Human brain organoid:trends,evolution,and remaining challenges

Advanced brain organoids provide promising platforms for deciphering the cellular and molecular processes of human neural development and diseases.Although various studies and reviews have described developments and advancements in brain organoids,few studies have comprehensively summarized and analyzed the global trends in this area of neuroscience.To identify and further facilitate the development of cerebral organoids,we utilized bibliometrics and visualization methods to analyze the global trends and evolution of brain organoids in the last 10 years.First,annual publications,countries/regions,organizations,journals,authors,co-citations,and keywords relating to brain organoids were identified.The hotspots in this field were also systematically identified.Subsequently,current applications for brain organoids in neuroscience,including human neural development,neural disorders,infectious diseases,regenerative medicine,drug discovery,and toxicity assessment studies,are comprehensively discussed.Towards that end,several considerations regarding the current challenges in brain organoid research and future strategies to advance neuroscience will be presented to further promote their application in neurological research.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

Advances and Applications of Brain Organoids

Understanding the fundamental processes of human brain development and diseases is of great importance for our health.However,existing research models such as non-human primate and mouse models remain limited due to their developmental discrepancies compared with humans.Over the past years,an emerging model,the“brain organoid”integrated from human pluripotent stem cells,has been developed to mimic developmental processes of the human brain and disease-associated phenotypes to some extent,making it possible to better understand the complex structures and functions of the human brain.In this review,we summarize recent advances in brain organoid technologies and their applications in brain development and diseases,including neurodevelopmental,neurodegenerative,psychiatric diseases,and brain tumors.Finally,we also discuss current limitations and the potential of brain organoids.

Modeling human neurodevelopmental diseases with brain organoids

Studying the etiology of human neurodevelopmental diseases has long been a challenging task due to the brain’s complexity and its limited accessibility.Human pluripotent stem cells(hPSCs)-derived brain organoids are capable of recapitulating various features and functionalities of the human brain,allowing the investigation of intricate patho-genesis of developmental abnormalities.Over the past years,brain organoids have facilitated identifying disease-associated phenotypes and underlying mechanisms for human neurodevelopmental diseases.Integrating with more cutting-edge technologies,particularly gene editing,brain organoids further empower human disease modeling.Here,we review the latest progress in modeling human neurodevelopmental disorders with brain organoids.

Brain organoid-on-chip system to study the effects of breast cancer derived exosomes on the neurodevelopment of brain

Early human brain development can be affected by multiple prenatal factors that involve chemical exposures in utero,maternal health characteristics such as psychiatric disorders,and cancer.Breast cancer is one of the most common cancers worldwide arising pregnancy.However,it is not clear whether the breast cancer might influence the brain development of fetus.Exosomes secreted by breast cancer cells play a critical role in mediating intercellular communication and interplay between different organs.In this work,we engineered human induced pluripotent stem cells(hiPSCs)-derived brain organoids in an array of micropillar chip and probed the influences of breast cancer cell(MCF-7)derived-exosomes on the early neurodevelopment of brain.The formed brain organoids can recapitulate essential features of embryonic human brain at early stages,in terms of neurogenesis,forebrain regionalization,and cortical organization.Treatment with breast cancer cell derived-exosomes,brain organoids exhibited enhanced expression of stemness-related marker OCT4 and forebrain marker PAX6.RNA-seq analysis reflected several activated signaling pathways associated with breast cancer,medulloblastoma and neurogenesis in brain organoids induced by tumor-derived exosomes.These results suggested that breast cancer cell-derived exosomes might lead to the impaired neurodevelopment in the brain organoids and the carcinogenesis of brain organoids.It potentially implies the fetus of pregnant women with breast cancer has the risk of impaired neurodevelopmental disorder after birth.

The generation and properties of human cortical organoids as a disease model for malformations of cortical development

As three-dimensional“organ-like”aggregates,human cortical organoids have emerged as powerful models for studying human brain evolution and brain disorders with unique advantages of humanspecificity,fidelity and manipulation.Human cortical organoids derived from human pluripotent stem cells can elaborately replicate many of the key properties of human cortical development at the molecular,cellular,structural,and functional levels,including the anatomy,functional neural network,and interaction among different brain regions,thus facilitating the discovery of brain development and evolution.In addition to studying the neuro-electrophysiological features of brain cortex development,human cortical organoids have been widely used to mimic the pathophysiological features of cortical-related disease,especially in mimicking malformations of cortical development,thus revealing pathological mechanism and identifying effective drugs.In this review,we provide an overview of the generation of human cortical organoids and the properties of recapitulated cortical development and further outline their applications in modeling malformations of cortical development including pathological phenotype,underlying mechanisms and rescue strategies.

