11.7T Diffusion Magnetic Resonance Imaging and Tractography to Probe Human Brain Organoid Microstructure

Abstract

Background: Human brain organoids are 3-dimensional cellular models that mimic architectural features of a developing brain. Generated from human induced pluripotent stem cells, these organoids offer an unparalleled physiologically relevant in vitro system for disease modeling and drug screening. In the current study, we sought to establish a foundation for a magnetic resonance imaging (MRI)-based, label-free imaging system that offers high-resolution capabilities for deep tissue imaging of whole organoids.

Methods: An 11.7T Bruker/89 mm microimaging system was used to collect high-resolution multishell 3-dimensional diffusion images of 2 induced pluripotent stem cell-derived human hippocampal brain organoids. The MRI features identified in the study were interpreted on the basis of similarities with immunofluorescence microscopy.

Results: MRI microscopy at ≤40 μm isotropic resolution provided a 3-dimensional view of organoid microstructure. T2-weighted contrast showed a rosette-like internal structure and a protruding spherical structure that correlated with immunofluorescence staining for the choroid plexus. Diffusion tractography methods can be used to model tissue microstructural features and possibly map neuronal organization. This approach complements traditional immunohistochemistry imaging methods without the need for tissue clearing.

Conclusions: This proof-of-concept study shows, for the first time, the application of high-resolution diffusion MRI microscopy to image 2-mm diameter spherical human brain organoids. Application of ultrahigh-field MRI and diffusion tractography is a powerful modality for whole organoid imaging and has the potential to make a significant impact for probing microstructural changes in brain organoids used to model psychiatric disorders, neurodegenerative diseases, and viral infections of the human brain, as well as for assessing neurotoxicity in drug screening.

Keywords: Differentiating rosette-like structures; Diffusion imaging microscopy; High-field MRI; Human brain organoids; Human induced pluripotent stem cells; Neuronal streamlines.

Figure 1

Characterization of 125-day-old brain organoids. (A–E, I,J) Immunostaining of 10-μm-thick sections of organoids with antibodies recognizing MAP2/TTR (A, B), FOXG1 (C), GABA (D), PROX1 (E), CTIP2 (I), and TBR1 (J). (F) Immunostaining of whole organoid for GRIK4. (G, H) Immunostaining of monolayer cultures of neurons generated from differentiating organoids dissociated at day 71 and cultured on Matrigel-coated plate for additional 19 days. Nuclei were counterstained with Hoechst 33342. Scale bar = 250 mm in panels (A–C, I, and J), 50 μm in panels (D, G, and H), 10 μm in panel (E), and 100 μm in panel (F).

Figure 2

Organoid sample holder for magnetic resonance imaging (MRI). (A) Organoid sample holder made from a 5-mm NMR tube and polyethylene (PE) tubing. (B, C) Pilot image of an organoid in phosphate-buffered solution (PBS) background. The PE tubing is observed in black contrast with the organoid in the center. (D, E) T2-weighted images after replacing PBS with Fluorinert. (D) Differentiating rosette-like structures with T2 hypointense contrast in the center of the organoid, with a small residual water droplet visible at the 7-o’clock position. (E) A structure presumably containing a choroid plexus and water.

Figure 3

Diffusion magnetic resonance imaging (dMRI) analyses. (A) Coronal views of dwi b0 and dMRI segmentation. In dMRI, this segmentation uses a standard color-coding convention, where blue voxels reflect anisotropic areas, and green/red voxels reflect isotropic areas. Here, to enhance the side-by-side comparison with the confocal imaging, the blue/green color coding was switched. (B) Side-by-side comparison of the dMRI segmentation and confocal imaging. (C) Orthogonal and 3-dimensional (3D) views of one differentiating rosette-like structure superimposed on the mean A0 image. (D) Coronal view shown in panel (C) is zoomed out to display interconnecting local tractography using color-coding convention in dMRI. (E) Reconstruction of whole organoid depicts streamlines using increasing range of streamline length. It is important to acknowledge that streamlines are a visual representation of connecting neighboring voxels with similar orientational diffusion anisotropy, such as voxels in the context of white matter tissue but not only (e.g., muscle tissue), and results are strongly dependent on the setting of main parameters. nqa, normalized quantitative anisotropy.