Differentiation Of Bone Marrow Mesenchymal Stem Cell And P19 Cell Toward Cardiomyocyte And Effect Of The Cell Transplantation For Repairing Infarcted Myocardium

Acute myocardial infarction (AMI) is a familiar and common disease that severely threatens the elder's health.At the present time, the ultimate treatment to regenerate the infarcted myocardium is lacking.Although dissolving the thrombus and catheterizing in time in 4-6 hours after disease breaks out can return the blood of the infarct-associated vessels and save the dying cardiomyocytes, the reperfusion therapy only can limit the infarcted size and have no effect on the infarcted myocardium and the ischemic cardiomyocytes of the majority patients who have infarcted when they go to see the doctor. Lacking the ability of differentiation, the infarcted myocardium can only be replaced by the fibroblasts to form the scar tissue and have no systolic function. Thus the patients are easy to suffer heart failure, even sudden death, and their life quality is also very poor. Under this condition, someone puts forward the notion of cellular cardiomyoplasty(CMM) or cell therapy. CMM, in which one delivers appropriate donor cells into the injured myocardium, targets the basic pathophysiology of ischemic myocardium and represents a novel means of augmenting the myocyte number and contractile function.Fetal cardiomyocytes, fetal stem cells, skeletal muscle cells which were known as satellite cells, and bone marrow mesenchymal stem cells(MSCs) have been studied for the cellular transplantation treatment. MSCs can be obtained from bone marrow repeatedly by bone marrow aspiration and proliferate rapidly in vitro. The multipotentiality of mesenchymal stem cells (MSCs) has been re-recognized for these years. MSCs in various species, including human, can be easily isolated from bone marrow, adapted to exvivo expansion,and both in vitro and in vivo differentiated into multilineage cells such as bone, cartilage, adipocytes, heptocytes,neurocytes, endothelial cells, marrow stroma cells, smooth muscle, skeletal muscle and cadiomyocytes, disregard of ethical concern and immunological rejection. Because of these characters, the clinical use of MSCs for CMM appears to be much more advantageous.Embryonal carcinoma cells (EC ceIls) resemble the embryonic stem cells (ES cells) in some aspects, for example in differentiating potential, ultrastructure, and cell surface antigen and biochemical characters and so on.In addition, compared with ES cells, EC cells keep the undifferentiated state when cu1tured without feeding layer and LIF culture condition. The culture and genic manipulation of EC cells are easy .While, EC cells can transform from malignant type to non-malignant type during the differentiating cultllre processes. So researches pay more attention to EC ce1ls now. Pl9EC cell line is a euploid multi-directions differentiating potential stem cell line which was isolated from C3H/He mice carcinoma by McBurney etc. In vitro culture, undifferentiated Pl9 ce1ls can growth without feeding 1ayer in single layer, quickly amplify and keep special markers of ES cells such as surface glycoprotein SSEA-l and transcriptional factor Oct-3. Pl9 cells can be induced to many kinds of ce1ls including cardiomyocyte, skeleton muscle cells and neurons by cu1turing in vitro and the differentiating process resemble the early differentiation of embryonic cells. Therefore Pl9 cells are often considered as vitro model system using for research of early development of cells.When Pl9 cells differentiated toward cardiomyocytes, the expression of related genes of development and basic electrophysiological characters resembled the early process of normal development of mice cardiomyocyte. It was considered as a good cell model in vitro used to study cardiomyocyte development, and the differentiation of Pl9 cells toward cardiomyocyte had got more attentions.Therefore, the studies were trying to induce MSCs and Pl9 cells differentiate toward cardiomyocytes in vitro by some methods and reagents.Observed them morphologic change during differentiation, and detected the expression of early transcription factor and cardiac specific genes. Through investigating the biological characters of them, search for a cultural method feasible for cardiomyocyte differentiation of MSCs and Pl9 cel1s, increase the differentiating ratio of cardiomyocytes, afford groundwork for establishing a set of high standard and the most feasible cultural condition, lay groundwork for