Cerebral organoids exhibit mature neurons and astrocytes and recapitulate electrophysiological activity of the human brain

Multiple protocols have been devised to generate cerebral organoids that recapitulate features of the developing human brain, including the presence of a large, multi-layered, cortical-like neuronal zone. However, the central question is whether these organoids truly present mature, functional neurons and astrocytes, which may qualify the system for in-depth molecular neuroscience studies focused at neuronal and synaptic functions. Here, we demonstrate that cerebral organoids derived under optimal differentiation conditions exhibit mature, fully functional neurons and astrocytes, as validated by immunohistological, gene expression, and electrophysiological, analyses. Neurons in cerebral organoids showed gene expression profiles and electrophysiological properties similar to those reported for fetal human brain. These important findings indicate that cerebral organoids recapitulate the developing human brain and may enhance use of cerebral organoids in modeling human brain development or investigating neural deficits that underlie neurodevelopmental and neuropsychiatric conditions, such as autism or intellectual disorders.

Neural lineage differentiation of human pluripotent stem cells:Advances in disease modeling

Brain diseases affect 1 in 6 people worldwide.These diseases range from acute neurological conditions such as stroke to chronic neurodegenerative disorders such as Alzheimer’s disease.Recent advancements in tissue-engineered brain disease models have overcome many of the different shortcomings associated with the various animal models,tissue culture models,and epidemiologic patient data that are commonly used to study brain disease.One innovative method by which to model human neurological disease is via the directed differentiation of human pluripotent stem cells(hPSCs)to neural lineages including neurons,astrocytes,and oligodendrocytes.Three-dimensional models such as brain organoids have also been derived from hPSCs,offering more physiological relevance due to their incorporation of various cell types.As such,brain organoids can better model the pathophysiology of neural diseases observed in patients.In this review,we will emphasize recent developments in hPSC-based tissue culture models of neurological disorders and how they are being used to create neural disease models.

脑类器官在创伤性脑损伤研究中的应用

创伤性脑损伤(traumatic brain injury,TBI)是由外力引起的脑正常功能破坏和/或脑组织病理性损伤。由于发生功能神经元不可逆的丧失和神经组织损伤,中枢神经系统在创伤后很难修复和再生,造成TBI患者预后存在严重后遗症。由于缺乏概括人脑特征的TBI模型,虽然有大量成功的临床前研究用于TBI治疗,但少有转化应用。脑类器官作为一种自我组装的3D组织,具有来自干细胞和器官特异性细胞类型的集合,能够在一定程度上模拟天然脑器官的结构和功能,应用于TBI研究,能够有效地解决人中枢神经系统组织获取的局限性以及人和动物之间的生物学特征不匹配问题。因此,本文通过对脑类器官的产生、特性及其在TBI模型中的应用进行综述,重点讨论了基于人多能干细胞(hPSCs)的脑类器官模拟体外TBI模型和嵌合动物TBI模型研究进展,以期为脑类器官应用于TBI损伤的研究及治疗提供新思路。

利用人脑类器官构建胶质瘤侵袭模型

目的:利用人脑类器官建立胶质瘤侵袭模型并用于评价胶质瘤细胞侵袭能力。方法:通过神经定向诱导分化形成人胚胎干细胞(embryonic stem cells,ESCs)来源的脑类器官;将人脑类器官与人胶质瘤干细胞(glioma stem cells,GSCs)共培养,构建胶质瘤侵袭模型;检测不同类型GSCs在脑类器官中的侵袭深度,评价其侵袭能力。结果:(1)人ESCs经诱导分化,于第31天形成大量神经上皮芽结构,免疫荧光染色显示神经干细胞特异性蛋白Sox2、Pax6和nestin表达,提示脑类器官形成;(2)与脑类器官共培养后,胶质瘤细胞侵入脑类器官中生长,成功建立胶质瘤侵袭模型;(3)不同类型GSCs在脑类器官中的侵袭深度存在差别。结论:本研究建立了胶质瘤侵袭类器官模型,该模型可用于评价GSCs的侵袭能力。

