Functional and molecular characterization of ex vivo cultured epiretinal membrane cells from proliferative diabetic retinopathy

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Abstract

Purpose

To characterize the cell surface marker phenotype and function of ex vivo cultured cells growing out of human epiretinal membranes (ERMs) from proliferative diabetic retinopathy (PDR).

Methods

All tissue collection complied with the Guidelines of the Helsinki Declaration. ERMs were obtained from vitrectomies due to intravitreal hemorrhage in PDR. Ex vivo cultivation under adherent conditions was performed in DMEM supplemented with FBS and the cell surface marker phenotype determined. Release of IL-6, IL-8 and TNFalpha was measured upon activation of the cells with TLR lingands and TNFalpha. The dynamics of the intracellular calcium was measured using fluorescent dye Fura-2 and imaged in response to mechano-stimulation.

Results

The cultivated ERMs formed proliferating cell monolayers when cultivated ex vivo. These cells were negative for endothelial markers (CD31, VEGFR2), partially positive for hematopoietic (CD34, CD47) and mesenchymal markers (CD90, PDGFRb, CD73) and negative for CD105. IL-6, IL-8 and TNFalpha secretion could be measured upon activation of the cells by LPS, Poly I:C and TNFalpha. Mechano-stimulation of the outgrowing cells induced intracellular calcium propagation representing their functional viability.

Conclusion

ERMs from PDR contain cells of hematopoietic and mesenchimal origin which have proliferative potential, release pro-inflammatory cytokines upon selective inflammatory stimulation and show functionality reflected through calcium dynamics upon mechano-stimulation.

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