Journal: bioRxiv

Article Title: Spontaneous differentiation across cell lineages of separate germ layer origin during progenitor cell-mediated regeneration of the central nervous system

doi: 10.1101/2025.11.24.690179

Figure Lengend Snippet: A. Schematic linear map of the AAV-SOX10-OLIG2-gRNA and AAV-mKate2 plasmid constructs. B. Schematic of the experimental overview. Animals received either AAV-SOX10-OLIG2-gRNA or AAV-mKate2 via tail vein injection, followed by doxycycline administration in drinking water from D12 to D16. At D14 post-injection, a focal lysolecithin-induced demyelinating lesion was created in the spinal cord. Remyelination was assessed at D40, three weeks after demyelination. C. Representative immunohistochemistry images of lesioned spinal cords at 21 dpl showing SOX10 + PRX + HA - cells (endogenously arising SCs) and SOX10 + PRX + HA + cells (OPC-derived SCs infected by the AAV) in both treatment groups. D. Quantification of HA+ and SOX10 + MPZ + HA - cells in the spinal cord lesion. E. Quantification of SOX10 + MPZ + HA + cells in the spinal cord lesion. F. Immunohistochemistry of the lesion area PRX, MPZ, and GFAP. G. Quantification of (E) shows a significant increase in the number of PRX + , MPZ + , and Sox10 + cells in animals treated with AAV-SOX10-OLIG2-gRNA compared to AAV-mKate2-treated animals. Values are presented as mean ± SD. (*p< 0.05, ns, not significant, by unpaired t-test). The above data was obtained from four biological replicates per group, with at least three fields of view quantified per replicate. H. Immuno-electron microscopy image showing the ultrastructure of an HA + SCs within the spinal cord. The SC body is pseudocoloured yellow, the associated axon in blue and the SC nucleus in red. The inset highlights immunogold particle labelling of HA in the SC nucleus.

Article Snippet: A second construct was generated using the same AAV backbone (AAV-CMVc-Cas9, Addgene #106431) and Sox10 U2 promoter, combined with rtTA, a T2A self-cleaving peptide, and WPRE elements (from pHR-EF1α-Tet-on 3G), together with mKate2 (from NFKBRp-mKate2-2xNLS-p2a-puroR, Addgene #82024) to enable fluorescent reporting of promoter activity.

Techniques: Plasmid Preparation, Construct, Injection, Immunohistochemistry, Derivative Assay, Infection, Immuno-Electron Microscopy

Journal: bioRxiv

Article Title: Spontaneous differentiation across cell lineages of separate germ layer origin during progenitor cell-mediated regeneration of the central nervous system

doi: 10.1101/2025.11.24.690179

Figure Lengend Snippet: A. Immunostaining of OLIG2, IBA1 and MBP in the lesion area. B. Quantification of the data presented in A. The bar graphs show the lesion size and the number of OLIG2 + , MBP + , and IBA1 + cells in animals receiving AAV-SOX10-OLIG2-gRNA or AAV-mKate2-treated animals. Values are expressed as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. C. Quantification of the spatial relationship between PRX + SCs and GFAP + astrocytes in the lesion site.

Article Snippet: A second construct was generated using the same AAV backbone (AAV-CMVc-Cas9, Addgene #106431) and Sox10 U2 promoter, combined with rtTA, a T2A self-cleaving peptide, and WPRE elements (from pHR-EF1α-Tet-on 3G), together with mKate2 (from NFKBRp-mKate2-2xNLS-p2a-puroR, Addgene #82024) to enable fluorescent reporting of promoter activity.

Techniques: Immunostaining