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December 09, 2017
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The overall goal of these differentiation cultures is to generate cerebellar cell lineages in 2D or 3D environments.These methods can help to answer basic questions regarding new developments or disease mechanisms underlying childhood brain disorders using patient derived stem cells.The main advantage of this technique is that it has a simple startup and maintenance with few factors and that it can be used as a base for testing more complex neural protocols.Demonstrating the procedure will be Lisa a technician from my laboratory.To set up the differentiation with modified method G-method of passing of hPSC’s transfer the required volume of neural maintenance medium to a sterile tube and supplement with 4 nanograms per milliliter FGF2 and ten micromolar rock inhibitor.Then, keep the medium tube at room temperature or in water bath at 37
Summary
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We describe a simplified 3D differentiation protocol for hPSCs, using defined medium and reduced growth factors, capable of generating cell aggregates with early neuroepithelial structures and positive for cerebellar-associated markers, as well as an optional 2D modification for differentiating cells as a monolayer to generate functional neurons.
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Cite this Article
Holmes, D. B., Heine, V. M. Streamlined 3D Cerebellar Differentiation Protocol with Optional 2D Modification. J. Vis. Exp. (130), e56888, doi:10.3791/56888 (2017).
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