selecting cardiomyocyte stem cells on a large scale.After coronary artery occlusion,MSCs labelled with colloidal gold were implanted into myocardial infarct site via topical injection.The MSCs growth state and the action to heart infarction by MSCs transplantation were studied to investigate the possibility of the clinical use of implanted MSCs for ischemic heart disease.The experiment included 3 parts.Part I:We examined the isolation, purification, expansion and differentiation of the rat MSCs into cardiomyocytes in vitroMSCs were isolated from the femora and tibiae of SD rats (3w, 15–25g) with wall-attaching cultivation. MSCs from bone marrow were cultured at 37°C in humid air with 5% CO2 in Iscove's modified Dulbecco's medium (IMDM), 10% fine fetal bovine serum ( FBS ), 1% L-glutamine, penicillin (100μg/ml)/streptomycin (100μg /ml). The medium was firstly changed 24 h later, after that one time every 3d. The morphology of MSCs was observed under a phase contrast microscope. Primary fibroblast-like MSCs were found to sparsely attach to the wall of culture dish 4-8 h after incubation, began to proliferate on day 2-4, and finally reached 80%-90% confluence on day 3-5. After a series of passages, the attached cells became morphological homogeneous without obvious hematopoietic cells, but they gradually became larger in size and flattened-shape cells increased.To induce rat MSCs differentiation, 48h after MSCs were isolated ,MSCs were cultured in differentiation-inducing medium composed of IMDM, 5,10,15μm 5-aza, 10% fine FBS and penicillin (100ug/ml)/streptomycin (100μg/ml) for 24 h and then transferred into the culture medium mentioned above.At the same time,we try on other inducer such as DMSO(0.6%,0.8%,1.0%),OT(1×10-7,3×10-7,5×10-7μmol/L),BMP-2(0.1,0.2,0.4ng/ml),FGF-2(0.8,1.0,1.2ng/ml)for24 h,96 h,72 h,72 h respectively and then transferred into the culture medium.Morphological observation, immunocytochemistry, RT-PCR were performed to identify whether there were cardiomyocytes in various kinds inducer induced MSCs. After inducer treatment, in 1-2 w some MSCs began to change from fibroblast-like morphology to rod- or ball-like shape with centric oval-shape mononucleus or multinuclei,representing the features of myotube-forming cells. These cells gradually proliferated and connected with adjoining cells in 3-4 w after the treatment. To confirm the cardiomyogenic differentiation of MSCs, MSCs clusters were immunocytochemically stained with desmin,α-sarcomeric actin and cTnT. The rates of the differentiated MSCs were calculated in each group. The number of cells stained positively in the 5μmol/L 5aza,1.0% DMSO,3×10-7μmol/L OT,0.2 ng/mL BMP-2 and 1.0 ng/mL FGF-2 was greater than that of other groups(P<0.01). Desmin,α-sarcomeric actin-positive cells made up higher of all MSCs than cTnT-positive cells .laser scanning confocal microscopy indicated that desmin(α-sarcomeric actin ) was signed red, cTnT was green.When they were both been showed,it changed to yellow. Transmission electron microscope showed that the induced cells had a cardiomyocyte-like ultrastructure: the nuclei were positioned in the center of the cell. a lot of mitochondria , rough endoplasmic reticulum and ribosome were founded in plasm, and paralleled myofilaments could be seen beside the adge of membrane. RT-PCR assessment showed that the differentiated cells began to express GATA4 from day 7 to day 28 of differentiation and began to expressαMHC from day 21 to day 28 of differentiation. GATA4 andαMHC expression increased obviously on day 21 and day 28 respectively(P<0.01). After MSCs were induced by BMP-2,gene expression were significantly higher than that of other groups at every time point (P<0.01).PartⅡ:Effect of BMP-2 and FGF-2 on cardiomyocyte differentiation of P19 cells in vitroThe cells were cultured in medium containing 0.2ng/ml BMP-2 and 1.0ng/ml FGF-2 in tissue dishes having a thin layer of soft agar. Cell masses were built after P19 cells were suspension cultured for 24 hours and then globular cell masses grew up every day and grew into EBs after 4 days.At 4th day cell aggregates/EBs were transferred to coverslips in 24 well plates, allowed to adhere and cultllred in medium without BMP-2 and FGF-2. The EBs were regular and the cores were observed to be darker and have high cell density by inverted microscope. After the EBs were transferred, cells grew from the border of EB and formed monolayered peripheral outgrowth. Central cells were undifferentiated cells, which were multilayered,smaller and had high nuclear-cytoplasmic ratio and grew faster. Some cells in the peripheral outgrowth became elongated,larger and radial.immunocytochemical staining result: metamorphotic cells in the peripheral outgrowth of experiment group expressedα-sarcomeric actin and cTnT.