体外诱导人胚胎干细胞定向分化为神经干细胞

目的:探讨体外定向诱导人胚胎干细胞(human embryonic stem cells,hESCs)生成高纯度神经干细胞(neural stem cells,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,分三阶段诱导hESCs定向生成NSCs.形态学观察结合免疫荧光细胞化学、流式细胞术和RT-PCR检测胚胎干细胞标志和神经干细胞标志;NSCs分化实验对所诱导的NSCs的分化潜能进行检测.结果:体外培养的hESCs在胚胎成纤维细胞饲养层上连续传代培养50代,仍保持SSEA-4,TRA-1-81阳性,表达Nanog基因,流式细胞术检测SSEA-4阳性表达率为83.44%;经三步法最终可诱导形成纯度高达90%以上的nestin阳性细胞,表达nestin基因,流式细胞术检测nestin阳性表达率为89.38%;诱导生成的细胞反复传代,仍表现为nestin阳性,并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:模拟体内神经分化过程的三步诱导法,可诱导hESCs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.

Study on differentiation of human embryonic stem cells into neural stem cells

Objective:To search for a method to induce human embryonic stem cells (hESCs) differentiating into neural stem cells (NSCs) in vitro. Methods:HESCs were induced to differentiate into NSCs by three-step differentiation under a condition simulating the microenvironment and different development stages of neural cells in vivo. The surface markers of hESCs and NSCs were detected by morphological observation, immunocytochemistry assay, flow cytometry and RT-PCR. The plasticity of NSCs was evaluated by differentiating test.Results:The hESCs retained expression of SSEA-4, TRA-1-81 proteins and Nanog genes after cultured for 50 passages. Flow cytometry revealed the positive rate of SSEA-4 was 83.44%.After induced by the three-step differentiation the purity of nestin-positive cells was higher than 90%. Flow cytometry revealed that the positive rate of nestin was 89.38%. The differentiated cells retained the characteristics of NSCs after repeated passaging and could be further induced into neurons, astrocytes and oligodendrocytes. Conclusion:The present three-step differentiation method can induce hESCs into high purity NSCs, while retaining the plasticity of stem cells.