Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.
Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.
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DOI：
10.1186/s13229-021-00413-1
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发表时间：
2021-01-22
期刊：
影响因子：
6.2
通讯作者：
Srivastava DP
中科院分区：
文献类型：
--
作者：
Adhya D;Chennell G;Crowe JA;Valencia-Alarcón EP;Seyforth J;Hosny NA;Yasvoina MV;Forster R;Baron-Cohen S;Vernon AC;Srivastava DP
无法在体内观察到相关的生物过程严重限制了人类神经发育的研究。适当的体外模型系统的进展，包括患者特异性的人类大脑类器官和人类皮质球体（hCSs），为这一问题提供了实用的解决方案。特别是，hcs是一种易于获得的方法，用于产生背端端脑命运的同质类器官，它概括了人类皮质发生的关键方面，包括神经玫瑰花的形成-与神经管相关的体外相关。这些神经源性小生境产生随后分化为神经元的神经祖细胞。在2D中分化诱导多能干细胞（hiPSCs）的研究已经将神经玫瑰花的非典型形成与神经发育障碍（如自闭症谱系疾病）联系起来。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的hCS或其他3D制备中对这些结构进行三维成像的能力。为了克服这一限制，我们试图优化一种方法来处理hCS，以最大限度地利用新型气束光片显微镜（ALSM）来获取具有早期发育时间点代表的hCS内部结构的高分辨率体积图像。通过共聚焦显微镜成像hCS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，与传统的共聚焦成像系统相比，ALSM的体积采集在速度和分辨率上都能通过完整的、未澄清的体外组织提供更好的成像。此外，优化的免疫组织化学和hcs的光学清除可以改善深度成像。这使得神经结内腔的形态可视化。我们提出了一种优化的方法，该方法利用了ALSM系统，可以在保持大视野的同时以高分辨率快速成像完整的3D脑类器官。这种成像方式可以应用于非清除和清除的体外人类大脑球体，这些球体来源于hiPSCs，用于精确检查其内部3D结构。该过程代表了一种快速、高效的方法，可以在3D中检查和量化健康和疾病状态下神经发育过程协调所需的关键结构的形成。我们认为这种方法将有助于在体外研究人类神经发育过程。
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.
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影响因子：
7.7
作者：
Knight GT;Lundin BF;Iyer N;Ashton LM;Sethares WA;Willett RM;Ashton RS
通讯作者：
Ashton RS
影响因子：
9.8
作者：
McQuin C;Goodman A;Chernyshev V;Kamentsky L;Cimini BA;Karhohs KW;Doan M;Ding L;Rafelski SM;Thirstrup D;Wiegraebe W;Singh S;Becker T;Caicedo JC;Carpenter AE
通讯作者：
Carpenter AE
影响因子：
64.5
作者：
Mariani J;Coppola G;Zhang P;Abyzov A;Provini L;Tomasini L;Amenduni M;Szekely A;Palejev D;Wilson M;Gerstein M;Grigorenko EL;Chawarska K;Pelphrey KA;Howe JR;Vaccarino FM
通讯作者：
Vaccarino FM
影响因子：
23.9
作者：
Bershteyn M;Nowakowski TJ;Pollen AA;Di Lullo E;Nene A;Wynshaw-Boris A;Kriegstein AR
通讯作者：
Kriegstein AR
影响因子：
2.9
作者：
Rakotoson, Irina;Delhomme, Brigitte;Oheim, Martin
通讯作者：
Oheim, Martin