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Clinical tests for commercialization II/III are taking place in Yonsei University Shinchon Severance Hospital, Wonju Christian Hospital and Inha University Hospital for acute myocardial infarction as part of the heart disorder drug research. Clinical tests are carried out by mass-cultivating mesenchymal stem cells derived from the patient's own body and inserting it back into the plexus coronarius using cardiac catheterization. We plan to apply for product authorization once objective data is derived from clinical tests of various facilities.
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- Study on the transplantation effect(medical effect) of human bone marrow-derived mesenchymal stem cells to myocardial infarction animal models(white rats)
This study cultivated human bone marrow-derived mesenchymal stem cells in test tubes and transplanted them to myocardial infarction models to observe treatment effects and reactions. The study was carried out using FCB-Pharmicell cells and took place in the circulatory internal medicine department of Yonsei Universiy Wonju Medical School. |
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Myocardinal infarction animal model
A sprague-dawley rat was anesthetized and laid on the lab desk. The wind pipe was opened and respiratory machine inserted to sustain breathing, and the 4th intercostal of the left heart was opened to expose the epicardium. The left anterior descending coronary artery 2-3mm away from the heart apex was ligated using 6.0 polypropylene stitching fiber. A state of myocardinal infarction was confirmed by observing the distal heart muscle in the ligated area turning white. |
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Myocardial infarction analysis - Measuring size of myocardial infarction through echocardiography and structure inspection
The size of the animal model's myocardial infarction was analyzed using echocardiography of rats that survived 4 weeks after the condition was induced. Anesthetic was injected into the abdominal cavity and a 12 MHz probe was used to measure M-mode, the dimension of the left heart and fractional shortening through schocardiography (VIVID 7, GE MEDICAL Co.) (Figure 1). After observing each white rat's heart by eye, the myocardial infarction area was opened at a 5mm thickness. It was put into 2 percent TTC liquid at 37 degrees centigrade for 15 minutes. The infarction area was confirmed by eye and put in 10 percent formalin liquid for 12 hours. Once the disease sample was made it was dyed using Hasson's trichrome and the infarction area and length was analyzed by morphological analysis. (Figure 2). |
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Figure 1. Artery ligation of white rat (right) and an example of
echocardiography after the ligation (left) |
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Figure2. Eye observation of white rat and the infarction area after dyeing
with Hasson's trichrome (The blue area is the dyed area) |
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Division, transplantation and effect analysis of mesenchymal stem cells
Mesenchymal stem cells were divided and cultivated in a T175 flask 4-5 times. Results of analyzing the SH2 (CD105) mesenchymal stem cells before the animal model transplantation using a flow cytometer, 95 percent (Figure 3) showed no signs of CD34, CD45, MHC class I or II (data not shown). |
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Figure 3. Results of SH-2 (CD105) analysis using flow cytometer |
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After inducing myocardial infarction, 0.3 ml of saline solution (control) or mesenchymal stem cells (cell treatment factor) were injected into the infarction area and cell treatment effects were analyzed for 4 weeks. The sets were divided into the control set and the cell treatment set. One set was to evaluate how much the blood vessel functions of the left heart would revive, and the other set used stem cells with GFP genes to see how much the mesenchymal stem cells spread in the infarction area. |
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Cell treatment effect analysis was carried out through echocardiography of rats that survived after 4 weeks. Ketamine was injected into the abdominal cavity and the rats were anesthetized. Then a 12 MHz probe was put to the chest and mitral valve, aortic valve, papillary muscle inspections were carried out in M mode and B mode, and picture of the parasternal long-axis view and parasternal short-axis view were analyzed. The left ventricular end-diastolic diameter and left ventricular end-systolic diameter were measured using the single-plan area-length mode, and the left ventricular fractional shortening was calculated. Anterior and posterior end-diastolic and end-systolic wall thickness and the left ventricular internal dimension was measured according to the leading-edge method og the American Society for Echocardiography. LVEDV, LVESV and :VEF were calculated using Simpson's single plane rule with the results of the long-axis view, and the analyses were carried out by researcher who were unaware of the control and treatment sets. |
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Dyeing was carried out by hematoxylin-eosin, Masson trichrom and von Willebrand factor dyes and immunofluorescent staining using particular antibodies of the myocardial cells, after extracting the hearts of the control set and cell treatment set. All statistical analysis measurements were written as (mean) ¡¾ SEM (standard error of the mean) and the capillary vessel density, LV function and other function measurements were analyzed statistically using the Student's t-test(P<0.05 was acknowledged as a significant statistic). |
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1) Experiment sets : In the first experiment the death rate of animals 4 weeks after the cell transplant was 28 percent (7/25) and there was no significant difference statistically between the control set and the treatment set. (control set 4/13, treatment set 3/12). 9 rats were used each for both the control and treatment sets, and the animal death rate for 2 weeks after the transplant was 30 percent (3/10) (Figure 4). |
