Extended Data Fig. 4: Cell sorting demonstrates that cells captured from the striatum did not migrate from the vSVZ.

a, Representative confocal images of the SVZ and striatum from a naive and a post-ischemic (2 dpi) mouse brain stained with TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling; labels apoptotic cells⁸⁶). Brain slides are counterstained with DAPI (blue). TUNEL⁺ nuclei (segmented with Cellpose⁶⁸) are shown in white. Scale bar: 100 µm. Bottom right: Quantification of TUNEL⁺ cells in the vSVZ and striatum of n = 5 mice depicted with 5 shapes (solid shapes: ischemic mice; outlined triangle: naive control; outlined square: sham-operated control; one-sided paired t test). b-c, FACS Strategy for sorting GLAST ⁺ cells from the vSVZ (b) and striatum (c). Striatal GLAST ⁺ cells correspond to astrocytes, while vSVZ GLAST ⁺ cells correspond to NSCs / vSVZ astrocytes. Dead cells and CD45/O4/Ter119 positive cells were excluded. d-e, The YFP intensity of GLAST ⁺ cells was captured by index sorting. Dot plots show the fraction of YFP⁺ cells from the GLAST ⁺ population, being higher in the vSVZ sample 2 dpi. Antigen names are written on each axis, followed by the filter used to measure the signal. FACS: Fluorescence-activated cell sorting. SSC-A: Side scatter area. FSC-A: forward scatter area. FSC-H: forward scatter height. RL-780/60-A: APC-Cy7 fluorochrome. VL-450/50-A: Pacific blue fluorochrome. RL-670/14-A: APC fluorochrome. YG-586/15-A: PE fluorochrome. f, FACS quantification of cells of the naive and post-ischemic vSVZ and striatum (n = 3 individual mice per condition). Shown is the percentage of YFP⁺ (TLX reporter) cells among astrocytes and NSC lineage cells after TAM injection. g-h, Neurosphere assay of cells isolated from the naive and post-ischemic vSVZ and striatum (n = 3 individual mice), quantified in h. Welch’s two-sided t-test. i-j, Immunofluorescence staining of DCX in the naive and post-ischemic vSVZ and striatum of TiCY mice, showing absence of YFP⁺DCX⁺ cells in the striatum. Arrows: YFP⁺DCX⁺ neuroblasts in the vSVZ; arrowheads: YFP-DCX⁺ neuroblasts in the striatum. j, Quantification of DCX⁺ cells in the striata of n = 6 individual mice (points). No YFP⁺DCX⁺ cells were observed. Bars and error bars in panels a, f, h, j represent mean and standard error.