体外构建胚胎干细胞生长分化的三维培养模型

Construction of a three dimensional culture model for inducing embryonic stem cells differentiation in vitro

目的以液态胶原为支架,小鼠胚胎干细胞(ESCs)为细胞模型,构建ESCs胶-原复合体,旨在尝试建立一种能够实现干细胞生长、分化的三维培养体系。方法提取鼠尾胶原,观察ESCs在自制胶原条带内增殖的形态特征,并测定葡萄糖/乳酸活性。以ESCs源心肌细胞为目的细胞,利用免疫组化、RT-PCR及电镜技术评价胶原条带内ESCs进行自发性分化的能力。结果ESCs在胶原条带所提供的三维培养体系内生长、增殖状态良好,且彼此间能够建立细胞连接。胶原条带内部分ESCs能够自发性分化为心肌细胞。该心肌细胞均可表达心肌蛋白cTn-T,心肌转录因子NKX2.5及肌球蛋白轻链MLC-2 vmRNA,且肌小节结构发育成熟。结论以液态Ⅰ型胶原为主体的支架材料可以为ESCs提供良好的生长基质,促进其组织化发育。该实验初步明确了体外构建干细胞生长分化三维模型的可行性。

Objective To simulate the three-dimensional （3-D） growth pattern of stem cells in vivo by a 3-D culture system in vitro constructed by rat tail collagen scaffold. Methods Circular strands were prepared by mixing suspended murine embryonic stem cells （mESCs） with rat tail collagen. Growth profile of mESCs within the collagen strand was observed with phase contrast microscopy. Their metabolic activity was evaluated by glucose/lactic acid contents. To evaluate the effect of 3D culture system on ESCs differentiation, ES-derived cardiomyocytes were detected by immunohistochemistry, RT-PCR and transmission electron microscopy respectively. Results ESCs grew well in the 3-D culture system constructed by rat tail collagen. Cell connections can be found in those cell clusters formed within collagen stands, which indicated that tissue-like cultures should be produced during the process of 3-D culture in vitro. ESCs cultured by 3-D collagen strand differentiated into cardiomyocytes spontaneously. Conclusion Collagen provides an ideal growth matrix for ESCs proliferation in vitro and promotes ESCs differentiation towards tissue-like structures. Thus, the three dimensional culture system constructed by rat tail collagen can be applied to study ESCs differentiation in vivo.