Organized Neurogenic-Niche-Like Pinwheel Structures Discovered in Spinal Cord Tissue-Derived Neurospheres.

Frontiers in cell and developmental biology (2020-01-11)
Francisco Javier Rodriguez-Jimenez, Eleonora Clemente, Victoria Moreno-Manzano, Slaven Erceg

The neurogenic niche of the subventricular zone (SVZ) in adult brain tissue takes the form of a pinwheel-like cytoarchitectural structure, with mono-ciliated astrocytes displaying neural stem cell (NSC) characteristics present in the core surrounded by ciliated ependymal cells. For the first time, we have demonstrated the formation of similar pinwheel structures in spinal cord and SVZ tissue-derived neurospheres cultured in vitro. To investigate whether the organization and integrity of these pinwheel structures depends on the appropriate organization of ciliated astrocytes and ependymal cells, we modified neurosphere cell arrangements via the application of the methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dc) or the antiviral drug ganciclovir (GCV) in transgenic mice expressing herpes simplex virus thymidine kinase from the GFAP promoter (GFAP-TK). Treatment of neurospheres with 5-aza-dc increased FoxJ1 expression, a crucial factor for ciliogenesis, by reducing methylation of the FoxJ1 CpG island. 5-aza-dc also increased the expression of the astrocyte marker GFAP and caused aberrant accumulation of ciliated astrocytes. However, the ablation of dividing astrocytes within neurospheres by GCV treatment led to an increase in the accumulation of ciliated ependymal cells, as evidenced by the increased expression of the ependymal cell markers Vimentin or CD24. While 5-aza-dc and GCV treatment differentially affected cell arrangement, both compounds significantly diminished the number of pinwheel structures present in neurospheres. Thus, we suggest that the ratio of ciliated astrocytes to ependymal cells plays a crucial role in the correct formation of the pinwheel structures in spinal cord tissue-derived neurospheres in vitro.

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Poly-L-lysine solution, 0.01%, sterile-filtered, BioReagent, suitable for cell culture
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Monoclonal Anti-Tubulin, Acetylated antibody produced in mouse, clone 6-11B-1, ascites fluid
Sodium bisulfite solution, purum, ~40%
DL-Glyceraldehyde 3-phosphate solution, 45-55 mg/mL in H2O
Anti-γ-Tubulin antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution

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