Astrocytes, which proliferate after brain injury, represent a promising target for cellular reprogramming due to their abundance and ability to support brain repair. In this study, we investigated the in vitro reprogramming of primary cortical astrocytes from neonatal rats into neuronal precursor cells (NPCs) using the transcription factors Oct4, Sox2, and Klf4 (OSK), delivered via lentiviral vectors. We designed a reporter system to trace the conversion of astrocytes to NPCs and neurons by using GFAP-driven iCre and Nestin- or Synapsin1-driven fluorescent reporters. After transduction, we observed morphological changes and the expression of neuronal markers in some cells, while many cells remained in a transitional state, expressing both astrocytic and neuronal features. Importantly, the study was not designed to quantify reprogramming efficiency or demonstrate full astrocyte-to-neuron conversion but rather to establish and evaluate a traceable reporter system. Our data suggest that OSK-mediated reprogramming in this in vitro model can initiate conversion of astrocytes to neuronal precursor-like cells, although the process is complex and incomplete within the one-week timeframe. We also highlight limitations in co-transduction efficiency and potential silencing of the reporter system during reprogramming. These findings provide an initial technical platform to explore astrocyte reprogramming in vitro and inform future studies aiming to refine these methods and apply them in vivo.

Transplanted neural precursor cells (NPCs) can support repair after spinal cord injury (SCI) but often face poor survival and limited differentiation within the hostile post-injury microenvironment. We tested whether subacute activation of Sonic hedgehog (Shh) signaling improves NPC engraftment and functional recovery over the duration of 6 weeks. Female Wistar rats underwent T9/T10 clip-contusion SCI and were assigned to vehicle, Shh-only, NPC-only, Shh + NPC, or sham groups (n = 8 per SCI arm, n = 5 sham, total n = 37). GFP + NPCs (4 × 10⁵) were grafted at 7 days post injury. Recombinant Shh or vehicle was delivered intrathecally via osmotic pumps for 7 days thereafter. Outcome measures included pathway activation (GLI1, SMO), NPC differentiation (NeuN, Olig2, Nestin, GFAP), astrogliosis (GFAP), CSPG deposition, immune cell infiltration (CD3, Iba1), apoptosis (cleaved Caspase-3), and locomotion (BBB, Gridwalk, CatWalk) over 6 weeks. Shh delivery increased GLI1/SMO expression and, when combined with NPCs, significantly elevated NPC-derived neurons and oligodendrocytes while preserving undifferentiated NPCs. Combined therapy reduced astrogliosis, CSPG deposition, T-cell and macrophage/microglial densities, cyst size and apoptotic cells compared with controls. These histological benefits were accompanied by improved locomotor scores in BBB, Gridwalk and selected CatWalk parameters. Our findings highlight the synergistic effects of Shh pathway activation and NPC transplantation in promoting neuroregeneration after SCI. The data support Shh as an adjunct to NPC transplantation for SCI repair.