Abstract

BackgroundThe 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.ResultsConventional 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.ConclusionWe 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.

Highlights

  • Human brain organoids represent a revolutionary step forward in our ability to investigate human neurodevelopment through the use of patient-specific human induced pluripotent stem cells to generate neural tissues [1,2,3]

  • Application of Airy‐beam light sheet microscopy (ALSM) with high resolution A particular advantage of Airy-beam light sheet microscope (ALSM) is that it has a wider field of view (FOV) than Gaussian or Bessel-based light sheet beams due to the properties of the propagationinvariant Airy beam [27] (Fig. 1a–c)

  • The Airy-beam’s characteristic asymmetric excitation pattern creates lobes spreading the beam across the FOV, lowering the overall light exposure to the sample resulting in 80% less photo-bleaching in comparison with the Gaussian beam [27]

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Summary

Introduction

Human brain organoids represent a revolutionary step forward in our ability to investigate human neurodevelopment through the use of patient-specific human induced pluripotent stem cells (hiPSC) to generate neural tissues [1,2,3]. Several studies using patient-derived hiPSCs or human embryonic stem cells (hESCs) have linked abnormal neural rosette development and subsequent neurogenesis with the pathophysiology of neurodevelopmental disorders. 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. 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

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