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Open Access Highly Accessed Methodology article

A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

Patricia García-Parra12*, Marcos Maroto3, Fabio Cavaliere4, Neia Naldaiz-Gastesi12, José Iñaki Álava5, Antonio G García3, Adolfo López de Munain2 and Ander Izeta1

Author Affiliations

1 Tissue Engineering Laboratory, Department of Bioengineering, Instituto Biodonostia, Hospital Universitario Donostia, San Sebastian, 20014, Spain

2 Neuroscience Area and CIBERNED, Instituto Biodonostia, Hospital Universitario Donostia, San Sebastian, 20014, Spain

3 Instituto Teófilo Hernando de I + D del Medicamento. Departamento de Farmacología y Terapeútica and Servicio de Farmacología Clínica del IIS Hospital Universitario de La Princesa, Facultad de Medicina, Universidad Autónoma de Madrid (UAM), Madrid, 28029, Spain

4 Department of Neuroscience and CIBERNED, University of Basque Country (UPV/EHU), Zamudio, 48170, Spain

5 Basque Culinary Center R&D, San Sebastian, 20009, Spain

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BMC Neuroscience 2013, 14:48  doi:10.1186/1471-2202-14-48

Published: 18 April 2013

Abstract

Background

The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells.

Methods

Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates.

Results

When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons.

Conclusions

We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.

Keywords:
Neural extracellular matrix; Subventricular zone; Neuronal culture; Neural progenitor cells; Dopaminergic differentiation