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Ruchi Shah, Tanya M Spektor, Vasu Punj, Sue Turjman, Sean Ghiam, Jiho Kim, Mehrnoosh Saghizadeh, Andrei A Kramerov, Alexander V Ljubimov; Differential DNA methylation between diabetic and non-diabetic human corneolimbal cells and limbal-derived iPSCs. Invest. Ophthalmol. Vis. Sci. 2019;60(9):934.
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© ARVO (1962-2015); The Authors (2016-present)
Diabetes mellitus appears to be an epigenetic disease with DNA methylation and histone acetylation changes. Diabetic corneal epithelial alterations include persistent defects and impaired wound healing, which may occur due to the dysfunction of limbal epithelial stem cells (LESC). Our purpose was to compare DNA methylation patterns of diabetic and non-diabetic limbal-derived induced pluripotent stem cells (iPSC) and primary LESC, and to identify differentially methylated diabetic markers that can be targeted for treatment.
DNA was extracted from 3 diabetic and 4 non-diabetic primary limbal epithelial cells (LEC) isolated from donor corneoscleral rims and enriched in LESC, and from 3 limbal-derived iPSC. After bisulfite conversion, DNA methylation analysis was performed using Illumina Infinium Methylation 450k Beadchips. Scratch wound healing was carried out in siRNA transfected or recombinant WNT5A (200ng/mL) treated LEC. Protein expression was determined in LEC and limbal tissue from 5 diabetic and 5 non-diabetic donors using Western blot and immunostaining.
DNA methylation analysis showed gene clusters with common significant methylation differences between non-diabetic and diabetic LEC. Conversely, another gene cluster showed similarity between diabetic iPSC and non-diabetic LEC but not with diabetic LEC. In the differentially methylated cluster, WNT5A gene was hypermethylated in diabetic LEC. This finding was validated by the decreased WNT5A expression in diabetic vs. non-diabetic limbus and LEC. siRNA knockdown of WNT5A expression in non-diabetic LEC showed decreased wound healing as compared to the control. In contrast, treatment of diabetic LEC with recombinant WNT5A resulted in accelerated wound healing as compared to the untreated control.
We report for the first time the similarity in the methylation patterns of certain genes in diabetic iPSC and non-diabetic primary LEC, indicating a possible way of normalizing diabetic LEC through iPSC generation. On the other hand, there is reproducible differential methylation of diabetic and non-diabetic primary LEC, confirming epigenetic changes in diabetic limbal epithelium. We have identified WNT5A as a new diabetic marker in the cornea with increased gene methylation and decreased protein expression, which can be modulated to accelerate diabetic epithelial wound healing.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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