Screening for axon regeneration promoters in injured hiPSC-derived cortical neurons

Over 20 million people worldwide live with spinal cord injury (SCI), yet effective strategies to promote central nervous system (CNS) repair remain limited, partly due to the poor intrinsic regenerative capacity of adult mammalian neurons. Human-induced pluripotent stem cells (hiPSCs)–derived cortical neurons offer a powerful platform to study neuronal regrowth in a human context. In this study, we investigated axon regrowth following traumatic CNS injury in an in vitro model using hiPSCs and identified genes associated with this regrowth. Excitatory cortical neurons were derived from six hiPSC-derived cortical neural progenitor cell lines. Following staining, 6-week-old neurons underwent simulated injury, and subsequent regrowth was quantified using ImageJ. Images were taken at pre-injury, 1 hour post-injury, and 168 hours post-injury. Bulk Ribonucleic Acid (RNA) sequencing was performed on neurons after 3 weeks, and gene expression of genes of interest, including intrinsic regulators of axon regeneration in mature mammals, was analyzed. Gene set enrichment analysis (GSEA) explored the biological pathways utilized in both growth and regrowth. The model produced consistent injury areas, and there was no statistically significant regrowth over time. Nevertheless, notable donor-specific recovery patterns were observed, enabling the identification of a distinct genetic signature for regrowth. Regrowth was positively correlated with the genes KLF7, KLF6, HDAC5, and NTRK2, and negatively correlated with RAB11A, DLK1, WNT1, and STK1. Growth rates were positively correlated with the expression of PTEN, WNT1, SNPH, PLPPR1, and DCLK2, and negatively correlated with LPAR1, ASCL1, ATF3, KLF4, and KLF6. Data-driven GSEA identified enrichment for RNA 5′-end processing and NADH dehydrogenase assembly, and negative enrichment for apoptotic and dopaminergic differentiation pathways. Regeneration was also correlated with the activation of RNA and protein synthesis programs, coupled with the suppression of microtubule bundle formation. This study established a viable in vitro model of traumatic CNS injury, revealing intrinsic genetic factors that promote axonal regeneration in traumatic CNS. Our findings highlight several candidate genes and biological pathways that may influence human CNS repair. Further validation in larger donor cohorts will be essential to refine these targets and future therapeutic strategies for SCI treatment.