Third- to fifth-generation human UCB-MSCs were induced to differentiate into CPCs using 5-Azacytidine (5-Aza)-containing medium, according to the process of Pham et al.Pham et al., 2014. Briefly, the cells were induced in Dulbecco’s-modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS); 1% penicillin/streptomycin; 10 μM 5-Aza; 50 ng/mL activin A; 0.1 mM ascorbic acid. After 24 h induction, cells were washed twice with phosphatebuffered saline (PBS) and cultured in Dulbecco’smodified Eagle’s medium supplemented with 15% FBS, 1% penicillin/streptomycin, 50 ng/mL activin A; 0.1 mM ascorbic acid, without 5-Aza. The medium was replaced every 3 days until day 30.

Male albino mice (25–30g) were anesthetized by thigh muscle injection with 0.022 g/mL ketamine until the ratio of ketamine anesthetic reached 87 mg/kg of body weight Kanno et al., 2002. Mice were intubated and mechanically ventilated, with pump speed 120–130 cycles/min, pump volume per cycle was 1 mL/100 g body weight. After left thoracotomy, ligation of the left anterior descending artery (LAD) was performed at the distal 1/3 of the coronary artery from the aorta to the heart apex Degabriele et al., 2004. After 3 min, CPCs were infused into the area of infarction, at a density 106 cells per 30 μL for each mouse. Then, the chest was closed, and the muscle layer was sewn tight and applied with antiseptic. The mouse was placed carefully into a clean case. Normal mice were used as positive controls, while LAD ligation plus PBS injection was used as a negative control.

Transplantation efficacy was assessed according to criteria, including blood pressure fluctuation, and the survival, migration, and influence of CPCs on the grafted heart tissue.

Mice blood pressure changes were monitored using a 58500 Blood Pressure Recorder (Ugo BasileSrl, Italy), 1–2 weeksbefore and after cell transplantation.

To facilitate with stress, mice were trained to be familiar with tail-cuff blood pressure measurements. The animals were anesthetized by placing them in an anesthesia closed chamber, which provided continuous supply of 5% pure oxygen at a rate 1 L/min of isoflurane, until coma was induced. The mouse tail was inserted into the blood pressure measuring ring with signal sensor. Systolic (SYS) and diastolic (DIA) blood pressure of the animals were measured and recorded following the manufacturer’s instructions (58600 Blood Pressure Recorder). Mean arterial pressure (MAP) was calculated by the following formula:

MAP = [(2 × DIA) + SYS] / 3

The animal heart was washed with PBS and fixed in 4% paraformaldehyde overnight. After sucrose cryoprotection, the tissue was moulded with optimum cutting temperature compound and cryosectioned into 10-μm-thick slices. The obtained slices were then treated with hematoxylin and eosin (H&E) stain, Trichrome stain, and immunohistochemistry.

H&E staining was performed to evaluate tissue and cell structures. Slices were immersed in a graded series of alcohol solutions as follows: 100% for 2 min, 90% for 2 min, 70% for 1 min, 50% for 1 min, and double- distilled water for 2 min. The surrounding tissue area was dried with absorbent paper, followed by an addition of a drop of hematoxylin stain onto the tissue slice. After 2 min, the slice was washed with distilled water. Two drops of eosin stain were added to the tissue slice for 30 s and the specimen was successively embedded again with: 50% alcohol for 1 min, 70% alcohol for 1 min, 90% alcohol for 1 min, and 100% alcohol for 1 min. Immersion oil was dispensed onto the slice followed by lamelle fixation.

Masson’s Trichromedye is commonly used to distinguish collagen in pathological cases. Three dyes are used to identify collagen, fibrin, and erythrocytes. Tissue was initially stained with Biebrich Scarlet acidic dye (light red), which has a binding preference to acidic components in the tissue. When the tissue was treated with phosphomolybdic acid, the red color was displaced from the collagen component. In addition, phosphomolybdic acid also generated links between collagen and light green molecules that tainted the collagen green.