脑类器官研究进展和伦理问题研究

类器官是由干细胞在体外构建的三维生物结构,它们部分地模仿了真实器官的功能。脑类器官是类器官研究中极其重要的分支部分,它可以通过运用多功能干细胞进行分化,在三维培养环境中形成所需的细胞类型,并经自组装成结构体,但目前尚无法完全复制真实人脑的结构和生理特征。而脑类器官的成熟可能形成意识,这使得脑类器官研究会面临道德地位的判定、知情同意等伦理问题。阐述了脑类器官的研究进展和未来发展方向,并从伦理原则规范、保障公众知情权利、法律监管等角度,对脑类器官研究中所面临的伦理问题提出了多维治理策略。通过对上述问题的探讨,为将来制定伦理原则指导脑类器官的研究应用提供参考。

三邻甲苯磷酸酯对人诱导多能干细胞来源的人脑类器官的毒性

目的观察三邻甲苯磷酸酯(TOCP)对人诱导多能干细胞来源的人脑类器官的神经毒性。方法培养约60 d的人脑类器官随机分为4组,正常对照组、TOCP 1,5和10 mmol·L^(-1)组,每组3个,孵育24 h。CTG法测定人脑类器官细胞存活率,TUNEL法检测人脑类器官细胞凋亡,倒置荧光显微镜实时记录人脑类器官的钙震荡信号,比色法检测总抗氧化能力(T-AOC),还原型谷胱甘肽(GSH)、丙二醛(MDA)、一氧化氮(NO)和过氧化氢(H_(2)O_(2))含量及超氧化物歧化酶(SOD)和谷胱甘肽还原酶(GSH-Px)活性。结果与正常对照组相比,TOCP 5和10 mmol·L^(-1)组人脑类器官细胞存活率显著降低(P<0.05);TUNEL显色表明,TOCP 5 mmol·L^(-1)组人脑类器官细胞凋亡增加(P<0.01);TOCP 5 mmol·L^(-1)组人脑类器官细胞出现钙震荡信号紊乱,表现为荧光强度持续增强、震荡幅度增加(P<0.05)。与正常对照组相比,TOCP 1,5和10 mmol·L^(-1)组人脑类器官H_(2)O_(2)含量均显著升高(P<0.01),10 mmol·L^(-1)组MDA含量显著升高(P<0.01),各组NO水平无显著变化。TOCP 1,5和10 mmol·L^(-1)组T-AOC显著降低(P<0.05,P<0.01),GSH含量下降(P<0.05,P<0.01);TOCP 5和10 mmol·L^(-1)组SOD和GSH-Px活性显著降低(P<0.05,P<0.01)。结论TOCP诱导人脑类器官细胞凋亡,降低人脑类器官的细胞存活率,具有显著神经毒性,其机制可能是通过破坏人脑类器官的钙稳态,使细胞内钙离子浓度升高,氧化应激反应加剧,从而介导了细胞凋亡等损伤发生。

人类3D大脑类器官研究进展

人类3D大脑类器官为研究人脑发育和神经系统疾病提供了新的模型。体外培育的人类3D大脑类器官主要是由人多能干细胞(human pluripotent stem cell,hPSC),包括人胚胎干细胞(embryonic stem cell,ESC)和诱导多能干细胞(induced pluripotent stem cell,iPSC)分化而来。iPSC重编程技术与3D大脑类器官技术相结合,可以获得来自患者的iPSC并分化成包括神经元及大脑类器官在内的几乎任何人体细胞或组织,是动物实验向临床试验转化的桥梁。本文回顾了从多能干细胞技术到3D大脑类器官诞生并发展的历程,介绍了以3D大脑类器官为工具构建脑发育和神经系统疾病的研究模型,讨论了大脑类器官在其他方面的应用和相关技术的研究进展,并分析了3D大脑类器官的局限性及其未来可能的发展方向。

培养皿中的大脑模型:人脑类器官研究的伦理问题探讨

人脑类器官技术为人类神经系统所特有的神经发育和疾病过程研究提供了具有生理相关性的模型,可用于大脑发育的研究和脑部疾病的预测。然而,这一技术的最新发展带来了新的伦理问题和潜在风险。通过概述由人脑类器官技术引发的意识风险方面、动物“人性化”和能力增强方面、人脑类器官的道德地位归属方面以及隐私保护与知情同意方面的伦理问题,提出道德预防性原则、进行伦理限制和伦理监督、设立特定伦理评估机构以及扩大公众参与和伦理讨论等对应举措,为人脑类器官的伦理治理提供一些思路和建议。

