Ischemic heart disease accounts for 50% of all cardiovascular deaths in the world. The progressive nature of ischemic heart disease and restenosis after percutaneous intervention have exhausted patients and their doctors. Researchers have investigated cell transplantation and considered it as an alternative treatment for heart disease1.
Stem cell research holds a hope for better treatment of many diseases particularly those like myocardial infarction, which are characterized by severe damage to tissue. In the last few years, a major effort has been made in an attempt to identify immature cells capable of differentiating into cell lineages different from the organ of origin to be employed for the regeneration of damaged heart2. So, regenerative medicine applies the basic stem cell knowledge to develop specific cells or tissue to replace the original cells or tissue that have been degenerated, injured or damaged by different processes. This is the basic concept of the promising cell and tissue based therapy that would have a potential to make many chronic disease as diabetes mellitus type one, myocardial infarction and others to be curable3. Embryonic stem cells and bone marrow stem cells have been extensively studied and dramatic advances have been made in their clinical application in heart failure of ischemic and non ischemic origin. The discovery that hematopoietic stem cells can acquire cell lineages different from the organ of origin has started a new intriguing scientific revolution4–13.
It is now accepted that an adherent population of cells isolated from bone marrow is multipotential progenitor cells, which can differentiate into muscle, cartilage, bone, fat and tendon14–19. Researchers reported an experimental application of bone marrow stem cells for the regeneration of the infracted heart16,17.
Other researchers demonstrated that autologous transplantation of bone marrow cells improved damaged heart function20.
Vimentin is an intermediate filament of protein that is present in the sarcoplasm of cardiac myocytes and plays a role in nuclear centration, organell movement and cell shape. Evaluation of vimentine is considered a monitor for cardio -protection against ischemia21.
The present study was designed to test the therapeutic effect and the healing capacity of autologous bone marrow stem cells and their role in regeneration of myocardium in cases of myocardial infarction in rabbit through assessment of vimentine content of the regenerating cardiomyocytes.
MATERIALS AND METHODS
Thirty adult male albino rabbits weighing (1.25-1.5kg) obtained from animal house of Cairo Faculty of Medicine. Rabbits were fed a basal diet of ordinary bread, vegetables and liberal supply of water and after acclimatization to the laboratory conditions they were divided into 2 major groups. Group I (10 rabbits) was considered as - ve control group and each animal received a single subcutaneous (s.c.) injection of the vehicle of isoproterenol (0.05% ascorbic acid dissolved in 0.9% Nacl)21.
Group II (20 rabbits) was considered as experimental group and divided into 2 equal subgroups (IIa, IIb) 10 rabbits each. Subgroup IIa was used as positive control and myocardial infarction was induced by receiving a single S.C. injection of isoproterenol (Fulka, Steinheim, Germany) 150 mg/kg. Subgroup IIb was treated as subgroup IIa but received intravenously (i.v.) undifferentiated bone marrow derived Mesenchymal Stem Cells (MSCS) in a dose of (1.4-1.6 X 106ml in 5ml sterile saline) 2 weeks after myocardial infarction22.
Isolation & preparation of mesenchymal stem cells (MSCS):
Bone marrow extracts contain heterogenous cell populations; MSCs represent a small fraction of total mono-nucleated cells within the bone marrow. Mesenchymal stem cells were obtained under complete aseptic condition using laminar flow22. Cells were incubated in flasks containing RPMI media (Gib6 AG. Germany) and resuspended in 4 ml of 20% of fetal calf serum, 10% antibiotic and antimycotic (Biochrom AG, Germany) and incubated in humidified 5% CO2 and 95% air atmosphere at 37°C for 24 hours.
Separation of mesenchymal stem cell from flask:
The supernatant was removed from flasks and the colonies of mesenchymal stem cell adherent to flasks borders were left for 10 days18, then harvested after being treated with 0.25% trypsin EDTA (Biochrom AG, Germany) rinsed with RPMI twice for 3 minutes. The conical tube was filled with suspention of culture media with trypsin and washed with RPMI centrifuged. Supernatent was removed and the precipitate was taken to assess viability.
