BM was collected from volunteers and the UCB units were from full term (38-40 weeks' gestation) deliveries; the latter were collected from the unborn placenta with obtaining the mother's informed consent for the use of their material for scientific purposes. MSCs were isolated as described previously with slight modification.7) Briefly, to isolate the mononuclear cells (MNC), each BM or UCB unit was diluted 1 : 1 with phosphate-buffered saline (PBS) and then it was carefully loaded onto Ficoll-Hypaque solution (Amersham, Germany). After density gradient centrifugation at 2,500 rpm for 30 minutes at room temperature, the MNCs were collected from interphase and they were washed twice with PBS. The MNCs were seeded at a density 10⁶ cells/cm² into 6-well culture plates (Falcon, Becton Dickinson, Heidelberg, Germany) in Dulbecco's modified Eagle's medium (DMEM) (Gibco-Invitrogen Corp., USA) that was supplemented by 10% fetal bovine serum (FBS; selected lots; Gibco-Invitrogen), 100 U/mL penicillin and 100 g/mL streptomycin (Sigma-Aldrich, USA). After overnight incubation at 37℃ in a humidified atmosphere containing 5% CO₂, the non-adherent cells were removed and fresh medium was added to the wells. The cultures were maintained, and the remaining nonadherent cells were removed by changing the media every 7 days. The culture wells were continuously screened to obtain the developing colonies of adherent cells. The cells were detached by incubating them for 5 minutes in 0.05% trypsin/EDTA (Gibco-Invitrogen, USA), and then they were harvested and replated at a density of 5,000 cells/cm²; these were then termed as passage 1 cells thereafter. Before transplantation into the rat heart, the MSCs were trypsinized, washed and labeled with 50 µg/mL of 4',6-diamidino-2'-phenylindole (DAPI) (Sigma, USA) for 30 minutes.

To analyze the cell-surface expression of typical marker proteins, the MSCs were labeled with anti-human antibodies against HLA-ABC, HLA-DR, CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105 and CD106 (Becton Dickinson, CA, USA). Mouse isotype antibodies served as the respective controls. Ten thousand labeled cells were acquired and these were analyzed by using a cytomics FC 500 flow cytometer (Beckman Coulter, Krefeld, Germany).

Osteogenic differentiation was achieved by culturing the cells in complete medium along with 0.1 µM dexamethasone, 10 mM β-glycerol phosphate and 50 µM ascorbate. Chondorogenic differentiation was induced by using medium containing 50 µM ascorbic acid, 0.1 µM dexamethazone, 10 ng/mL TGF-β, 40 µg/mL L-proline and 100 µg/mL sodium pyruvate. Adipogenic differentiation was induced by the addition of 0.5 mM isobutylmethylxanthine and 1 µM dexamethasone.

This study was reviewed and approved by the Chonnam National University Institutional Animal Care and Use Committee.

Male Sprague Dawley rats (200-200 g each, Jung Ang Animals, Korea) were anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). Myocardial ischemia-reperfusion injury was induced by temporary ligation of the proximal left anterior descending coronary artery (LAD) for 30 minutes and this was followed by release. The MSCs were directly injected into the peri-infarct areas of the myocardium just before the reperfusion. The sham group rats received PBS (n=15) and the MSC group rats received UCB-MSCs (n=15) or BM-MSCs (n=15) (1×10⁶ diluted in 100 µL of PBS).

The left ventricular (LV) function was assessed by performing transthoracic echocardiography at 2 weeks after surgery with a 7.5-MHz sector scan probe. The rats were anesthetized and placed in the supine position and their chests were shaved. M-mode echocardiograms that were guided by two-dimensional long-axis images were obtained. The LV percent fractional shortening (FS) was calculated as 100×(LVEDD-LVESD)/LV, and the LV ejection fraction (EF) was calculated as 100×(stroke volume/diastolic volume) where the LVEDD is the LV end diastolic diameter and the LVESD is the LV end systolic diameter.

After echocardiography, the rats were sacrificed and their hearts were immediately removed and frozen in liquid nitrogen for immunofluorescent study. Immunofluorescent staining was carried out on the 10-µm frozen ventricular sections.

The samples were incubated overnight with antibodies against α-sarcomeric actin (Sigma, USA), CD31 (BD Science), von Wilebrand factor (Sigma, USA) and connexin 43 (Sigma, USA). The following secondary antibodies were used: anti-rabbit IgG coupled to Alexa Fluor 488 and anti-mouse IgG coupled to Alexa Fluor 594 (InVitrogen, USA). The images were obtained with using a multitracking laser scanning confocal microscope (200×). The fluorochromes were detected at 488 nm and 543 nm.

To compare the gene expression profile of the UCB-MSCs and BM-MSCs, we performed expression analysis by using an Illumina expression chip 24 k (Macrogen, Korea).

