Multiscale 3D phenotyping of human cerebral organoids

Abstract

Brain organoids grown from human pluripotent stem cells self-organize into cytoarchitectures resembling the developing human brain. These three-dimensional models offer an unprecedented opportunity to study human brain development and dysfunction. Characterization currently sacrifices spatial information for single-cell or histological analysis leaving whole-tissue analysis mostly unexplored. Here, we present the SCOUT pipeline for automated multiscale comparative analysis of intact cerebral organoids. Our integrated technology platform can rapidly clear, label, and image intact organoids. Algorithmic- and convolutional neural network-based image analysis extract hundreds of features characterizing molecular, cellular, spatial, cytoarchitectural, and organoid-wide properties from fluorescence microscopy datasets. Comprehensive analysis of 46 intact organoids and ~ 100 million cells reveals quantitative multiscale “phenotypes" for organoid development, culture protocols and Zika virus infection. SCOUT provides a much-needed framework for comparative analysis of emerging 3D in vitro models using fluorescence microscopy.

Perspective: Extending the Utility of Three-Dimensional Organoids by Tissue Clearing Technologies

“…A recent study proposed that 3D information relevant to the pathophysiological processes and responses to perturbations (e.g., drug administration) is needed for further quantitative and comprehensive analysis of these in vitro disease models. Chung and colleagues developed a prominent SCOUT framework for whole 3D organoid phenotyping by clearing-and LSFM imagingbased multiple feature acquisition and atlas-independent analysis (Albanese et al, 2020). The use of SCOUT successfully extracted multiple features from the 3D dataset relevant to the developmental stages of the organoid and differences in protocols.…”

“…For example, Paşca and colleagues clearly and quantitatively reproduced cortico-striatal projections in human induced PSC (iPSC)-derived cortico-striatal assembloids with the use of the latest CUBIC-L/R procedure (Miura et al, 2020). Chung and colleagues applied SHIELD technology to single-cell and cytoarchitecture combined with multiple labeling methods for analysis of organoids (Albanese et al, 2020). ExM (Chen et al, 2015) enables super-resolution imaging together with improved clearing and staining results.…”

“…Moreover, for staining of human cerebral organoids, Chung and colleagues applied a modified eFLASH protocol that enables homogeneous staining of 8-10 whole organoids simultaneously in 1-2 days (Yun et al, 2019). The versatile staining ability of their SCOUT method further supports system-level analysis of the framework of 3D organoids (Albanese et al, 2020).…”

“…In addition, 3D reconstitution by serial sectioning is arduous due to the fragility of organoids, which leads to deformation and fracturing of the sample, resulting in insufficient resolution, low contrast, and lack of internal 3D characterization (Pampaloni et al, 2013;Renner et al, 2017). A biased sampling of twodimensional sections may result in inaccurate quantitative data with large standard deviations (Albanese et al, 2020).…”

The Effects of Environmental Adversities on Human Neocortical Neurogenesis Modeled in Brain Organoids

“…The consensus is that Zika virus reduces proliferation in NPCs, induces selective cell death in NPCs, and, finally, decreases the size of the organoid ( Sutarjono, 2019 ). Later, 3D image analysis of the morphology of the organoids infected by Zika revealed a decreased number of VZ-like areas as well as the number of SOX2-positive RGs and TBR1-positive layer VI neurons ( Albanese et al, 2020 ). However, the same analysis found a decrease in size of ventricle-like cavities, which is inconsistent with clinical observations of ventriculomegaly characteristic to Zika virus-induced microcephaly ( De Fatima Vasco Aragao et al, 2016 ; Albanese et al, 2020 ).…”