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

doi:10.3969/j.issn.1674-8115.2022.01.006

上海交通大学学报(医学版) ›› 2022, Vol. 42 ›› Issue (1): 36-43.doi: 10.3969/j.issn.1674-8115.2022.01.006

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¹(), 任兆瑞¹^,², 颜景斌¹^,²()

^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

收稿日期:2021-09-27 出版日期:2022-01-28 发布日期:2022-02-18
通讯作者: 颜景斌 E-mail:www.liuyanna@163.com;m18917128323@163.com
作者简介:刘艳娜（1985—），女，研究实习员，硕士；电子信箱：www.liuyanna@163.com。
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)

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

Yanna LIU¹(), Zhaorui REN¹^,², Jingbin YAN¹^,²()

^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

Received:2021-09-27 Online:2022-01-28 Published:2022-02-18
Contact: Jingbin YAN E-mail:www.liuyanna@163.com;m18917128323@163.com
Supported by:
National Natural Science Foundation of China(81971421);Shanghai Key Clinical Specialty Project(shslczdzk05705)

摘要/Abstract

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

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

中图分类号:

R331

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.

Yanna LIU, Zhaorui REN, Jingbin YAN. Enhancement of BMP4 inhibitor DMH1 on the efficiency of BiSF in human iPSC-induced differentiation into neurons[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(1): 36-43.

图/表 6

表1 qRT-PCR检测相关基因的引物序列

Tab 1 Genes and primer sequences used for qRT-PCR analysis

Primer name	Forward （5'→3'）	Reverse （5'→3'）
GAPDH	TTCCAAGCATAAAAGAACGG	TACATCCTCATCACCACCCA
NANOG	TTTGTGGGGCTGAAGAAACT	AGGGCTGTCCTGAATAAGCAG
OCT4	GACGCCATCAACACCGAGTT	CTTTGTCGTTGGTTAGCTGGT
Nestin	CAGCGTTGGAACAGAGGTTGG	TGGCACAGGTGTCTCAAGGGTAG
PAX6	ATGTGTGAGTAAAATTCTGGGCA	GCTTACAACTTCTGGAGTCGCTA
MAP2	CCACCTGAGATTAAGGATCA	GGCTTACTTTGCTTCTC

图1 hiPSC形态及多能性的鉴定Note： A. Immunofluorescence analysis of SSEA1 (green fluorescence). B. Immunofluorescence analysis of TRA-1-60 (red fluorescence), and nuclei stained positive for DAPI (blue fluorescence).

Fig 1 hiPSC morphology and pluripotency identification

图2 诱导分化第9日镜下观察细胞形态Note： A. Cell morphologies of BiSF+DMH1 group. B. Cell morphologies of BiSF group. C. Cell morphologies of natural differentiation group.

Fig 2 Cell morphologies were observed under microscope on day 9 of differentiation

图3 NSC标志物nestin的表达及细胞的增殖情况Note： A. Immunofluorescence analysis of nestin (red fluorescence), and nuclei stained positive for DAPI (blue fluorescence). B. Cell proliferation was examined by CCK-8 assay. ①P=0.000, ②P=0.008, ③P=0.020, ④P=0.002.

Fig 3 Marker for NSC nestin and proliferation of NSCs

图4 诱导第13日多能性基因及NSC相关基因表达水平比较Note： A. mRNA expression levels of NANOG. B. mRNA expression levels of OCT4. C. mRNA expression levels of nestin. D. mRNA expression levels of PAX6. ①P=0.000, ②P=0.019, ③P=0.011.

Fig 4 mRNA expression levels of pluripotency-related genes and NSC-related genes on day 13 of differentiation

图5 神经元特异性标志物的检测Note： A. Immunofluorescence analysis of βⅢ-tubulin (red fluorescence), and nuclei stained positive for DAPI (blue). B. Percentage of red fluorescence cells. C. Relative MAP2 mRNA expression level (hiPSC as the control). ①P=0.003, ②P=0.006.

Fig 5 Detection of neuron-specific markers

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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

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

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