Sprague-Dawley rats from Orient Bio, Inc. (Seongnam, Korea) were handled according to the Association for Assessment and Accreditation of Laboratory Animal Care International system. Animal experiments conformed to the International Guide for the Care and Use of Laboratory Animals, and experimental procedures were examined and approved by the Animal Research Committee of Yonsei University College of Medicine (Seoul, Korea). All rats were allowed free access to food and water. The room was kept at a constant temperature (22±2°C) and relative humidity (50±10%) on a 12-h light/dark cycle. The total number of rats used was 56: 10 rats used for MSC isolation (weight, 100±5 g; male; 4 weeks of age) and 46 rats used for MI model (weight, 270±10 g; male; 8–9 weeks of age).

The pEF1/His C::IGF-1::EGFP selection vector was constructed by inserting rat IGF-1 and enhanced green fluorescent protein (EGFP) DNA into the pEF1/His C vector (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). In the first step, total RNA was extracted from rat MSCs using an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany). The MSCs cDNA was synthesized by using Promega RT reagents (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Briefly, RNA with oligo(dt) primer and Nuclease-free water was preheated for 5 min at 70°C, and immediately chilled in 4°C for at least 5 min. Reverse transcription reaction mix (nuclease-free water, ImProm-II™ 5X Reaction Buffer, MgCl₂, dNTP Mix, Recombinant RNasin^® Ribonuclease Inhibitor, ImProm-II™ Reverse Transcriptase) was added to RNA and primer mix. This mix was annealed at 25°C for 5 min, extended at 42°C for 1 h, inactivated reverse transcriptase at 70°C for 15 min. Rat IGF-1 cDNA was amplified by polymerase chain reaction (PCR) with DNA polymerase (Takara Bio, Inc., Otsu, Japan) using rat MSCs cDNA as a template. The forward and reverse primers for IGF-1 contained EcoRI and XbaI restriction sites, respectively (Table I). The restriction site for EcoRI was 5′-GAATTC-3′ and the restriction site for XbaI was 5′-TCTAGA-3′. The PCR thermocycling conditions were 95°C for 5 min, 32 cycles of 95°C for 1 min, 65°C for 1 min 30 sec, 72°C for 40 sec, and then a final extension step at 72°C for 10 min. The amplified PCR product was purified and digested with EcoRI and XbaI restriction enzymes. The pEF1/His C::IGF-1 vector was constructed by inserting the digested IGF-1 fragment into the pEF1/His C vector. Expression of IGF-1 from pEF1/His C::IGF-1 was confirmed in H9c2 cells (KCLB no. 21446; Korean Cell Line Bank, Seoul, Korea) by reverse transcription (RT)-PCR. The H9c2 cells were grown in DMEM (WELGENE, Inc., Gyeongsan, Korea) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% antibiotics (Gibco; Thermo Fisher Scientific, Inc.) in humidified atmosphere at 37°C with 5% CO₂. Subcloning was performed by inserting EGFP DNA into the pEF1/His C::IGF-1 vector; the DNA was isolated from the pEGFP-C1 vector (Takara Bio, Inc.) by digestion with AseI and MluI restriction enzymes. A gap-filling DNA precipitation step was conducted followed by DNA ligation (T4 ligase; New England BioLabs, Inc., Ipswich, MA, USA). This cloned sequence of the pEF1/His C::IGF-1::EGFP vector was confirmed by digesting with restriction enzymes and sequencing (Cosmo Genetech Co., Ltd., Seoul, Korea).

