title: Functional identification of cell phenotypes differentiating from mice retinal neurospheres using single cell calcium imaging.
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 Ca(2+) 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. Ca(2+) 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.
authors.affiliation: Neuroprotection and Neurogenesis in Brain Repair, Center for Neurosciences and Cell Biology of Coimbra, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal.
authors.forename: R A
authors.initials: RA
authors.lastname: De Melo Reis
authors.forename: C S
authors.initials: CS
authors.lastname: Schitine
authors.forename: A
authors.initials: A
authors.lastname: Köfalvi
authors.forename: S
authors.initials: S
authors.lastname: Grade
authors.forename: L
authors.initials: L
authors.lastname: Cortes
authors.forename: P F
authors.initials: PF
authors.lastname: Gardino
authors.forename: J O
authors.initials: JO
authors.lastname: Malva
authors.forename: F G
authors.initials: FG
authors.lastname: de Mello
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D019070:Cell Lineage / Q000187:drug effects; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology; D000089662:Doublecortin Protein; D021621:Imaging, Three-Dimensional / Q000379:methods*; D051379:Mice; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010641:Phenotype; D012160:Retina / Q000166:cytology*; D059010:Single-Cell Analysis / Q000379:methods*; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D015415:Biomarkers; D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; C000714847:Dcx protein, mouse; D000089662:Doublecortin Protein; D002118:Calcium