Development of a high throughput fluorometric assay with small-scale Neurosphere cultures for the identification of factors that promote oligodendrogenesis

MOLINARI YAMILA A.; BYRNE AGUSTIN J; FRANCO PAULA G.

Ciudad de Buenos Aires

Congreso; XXXVI Reunion Anual de la Sociedad Argentina de Investigacion en Neurociencia; 2021

Sociedad Argentina de Neurociencias (SAN)

In demyelinating diseases, like Multiple Sclerosis, myelin is progressively lost due to oligodendrocyte damage. Since the identification of undifferentiated neural stem cells (NSC) with the ability to regenerate neural lineages, new potential alternatives have emerged for these pathologies.In this work, by using transgenic mouse strains expressing green fluorescent protein (GFP), we developed a small-scale, high-throughput cell culture fluorometric assay that allows the identification of factors that could enhance oligodendrogenesis from NSC. Dissociated neurospheres from ACT::GFP mice were used to optimize fluorometric detection of DNA content by Höechst staining and quantification of viable cells by GFP expression, using a multiplate fluorometer reader. Dissociated neurospheres from CNP::GFP mice, expressing GFP under 2´,3´-Cyclic-nucleotide 3´- phosphodiesterase (CNPase) promoter, were used for the detection of oligodendroglial lineage by the same fluorometric method. We found a 45% increment in GFP fluorescence after treatment with prooligodendrogenic factor PDGF-BB respect to controls, indicating that this method effectively detects the activation of oligodendrogenesis from NSC. This novel approach could be used for the screening of large numbers of factors or drugs at different doses for the identification of possible candidates that promote oligodendrogenesis from NSC that could be used for the development of new therapeutical approaches for demyelinating diseases.