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.

Differentiation and maturation of oligodendrocytes in human three-dimensional neural cultures

“…PBS was used to wash off the primary antibodies and the cryosections were incubated with secondary antibodies in PBS with 5% NDS for 1 hour. The following primary antibodies were used for immunofluorescence: anti-OLIG2 45 (rabbit, 1:500, Millipore, AB9610), anti-NKX2–2 46 (mouse, 1:200, DSHB, 74.5A5), anti-PDGFRα 47 (rabbit, 1:500, Santa Cruz, sc-338), anti-O4 48 (mouse, 1:500, R&D systems, MAB1326), anti-O1 49 (mouse, 1:200, R&D systems, MAB1327), anti-MBP 50 (rat, 1:300, Millipore, MAB386), anti-GFAP 51 (rabbit, 1:1000, Agilent DAKO, Z0334), anti-Neurofilament-H 200K 52 (mouse, 1:500, Abcam, AB7795), anti-GABA 53 (rabbit, 1:1000, Sigma, A2052), anti-MAP2 54 (guinea pig, 1:2500 on coverslips, 1:10000 on cryosections, Synaptic Systems, 188004). Alexa Fluor dyes (Life Technologies) were used at 1:1000 dilution on cryosections and 1:2000 on coverslips for amplification of the signal.…”

Using Advanced Cell Culture Techniques to Differentiate Pluripotent Stem Cells and Recreate Tissue Structures Representative of Teratoma Xenografts

“…Similar strategies are used in ex vivo organotypic slice culture and to extend the differentiation, complexity, and longevity of neurospheres ( Nestor et al, 2013 ; Giandomenico et al, 2019 , 2021 ); indeed, the use of complex 3D culture strategies combining biomaterial scaffolds with PSCs in order to direct and enhance their differentiation has expanded significantly over the last decade ( Willerth and Sakiyama-Elbert, 2019 ). Initial studies were carried out using a well characterised EC cell line ( Przyborski, 2001 ; Clarke et al, 2017 ), due to shared characteristics with ES cell populations ( Andrews et al, 2005 ) but simpler culture requirements, allowing for large numbers of EBs to be generated during method optimisation. To assess the impact of seeding EBs onto the scaffold on overall viability, cell death within the structures was analysed using a caspase 3/7 dye [upstream in the apoptotic cascade ( Walsh et al, 2008 )] and TUNEL staining (DNA breaks are a marker of later stages of apoptosis).…”

Review of high-content screening applications in toxicology

“…Many protocols detail the preparation of different neuronal types and specific workflows for utilizing them in HCS [59]. Three dimensional (3D) models have been developed and used in neurite outgrowth assays for predicting neurotoxicity, such as the human pluripotent stem cell-derived 3D model [60], the 3D neurospheres [61], and the 3D dopaminergic neuronal model [62]. The 3D neurospheres from human neural progenitor cells mimic basic processes of brain development and are useful for identification of developmental neurotoxicity hazard.…”