To compare the mRNA expression levels in cell-injected animals and the control rats, their myocardial specimens were pulverized and then homogenized in Trizol according to the manufacturer's instructions. cDNA was synthesized to perform reverse transcriptase-polymerase chain reaction (RT-PCR). The primers we used for cardiac ankyrin repeat protein (CARP) were F-ACCGCTATAA GATGATCCGA and R-AATGAAGCTCTGCTCACCAG, and the primers we used for β-actin were, F-GACTACCTCATGAAGATC and R-GATCCACATCTGCTGGAA.

The cells were extracted to separate them on 8% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then they were transferred to PVDF membranes. The blots were incubated with antibodies against CARP (Santa Cruz, USA) and β-actin (Sigma, USA), and the signals were detected by a LAS-3000 (FUJI, Japan).

The BM-MSCs, UCB-MSCs and the CARP or its scrambled control small interfering ribonucleic acid (siRNA)-transfected UCB-MSCs were cultured on 12-well plates. The confluent cell monolayers were scratched using sterilized 200-µL pipette tips and they were washed with serum free media and then further incubated. The images of the cells were captured at 0 hour, 6 hours and 24 hours after the wounding.

The MSCs were cultured in DMEM supplemented with 1% FBS for 36 hours, and then the cytokine levels in the culture supernatant were detected using the Human Cytokine Antibody Array V (RayBiotech, USA) per the manufacturer's protocol. Briefly, the supernatants were incubated for 2 hours with membranes that were arrayed with antibodies against 79 cytokines. After washing them twice, the membranes were incubated for 2 hours with biotin-conjugated primary nti-cytokine antibodies and then the membranes were washed twice. The membranes were then incubated with horseradish peroxidase-conjugated streptavidin for 1 hour, washed twice and they were placed in the detection buffer for a few minutes. The signals were detected by a LAS-3000 (FUJI,Japan).

Each experiment was performed at least three times. The data is presented as means±SDs. Differences were analyzed by ANOVA. The statistical significance of the differences was accepted at the level of p<0.05.

The MSCs were characterized by analysis of their surface markers and by the differentiation study. On the cytomeric analysis, both groups of MSCs were positive for CD29, CD44, CD90 and HLA-ABC, while they were both negative for CD13, CD34 and CD106 (Fig. 1). Osteogenic, chondrogenic and adipogenic differentiation were successfully induced (Fig. 1).

To compare the therapeutic capacity of MSCs for repairing the ischemia/reperfusion damage, we injected MSCs into the IR injured rats. Echocardiography was performed at two weeks after IR injury. As shown in Fig. 2, the EF and FS were significantly increased in the UCB-MSCs group and the BM-MSCs group compared with those values of the IR control group (p<0.01). The EF was 79.8±4.6% in the UCB-MSCs group and the EF was 76.1±5.1% in the BM-MSCs group (the IR control: 56.6±6.8%). The FS was 43.9±5.0% in the UCB-MSCs group and it was 40.6±6.3% in the BM-MSCs group (IR control: 26.2±4.2%). The echocardiographic findings did not show any statistical difference between the UCB-MSCs and BM-MSCs groups. The myocardial IR significantly increased the end-diastolic and end-systolic dimensions and it significantly reduced their respective FS and EF.

DAPI-tagging MSCs were detected in the peri-infarct zone at 2 weeks after injection. Immunofluorescence staining showed positively stained cells for α-sarcomeric actin, CD31, von Wilebrand factor and connexin43 in the UCB-MSCs group. On the other hand, only a few cells or none were stained positively in the BM-MSCs group (Fig.3).

The microarray data was examined and the ranking of the genes is shown in Table 1 and 2, which demonstrates the highly expressed genes. Table 3 shows the genes that were more highly expressed in the UCB-MSCs than in the BM-MSCs. This result provided the putative candidate genes to explore for explaining the UCB-MSCs properties as compared with the BM-MSCs. In Fig. 4, a pie chart represents the distribution of the classifications of complementary deoxysibonucleic acid (cDNAs) that were expressed in the UCB-MSCs (A) and BM-MSCs (B). Among the genes that were highly expressed in the UCB-MSCs, we choose CARP and the CARP mRNA level was determined by RT-PCR in the UCB-MSCs and BM-MSCs.

The wound scratching assay was used for the assay of cell migration and motility. Acceleration of wound closure by the UCB-MSCs was observed, as compared to that by the BM-MSCs. To examine whether CARP, which is highly and exclusively expressed in UCB-MSCs (Fig. 5A), contributes to the UCB-MSCs' motility, the CARP was knocked down by using siRNA transfection to the UCB-MSCs. Knock down of the CARP protein level in the siRNA-transfected UCB-MSCs was confirmed by Western blotting, as compared with the non-treated UCB-MSCs (Fig. 5B). The CARP siRNA-transfected UCB-MSCs showed significantly decreased migration of cells into the wounded area (Fig. 5C).

The expression levels of angiogenin, insulin-like growth factor binding protein (IGFBP)-6 and osteoprotegerin were lower in the UCB-MSCs, whereas the levels of most cytokines were similar and that of IGFBP-1 was higher in the UCB-MSCs, as compared to the BM-MSCs (Fig. 6).