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目的建立无血清无饲养层人胚胎干细胞(hESCs)胶质前体细胞分化体系。方法将生长在层黏连蛋白包被的培养板上的人胚胎干细胞,悬浮培养诱导拟胚体(EBs)形成,将25d的EBs转移至含有胰岛素、5μg/L碱性成纤维细胞生长因子(bFGF)、20μg/L表皮生长因子(EGF)和5μg/L三碘甲状腺素(T3)的胶质细胞分化培养基贴壁培养7～10d,诱导胶质前体细胞分化。收集成纤维样分化细胞,采用RT-PCR法检测胶质前体细胞早期基因的表达,细胞免疫荧光染色检测胶质前体细胞标记的表达。结果分化得到的细胞呈长梭形,表达神经胶质前体细胞标记NG2和血小板衍生生长因子受体α(PDGFRα),阳性率分别为37.2%和47.6%。该前体细胞可进一步分化为星形胶质细胞和少突胶质细胞,未见神经元。结论含有胰岛素、bFGF、EGF和T3的胶质细胞分化培养基可诱导hESCs向胶质前体细胞分化。
Objective To establish a serum-free feeder-derived human embryonic stem cells (hESCs) glial precursor cell differentiation system. Methods Human embryonic stem cells (ESCs) cultured in laminin-coated plates were induced to form EBs by suspension culture. EBs of 25 days were transferred to medium containing insulin, 5μg / L basic fibroblast growth factor bFGF, 20μg / L epidermal growth factor (EGF) and 5μg / L triiodothyronine (T3) were cultured for 7-10 days to induce differentiation of glial precursor cells. The fibroblast-like differentiated cells were collected. The expression of early genes of glial precursor cells was detected by RT-PCR. The expression of glial precursor cells was detected by immunofluorescence staining. Results The differentiated cells were long fusiform and expressed NG2 and PDGFRα, the positive rates were 37.2% and 47.6%, respectively. The precursor cells can be further differentiated into astrocytes and oligodendrocytes, no neurons. Conclusion Glial differentiation medium containing insulin, bFGF, EGF and T3 can induce hESCs to differentiate into glial precursor cells.