Assessment of Cell viability by microscopy:
Cell viability was detected using trypan blue stain and counted by hemocytometer.
One million cells per 5 ml conical tube were incubated as per the manufacturer's instructions at room temperature for 10 minutes with monoclonal antibodies labeled with Phycoerythrin (PE) against one of CD34 or CD4423. Flow cytometry was used as an additional mean of characterization of MSC preparation.
Transfection procedure (cell tagging):
According to some authors undifferentiated stem cells were transfected 24 h before transplantation24. Examination of the transfected cells in the tissue of rabbits injected with stem cells was done after sacrifaction. Specimens from lower left ventricular wall were taken and examined under fluorescent microscope.
Animals were anesthetized by ether and sacrificed 2 weeks post-implantation. The hearts were perfused with 4% paraformaldehyde and samples were collected from the lower left ventricle. Paraffin sections of 4μm thickness were prepared and stained with H&E25.
Paraffin blocks were prepared from specimens already fixed in 4% paraformaldehyde. Sections were dewaxed in xylene, rehydrated and pretreated with 3% hydrogen peroxide for blocking of endogenous peroxidase activity. Microwave -assisted antigen retrieval was then performed for 20 minutes. Sections were incubated over night at 4°C with monoclonal anti-vimentin anti body ready for use (optimum dilution) (−3 clone vim 3b 4) mouse M antibody IgGA - Kappa from Lapvision Inc., Fremont, CA, USA. After washing with PBS, sections were incubated with biotinylated IgG and then with strept avidin-peroxidase conjugate (Zymed Crop). Sections were then washed with PBS and incubated with diaminobenzidine (DAB) for 5 minutes to detect immunoreactivity and counter stained with MayER's haematoxylin and examined microscopically.
For the morphometric study:
The image analyzer Leica Q 500 MC program in the Histology Department, Faculty of Science Alazhar University was used. Data obtained from image analyzer were subjected to statistical analysis using student t- test.
Assessment of cell viability:
The viability of mesenchymal stem cells was confirmed by trypan blue exclusion. The live cells were not stained and the dead cells were stained blue (Fig. 1).
Analysis for immunophenotype of MSCS preparations was done to assess purity. The cells were negative for CD34 and positive for CD44 mesenchymal markers (Fig. 2).
Fluorescent Microscopic Evaluation:
Fluorescence microscopy image demonstrating the green fluorescence of mesenchymal stem cells labeled with enhanced green fluorescent protein in vivo two weeks after implantation (Fig. 3).
Group 1 (-ve control):
Cardiac muscle cells of the control group showed normal histological architecture. Cardiac myocytes exhibited acidophilic sarcoplasm and centrally located nuclei (Fig. 4).
Subgroup IIa (+ve control):
Cardiac myocytes appeared paler with faint sarcoplasm. Areas of necrosis with vacuolated cells and pyknotic nuclei. Some cells appeared fragmented (Fig. 5).
Subgroup IIb (received autologous mesenchymal stem cells):
Few vacuolations of cardiac myocytes with normal cells were detected. The nuclei appeared central with nearly normal histological pattern (Fig. 6).
Group I (-ve control):
Immunostaining for vimentin showed evident brown cytoplasmic staining in the cardiomyocytes, smooth muscles of blood vessels and vascular endothelial cells (Figs. 7,8).
Subgroup IIa (+ve control):
A faint brown staining of sarcoplasm of cardiac myocytes, smooth muscles and endothelial cells of blood vessels was detected compared to the control denoting weak immunoreaction for vimentin (Fig. 9).
The brown immunoreaction for vimentin was increased compared to the previous group in the sarcoplasm of cardiac myocytes, smooth muscles and endothelial cells of the newly formed small blood vessels (Figs. 10,11).
