Differentiation Characteristics Of Rabbit Bone Marrow-derived Mesenchymal Stem Cells Induced By Cardiac Microenvironment

In recent years, there has been a breakthrough in the field of cardiovascular therapeutics, named as cellular cardiomyoplasty (CCM), which is an outside intervention to support the reparative process in the heart through transplantation of stem/progenitor cells or cardiac cells, or mobilizing peripheral blood or bone marrow-derived stem cells into the site of cardiac injury. CCM with stem cells is a possible option to reverse the deleterious hemodynamic and neurohormonal effects after MI;various preclinical animal studies show the potential of stem cells to regenerate myocardium, improve perfusion of the infarcted area and improve cardiac function. Some clinical studies indicate that implanted stem cells could transdifferentiate into cardiomyocytes, vascular smooth muscle cells and endothelial cells, decrease apoptosis of hypertrophic myocytes in the peri-infarction region, decrease size of fibrosis scar, enhance myocardial blood flow so as to improve perfusion, rescue hibernating myocardium, excrete cytokines or growth factors causing proliferation of endogenous cardiac myocytes and enhance resident cardiac stem cell function.Recently, bone marrow stromal cells(MSCs)have been considered as a proming candidate because of their some advantages such as easiness to obtain by a simple routine bone marrow aspiration, ability to self-renew. Bone marrow mesenchymal stem cells(MSCs)are capable of self-replication and renewal. Under the certain condition they will differentiate into multiple lineages cells, including osteoblast, chondrocyte, adipocyte, eurocyte, uscle cells and so on. A lot of evidence have indicated that MSCs engrafted into the ischemic myocardium will regenerate the necrotic cardiomyocyte and endothelium, even the Purkinje fiber, promote the perfusion through secretion of many growth factors, improve the heart function post-infarction. The clinical therapy based on the utilizing of MSCs pluropotential has become the new hope for numerous chronic heart failure patients. Many species MSCs'differentiation into cardiomyocyte-like cells can be induced in vitro. After the inducement, MSCs have adopted some of the cardiomyocyte's phenotype, with the expression of the cardiac specific proteins, and even beat spontaneously. But there is not enough experiment on human MSCs'inducement research. We are lack of the corresponding proof about the changes of morphology, histology and molecular biology of the cells during the differentiation after the inducement.Data derived from animal experimental study and clinical observation have demonstrated the feasibility of MSCs transplantation therapy for myocardium injury. After injection into the damaged region, MSCs incorporated and intergrated into native myocardial fibers and expressed cardiac specific markers and resulted in cardiac function improvement. However, some other research group reported that the graft cells do not express cardiac markers unless MSCs treated by 5-azacytidine pre-transplantation. Therefore, more experiment evidence should be provided to confirm the hypothesis that MSCs could undergo milieu-dependent differentiation in the host heart tissue. However, the precise understanding of the molecular control on this differentiation needs some further experimental work. It presumed that tissue environment dictated the fate changes of MSCs and MSCs trans-differentiated into the specified cells which surrounding them. There may give rise to another question whether MSCs undergo the same fate in co-culture system with cardiomyocytes in vitro as them in vivo or not. It is well-known that the number of cardiomyocytes only is one third of the whole cell population in heart, however, which type of these cells guide thereprogram of MSCs into cardiomyocytes?However, the claim that adult stem cells can be converted into multiple unexpected cell types is also questioned by some experts. To give a further investigation on the potential cardiomyogenic differentiation of MSCs and explore a new strategy for the therapy of myocardial infarction with cellular transplantation and gene engineering, the present study was performed in several parts as following:To observe morphology and growing status of Bone Marrow-derived Rabbit Mesenchymal Stem Cells (MSCs) in vitro,make up an optimum method of culture and isolation of MSCs,research cellular characteristic of MSCs and established foundation of cardiovascular therapeutic developments,the methods and conditions of harvesting,isolating,cultivating and identifying MSCs were studied. The Investigation on the Cardiomyogenic Differentiation of MSCs in a"Cardiac-like"Milieu in vitro to investigate the cardiomyogenic of MSCs when exposed to a"cardiac-like"milieu in vitro. MSCs was marked with 4, 6-diamidino-2-phenylindole(DAPI), and Primary cardiac myocyte cultures were prepared by enzymatic dissociation of ventricular tissue from neonatal rabbit according to methods described previously. To increase the cardiac myocyte density,a two-step differential attachment technique was used. After dispersed cells were incubated on culture dishes for 90 min at 37°C in 5% CO2 incubator,non-attached viable cells were collected and incubated on another culture dishes at 37°C in 5% CO2 incubator. Prepared MSCs were planted into the cardiomyocytes dishes to