一种利用STO饲养层细胞制备拟胚体的新方法

A Novel Method to form Embryoid Bodies Using STO Cells as Feeder Layers

建立了一种利用STO饲养层细胞制备拟胚体的新方法。该方法选用生长至80%饱和密度的STO细胞,经丝裂霉素C(10 mg/ml)处理4 h后以8×104 cm-2的密度接种培养12 h,制备饲养层,再将ES-D3细胞以1×104 cm-2的密度接种其上,首先用含mLIF的DMEM培养液培养24 h,再更换拟胚体诱导培养液,5～9天后获得了各成熟阶段的拟胚体。形态结构和分化潜能等研究表明,该方法制备的拟胚体结构典型,具有产生3个胚层谱系来源的功能细胞的潜能。与传统拟胚体制作方法如悬滴培养法相比,具有操作简便,拟胚体形成率高,重复性好等优点,是开展哺乳动物早期胚胎发育和干细胞分化研究的理想工具。

Embryoid bodies, which are similar to post-implantation embryonic tissues, provide a model forthe study of embryo development and stem cell differentiation. We describe here a novel method to form embryoidbodies using STO cells as feeder layers. When approx 80% confluent, STO cells are treated with mitomycin C (10g/ml in DMEM) for 4 h and cultured in the concentration of 8104 cm2 for another 12 h before use. Embryonicstem cells are suspended in the ES medium consisting of mLIF and seeded on the previously prepared inactivatedSTO feeder layers in the concentration of 1104 cm2. After 24 h, replace the medium with ES medium withoutmLIF. Various embryoid bodies are formed after 59 days. Studies of the morphology and differentiating potentialof these embryoid bodies suggest that they are typical in structure and they can generate derivatives of all threeembryonic germ layers. Compared with the traditional embryoid body forming method such as the hanging dropsculture, our method has a prominent advantage of performing conveniently. Moreover, the efficiency of embryoidbody formation is greater than traditional methods. Thus, this new method of forming embryoid bodies provides anappropriate tool for the study of mammal embryo development and embryonic stem cell differentiation.