A layered approach to secure the safety and therapeutic efficacy of hiPSC-derived functionalized neuronal tissueoid for repair after complete spinal cord lnjury in rats

The functional integration of donor neurons with host neural pathways is a critical treatment for spinal cord injury (SCl). We constructed human induced pluripotent stem cell (hiPSC)-derived neuronal tissueoids (Ntoids) transfected with thymidine kinase (TK) to transplant donor neurons into SCl rats. Ntoids were produced after 28-days culture in a 3-dimensional gelatin sponge scaffold and possessed neuronal cells with synaptic transmission and neural network property in vitro . Ganciclovir, a TK substrate, depleted proliferative cells in Ntoids without affecting neuronal tissueoid formation, leaving non-mitotic and functionalized neuronal cells for transplantation. Ntoid-treated SCl rats achieved significantly better functional recovery in paralyzed hindlimbs and improved electrophysiological performance. Eight weeks after transplantation, remarkable surviving of donor cells suffused the entire injury/graft site and did not present discernible long-distance cell migration, leading to intensive innervation of the injury/graft site. Ntoids possessed cholinergic hiPSC-derived neurons that survived at the injury/graft site, partially integrating with regenerating nerve fibers and neurons via synapse-like contacts. Some donor cells differentiated into astrocytes and formed a chimeric blood/spinal cord barrier with the host's vascular epithelium. Ntoids represent a structural and functional neuronal relay to restore functionality in paralyzed limbs. An additional assembloid model also presented robust synapse formation between human brain organoid and Ntoid. These findings support the safety and efficacy of the transplantation of neuronal tissue with safety-regulatory mechanisms to treat SCI and provide a framework for future clinical application using tissue-engineered Ntoid.