| As a proliferative cell line, embryonic stem cells (ESCs), capable of propagating in undifferentiated state, have the potential to form various kinds of adult tissue cells. It is verified that ESCs can differentiate into cardiomyocytes (CMs) with excitability and spontaneous contractions under appropriate conditions. These cells have the similar structure and function to in vivo CMs, and even the same ion channels to control pacing. These features of ESCs suggest it is possible to apply cellular transplant and tissue repair to the therapy of heart diseases. However, since the molecular mechanisms and signal pathway of ESC differentiation are still unclear, it is hard to control the process of its differentiation. The recent studies demonstrate the low differentiation rate of ESC, that is, only few embryoid bodies (EB) can differentiate into the beating CM. As some receptors can be detected in the ESC differentiation, and the expressions of oxytocin and receptors in cardiomyocytes, oxytocin arouses great interests as a new inductor. However, although oxytocin does increase the beating efficiency of EB in ESC differentiation, it causes the increased differentiation only at early stage and no improvement in ultrastructure of differentiated cells. To further study the ESC differentiation mechanism and the effect of microenvironment on the differentiation, we studied the effect of the interaction between cell and cell, and cell and matrix on the differentiation of ESC into ESCM, especially at the later stage. Our study is to clarify the mechanism of the promoted differentiation of ESC into CM.Our study can be divided into two parts:Part 1. The effect of oxytocin on the differentiation of CGR8 cells. In this part, the classical induction of ESC was conducted, that is, EB from the hanging-drop culture of ESC was induced to differentiate, and then cells were treated with 10-7M oxytocin. The structures and functions of differentiated cells (ESC-derived CM, ESCM) in two groups (experimental group and control) were observed and analyzed. The results showed the formation of EB in the induction with the treatment of oxytocin and the appearance of spontaneous beating cells on the 8th day. The expressions of MLC-2V and GATA4 increased, suggesting the promoting effect of oxytocin on the differentiation of ESC into CM at the early stage. The immunohistochemistrical results on the 10th day showed thatα-sarcomeric actin and cTnT were expressed in cardiomyocytes in the experimental group, and with the treatment of oxytocin, the distribution of the expression ofα-sarcomeric actin and cTnT was more similar to that of primary CM. The results demonstrate that oxytocin can promote the early differentiation of ESC into CM in mouse.Part 2. The effects of 3-D culture and co-culture on the differentiation of ESC into CM induced by oxytocin. The structures and functions of the differentiated cells in each group (common group, 3D group, co-culture group and 3D plus co-culture group) were observed and analyzed. It was observed oxytocin could promote the differentiation of ESC with the unsatisfactory results (the average of beating EBs was 17.76±5.99% and the autorhythmicity of excitement contraction could not maintain for a long period of time). On the 18th day, the number of beating EBs started to decrease significantly. In the 3-D culture, the average of beating EBs was 22.53±3.75% and the autorhythmicity of excitement contraction maintained for longer period than that in the 2-D culture. Surprisingly enough, in vitro simulative microenvironment of heart, the number of beating EBs was increased significantly and the autorhythmicity of excitement contraction maintained for longer period. The ultrastructure of ESCM showed the typical sarcomere and intercellular intercalary disc (fascia adherens, demonsomes and gap junctions) in vitro simulative microenvironment of heart. We suppose that the primary CM may promote ESCM to form more full-grown sarcomere, which provides the base for ESCM to have the function of CM.The results of our study show that the induction of oxytocin contributes to the early differentiation of ESC; the cardiac microenvironment is the one of the key factors to promote the differentiation of ESC; 3-D culture plus co-culture can improve the differentiation of oxytocin-induced ESC at the later stage, contributing to the oriented differentiation of ESC into CM and the longer period of autorhythmicity of excitement contraction. All these factors may be associated with the interaction of cell and cell or cytokines secreted by the primary CM.In short, our study shows that 3-D culture plus co-culture can significantly promote the differentiation of ESC into CM with excitability and spontaneous contraction, which provides a new method to control specific differentiation of ESC in vitro and plays an important role in the application of ESC in tissue engineering and medical regeneration. |
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