MSCs were isolated from 4-week-old male Sprague Dawley rats (weight, 100±5 g) as previously described (10). Rat bone marrow (BM)-derived cells were flushed out from the femur and tibia using DMEM (WELGENE, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution (Gibco; Thermo Fisher Scientific, Inc.). The medium was collected and centrifuged at 596 × g for 5 min at room temperature (RT), and cells were loaded onto a Ficoll-Paque density gradient medium (GE Healthcare Life Sciences, Uppsala, Sweden). Following centrifugation (596 × g, 30 min, RT), the middle layer containing mononuclear BM cells was removed, washed and seeded in culture dishes at 1×10⁶ cells per 100 cm². Following incubation for 72 h in humidified atmosphere at 37°C with 5% CO₂, adherent cells were washed and fresh MSC medium was added. MSCs were identified using a flow cytometer (FACSVerse; BD Biosciences, Franklin Lakes, NJ, USA) and FACSuite software (version 1.0.6; BD Biosciences) based on the presence of cell surface markers. Resuspended single cells were labeled with antibodies against surface markers, including anti-cluster of differentiation (CD)29-fluorescein isothiocyanate (FITC; cat. no. 102205; 1:50), anti-CD54-phycoerythrin (PE; cat. no. 202405; 1:80), anti-CD90-FITC (cat. no. 202504; 1:200), anti-CD45-PE (cat. no. 202207; 1:80), and anti-CD49d-FITC (cat. no. 200103; 1:200) antibodies (BioLegend, Inc., San Diego, CA, USA) in ice for 1 h. MSCs labeled with antibodies were sorted by flow cytometry.

MSCs were transfected with the non-viral carrier PAM-ABP (supplied by Professor Minhyung Lee) and selection vector complexes at different concentrations of pEF1/His C::IGF-1::EGFP (0, 0.5, 1, 2, 4, 8 and 16 µg). Complexes of plasmid DNA and PAM-ABP were mixed based on weight ratio (1:5) and were used to treat MSCs. At 4 h after transfection, MSCs were washed and the medium was replaced with fresh complete medium. Transfected MSCs were continuously incubated for 24 or 48 h.

Cells transfected with pEF1/His C::IGF-1::EGFP were harvested 24 or 48 h following transfection, and total RNA was extracted using an RNeasy Mini kit (Qiagen GmbH). cDNA was synthesized by using Promega RT reagents (Promega Corporation) according to manufacturer's protocol. Briefly, RNA with oligo(dt) primer and nuclease-free water was preheated for 5 min at 70°C and immediately chilled in 4°C for at least 5 min. Reverse transcription reaction mix (nuclease-free water, ImProm-II™ 5X Reaction Buffer, MgCl₂, dNTP Mix, Recombinant RNasin^® Ribonuclease Inhibitor, ImProm-II™ Reverse Transcriptase) was added to RNA and primer mix. This mix was annealed at 25°C for 5 min, extended at 42°C for 1 h with inactivated reverse transcriptase at 70°C for 15 min. RT-PCR was performed with an AmfiEco kit (GenDEPOT, Katy, TX, USA). Primer sequences targeting rat IGF-1 and GAPDH are listed in Table I. The PCR cycling conditions of rat IGF-1 were 95°C for 5 min, 28 cycles of 95°C for 1 min, 65°C for 1 min 30 sec, 72°C for 40 sec, and then a final extension step at 72°C for 10 min. Thermocycling conditions of GAPDH were 95°C for 5 min, 28 cycles of 95°C for 1 min, 60°C for 1 min, 72°C for 40 sec, and then a final extension step at 72°C for 10 min. PCR products were loaded in 1.2% agarose gel containing red-safe (iNtRON Biotechnology, Seongnam, Korea). The results were scanned and visualized with CoreBio i-MAX^™ gel imager analysis system (CoreBiosystem, Seoul, Korea).

Conditioned medium from transfected MSCs was collected 24 or 48 h following incubation and analyzed using a rat IGF-1 Quantikine ELISA kit (cat. no. MG100; R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Serum-free low-glucose Dulbecco's modified Eagle's medium (WELGENE, Inc.) was used as a negative control. All samples were assayed in triplicate, and the optical density was measured at 450 nm.

FACS analysis was performed to isolate transfected MSCs using a FACSAria III flow cytometer (BD Biosciences). At 24 h following transfection, transfected MSCs were harvested, washed and resuspended in FACS buffer (2 mM EDTA, 25 mM HEPES, and 1% FBS in 1X PBS). MSCs transfected with pEF1/His C::IGF-1::EGFP were identified based on green fluorescence using a FITC filter (488 nm). Data were analyzed with FACSDiva software (version 3.1.3; BD Biosciences).

