A human corticospinal organoid-slice connectoid model informs enhancer strategies for post-injury axon regrowth.

Axon elongation in the mammalian central nervous system (CNS) declines during development, limiting regenerative capacity after birth. Intrinsic regulators of this process are promising repair targets, as immature axons can regrow in tissues otherwise not conducive to regeneration. Yet the precise timing and mechanisms underlying the cessation of axon growth in the human CNS remain unresolved. Here, we developed a three-dimensional human corticospinal motor organoid-slice connectoid platform mimicking the developmental axon elongation program and its subsequent restriction through maturation. Cortical and spinal slices establish functional connections while remaining spatially segregated, enabling cortical cell-type-specific observations without direct confounding effects by spinal cells. Using single-cell transcriptomics, computational analyses, axon regrowth assays, and live imaging, we identified transcriptional alterations contributing to decreased axon growth in maturing human cortical projection neurons. We further demonstrate that this decline can be reversed using compounds and repurposable drugs targeting a maturation-associated transcriptional shift, promoting post-injury axon repair.