Tissue specimens were immersed in a graded series of alcohol solutions as follows: 100% for 1 min, 90% for 1 min, and 80% for 1 min. Samples were washed gently twice with distilled water, followed by immersion in Bouin-containing solution overnight at 56 °C. Samples were rinsed with water to remove traces of piric acid (yellow) and soaked in Weigert’s iron hematoxylin solution for 10 min. Samples were gently washed continuously with distilled water for 30 s. Next, they were embedded in Biebrich scarlet solution for 1 min. The slices were washed quickly again with distilled water and immersed in 5% phosphomolybdic acid solution for 30 min. Then, samples were transferred and bathed in the light green solution for 10 min. The samples were rinsed quickly with distilled water and immersed in 0.5% acetic acid solution for 2 min. The samples were immersed in a series of graded alcohol solutions as follows: 95% for 1 min × 2, followed by 100% for 1 min × 2. Immersion oil was dropped onto the slices, followed by lamelle fixation. Trichromestained sections were identified and collagen structures were evaluated by ImageJ software.

Cells grown on coverslips (Santa Cruz Biotechnology, Dallas, TX, USA) were prepared for immunocytochemistry as follows: cells were fixed in % paraformaldehyde solution for 15 min; permeabilized with 0.1% Triton X-100; washed three times in PBS; blocked with BSA; and incubated with a primary human antibody alpha-actinin (Abcam 1:400) or human Troponin T (Abcam 1:400) overnight at 4 °C. After washing, the samples were treated with goat anti-rabbit IgG secondary antibodies (Abcam 1:400) and Hoechst 33342 (Sigma 1:400) for 45 min at room temperature. The samples were rinsed three times with PBS, mounted, and observed under a fluorescent microscope (Zeiss Axiovert).

Tissue sections were fixed and stained with Hoechst 33342 (Sigma, 1:400) and antibodies specific for human proteins of interest, Troponin T (Abcam, 1:200) and alpha-actinin (Abcam, 1:200), according to the manufacturer’s protocol. Slides containing tissue sections were washed briefly for 2 × 5 min in PBS. Tissue sections were blocked with 5% BSA at room temperature for 30 min and incubated with primary antibodies at room temperature for 60 min. After washing, slides were then treated with secondary antibodies at room temperature for 60 min in the absence of light to avoid optical bleaching. The samples were washed 4 × 5 min in PBS, coated with polyvinyl alcohol solution, and covered by lamelle. Their images were observed and recorded by fluorescence microscopy.

Before transplantation, CPCs were tested for the expression of human Sox2 and Oct4 genes, which are used to characterize tumor formation, using reverse transcription-polymerase chain reaction (RT-PCR). Human glyceraldehyde 3-phosphate dehydrogenase (hGAPDH) was used as an internal control.

At 14 days post-transplantation, total RNA from hearts of control animals or test animals was collected to examine the tumor formation capacity of infused cells in vivo.

Total RNA was extracted from CPCs prepared for transplantation, human MCF7 breast cancer cells, and heart tissue samples 14 days post-transplantation using Easy Blue Reagent (Intron), following the manufacturer’s instructions. cDNA was synthesized from RNA using the Superscript II Reverse Transcriptase (Invitrogen), according to the manufacturer’s procedure. Next, PCR was performed using Taq DNA Polymerase (Takara, Shiga, Japan). The primers of the examined genes are listed in Table 1 .

Gel electrophoresis was performed on RT-PCR products on 2% agarose gels, at 100 V, for 60 min. A 100-bp DNA ladder was used for all products. The results were analyzed by electrophoresis using the Gel Doc IT system (UVP).

Data were statistically interpreted using GraphPad Prism software version 6.

UCB-MSCs were induced in medium containing 5-Aza. The induced cells were checked by ICC staining with antibodies specific for human cardiac muscle cells, including human alpha-actinin and human troponin T. The results indicated that a phenotypic change occurred in the induced cells. Originally, the population exhibited the morphology of MSCs, which are mononuclear and spindle shaped; then, they transformed into multinuclear cells, which are similar to those of cardiac muscle cell lineages. Additionally, they also expressed both specific markers of cardiac muscle cells, human alpha-actinin and human troponin T ( Figure 1 ).

To assess the effect of cell transplantation in the mouse model of myocardial ischemia, changes in blood pressure before and after transplantation were recorded through a dedicated system ( Figure 2 ). The collected data were processed using statistical software.

