背景:第一个人脑类器官协议是在过去十年的开始提出的,从那以后,该领域见证了许多新的大脑区域特定模型的发展,以及随后的协议调整和修改。关于大脑类器官技术的大量数据可能对该领域的新科学家来说是压倒性的,因此降低了其可访问性。这里,我们旨在通过系统地审查人脑类器官出版物,为该领域的新研究人员提供实用指南。
方法:选择了2010年至2020年之间发表的文章,并将其分类为脑类器官应用。进一步分析了描述神经发育研究或新型类器官模型的方案的脑类器官的培养持续时间。类器官生成关键方面的协议比较,并进行功能表征分析。然后,我们总结了针对不同模型采取的方法,并分析了用于实现类器官区域化的小分子和生长因子的应用。最后,我们分析了类器官细胞类型组成的文章,每个细胞类型报告的时间点,以及用于表征不同细胞类型的免疫荧光标记。
结果:钙成像和膜片钳分析是脑类器官中最常用的神经元活性测定。神经活动显示在所有分析的模型中,然而网络活动已经过时了,模型,和测定依赖性。背侧前脑类器官的诱导主要通过组合的(双重)SMAD和Wnt信号传导抑制来实现。腹侧前脑类器官诱导与双重SMAD和Wnt信号抑制,以及Shh途径的额外激活。脑类器官和背侧前脑模型在第35天和第60天之间呈现大多数细胞类型。84天,背侧前脑类器官含有星形胶质细胞和潜在的少突胶质细胞。免疫荧光分析显示多种细胞类型的非排他性标志物的细胞类型特异性应用。
结论:我们提供了一个容易获得的人脑类器官培养概述,这可能有助于那些研究大脑类器官的人定义他们选择的模型,文化时间,功能测定,分化,和表征策略。
The first human brain organoid protocol was presented in the beginning of the previous decade, and since then, the field witnessed the development of many new brain region-specific models, and subsequent protocol adaptations and modifications. The vast amount of data available on brain organoid technology may be overwhelming for scientists new to the field and consequently decrease its accessibility. Here, we aimed at providing a practical guide for new researchers in the field by systematically reviewing human brain organoid publications.
Articles published between 2010 and 2020 were selected and categorised for brain organoid applications. Those describing neurodevelopmental studies or protocols for novel organoid models were further analysed for culture duration of the brain organoids, protocol comparisons of key aspects of organoid generation, and performed functional characterisation assays. We then summarised the approaches taken for different models and analysed the application of small molecules and growth factors used to achieve organoid regionalisation. Finally, we analysed articles for organoid cell type compositions, the reported time points per cell type, and for immunofluorescence markers used to characterise different cell types.
Calcium imaging and patch clamp analysis were the most frequently used neuronal activity assays in brain organoids. Neural activity was shown in all analysed models, yet network activity was age, model, and assay dependent. Induction of dorsal forebrain organoids was primarily achieved through combined (dual) SMAD and Wnt signalling inhibition. Ventral forebrain organoid induction was performed with dual SMAD and Wnt signalling inhibition, together with additional activation of the Shh pathway. Cerebral organoids and dorsal forebrain model presented the most cell types between days 35 and 60. At 84 days, dorsal forebrain organoids contain astrocytes and potentially oligodendrocytes. Immunofluorescence analysis showed cell type-specific application of non-exclusive markers for multiple cell types.
We provide an easily accessible overview of human brain organoid cultures, which may help those working with brain organoids to define their choice of model, culture time, functional assay, differentiation, and characterisation strategies.