脑类器官在再生医学中的研究进展

doi:10.12211/2096-8280.2023-102

合成生物学

• 特约评述 •

脑类器官在再生医学中的研究进展

洪源¹^,²^,³, 刘妍¹^,⁴^,⁵

^1.南京医科大学姑苏学院，南京 211166
^2.苏州市立医院，苏州 215006
^3.南京医科大学附属苏州医院，苏州 215006
^4.南京医科大学生殖医学与子代健康全国重点实验室，南京 211166
^5.南京医科大学药学院干细胞与神经再生研究所，南京 211166

收稿日期:2023-12-01 修回日期:2024-05-29 出版日期:2024-06-03
通讯作者: 刘妍
作者简介:洪源（1995—），男，博士，研究方向为利用人脑类器官模型开展抑郁症病理机制研究。E-mail：justhongyuan@njmu.edu.cn
刘妍（1981—），女，教授，博士生导师，研究方向为利用人脑类器官开展神经疾病机制及细胞移植治疗研究。E-mail：yanliu@njmu.edu.cn
基金资助:
国家重点研发计划(2021YFA1101800);国家自然科学基金(82171528);南京医科大学姑苏学院博士后科研项目(GSBSHKY202307)

Research progress of brain organoids in regenerative medicine

Yuan HONG¹^,²^,³, Yan LIU¹^,⁴^,⁵

^1.Gusu School，Nanjing Medical University，Nanjing 211166，Jiangsu，China
^2.Suzhou Municipal Hospital，Suzhou 215006，Jiangsu，China
^3.The Afflicated Suzhou Hospital of Nanjing Medical University，Suzhou 215006，Jiangsu，China
^4.State Key Laboratory of Reproductive Medicine and Offspring Health，Nanjing Medical University，Nanjing 21166，Jiangsu，China
^5.Institution of Stem Cells and Neuroregeneration，Nanjing Medical University，Nanjing 211166，Jiangsu，China

Received:2023-12-01 Revised:2024-05-29 Online:2024-06-03
Contact: Yan LIU

摘要/Abstract

摘要：

脑类器官是一种基于人多能干细胞的三维体外模型，能够模拟人脑的细胞异质性、结构和功能。再生医学是一个多学科交叉的领域，致力于应用工程学和生物学手段修复因年龄、疾病或外伤而受损的组织或器官。脑类器官技术作为再生医学领域的一种重要手段，具有广阔的应用前景和重要的科学意义。近年来，利用组织工程和诱导因子分化技术，研究人员成功构建出不同脑区的脑类器官模型，可用于模拟脑损伤或修复病变组织。本文将系统介绍包括大脑皮层、海马、纹状体、中脑、丘脑及下丘脑、小脑和视网膜在内的脑区特异类器官构建技术的最新进展，总结其在再生医学领域中的应用，并概括当前脑类器官应用面临的挑战，如异质性大、缺乏脉管系统和成熟度较低等。这将加深对人类大脑的理解，并增强脑类器官在基础研究和临床研究中的进一步应用。

关键词: 脑类器官, 再生医学, 个体化医疗, 疾病模型, 药物筛选

Abstract:

The brain, as the epicenter of human intelligence, sensation, and motor coordination, represents the pinnacle of biological complexity. Despite its critical role, the availability of live human brain tissue for research is fraught with challenges, impeding advancements in our understanding of the nervous system. Brain organoids are sophisticated three-dimensional cultures derived from human pluripotent stem cells that emulate the diverse cellular composition, structural intricacies, and functional attributes of the human brain. These organoids eclipse traditional two-dimensional cultures and animal models in mirroring the brain's spatial organization and cellular interplay, bolstered by a genetic congruence with their human counterparts. This congruence renders them particularly adept at modeling neuropsychiatric conditions and pioneering cell-based therapeutic interventions. Regenerative medicine, a confluence of engineering and biological sciences, endeavors to restore tissues and organs compromised by aging, disease, or trauma. However, the field grapples with limitations stemming from the scarcity of samples and ethical quandaries. Brain organoid technology emerges as a formidable asset in this domain, offering expansive potential and profound implications for scientific inquiry. Recent strides have seen the successful assembly of organoid models representing various brain regions through the application of tissue engineering and directed differentiation. These models hold promise for simulating neuropathological states and facilitating tissue repair. This article meticulously surveys the cutting-edge methodologies for constructing organoids specific to brain regions such as the cerebral cortex, hippocampus, striatum, midbrain, thalamus, hypothalamus, cerebellum, and retina. It delineates the principal applications of brain organoids in regenerative medicine, encompassing injury simulation, exploration of inter-regional and multi-lineage cellular dynamics, drug efficacy and toxicity assessments, and the potential for organoid transplantation. Furthermore, the review addresses the prevailing obstacles in the application of brain organoids, notably their pronounced variability, absence of vascularization, and developmental immaturity. In essence, this review seeks to illuminate the organoid generation techniques tailored to discrete brain territories and their significance in regenerative medicine's landscape. By probing into research poised to surmount the limitations of current models, it aspires to broaden the horizons for brain organoids in both foundational research and clinical applications.

