Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Author Hayashi, Mirian A. F. Autor UNIFESP Google Scholar
Guerreiro, Juliano R. Google Scholar
Cassola, Antonio C. Google Scholar
Lizier, Nelson F. Autor UNIFESP Google Scholar
Kerkis, Alexandre Google Scholar
Camargo, Antonio C. M. Google Scholar
Kerkis, Irina Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Ctr Toxinol Aplicada CEPID
Universidade de São Paulo (USP)
Inst Butantan
Atividades Vet Ltda Genet Aplicada
Abstract Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). the AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. the expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. in contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.
Language English
Sponsor Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Date 2010-12-01
Published in Tissue Engineering Part C-methods. New Rochelle: Mary Ann Liebert Inc, v. 16, n. 6, p. 1493-1502, 2010.
ISSN 1937-3384 (Sherpa/Romeo, impact factor)
Publisher Mary Ann Liebert Inc
Extent 1493-1502
Origin http://dx.doi.org/10.1089/ten.tec.2009.0788
Access rights Closed access
Type Article
Web of Science ID WOS:000284627000027
URI http://repositorio.unifesp.br/handle/11600/33148

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