快速聚集无血清悬浮培养法诱导BALB/c小鼠胚胎干细胞分化为大脑皮质椎体神经元

目的以往的研究多集中于胚胎干细胞(ESCs)经外部因素的诱导而转化为神经元,本研究拟探讨一种新的诱导ESCs定向分化方法——快速聚集无血清悬浮培养法(SFEBq)。方法 BALB/c小鼠囊胚内细胞团(ICM)消化成单个细胞,培养传代并鉴定后,应用改进的SFEBq诱导小鼠胚胎干细胞(mESCs)向神经细胞选择分化,并应用免疫荧光染色和流式细胞仪检测Bf1+和Emx1+神经前体细胞的比例,RT-PCR检测不同分化阶段的细胞标志基因表达。结果①分离培养得到的mESCs生长状态良好;②SFEBq使mESCs自主发生神经细胞,无需外部诱因,细胞种植量为3 000个/孔时,细胞Bf1+和Emx1+相对表达量最高;③mESCs按照一个先神经元-后胶质细胞的顺序有效分化,与体内胚胎神经组织发育先后顺序一致,且诱导的神经前体细胞可分化为皮质椎体神经元。结论建立了一种改良的高效ESCs诱导转化的神经元的SFEBq。

Directed Differentiation of Telencephalic Cautical Neurons from Mouse Embryonic Stem Cells by Serum-free Culture of Embryoid Body-like Aggregates with Quick Reaggregation

Aim To introduce a new different approach of embryonic stem cells （ESCs） induced into neuron cells.Methods Blastocyst inner cell mass of BALB/c mice were dislodged into single cells,and cultured,identified.The mouse embryonic stem cells differentiated into neural cells with serum-free culture of embryoid body-like aggregates with quick reaggregation （SFEBq） method.The proportion of cells with Bfl＋ and Emxl ＋ were detected by using immunofluorescence staining and flow cytometry.The mark genes expression at different differentiation stages were detected by RT-PCR.Results ① Cultured mESCs were in good condition.② Embryonic stem cells could be differentiated into neural cells independently through SFEBq method without extemal factors.In addition,Bfl ＋ and Emx 1 ＋ relative expression of cells were the highest when cell cultivation was 3 000/hole.③ mESCs differentiated effectively in an order of neuron-glia cell,which was consistent with the order of embryonic neural tissue development in vivo.Also,the precursor cells can be induced into vertebral cortex neurons.Conclusion An efficient directed differentiation approach of embryonic stem cells may be established.