Key words: brain organoids, regenerative medicine, personalized medicine, disease model, drug screening

中图分类号:

洪源, 刘妍. 脑类器官在再生医学中的研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2023-102.

Yuan HONG, Yan LIU. Research progress of brain organoids in regenerative medicine[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2023-102.

图/表 1

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脑区类型	年份	课题组	分化方法	参考文献
大脑皮层	2008	Yoshiki Sasai	PSCs在含有Dkk -1和Lefty-1的神经分化培养基中自组织形成SFEBq培养物。	[26]
	2013	Juergen A. Knoblich	先将PSCs重聚成拟胚体，并生成神经外胚层，随后将其嵌入Matrigel，在没有外源性生长因子的条件下悬浮培养。	[12]
	2015	Sergiu P. Paşca	hiPSCs形成拟胚体后使用Dorsomorphin和SB431542进行神经诱导分化，6天后转移至含FGF2和EGF的培养基中，体外培养第25天更换成BDNF和NT3。	[14]
	2016	Hongjun Song & Guo-li Ming	iPSCs重聚的拟胚体先在dorsomorphin和A-83的条件下培养7天，并用基质胶包埋后添加CHIR99021、WNT3A和SB431542因子培养一周后转移至微型旋转生物反应器继续培养。	[15]
海马体	2015	Yoshiki Sasai	hESCs重聚形成SFEBq后，添加IWR1e和 SB431542培养18天，随后加入CHIR和BMP4诱导海马体分化。	[27]
纹状体	2020	Sergiu P. Paşca	hiPSCs形成拟胚体后，添加DMH1和SB431542培养6天，随后在第6-22天添加Activin A、IWP2和SR11237促进纹状体的分化。	[28]
纹状体	2022	Lixiang Ma	PSCs重聚后在含LDN-193189和SB431542的培养基中培养10天，随后在purmorphamine的诱导下培养至第25天。	[29]
中脑	2016	Huck-Hui Ng	hESCs重聚成拟胚体后第4天开始添加SHH-C25II和FGF8诱导中脑分化命运，神经外胚层出现后包埋类器官，转移至低吸附六孔板中培养。	[30]
中脑	2016	Hongjun Song & Guo-li Ming	iPSCs重聚后加入SHH、FGF-8、SB431542、LDN193189和CHIR99021诱导中脑命运，并在第14天时将其转移至微型旋转生物反应器继续培养。	[15]
丘脑和下丘脑	2016	Hongjun Song & Guo-li Ming	iPSCs重聚后先加入SB431542 和LDN193189，分化第4至7天，加入WNT3A，SHH和purmorphamine诱导下丘脑命运，随后持续添加FGF2和CNTF促进类器官成熟。	[15]
	2017	In-Hyun Park	hESCs重聚后添加SB431542、LDN193189和insulin促进尾侧的神经诱导，第8天开始添加PD0325901和BMP7诱导丘脑分化。	[31]
	2021	Hongjun Song & Guo-li Ming	hiPSCs经过双SMAD抑制诱导神经外胚层命运，同时加入IWR1-endo、SAG、PMA和SHH促进弓状核分化，第12天开始与小鼠下丘脑星形胶质细胞共培养。	[32]
小脑	2015	Yoshiki Sasai	将hESC重聚为SFEBq，第2至14天在含有胰岛素和SB431542的培养基中，持续添加重组人FGF2，并在后期加入FGF19和SDF1促进极性结构的形成。	[33]
小脑	2024	Giorgia Quadrato	hiPSCs重聚后第0至16天加入SB431542、Noggin、FGF8b和CHIR99021诱导后脑分化命运，并在第30天开始加入T3和BDNF促进后脑成熟，加入SDF1a完成小脑模式化。	[34]
视网膜	2012	Yoshiki Sasai	hESCs重聚形成SFEBq后，在 IWR1e、FBS、SAG和CHIR99021的诱导下，自组织形成视网膜类器官。	[35]
	2014	M Valeria Canto-Soler	将hiPSCs重聚后，分化第7天时用基质胶包埋，并在第4周手动分离神经视网膜结构域，从第42天开始向培养基中添加FBS、Taurine和GlutaMAX。在培养过程中，每天都需添加RA以促进光感受器的成熟。	[36]

