gms | German Medical Science

Objective: The main drivers for persisting neurological symptoms after CNS injury are secondary pathophysiological changes, such as astrogliosis and glial scarring, inhibiting axonal regeneration and functional recovery. Thus, targeting the astroglial scar has become a promising experimental treatment approach. Our current project aims to convert primary rat spinal cord astrocytes to neuronal precursor cells (NPCs) in vitro, providing a proof of concept that could be translated to CNS injury models in vivo.

Methods: Astrocytes were isolated from the spinal cord of rat pups (N1-2) and subjected to anti-GLAST MACS-sorting. Purified primary astrocytes (P2) were then transduced with a custom-designed set of lentiviral vectors with the following properties: 1. Indication of successful transfection by expression of the reporter fluorescent protein BFP-HA tag under the control of an astrocyte-specific GFAP promoter. 2. Cellular conversion by overexpression of the transcription factors Oct4, Sox2, and Klf4 (OSK). 3. Indication of successful conversion in the presence of GFAP-BFP-T2A-iCre by flipping and expression of the double-floxed reporter fluorescent proteins Scarlet or GFP, driven by either a Synapsin-1 or Nestin promoter. After seven days, cells were fixed and subjected to immunofluorescence staining and confocal imaging. The phenotype of the converted cells was characterized and compared between groups.

Results: A primary cell culture of purified rat spinal cord astrocytes, characterized by GLAST and GFAP-expression, could be successfully established. Using a molecular cloning approach, the custom-designed set of lentiviruses with the ability to transduce GFAP positive astrocytes (BFP-HA tag expression) and to allow continuous evaluation of their conversion to NPCs (an increase of the Scarlet/GFP expression) was created. One week after viral transduction, the phenotype of the astrocytes had changed, as indicated by their decreased expression of astrocytic markers and increased expression of Nestin.

Conclusion: Our experiments suggest that the conversion of astrocytes to NPC-like cells with a lentiviral vector overexpressing OSK is feasible in vitro. The design of the vector system allows for specific targeting of astrocytes and continuous assessment of the transfection efficiency. Converting the astroglia scar to a more neuroregenerative tissue after CNS injuries in vivo might thus come within therapeutic reach.