Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging |
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Authors: | R?A?De?Melo Reis C?S?Schitine A?Kofalvi S?Grade L?Cortes P?F?Gardino J?O?Malva Email author" target="_blank">F?G?de?MelloEmail author |
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Institution: | 1.Neuroprotection and Neurogenesis in Brain Repair, Center for Neurosciences and Cell Biology of Coimbra, Faculty of Medicine,University of Coimbra,Coimbra,Portugal;2.Neuromodulation and Metabolism, Center for Neurosciences and Cell Biology of Coimbra, Faculty of Medicine,University of Coimbra,Coimbra,Portugal;3.Laboratório de Neuroquímica,Instituto de Biofísica Carlos Chagas Filho, CCS, UFRJ,Rio de Janeiro,Brazil |
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Abstract: | Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be
used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific
drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By
using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated
retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
(AMPA) stimulated Ca2+ transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell
adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response
to muscimol. Ca2+ fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate
the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic
factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl
or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing
GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets
and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal
diseases. |
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