Functional identification of cell phenotypes differentiating from mice retinal neurospheres using single cell calcium imaging

Cell Mol Neurobiol. 2011 Aug;31(6):835-46. doi: 10.1007/s10571-011-9673-6. Epub 2011 Mar 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Brain-Derived Neurotrophic Factor / pharmacology
  • Calcium / metabolism*
  • Cell Differentiation* / drug effects
  • Cell Lineage / drug effects
  • Ciliary Neurotrophic Factor / pharmacology
  • Doublecortin Protein
  • Imaging, Three-Dimensional / methods*
  • Mice
  • Neuroglia / cytology
  • Neuroglia / drug effects
  • Neuroglia / metabolism
  • Neurons / cytology*
  • Neurons / drug effects
  • Neurons / metabolism
  • Phenotype
  • Retina / cytology*
  • Single-Cell Analysis / methods*
  • Spheroids, Cellular / cytology*
  • Spheroids, Cellular / drug effects
  • Spheroids, Cellular / metabolism
  • Stem Cells / cytology
  • Stem Cells / drug effects
  • Stem Cells / metabolism

Substances

  • Biomarkers
  • Brain-Derived Neurotrophic Factor
  • Ciliary Neurotrophic Factor
  • Dcx protein, mouse
  • Doublecortin Protein
  • Calcium