The Journal of Neuroscience, November 30, 2005, ():
Overexpression of the Epidermal Growth Factor Receptor Confers Migratory Properties to Nonmigratory Postnatal Neural Progenitors
J. Neurosci. Aguirre et al.
25: 11092
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure S1. CNP-EGFP+ cells that migrate towards EGF-heparin beads are multipotential. (a1) SVZ explants from the CNP-EGFP mouse were co-cultured with EGF-soaked heparin beads for 72hr. Dotted lines indicate the edges of the SVZ explants. (a2) After 72hr in culture, the explant was scraped off to leave migratory CNP-EGFP+ cells attached to the culture dish. (b) These cells were FACS-purified and re-plated to generate neurospheres. After 6-7 days, neurospheres were plated in differentiation medium (Aguirre and Gallo, 2004), and stained with anti-Map2 (neurons) and Gal-C (oligodendrocytes) (c), or anti-Map2 (neurons) and anti-GFAP (astrocytes) (d), to demonstrate multipotentiality. Scale bars = 50µm (a, c and d), and 300 µm (b).
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Supplementary Figure S2. The cell viability and proliferation rate of SVZ NG2+ cells were not modified during the time frame of the microchemotaxis chamber migration assay. SVZ NG2+ cells were FACS-purified from the CNP-EGFP mouse at P8, plated on coverslips (with or without EGF) and pulse-labeled with BrdU for 12h. Cells were then processed for immunocytochemistry with anti-NG2 and anti-BrdU antibodies. Cells were also stained for TUNEL, to determine cell viability. (a and b) BrdU incorporation in EGFP+ cells cultured in the absence or in the presence of 10ng/ml EGF. (c and d) BrdU incorporation in NG2+/EGFP+ cells cultured in the absence or in the presence of 10ng/ml EGF. (c2-c5 and d2-d5) Examples of individual NG2+/EGFP+/BrdU+ cells. (e and f) TUNEL assay in untreated (Neg) and DNAse-treated (Pos) EGFP+ cells. (g and h) TUNEL staining of NG2+/EGFP+ cells cultured in the absence or in the presence of 10ng/ml EGF. Note that NG2-staining is not shown. Arrows indicate double-positive cells. (g2-g5 and h2-h5) Examples of individual EGFP+/TUNEL+ cells. Scale bars = 20µm (a), and 50 µm (c and d).
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Supplementary Figure S3. Cortical NG2+/DiI+ cells purified from a C57BL/6 P8 wt mouse do not migrate after transplantation into the lateral ventricle. Cortical NG2+ cells were FACS-purified from P8 C57BL/6 wt mice, labeled with DiI and then transplanted in the LV of P4 wild-type host mice. (a) At 1 WAT cells displayed very limited migration. A small percentage of DiI+ cells were found in the SCWM (a1). However, the majority of the graft-derived DiI+ cells were found attached to the parenchyma of the LV (a3). A large percentage of DiI+ cells were found in the aSVZ (a2) and in the fimbria (a4). Scale bars = 300µm (a), and 50µm (a1-a4).
