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

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.

Dielectrophoretic Micro-Organization of Chondrocytes to Regenerate Mechanically Anisotropic Cartilaginous Tissue

Recently, many studies have focused on the repair and regeneration of damaged articular cartilage using tissue engineering. In tissue engineering therapy, cells are cultured in vitro to create a three-dimensional (3-D) tissue designed to replace the damaged cartilage. Although tissue engineering is a useful approach to regenerating cartilage, mechanical anisotropy has not been reconstructed from a cellular organization level. This study aims to create mechanically anisotropic cartilaginous tissue using dielectrophoretic cell patterning and gel-sheet lamination. Bovine chondrocytes were patterned in a hydrogel to form line-array cell clusters via negative dielectrophoresis (DEP). The results indicate that the embedded chondrocytes remained viable and reconstructed cartilaginous tissue along the patterned cell array. Moreover, the agarose gel, in which chondrocytes were patterned, demonstrated mechanical anisotropy. In summary, our DEP cell patterning and gel-sheet lamination techniques would be useful for reconstructing mechanically anisotropic cartilage tissues.

A Scribble-E-cadherin complex controls daughter cell patterning by multiple mechanisms

The fate of the two daughter cells is intimately connected to their positioning, which is in turn regulated by cell junction remodelling and orientation of the mitotic spindle. How multiple cues are integrated to dictate the ultimate patterning of daughters is not clear. Here, we identify novel mechanisms of regulation of daughter positioning in single MCF10A cells. The polarity protein, Scribble, links E-cadherin to NuMA and Arp2/3 signalling for sequential roles in daughter positioning. First Scribble transmits cues from E-cadherin localised in retraction fibres to control orientation of the mitotic spindle. Second, Scribble re-locates to the junction between the two daughters to allow a new E-cadherin-based-interface to form between them, influencing the width of the nascent daughter-daughter junction, generation of filopodia and subsequent cell patterning. Thus, E-cadherin and Scribble dynamically relocate to different intracellular sites during cell division to orient the mitotic spindle and control placement of the daughter cells after cell division.