Figure 2.

Isolation of NSCs from adult Vegfr3:YFP mice. (A) YFP-positive and YFP-negative cells from the periventricular zone of Vegfr3:YFP mice were FACS-sorted. (B) The cells were tested for their ability to generate primary neurospheres in the presence of bFGF/EGF (2 × 10⁴ cells per well in 24-well plates). Histograms show that YFP-positive and YFP-negative cells form primary neurospheres and include a subset of 0.5% of cell-forming spheres (n = 6). (C) YFP-positive and YFP-negative cells from primary neurospheres are multipotent, differentiating into TuJ1⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes after growth factor removal. Quantification is shown in the histogram (n = 3). (D) YFP⁺/EGFR⁺, YFP⁺/EGFR⁻, YFP⁻/EGFR⁺, and YFP⁻/EGFR⁻ subpopulations isolated after labeling with EGF-647 are tested for their capacity to self-renew and generate secondary (2^ary NS) and tertiary (3^ary NS) neurospheres. Histogram shows the increase in the number of neurosphere cells (NS-cells) relative to the number of plated cells. Only EGFR⁺/VEGFR-3⁺ cells generate numerous secondary neurospheres (2^ary NS) and tertiary neurospheres (3^ary NS) (n = 4), even forming neurospheres after six passages (6^ary NS) (data not shown), and thus correspond to the majority of NSCs. (E) Primary neurospheres cultured in the presence of VEGF-C (50 ng/mL) and 31-C1 (a mouse VEGFR-3 function-blocking Ab, 5 μg/mL). The number of neurosphere formed (NS formed) is expressed as a percentage of the control. The 31-C1 Ab blocks the VEGF-C-induced amplification of neurospheres (n = 3). (F) Dissociated neurosphere cells are counted and TUNEL-labeled in control or VEGF-C-containing (50 ng/mL) medium (n = 3). (G) Increased proliferation of YFP⁺/EGFR⁺ NSCs following VEGF-C treatment (50 ng/mL). Cells were pulsed with BrdU (10 μM) for 24 h and fixed after 2 d in vitro (n = 3). Bar: C, 20 μm. Error bars indicate SEM. (*) P < 0.05; (***) P < 0.001, Student's t-test.