April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
DNA methylation profiling of limbal- and fibroblast-derived iPSC suggests retention of some epigenetic signatures of parental cells
Author Affiliations & Notes
  • Tanya M Spektor
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Mehrnoosh Saghizadeh
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Dhruv Sareen
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Loren Alexis Ornelas
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Vincent Funari
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Jie Tang
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Vasu Punj
    Epigenome Center, University of Southern California, Los Angeles, CA
  • Yaron S Rabinowitz
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Clive Svendsen
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
  • Alex Ljubimov
    Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA
    6David Geffen School of Medicine, UCLA, Los Angeles, CA
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 6038. doi:
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      Tanya M Spektor, Mehrnoosh Saghizadeh, Dhruv Sareen, Loren Alexis Ornelas, Vincent Funari, Jie Tang, Vasu Punj, Yaron S Rabinowitz, Clive Svendsen, Alex Ljubimov; DNA methylation profiling of limbal- and fibroblast-derived iPSC suggests retention of some epigenetic signatures of parental cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6038.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Limbal epithelial stem cell (LESC) deficiency represents a significant clinical problem especially in bilateral cases. Induced pluripotent stem cells (iPSC) may be a promising source of LESC allowing standardized and continual propagation and banking. Our objective was to find out whether iPSC would retain some DNA methylation signatures of parental cells, which could facilitate their redifferentiation into the original cells.

Methods: LESC obtained from donor corneoscleral rims and human skin fibroblasts were reprogrammed using Yamanaka’s episomal vectors to generate iPSC. Obtained clones expressed stemness markers, had normal karyotype, and produced teratomas in nude mice. DNA extracted from cells underwent bisulfite conversion and cleanup for methylation analysis using Qiagen Kits. Illumina Infinium Methylation 450k Beadchips were used to generate DNA methylation profiles of limbal-derived, fibroblast-derived iPSC and parental cell lines. Unique numbers of hyper- and hypomethylated genes were identified globally and for LESC-associated genes.

Results: iPSC clones expressed stemness markers, had diploid karyotypes, and produced teratomas in nude mice representing three germ layers. Analysis of Venn diagrams showed that in comparison to parental LESC, limbal-derived iPSC had about 55% fewer hyper- or hypomethylated genes compared to parental fibroblasts. Fibroblast-derived iPSC had a similar tendency. Hierarchical analysis identified distinct clusters of genes in both limbal-derived and fibroblast-derived iPSC whose methylation profiles became more similar to parental cells upon growth on denuded human amniotic membrane resembling limbal basement membrane.

Conclusions: The data suggest that iPSC differentiation to a desired lineage may be facilitated by their generation from the same tissue. This may be related to preservation of parental tissue epigenetic methylation signatures in iPSC and use of biological substrata similar to the natural niche of parental cells.

Keywords: 480 cornea: basic science  
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