Symmetry breaking of hPSCs in micropattern generates a polarized spinal cord-like organoid (pSCO) with dorsoventral organization

Abstract

Brain organoid research is advancing, but generation of organoids with proper axis formation, which could lead to spatially ordered structures for complex brain structure and function, still remains a challenge. Axis formation and related spatial cell organization in the CNS are initiated by the symmetry breaking during the early embryo development. It has been demonstrated that the geometrically confined culture of human pluripotent stem cells (hPSCs) can be used to induce symmetry breaking and regionalized cell differentiation. In this study, we generated a polarized spinal cord organoid with a self-organized dorsoventral (DV) organization, using 2D cell patterning by geometric confinement. Initially, the application of caudalization signals to hPSCs promoted the regionalized cell differentiation along the radial axis and sprouting-like protrusion morphogenesis in cell colonies confined to ECM protein micropatterns. Detachment of colonies turned them into extended spinal cord-like organoids which maintained center- and edge-derived two poles. Further analyses including single cell RNA sequencing and spatial transcriptome analysis unveiled that these organoids contained rich repertoire of developing spinal cord cells and exhibited the spatially ordered DV domain formation along the long axis without external organizing signals. Modulation of BMP and Shh signaling can control the extent of DV coverage in organoids following the principles of embryo patterning. Our study provides a simple, and precisely controllable method to generate spatially-ordered organoids for understanding of biological principles of cell patterning and axis formation during neural development.

Bioengineering the human spinal cord

“…Mesodermal cells form a radial layer on the outer edge of the micropatterned colony, such that when detachment occurs, they remain localized to a single side of the organoid and act as a source for BMP signaling. Staining, single cell RNA-seq, and spatial RNAseq data confirm that the resultant BMP4 gradient generates discrete dorsal through ventral progenitor domains except for the pMN and p3 domains, which likely require greater SHH than the organoids are able to produce endogenously (Seo et al, 2021). Kazbrun et al demonstrate that stem cells reproducibly fold into an organized 3D structure with a single lumen when a layer of matrigel is added to their micropatterned substrates (Karzbrun et al, 2021).…”

“…Micropatterned arrays in 2D can also be used as a template for 3D organoids. After attempting to caudalize hPSCs on micropatterned arrays, Seo et al show that tissues detach to produce elongating spinal organoids that are organized along the dorsoventral axis (Seo et al, 2021). Mesodermal cells form a radial layer on the outer edge of the micropatterned colony, such that when detachment occurs, they remain localized to a single side of the organoid and act as a source for BMP signaling.…”

Spinal Cord Organoids to Study Motor Neuron Development and Disease

“…An interesting approach to polarize SCOs has been reported by culturing hiPSCs in 2D in a geometrical confinement, analogous to symmetry breaking during development [ 223 ]. This method was based on previous studies demonstrating the potential of micropatterned cells to form regionalized signaling centers, resembling the embryonic organization hubs [ 224 , 225 , 226 ].…”

“…This method was based on previous studies demonstrating the potential of micropatterned cells to form regionalized signaling centers, resembling the embryonic organization hubs [ 224 , 225 , 226 ]. However, the SCOs shown in the study contained a prominent dorsal cell cluster expressing BMP while the ventral cluster expressed relatively low levels of SHH [ 223 ], suggesting that further optimization is required to generate both organizing cell clusters equally. Sandwich-like advanced hydrogel structures [ 227 ] or beads loaded with morphogens inserted into ECMs of choice [ 228 ] are alternative polarizing techniques that rely on the passive diffusion of molecules from the gel into the organoid and can be utilized to provide different morphogens from opposing sides.…”

Tensorial tomographic Fourier ptychography with applications to muscle tissue imaging

“…Creating high-resolution volumetric representations of the polarization properties of these samples is essential to studying biology and pathology. While tomographic imaging methods such as confocal-based approaches have been developed in the past to image muscle tissue and neural organoids for instance, 35 , 36 there remain relatively few microscopic techniques to jointly capture quantitative phase and anisotropy across a large 3D volume at high resolution. One very recent study attempted to create 3D anisotropy maps using off-axis LED illumination but did not provide tomographic permittivity matrix reconstructions 37 …”