上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2022 , Vol. 42 >Issue 1: 36 - 43

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.01.006

论著 · 基础研究

BMP4抑制剂DMH1对BiSF法诱导人iPSC向神经元分化效率的提升作用

  • 刘艳娜 ,
  • 任兆瑞 ,
  • 颜景斌
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  • 1.上海市儿童医院/上海交通大学附属儿童医院上海交通大学医学遗传研究所，上海 200040
    2.上海市胚胎与生殖工程重点实验室/国家卫健委医学胚胎分子生物学重点实验室，上海 200040
刘艳娜（1985—），女，研究实习员，硕士；电子信箱：www.liuyanna@163.com。
颜景斌，电子信箱：m18917128323@163.com。

收稿日期: 2021-09-27

  网络出版日期: 2022-01-28

基金资助

国家自然科学基金面上项目(81971421);上海市临床重点专科项目(shslczdzk05705)

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Enhancement of BMP4 inhibitor DMH1 on the efficiency of BiSF in human iPSC-induced differentiation into neurons

  • Yanna LIU ,
  • Zhaorui REN ,
  • Jingbin YAN
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  • 1.Institute of Medical Genetics, Shanghai Jiao Tong University; Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China
    2.NHC Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology; Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China
YAN Jingbin, E-mail: m18917128323@163.com.

Received date: 2021-09-27

  Online published: 2022-01-28

Supported by

National Natural Science Foundation of China(81971421);Shanghai Key Clinical Specialty Project(shslczdzk05705)

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摘要

目的·通过对已有神经诱导方法BiSF的改进，提高人诱导多能干细胞（human induced pluripotent stem cell，hiPSC）定向诱导分化为神经元的效率。方法·当hiPSC生长到75%融合度时启动诱导，在BiSF诱导方法的基础上加入BMP4抑制剂4-［6-（4-异丙氧基苯基）吡唑并［1，5-a］嘧啶-3-基］喹啉（4-［6-［4-（1-methylethoxy）phenyl］pyrazolo［1，5-a］pyrimidin-3-yl］quinoline，DMH1），镜下观察细胞形态，实时荧光定量PCR（quantitative real-time PCR，qRT-PCR）和免疫荧光检测神经干细胞（neural stem cell，NSC）相关基因的表达，细胞计数试剂盒8（cell counting kit-8，CCK-8）方法比较2种诱导方法获得细胞的增殖情况。将NSC样细胞进一步向神经元诱导分化，通过qRT-PCR及免疫荧光比较2种诱导方法的诱导效率。结果·镜下观察发现BiSF+DMH1诱导的第9日细胞团周围出现较多的梭形细胞，BiSF诱导的细胞出现少量梭形细胞，且细胞团灰暗不规则。CCK-8结果显示BiSF+DMH1诱导组的NSC样细胞的增殖能力在第2日开始高于BiSF诱导组（P=0.000）。诱导分化第13日BiSF+DMH1组的细胞nestin、PAX6表达量高于BiSF组（P=0.019，P=0.011）。免疫荧光结果显示BiSF+DMH1诱导分化组中神经元特异性标记βⅢ微管蛋白（βⅢ-tubulin）阳性的细胞比例高于BiSF诱导组（P=0.003）。qRT-PCR结果证明BiSF+DMH1组MAP2的相对表达水平明显高于BiSF组（P=0.006）。结论·DMH1能显著提高hiPSC向神经元诱导分化的效率。

关键词： 人诱导多能干细胞; 神经干细胞; 分化; 神经元

本文引用格式

刘艳娜 , 任兆瑞 , 颜景斌 . BMP4抑制剂DMH1对BiSF法诱导人iPSC向神经元分化效率的提升作用[J]. 上海交通大学学报（医学版）, 2022 , 42(1) : 36 -43 . DOI: 10.3969/j.issn.1674-8115.2022.01.006

Abstract

Objective

·To obtain an efficient way of promoting induced human pluripotent stem cells (hiPSCs) to differentiate into neurons by improving existing methods BiSF.

Methods

·Induction was initiated when hiPSCs reached 75% fusion, and BMP4 inhibitor (4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline,DMH1) was added based on this induction method. The growth state was observed under microscope, and the expression of neural stem cell (NSC)-specific genes was quantitatively detected by quantitative real-time PCR (qRT-PCR) and immunofluorescence analysis. The proliferation level of the induced cells was determined by cell counting kit-8 (CCK-8). Then the NSC-like cells were further induced into neurons, and the ability of differentiation was detected by qRT-PCR and immunofluorescence.

Results

·Microscopically, it was found that more spindle cells appeared around the cell mass of BiSF+DMH1 group on day 9, and a small amount of spindle cells appeared in the BiSF group with irregular gray cell clusters. CCK-8 growth curve showed that the cells derived from method BiSF+DMH1 were with a significantly higher proliferation on the next day (P=0.000). The cells derived from method BiSF+DMH1 achieved higher expression of nestin and PAX6 (P=0.019, P=0.011). The number of neurons with positive neuron-specific marker βⅢ-tubulin in the BiSF+ DMH1 group was significantly higher than that in BiSF group (P=0.003). The results of qRT-PCR showed that the relative expression of MAP2 in the BiSF+DMH1 group was significantly higher than that in the BiSF group (P=0.006).

Conclusion

·The synergistic effect of DMH1 can significantly improve the efficiency of hiPSCs to differentiate into neurons.

Key words： human induced pluripotent stem cell; neural stem cell; differentiation; neuron

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