establish a co-culture system. Supernatant liquid of the cardiomyocytes added into the MSCs dishes and MSCs concultured with fibroblast as control. The morphologic changes of MSCs were observed and the cardiac specific proteins were detected by Immunocytochemical staining, RT-PCRand Western-blotting analysis. To explore electrophysiological characteristics of Cardiomyogenic Differentiation of MSCs in a"Cardiac-like"Milieu in vitro both in normal and hypoxia condition under whole cell recording mode of patch clamp technique.The results were as follows:1.After 24 hours in primary culture, the cells adhered to the plastic surface. After 2-3 days, the cells were shuttle-shape. The cells began to form a few clones at day 8~12. The cells fused into monolayer and the shuttle-shape cells became long, obvious directivity and eddy-shape. After 24 hours, the cells of passage adhered to the plastic surface, extended to become shuttle-shape and began to proliferate rapidly. After 7~10 days, the cells overspread the culture bottle bottom, and kept the same figure characteristics of primary culture. With passage's number increasing, the cells were purified and the shuttle-shape of them changed to plainness and bounty, cytokinesis decreased, and cytoplasm was loosen and there were vacuoles in cytoplasm. When came to the tenth passage, the part of the cells became round, refraction of them inceased and some of them died. With passage's number increasing, the clone formation ratio decreased gradually. After the ten passage, the new clones weren't found.2.The gradient centrifugation combinded adherence culture can be used as an efficient method to obtain the purified rabbit mesenchymal stem cells at the third passage. A standardized technique platform was successfully established for isolation, cultivation, purification, identification and amplification of rabbit MSCs. Highly homologous MSCs were isolated and obtained . They may be the cell source for extended experiments, because they maintained stable biological characteristics at even passage 10 in undifferentiation state.3.In certain condition,rabbit MSCs can be differentiated into osteogenic cells and adipogenic cells.4.The experimental results showed that the MSCs cultured of all groups did not show any contraction within 16 days. However, in coculture groups, after 24 hours, we observed that some spindle-like or triangle-like MSCs became adjacent to the beating cardiomyocytes and after 48 hours, the MSCs grew in colony and MSCs did not beat. We also found that the MSCs cocultured with cardiomyocytes grew slowly(data not shown). Especially, with time on, more and more MSCs appeared depauperate and even dead.5.It was interesting to find that the coculture of rabbit MSCs and neonatal rabbit cardiomyocyte could induce MSCs to express cardiac specific protein. Immunofluorescence examination and RT-PCR analysis clearly detected cardiac myosin heavy chain and cardiac-specific troponin I in the coculture of rabbit MSCs and neonatal rabbit cardiomyocyte,whereas did not find in Supernatant liquid of the cardiomyocytes added into the MSCs dishes and MSCs concultured with fibroblast. Western-blotting analysis could also detect the expressions of troponin in the coculture of rabbit MSCs and neonatal rabbit cardiomyocyte , but could not detect it's expressions in the other groups.6.To study the electrophysiological characteristics of ion channels and action potential of MSCs cocultured with cardiomyocytes of rabbit under whole cell recording mode of patch clamp technique. The functional expression of ion channels occurred during MSCs differentiation, the characteristics of sodium and calcium channels and action potential of MSCs 1w after cocultured with cardiomyocytes were very similar to normal cardiomyocytes.7.In hypoxia condition , the changes of the ICa and INa densities and characteristics of their current-voltage curves were very similar to normal cardiomyocytes under whole cell recording mode of patch clamp technique. The width depressed and the time course shortened of action potential were similar to normal cardiomyocytes. The peak value of sodium channels MSCs 1w after cocultured with cardiomyocyte in hypoxia condition significantly decreased compared with normal MSCs cocultured with cardiomyocytes,while the peak of calcium channels did not show significant changes.In this study, the mature methods of isolation and culture of MSCs was set up and build a foundation for a further investigation on the growth kinetics, differentiation of MSCs and their application value in cell transplantation. This study proved that a"cardiac-like"milieu in vitro could initiate the cardiomyogenic differentiation of MSCs;Importantly, with this coculture experiment as a useful research method in vitro, we may directly observe the reciprocity between MSCs and cardiomyocytes more conveniently in vitro and carry out a further investigation on the differentiation mechanism of MSCs thereafter. This study also showed that to obtain cardiomyocyte phenotype in vitro, direct contact of MSCs with cardiomyocytes is essential. This study, for the first time, has demonstrated the changes in electrophysiological characteristics of coculturede MSCs in hypoxia condition condition, and has provided relevant experimental data. Cell transplantation, as a revolutionary idea, has opended a new exciting perspective for the treatment of infarcted myocardium.