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.

中文翻译：

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应用艾里束光片显微镜检查 3D hiPSC 衍生的人类皮质球体的早期神经发育结构


无法观察体内相关生物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（包括患者特异性人脑类器官和人皮质球体（hCS））的进步为这一问题提供了务实的解决方案。特别是，hCS 是一种生成背侧端脑命运同质类器官的可行方法，它概括了人类皮质生成的关键方面，包括神经玫瑰花结的形成——神经管的体外相关物。这些神经源性生态位产生神经祖细胞，随后分化为神经元。二维分化诱导多能干细胞 (hiPSC) 的研究表明，神经花结的非典型形成与自闭症谱系疾病等神经发育障碍有关。然而，到目前为止，该领域的传统组织制备方法限制了在完整 hCS 或其他 3D 制备物中对这些结构进行三维成像的能力。为了克服这一限制，我们寻求优化处理 hCS 的方法，以最大限度地利用新型艾里束光片显微镜（ALSM）来获取代表早期发育时间点的 hCS 内部结构的高分辨率体积图像。通过共焦显微镜对 hCS 进行成像的传统方法在有效成像完整球体的能力方面受到限制。相反，与传统的共焦成像系统相比，ALSM 的体积采集可通过完整的、未澄清的体外组织在速度和分辨率方面提供卓越的成像。此外，hCS 的优化免疫组织化学和光学透明化改善了深度成像。 这使得神经玫瑰花结内腔的形态可视化。我们提出了一种优化的方法，该方法利用 ALSM 系统，可以以高分辨率快速成像完整的 3D 大脑类器官，同时保留大视野。这种成像方式可应用于来自 hiPSC 的非透明和透明体外人脑球体，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，可在 3D 状态下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。我们认为这种方法将有助于体外研究人类神经发育过程。