The Differentiation Of Human Mesenchymal Stem Cells Into Cardiomyocytes In Vitro

Backgrounds and aims:In recent years, research of basic study and clinic therapy of acute and chronic ischemic heart disease are developing greatly, especially in medication and intervention therapy. The mortality rate of acute myocardial infarction decreases, but disability rate of heart failure after MI increases incidences. The clinic therapy measures in common use now are medication, internal medicine intervention treatment, and surgical treatment. These measures most effects are improving local blood circulation, rescuing dead cardiac muscle, enhancing heart function, they can't reforge the coronary artery and regenesis the infarct cardiac muscle. Studies and applications of stem cells provide a novel treatment to cardiac infarct illness.Cellular therapy has been a new strategy to many tissue necrituc diseases. By transplant functional cells, it can substitute, repair and strengthen the biologic function of tissues or organs. Cellular transplantation and myocardial regeneration are the hotspot of today's study. Bone marrow mesenchymal stem cells become the most important research area because of their particular biologic characteristics. There are two types of stem cells in bone marrow, one is hematopoietic stem cells (HSCs), the other is mesenchymal stem cells(MSCs). BM-MSCs are non-hematopoietic cell group in bone marrow stroma, which derive from mesoderm.They have strong ability of self-reproduction, can produce not only hematopoietic cells and hematopoietic stromal cells, but also mesoblast and ectoderm cells cells. The current studies discovered that MSCs can differentiate into cardiomyocytes in vivo and in vitro. MSCs may be the perfect cell source for cellular therapy to ischemic heart disease base on their two advantages. First, it is convenient to harvest MSCs and the process does little harm to body. Second, self stem cells derived organs are not restricted by MHC. Therefore, MSCs have great prospects in clinic application. This task is to culture human bone marrow mesenchymal stem cells, study part of their biologic characteristics, observe the effect of 5-acacytine to MSCs, and find the influence factors of MSCs induced to cardiomyocytes. It will provide a basis for farther studies on animal transplantation and clinic investigation.Materials and methods:Bone marrow cells are collected from healthy volunteers aged from 8 to 23 years old. Bone marrow mononuclear cells were isolated form bone marrow by density gradient centrifugation method, and then purified by different ability of adhibition. Standard of passage must be held strictly. When cells proliferated reaching to 80% culture flask's undersurface, it must be digested and passage. Cell culture system is low glucose DMEM contain 10% fetal bovine serum (FBS). When MSCs reached logarithmic stage at passage 3, they were induced by 10μmol/L 5-azacytine for 24 hours and then cultured by DMEM-LG contain 5% FBS. Cells were digested and re-inoculated into 24-well plates with glass coverslips, the glass coverslips were treated with 1μg/mL fibronectin beforehand. Cells were harvested at 28 days after inducement, and immunocytochemistry method were used to identify antigen of muscle tissue and cardiac muscle, such as a-Actin, Desmin and cardiac special troponin T(cTnT).Result:1. The human bone marrow cells separated by density gradient centrifugation method had homogenous size and cell viability is higher than 95% tested by trypan blue staining. CD29 and CD44 were examined by flow cytometric analysis atpassage 3, the positive rate was 90.6% and 91.5% respectively. It confirmed the high purity of MSCs.2. Freshly separated bone marrow cells were little and round. MSCs began to adhere within 24 hours and the number of adherent cells increasing with time. MSCs had similar fibroblastic morphology, proliferated in colony, and arrayed along the cellular long axis in monolayer. By the time of ten days, MSCs reached logarithmic stage and clones connected with each other. MSCs get passage standard at the third week.3. Period of passage culture was short than primary culture. The first 24 to 36 hours was stagnant period; time from that to 3 to 5 days was log phase, MSCs expanded vastly; then followed by a confluent growth-arrested phase. During the log phase of growth, cells proliferated with a population doubling time of 33 hours. Most samples showed caducity over ten passages.4. Cellular morphology became big, arrayed consistently and proliferated slowly after induced by 10μmol/L 5-azacytine. When at 25th day, cells were digested and re-inoculated into wells with glass coverslips. Growing on the glass coverslips cell missed became multiplicate.5. Cells were identified the antigen of muscle tissue and cardiac muscle by immunocytochenistry method at 28 days after inducement. Results showed that part of the inducing cells were positive stained for a-Actin, Desmin 和 cTnT. The positive rate was 20%, 19% and 10% respectively. There was negative expression in MSCs with the same number of days which hadn't been induced by 5-azacytine.Conclusions:1. The density gradient centrifugation method is fit for the separation of human bone marrow mesenchymal stem cells and the activity is good. The purity is high through purified by different ability of adhibition.2. The DMEM low glucose medium including 10% fetal bovine serum is fit for the culture and amplification of human bone marrow mesenchymal stem cells in vitro.3. 5-azacytine can induce human bone marrow mesenchymal stem cells which are purified in vitro culture into cardiomyocytes and induced cells express muscle tissue proteins a-Actin and Desmin as well as cardiac special troponin T.4. The DMEM low glucose medium including 10% fetal bovine serum is fit for the culture research of human bone marrow mesenchymal stem cells induced to cardiomyocytes in vitro.