Figure 2.

TSP1 deficiency impairs NPC proliferation in vitro. A) Phase contrast representative images showing primary neurosphere cultures derived from SVZ of WT and TSP1^−/− mice at different time points in vitro. B–E) Quantification of

average number (B), diameter (C), self-renewal (neurospheres/plated cells×100) (D), and amplification (live cells from neurospheres/plated cells) of P1 neurospheres obtained from WT and TSP1^−/− mice (E). Data are means ± se from ≥3 independent experiments; ≥4 mice/group. F) TSP1^−/− NPC proliferation and sphere formation were terminally impaired at P7, as compared with WT counterparts. G) Phase-contrast representative images showing primary neurosphere cultures derived from SVZ of WT and TSP1^−/− p14 pups during P3 in vitro. H) No difference in self-renewal capacity was observed for NPCs obtained from TSP1^−/− and WT p14 mice. I) FACS histograms for dissociated WT or TSP1^−/− NPCs from P5 labeled for CD133 and CD24. J) Significant reduction in CD133^hiCD24^lo early progenitor cells is evident in TSP1^−/−-derived NPCs. K) To examine proliferation of NPCs, neurospheres from WT and TSP1^−/− mice were incubated with BrdU (10 μM) for 8 h and labeled with anti-Nestin (red) and anti-BrdU (green) antibodies. L) Average percentage of BrdU⁺ cells of total Nestin⁺ cells from 3 independent experiments; ≥40 neurospheres/group. M) To examine apoptosis, neurospheres from WT and TSP1^−/− mice were TUNEL labeled. Micrographs are representative. N) Average percentage of TUNEL⁺ cells of total cells from ≥3 independent experiments; ≥40 neurospheres/group. O) To determine whether TSP1 affects cell cycle progression, dissociated WT and TSP1^−/− NPCs were analyzed by FACS for DNA content using DRAQ-5. Cell cycle histograms are representative. P) Average percentages of G1-, S-, and G2-phase cells from 3 independent experiments. Q–T) P3 neurospheres derived from TSP1^−/− mice were treated with 2 μg/ml of purified TSP1 for 72 h. Q) Representative phase-contrast images showing primary neurospheres from TSP1^−/− mice at P3 in vitro with and without treatment with purified TSP1. R–T) Quantification of average number (R), diameter (S), and amplification (T) (live cells from neurospheres/plated cells) of TSP1^−/− P3 neurospheres treated with purified TSP1 or vehicle. All data are means ± se from ≥3 independent experiments; ≥4 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001; Student’s t test. Scale bars = 200 μm (A); 100 μm (F, G, Q); 20 μm (K, M).