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Keywords:

  • cones;
  • cytokines;
  • retinal spheroids;
  • rods;
  • three-dimensional cell culture

Abstract

The role(s) of basic fibroblast growth factor (bFGF, FGF-2) in the differentiation and survival of photoreceptor (PR) cells was investigated in three-dimensional reaggregated histotypic spheres, derived from dispersed cells of the embryonic day 6 chicken embryo retina. Novel data processing methods are introduced to reliably quantify sphere sizes and spatial distributions of immunochemical signals in spheroids. Supplementation with 25 ng/mL FGF-2 increased cell proliferation, detected by bromodeoxyuridine uptake, and growth of spheroids. Immunochemical studies showed that FGF-2 decreased the number of visinin-positive and XAP-1-positive cells, including the total PR pool from early precursor until mature states, whereas the number of Pax6-positive amacrine cells was strongly increased. Notably, the relative number of PR precursors as detected by an Islet2 antibody was increased. The further differentiation of both red/green cones and then rods, as detected by CERN-906 and CERN-901 antibody binding, was much delayed. In contrast, blocking system-inherent FGF-2 by suramin showed opposite effects. Addition of both FGF-2 plus suramin resulted in nearly normal levels of PR differentiation. Terminal deoxynucleotidyl transferase dUTP nick end labelling histochemistry showed that PR apoptosis, which generally progresses with the age of spheres, was strongly increased by suramin treatment. These results suggest that in a three-dimensional retinal tissue context, FGF-2 restricts the pool of PRs in favour of cells of the inner retina, increases and maintains their precursor pool, delays their differentiation, and also protects them from apoptosis.