中国病理生理杂志Issue(4)：763-768,6.DOI:10.3969/j.issn.1000-4718.2014.04.034
小鼠来源诱导多能干细胞定向分化为神经元的体外实验研究
Differentiation of neurons from mouse induced pluripotent stem cells in vitro
摘要
Abstract
AIM:To study the induction method of mouse induced pluripotent stem cells (iPSCs) that differ-entiate into neurons in vitro.METHODS:Mouse iPSCs were cultured in non-adherent culture dishes for 2 d to form em-bryoid bodies (EBs).The EBs were cultured for consecutive 2 d in the presence of retinoic acid (RA), and then were plated in the serum-free medium for adherent culture .Seven days later , Pasteur pipette was used to detach the differentia-ted cells around adherent EBs into “fragment” cell colonies with the help of dissecting microscopes , and these“fragments”were transferred to culture dishes with neural stem cell medium .Another 7 days later, the cells were plated onto the culture dishes using differentiation medium containing fetal bovine serum ( FBS) and RA.The morphological changes of the cells were observed under inverted microscope .The iPSCs markers Oct4, Sox2 and SSEA1, the neural stem cell (NSC) marker nestin, the neuronal marker microtubule-associated protein 2 (MAP-2), the astrocyte marker glial fibrillary acidic protein ( GFAP) and oligodendrocyte marker myelin basic protein ( MBP) were detected by immunofluorescence method .The mR-NA expression of GFAP, nestin,β3-tubulin, MAP-2 and MBP was detected by RT-RCR.MAP-2 gene sequence was iden-tified.The proportions of NSCs differentiated from iPSCs and neurons from NSCs were detected by flow cytometry .RE-SULTS:Mouse iPSCs strongly expressed Oct4, Sox2 and SSEA1, and formed spherical EBs by suspended culture .The EBs were induced by RA and serum-free medium in adherent culture for 2 d, and rosette structure was observed under the microscope .“Fragments” separated by Pasteur pipette from the rosette structure formed neurosphere-like colonies .After the colonies were cultured in adherent condition for 5 d to 7 d in the presence of RA and FBS , the typical neurite was ob-served under the microscope .The neurospheres expressed nestin and their differentiated derivatives expressed MAP -2, GFAP and MBP, respectively.RT-PCR analysis and gene sequencing showed that the neurons were induced successfully . The results of flow cytometry demonstrated that 63.93%±1.47%of iPSCs differentiated into NSCs and 21.4%±1.70%of NSCs differentiated into neurons .CONCLUSION: Mouse iPSCs proliferate stably and differentiate into neurons in vitro, which provide a reliable source for the treatment of spinal cord injury .关键词
小鼠/诱导多能干细胞/神经元/定向分化/机械分离Key words
Mice/Induced pluripotent stem cells/Neurons/Directional differentiation/Mechanical separation分类
生物科学引用本文复制引用
庞卯,杨阳,刘斌,张良明,戎利民..小鼠来源诱导多能干细胞定向分化为神经元的体外实验研究[J].中国病理生理杂志,2014,(4):763-768,6.基金项目
国家自然科学基金资助项目（ No．31170947）；广东省自然科学基金资助项目（ No．S2012020011099； No． S2013010016413）；广州市科技计划 （）
