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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 the method of inducing embryonic stem cells (ESCs) to generate high purity neural stem cells (NSCs) in vitro. Methods Different stages and microenvironments of neural cell differentiation and development in vivo were simulated. The brain astrocytes were used as the supporting cells. All-trans retinoic acid (ATRA) was used to induce ESCs into NSCs. Morphological observation and teratoma formation assay were used to identify the ESCs. Morphological observation was performed to detect the expression of embryonic stem cell marker OCT-4 and neural stem cell marker Nestin protein by immunocytochemistry, flow cytometry and RT-PCR. Gene expression on the whole process of induction of dynamic monitoring; NSCs differentiation test on the induction of differentiation potential of NSCs were detected. Results (1) The ESC-D3 cells cultured in vitro were continuously subcultured on the feeder layer of embryonic fibroblasts and maintained their ability to differentiate into the three germ layers. (2) The expression of OCT-4 gradually decreased and disappeared as the induction, And gradually increased. The three-step method can induce the formation of more than 90% of Nestin-positive cells. (3) The induced cells repeatedly passaged in NSCs selective medium and still showed the ability of Nestin-positive and neurosphere-forming , Can be further differentiated into neurons, astrocytes and oligodendrocytes in serum-containing media. Conclusion Astrocytes can induce ESCs to produce higher purity of NSCs by using astrocyte as the induction matrix and simulating the process of neural differentiation in vivo, and can maintain the characteristics of stem cells and further differentiation.