (non-infarcted) rats. C: T2* weighted MRI images showing cell retention 24 h after delivery of CDCs into rats after myocardial infarction. The presence of iron created signal void (dark area; pointed with red arrows). D: Fluorescent imaging the presence of RFP-labeled CDCs (red) in hearts and lungs. E: SRY qPCR quantitation of cell retention. All experiments were run in triplicates. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

infused) hearts (Fig. 7A). In contrast, the two cell-treated groups exhibited attenuated LV remodeling and better heart morphology. The protective effect was greatest in the FHPeCDC þ magnet group, which had more viable myocardium in the risk region (Fig. 7B) and smaller scar sizes (Fig. 7C) than the FHPeCDC group. To investigate whether improved cell engraftment translated to better functional outcomes, left ventricle ejection fraction (LVEF) was assessed by echocardiography at baseline (24 h after I/R and treatment) and 3 weeks afterward. LVEFs at baseline did not differ between treatment groups, indicating a comparable degree of initial injury (Fig. 7D). Over the next 3 weeks, LVEF declined progressively in the control group, but not in the FHPeCDC-treated animals. Notably, the FHPeCDC þ magnet group exhibited better therapeutic outcome, with LVEF superior to the FHPeCDC group (p < 0.05) at 3 weeks. The boost in cell retention/engraftment does translate into better heart morphology and greater functional benefit.

3.6. Cell engraftment and angiogenesis All animals were sacrificed at 3 weeks after the echocardiography session and hearts were excised and cryosectioned for histological analysis of cell engraftment and capillary density. A greater number of RFPPOS cells were evident in the FHPeCDC þ magnet group than in the FHP-CDC group, suggesting the superior cell engraftment boosted by magnetic targeting persisted at least into 3 weeks after transplantation (Fig. 8). Mounting evidence has shown that indirect regeneration plays a crucial role in cell therapy, as oppose to direct regeneration (cell engraftment and differentiation into mature cardiovascular cells) [16]. It has been reported that one of the major indirect regenerative effects from CDCs is stimulation of angiogenesis, which can be evaluated by counting vWFPOS capillary structures in the heart [16]. Infusion of FHP-CDCs promoted angiogenesis (as compared to Control) while the greatest capillary density was seen from the FHPeCDC þ magnet group (Fig. 9).

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Fig. 6. In vivo toxicity of FHPeCDCs in post-MI rat hearts. A: Representative Prussian blue staining of rat heart sections 3 days after injection of FHPeCDCs. Iron was shown in blue and all nuclei were shown in red. B: A confocal image of the neighboring tissue section of Panel A, showing engrafted CDCs (red) and macrophages (green). “L” denoted a blood vessel lumen as a reference point. C & D: Quantitation of RFPPOS injected FHPeCDCs and CD68POS macrophages. E & F: ELISA measurement of serum transferrin and ferritin. n ¼ 3 animals per group. Scale bars ¼ 200 mm * indicates p < 0.05 when compared to the FHPeCDC group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Discussion We are in an era of explosion in pre-clinical and clinical studies on stem cell therapies for cardiac regeneration [17]. To deliver stem cells to the heart, intravascular routes such as intracoronary infusion have been proved to be safe in numerous clinical trials to deliver stem cells into the infarct area [11]. However, a significant disadvantage of intracoronary cell infusion is the extremely low cell retention rate, due at least in part to a strong wash-out effect from cardiac contraction and venous drainage [2]. We and other groups have shown that magnetic targeting can effectively increase shortterm cell retention and long-term cell engraftment and functional benefit. In order to facilitate magnetic targeting, cells usually need

to be labeled with clinical-grade SPIONs. However, ferumoxides (Feridex®) and similar SPIONs were removed from the market thus, halting the progress towards translating this approach to label and track cells by MRI for clinical trials. Recently, ferumoxytol (Fareheme®), a semi-synthetic carbohydrate non-dextran-coated ultrasmall SPION (USPION), has been approved for the treatment of iron deficiency anemia in chronic kidney disease [18]. Ferumoxytol is also under experimental and clinical investigations as a MRI contrast agent [19]. Ferumoxytol alone or in combination with protamine does not effectively label cells very well [10]. A recent study has shown the combination of three FDA-approved drugs heparin, protamine and ferumoxytol led to great cell-labeling efficiency [10]. Furthermore, MRI revealed dynamic in vivo

