Long-lasting structural and functional maturation in transplanted human neurons reprogrammed from glial progenitor cells

Direct reprogramming of brain-resident glial progenitor cells (GPCs) into induced neurons (iN) shows great promise for future regenerative therapies for brain diseases. This type of cell conversion is facilitated by introducing lineage-specific transcription factors or small molecules to somatic cells, and can even be achieve in vivo , targeting resident glia cells in the brain for in vivo conversion. While this have shown promising results in animal models, a crucial step is to show the long-term survival and maturation of human reprogrammed cells in vivo. However, this remains largely unexplored partly due to the inaccessible cell source of GPC and difficulty in transplanting converted neurons. In this study, we assessed human GPCs derived iNs for their long-term structural and functional neuronal maturation after transplantation to the immunodeficient mouse brain. GPCs were transduced with established transcription factor cocktail and transplanted to the medial prefrontal cortex as three-dimensional cultures to improve survival and integration upon transplantation. Our results reveal survival of hiNs, for up to 10 months post-transplantation (MPT) and wide distribution across local and proximal functionally connected brain regions. Morphological analysis revealed a gradual neuronal maturation over time, characterized by increase in cellular complexity and expression of neuronal markers. Supporting these findings, electrophysiological recordings demonstrated progressive functional maturation up to 5 MPT with increase in sodium and potassium currents, and the ability to generate multiple evoked and spontaneous action potentials, validating a neuronal phenotype. Importantly, these changes were absent in control grafts of non-converted hGPCs that retained their glia identity. Our findings indicate that hGPCs derived iNs maintain a stable neuronal phenotype that persist in the mouse brain for extended periods. These cells exhibit survival and remarkable adaptability to the host environment, enabling both structural and functional maturation over time. These results highlight their potential as a promising strategy for neural replacement therapies aimed at restoring damaged circuits and promoting brain repair. Highlights • Human neurons reprogrammed from glia progenitor cells survive long term upon transplantation in the immunodeficient mouse brain. • The human induced neurons migrate to local and distal connected brain regions within prefrontal cortex circuitry. • Grafted neurons gradually develop neuronal complex morphology and electrophysiological functional properties in the mouse brain.