Functional and molecular characterization of ex vivo cultured neuronal- and glial- like cells from internal limiting membrane

Abstract

Purpose: To test the proliferating potential and characterize structurally and functionally ex vivo cultured cells growing out of human internal limiting membranes (ILMs).

Methods: All tissue collection complied with the Guidelines of the Helsinki Declaration and was approved by the National Medical Ethics Committee of Slovenia. ILMs were obtained from uneventful vitrectomies from cases with idiopathic epiretinal membrane. Ex vivo cultivation under adherent conditions in DMEM supplemented with FBS was performed and followed up to 6 days. The dynamics of the intracellular calcium was measured using fluorescent dye Fura-2 (excitation: 360 and 380 nm) and imaged in response to pharmacological stimulation by acetylcholine (ACh). The sequence of images was acquired using a WinFluor software and CCD camera. Morphological and immunocytochemical characterization of the cells was also carried out.

Results: The cells from the ILMs formed sphere-like structures when cultured ex vivo. The diameter of the spheres increased by 5% at day 6 and kept an increasing tendency over a month. The cells growing out of the ILM spheres had a mainly glial- and some neuronal- like morphology. Stimulation of these cells with ACh induced intracellular calcium propagation in the neuronal-like cells from the soma to the dendrites resembling action potential, while in the glial-like cells the calcium did not propagate. Immunocytochemistry confirmed the glial and neuronal distribution of the cells.

Conclusions: ILMs contain cells of neuronal- and glial- like origin which has proliferative potential, show functionality reflected through calcium dynamics upon ACh stimulation, and corresponding molecular phenotype.

Keywords: 562 inner retina dysfunction: biochemistry and cell biology • 691 retina: proximal (bipolar, amacrine, and ganglion cells)