Astrocyte‐to‐neuron reprogramming for spinal cord repair

Abstract

Traumatic spinal cord injury (SCI) disrupts neural pathways and results in the devastating tissue damage and loss of function, with subsequent reactive gliosis and tissue scarring that limits further loss, but also limits endogenous repair. One therapeutic strategy gaining some attention in recent years is cellular reprogramming, which harnesses advances in cellular engineering to directly convert cells from one phenotype to another. The present work explores strategies for converting either mature or developing astrocytes into neurons. Our initial experiments have begun exploring viral targeting of glial cells to promote doxycycline‐inducible overexpression of neurogenic transcription factors (TFs) Ascl1 or a combination of microRNAs (miRs) miR124, miR9/9*, for neuronal conversion. Our primary goal was to establish optimal conditions for consistent and efficient reprogramming of developing spinal glia and mature reactive spinal astrocytes.Primary astrocyte cultures were generated from postnatal days 2‐3 (P2‐3) rat cortex and spinal cord, and then were activated with TGFβ (Transforming growth factor beta) one day prior to viral transduction. Fifteen days following doxycycline administration, astrocytes took on a typical neuronal morphology. Immunocytochemistry confirmed that these cells were positive for neuronal markers MAP2, NeuN and TAUR1, and negative for GFAP. Multielectrode array recordings of converted cells revealed spontaneous firing consistent with neuronal activity.These preliminary results confirm that cortical and spinal astrocytes can be efficiently reprogrammed into neurons by either Ascl1 or miRs. Advances in cellular reprogramming are providing novel ways to manipulate the cytoarchitecture of the injured cervical spinal cord. Our ongoing work is now using this same approach to modify developing glial precursors that can be harness for their pro‐reparative properties for spinal cord repair.