Laminin enhances the growth of human neural stem cells in defined culture media
1 Department of Pathology, University of Cambridge, Cambridge, UK
2 Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, UK
3 Laboratory of Neurosciences, National Institute on Aging, Baltimore, USA
4 Laboratory for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, UK
5 MRC Centre for Regenerative Medicine, Queen's Medical Research Institute, Edinburgh, UK
BMC Neuroscience 2008, 9:71 doi:10.1186/1471-2202-9-71Published: 23 July 2008
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Comparison of the effect different laminin concentrations on mouse neurosphere numbers. Mouse neurospheres were dissociated and plated at 500 cells/well in medium containing 5–20 μg/ml laminin. After 7 days, the number of neurospheres formed was quantified. Compared to wells with no added laminin, significant increases were observed for all concentrations (p < 0.01). However, whilst 10 μg/ml laminin produced significantly more neurospheres than 5 μg/ml (p < 0.01), no significant difference was found between 10 or 20 μg/ml laminin. Therefore, 10 μg/ml laminin was used for all experiments on human cells. Statistics were determined by a one-way ANOVA with Tukey post-test, with 8 technical replicates performed within a single experiment.
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Laminin increases mouse neurosphere formation in an α6β1-dependent manner. (A) Mouse neurospheres were dissociated and plated in medium containing 10 μg/ml laminin ('Medium+Laminin'), resulting in augmented primary neurosphere formation after 7 days in culture. (B) However, this difference was not maintained upon passaging in medium alone (secondary neurosphere formation). (C) Graph demonstrating that the effect of laminin on primary neurosphere formation is mediated by integrin β1. In medium alone, neither an isotype control antibody ('Iso. cont.') nor an integrin β1-blocking antibody ('β1 block'; 1 μg/ml) affected neurosphere formation. Significantly, though, in the presence of laminin the β1 integrin-blocking antibody reduced neurosphere formation to control levels. (D) Similarly, an α6 blocking antibody ('α6 block'; 20 μg/ml) inhibited the effect of laminin ('Laminin') on neurosphere formation, although blocking another laminin-binding integrin, α7 ('α7 block'; 10 μg/ml) did not. ITC, isotype control (A) *p < 0.0001, as determined by Student's t-test; n = 3. (C, D) *p < 0.001, as determined by a one-way ANOVA with Tukey post-test; n = 3.
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TS2/16 does activate integrin beta 1 signalling. (A) Platelet adhesion assay, demonstrating that the activating-β1 integrin antibody, TS2/16, stimulates adhesion to pepsin-digested collagen I at the concentrations of 5 μg/ml, relative to medium alone or to an isotype control antibody. (B) Western blot showing that 10 μg/ml TS2/16 stimulates focal adhesion kinase (FAK) phosphorylation on tyrosine 397 (pY397) after 10 min of treatment. 'Iso. Cont.', isotype control antibody (10 μg/ml). *p < 0.01, relative to the isotype control or medium alone, as determined by a one-way ANOVA with Tukey post-test; n = 2.
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