Experimental MI was induced in male Sprague Dawley rats at 8–9 weeks of age (weight, 270±10 g) as previously described (10). Rats were anesthetized by intraperitoneal injection of Zoletil (30 mg/kg; Virbac Corporation, Carros, France) and Rompun (10 mg/kg; Bayer, Leverkusen, Germany) and ventilated with positive pressure (180 ml/min; Harvard Apparatus, Holliston, MA, USA). The heart was exposed through a 2-cm incision in the left lateral costal rib. The left anterior descending (LAD) artery was ligated with 6-0 prolene (Ethicon, Inc., Cincinnati, OH, USA) below the left atrium for 1 h. Reperfusion and intramyocardial injection of 100 µl PBS (n=10) was performed, and control (MSCs without transfection; n=10), unsorted (MSC transfection-unsorted; n=13), or sorted (MSC transfection-sorted; n=13) MSCs (1×10⁶ cells in 100 µl PBS) were delivered to three or four different sites in the border zone. At 2 weeks following transplantation, the animals were re-anesthetized and sacrificed for histological examination.

To analyze MSC engraftment (or localization) within the infarcted myocardium, the heart was perfused, fixed in 10% formalin solution overnight at 4°C, embedded in Optimal Cutting Temperature compound (Sakura, Zoeterwoude, The Netherlands), frozen on dry ice and cut into transverse sections (10 µm) with a cryostat that were mounted with mounting medium containing DAPI (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). DAPI and EGFP fluorescence was detected by confocal fluorescence microscopy (magnifications, ×100 and ×400).

To confirm IGF-1 expression in MSCs transfected with pEF1/His C::IGF-1::EGFP in ischemic hearts, frozen tissue sections were permeabilized with PBS containing Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), quenched with 100% methanol and 30% H₂O₂, washed with PBS, and blocked with 3% bovine serum albumin (GenDEPOT, Katy, TX, USA) at RT for 1 h. Cryosections were incubated with primary antibody against IGF-1 (cat. no. sc-1422, 1:50; Santa Cruz Biotechnology, Inc.) in blocking solution at RT for 2 h followed by Cy5.5 donkey anti-goat secondary antibody (cat. no. sc-45102, 1:200; Santa Cruz Biotechnology, Inc.) at RT for 1 h. The sections were mounted in mounting medium containing DAPI and examined under a fluorescence microscope (Olympus Corporation, Tokyo, Japan) for detection of DAPI and Cy5.5 fluorescence (magnification, ×100).

Myocardial infarct size was measured by staining with TTC (Sigma-Aldrich; Merck KGaA). Hearts isolated from rats were incubated in 1% TTC for 15 min at 37°C, cut into transverse sections, and imaged with a digital camera (Samsung Electrics Co., Ltd., Suwon, Korea). Infarct size was measured by calculating the ratio of the cumulative infarcted area to that of the entire left ventricle using ImageJ software (version 1.51j8; National Institutes of Health, Bethesda, MD, USA).

Fibrosis in the heart following MI was evaluated by Masson's trichrome staining (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. In brief, sections were stained with Bouin's solution at 56°C for 15 min, Weigert's iron hematoxylin solution for 15 min and Blebrich scarlet-acid fuchsin for 5 min. The sections were placed in working phosphotungstic/phosphomolybdic acid solution for 5 min, Aniline blue solution for 5 min, and 1% acetic acid for 2 min. Stained sections were mounted with Permount (Thermo Fisher Scientific, Inc.) and scanned to digitalize at ×200 magnification using an Aperio AT2 (Leica Biosystems, Wetzlar, Germany).

Apoptotic cells in paraffin-embedded heart tissue sections were detected with the TUNEL assay using a commercial kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer's protocol. Briefly, the paraffin-embedded heart tissue sections were pretreated with proteinase K (20 µg/ml) for 15 min, quenched in 3% H₂O₂ for 5 min at RT, and immediately equilibrated. The tissue sections were reacted with the TdT enzyme at 37°C for 1 h, and incubated with digoxigenin conjugated nucleotide substrate at 37°C for 30 min. Then, the heart sections were stained with 3,3-diaminobenzene (DAB) for 5 min, counterstained with hematoxylin for a short time, mounted with Permount (Thermo Fisher Scientific, Inc.). A total of 5 animals per group were analyzed. For each animal sample, apoptotic cells were counted in at least five different regions of the stained slide (magnification, ×100 and ×400).