α-sarcomeric actin-positive cells made up higher of all MSCs than cTnT-positive cells .LSCM result: after stimulated by laser,α-sarcomeric actin was signed red in the cells of the peripheral outgrowth, cTnT was green. When they both showed, it changed into yellow. Under transmission electron microscopy, undifferentiation P19 cells had a large nucleus and plenty of cell organ which distributed around nucleus, and there were abundance hepatin granule. The differentiated cells derived from Pl9 cells had the characters of cardiomyocyte in early stage: there was a single orbicular-ovate cell nucleus and plenty of cell organs, including chondriosomes,endoplasmic reticulum and golgiosomes. paralleled myofilaments regularly arranged in the cytoplasm without forming sarcomeres.PartⅢ: we demonstrated the possibility of survival,migratory,differentiation and the effect to left ventricular function of rat MSCs in infarcted myocardiumThe models of injured hearts were induced by left coronary artery ligation. The rats were randomly divided into 3 groups according to the means dealing with MSCs and the components injected into myocardium. GroupA composed of twelve rats ,the rats received exogenous transplantation of MSCs after myocardial infarction.Group B composed of twelve rats , the rats received exogenous transplantation of MSCs induced by 5-aza after myocardial infarction. Group C composed of twelve rats is Acute Myocardial Infarction control group, the rats was injected 100μl IMDM after myocardial infarction.Changes of cell morphology of transplanted cells were observed in 2th week and 4th week after transplantation by HE stainning. The proteins of cTnT andα-sarcomeric actin were detected by immunocytochemistry.HE stainning showed that transplanted cells could survive in area of inferepicardium,MI and beside normal myocardium.Immunocytochemistry results: (1) In the site of transplantation, some of the cells shown positive expression ofα-sarcomeric actin and the number of positive cells was more in 4th week than that in 2th week. (2) In 2th week, the cTnT positive cells was very little,in 4th week,cTnT showed positive expression in some of the transplanted cells and many of the positive cells located beside normal myocardium. The results showed that some of the MSCs possessed the features of myocyts in 2th week and some of myocyts possessed the features of cardiomyocytes in 4th week.In 4th week after transplantation, index of hemodynamics of heart of three groups were tested.The results show that: (1) The difference in Cardiac function between cells transplantation groups(Aand B) and the control group(C) has remarkable statistical significance(P<0.01) at 4 week after intramyocardiac injection. There were no significant difference between group A and group B. (2) Compared with control group, LVEDP was lower in treatment group than control group (P<0.01), LVSP and±dp/dtmax were higher in treatment group than control group (P<0.01). These results indicate:The exogenous MSCs transplantation may be a novel approach to improve ventricular function in transmural infarct.summary1 MSCs from rat bone marrow can be differentiated into cardiac-like muscle cells with 5-aza,DMSO,OT,BMP-2,FGF-2 inducement in vitro. Electron microscopy and immunocytochemistry revealed that early differentiated cells exhibited characteristic consistent with cardiomyocyte.2 The Changing expression of GATA4 in 5-aza,DMSO,OT,BMP-2,FGF-2 induced MSCs with induced time also suggests that it might involve in transcriptional regulation of the differentiation from MSCs to cardiomyocyte-like cells.3 Combination of BMP-2,FGF-2 exposure and suspension culture could effectively promote Pl9 cells aggregation, form cystoid EBs, and induced cardiomyocyte differentiation.4 The exogenous rat MSCs can survive, migrate, differentiate into cardiac-like cells in the rat injured hearts.5 MSCs were implanted into ischemic myocardium. The fate of implanted MSCs were determined by external signals from the microenvironment.MSCs were demonstrated myogenic difertentiation under myocardial environment and were able to improve the cardiac function. When transplanted into infarcted hearts, 5-aza-induced MSCs have no advantage over uninduced MSCs.