脑区类型	年份	课题组	分化方法	参考文献
大脑皮层	2008	Yoshiki Sasai	PSCs在含有Dkk -1和Lefty-1的神经分化培养基中自组织形成SFEBq培养物。	[26]
	2013	Juergen A. Knoblich	先将PSCs重聚成拟胚体，并生成神经外胚层，随后将其嵌入Matrigel，在没有外源性生长因子的条件下悬浮培养。	[12]
	2015	Sergiu P. Paşca	hiPSCs形成拟胚体后使用Dorsomorphin和SB431542进行神经诱导分化，6天后转移至含FGF2和EGF的培养基中，体外培养第25天更换成BDNF和NT3。	[14]
	2016	Hongjun Song & Guo-li Ming	iPSCs重聚的拟胚体先在dorsomorphin和A-83的条件下培养7天，并用基质胶包埋后添加CHIR99021、WNT3A和SB431542因子培养一周后转移至微型旋转生物反应器继续培养。	[15]
海马体	2015	Yoshiki Sasai	hESCs重聚形成SFEBq后，添加IWR1e和 SB431542培养18天，随后加入CHIR和BMP4诱导海马体分化。	[27]
纹状体	2020	Sergiu P. Paşca	hiPSCs形成拟胚体后，添加DMH1和SB431542培养6天，随后在第6-22天添加Activin A、IWP2和SR11237促进纹状体的分化。	[28]
纹状体	2022	Lixiang Ma	PSCs重聚后在含LDN-193189和SB431542的培养基中培养10天，随后在purmorphamine的诱导下培养至第25天。	[29]
中脑	2016	Huck-Hui Ng	hESCs重聚成拟胚体后第4天开始添加SHH-C25II和FGF8诱导中脑分化命运，神经外胚层出现后包埋类器官，转移至低吸附六孔板中培养。	[30]
中脑	2016	Hongjun Song & Guo-li Ming	iPSCs重聚后加入SHH、FGF-8、SB431542、LDN193189和CHIR99021诱导中脑命运，并在第14天时将其转移至微型旋转生物反应器继续培养。	[15]
丘脑和下丘脑	2016	Hongjun Song & Guo-li Ming	iPSCs重聚后先加入SB431542 和LDN193189，分化第4至7天，加入WNT3A，SHH和purmorphamine诱导下丘脑命运，随后持续添加FGF2和CNTF促进类器官成熟。	[15]
	2017	In-Hyun Park	hESCs重聚后添加SB431542、LDN193189和insulin促进尾侧的神经诱导，第8天开始添加PD0325901和BMP7诱导丘脑分化。	[31]
	2021	Hongjun Song & Guo-li Ming	hiPSCs经过双SMAD抑制诱导神经外胚层命运，同时加入IWR1-endo、SAG、PMA和SHH促进弓状核分化，第12天开始与小鼠下丘脑星形胶质细胞共培养。	[32]
小脑	2015	Yoshiki Sasai	将hESC重聚为SFEBq，第2至14天在含有胰岛素和SB431542的培养基中，持续添加重组人FGF2，并在后期加入FGF19和SDF1促进极性结构的形成。	[33]
小脑	2024	Giorgia Quadrato	hiPSCs重聚后第0至16天加入SB431542、Noggin、FGF8b和CHIR99021诱导后脑分化命运，并在第30天开始加入T3和BDNF促进后脑成熟，加入SDF1a完成小脑模式化。	[34]
视网膜	2012	Yoshiki Sasai	hESCs重聚形成SFEBq后，在 IWR1e、FBS、SAG和CHIR99021的诱导下，自组织形成视网膜类器官。	[35]
	2014	M Valeria Canto-Soler	将hiPSCs重聚后，分化第7天时用基质胶包埋，并在第4周手动分离神经视网膜结构域，从第42天开始向培养基中添加FBS、Taurine和GlutaMAX。在培养过程中，每天都需添加RA以促进光感受器的成熟。	[36]

脑类器官在再生医学中的研究进展

Research progress of brain organoids in regenerative medicine

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