In normal mice, the MAP was approximately 111 mmHg Tiemann et al., 2003. Healthy mice (n = 10) were used as a positive control with stable blood pressure (DO = 111.1 mmHg; 07 = 111.0 mmHg; 014 = 111.1 mmHg). However, MAP in the LAD ligation and PBS-injected mice (PBS group) (n = 10) tended to decrease over the survey period (DO = 111.0 mmHg; 07 = 104.8 mmHg; 014 = 103.5 mmHg). This result suggested that heart activity was decreased in the PBS group. Meanwhile, the MAP in the CPC-transplanted group (n = 10) was decreased compared with the control group (DO = 111.0 mmHg; 07 = 110.1 mmHg; 014 = 109.1 mmHg); however, the decrease was evidently less than that of the PBS group.

To assess the effects of grafted CPCs in the mouse model, animals were divided into three groups. The control group consisted of normal healthy mice; the PBS group consisted of mice receiving LAD ligation and PBS injection; and the CPC group comprised mice receiving LAD ligation and CPC transplantation.

The heart tissue was fixed and cryo-sectioned onto slides (lO-f-lm-thick slices). Slides were then stained with H&E for tissue damage assessment. Results showed that heart tissue in the control group remained intact and exhibited no sign of inflammation. The left ventricle wall of the control group was normal, while it was thinner in the PBS group. Heart tissue of the PBS group exhibited more loss of crossstriations, with significantly shrunken bands and nuclear karyolysis in heart cells (green arrow, Figure 3 ). Some neutrophils infiltrated the heart muscle and fibers appeared rippled in the PBS group (area circled in blue, Figure 3 ). However, no fracture or damage of the heart tissue was observed on the CPC group. In addition, the nucleus was located in the center of the cell (yellow arrows, Figure 3 ), pale pink plates were also observed and the left ventricle wall was not as thin as the PBS group ( Figure 3 ).

To assess scar formation in the damaged heart tissue and effects of CPCs on the process, mice hearts were collected for Trichrome staining. Results showed that the myocardium of the control group did not experience fibrosis ( Figure 4a , Figure 4d , Figure 4g ), while the PBS group formed broad blue regions of fibrosis when stained with Trichrome ( Figure 4b , Figure 4e , Figure 4h ). No sign of fibrosis was observed in the myocardium of the CPC group compared with the PBS group ( Figure 4c , Figure 4f , Figure 4i ).

To investigate the fate of the infused CPCs in the damaged heart tissue of experiment animals, mouse heart tissue samples were collected, frozen, and sectioned. Slices were then stained with antibodies, which were specific for human heart cells, including human alpha-actinin and human troponin T.

The results of tissue staining indicated that heart tissues of the control and PBS groups were negative for staining with these specific antibodies ( Fig. 5a, d, g, j, b, e, h, k ). Meanwhile, some areas of CPC-transplanted heart tissue exhibited positive staining ( Fig. 5c, f, i, l ), indicating that grafted CPCs survived and expressed both human Troponin T (green) and human alpha-actinin (red). These cells not only did not form scar tissue but they also prevented or reduced tissue damage caused by ischemia. Therefore, the left ventricular walls of CPC-transplanted mice were relatively thick compared with normal mice. In contrast, mice in the PBS group exhibited thin ventricular walls and scar formation.

To test for the tumor-forming ability of the transplanted cells prior to transplantation, CPCs were evaluated for human cancer gene expression, including human Sox2 (hSox2) and human Oct4 (hOct4). The human breast cancer MCF7 cell line was used as a positive control ( Figure 6 ) and distilled water was used as a negative control. The results showed that the CPCs did not express hOct4 and hSox2 compared with MCF7 cells ( Figure 7a ).

To assess whether CPC survival and tumorigenesis in mouse cardiac tissue post-transplantation, CPC-transplanted tissue was collected. Total RNA was extracted from heart tissue for further analysis of hSox2 and hOct4 expression. RNA from normal mouse heart was used for comparison. MCF7 cells and distilled water controls were treated with the same procedure. The results showed that transplanted CPCs existed in grafted tissues and the tumor genes hSox2 and hOct4 were not expressed ( Figure 7b ).