Inhibition of IGF-1Rα affects the differentiation fate of rat optic cup-derived retinal stem cells to retinal ganglion cells in vitro

Abstract

Aim: To explore the impact of insulin-like growth factor-1 receptor α (IGF-1Rα) on the differentiation fate of optic-cup-derived retinal stem cells (OC-RSCs) into retinal ganglion cells (RGCs) in vitro.

Methods: OC-RSCs were isolated from optic cups of rats on embryonic day 12.5, and high-purity OC-RSCs were obtained by conditioned culture and passage. Differentiation of OC-RSCs into RGCs under different serum concentrations was examined using flow cytometry, and the serum concentration with high interference with differentiation ratio was selected. Furthermore, the effect of blocking IGF-1Rα on the differentiation of OC-RSCs into RGCs was analyzed through immunocytochemistry and Western blotting.

Results: Immunohistochemical analysis revealed IGF-1Rα was highly expressed in rat embryos at day 12.5. OC-RSCs were isolated and purified, and high-purity OC-RSCs were obtained. When 2.5% serum was administered, the ratio of differentiated RGCs (Thy-1.1 positive) decreased significantly, and the results of immunoblotting also confirmed the blockade of IGF-1Rα reduced Thy-1.1 protein expression.

Conclusion: IGF-1Rα blocking can reduce the differentiation of OC-RSCs into RGCs.

Keywords: differentiation fate; insulin-like growth factor-1 receptor α; rat optic cup; retinal ganglion cell; retinal stem cells.

Figure 1. Expression of insulin-like growth factor-1 receptor alpha (IGF-1Rα) in the developing retina and OC-RSCs

A: Embryonic day 12.5; B: Embryonic day 13.5; C: Embryonic day 15.5; D: Fluorescence detection of IGF-1Rα expression on the surface of OC-RSCs; E: FCM of IGF-1Rα expression on the surface of OC-RSCs. A-C, red, IGF-1Rα, bar=100 µm; D, green, IGF-1Rα, bar=20 µm. OL: Outer layer; IL: Inner layer; FCM: Flow CytoMetry; OC-RSC: Optic-cup-derived retinal stem cells.

Figure 2. Morphology and identification of OC-RSCs

A: Passage 0; B: Passage 3; A and B: bar=100 µm; C: Co-expression of CHX10 and BrdU markers; D: Co-expression of PAX6 and BrdU markers, C and D, bar=20 µm. CHX10: Ceh Homeobox 10; PAX6: Paired box 6; BrdU: Bromodeoxyuridine; OC-RSC: Optic-cup-derived retinal stem cells; DAPI: 4′,6-diamidino-2-phenylindole.

Figure 3. Differentiation of OC-RSCs into RGCs under different serum concentrations

A: Flow cytometry analysis of OC-RSCs differentiation; B: Statistical chart of differentiation of OC-RSCs under different serum concentrations (n=6), ^aP<0.05; ^bP<0.01; ^cP<0.001; ^dP<0.0001; ns: No significance. FBS: Fetal bovine serum; OC-RSC: Optic-cup-derived retinal stem cells; RGCs: Retinal ganglion cells; FITC-H: Fluorescein isothiocyanate.

Figure 4. Immunocytochemical analysis of the IGF-1Rα antibody inhibiting the differentiation of OC-RSCs into RGCs for 7d

A-C: No IGF-1Rα antibody was added at a serum concentration of 2.5%; B: IGF-1Rα antibody 1 µg/mL was added at a serum concentration of 2.5%; green, Thy 1.1; red, PI staining; bar=20 µm. IGF-1Rα: Insulin-like growth factor-1 receptor alpha; OC-RSC: Optic-cup-derived retinal stem cells; RGCs: Retinal ganglion cells; Thy 1.1: Thymus cell antigen 1.1; PI: Propidium iodide.

Figure 5. Western blot analysis of the IGF-1Rα antibody inhibiting the differentiation of OC-RSCs into RGCs for 7d

A: Western blot analysis of the RGCs marker Thy-1.1; B: Statistical analysis of Western blot of the RGCs marker Thy-1.1 (n=3), ^cP<0.001; C: Gray value analysis of Western blot of RGCs marker Thy-1.1. IGF-1Rα: Insulin-like growth factor-1 receptor alpha; Ab-IGF-1Rα: Antibody insulin-like growth factor-1 receptor alpha; OC-RSC: Optic-cup-derived retinal stem cells; RGCs: Retinal ganglion cells; Thy 1.1: Thymus cell antigen 1.1.