Extended Data Fig. 1: Aged NSPCs exhibit reduced potential for differentiating into neurons compared to young NSPCs.
a, Confocal microscopy images showing immunofluorescence co-labeling of DCX and BrdU in the coronal SVZ sections from young and aged mice. Scale bar, 40 μm. The quantification of DCX and BrdU double-positive cells was performed in both the lateral and dorsolateral SVZ for each section. A total of four sections per mouse, spaced at 120 μm intervals along the rostrocaudal axis of the SVZ, were used for cell counting. Seven mice per young group. Six mice per aged group. The average number of DCX and BrdU double-positive cells per group was plotted in the chart on the right panel. P = 0.0009. **** P < 0.001. b, Immunofluorescence labeling of SOX2 in NSPCs isolated from the young and aged SVZ, respectively. Primary NSPC-derived neurospheres were dissociated into single cells for staining. Nuclei were stained with DAPI. Scale bar, 200 μm. Quantification data were plotted, showing the percentage of SOX2-positive cells among DAPI-positive cells counted from 10 microscopic fields per age group. ns: not significant.c, Immunofluorescence labeling of NESTIN in NSPCs isolated from the young and aged SVZ, respectively. The staining and quantification methods used are identical to those described in (b). d, The size of primary NSPC-derived neurospheres in young and aged groups. Neurospheres were imaged after 6 days of culture, and their diameters were measured through ImageJ software. n = 80. P = 3.05E-15. **** P < 0.001. e, Luminescent cell viability analysis of young and aged NSPCs cultured from Day 1 to Day 4. n = 3 independent experiments. Day1: P = 0.2436, Day2: P = 0.0012, Day3: P = 0.0013, Day4: P = 0.0003. *** P < 0.005. **** P < 0.001. ns: not significant. f, Cell death analysis by propidium iodide (PI) staining. Scale bar, 200 μm. Quantification data were plotted, showing the percentage of PI-positive cells among Hoechst-positive cells counted from 10 microscopic fields per age group. g, Neural differentiation starting from NSPC-derived neurosphere as neurite outgrows. After 2 days of differentiation, cells were fixed and subjected to immunofluorescence labeling of neuron marker TUJ1. The number of TUJ1+ differentiated cells with neurites that migrated away from the neurosphere was counted. Scale bar, 200 μm. Data were plotted with the percentage of TUJ1 versus DAPI-positive cells with neurites in each neurosphere differentiation. n = 10. P = 6.71E-05. h, Immunofluorescence labeling of neuron marker MAP2 four days post-differentiation. The number of MAP2+ differentiated cells that migrated away from the neurosphere was counted. Scale bar, 200 μm. n = 8 in young group. n = 13 in aged group. P = 4.83E-06. i, Immunofluorescence labeling of astrocyte marker GFAP four days post-differentiation. The number of GFAP+ differentiated cells that migrated away from the neurosphere was counted. Scale bar, 200 μm. Data were plotted with the percentage of GFAP-positive cells with astrocytic extensions versus DAPI-positive cells in each neurosphere differentiation. n = 13 per group. Data analysis for g-i: P = 3.99E-07. *** P < 0.005. **** P < 0.001. Error bars are mean ± SD in all graphs. Two-sided Student’s t-test for statistical analysis in all graphs.