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Fig. 7. Morphometric heart analysis and cardiac function. A: Representative Masson's trichrome-stained myocardial sections 3 weeks after treatment. Scar tissue and viable myocardium are identified by blue and red color, respectively. B & C: Quantitative analysis of viable myocardium and scar size from the Masson's trichrome-stained heart sections (n ¼ 5 animals per group). D & E: Left ventricular ejection fraction (LVEF) measured by echocardiography at baseline and 3 weeks after cell injection (n ¼ 7 animals per group). * indicates p < 0.05 when compared to Control. ** indicates p < 0.05 when compared to any other groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

distribution of ferumoxytol-labeled neural stem cells correlated with histological analysis, suggesting the clinical use of ferumoxytol labeling of cells for post-transplant MRI visualization and tracking [20]. In the present study, we used ferumoxytol to label heart stem cells and then apply magnetically-targeted cell therapy in a rat model of myocardial infarction. The goal is to see whether ferumoxytol labeling can facilitate magnetically-targeted intracoronary delivery of heart stem cells and enhance the therapeutic benefit of cell transplantation while causing no toxicity. Our lab has been studying cardiosphere-derived cells (CDCs) for its cardiac regeneration potency in both pre-clinical animal models and a recently completed CADUCEUS trial [11]. Because our ultimate goal is to translate the idea into the clinic, we performed

in vitro cytotoxicity testing in human CDCs. Human CDCs can be efficiently labeled with Feraheme using the FHP method (Fig. 1) and such labeling is nontoxic to the cells (Figs. 2 and 3). We then switched to FHP-labeled rat CDCs and tested the safety and efficacy of magnetic targeting in a rat model of ischemia/reperfusion. Magnetic targeting boosted acute cell retention, as indicated by MRI, FLI, and PCR (Fig. 4). We have reported that magneticallytargeted intracoronary delivery of 500,000 iron-labeled CDCs into a rat heart is safe while the dose of 1,000,000 cells caused microembolic injury [5]. A recent study from Huang et al. also reported that magnetically-targeted intracoronary delivery of 1,000,000 mesenchymal stem cells (MSCs) caused troponin leakage and had no functional benefit in rats with ischemia-reperfusion injury [7].

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Fig. 8. Long-term cell engraftment enhanced by magnetic targeting. AeC: Representative fluorescent micrographs showing RFPPOS cells (injected CDCs and their progeny) in the Control, FHPeCDC and FHPeCDC þ magnet groups. D: Quantification of engraftment (RFPPOS cells). Bar ¼ 100 um.). * indicates p < 0.05 when compared to the FHPeCDC group.

These datasets warrant the importance to perform a dose-finding study and determine the maximum safety dose for future large animal studies on magnetically-targeted cell infusion. 3 days after treatment, the Prussian blue signal correlated with the engraftment signal (RFP) (Fig. 5). Previous report suggests signal from ferumoxides SPIONs either by MRI or Prussian blue staining is not reliable to trace stem cell fate in vivo, rationalizing SPIONs exocytosed by injected stem cells and/or left over from cell death may be phagocytosed by macrophages and create false positive engraftment signal [15]. However, that study was performed at 3 weeks after cell injection. Also, the in vivo metabolic fate of ferumoxytol may be different from that of ferumoxides. Our results suggest the ferumoxytol signal can predict cell engraftment at least up to day 3 after injection. Future studies are needed to define the time window when ferumoxytol labeling can be reliably used for tracking stem cells in vivo. We then asked whether FHP labeling and/or magnetic targeting would increase cardiac inflammation and cause iron overload in those rats. We found there is no incremental inflammation or iron overload induced by FHP labeling and/or magnet targeting: the tissue density of CD68POS macrophages and serum concentrations of ferritin and transferrin were indistinguishable in all three treatment groups. We reason this is probably due to the fact that the administered iron quantity for each animal is small (~0.5 mg per animal based on our FHP labeling protocol), compared to the total body iron store of the animal (~10 mg). However, it is worth noting that localized injection of iron particles may cause iron overload to the heart rather than to the whole body. This warrants future investigations on the specific cardiotoxic effects of ironlabeled stem cells and magnetic-targeted cell delivery. The boost

