Study On Cardiac Differentiation Of Embryoid Bodies And Its Molecular Mechanism

Embryoid bodies, comparable in their morphology with that of the early developmental stages of mammalian embryos, possess globular structures. They are the ideal materials to study early differently developmental stages of embryos and to explore the differentiation organisms of biology in vitro. Because of this it offers hope for the regenerative medicine. Embryonic stem cells can keep the undifferentiated state in vitro via the addition of differentiated inhibitor like trophoderm and leukaemia inhibitory factor. The process of differentiation will switch on after the removal of inhibitors. What’s more, suspension culture of embryonic stem cells or induced pluripotent stem cell in vitro will lead to the formation of three dimensional globular structures which are known as embryoid bodies. Embryoid bodies can differentiate into all three primary germ layers, which have a remarkable resemblance to normal early primate embryos in vivo. The heart is one of the first identifiable tissues to develop in vertebrate embryos, so it’s very effective to study cardiac differentiation via embryoid bodies. The complicated operational process, the heterogeneity of embryoid bodies and the different size of EBs which influence the synchrony of differentiation process significantly restrict the development of traditional hanging drop method. Even so, the majority of preparation methods are still derived from hanging drop method. In this study we found a new system which can promote the quality of EBs compared to the traditional preparation process from the uniformity of morphology to the efficiency of cardiac differentiation. Operational sequence is very simple and convenient. This system is not only suitable for the system of mechanical automation, but also fits the system of large-scale drug screening via the material from stem cells.In this study, mouse embryonic stem cell line and induced pluripotent stem cell were used for the materials to prepare the embryoid bodies. Fortunately, we discovered that a culture plate which was named PCR Microplate from corning company could enhance the efficiency of embryoid bodies’ preparation by virtue of centrifugation. It got rid of the adverse effect of aggrewells because of its shoal grooves. iPS cells used in my research are induced by mouse embryonic fibroblasts, which has proved by different methods, such as:RT-PCR, real time PCR, AP dying, immunocytochemistry, embryoid bodies differentiation, tertom, to possess all characters of iPS cells. There is little difference founded with the embryoid bodies derived from D3 cell during the process of preparation. During the preparation process of embryoid bodies using PCR microplate, we also investigated some other elements, such as:original density, centrifugal force, which might affect the quality of embryonic bodies. After that, we gradually refined the procedure of preparation to produce ideal embryonic bodies. And then we found that the ratio of myocardial beating of EBs which were through centrifugal processing was higher than static EBs. For another hand, we also assayed the expression of marker genes and embryonic stem-related genes which indicated the good state of EBs. Wnt action is complex, however, and may inhibit or promote differentiation depending on spatio-temporal context and whether the canonical signaling pathway is activated. We found the gravity can influence the expression of β-catenin which enhanced the ratio of myocardial beating significantly.My study on new preparation method can offer original materials for researchers to study cardiac development. It not only possesses good synchronicity and reproducibility traits, but also can be taken example by iPS to explore cell therpy on heart disease.