Quantitative data are expressed as the mean ± standard deviation of 5 independent experiments. Differences between groups were evaluated by one-way analysis of variance followed by Kruskal-Wallis test and Dunn's multiple comparison post-hoc tests. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS v.18.0 software (SPSS, Inc., Chicago, IL, USA) and PRISM v.5.01 (GraphPad Software, Inc., La Jolla, CA, USA).

The pEF1/His C::IGF-1::EGFP selection vector was constructed by inserting rat IGF-1 cDNA and EGFP DNA into the pEF1/His C vector (Fig. 1A and B). The sequence of the cloned vector (pEF1/His C::IGF-1 vector; 6,645 bp) was confirmed by gene sequencing (data not shown). Expression of IGF-1 from pEF1/His C::IGF-1 was confirmed in H9c2 cells by RT-PCR (0–4 µg; Fig. 1C). The EGFP gene was subsequently inserted into the pEF1/His C::IGF-1 vector (Fig. 1D). The final size of the cloned vector pEF1/His C::IGF-1::EGFP was 6,121 bp.

Rat primary BM-MSCs exhibited a spindle shape (data not shown). To characterize the MSC phenotype, BM-MSCs were labeled for positive and negative cell surface markers. Notably, BM-MSCs were positive for MSC markers, including CD29 (99.8%), CD54 (96.3%) and CD90 (99.7%), and were negative for CD45 (0.34%) and CD49d (0.42%; Fig. 2A). The efficiency of MSC transfection with the non-viral carrier PAM-ABP was assessed by FACS based on the percentage of MSCs expressing EGFP. The efficiency of transfection of pEF1/His C::IGF-1::EGFP and PAM-ABP in MSCs was ~20%. Subsequent to sorting, the efficiency was increased to 95.1% by EGFP selection, corresponding to a 4.8-times increase in the purity of transfected MSCs (Fig. 2B and C).

To determine the optimal concentration of pEF1/His C::IGF-1::EGFP for transfection in rat BM-MSCs, different amounts were transfected (0–16 µg). The intensity of EGFP fluorescence was concentration-dependent and was maximal at 8 µg following 24 and 48 h (Fig. 3A). IGF-1 mRNA expression level was additionally increased in a concentration-dependent manner as detected by RT-PCR, reaching a maximum value at 8 µg after 48 h (Fig. 3B and C). IGF-1 protein expression levels in conditioned culture medium from transfected MSCs were measured by ELISA. Conditioned medium from pEF1/His C::IGF-1::EGFP-transfected MSCs exhibited higher expression levels of IGF-1 compared with the negative control (0 µg; Fig. 3D). IGF-1 secretion was enhanced at concentrations ≤8 µg and began to decline at 24 and 48 h following transfection (Fig. 3D). Furthermore, the amount of IGF-1 secreted from transfected MSCs at 48 h post-transfection was increased compared with 24 h following transfection.

The green fluorescence associated with pEF1/His C::IGF-1::EGFP-transfected MSCs was detected in rats injected with unsorted and sorted cells. Transfected MSCs were more abundant in the sorted group compared with the unsorted group (Fig. 4A). The pEF1/His C::IGF-1::EGFP-transfected MSCs were localized in the myocardium of the ischemic left ventricle.

Red fluorescence corresponding to IGF-1 expression was detected in the myocardial layer of unsorted and sorted groups (Fig. 4B). IGF-1 expression levels were increased in the sorted group compared with the unsorted group. Therefore, the sorting system enhances the efficiency of gene delivery.

The therapeutic efficacy of the system was evaluated through histological analysis of the infarcted tissue by TTC and Masson's trichrome staining. Infarct size was decreased in the sorted group compared with the PBS and MSC groups (P<0.05; Fig. 5A and B). Although the infarct size in the sorted was half of that of the unsorted group, the difference was not statistically significant (Fig. 5B). Masson's trichrome staining demonstrated that fibrotic area of the myocardial layer was significantly decreased in the sorted group compared with the unsorted group (P<0.05; Fig. 5C and D).

Analysis of the apoptotic cell ratio revealed that apoptosis was decreased in the sorted group compared with the PBS, MSC and unsorted groups (P<0.05, P<0.01; Fig. 5E and F).