title: A robust and reproducible human pluripotent stem cell derived model of neurite outgrowth in a three-dimensional culture system and its application to study neurite inhibition.
abstract: The inability of neurites to grow and restore neural connections is common to many neurological disorders, including trauma to the central nervous system and neurodegenerative diseases. Therefore, there is need for a robust and reproducible model of neurite outgrowth, to provide a tool to study the molecular mechanisms that underpin the process of neurite inhibition and to screen molecules that may be able to overcome such inhibition. In this study a novel in vitro pluripotent stem cell based model of human neuritogenesis was developed. This was achieved by incorporating additional technologies, notably a stable synthetic inducer of neural differentiation, and the application of three-dimensional (3D) cell culture techniques. We have evaluated the use of photostable, synthetic retinoid molecules to promote neural differentiation and found that 0.01 μM EC23 was the optimal concentration to promote differentiation and neurite outgrowth from human pluripotent stem cells within our model. We have also developed a methodology to enable quick and accurate quantification of neurite outgrowth derived from such a model. Furthermore, we have obtained significant neurite outgrowth within a 3D culture system enhancing the level of neuritogenesis observed and providing a more physiological microenvironment to investigate the molecular mechanisms that underpin neurite outgrowth and inhibition within the nervous system. We have demonstrated a potential application of our model in co-culture with glioma cells, to recapitulate aspects of the process of neurite inhibition that may also occur in the injured spinal cord. We propose that such a system that can be utilised to investigate the molecular mechanisms that underpin neurite inhibition mediated via glial and neuron interactions.
authors.affiliation: Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
authors.forename: Kirsty E
authors.initials: KE
authors.lastname: Clarke
authors.affiliation: Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tams
authors.affiliation: Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Henderson
authors.affiliation: Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK.
authors.forename: Mathilde F
authors.initials: MF
authors.lastname: Roger
authors.affiliation: Department of Chemistry, Durham University, South Road, Durham DH1 3LE, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Whiting
authors.affiliation: Department of Biosciences, Durham University, South Road, Durham DH1 3LE, UK; ReproCELL Europe Ltd., NETPark Incubator, Thomas Wright Way, Sedgefield TS21 3FD, UK. Electronic address: stefan.przyborski@durham.ac.uk.
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Przyborski
mesh_terms: D001565:Benzoates / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D018920:Coculture Techniques / Q000379:methods; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D055495:Neurogenesis / Q000187:drug effects / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000502:physiology*; D013764:Tetrahydronaphthalenes / Q000494:pharmacology
chemical_list: D001565:Benzoates; C542239:EC23 compound; D013764:Tetrahydronaphthalenes
keywords: 3D culture; Cell-differentiation; Neuritogenesis; Neuronal; Retinoid acid; Stem cell