in short-term cell retention translated into better heart morphology and greater functional benefit at 3 weeks (Fig. 6). Such engraftment-benefit or doseebenefit relations were confirmed in early reports [21]. We know that CDCs and other stem cell types exhibit their therapeutic benefit by both direct regeneration and other indirect mechanisms [16]. Here we found magnetic targeting increased long-term cell engraftment (Fig. 7) and capillary densities (Fig. 8) in the post-MI heart. This attests the enhanced therapeutic benefit from magnetic targeting may also come from both direct and indirect regeneration. Clinical translation of this approach requires a magnetic field that is compatible with patient care and clinical settings. Mounting a large magnet on the patient's chest is generally not feasible. Nevertheless, we have proved that only a short period of time is needed for the magnet to be on (e.g. 10 min). Additionally, more sophisticated magnetic focusing machines are currently under development [22]. 5. Conclusion In summary, the present study confirms the safety and efficacy of ferumoxytol labeling of heart stem cells and magnetically-targeted stem cell delivery in a small animal model of myocardial infarction. The approach described here is generalizable to any cell type that can be delivered via the intracoronary route. With further pre-clinical optimization, this approach may improve the outcome of current cell therapy for ischemic cardiomyopathy. Disclosure The authors report no financial conflicts.

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Fig. 9. Angiogenesis at 3 weeks after treatment. AeC: Representative fluorescent micrographs showing wWFPOS capillary structures the Control, FHPeCDC and FHPeCDC þ magnet groups. D: Quantification of capillary structures. Bar ¼ 100 um.). * indicates p < 0.05 when compared to the FHPeCDC group. * indicates p < 0.05 when compared to Control. ** indicates p < 0.05 when compared to any other groups.

Acknowledgment This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor's Faculty Excellence Program, The Education Department of Henan Province Science and Technology Key Project 13A320623, and National Natural Science Foundation of China H020381370216. The authors thank O. Marks, X. Zhang, J. Shipley-Phillips, and E. Johannes for technical assistance. The authors also thank J. Piedrahita and J. Horowitz for helpful discussions and lab equipment support. References [1] Wollert KC, Drexler H. Clinical applications of stem cells for the heart. Circ Res 2005;96:151e63. [2] Al Kindi A, Ge Y, Shum-Tim D, Chiu RC. Cellular cardiomyoplasty: routes of cell delivery and retention. Front Biosci 2008;13:2421e34. [3] Terrovitis JV, Smith RR, Marb an E. Assessment and optimization of cell engraftment after transplantation into the heart. Circ Res 2010;106: 479e94. [4] Cheng K, Li TS, Malliaras K, Davis DR, Zhang Y, Marban E. Magnetic targeting enhances engraftment and functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction. Circ Res 2010;106: 1570e81. [5] Cheng K, Malliaras K, Li TS, Sun B, Houde C, Galang G, et al. Magnetic enhancement of cell retention, engraftment, and functional benefit after intracoronary delivery of cardiac-derived stem cells in a rat model of ischemia/reperfusion. Cell Transplant 2012;21:1121e35. [6] Huang Z, Pei N, Wang Y, Xie X, Sun A, Shen L, et al. Deep magnetic capture of magnetically loaded cells for spatially targeted therapeutics. Biomaterials 2010;31:2130e40.

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