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. 2017 Jun;106:74-84.
doi: 10.1016/j.neuint.2016.12.009. Epub 2016 Dec 21.

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

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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 et al. Neurochem Int. .
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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.

Keywords: 3D culture; Cell-differentiation; Neuritogenesis; Neuronal; Retinoid acid; Stem cell.

Figures

Image 1
Fig. 1
Fig. 1
Induction of stem cell differentiation by retinoic acid. Representative phase contrast images of stem cells cultured as 2D monolayers (A) and subsequently treated with ATRA (B), EC23 (C) and AH61 (D) for 7 days. Flow cytometry (E) analysis of cellular expression of the stem cell marker, SSEA-3 (E) and the early neuronal marker, A2B5 (F) from 2D monolayers treated with retinoid compounds for 7 days (data represent mean ± SEM, n = 3). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Analysis of gene expression of transcription factors; Nanog (G), Oct4 (H) and PAX6 (I) measured by Q-PCR. Expression is relative to the untreated control (data represent mean ± SD, n = 3). Scale bars: 200 μm.
Fig. 2
Fig. 2
Development of an efficient method for the quantification of neurite outgrowth. Representative images showing TUJ-1 positive neurites (green) from neurospheres have been traced using image J software (white). All neurites from each neurosphere were counted (A) and compared with a sampling method (B) (see text for further details). Scale bars: 500 μm. Quantification of the number of neurites per neurosphere (C) and neurite density (D) for neurospheres differentiated with a range of concentrations of ATRA (0.001 μM-10 μM), and quantified using sampling method (data represent mean ± SEM, n = 2–8). No significant (ns) difference was found between counting all neurites (fully counted) and the more efficient sampling method developed. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Induction of neural differentiation using natural and synthetic retinoids. Representative confocal images of neurospheres (A) differentiated with 0.01 μM ATRA (a,d), EC23 (b,e) and AH61 (c,f) and subsequently cultured in 2D. TUJ-1 positive neurites are highlighted in green, and nuclei in blue. Scale bars: 200 μm. High magnification image of neurites from a typical neurosphere differentiated with 0.01 μM EC23 (e’). Scale bar: 100 μm. Quantification examining the number of neurites per neurosphere (B) (data represent mean ± SEM, n = 15–24), and the number of neurites per μm of aggregate circumference, neurite density (C) (data represent mean ± SEM, n = 15–24). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Characterisation of neuronal differentiation through the stages of neurite outgrowth from neurospheres over 20 days in 2D culture. Expression over time of nestin (A–D), neurofilament-L (E–H), neurofilament-M (I–L), neurofilament-H (M–P), and TUJ-1 (Q–T). Scale bars: (A–P): 50 μm, (Q–T): 100 μm.
Fig. 5
Fig. 5
Development of a novel 3D model of neurite outgrowth. Scanning electron micrograph demonstrating the internal structure of the scaffold membrane (A) with voids of 40 μm and interconnecting windows of 13 μm. Photograph of Alvetex® Scaffold 12-well inserts (B). Extensive neurite outgrowth can be observed within the 3D scaffold (C) with TUJ-1+ positive neurites staining green and nuclei blue, imaged using confocal microscopy. The majority of the neurosphere remains on top of the scaffold membrane (D) with neurites growing into the 3D material. Neurites can be visualized on the bottom of the scaffold (E), having grown through the 200 μm thick Alvetex® Scaffold membrane. Scale bars: 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Development of a novel 3D co-culture model to study the interactions between neurons and glial cells. Representative heamatoxylin and eosin staining of U118MG cells cultured alone within a 3D scaffold (A) (scale bar: 100 μm), along with CSPG (B) and brevican (C) immunostaining (scale bars: 50 μm). In the absence of co-cultured glioma cells, neurites sprout from the neurospheres and growth can be observed through the scaffold and visualized from the bottom of the membrane (D a-c). During co-culture, the glioma cells inhibit neurite outgrowth. Neurites grow over the surface of the neurosphere (D e) and do not penetrate the scaffold in the presence of the glioma cells (D f-h). Cross-sections of both control and co-cultured neurospheres were stained both with the neuronal marker TUJ-1+ (D c,g) and with haematoxylin and eosin staining (D d,h). Glioma cell induced neurite inhibition can be suppressed by application of the ROCK inhibitor, Y-27632 and significant numbers of neurites penetrate the scaffold as visualized from the bottom of the scaffold (E). Quantification of neurite outgrowth from neurospheres cultured with and without glioma cells and with the addition of Y-27632 (F) (data represent mean ± SEM, n = 3). Scale bars: (D a,b,e,f, E): 250 μm, (D c,g,d,h): 100 μm.

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