Optical density for valentine positive immune reactivity showing a significant drop of the reaction in the vascular wall and cardiac muscle sarcoplasm with a mean of -28.4681 and 29.1306 compared to the control and followed by a significant elevation in the group injected with undifferentiated mesenchymal cells (-25.5425 and 41.46545).
Advances in the past five decades including risk factor modification, use of β-blockers and effective percutaenous intervention, have reduced the mortality and morbidity of ischemic heart disease. However, ischemic heart disease still accounts for 50% of all cardiovascular deaths and nearly 40% of heart failure incidents. Bone marrow mesenchymal stem cells (MSCS) reside in bone marrow and are multipotent cells that can differentiate into lineages of mesenchymal tissues such as bone, cartilage, fat, tendon and muscle26–28.
Many researchers have investigated (MSCS) cell transplantation as an alternative treatment to acquire cardiac cell lineages and reconstitute the myocardium lost after infarction29 infarction promotes the initiation of a repair process that leads to the formation of a scar which does not posses the biochemical, physical and functional properties of the uninjured tissue and negatively affect the performance of the heart2,4–6,10–13.
Mesenchymal stem cells are used in regenerative medicine for a primary objective which is the complete structural and functional recovery of the damaged organ18,30–32 including spinal cord injury and myocardial infarction. Several in vitro studies have shown that bone marrow - derivedd stem (BM-MSCS) cells could be programmed to become cardiac myocytes33.
In the present study, (BM-MSCS) were isolated, cultured and characterized. The immunophenotype of (BM-MSCS) is negative for CD34 which indicated that they were not hematopoietic stem cells and positive for CD4429.
Separated bone marrow mononuclear cells were transplanted by intravenous injection as the safety of the approach has been documented and intra-cardiac injection of cells in the infracted wall is problematic29.
Spontaneous mobilization of the transplanted cells occurs after infarction. These circulating cells home to the region of injury rescuing the dead tissue.
BM-MSCS were marked with green florescence protein to be detected easily by florescence microscopy. Immunoreactions were avoided because autologous cells were used in the present study34.
Administration of a single overdose of isoproterenol (150 mg/kg s.c) evoked myocardial infarction1 while myocardial regeneration in the group IIb (received BM - MSCS) was denoted by histological examination.
In H&E stained sections; the affected group showed cardiac myocyte with vacuolated sarcoplasm and areas of necrosis35,21 which decreased in the group IIb (received i.v. injections of BM-MSCS). The possibilities of the positive outcome of (BM-MSCS) administration were due to de novo formation of myocytes and vascular structures and activation of resident progenitor cell via a paracrine effect mediated by the implanted cells17,34,36–44.
Vimentin is an intermediate protein that has been thought to play a role in nuclear centration, organell movement and maintenance of cell shape. Assessment of vimentin content explored cardioprotection against ischemia. This was demonstrated immunohistochemically where they can be detected in the sarcoplasm of cardiac myocytes, vascular smooth muscles and endothelial cells36,45.
In the present work, immunohistochemical staining of the treated animals showed relatively increased sarcoplasmic immuno-reaction for vimetnin in cardiac myocytes, smooth muscles and endothelial cells of blood vessels compared to those of the affected animals which showed faint cytoplasmic reaction. The vimentin protein was more expressed in the control animals compared to the experimental groups.
Bone marrow mesnchymal stem cells are easily isolated and cultured. Transplantation of autologous undifferentiated mesnchymal stem cells i.v. in rabbit could be an effective method for myocardial regeneration after infarction. So, (BM-MSCS) autologus transplantation could be considered a successful line for treatment of cardiac degenerative diseases as myocardial infarction.
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Keywords:© 2010 The Egyptian Journal of Histology
Isopreterenol; auto logustransplantation; stem cells; myocardial infarction.