DIFFERENTIAL EFFECTS OF KETAMINE AND (2R,6R)-HYDROXYNORKETAMINE ON EARLY NEURAL TUBE DEVELOPMENT IN HUMAN IPSC-DERIVED ORGANOIDS

Treatment-resistant depression during pregnancy presents a clinical challenge, as individuals unresponsive to SSRIs may require alternative interventions during embryonic brain development. Ketamine is an effective antidepressant but raises concerns about early neurodevelopmental disruption, including neural tube formation. Here, we used human iPSC-derived neural tube organoids to compare the developmental impact of ketamine and its major metabolite, hydroxynorketamine [(2R,6R)-HNK], at early developmental stages corresponding to neural tube formation.

Neural tube (NT) organoids were exposed to two different concentrations (20μΜ and 100μM) of (2R,6R)-HNK during neural tube formation. Organoids exposed to 100 µM ketamine exhibited reduced growth, decreased neural progenitor proliferation, malformed neural tubes with enlarged lumens, and disrupted overall morphology, indicating impaired neuroepithelial organization. Microtubule networks marked by acetylated α-tubulin were markedly disorganized, suggesting cytoskeletal destabilization required for progenitor coordination. Functional calcium imaging revealed that ketamine suppressed spontaneous calcium transients, consistent with impaired basal excitability and disrupted calcium-dependent developmental signaling. Furthermore, quantitative RT-PCR (qRT-PCR) revealed altered expression of key genes involved in neural tube formation.

In contrast, (2R,6R)-HNK preserved organoid size, neural tube morphology, microtubule organization, gene expression levels and calcium transients even at high concentrations. These results indicate a lower risk of interfering with early neural tube formation compared with ketamine.

Together, our findings highlight human neural organoids as a platform for assessing developmental safety of antidepressant candidates and suggest that (2R,6R)-HNK may represent a safer alternative to ketamine for treating SSRI-resistant depression during pregnancy, minimizing the potential risk of neural tube defects in the developing embryo.