Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.
Autism Research Centre, Department of Psychiatry, University of Cambridge, Cambridge, UK.
Mol Autism. 2021 Jan 22;12(1):4. doi: 10.1186/s13229-021-00413-1.
The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.
Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.
We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.
无法在体内观察相关的生物学过程极大地限制了人类神经发育研究。适当的体外模型系统的进步，包括患者特异性人脑类器官和人类皮质球体（hCS），为解决这个问题提供了一个切实可行的方法。特别是，hCS 是一种可获得的方法，可生成同源背侧端脑命运的同质类器官，这些类器官可重现人类皮质发生的关键方面，包括体外神经玫瑰花结的形成-神经管的对应物。这些神经发生龛产生神经祖细胞，随后分化为神经元。在 2D 中分化诱导多能干细胞（hiPSC）的研究将神经玫瑰花结的非典型形成与自闭症谱系障碍等神经发育障碍联系起来。然而，迄今为止，该领域中常规的组织制备方法限制了在完整的 hCS 或其他 3D 制剂中以三维方式对这些结构进行成像的能力。为了克服这一限制，我们一直在寻求优化一种处理 hCS 的方法，以最大限度地利用新型 Airy-beam 光片显微镜（ALSM）获取具有代表性的早期发育时间点的内部结构的高分辨率体积图像。
通过共聚焦显微镜对 hCS 进行成像的常规方法在对完整球体进行有效成像的能力方面受到限制。相比之下，通过 ALSM 进行的体积采集在速度和分辨率方面均优于传统共聚焦成像系统，可在不进行澄清的情况下对完整的体外组织进行更好的成像。此外，hCS 的优化免疫组织化学和光学澄清提高了深度成像。这允许可视化神经玫瑰花结内部腔的形态。
我们提出了一种优化的方法，该方法利用 ALSM 系统可以快速以高分辨率对完整的 3D 脑类器官进行成像，同时保持较大的视野。这种成像方式可应用于源自 hiPSC 的未经澄清和已澄清的体外人脑球体，以精确检查其内部 3D 结构。该过程代表了一种快速、高效的方法，可以在 3D 中检查和量化协调健康和疾病状态下神经发育过程所需的关键结构的形成。我们假设，这种方法将有助于体外研究人类神经发育过程。
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