The barrier function, autophagy and inflammatory responses are similar under diabetic stimuli in retinal pigment epithelial cells derived from human induced pluripotent stem cells from the diabetic patients and the normal controls

Abstract

Purpose : In diabetic retinopathy the high blood glucose induces alterations in retinal function and can lead to visual impairment. In previous studies with immortalised retinal pigment epithelial (RPE) cell lines diabetic stimuli i.e. high glucose, cytokines and hypoxia showed alterations in the barrier properties. Here we studied the effects of high glucose and high insulin concentration on the functionality of normal control or diabetic human induced pluripotent stem cell (hiPSC) RPE cells.

Methods : The hiPSC-RPE cell lines derived from type 1 diabetic (T1D, 04511.WTS), type 2 diabetic (T2D, 08002. DMS, 08102.DMS, 08203.DMS) and normal control (CTRL, EURCCS_10802, EURCCS_10902, EURCCS_10212) patients were grown for 4-6 weeks under high or low glucose, and high or low insulin and subjected to cytokine stimulation (H₂O₂ or tumour necrosis factor α (TNFα)) or autophagic stimulation (AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), starvation or resveratrol. The barrier function of the epithelium was analysed with permeability assays, gene expαression with PCR, morphology with immunofluorescence (IF) and electron microscopy (EM), MMP2 and MMP9 activity with zymography and autophagic activity with Western blotting (WB).

Results : All the cell lines acquired RPE specific morphology, and gene expression profile under high and low glucose and high and low insulin. There were no statistically significant alterations in barrier function or tight junction morphology or IF staining in any cell lines in above mentioned stimuli. The proMMP2 was reduced after H₂O₂ stimulation and proMMP9 was shown to be upregulated under TNFα exposure. Whereas AICAR and resveratrol induced cell type independent but glucose and insulin concentration dependent variation on the p62 or LC3 expression.

Conclusions : The glucose and insulin concentration, as well as cytokine and autophagy stimulation induced alteration on cellular functions, but these were similar in all cell lines. This data thus suggest that the hiPSC-RPE T1D and T2D cells function similarly in respect of morphology, barrier function, autophagic or matrix metalloproteinase activity when compared to hiPSC-RPE WT cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.