类脑器官在环境毒理学中的应用

自19世纪以来,模式生物的出现帮助研究人员进一步了解细胞信号通路,确定药物靶点以及进行环境毒理学的研究。外源性化学物质,如污染物、药物和工业化学品,可能会影响大脑的生物过程或其功能,并最终导致神经疾病。然而人脑是一个复杂的、组织严密的器官,在很多方面与模式生物有着根本的区别。由于物种的差异,动物模型可能不能完全的模拟人脑以测试环境污染物的神经毒性。由人多能干细胞诱导而来的人类脑器官在基因组、转录组以及代谢组水平更贴合人体的真实情况,提供了测试以及预测环境污染物神经毒性的有效平台,成为研究神经毒理学的新模型。本文综述了类脑器官技术的新进展及其在评估环境毒物方面的应用,以期为类脑器官在环境神经毒理学应用提供新见解。

人源脑类器官模型在孤独症相关神经发育缺陷研究中的应用与进展

孤独症谱系障碍(autism spectrum disorder,ASD)是一种以社交障碍及刻板行为为核心症状的神经发育疾病。其受遗传和环境等多种因素影响,病因复杂、患者异质性极高,这给疾病机制研究和治疗靶点的研究造成了阻碍。3D人脑类器官是由多能干细胞(pluripotent stem cell,PSCs)经悬浮培养及诱导分化后自组织形成的器官样三维组织,其可携带患者完整的遗传信息,可在体外模拟胚胎的早期脑发育过程并反映病理过程。脑类器官是研究孤独症谱系障碍的理想模型与平台,该文将对近年来人脑类器官在孤独症研究中的进展与成果作综述。

Brain physiome:A concept bridging in vitro 3D brain models and in silico models for predicting drug toxicity in the brain

In the last few decades,adverse reactions to pharmaceuticals have been evaluated using 2D in vitro models and animal models.However,with increasing computational power,and as the key drivers of cellular behavior have been identified,in silico models have emerged.These models are time-efficient and cost-effective,but the prediction of adverse reactions to unknown drugs using these models requires relevant experimental input.Accordingly,the physiome concept has emerged to bridge experimental datasets with in silico models.The brain physiome describes the systemic interactions of its components,which are organized into a multilevel hierarchy.Because of the limitations in obtaining experimental data corresponding to each physiome component from 2D in vitro models and animal models,3D in vitro brain models,including brain organoids and brain-on-a-chip,have been developed.In this review,we present the concept of the brain physiome and its hierarchical organization,including cell-and tissue-level organizations.We also summarize recently developed 3D in vitro brain models and link them with the elements of the brain physiome as a guideline for dataset collection.The connection between in vitro 3D brain models and in silico modeling will lead to the establishment of cost-effective and time-efficient in silico models for the prediction of the safety of unknown drugs.

Human stem cell modeling of neuropsychiatric disorders:from polygenicity to convergence

Neuropsychiatric disorders(NPD)are prevalent and devastating,posing an enormous socioeconomic burden to modern society.Recent genetic studies of NPD have identified a plethora of common genetic risk variants with small effect sizes and rare risk variants of high penetrance.While exciting,there is a pressing need to translate these genetic discoveries into better understanding of disease biology and more tailored clinical interventions.Human induced pluripotent stem cell(hiPSC)-derived 2D and 3D neural cultures are becoming a promising cellular model for bridging the gap between genetic findings and disease biology for NPD.Leveraging the accessibility of patient biospecimen to convert into stem cells and the power of genome editing technology to engineer disease risk variants,hiPSC model holds the promise to disentangle the disease polygenicity,model genetic interaction with environmental factors,and uncover convergent gene pathways that may be targeted for more tailored clinical intervention.

人脑类器官的道德地位问题

人脑类器官(human brain organoids)能够突破传统疾病研究模型的瓶颈,成为探索人类大脑发育、脑部相关疾病发生机制以及药物筛选和基因治疗的优质模型。然而,在给神经科学领域带来革命性变化的同时,人脑类器官也将引发诸多伦理新问题,其中道德地位评估尤为关键。拥有利益的实体才拥有道德地位,而意识决定其利益诉求。与人脑类器官道德地位相关的意识有现象意识、通达意识和自我意识,应设置最低限度现象意识、通达意识和类人自我意识三个观测点,以评估人脑类器官的道德地位并规范相应研究行为。