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

Kirsty E Clarke,Daniel M Tams,Andrew P Henderson,Mathilde F Roger,Andrew Whiting,Stefan A Przyborski

doi:10.1016/j.neuint.2016.12.009

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.

Highlights

During neuronal development, dynamic processes involving cytoskeletal remodelling occur, known as neuritogenesis
Impaired neurite outgrowth has been implicated in pathologies including central nervous system trauma (Chen et al, 2000; Prinja et al, 2000; Silver and Miller, 2004; McKeon et al, 1991), Down's Syndrome (Bahn et al, 2002; Murtomaki et al, 1992), schizophrenia (Ozwki et al, 2003) and neurodegenerative diseases such as Alzheimer's (Petratos et al, 2008; Postuma et al, 2000) and Parkinson's Disease (Takenouchi et al, 2001)
As neurite inhibition may be common to such a large variety of neurological disorders, it highlights the need for a well characterised and physiologically representative model of the process that can screen the ability of small molecules to recover such inhibition and provide potential therapeutic strategies

Summary

Introduction

Dynamic processes involving cytoskeletal remodelling occur, known as neuritogenesis. The remodelling of the actin cytoskeleton drives protrusion and motility of the growth cone; actin dynamics play an important role during neuritogenesis (Dent et al, 2011). K.E. Clarke et al / Neurochemistry International 106 (2017) 74e84 neurodegenerative diseases (Petratos et al, 2008; Postuma et al, 2000; Takenouchi et al, 2001; Winner et al, 2011). Clarke et al / Neurochemistry International 106 (2017) 74e84 neurodegenerative diseases (Petratos et al, 2008; Postuma et al, 2000; Takenouchi et al, 2001; Winner et al, 2011) This highlights the importance of in vitro models of neuritogenesis to enhance our understanding of the process and to screen potential therapeutic molecules

Methods

Results

Conclusion