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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。
OBJECTIVE Previous studies focused on the transformation of embryonic stem cells (ESCs) into neurons induced by external factors. In this study, we aimed to explore a new method for inducing directional differentiation of ESCs, fast serum-free suspension culture (SFEBq). Methods The blastocyst (ICM) cells of BALB / c mice were digested into single cells. After subculture and identification, the cultured embryonic stem cells (mESCs) were induced to differentiate into neurons with modified SFEBq. Immunofluorescence staining and flow cytometry Cytometry was used to detect the ratio of Bf1 + and Emx1 + neural precursor cells. RT-PCR was used to detect the expression of cell marker genes at different stages of differentiation. Results ① The growth of mESCs obtained by isolation and culture was good. ②SFEBq allowed mESCs to autonomously produce nerve cells without external inducement. The highest expression of Bf1 + and Emx1 + was observed when the cells were seeded at 3 000 cells / The order of the glial cells is effectively differentiated, which is in accordance with the order of the embryonic nervous tissue development in vivo. The induced precursor cells can differentiate into cortical vertebral neurons. Conclusion An improved and efficient ESCs-induced neuronal SFEBq was established.