Reprogramação de células astrogliais em neurônios utilizando pequenas moléculas orgânicas

Abstract

The reprogramming of different specialized cell types into others has been widely studied in recent years. More specifically, the generation of induced neurons (iNs) from other differentiated cells is applied to study the molecular mechanisms of neuronal differentiation, to generate humanized models of neurological and psychiatric diseases, as well to obtain iNs that can be used in cell therapies for neurodegenerative diseases or acute lesions in the central nervous system. The first type of non-progenitor cell converted to iNs through expression of an exogenous gene was astroglia. Then, non-neural cells, such as fibroblasts and hepatocytes, were also reprogrammed into iNs by genetic manipulation. More recently, the use of small molecules, capable of interfering with specific intracellular signaling cascades, has been proposed as an alternative for the reprogramming of differentiated cells in iNs without direct gene manipulation. However, it is still unclear what is the best combination of small molecules to reprogram astroglial cells isolated from the postnatal brain. In this work, we evaluated the possibility of reprogramming astrocytes in iNs, using a combination of small molecules previously used to reprogram embryonic fibroblasts. For this, astrocytes isolated from the neocortex and cerebellum of postnatal mice were cultured and exposed to small molecules for 8-20 days. After this period, the culture cell phenotype was evaluated by immunocytochemistry, RTqPCR and time-lapse video-microscopy. We evaluated aspects such as the expression of mRNA and specific proteins of neurons and astrocytes, morphology, cell survival and proliferation. Our results indicate that only a fraction of the cultured astrocytes acquire typically neuronal characteristics when exposed to small molecules. In addition, some of these cells maintain astrocytic properties, indicating an incomplete reprogramming state. Time-lapse video-microscopy analyzes also indicate that treatment with small molecules leads to increased cell death, which could contribute to the low conversion rate in iNs observed. Alternatively, the combination of molecules used may not be the most suitable to reprogram astrocytes into iNs, indicating the need for different combinations for this purpose.