小分子化合物组合改善多能干细胞向脑类器官定向分化的效率

doi:10.16352/j.issn.1001-6325.2024.06.0786

基础医学与临床 ›› 2024, Vol. 44 ›› Issue (6): 786-792.doi: 10.16352/j.issn.1001-6325.2024.06.0786

小分子化合物组合改善多能干细胞向脑类器官定向分化的效率

朱宛宛^#, 周金娟^#, 修建波^*

中国医学科学院基础医学研究所北京协和医学院基础学院重大疾病共性机制研究全国重点实验室,北京 100005

收稿日期:2023-03-18 修回日期:2024-04-12 出版日期:2024-06-05 发布日期:2024-05-24
通讯作者: ^*xiujianbo@ibms.cams.cn
作者简介:^#对本文有相同贡献
基金资助:
国家自然科学基金(82101545);中国医学科学院医学与健康科技创新工程(2022-I2M-1-002,2022-I2M-2-001);中央高水平医院临床科研业务费(2022-PUMCH-D-002)

Small molecule compounds improve the targeted differentiation efficiency of cerebral organoids from pluripotent stem cells

ZHU Wanwan^#, ZHOU Jinjuan^#, XIU Jianbo^*

State Key Laboratory of Common Mechanism Research for Major Diseases, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2023-03-18 Revised:2024-04-12 Online:2024-06-05 Published:2024-05-24
Contact: ^*xiujianbo@ibms.cams.cn

摘要/Abstract

摘要： 目的优化多能干细胞(PSCs)定向分化为脑类器官的条件,提高人脑类器官的分化效率。方法利用人胚胎干细胞(hESCs)系H9向人脑类器官诱导分化体系,在早期脑类器官分化到神经祖细胞的发育阶段添加小分子化合物组合,通过形态学观察不同分化阶段脑类器官中的神经祖细胞形成的效率、细胞凋亡的情况以及分化成神经元的效率,通过RT-qPCR检测标志性基因的表达量,综合评估添加的小分子化合物组合对脑类器官形成的影响。结果在脑类器官神经祖细胞发育的关键阶段(1 d～14 d),培养基中依次添加多索啡(dorsomorphine)、A83-01、GSK-3β抑制剂CHIR99021和SMAD抑制剂SB-431542,可使神经祖细胞阶段的相关标志性基因的表达量显著提高,促进特异性神经管样结构形成,脑类器官中心区域的细胞凋亡减少。结论通过使用上述4种小分子化合物组合,可明显提高早期脑类器官的形成效率,减少脑类器官中的细胞凋亡、促进神经元形成,减少培养过程中的组织结构异质性。

关键词: 多能干细胞, 小分子化合物, 神经分化, 脑类器官

Abstract: Objective To optimize the conditions and improve the efficiency for the differentiation of pluripotent stem cells (PSCs) into cerebral organoids. Methods Based on the induction differentiation system for inducing human embryonic stem cells (hESCs) H9 towards cerebral organoids, a combination of small molecule compounds was added to the developmental stage of neural progenitor cells in the early stage of cerebral organoid differentiation, and the efficiency of neural progenitor cell formation, apoptosis and differentiation into neurons in cerebral organoids in the differentiation stage were observed through morphology, the expression of marker genes was detected by RT-qPCR and the effect of small molecule compound combination on cerebral organoids was comprehensively evaluated. Results At the critical stage of neural progenitor cell development (1 d-14 d) of cerebral organoids, dorsomorphine, A83-01, GSK-3β inhibitor CHIR99021 and SMAD inhibitor SB-431542 were successively added to the medium. It could significantly increase the expression of marker genes in the neural progenitor cell stage, promote the formation of specific neural tube-like structures and reduce apoptosis in the central region of cerebral organoids. Conclusions By using the combination of four small molecule compounds, the formation efficiency in early cerebral organoids can be significantly improved, apoptosis in cerebral organoids can be reduced, neuronal formation can be promoted, and tissue structure heterogeneity in the culture process can be reduced.

Key words: pluripotent stem cells, small molecule compounds, neural differentiation, cerebral organoids

中图分类号:

朱宛宛, 周金娟, 修建波. 小分子化合物组合改善多能干细胞向脑类器官定向分化的效率[J]. 基础医学与临床, 2024, 44(6): 786-792.

ZHU Wanwan, ZHOU Jinjuan, XIU Jianbo. Small molecule compounds improve the targeted differentiation efficiency of cerebral organoids from pluripotent stem cells[J]. Basic & Clinical Medicine, 2024, 44(6): 786-792.

