Using Primary Neurosphere Cultures to Study Primary Cilia
| Autor: | Saikat Mukhopadhyay, Bandarigoda N. Somatilaka, Issei S. Shimada, Hemant B. Badgandi |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
General Chemical Engineering Cellular differentiation Cell Culture Techniques Biology Article General Biochemistry Genetics and Molecular Biology Receptors G-Protein-Coupled Mice 03 medical and health sciences Neural Stem Cells Ciliogenesis Neurosphere Animals Cilia Progenitor cell Sonic hedgehog Cells Cultured Neurons General Immunology and Microbiology General Neuroscience Brain Cell Differentiation Embryonic stem cell Hedgehog signaling pathway Neural stem cell Cell biology 030104 developmental biology biology.protein Neuroglia Neuroscience Adenylyl Cyclases Signal Transduction |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/55315 |
| Popis: | The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons. |
| Databáze: | OpenAIRE |
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