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This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.
Cite this Article
Currle, D. S., Hu, J. S., Kolski-Andreaco, A., Monuki, E. S. Culture of Mouse Neural Stem Cell Precursors. J. Vis. Exp. (2), e152, doi:10.3791/152 (2007).
- Mouse neural precursors (NPCs) were isolated from E12.5 embryo cortex.
- Skin and mesenchymal layers were removed from dissected telencephalic vesicles.
- Vesicles were incubated in 0.05% trypsin with 0.02% EDTA and 0.2% BSA in HBSS for 20 minutes at 37°C.
- Trypsinization was stopped by an equal volume of 1 mg/ml soybean trypsin inhibitor (Sigma #T6522) in HBSS.
- Tissue digests were dissociated using several rounds of trituration with fire-polished Pasteur pipettes
- Cells were washed once with 0.2% BSA in HBSS and plated at 50,000 cells/ml on laminin-coated coverslips in media with 20 ng/ml EGF, 10 ng/ml FGF2 (R&D Systems or Peprotech), and 2 ug/ml heparin (Sigma).
Great advances in our understanding of CNS development and stem cell biology have been made possible by our ability to harvest, isolate and culture embryonic neural stem cells. This video demonstrates the dissection of E12.5 mouse cerebral cortex and the subsequent disaggregation and culturing of embryonic neural stem cells. Many other other similar methods have been successfully employed by other investigators.
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- Currle, D., Cheng, X., Hsu, C., Monuki, E. Direct and indirect roles of CNS dorsal midline cells in choroid plexus epithelial formation. Development. 132, (15), 3549-3559 (2005).
- Flanagan, L., Rebaza, L., Derzic, S., Schwartz, P., Monuki, E. Regulation of human neural precursor cells by laminin and integrins. J Neurosci Res. 83, 845-856 (2006).
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