Development of novel human cellular models for neurotoxicity studies

Dissertation to obtain master degree in Genética Molecular e Biomedicina

Information currently available on neurotoxicity of chemicals is scarce and there are a growing number of new compounds to be tested. Therefore, new strategies are necessary to identify neurotoxic agents with speed, reliability and respect for animal welfare. The limited availability of primary human brain cells means that there is a need for human cell lines that reliably model human neurons and astrocytes. Despite the advances in stem cell research, numerous challenges must be overcome before this technology can be widespread used, such as low differentiation efficiency. Human pluripotent embryocarcinoma NTera2/cloneD1 (NT2) cell line is an alternative cell source from which neurons and astrocytes can be derived in vitro. The aim of this work was to develop scalable and reproducible novel human cellular models using NT2 cells as source of differentiated neural phenotypes. A 2D culture system for astrocytic differentiation was implemented. After 4 weeks of differentiation with retinoic acid followed by 5 weeks maturation with mitotic inhibitors, astrocytes obtained expressed vimentin, GFAP, S100- and GLT-1 as characterized by immunodetection and qRT-PCR. Then, a 3D culture approach was adopted, using stirred suspension culture systems, in which cell-cell and cell-extracellular matrix interactions occur, mimicking better the in vivo situation. NT2 cells, inoculated as single cells, spontaneously aggregated without compromising their pluripotency. Optimization of stirring rate allowed control of aggregate size along time. After 3 weeks of RA treatment and 2 weeks of maturation, neurons expressing βIII-tubulin, MAPs and synaptophysin and astrocytes expressing vimentin, GFAP, S100- and GLT-1 were detected, as characterized by immunodetection and qRT-PCR. Furthermore, astrocytes presented a 2.5-fold higher yield than that observed in 2D culture systems. Results showed that NT2 differentiated cells are promising models for neurotoxicity testing. Furthermore, the 3D culture systems developed herein can contribute to increase the relevance of these studies, recapitulating human neuron-astrocyte interactions in a 3D cellular context.

Fundação para a Ciência e Tecnologia - PTDC/EEB-BIO/112786/2009