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Figure 4 . Animal testing experiment flow chart |
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2) The effect of cell transplant treatment on LV function: There was no significant difference between the LVESD of the control set(6.8 ¡¾ 2.1 vs. 7.2 ¡¾ 1.9 mm) and the cell treatment set(9.1 ¡¾ 2.4 vs. 9.7 ¡¾ 2.9 mm) (p=NS). However fractional shortening 4 weeks after the transplant showed that the cell treatment set significantly improved compared to the control set(22.1 ¡¾ 3.9% vs. 39.2 ¡¾ 4.5%; p=0.023, Figure 5). The cell treatment set(48.1 ¡¾ 5.7%) also improved significantly compared to the control set(48.1 ¡¾ 5.7%) in ejection fraction (Table 1). |
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Figure 5. Echocardiography results of the control set (A) ad cell treatment set (B). Left heart movement is active in (A) but very much reduced in (B). LV fractional shortening was found to be significantly higher in the cell treatment set compared to the control set.. |
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[Table 1]. Echocardiographic Data |
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control group
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MSC group
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p value |
LV end-diastolic diameter (mm)
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9.7 ¡¾ 2.9
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9.1 ¡¾ 2.4
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0.73
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| LV end-systolic diameter (mm) |
7.2 ¡¾ 1.9
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6.8 ¡¾ 2.1
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0.87
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| LV diastolic ID (mm) |
7.8 ¡¾ 0.2
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7.3 ¡¾ 0.3
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0.10
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| LV systolic ID (mm) |
3.8 ¡¾ 0.3
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4.6 ¡¾ 0.3
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0.053
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| LV fractional shortening (%) |
22.1 ¡¾ 3.9
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39.2 ¡¾ 4.5
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0.023
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| LV ejection fraction (%) |
35.2 ¡¾ 3.1
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48.1 ¡¾ 5.7
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0.012
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| Septum thickness (mm) |
1.91 ¡¾ 0.01
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1.88 ¡¾ 0.03
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0.051
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| LV end-diastolic volume (mL) |
0.73 ¡¾ 0.03 |
0.63 ¡¾ 0.04 |
0.34 |
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LV: left ventricular, ID: internal diameter |
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3) The effect of cell transplantation on neovascularization : Change in vascular density was measured and neovascularization of the control set and cell treatment set was measured. The cell treatment set clearly increased compared to the control set in the border zone of the scar(8.9 ¡¾ 1.1 vs. 4.6 ¡¾ 0.6, capillaries per high-power field, p=0.021, Figures 6 and 7) , but there was no difference between the two sets in the scar zone(2.7 ¡¾ 1.5/hpf vs. 2.7 ¡¾ 1.0/hpf, n=7). The blood vessel with new positive cells shown in the von Willebrand factor confirms that mesenchymal stem cells contributed to making the blood vessel. (Figure 6). |
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Figure 6. Picture of cell transplanted to heart 4 weeks after mesenchymal stem cell transplant from human to animal model's left coronary artery occlusion. Vascular density is clearly higher in (A) than (B) (P=0.021). More vWF positive cells in the cell treatment set (C; x 400 magnification) |
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Figure 7. Vascular density is clearly higher in the border area of the infarction area where many cells were transplanted. |
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4) Confirmation of transplanted cells in myocardium after cell transplantation : Whether or not transplanted mesenchymal stem cells spread to the infarction area was confirmed by whether or not GFP was revealed. Cells with visible GFP were found in the cell treatment set (Figure 8) and we could confirm positive cells in GFP and myocardial cells' alpha-actin existed in the border area of the infarction area. |
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Figure 8. MT Masson trichrome) dye showing that transplanted cells exist mainly in the from border area of the myocardinal infarction (A; x 100 magnification, the blue area shows the fiber structure in the myocardinal infarction). Immunohistochemical staining shows that transplanted mesenchymal stem cells show alpha-actin (B' x 100 magnification). Cells showing GFP exist in myocardinal infarction area (C; x 400 magnification). Vascular density clearly increased in the border area of the infarction area where many cells were transplanted. |
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Heart functions improved when human mesenchymal stem cells were cultivated and amplified and inserted into myocardial infarction white rat models. This means transplanted mesenchymal stem cells provoked making of blood vessels around the infarction or branched into myocardial cells that revealed protein particular to myocardial cells. Therefore the results lead to a prediction that mesenchymal stem cell treatment has the effect of heart function recovery for patients of myocardial infarction. |
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