参考文献 14

[1]	Liu G, David BT, Trawczynski M, et al. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications[J]. Stem Cell Rev Rep, 2020, 16: 3-32.
[2]	Yamanaka S. Pluripotent stem cell-based cell therapy-promise and challenges[J]. Cell Stem Cell, 2020, 27: 523-531.
[3]	Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly[J]. Nature, 2013, 501: 373-379.
[4]	Sakaguchi H, Kadoshima T, Soen M, et al. Generation of functional hippocampal neurons from self-organizing human embryonic stem cell-derived dorsomedial telencephalic tissue[J]. Nat Commun, 2015, 6: 8896. doi: 10.1038/ncomms9896
[5]	Andrews MG, Kriegstein AR. Challenges of organoid research[J]. Annu Rev Neurosci, 2022, 45:23-39.
[6]	Nusse R, Clevers H. Wnt/β-catenin signaling, disease, and emerging therapeutic modalities[J]. Cell, 2017, 169: 985-999.
[7]	Qian X, Nguyen HN, Song MM, et al. Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure[J]. Cell, 2016, 165: 1238-1254.
[8]	Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science, 1998, 282: 1145-1147.
[9]	Lancaster MA, Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells[J]. Nat Protoc, 2014, 9: 2329-2340.
[10]	Rosebrock D, Arora S, Mutukula N, et al. Enhanced cortical neural stem cell identity through short SMAD and WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells[J]. Nat Cell Biol, 2022, 24: 981-995.
[11]	He J, Zhang X, Xia X, et al. Organoid technology for tissue engineering[J]. J Mol Cell Biol, 2020, 12: 569-579.
[12]	Mansour AA, Gonçalves JT, Bloyd CW, et al. An in vivo model of functional and vascularized human brain organoids[J]. Nat Biotechnol, 2018, 36: 432-441.
[13]	Shi Y, Sun L, Wang M, et al. Vascularized human cortical organoids (vOrganoids) model cortical development in vivo[J]. PLoS Biol, 2020, 18: e3000705. doi:10.1371/journal.pbio.3000705.
[14]	Hubert CG, Rivera M, Spangler LC, et al. A three-dimensional organoid culture system derived from human glioblastomas recapitulates the hypoxic gradients and cancer stem cell heterogeneity of tumors found in vivo[J]. Cancer Res, 2016, 76: 2465-2477.

小分子化合物组合改善多能干细胞向脑类器官定向分化的效率

Small molecule compounds improve the targeted differentiation efficiency of cerebral organoids from pluripotent stem cells

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 14

相关文章 8

编辑推荐

Metrics

本文评价

[1]	王靓, 夏隆阔, 张建民. 脑类器官在药物研发中的研究进展[J]. 基础医学与临床, 2024, 44(9): 1208-1213.
[2]	闫煜聪, 杨淑春, 贾玉艳, 黄粤. 通过定向内胚层的高效分化提高多能干细胞体外生成肝细胞样细胞的效率[J]. 基础医学与临床, 2023, 43(7): 1045-1052.
[3]	周化腾, 李长明, 王利祥, 胡劲涛, 杜伟斌, 章何陋, 任伟凡. 人尿液细胞来源的诱导多能干细胞定向分化为毛囊干细胞[J]. 基础医学与临床, 2020, 40(11): 1468-1473.
[4]	周芳颖刘玉琴. 饲养层细胞作用机制研究进展[J]. 基础医学与临床, 2019, 39(7): 1066-1070.
[5]	王记陆玉琴徐鑫纪召娟. 抑瘤素M促进小鼠诱导多能干细胞向心肌分化[J]. 基础医学与临床, 2017, 37(10): 1401-1406.
[6]	李慧文于树夔郝飞牛含虚徐群渊段德义. 胰岛素样生长因子结合蛋白-4对大鼠骨髓间充质干细胞增殖和神经分化的影响[J]. 基础医学与临床, 2016, 36(4): 513-519.
[7]	林真沈晓丽. 转导特定基因重编程体细胞为多能干细胞[J]. 基础医学与临床, 2010, 30(2): 206-208.
[8]	朱宛宛吴迪李宁邹春林徐艳玲关云谦张愚. 食蟹猴胚胎干细胞的培养及向神经细胞诱导分化[J]. 基础医学与临床, 2009, 29(7): 709-715.