体外三步法诱导胚胎干细胞定向生成神经干细胞的实验研究

The study of efficient production of neural stem cells from embryonic stem cells in vitro by three-step differentiation

目的探讨体外定向诱导胚胎干细胞(embryonicstemcells,ESCs)生成高纯度神经干细胞(neuralstemcells,NSCs)的方法。方法模拟体内神经细胞分化发育的不同阶段及微环境,以脑星形胶质细胞为支持细胞,用全反式维甲酸(All-transretinoicacid,ATRA)等分三阶段诱导ESCs定向生成NSCs。形态学观察及畸胎瘤形成试验对ESCs的全能性进行鉴定;形态学观察结合免疫组织化学、流式细胞术和RT-PCR检测胚胎干细胞标志OCT-4和神经干细胞标志Nestin蛋白和(或)基因表达对诱导全过程进行动态监测;NSCs分化试验对所诱导的NSCs的分化潜能进行检测。结果⑴体外培养的小鼠ESC-D3细胞在胚胎成纤维饲养层细胞上连续传代培养,仍保持向三胚层分化的能力;⑵随着诱导的进行,OCT-4表达逐渐减弱并消失,而Nestin表达逐渐增强,经三步法最终可诱导形成纯度高达90%以上的Nestin阳性细胞;⑶所诱导生成的细胞在NSCs选择性培养基中反复传代,仍表现为Nestin阳性和具有生成神经球的能力,在含血清培养基中可进一步分化为神经元、星形神经胶质细胞和少突胶质细胞。结论采用星形胶质细胞作为诱导基质,模拟体内神经分化过程的三步诱导法,可诱导ESCs生成较高纯度的NSCs,并能较好维持其干细胞特性和具有进一步分化的能力。

Objective To investigate a method of efficient production of neural stem cells (NSCs) from embryonic stem cells (ESCs) in vitro . Methods Mouse ESC line-D3 cells were used to differentiate into NSCs by means of three-step differentiation induced by all-trans retinoic acid (ATRA) and other factors under the condition of mocked microenvironment and different development stage of neural cells in vivo. The totipotency of ESCs were identified by the observation of cells′ morphology and the formations of teratoma in SCID mice . The cells′ differentiation was evaluated continuously by the morphological observation, immuno-histochemistry assay, flow cytometry and detection of the stem cell surface markers using RT-PCR, such as OCT-4 and Nestin. The plasticity of neural stem cells was detected by the differentiating test. Results ⑴ The ESC-D3 cells could keep the ability of differentiating into cellular derivations of all three primary germ layers after continuous passage culture. ⑵ With the expression of OCT-4 reduction and disappearance , the expression of Nestin was increased step by step , and at last the positive value of the Nestin was up to 90% in the differentiated cells by the three-step differentiation. ⑶ The differentiated cells could keep the characteristics of NSCs after continuous passage culture and could be further induced into neurons,astrocytes and oligodendrocytes. Conclusions Through mimic the microenvironment and different development stage of the neural cells in vivo, the ESCs could be induced into NSCs with high purification and keep the plasticity of stem cells by three-step differentiation using the astrocytes as the feeder.