Three-dimensional (3D) brain-like models such as neurospheres and organoids recapitulate key aspects of human cortical development, providing valuable platforms for studying neurogenesis, disease mechanisms, and translational applications. However, their characterization has traditionally relied on two-dimensional (2D) histology, which fails to capture spatially clustered populations and complex cytoarchitecture. Tissue-clearing methods combined with advanced imaging now enable volumetric analyses that preserve the native 3D organization of neural cells. We applied the iDISCO+ clearing protocol to human iPSC-derived neurospheres and performed whole-mount immunolabeling followed by light-sheet fluorescence microscopy. Quantitative analyses of cell-type composition were benchmarked against conventional 2D cryosectioning. Temporal studies were performed from day 25 to day 60 of differentiation using layer-specific cortical markers (BRN2, CTIP2, and FOXP2) to assess the dynamics of superficial and deep cortical neuron generation. Finally, the impact of 3D analysis on marker co-localization was evaluated using spot-based spatial quantification of CTIP2 and COUP-TF1 expressions. Benchmarking volumetric quantification against conventional histology revealed that both methods produced comparable estimates for broadly distributed markers such as Ki67 and CTIP2. In contrast, 2D analysis substantially underestimated clustered populations, with SATB2+ and especially FOXP2+ neurons detected at significantly higher proportions in 3D. Building on this, temporal analysis of 3D-cleared neurospheres demonstrated a marked expansion of BRN2+ superficial neurons between day 25 and day 60, a robust late-stage increase in CTIP2+ deep-layer neurons, and a maturation-dependent rise of FOXP2+ layer VI neurons. In addition, 3D analysis enabled robust quantification of CTIP2+/COUP-TF1+ co-expressing neurons, revealing significantly higher estimates than 2D sectional analysis by integrating spatial information across the entire neurosphere volume. These dynamics underscore the capacity of 3D volumetric imaging to capture both homogeneous and spatially restricted neuronal populations, providing a faithful readout of cortical layer specification during neurosphere differentiation. Our study demonstrates that iDISCO+ combined with light-sheet microscopy provides a powerful and reliable approach for quantitative, multiscale characterization of neurospheres. By preserving spatial integrity and enabling volumetric assessment of both single-marker expression and co-localization, this approach reduces biases inherent to 2D sectioning and improves the accuracy of temporal and spatial analyses in 3D neural models. While limitations remain regarding antibody penetration and antigen sensitivity, volumetric approaches are essential for advancing the accuracy and reproducibility of 3D neural model analyses.
Retho et al. (Fri,) studied this question.